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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #358601
Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Detection and identification of a protein biomarker in antibiotic resistant Escherichia coli using intact protein LC offline MALDI-MS and MS/MS

Author
item MAUS, ANTHONY - University Of Wyoming
item BISHA, BLEDAR - University Of Wyoming
item Fagerquist, Clifton - Keith
item BASILE, FRANCO - University Of Wyoming

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/4/2019
Publication Date: 11/12/2019
Citation: Maus, A., Bisha, B., Fagerquist, C.K., Basile, F. 2019. Detection and identification of a protein biomarker in antibiotic resistant Escherichia coli using intact protein LC offline MALDI-MS and MS/MS. Journal of Applied Microbiology. 128(3):697-709. https://doi.org/10.1111/jam.14507.
DOI: https://doi.org/10.1111/jam.14507

Interpretive Summary: The spread of antimicrobial resistance (AMR) across pathogenic and non-pathogenic bacteria is an increasing threat to public health. Methods are needed to rapidly identify bacteria, their virulence factors and the factors that enable them to resist antibiotic exposure. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) is now widely used worldwide in clinical laboratories to identify bacteria. This technology has been extended to the analysis of specific proteins produced by bacteria that may be associated with pathogenicity and AMR. Off-line liquid chromatography (LC) coupled with MALDI-ToF-ToF tandem mass spectrometry (MS/MS) is one approach to detect and identify a greater proportion of the bacterial proteome than is possible from an unfractionated sample. Using this approach a protein biomarker (HU) was identified that is highly correlated with antibiotic-resistant strains of Escherichia coli. The HU gene appears to be located in a circular piece of DNA (plasmid) in the host E. coli, and this plasmid also carries beta-lactamase genes that confer resistance to beta-lactam antibiotics. This is the first example of using LC-MALDI-ToF-ToF for biomarker discovery in microorganisms.

Technical Abstract: The identification and differentiation of antibiotic resistant bacteria by MALDI-TOF-MS profiling is not always feasible and remains a challenge due to the inability of this approach to detect unique protein biomarkers associated with this trait. To expand the detectable proteome in antibiotic-resistant bacteria, we developed an offline LC protein separation/fractionation prior to MALDI-ToF-MS analysis and applied it for the analysis of several antibiotic-resistant Escherichia coli isolates. Results show that this methodology increases the number of detected protein signals in the typically analyzed mass regions (m/z 3,000-20,000) by a factor of 13. Using the developed LC-MALDI-ToF-MS protocol in conjunction with supervised principal components analysis (sup-PCA), we identified a protein biomarker at m/z 9355, which exhibited the strongest correlation to beta-lactam resistance among the E. coli bacteria tested. Implementing a top-down proteomic approach using MALDI-ToF/ToF-MS, the pre-fractionated protein biomarker was identified as a DNA-binding HU protein, most likely translated from the blaCMY-2 gene (encoding AmpC-type beta-lactamase) in the incompatibility plasmid complex A/C (IncA/C). Our results demonstrate the utility of LC-MALDI-MS and MS/MS to extend the number of proteins detected and perform MALDI-accessible biomarker discovery in microorganisms.