titel;

RAAKpubliekSingle-cell MALDI TOF

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2Conventional Mass Spec vs BiosparQ workflows Conventional Mass Spec requires 12 72 hrs cultivation prior to analysis

BiosparQ analysis is direct from sample using patented single cell MALDI TOF Mass Spec

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System overview3System overview

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Single cell dispenserFunction: introduce sample with MALDI coating materials into system (pico-liter droplets containing 0, 1 or 2 bacterial cells) 4System overview

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Particle generatorTransport droplets towards beam generator and let volatiles evaporate from droplet

5System overview, quick reminder

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Aerosol Beam GeneratorFunction: Transport coated cells towards ionization chamber (vacuum) 6System overview

Individually free flying (400m/s) and aligned MALDI coated cells directed towards ionization (on the fly)

Particle velocity correlates with (aerodynamic) size of particle

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Mass spectrometerFunction: Generate bacterial fingerprint for all individual cells that pass the ionization chamber (M/Z spectrum)7System overview

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8ResultsSingle-cell MALDI TOF Mass spectra

Mass spectrometerFunction:Generates mass spectra for all individual cells that enter the ionization chamberAccumulation of 10-100 cells seems sufficient for ID

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Spectrum quality (accumulated)Sample composition (cel selection not implemented yet: accumulated spectrum also contains empty shots)Particle morphologyFluid phase compositionDrying speed9ResultsSpectrum variability

Good discrimination between e-coli and Serratia strainsMutual discrimination between Serratia strains must be improvedReduce variability of (single-particle) spectra = reduce variability in particle morfologyFind the optimal combination of peaks used (classifier)10Deconvolution of mixed samples

Single-particle MALDI-TOF Mass Spectrometer installed and functional1st protocols developed, 1st MALDI fluid recipies developedInstrument produces single-particle spectraSignature quality critically dependent on particle morphologyParticle morphology varies, signature quality varies

Signature quality of accumulated spectra sufficient for ID at species levelInformation content of single-particle spectra sufficient for classification species/genus levelClassification efficiency again critically dependent on particle morphologyEfficiency can be improved further by development of dedicated classifiers

11Conclusions

Innovative Molecular Diagnostics

Single cell MALDI-TOF based identification of strains obtained from hospitalized patients

H. E. Dekter, C. C. Orelio, R.R. Parchen, F. Koyuncu, A. Van der Zee, G.A. Parlevliet, G.C. de Valk, W. B. van LeeuwenSession OS32 New frontiers in MALDI-TOF

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Single cell MALDI-TOF Mass Spectroscopy

Analysis directly from clinical sample without culture step Identification and typing of bacterial species

Quantitative analysis of mixed bacterial populations.

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Single cell MALDI-TOF Mass Spectrometer

25th ECCMID, 25 28 April 2015

Sample introduction

Matrix application

Ionization

Individually free flying (400m/s) and aligned MALDI coated cells directed towards ionization

Standardized MALDI-fluid recipe developed (based on DMT as MALDI matrix)

: Generate bacterial fingerprint for all individual cells that pass the ionization chamber (M/Z spectrum)

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Cell by cellData-acquisition

Acquired cells151015202530354045200

Accumulation of 10-100 cells seems sufficient for ID

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Objective

In this pilot study we compared the results of a single-cell MALDI-TOF platform with a molecular reference typing method (AFLP) to type 10 Serratia marcescens strains, collected from patients hospitalized at secondary care hospital in Leiden (Netherlands).

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Results: Mass Spectra of 10 S. marcescens strains

25th ECCMID, 25 28 April 2015

Accumulated spectra composed from single-cell spectraBelow 8000 Da mostly coinciding peaks within speciesAbove 8000 Da varying peak positionbetween strains

Coinciding peaksVarying peaks

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Cluster analysis : MALDI Mass Spectra vs. AFLP

Two most similar mass spectra (1302500636 & 1307501833) coincide with the two most related strains according to AFLP

The cluster that contains the two related strains coincides with the corresponding cluster of mass spectra as wellMALDIAFLP

Wat voor soort cluster analysis? Similarity? Pearson etc? Wat is het % overeenkomst?

Lange nummers => kan dat niet korter? Is nl niet relevant

Ik vind het geen overzichtelijke manier om de data te presenteren18

ConclusionThe reference method (AFLP) and the single cell MALDI-TOF showed that the same two S. marcescens strains were highly associated.

This is the first report that describes the use of single cell MALDI-TOF for adequate characterization of S. marcescens on the strain level.

No culture, rapid, simple, cheap.

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Future perspectivesSingle cell MALDI-TOF is a new diagnostic platform for infectious diseases.

Identification of strains can be done without culturing/isolation, so the time-to-ID is limited and it requires minimum lab infrastructure.

Quantitative analysis of a total bacterial population(microbiota).

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Single Cell MALDI-TOF In the blink of an eye

AcknowledgmentsInnovative Molecular Diagnostics:Claudia OrelioMaarten MorsinkLaura CoenenSeydi TektasWillem van LeeuwenBiosparQ:Rene ParchenMarco Le KluseFahret KoyuncuGerold de ValkMaasstad Hospital:- Anneke van der ZeeBronovo Hospital:- Gerard ParlevlietErasmus MC:Ren te Witt

Funded by Ministery of Education and Science

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