Identifikation von Mikroorganismen mittels MALDI-TOF Massenspektrometrie - Schnelle und günstige Charakterisierung von Bakterien und Pilzen

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1 Identifikation von Mikroorganismen mittels MALDI-TOF Massenspektrometrie - Schnelle und günstige Charakterisierung von Bakterien und Pilzen Valentin Pflüger Guido Vogel Dominik Ziegler André Strauss Bernard Jenni 5. November, 2011 Basel

2 Identification of biological systems morphology Microscopy, smell, color etc. immunological mono- or polyclonal antibodies biochemical metabolic capacities ID molecular specific-pcr, 16S-Seq., RT-PCR SNP, MLST, RFLP, VNTR, PFGE ?

3 Modern approaches in taxonomy: proteomics & genomics protein proteome DNA fingerprint Mass (m/z) V o y a g e r S p e c # 1 = > A d v B C ( 3 2, 0. 5, 1. 0 ) = > N F 0. 7 [ B P = , ] DNA genome % Intensity ID species definition & determination mass fingerprint

4 MALDI-TOF MS: history Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry developed in 1980 s by Karas & Hillenkamp and Tanaka et al. first commercial apparatus in 1991

5 MALDI-TOF MS Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry Mass Spectrometry: MALDI-TOF the MS velocity of the ion depends on the mass-to-charge (m/z) ratio Detector for linear mode Time-Of-Flight: ions acceleration (electric field) and time-of-flight to the detector is recorded ~ 200 cm Laser Desorption/Ionization: matrix ionization (laser pulses) and partial transfer of its (+) charge to the analytes Matrix-Assisted: sample embedded in a matrix, avoid destruction Axima Confidence by the laser facilitate vaporisation and ionization

6 IC-MALDI-TOF MS mass ranges and applications %Int. 24 mv[sum= 2382 mv] Profiles Smooth Av Intact Cell MALDI-TOF MS Late 90`s Direct application of whole cells DNA, fatty acids, sugars metabolites enzymes & enzyme complexes matrix structural proteins & polymers 0 m/z reflector linear

7 40 m/z 80 IC-MALDI-TOF MS of bacteria differences in peak patterns Pantoea agglomerans Acinetobacter lwoffi Burkholderia cepacia Raoultella ornithinolytica Staphylococcus aureus Escherichia coli 4000 m/z 8000 distinctly different peak patterns when analysing different taxa

8 Available commercial Systems

9 Workflow of Step 1 Sample preparation: Smear-methode FlexiMass-Target with 48 positions colony selection and transfer of cells addition of 0.5 µl Matrix solution suitable for bacteria, yeasts and filamentous fungi

10 Workflow of Step 2 Measurement loading samples to AXIMIA FlexiMass target holder 4 X automated spectrum acquisition ~20 sec [c].2B m/z

11 Workflow of Step 3 Identification with SuperSpectra matching to SuperSpectra computing sum of peak weights ranking matching SuperSpectra check for conflicting significant results delivering result with confidence value m/z

12 Workflow of Step 4 Identification with SuperSpectra relative intensity Trichophyton_rubrum_1 Superspectrum matching fingerprints of two clinical isolates to SuperSpectra best match with T. rubrum_1 SuperSpectrum (containing 44 peaks): # 539: 39 matches, Σ 1036 points # 582: 37 matches, Σ 1011 points m/z from Erhard et al. 2007, Exp. Dermatol. both isolates identified as Trichophyton rubrum with 99% confidence

13 Workflow of Step 5 Comparison tools m/z comparison to mass spectral patterns of reference spectra in the database cluster analysis of selected reference and sample mass spectra

14 Evaluation of I result summary no spectrum: 1% no identification: 3% identification with 80-90% confidence: 6% identification with 90-99% confidence: 90% high hit rate in daily routine after a very short training phase SARAMIS is readily applicable in routine analysis

15 Identification of different bacterial taxa Cronobacter spp.

16 Identification of different bacterial taxa Yersinia enterocolitica biotypes

17 E. mallotivora E. papayae E. papayae E. psidii E. billingae E. amylovora E. toletana MALDI-TOF MS: Plant pathogens Erwinia ssp. identification 0.08 E. aphidicola E. persicina Erwinia amylovora causative agent of fireblight Rosacea affected E. tasmaniensis Interventions with streptomycin rpsl gene: 30S S12 protein E. piriflorinigrans E. pyrifoliae

18 MALDI-TOF MS: Erwinia amylovora rpsl allels for streptomycin resistance %Int mv 0.3 mv S12, wt S12, +28 Da, Lysin Arginin m/z

19 MALDI-TOF MS spectra database modules Dermatophytes: clinical relevant species Trichophyton ssp, Microsporum ssp. 24 species in module based on ITS1 and 2 taxonomy >85 % ID in clinical routine samples M. fulvum M. gypseum M. persicolor M. audouinii M. canis M. praecox M. racemosum 0.09 Microsporum ssp. T. erinacei T. terrestrae T. tonsurans T. megnini T. interdigitiale T. violaceum T. verucosum T. rubrum 0.1 Trichophyton ssp.

20 MALDI-TOF MS spectra database modules Clinical yeasts : Candida ssp. 34 Candida species in database >90% ID in clinical routine samples identification from positive blood cultures possible

21 MALDI-TOF MS spectra database modules Environmental fungi: Air- Surface, and Food-samples high species diversity classification of references ID on genus level focus on relevant species 60% ID in indoor air samples Penicillium ssp. Aspergilllus ssp. Fusarium ssp.

22 0.09 MALDI-TOF MS: differentiation of fungal species Verticillium spp. Verticillium tricorpus Gibellulopsis nigrescens Verticillium longisporum Acrostalagmus luteoalbus Musicillium theobromae Verticillium albo-atrum Plectosphaerella cucumerina Verticillium dahliae

23 IC MALDI-TOF MS of cell-lines Differentiation of cell-lines NK3.3 Homo sapiens WIL2S Raji Hela CMT93 RAW264 SF21 Mus musculus Spodoptera frungiperda Identification of specific marker proteins on the species level Independent of passage number and medium supplements SF9 H5 Trichoplusia ni Drosophila melanogaster Differentiation of lineages within the same species CRL1963

24 MALDI-TOF MS for the characterization of insects Ceratopogonidae, Culicoides spp.(biting midges) Vector for Bluetongue virus Current Taxonomy/Identification Morphological and molecular (RT-PCR) Distinguish between sibling species of the fruit fly Drosophila melanogaster (Campbell, 2005) Distinguish three species from three genera of plant-sucking aphids (Perera et al, 2005) Establish phylogenetic relationships among 13 species of Drosophila flies (Feltens et al, 2010)

25 MALDI-TOF MS for characterization of insects Ceratopogonidae, Culicoides spp.(biting midges) C. obsoletus Identification and discrimination of 14 Culicoides spp. was possible C. scoticus C. dewulfii C. imicola Fast and reliable sample preparation Head-Thorax for MALDI-TOF MS Abdomen for multiplex PCR and further analysis 1-3 mm C. pulicaris C. punctatus

26 0 MALDI-TOF MS: plant differentiation Zea mays strain differentiation using disected and extracted embryos relativ identity Amadeo LG LG %Int mv[sum= mv] Profiles Smooth Av disection of seed extraction of embryo acquisition of peptide mass fingerprint LG Ricardinio popcorn sweetcorn m/z

27 Peptide mass fingerprinting using MALDI-TOF MS Take home message Peptide mass fingerprinting using MALDI-TOF MS is a valuable tool for the fast and cost effective identification and characterization of diverse biological systems. Mabritec is a service provider and R&D partner in the field of MALDI-TOF mass spectrometry and has an outstanding experience in applying this technology to a wide range of biological questions.

28 Collaborators Agroscope Changins Wädenswil B. Duffy, J. Poithier, F. Rezzonico Agroscope Reckenholz Tänikon ART Franco Widmer, Mireille Dessimoz Institut für Parasitologie Zürich A. Mathis, C. Kaufmann, I. Steinmann ABC Labor Spiez N. Schürch, M. Wittwer Swiss TPH C. Lengeler, P. Müller Instituto cantonale di Microbiologia O. Petrini, M. Tonolla, Xavier Perret

29 Thank you