A Novel Peptidomic Approach to Strain Typing of Clinical Acinetobacter baumannii Isolates Using Mass Spectrometry

Abstract

Background: Acinetobacter baumannii is a common nosocomial pathogen and strain-typing methods play an important role in hospital outbreak investigations and epidemiologic surveillance. We describe a method for identifying strain-specific peptide markers based on LC-MS/MS profiling of digested peptides. This method classified a test set of A. baumannii isolates collected from a hospital outbreak with discriminatory performance exceeding that of MALDI-TOF mass spectrometry.

Methods: Following the construction of a species "pan-peptidome" by in silico translation and digestion of whole genome sequences, a hypothetical set of genome-specific peptides for an isolate was constructed from the disjoint set of the pan-peptidome and the isolate's calculated peptidome. The genome-specific peptidome guided selection of highly expressed genome-specific peptides from LC-MS/MS experimental profiles as potential peptide markers. The species specificity of each experimentally identified genome-specific peptide was confirmed through a Unipept lowest common ancestor analysis.

Results: Fifteen A. baumannii isolates were analyzed to derive a set of genome- and species-specific peptides that could be used as peptide markers. Identified peptides were cross-checked with protein BLAST against a set of 22 A. baumannii whole genome sequences. A subset of these peptide markers was confirmed to be present in the actual peptide profiles generated by multiple reaction monitoring and targeted LC-MS/MS. The experimentally identified peptides separated these isolates into 6 strains that agreed with multilocus sequence typing analysis performed on the same isolates.

Conclusions: This approach may be generalizable to other bacterial species, and the peptides may be useful for rapid MS strain tracking of isolates with broad application to infectious disease diagnosis.

© 2016 American Association for Clinical Chemistry.

Figures

Figure 1

MALDI-MS spectra for 4 strain D (red) isolates, a group of 15 strain A, B and C (black) isolates, 1 strain E (green) isolate and 1 strain F (blue) isolate. In the inserted graph, the scale of peak intensity of strain F (blue) was rescaled 10× from original data to show the peak shift in strain E.

Figure 2

Peptidome analysis to detect genome-specific and species-specific peptides using A. baumannii as an example. High confidence peptides detected experimentally by LC-MS/MS were compared with genome specific peptides to identify a set of detected genome-specific peptides. Lowest common ancestor (LCA) was then used to search species-specific peptides. MRM or targeted LC-MS/MS was used to validate the peptide marker experimentally.

Figure 3

Peptidome similarities of 22 NIH A. baumannii isolates and 7 other randomly selected strains using their genome data. The graph was generated by Peptidome analysis by Unipept. Strain A, B, C and D were clearly separated theoretically by comparing their tryptic peptidome differences.

Figure 4

Relative integrated peak intensities of three peptide markers measured by MRM LC-MS (Figures 4A.1 to 3) or targeted LC-MS/MS (Figure 4B.1 to 3) from 15 ABNIH isolates (strains A, B and C) shown on the X-axis. The blue bars are the integrated peak intensities of labeled peptides or standards and the red bars are the detected peptides from ABNIH isolates.