Efficient Detection of Carbapenemase Activity in Enterobacteriaceae by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Less Than 30 Minutes

Abstract

The recognition of carbapenemase-producing Enterobacteriaceae (CPE) isolates is a major laboratory challenge, and their inappropriate or delayed detection may have negative impacts on patient management and on the implementation of infection control measures. We describe here a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based method to detect carbapenemase activity in Enterobacteriaceae. After a 20-min incubation of the isolate with 0.5 mg/ml imipenem at 37°C, supernatants were analyzed by MALDI-TOF in order to identify peaks corresponding to imipenem (300 Da) and an imipenem metabolite (254 Da). A total of 223 strains, 77 CPE (OXA-48 variants, KPC, NDM, VIM, IMI, IMP, and NMC-A) and 146 non-CPE (cephalosporinases, extended-spectrum β-lactamases [ESBLs], and porin defects), were tested and used to calculate a ratio of imipenem hydrolysis: mass spectrometry [MS] ratio = metabolite/(imipenem + metabolite). An MS ratio cutoff was statistically determined to classify strains as carbapenemase producers (MS ratio of ≥0.82). We validated this method first by testing 30 of our 223 isolates (15 CPE and 15 non-CPE) 10 times to calculate an intraclass correlation coefficient (ICC of 0.98), showing the excellent repeatability of the method. Second, 43 strains (25 CPE and 18 non-CPE) different from the 223 strains used to calculate the ratio cutoff were used as external controls and blind tested. They yielded sensitivity and specificity of 100%. The total cost per test is <0.10 U.S. dollars (USD). This easy-to-perform assay is time-saving, cost-efficient, and highly reliable and might be used in any routine laboratory, given the availability of mass spectrometry, to detect CPE.