Rapid detection of carbapenem resistance via MALDI-TOF MS using an innovative broth microgrowth assay.

Abstract

Objectives: Carbapenem-resistant Enterobacterales (CRE) are a growing threat to human health worldwide. This study aimed to develop a novel method, the broth microgrowth assay, for the rapid identification of CRE from culture isolates using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Methods: A total of 80 isolates, including carbapenem-resistant Escherichia coli (CRECO, n = 28), carbapenem-sensitive Escherichia coli (CSECO, n = 28), carbapenem-resistant Klebsiella pneumoniae (CRKP, n = 12), and carbapenem-sensitive Klebsiella pneumoniae (CSKP, n = 12) were collected for this study. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were measured for all the isolates via the broth microdilution method. Carbapenem resistance (R) and susceptibility (S) were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) Performance Standards for Antimicrobial Susceptibility Testing, M100, 34th Edition. These isolates were incubated both with carbapenem antibiotics (imipenem and meropenem) as a detection site and without antibiotics as a growth control site at 35°C for 1 and 2 h, respectively. Following incubation, the mixtures were centrifuged, and the supernatant was pipetted off. The remaining sediment was subsequently applied to the MALDI-TOF MS target plate, and the residual broth was subsequently absorbed via sterile filter paper. Identification of the isolates was performed via the VITEK MS system. The test was deemed valid only if the sample without antibiotics (growth control) resulted in successful species identification (confidence level > 60.0%). Percentage of isolates that successfully grew without antibiotics and were identified by MALDI-TOF MS, which is referred to as the growth efficiency. CRE were distinguished if the microorganisms could be successfully identified.

Results: After 1 h of incubation with imipenem or meropenem, the growth efficiencies of E. coli and K. pneumoniae were 83.93% and 66.67%, respectively. For Escherichia coli, the sensitivity and specificity of imipenem resistance prediction by MALDI-TOF MS were 82.14% and 100%, respectively. Conversely, meropenem demonstrated a sensitivity of 89.29% and a specificity of 100%. When Klebsiella pneumoniae was examined, both imipenem and meropenem had sensitivity and specificity values of 83.33% and 100%, respectively. After the incubation time was extended to 2 h, both antibiotics achieved perfect sensitivity and specificity of 100%, coupled with a growth efficiency of 100% for both bacterial strains.

Conclusion: Combining the broth microgrowth assay with MALDI-TOF MS offers a rapid and accurate approach to identifying CRE, thus facilitating the swift selection of appropriate antibiotics.