EDTA-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Metallo-β-Lactamase-Producing Enterobacteriaceae

Abstract

The increase in the prevalence and impact of infections caused by carbapenemase-producing Enterobacteriaceae is a global health concern. Therefore, rapid and accurate methods to detect these organisms in any clinical microbiology laboratory, including those in resource-limited settings, are essential to prevent and contain their spread. It is also important to differentiate between serine- and metal-dependent carbapenemases elaborated by carbapenemase-producing isolates for epidemiologic, infection control and prevention, and therapeutic purposes. Here, we describe the development and evaluation of the EDTA-modified carbapenem inactivation method (eCIM), an assay for discriminating between serine- and metal-dependent (i.e., metallo-β-lactamases [MBLs]) carbapenemases when used in conjunction with the modified carbapenem inactivation method (mCIM). The eCIM had an overall sensitivity and specificity of 100% and was adopted by the Clinical and Laboratory Standards Institute as a method to use in combination with the mCIM to identify MBL-producing Enterobacteriaceae.

Keywords: EDTA-modified carbapenem inactivation method; MBL; carbapenemase; carbapenemase-producing Enterobacteriaceae; eCIM; mCIM; metallo-β-lactamase; modified carbapenem inactivation method; phenotypic detection.

FIG 1

Schematic diagram illustrating the mCIM and eCIM. (A) A 1-µl loopful of test organism with suspected carbapenemase activity is resuspended in two tubes containing 2 ml of TSB. One tube is devoid of EDTA (mCIM), while the other is supplemented with EDTA (eCIM). (B) A meropenem disk (10 µg) is submerged in each tube, and the tubes are incubated without shaking at 35°C in ambient air for 4 h ± 15 min. (C) After 4 h, the disks are removed from the tubes and placed on Mueller-Hinton agar plates upon which a carbapenem-susceptible reporter strain (E. coli ATCC 25922) has been freshly applied. The plates are incubated at 35°C in ambient air for 18 to 24 h before the zone sizes are recorded. Note: in the absence of a positive mCIM result the eCIM result is not applicable.

FIG 2

Photograph of Mueller-Hinton agar plates inoculated with a carbapenem-susceptible isolate (E. coli ATCC 25922) to which meropenem disks have been applied after disks were incubated with select isolates in the absence or presence of EDTA. (A) no EDTA; (B) 0.1 mM EDTA; (C) 5 mM EDTA. 1706, negative control (K. pneumoniae ATCC BAA-1706, carbapenemase negative); 1705, serine carbapenemase positive control (K. pneumoniae ATCC BAA-1705, KPC positive); 2146, metallo-β-lactamase positive control (K. pneumoniae ATCC BAA-2146, NDM positive).

FIG 3

Percentage of unhydrolyzed meropenem after 4 h of incubation of TSB inoculated with select isolates—1706, negative control (K. pneumoniae ATCC BAA-1706, carbapenemase negative); 1705, serine carbapenemase positive control (K. pneumoniae ATCC BAA-1705, KPC positive); and 2146, metallo-β-lactamase positive control (K. pneumoniae ATCC BAA-2146, NDM positive)—in the absence or presence of EDTA. No EDTA (black bars), 0.1 mM EDTA (dark gray bars), and 5 mM EDTA (light gray bars) are shown. All results represent the means of three independent experiments, with the error bars indicating the standard deviations of the mean.