Name: subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)
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Description: Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

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 Universita&#776;t Go&#776;ttingen.

1. Prepare a Safe Workplace by Considering Biosafety Conditions
<ol>
	<li>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.</li>
	<li>Regardless of the biohazard classification of the specific micr...

<ol>
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J., Tsai, F. J., Ho, C. M., Liu, Y. C., Chen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+biomarker+discovery+for+identification+of+methicillin-resistant+and+vancomycin-intermediate+Staphylococcus+aureus+strains+by+MALDI-TOF.">Peptide biomarker discovery for identification of methicillin-resistant and vancomycin-intermediate Staphylococcus aureus strains by MALDI-TOF.</a> Anal Chem. 84 (13), 5685-5692 (2012).</li><li>Novais, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF+mass+spectrometry+as+a+tool+for+the+discrimination+of+high-risk+Escherichia+coli+clones+from+phylogenetic+groups+B2+(ST131)+and+D+(ST69,+ST405,+ST393).">MALDI-TOF mass spectrometry as a tool for the discrimination of high-risk Escherichia coli clones from phylogenetic groups B2 (ST131) and D (ST69, ST405, ST393).</a> Eur J Clin Microbiol Infect Dis. , (2014).</li><li>Matsumura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+extended-spectrum-beta-lactamase-producing+Escherichia+coli+ST131+and+ST405+clonal+groups+by+matrix-assisted+laser+desorption+ionization-time+of+flight+mass+spectrometry.">Detection of extended-spectrum-beta-lactamase-producing Escherichia coli ST131 and ST405 clonal groups by matrix-assisted laser desorption ionization-time of flight mass spectrometry.</a> J Clin Microbiol. 52 (4), 1034-1040 (2014).</li><li>Christner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+MALDI-TOF+Mass+Spectrometry+Strain+Typing+during+a+Large+Outbreak+of+Shiga-Toxigenic+Escherichia+coli.">Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli.</a> PLoS One. 9 (7), e101924 (2014).</li><li>Zautner, A. E., Masanta, W. O., Weig, M., Gro&#223;, U., Bader, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry-based+PhyloProteomics+(MSPP):+A+novel+microbial+typing+Method.">Mass Spectrometry-based PhyloProteomics (MSPP): A novel microbial typing Method.</a> Scientific Reports. 5, (2015).</li><li>Dasti, J. I., Tareen, A. M., Lugert, R., Zautner, A. E., Gross, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Campylobacter+jejuni:+a+brief+overview+on+pathogenicity-associated+factors+and+disease-mediating+mechanisms.">Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms.</a> Int J Med Microbiol. 300 (4), 205-211 (2010).</li><li>Zautner, A. 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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...

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 biomarker ions for a particular subgroup of strains in Campylobacter jejuni4, Clostridium difficile5, Salmonella enterica ssp. enterica serovar Typhi6, Staphylococcus aureus7-9, and Escherichia coli10-12.
The combination of several variable biomarker masses corresponding to allelic isoforms offers the option for deeper subtyping. Previously, we successfully implemented a method to convert these variations in mass profiles into meaningful and reproducible phyloproteomic relations called mass spectrometry based phyloproteomics (MSPP) on a C. jejuni ssp. jejuni isolate collection13. MSPP can be used a mass spectrometric equivalent to DNA sequence based subtyping techniques like multilocus sequence typing (MLST).
Campylobacter species are the leading cause of bacterial gastroenteritis worldwide14,15. As a consequence of Campylobacteriosis post-infectious sequela, namely, Guillain Barr&#233; Syndrome, reactive arthritis and inflammatory bowel disease can arise16. The main sources of infection are contaminated livestock meat from chicken, turkey, swine, cattle, sheep and ducks, milk and surface water15,17. Therefore, regular epidemiological surveillance studies in the context of food safety are necessary. MLST is the &#34;gold standard&#34; in molecular typing for Campylobacter species18. Because the Sanger-sequencing based MLST method is labor intensive, time consuming and relatively expensive, MLST typing is restricted to relatively small isolate cohorts. Therefore, there is a need for cheaper and faster subtyping methods. This need could be met by mass spectrometric methods like MSPP.
This paper presents a detailed protocol for MSPP-typing using a collection of Campylobacter jejuni ssp. doylei isolates and comparison of its potential with MLST.

<table border="0" cellpadding="0" cellspacing="0" width="1348">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">acetonitrile</td>
			<td style="width:663px;">Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">34967</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoflex III TOF/TOF 200 system</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">GT02554 G201</td>
			<td>Mass spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">bacterial test standard BTS</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">BioTools 3.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>263564</td>
			<td>Software Package</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Bruker IVD Bakterial Test Standard</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">8290190</td>
			<td>5 tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8843</td>
			<td>ATCC 49349;IMVS 1141;NCTC 11951;strain 093</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9143</td>
			<td>Goossens Z90</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG7790</td>
			<td>ATCC 49350;CCUG 18265;Kasper 71;LMG 8219;NCTC 11847</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9243</td>
			<td>Goossens N130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8871</td>
			<td>NCTC A603/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG9255</td>
			<td>Goossens B538</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Campylobacter jejuni subsp. doylei isolate&#160;</td>
			<td>Belgium coordinated collection of microorganisms/Laboratory of Microbiology UGent BCCM/LMG Ghent, Belgium</td>
			<td>LMG8870</td>
			<td>NCTC A613/87</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Columbia agar base&#160;</td>
			<td>Merck, Darmstadt, Germany</td>
			<td style="width:119px;">1.10455 .0500</td>
			<td>500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Compass for FlexSeries 1.2 SR1</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>251419</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">defibrinated sheep blood&#160;</td>
			<td>Oxoid Deutschland GmbH, Wesel, Germany</td>
			<td>SR0051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">ethanol</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">02854 Fluka</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">formic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">HCCA matrix</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td style="width:119px;">604531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Kimwipes paper tissue</td>
			<td style="width:663px;">Kimtech Science via Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MALDI Biotyper 2.0</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>259935</td>
			<td>Software Package</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Mast Cryobank vials</td>
			<td>Mast Diagnostica, Reinfeld, Germany</td>
			<td style="width:119px;">CRYO/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">MSP 96 polished steel target</td>
			<td style="width:663px;">Bruker Daltonics, Bremen, Germany</td>
			<td>224989</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Mini Kit&#160;</td>
			<td style="width:663px;">Qiagen, Hilden, Germany</td>
			<td style="width:119px;">51304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">recombinant human insulin</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">I2643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">T6508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">water, molecular biology-grade</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">W4502</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtyping of campylobacter jejuni ssp. doylei isolates using mass spectrometry-based phyloproteomics (mspp)

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.

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...

Watch this Scientific Journal Video about Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics MSPP at JoVE.com

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

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).

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

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. ...

The overall goal of this mass spectrometric procedure, called MSPP or Mass Spectrometry-based PhyloProteomics, is to sub-type bacteria at the strain level. This method can be used to answer key questions in the fields of Microbiology, Epidemiology and Hygiene. For example, it can be used to analyze outbreak situations by visualizing phyloprotomic relatedness.Compared to other typing methods, MSPP is cheap per sample, and very fast. Nosocomial infections are an issue of increasing importance, and we hope that our MSPP scheme can be extended towards epidemiologic typing of other bacteria, or even fungi. Our MSPP scheme provides insight into relatedness of strains, so for the prediction of relevant phenotypes, such as drug resistance, or even virulence, it does make sense to apply it where these phenotypes correlate with specific genotypes.To begin this procedure, prepare two tubes of matrix solution by dissolving 2.5 milligrams of HCCA in 250 microliters of an organic solvent mixture, a 50%isonitrile. Add recombinant human insulin as an internal calibrate to one of them, then 47.5%double distilled water and 2.5%trifluoroacetic acid. Next, spread a pinhead sized amount of a colony from the reference strain, directly onto a maldi target plate.For determination of the exact mass of the calibration peak, place on microliter of test standard on another spot. Overlay each spot of bacteria and standard with 1 microliter of HCCA spiked matrix solution and leave them to crystallize at room temperature. For each strain to be analyzed, repeat the smear preparation.Overlay each spot with 1 microliter of spiked HCCA matrix solution and leave to crystallize at room temperature. Alternatively, proteins can be extracted from difficult to lie cells. For this, harvest approximately 5 colonies from an Agar plate culture with an inoculation loop.Suspend them in 300 microliters of double distilled water in a 1.5 milliliter reaction tube, and mix them thoroughly by repeated pipetting until the bacterial colonies are completely suspended. Then add 900 microliters of absolute ethanol and mix. Centrifuge the sample at 13, 000 g's for 1 minute.After drying the pellet, re suspend it by pipetting up and down in 50 microliters of 70%formic acid. Then, add 50 microliters of aceto-nitrile and mix. Remove the debris by centrifugation at 13, 000 g's for 2 minutes.When finished, transfer 1 microliter of the supernamed onto a sample position on a maldi target plate. After allowing the sample to dry, overlay each spot with one microliter of HCCA matrix solution and leave to crystallize at room temperature. At this point, perform MALDI-TOF analysis by gathering 600 spectra in 100 shot steps for each spot.First, click on the auto execute tab in the configuration software of the mass spectrometer. Open the method by left clicking the method button and choosing the method from the pull down menu. Next, left click the edit button to open the auto execute method editor.Under the accumulation tab, set the satisfactory shot value to 100 and the sum up value to 600. For each spectrum, choose the calibrant peak from the list, click automatic assign'and press OK'After obtaining mass spectra from each spot, experimentally determine the exact mass of the calibration peak, by opening a spectrum with the spectrum browser and finding the peak at the expected mass as compared to a spectrum without calibrant. Check that the calibrant peak is not obscured by any other biomarker of the organism of interest.From the genome sequencing data of the reference strain, calculate the theoretical monoisotopic molecular weight of each of the encoded proteins by translating the DNA sequence into the corresponding amino acid sequence. Upload file'or copy paste'this protein sequence into the input box of the appropriate bioinformatics resource panel, and compute the theoretical isoelectric point and molecular weight. Copy the results into a spreadsheet with one column containing the gene identifier and another column the molecular weight.Sort the rows by calculated molecular weight, to facilitate looking up masses. Insert an additional column into the spreadsheet for the molecular weight of the demathianinated form, subtracting 135 dautons from the mono isotopic molecular weight. Assign each major measured biomarker mass to the calculated masses from the reference strain, by looking up the measured mass from the genome table.In another spreadsheet, record the mass and identifier for each biomarker ion. Now calibrate the mass spectra obtained from the collection isolates as done for the reference strain. For one isolate per row, identify variant biomarkers in the mass spectra, record the measured mass for each biomarker and the predicted isoform in the respective columns.The MALDI-TOF mass spectrum of C-Jejuni Subspecies doylei ATCC 49349 contains 14 singularly charged biomarker ions that could be identified by comparison of the calculated molecular masses with the reference spectrum. Within the collection, varying iso forms were detected for L32 without methionine, L33, and the hypothetical protein encoded by gil152939117, S20 M, and S15 M.The remaining masses assigned to biomarker ions were invariable in the tested isolates. The amino acid substitution corresponding to the mass shift has been identified by PCR amplification and sanger sequencing of the particular gene using the primers listed here.The amino acid sequences of all biomarker ions included in the MSPP scheme, as well as the detected alelic isoforms are indicated here. The MLST based UPGMA dendrogram constructed from the examined C jejuni subspecies doylei isolates, shows that each isolate belongs to a different MLST sequence type. And strain LMG7790 has the longest phylogenetic distance.As seen with the MLST alaysis, isolate LMG7790 has the longest phyloprodeomic distance in the MSPP analysis of the examined C jejuni subspecies doylei isolates. Once mastered, this technique can be done within one day if it's performed properly. While following this procedure, it's important to remember the toxicity of some substances used during the experiment.For example, aceto-nitrile can cause skin irritation. Additionally, to MSPP other methods like DNA sequencing, can be performed to analyze further parameters, like the presence of resistance genes. The development of this technique paves the way for researchers in the field of microbiology to explore photogeny.After watching this video, you should have a good understanding of how MSPP works and how it can be applied to specific questions. Don't forget, that working with violent pathogens can be extremely hazardous and precautions such as S2 lab equipment should always be taken.

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