Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae

Abstract

The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories.

Keywords: Enterobacteriaceae; antimicrobial susceptibility testing; bacterial antibiotic resistance; bacteriological techniques; carbapenemase; carbapenems.

FIG 1

Distribution of mCIM zone diameter measurements. Displayed are the numbers of mCIM readings with specific zone diameter measurements during the second stage, nine-laboratory study, in which 27 isolates with carbapenemase genes and 34 isolates without carbapenemase genes were tested, generating a total of 549 mCIM results. Among the 243 mCIM results for isolates with carbapenemase genes, a total of 3 exhibited multiple small bacterial colonies within the zone of inhibition around the disk; zone size data for these 3 readings are not displayed. Isolates with carbapenemase genes (black bars) and isolates without carbapenemase genes (gray bars) are indicated.

FIG 2

Example of pinpoints within mCIM inhibition zone. During mCIM testing of some isolates, multiple small colonies were observed growing throughout the zone of inhibition of the 10-μg meropenem (MEM) disk; results were interpreted as positive when the zone of confluent growth inhibition measured ≤18 mm in diameter and as indeterminate when the zone measured ≥19 mm in diameter.

FIG 3

Reading and interpretation of mCIM results. (A) A 22-mm zone of inhibition of growth of the carbapenem-susceptible indicator organism is present around the meropenem (MEM) disk that was incubated with the negative-control organism (N), while no zone of inhibition is present around the MEM disks that were incubated with the positive-control organism (P) or the test organism (T). (B) Closer examination of the zone around the MEM disk incubated with the negative-control organism reveals a narrow ring of growth abutting the disk, which represents carryover of the test organism from the tryptic soy broth; this growth is ignored when interpreting mCIM results.