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

Summary

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).

Abstract

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.

Introduction

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 microbiology^1,2. 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....

Protocol

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 microorganism, regard all materials that came in contact with the infectious agent as infectious waste that must be autoclaved before disposal. Respect re....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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