Rapid LC-MS/MS detection of different carbapenemase types in carbapenemase-producing Enterobacterales.

Klebsiella pneumoniae carbapenemase (KPC)-2, metallo-beta-lactamases (MBL), and oxacillinase (OXA)-48-like carbapenemases are considered the most important carbapenemases. In vitro studies have demonstrated that the carbapenemase activity of KPC-2 and MBL can be inhibited by 3-aminophenylboronic acid and ethylenediaminetetraacetic acid (EDTA), respectively. Understanding the carbapenemase types expressed in carbapenem-resistant Enterobacterales (CRE) is of great significance to clinical therapies. Liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) is fast, stable, and specific; and is considered the gold standard method for measuring small molecules. In this study, we developed carbapenemase inhibition tests combined with LC-MS/MS to rapidly identify carbapenemase types. A total of 295 clinical isolates were examined, including 212 KPC-2 producers, 29 MBL producers, 15 OXA-48-like producers, 3 KPC-2 + OXA-232 producers, and 36 carbapenem-sensitive strains. We used LC-MS/MS to determine the carbapenemase types by measuring the ratio of the hydrolyzed meropenem peak areas in the presence and absence of different inhibitors. The sensitivity and specificity of LC-MS/MS in detecting single KPC-2 producers were 97.64% and 100.00%, respectively, and 96.55% and 100.00% for MBL producers, respectively. In addition, the sensitivity and specificity of LC-MS/MS in detecting single OXA-48-like producers were both 100.00% when extending incubation time up to 2.5 h. LC-MS/MS showed excellent agreement in detecting carbapenemase types using the modified carbapenem inactivation (mCIM)/EDTA-carbapenem inactivation assay (eCIM) (kappa = 0.93 for serine carbapenemases and kappa = 0.98 for MBL carbapenemases). In this study, LC-MS/MS demonstrated excellent detection of different carbapenemase types, showing potential reliability in future clinical applications.