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.

Clinical and laboratory considerations for the rapid detection of carbapenem-resistant Enterobacteriaceae

“…This is possible because hydrolysis of carbapenem b-lactam rings results in a measurable mass increase that can be differentiated from the native carbapenem mass on the MALDI TOF MS. Interestingly, longer incubation times are associated with false-negative results, possibly because the degradation products are further decomposed to a lighter mass that is not visible in the queried spectra of the assay. 37 Conversely, if the carbapenemase has only weak carbapenem hydrolytic activity, as is seen with the OXA-48-like enzymes, extensive incubation may be required (i.e. > 24 hours) before an appreciable change in the carbapenem spectrum is observed.…”

Mass spectrometry: a revolution in clinical microbiology?

“…Basically, the MS follows the enzymatic hydrolysis of the antimicrobial agent. The sensitivity and specificity of this approach is high (97 % and 98 % , respectively) [21] and the results are available in < 3 h [95] . However, this approach must be validated in routine laboratories to consider replacing the conventional techniques (such as cefoxitin disks, or PCR to detect the mecA gene).…”

“…Recently some resistance markers to one or more antimicrobial agents have been detected by MALDI-TOF MS. Reports suggest that MS has the ability to differentiate methicillin-susceptible S. aureus from methicillinresistant S. aureus (MRSA) strains [93,94] , and also detect carbapenem resistance activity based on the detection of degradation of β -lactam antimicrobials [20,21,95] . Basically, the MS follows the enzymatic hydrolysis of the antimicrobial agent.…”

Evaluation of Intact Cell Matrix-Assisted Laser Desorption/Ionization Timeof- Flight Mass Spectrometry for Capillary Electrophoresis Detection of Controlled Bacterial Clumping

“…In screening analysis of microbial cells the use of these two matrices is common [23][24][25][26][27][28][29][30][31][32]. However, the spectra recorded with different matrices had different distribution of peaks ( Figure 3B, 3C).…”

“…Therefore, it was impossible to ionize a sufficient number of proteins. DHB matrix has been reported to facilitate cell wall disruption and protein extraction [1,14,[27][28][29][30][31][32].…”