Clinical evaluation of advanced MALDI-TOF MS for carbapenemase subtyping in Gram-negative isolates

The spread of carbapenemase-producing Enterobacterales (CPE) is emerging as a significant clinical concern in tertiary hospitals and, in particular, long-term care facilities with deficiencies in infection control. This study aims to evaluate an advanced matrix-assisted laser desorption/ionization (A-MALDI) mass spectrometry method for the identification of carbapenemases and further discrimination of their subtypes in clinical isolates. The A-MALDI method was employed to detect CPE target proteins. Enhancements were made to improve detectability and mass accuracy through the optimization of MALDI-TOF settings and internal mass calibration. A total of 581 clinical isolates were analyzed, including 469 CPE isolates (388 Klebsiella pneumoniae carbapenemases [KPC], 51 NDM, 40 OXA, and 2 GES) and 112 carbapenemase-negative isolates. Clinical evaluation of the A-MALDI demonstrated 100% accuracy and precision in identifying all the collected CPE isolates. Additionally, A-MALDI successfully discriminated individual carbapenemase subtypes (KPC-2 or KPC-3/KPC-4, OXA-48 or OXA-181 or OXA-232, GES-5 or GES-24) and also differentiated co-producing carbapenemase strains (KPC and NDM, KPC and OXA, KPC and GES, and NDM and OXA), attributed to its high mass accuracy and simultaneous detection capability. A-MALDI is considered a valuable diagnostic tool for accurately identifying CPE and carbapenemase's subtypes in clinical isolates. It may also aid in selecting appropriate antibiotics for each carbapenemase subtype. Ultimately, we expect that the A-MALDI method will contribute to preventing the spread of antibiotic resistance and improving human public health. IMPORTANCE A-MALDI clearly demonstrated excellent ability to identify CPEs such as KPC, NDM, OXA, and GES when carbapenemase is present in the strain (100% accuracy and precision). The method also successfully discriminated carbapenemase subtypes and simultaneous detection of co-producing multiple carbapenemases in a single strain. This is the first report for simultaneous and multiple detection of intact carbapenemases of KPC, NDM, OXA, and GES using matrix-assisted laser desorption/ionization mass spectrometry in a clinical isolate.