High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS

Abstract

Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the bla KPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the bla KPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPCnegative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.

Molecular phenotyping approaches for the detection and monitoring of carbapenem-resistant Enterobacteriaceae by mass spectrometry

“…Controversially, Sidjabat et al observed changes in the proteome in the presence of drug only, with the drug-negative proteome reflecting that of the susceptible control [52] . However, studies employing targeted methods suggest that resistant and susceptible phenotypes differ even in the absence of drug stress [53] , [54] , [55] , [56] .…”

“…The diagnostic performance of the peptides resulted in an overall accuracy of 100 % with a turnaround time <90 min. Lovison et al later adapted and validated the method and reported a sensitivity of 96.07 % and specificity of 100 % [56] . Similar protocols have been published for the detection of NDM and OXA-48-like CP isolates [53] , [55] .…”