Direct detection of intact Klebsiella pneumoniae carbapenemase variants from cell lysates: Identification, characterization and clinical implications

Abstract

Introduction: Carbapenemase-producing organisms (CPOs) are a growing threat to human health. Among the enzymes conferring antibiotic resistance produced by these organisms, Klebsiella pneumoniae carbapenemase (KPC) is considered to be a growing global health threat. Reliable and specific detection of this antibiotic resistance-causing enzyme is critical both for effective therapy and to mitigate further spread.

Objectives: The objective of this study is to develop an intact protein mass spectrometry-based method for detection and differentiation of clinically-relevant KPC variants directly from bacterial cell lysates. The method should be specific for any variant expressed in multiple bacterial species, limit false positive results and be rapid in nature to directly influence clinical outcomes.

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.

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.

Keywords: ATCC, American type culture collection; BLAST, basic local alignment search tool; CDC, Centers for Disease Control and Prevention; CPO, carbapenemase-producing organisms; CSD, charge state distribution; Carbapenem-resistant Enterobacteriaceae; Carbapenemase-producing organisms; ESI, electrospray ionization; KPC, Klebsiella pneumoniae carbapenemase; Klebsiella pneumoniae carbapenemase; LC, liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MW, molecular weight; Mass Spectrometry; PCR, polymerase chain reaction; TOF, time-of-flight; Tandem mass spectrometry; m/z, mass-to-charge ratio.

Fig. 1

Fragmentation spectrum of the N-terminal tryptic peptide of KPC-2. Sequence ladder produced using Prosight lite .

Fig. 2

Chromatographic separation of intact proteins from cell lysates show (a) the total ion current from all ionized species, and (b) the extracted ion chromatogram corresponding to KPC. Mass spectra of intact KPC-2 showing (c) the CSD of the protein; (d) expanded view of the isotopic distribution within a single charge state, average isotopic spacing of 0.0528 m/z. The theoretical m/z for the + 19 charge state, 1512.4673, is depicted with a dashed vertical line; (e) deconvoluted mass of KPC-2 from (c).

Fig. 3

Fragmentation of KPC-2 showing (a) the range from m/z 400 to 1800, sequence coverage of KPC-2 showing cleavage sites, with a 4x magnification from m/z 400 to 1000; (b) expanded view of m/z 1450–1750 range of the fragmentation spectrum.

Fig. 4

Comparison of strains with and without KPC-4. Extracted ion chromatogram (EIC) centered on the y₇⁺ fragment of KPC-4 from (a) DH5α carbapenem-susceptible E. coli lysate, (c) lysate from DH5α E. coli cells following transformation of a KPC + plasmid, and (e) cell lysate from KPC-4-producing K. pneumoniae, #104 from the CDC and FDA AR isolate Bank, from which the KPC + plasmid was isolated. The corresponding MS/MS spectra presenting (b) no evidence for KPC from the DH5α lysate, and fragment ions of KPC-4 from lysates of (d) DH5α KPC + and (f) the original KPC-producing isolate. MS spectra of the intact KPC-4 proteins from both (d) and (f) were also identified and are provided as supplemental information (Fig. S6).

Fig. 5

Comparison of MS/MS spectra between KPC variants. Fragmentation spectra of (a) KPC-2, C. freundii, CDC and FDA AR Bank #116, (b) KPC-3, K. ozaenae CDC and FDA AR Bank #96, and (c) KPC-4, E. coli CDC and FDA AR Bank #104. (d) KPC-5, P. aeruginosa CDC and FDA AR Bank #90.