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Genetics

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP)

Published: October 30th, 2016

DOI:

10.3791/54165

1Institut für Medizinische Mikrobiologie, Universitätsmedizin Göttingen

Mass spectrometry-based phyloproteomics (MSPP) was used to type a collection of Campylobacter jejuni ssp. doylei isolates at the strain level in comparison to multilocus sequence typing (MLST).

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms.

Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations.

The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

During the last decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has advanced to be a highly valued standard method for microbial genus and species identification in clinical microbiology1,2. Species identification is based on the recording of small protein fingerprints of intact cells or cell lysates. The typical mass range for a mass spectrometer used in routine clinical microbiology is 2-20 kDa. Additionally, the resulting spectra can be used to discriminate strains at the below-species and below-subspecies level3. Early pioneering studies have identified specific biomark....

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1. Prepare a Safe Workplace by Considering Biosafety Conditions

  1. Become familiar with the laboratory and safety regulations that are of relevance for working with microorganisms. Most human pathogenic microorganisms must be handled at biosafety level 2 conditions but some, such as Salmonella enterica serovar Typhi, require biosafety level 3. Information on level of handling each pathogen can be accessed at www.cdc.gov/biosafety.
  2. Regardless of the biohazard classification of the specific micr.......

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Previously, we successfully established a MSPP scheme for C. jejuni ssp. jejuni13. Here, we aimed to extend the method to the sibling subspecies C. jejuni ssp. doylei. In this specific setting, seven C. jejuni ssp. doylei isolates were acquired from the Belgian collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium. All seven isolates used for our analyses were of human origin. The genome-seque.......

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The most critical step in the establishment of an MSPP scheme is the unequivocal genetic determination of biomarker ion identities. If it is not possible to identify a biomarker undoubtedly, then it should be excluded from the scheme13.

The C. jejuni ssp. doylei scheme includes 14 different biomarker ions. These are 5 less compared to the C. jejuni ssp. jejuni MSPP scheme13.The most significant difference between the detectable C. j.......

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We are grateful to Hannah Kleinschmidt for excellent technical support. This paper was funded by the Open Access support program of the Deutsche Forschungsgemeinschaft and the publication fund of the Georg August Universität Göttingen.

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Name Company Catalog Number Comments
acetonitrile Sigma-Aldrich, Taufkirchen, Germany 34967
Autoflex III TOF/TOF 200 system Bruker Daltonics, Bremen, Germany GT02554 G201 Mass spectrometer
bacterial test standard BTS Bruker Daltonics, Bremen, Germany 604537
BioTools 3.2 SR1 Bruker Daltonics, Bremen, Germany 263564 Software Package
Bruker IVD Bakterial Test Standard Bruker Daltonics, Bremen, Germany 8290190 5 tubes
Campylobacter jejuni subsp. doylei isolate  Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium LMG8843 ATCC 49349;IMVS 1141;NCTC 11951;strain 093
Campylobacter jejuni subsp. doylei isolate  Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium LMG9143 Goossens Z90
Campylobacter jejuni subsp. doylei isolate  Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium LMG7790 ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847
Campylobacter jejuni subsp. doylei isolate  Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium LMG9243 Goossens N130
Campylobacter jejuni subsp. doylei isolate  Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium LMG8871 NCTC A603/87
Campylobacter jejuni subsp. doylei isolate  Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium LMG9255 Goossens B538
Campylobacter jejuni subsp. doylei isolate  Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium LMG8870 NCTC A613/87
Columbia agar base  Merck, Darmstadt, Germany 1.10455 .0500 500 g
Compass for FlexSeries 1.2 SR1 Bruker Daltonics, Bremen, Germany 251419 Software Package
defibrinated sheep blood  Oxoid Deutschland GmbH, Wesel, Germany SR0051
ethanol Sigma-Aldrich, Taufkirchen, Germany 02854 Fluka
formic acid Sigma-Aldrich, Taufkirchen, Germany F0507
HCCA matrix Bruker Daltonics, Bremen, Germany 604531
Kimwipes paper tissue Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany Z188956
MALDI Biotyper 2.0 Bruker Daltonics, Bremen, Germany 259935 Software Package
Mast Cryobank vials Mast Diagnostica, Reinfeld, Germany CRYO/B
MSP 96 polished steel target Bruker Daltonics, Bremen, Germany 224989
QIAamp DNA Mini Kit  Qiagen, Hilden, Germany 51304
recombinant human insulin Sigma-Aldrich, Taufkirchen, Germany I2643
trifluoroacetic acid Sigma-Aldrich, Taufkirchen, Germany T6508
water, molecular biology-grade Sigma-Aldrich, Taufkirchen, Germany W4502

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