Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Abstract

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.

Fig. 1.

The chemical structures of meropenem, 383.464 Da (A), its disodium salt, 427.422 Da (B), a meropenem degradation product with a disrupted amide bond, 401.483 Da (C), and its trisodium salt, 467.420 Da (D).

Fig. 2.

Mass spectra of meropenem (detected mass 382.975 m/z) and its sodium salts (405.193 m/z and 427.428 m/z) (A) and meropenem with a disrupted amide bond (401.072 m/z) and its sodium salts (423.282 m/z, 445.583 m/z, and 467.839 m/z) (B). Peaks corresponding to the tested molecules are indicated with arrows. Units on the y axes represent relative intensity.

Fig. 3.

Mass spectra of the meropenem hydrolysis assay of a non-carbapenemase-producing isolate (A) and a carbapenemase (KPC-2)-producing isolate (B). Units on the y axes represent relative intensity.

Fig. 4.

Mass spectra of special interest: unreliable spectrum (A) and spectrum obtained after meropenem digestion by some carbapenemase-producing strains (KPC-2) (B). Analysis represented by the spectrum shown in panel A had to be repeated; using flexAnalysis 3.0 software with a signal-to-noise threshold of 1, the peak at ca. 383 m/z in the spectrum shown in panel B was detected. This peak may be interpreted as noise. No peak at 405 m/z was detected. Therefore, this strain was correctly interpreted as carbapenemase producer.