A sensitive and specific phenotypic assay for detection of metallo-β-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin

Abstract

Enterobacteriaceae producing carbapenemases, such as KPC or metallo-β-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 μg of EDTA, 1000 μg of dipicolinic acid (DPA), 600 μg of aminophenylboronic acid (APBA), or 750 μg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum β-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added β-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.

Comparative Evaluation of a Chromogenic Agar Medium, the Modified Hodge Test, and a Battery of Meropenem-Inhibitor Discs for Detection of Carbapenemase Activity in Enterobacteriaceae

“…MHT and Colorex KPC plates detected 4 carbapenemase producers (Table 1), but 2 plasmid-mediated AmpC producers also grew on chromogenic agar. Using the inhibitor approach, strong potentiation with M-DPA (⌬ of 6 to 12 mm) was observed among 3 MBL producers (2 VIM-1 and 1 NDM-1), but the OXA-48-producing strain could not be detected with this method; this limitation was also previously reported (7).…”

“…MHT was performed using both meropenem and ertapenem discs (Oxoid). Since our previous tests showed similar results for both meropenem and imipenem discs combined with dipicolinic acid (DPA), aminophenylboronic acid (BA), or cloxacillin (CLX) (data not shown), the discs used in this study ( ) and the interpretation of the assays were based on a recent study by Giske et al (7). Briefly, an increase of 4 mm in the inhibition zone for M-BA and 5 mm for M-CLX or M-DPA compared with the zone when the meropenem disc was used alone were considered to be positive results.…”

“…Several approaches for the detection of these microorganisms have been described, including the modified Hodge test (MHT) (3), chromogenic agars (1,13,18), and the use of carbapenem discs with the addition of specific ␤-lactamase inhibitors (7,9,10,15,16,19,22). In this study, we compared these 3 approaches with a panel of characterized Enterobacteriaceae.…”

Phenotypic and Molecular Characterization of Plasmid Mediated AmpC among Clinical Isolates of Klebsiella pneumoniae Isolated from Different Hospitals in Tehran

“…The increasing inhibition zone at least 5mm around meropenem (10µg) plus 600 µg/disc boronic acid and meropenem (10µg) plus 750 µg/disc cloxacillin simultaneously in comparison to meropenem (10µg) alone. The stock of boronic acid was prepared 30mg/ml and the stock of clocacillin was prepared 37.5 mg/ml [12].…”

Carbapenemase-producing Klebsiella pneumoniae in the Czech Republic in 2011

“…All isolates were tested for carbapenemase production by MALDI-TOF mass spectrometry (MS) meropenem hydrolysis assay [9,10]. Phenotypic identification of carbapenemases was performed by an inhibitor-based method [11]. Species identification was performed using a MALDI Biotyper Version 3.0 (Bruker Daltonik GmbH., Bremen, Germany).…”