Assessment of an LC coupled with tandem MS (LC-MS/MS) tool in the detection of antimicrobial resistance mechanisms in Enterobacterales.

Objectives: To assess a new MS approach in detection of antimicrobial resistance (AMR) mechanisms in Enterobacterales and its usefulness in the routine practice of a national reference centre for carbapenemase-producing enterobacteria (CPE).

Methods: A tool utilizing LC coupled with tandem MS (LC-MS/MS) in multiple reaction monitoring mode was tested in the direct and concurrent detection of 13 AMR enzymes. These comprised carbapenemases, extended-spectrum β-lactamases (ESBLs), AmpC-like cephalosporinases, aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylases. The study included 357 Enterobacterales isolates largely from Polish hospitals (2000-23), including 292 CPE, collected by the National Reference Centre for Susceptibility Testing. The isolates were short-read sequenced to determine their resistomes, and subjected to MIC evaluation.

Results: The number of target AMR genes in the isolates was 1128 without defective and duplicated copies. When possible to deduce, the MIC patterns indicated activity of most of the genes/enzymes. The LC-MS/MS assay showed >95% sensitivity in the identification of KPC-, NDM-, VIM-, IMP- and OXA-48-type carbapenemases, CTX-M-like ESBLs, CMY-2- and DHA-type AmpCs, Aac(3)-II- and Aac(6')-Ib-like AMEs and the ArmA 16S rRNA methylase, but only 66.7% and 44.4% of GES-type ESBLs/carbapenemases and RmtB/C-like methylases, respectively. Detection specificity was almost 100% for all of the enzymes.

Conclusions: The study demonstrated good performance of the assay against multiple clinically-relevant AMR mechanisms, including main carbapenemase types, and its usefulness in a CPE reference centre was confirmed. However, further studies, e.g. with other bacterial populations, AMR mechanisms and clinical samples, are necessary to characterize its full diagnostic potential.