Rapid Detection and Characterization of Carbapenemases in Enterobacterales with a New Modified Carbapenem Inactivation Method, mCIMplus

Abstract

The worldwide emergence and spread of antimicrobial resistance in Gram-negative bacteria are severely limiting therapeutic options and thus constitute a major public health threat. The timely accurate detection of carbapenemase producers and the determination of carbapenemase class according to the Ambler classification can guide antimicrobial therapy and facilitate infection control measures. A modified version of the carbapenemase inactivation method (CIM), mCIM, was described and approved by the CLSI in 2017. We evaluated the performance of a faster new mCIM-based assay, mCIMplus, which can detect carbapenemase activity within 8 h and characterize the carbapenemase according to the Ambler classification in 20 h. A panel of 137 isolates producing carbapenemases (GES, IMP, KPC, NDM, OXA-48, OXA-48-like, and VIM enzymes) and 22 non-carbapenemase-producing isolates was used to evaluate the performance of mCIMplus. We evaluated the detection of carbapenemase activity at 8 and 20 h. Carbapenemase class was determined, with specific inhibitors, at 20 h. The sensitivities of mCIMplus were 99.3% at 8 h and 98.5% at 20 h. Its specificity was 100% regardless of culture time. Based on a decision algorithm, this test successfully identified the carbapenemase class for 98.4% of the tested isolates (127/129). Characterization was correct for 100, 95, and 100% of Ambler class A, B, and D isolates, respectively. This test can, therefore, be used to detect carbapenemase activity within 8 h and to determine carbapenemase class within 20 h. It constitutes a very affordable (<€1 per isolate) and reliable technique requiring only basic laboratory equipment.

Keywords: Ambler classification; EDTA; Enterobacterales; NDM; OXA-48; carbapenemase; carbapenemase inactivation method; mCIM; phenotypic detection; phenylboronic acid.

FIG 1

Decision algorithm of the mCIMplus test for detecting the carbapenemase produced by a test isolate and determining the class of enzyme to which it belongs. A 10-μg meropenem disk (MEM) was used, in the presence or absence of a specific carbapenemase inhibitor. CPE, carbapenemase-producing Enterobacterales; EDTA, ethylenediaminetetraacetic acid (a class B carbapenemase inhibitor); PBA, phenylboronic acid (a class A carbapenemase inhibitor); TSB, tryptic soy broth; Ø, inhibition zone diameter.

FIG 2

Example of the carbapenemase detection test with reading at 8 h and of carbapenemase characterization with reading at 20 h for the mCIMplus test. Black dotted lines represent the inhibition zone circumferences. (A) Presence of carbapenemase (SL75, NDM positive) at 8 h. The culture of E. coli ATCC 25922 grows with an inhibition zone diameter of ≤10 mm around the meropenem disk (MEM). (B) Absence of carbapenemase (T3, absence of carbapenemase) at 8 h. An inhibition zone diameter of >10 mm is observed. (C) Identification of a class B carbapenemase (SL77, NDM positive) at 20 h. The culture of E. coli ATCC 25922 grows in contact with the meropenem disk incubated with phenylboronic acid (PBA), and an inhibition zone diameter of >10 mm is observed around the meropenem disk incubated with EDTA. (D) Identification of a class D carbapenemase (SL3, OXA-48 positive) at 20 h. The culture of E. coli ATCC 25922 grows in contact with meropenem disks incubated with either PBA or EDTA.