Product(s) used in this publication: Reference Peptides for Targeted Proteomics - SpikeTides™ & SpikeMix™
Phenotypic detection of the OXA-48-type class D β-lactamases in Enterobacteriaceae is challenging. We describe a rapid (less than 90 min) assay for the identification of OXA-48 family carbapenemases in sub-cultured bacterial isolates based on a genoproteomic approach. Following in silico trypsin digestion to ascertain theoretical core peptides common to the OXA-48 family, liquid chromatography tandem mass spectrometry (LC-MS/MS) data-dependent acquisition was used to identify candidate peptide markers. Two peptides were selected based on performance characteristics; ANQAFLPASTFK, a core peptide common to all 12 OXA-48 family β-lactamase members, and YSVVPVYQEFAR, a highly specific peptide common to 11 of 12 OXA-48 family proteins providing the basis for an LC-MS/MS multiple reaction monitoring assay. An accuracy assessment was performed that contained 98 isolates, 26 of which were OXA-48 positive. Two additional specificity assessments were performed containing a mixture of isolates positive for OXA-48, KPC, NDM, VIM, and IMP carbapenemases. A combination of expert rules and expert judgement were applied by blinded operators to identify positive isolates. All isolates containing an OXA-48 family carbapenemase across all three test sets were correctly identified with no false positives, demonstrating 100% sensitivity (95% confidence interval [CI], 91.2-100%) and 100% specificity (95% CI, 96.2-100%) for the assay. These findings provide a framework for an LC-MS/MS-based method for the direct detection of OXA-48 family carbapenemases from cultured isolates that may have utility in predicting carbapenem resistance and tracking hospital outbreaks of OXA-48-carrying organisms.