Parallel Reaction Monitoring Mass Spectrometry for Rapid and Accurate Identification of beta-Lactamases Produced by Enterobacteriaceae

The increasing spread of drug-resistant bacterial strains presents great challenges to clinical antibacterial treatment and public health, particularly with regard to beta-lactamase-producing Enterobacteriaceae. A rapid and accurate detection method that can expedite precise clinical diagnostics and rational administration of antibiotics is urgently needed. Targeted proteomics, a technique involving selected reaction monitoring or multiple reaction monitoring, has been developed for detecting specific peptides. In the present study, a rapid single-colony-processing procedure combined with an improved parallel reaction monitoring (PRM) workflow based on HRAM Orbitrap MS was developed to detect carbapenemases (Klebsiella pneumoniae carbapenemase, KPC; imipenemase, IMP; Verona integron-encoded metallo-beta-lactamase, VIM; New Delhi metallo-beta-lactamase, NDM; and oxacillinase, OXA), extended spectrum beta-lactamases (TEM and CTX-M), and AmpC (CMY-2) produced by Enterobacteriaceae. Specific peptides were selected and validated, and their coefficients of variation and stability were evaluated. In total, 188 Enterobacteriaceae strains were screened using the workflow. Fourteen out of total 19 peptides have 100% specificity; three peptides have specificity >95% and two peptides have specificity ranged from 74 similar to 85%. On the sensitivity, only nine peptides have 95 similar to 100% sensitivity. The other 10 peptides have sensitivity ranged from 27 similar to 94%. Thus, a screening method based on peptide groups was developed for the first time. Taken together, this study described a rapid extraction and detection workflow for widespread b-lactamases, including KPC, IMP, VIM, NDM, OXA, CMY, CTX-M, and TEM, using single colonies of Enterobacteriaceae strains. PRM-targeted proteomics was proven to be a promising approach for the detection of drug-resistant enzymes.