Conclusion
Direct detection of these enzymes contributes to the understanding of occurrence and spread of these antibiotic-resistant organisms. The ability to detect intact KPC variants via a simple LC-MS/MS approach could have a direct and positive impact on clinical therapy, by providing both direction for epidemiological tracking and appropriate therapy.
Methods
Lysates obtained directly from bacterial colonies were used for intact protein detection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Bottom-up and top-down proteomic methods were used to characterize the KPC protein targets of interest. Comparisons between KPC-producing and KPC-non-producing isolates from a wide variety of species were also performed.
Results
Characterization of the mature KPC protein revealed an unexpected signal peptide cleavage site preceding an AXA signal peptide motif, modifying the molecular weight (MW) of the mature protein. Taking the additional AXA residues into account allowed for direct detection of the intact protein using top-down proteomic methods. Further validation was performed by transforming a KPC-harboring plasmid into a negative control strain, followed by MS detection of the KPC variant from the transformed cell line. Application of this approach to clearly identify clinically-relevant variants among several species is presented for KPC-2, KPC-3, KPC-4 and KPC-5.