Environmental Genomics and Systems Biology Institute of Natural Resource Sciences Zurich University of Applied Sciences, Wädenswil, Switzerland

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1 Environmental Genomics and Systems Biology Institute of Natural Resource Sciences Zurich University of Applied Sciences, Wädenswil, Switzerland MALDI-TOF MS for microorganism identification: from pattern recognition towards marker based approaches - The example of plant pathogenic Pseudomonas isolates Joël F. Pothier M. Ruinelli F. Foucault, V. Pflüger FA

2 Environmental Genomics and Systems Biology Institute of Natural Resource Sciences Zurich University of Applied Sciences, Wädenswil, Switzerland MALDI-TOF MS for microorganism identification: from pattern recognition towards marker based approaches - The example of Streptococci (and plant pathogenic Pseudomonas isolates) Joël F. Pothier M. Ruinelli F. Foucault, V. Pflüger FA

3 MALDI-TOF MS principle Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry Mass Spectrometry: the velocity of the ion depends on the mass-to-charge (m/z) ratio % intensity Time-Of-Flight: ions acceleration (electric field) and time-of-flight to the detector is recorded Pulsed laser ~337nm m/z To MS detector Ions Laser Desorption/Ionization: matrix ionization (laser pulses) and partial transfer of its (+) charge to the analytes sample + matrix Matrix-Assisted: sample embedded in a matrix, - avoid destruction by the laser - facilitate vaporisation and ionization Ionization Separation Detection slide Environmental Genomics and Systems Biology, IUNR, ZHAW 3

4 What does a MALDI-TOF MS look like? A MALDI-TOF Mass Spectrometer Not a MALDI-TOF Mass Spectrometer X Axima Performance (Shimadzu) Environmental Genomics and Systems Biology, IUNR, ZHAW 4

5 Current bacterial identification methods morphology color, smell, microscopy, etc. molecular Specific PCR, 16S seq., MLSA, MLST, ANI, SNP, RFLP, REP-PCR, etc. immunological mono- or polyclonal antibodies ID biochemical metabolic capacities and resistance to antibiotics ? Environmental Genomics and Systems Biology, IUNR, ZHAW 5

6 Voyager Spec #1=>AdvBC(32,0.5,1.0)=>NF0.7[BP = , 2117] Mass (m/z) Modern microbial taxonomy: genomics & proteomics bacterial cell protein DNA proteome species definition & determination genome % Intensity mass fingerprint DNA fingerprint Environmental Genomics and Systems Biology, IUNR, ZHAW 6

7 Gold standards for identification & phylogeny Specific PCR: Portfolio of different methods Primer design Metabolic profiling: Variability within taxa High influence of growth Low resolution Fatty acid profiling: Low resolution Sequencing (locus / MLSA): Multiple primers design to amplify all strains Requires sequencing Expensive Fingerprints (REP, BOX, ERIC, etc.): Low reproducibility Comparison Environmental Genomics and Systems Biology, IUNR, ZHAW 7

8 Gold standards vs. IC MALDI-TOF MS Specific PCR: Portfolio of different methods Primer design Metabolic profiling: Variability within taxa High influence of growth Low resolution Fatty acid profiling: Low resolution Sequencing (locus / MLSA): Multiple primers design to amplify all strains Requires sequencing Expensive IC MALDI-TOF: Time of preparation/analysis < 30 min No primer optimization needed Down to species or in individual cases even to subspecies and strain-level High reproducibility Relatively low cost per analysis but: - Requires (sufficient) bacterial growth - High initial investment for the machine Fingerprints (REP, BOX, ERIC, etc.): Low reproducibility Comparison Environmental Genomics and Systems Biology, IUNR, ZHAW 8

9 IC-MALDI-TOF MS workflow is easy and fast (1) (3) Transfer Add matrix RT crystallisation (2) Acquire mass spectra automated ~20s Identification or phylogeny % Intensity Mass (m/z) Environmental Genomics and Systems Biology, IUNR, ZHAW

10 4000 m/z IC-MALDI-TOF MS of bacteria: different taxa produce distinct peak patterns Pantoea agglomerans Acinetobacter lwoffi Burkholderia cepacia Raoultella ornithinolytica Staphylococcus aureus Escherichia coli 4000 m/z 8000 Environmental Genomics and Systems Biology, IUNR, ZHAW

11 Several peaks can be associated to ribosomal proteins 30S: about 21 subunits 50S: about 31 subunits (Demirev et al. 2001, Welker et al. 2011) Ribosomal proteins are: constitutively expressed and therefore abundant in detectable amounts (independently of sample preparation and culturing conditions) highly conserved and therefore allow a highly accurate species identification in general smaller than 20kDa, making small changes easily detectable by MALDI-TOF MS Environmental Genomics and Systems Biology, IUNR, ZHAW 11

12 For example, let s take two samples Environmental Genomics and Systems Biology, IUNR, ZHAW 12

13 Same residues in different order = not informative Transcription DNA ATGATTTGCCAT GCGGAACTGTAA ATGATTTGCCAT GAACTGGCGTAA Translation RNA AUGAUUUGCCAU GCGGAACUGUAA AUGAUUUGCCAU GAACUGGCGUAA Protein MICHAEL* MICHELA* Ionisation Protein mass m/z Da m/z Da Mass spectrum Environmental Genomics and Systems Biology, IUNR, ZHAW 13

14 Similar residues = not informative ATGATTTGCCATGCGGAACTGGGCGAAACCGCG[G/C]A[G/A]TAA MICHAELGETAZ* m/z Da Transcription DNA ATGATTTGCCATGCGGAACTG GGCGAAACCGCGGAATAA ATGATTTGCCATGCGGAACTG GGCGAAACCGCGCAGTAA Translation RNA AUGAUUUGCCAUGCGGAACUG GGCGAAACCGCGGAAUAA AUGAUUUGCCAUGCGGAACG GGCGAAACCGCGCAGUAA Protein MICHAELGETAE* MICHAELGETAQ* Ionisation Protein mass m/z Da m/z Da % Mass spectrum Environmental Genomics and Systems Biology, IUNR, ZHAW 14

15 C-terminal variation = informative Transcription DNA ATGATTTGCCATGCGGAACTG GGCGAAACCGCGTAA ATGATTTGCCATGAACTG GCGCGCATTAACTAA Translation RNA AUGAUUUGCCAUGCGGAACU GGGCGAAACCGCGUAA AUGAUUUGCCAUGAACUG GCGCGCAUUAACUAA Protein MICHAELGETA* MICHELARUIN* Ionisation Protein mass m/z Da m/z Da % Mass spectrum Environmental Genomics and Systems Biology, IUNR, ZHAW 15

16 Missense mutation = very informative Transcription DNA ATGATTTGCCATGAACTGGCGC GCATTAACGAACTGCTGATTTAA ATGATTTGCCATGAACTGGTGCG CATTAACGAACTGCTGATTTAA Translation RNA AUGAUUUGCCAUGAACUGGCGC GCAUUAACGAACUGCUGAUUUAA AUGAUUUGCCAUGAACUGGUGC GCAUUAACGAACUGCUGAUUUAA Protein MICHELARUINELLI* MICHELVRUINELLI* Ionisation Protein mass m/z Da m/z Da % Mass spectrum Environmental Genomics and Systems Biology, IUNR, ZHAW 16

17 Voyager Spec #1=>AdvBC(32,0.5,1.0)=>NF0.7[BP = , 2117] Mass (m/z) Modern microbial taxonomy: genomics & proteomics bacterial cell protein DNA proteome species definition & determination genome % Intensity mass fingerprint DNA fingerprint Environmental Genomics and Systems Biology, IUNR, ZHAW 17

18 PAPMID TM database (Putatively Assigned Protein Masses for Identification) Taxonomic tools PAPMID TM Hybrid plot features E. coli K12 DH10B L L L L SNP Codon 43 Weight (Da) AAA K AGA K43R ACA K43T AAY K43N Compare functions The PAPMID TM database: Contains the relevant reference information of > genomes covering ~3 200 bacterial species Is independent of the MALDI-TOF MS instrument type and enables automated spectra quality control Allows phylogenetic interpretation of acquired bacterial spectral data Environmental Genomics and Systems Biology, IUNR, ZHAW

19 Application to streptococci Over 50 species are recognized in the genus Divided into Lancefield groups Divided into 6 groups based on 16S rdna seq. Several species are medically relevant: Several new species described: S. infantis, S. tigurinus, S. orisratti, etc. ongoing in Group D streptococci PAPMID: 792 genomes available, covering 34 species S. pneumoniae vs. S. mitis blind test with 61 isolates (17 S. pneumoniae) 100% specificity. Applied in daily routine. Environmental Genomics and Systems Biology, IUNR, ZHAW 19

20 Application to streptococci S._mitis_11_5 S._mitis_NCTC_12261 S._mitis_13_39 S._pseudopneumoniae_ATCC_BAA-960 S._pseudopneumoniae_SK674 S._pneumoniae_801 S._pneumoniae_N.A. S._pneumoniae_1542 S._oralis_SK100 S._oralis_SK10 S._oralis_ATCC_49296 S._tigurinus_1366 S._tigurinus_AZ_3a S._infantis_ATCC_ S._infantis_X S._infantis_SK1076 S._peroris_ATCC_ S._parasanguinis_CC87K S._parasanguinis_F0405 S._parasanguinis_ATCC_903 S._australis_ATCC_ S._mutans_1ID3 S._mutans_1SM1 S._mutans_2ST1 S._vestibularis_ATCC_49124 S._vestibularis_F0396 S._salivarius_K12 S._salivarius_SK126 S._thermophilus_ASCC_1275 S._thermophilus_MN-ZLW-002 S._thermophilus_1F8CT S._gallolyticus_ATCC_43143 F-1867 S._gallolyticus_subsp._gallolyticus_ S._pasteurianus_ATCC_43144 JCM_534 S._equinus_ATCC_ S._macedonicus_N.A. S._equinus_ S._equinus_ATCC_9812 S._infantarius_subsp._infantarius_A S._lutetiensis_033 S._intermedius_ATCC_27335 S._intermedius_BA1 S._intermedius_F0395 S._anginosus_N.A. S._anginosus_SK1138 S._anginosus_subsp._whileyi_CCUG_ S._sanguinis_ATCC_29667 S._sanguinis_CC94A S._macacae_NCTC_11558 S._dysgalactiae_subsp._dysgalactiae_ S._dysgalactiae_subsp._equisimilis_1 S._dysgalactiae_subsp._equisimilis_R S._canis_FSL_Z3-227 S._ictaluri_ S._pyogenes_ABC S._pyogenes_ABC S._pyogenes_ABC S._iniae_N.A. S._iniae_SF1 S._iniae_IUSA1 S._urinalis_ S._urinalis_FB127-CNA-2 S._pseudoporcinus_LQ_ S._pseudoporcinus_SPIN_20026 S._porcinus_str._Jelinkova_176 S._parauberis_NCFD_2020 S._suis_05HAS68 S._suis_A7 S._suis_89_1591 S._downei_F Environmental Genomics and Systems Biology, IUNR, ZHAW 20

21 Example of application: Streptococcus agalactiae S. agalactiae is an ubiquitous zoonotic Gram-positive bacterium Reported isolation from a variety of hosts: humans of all age classes, cattle and camels, several fish species and a range of aquatic animals (frogs, crocodiles, seals, dolphins) Commensal of the digestive and genitourinary tracts of humans Has emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Environmental Genomics and Systems Biology, IUNR, ZHAW 21

22 S. agalactiae multilocus sequence typing Population snapshot based on all ST s described ( Five main clonal complexes Almost 80% of reported invasive neonatal infections due to one main highly virulent clonal complex (CC17/ST17) The human CCs 19, 1, 10 and 17 are recently all linked but the founder STs are still dominating over time Environmental Genomics and Systems Biology, IUNR, ZHAW 22

23 Genomes in PAPMID database vs. isolates in MALDI-TOF 58 Environmental Genomics and Systems Biology, IUNR, ZHAW 23

24 S. agalactiae ribosomal proteins in MALDI-TOF MS Protein mass: dominant variants From the 57 ribosomal proteins present in S. agalactiae genome, 32 can be reproducibly detected 3 detectable mass shifts of ribosomal proteins in 7 reference strains Dominant protein masses can be used for internal calibration Environmental Genomics and Systems Biology, IUNR, ZHAW 24

25 Ribosomal protein mass variability is linked to main CCs L13 L35 S8 Variability (400 ppm accuracy) Environmental Genomics and Systems Biology, IUNR, ZHAW 25

26 Ribosomal protein mass profiles define 20 lineages Variability (400 ppm accuracy) Environmental Genomics and Systems Biology, IUNR, ZHAW 26

27 Validation statistics of S. agalactiae subtyping Full in silico mass set allowed subtyping of 239 isolates (38 STs) Environmental Genomics and Systems Biology, IUNR, ZHAW 27

28 Example of application: root nodule bacteria Tested with 116 genome sequenced strains A subset of 13 ribosomal protein biomarkers is sufficient for identification Successfully validated on unknown isolates (n>100) from Ivory Coast pigeon pea nodules Environmental Genomics and Systems Biology, IUNR, ZHAW 28

29 Example of application: root nodule bacteria Bradyrhizobium sp. WSM 2793 Ensifer fredii NGR234 Microvirga lotononidis WSM3557 Environmental Genomics and Systems Biology, IUNR, ZHAW 29

30 Example of application: root nodule bacteria 16S rrna MLSA (n=5; bp) ribo MLSA (n=13; bp) MALDI Environmental Genomics and Systems Biology, IUNR, ZHAW 30

31 Application to Pseudomonas isolates biocontrol Very diverse genus Mulet et al. 2010: MLST phylogeny of Pseudomonas sp. plant pathogens soil bioremediation human pathogen Over 218 species are recognized in the genus, 18 subspecies PAPMID: genomes available, covering 75 species Possible to identify successfully: - Pseudomonas fluorescens isolates - Pseudomonas viridiflava isolates - Pseudomonas syringae isolates Environmental Genomics and Systems Biology, IUNR, ZHAW 31

32 Conclusions and outlooks Establishment of a centralized database for quick bacterial identification based on genome derived data Validation with 100 clinically relevant bacterial species (n = 1 000) with 99.2% specificity and 97.6% sensitivity Confident discrimination of closely related bacterial species High potential for bacterial subtyping and phylogenetic interpretation of spectral data Possibility to detect some antibiotic resistances that reside on ribosomal proteins Allows high coverage for environmental and rare species PAPMID can be easily extended with new genome data Environmental Genomics and Systems Biology, IUNR, ZHAW 32

33 Acknowledgments MABRITEC AG (Riehen, CH): D. Ziegler, G. Vogel ZHAW (Wädenswil, CH): M. Gétaz, M. Palmisano, F. Rezzonico Istituto cantonale di microbiologia (Bellinzona, CH): C. Benagli, M. Tonolla Spiez Labor (Spiez, CH): M. Wittwer Research Insitute of Horticulture (Skierniewice, PL): M. Kałuzna, J. Puławska COST FA 1104 Sustainable production of high-quality cherries for the European market : J. Quero-Garcia, D. Calvi, C. Mounier... thank you for you attention FA 1104 joel.pothier@zhaw.ch Environmental Genomics and Systems Biology, IUNR, ZHAW 33

34 FA 1104 COST 1104 Training School Molecular diagnostics of bacterial diseases September 2015, Wädenswil Environmental Genomics and Systems Biology, IUNR, ZHAW 34

35 Environmental Genomics and Systems Biology, IUNR, ZHAW 35