Freiburg, 12.06.2007
Mass spectrometry, an old method with new applications
Dr. Markus Kostrzewa
Bruker Daltonik GmbH
Uppsala, November 7th 2007
Freiburg, 12.06.2007
MALDI BioTyper Reliable identification of microorganisms on species and subspecies level using MALDI-TOF MS fingerprinting
Freiburg, 12.06.2007
From sample to fingerprint
Bioinformatics
Application examples
Freiburg, 12.06.2007
From sample to fingerprint
Freiburg, 12.06.2007
MALDI-TOF MS microorganism identification
Identified species
Select a colony
Prepare onto a MALDI target plate
Unknown microrganism
?
Data interpretation
Generate MALDI-TOF profile spectrum
Freiburg, 12.06.2007
Target/Acceleration
Time-of-Flight Molecular Mass Desorption/Ionisation
Detector Drift Region
m/z
a.i.
Mass
Spectra
Laser
MALDI-TOF mass spectrometry
Freiburg, 12.06.2007
MALDI-TOF instruments
ultraflex
autoflex
microflex
MALDI-TOF mass spectrometry
Freiburg, 12.06.2007
Sample preparation
• Direct “cell smear“ method
most simple method, applicable to many bacteria
• Organic solvent extraction
improved quality for difficult bacteria, yeast, fungi
• Mechanical cell disruption (e.g. sonication)
In case of very ridgid cell walls
Compatibility of different procedures
Protocols for inactivation and shipment of microorganisms are available
Freiburg, 12.06.2007
E.coli 4
36
4.0
6
53
80
.64
62
54
.64
6
31
5.4
9
50
96
.01
71
57
.65
7
27
3.8
7
64
10
.90
78
70
.62
83
68
.99
0
1000
2000
3000
4000
5000
Inte
ns. [a
.u.]
4000 4500 5000 5500 6000 6500 7000 7500 8000
m/z
ribosomal Protein m/z
RL36 4364,33
RS32 5095,82
RL34 5380,39
RL33meth. 6255,39
RL32 6315,19
RL30 6410,60
RL35 7157,74
RL29 7273,45
RL31 7871,06
RS21 8368,76
MALDI-TOF MS profile spectrum Positive linear mode
Mass range 2-20 kDa
Freiburg, 12.06.2007
Filamentous fungi, yeast, gram+ and gram- bacteria
Aspergillus fumigatus
0
1000
2000
3000
Inte
ns.
[a.u
.]
Bacillus subtilis
0
2000
4000
6000
8000
Inte
ns.
[a.u
.]
Candida albicans ATCC 10231
0.0
0.2
0.4
0.6
0.8
1.0
Escherichia coli DH5alpha
0
500
1000
1500
2000
2500
3000 4000 5000 6000 7000 8000 9000 10000 m/z
Broad applicability of MALDI-TOF MS profiling
Freiburg, 12.06.2007
Psdm. oleovorans B396_Medium 360
0
1000
Psdm. oleovorans B396_Medium 464
0
1000
Psdm. oleovorans B396_Medium 53
0
1000
Psdm. oleovorans B396_Medium 65
0
1000
Psdm. oleovorans B396_Medium 98
0 500
1000
Psdm. oleovorans B396_MRS10
0 1000 2000
Psdm. oleovorans B396_YPD
0 1000 2000
4000 5000 6000 7000 8000 9000 10000 11000 m/z
Pseudomonas oleovorans grown on different media
Low influence of culture conditions
Freiburg, 12.06.2007
Bioinformatics
Freiburg, 12.06.2007
MALDI BioTyper 2.0 - GUI
Unknown samples
Match against microbial reference database
Identification result
Freiburg, 12.06.2007
MALDI BioTyper 2.0 – realtime analysis
Wizard guiding through setup from measurement to data analysis
BioTyper Automation Control Wizard
Define Project
Analyte Placement
Select Methods
Start
MALDI BioTyper 2.0: realtime Analysis
• Start Automation Control Wizard.
• Use a SOP and do not bother with instrument settings.
• Results available directly after measurement
Freiburg, 12.06.2007
Color-coded identification result.
MALDI BioTyper 2.0 – result table
Freiburg, 12.06.2007
MALDI BioTyper - score based pattern matching
Calculation of a matching score based on:
Rel Score % matches of the reference spectrum
(e.g. 6 / 10 = 0.6)
Rel P-Num. % matches of the unknown spectrum
(e.g. 6 / 20 = 0.3)
I-Corr. value of intensity correlation
Unknown microorganism is matched against each Main spectrum in a library. Ranking according to matching score, threshold for ID
Robust standard method for species ID
Freiburg, 12.06.2007
Pseudo-Gel view
M/Z [Da]
File
no.
4000 5000 6000 7000 8000 9000 10000 11000 12000
1 2 3 4 5 6 7 8 910111213141516171819202122232425262728293031323334353637383940
Neisseria meningitidis serotypes
A
W135
X
Y
How about subtyping?
Pseudo-Gel view
M/Z [Da]
File
no.
6100 6200 6300 6400 6500 6600 6700 6800 6900 7000 7100
1 2 3 4 5 6 7 8 910111213141516171819202122232425262728293031323334353637383940
Freiburg, 12.06.2007
MALDI BioTyper – weighted pattern matching
Identification Results weighted
Detected Species log(Score)
--------------------------------------------------------------
Sp. 1 Serogruppe_A 2.677
Serogruppe_Y 2.150
Serogruppe_W135 2.044
Serogruppe_X 2.026
Sp. 2 Serogruppe_W135 2.339
Serogruppe_Y 2.123
Serogruppe_X 1.784
Serogruppe_A 1.571
Sp. 3 Serogruppe_X 2.665
Serogruppe_W135 2.033
Serogruppe_Y 1.902
Serogruppe_A 1.136
Sp. 4 Serogruppe_Y 2.294
Serogruppe_W135 2.126
Serogruppe_X 1.958
Serogruppe_A 1.617
Neisseria meningitidis serogroups
Correct identification of subspecies through weighting of specific peaks.
Expansion of pattern matching towards subspecies detection.
Freiburg, 12.06.2007
Correlation analysis
Composite Correlation Index Map
Species
Specie
s
2 4 6 8 10 12
2
4
6
8
10
12
Color code:
dark red – highest correlation
dark blue – lowest correlation
Correlation analysis of different Salmonella enterica serovars: Correlation analysis according to Arnold & Reilly, RCMS, 1998, modified
1. 1849_Hadar_VAB
2. 371_enteritidis_VAB
3. 042_typhimurium_O5_VAB
4. 104_enteritidis_VAB
5. 123_typhimurium_O5_VAB
6. 163_Virchow_VAB
7. 188_Dublin_VAB
8. 202_Infantis_VAB
9. 242_Infantis_VAB
10. 285_Virchow_VAB
11. 506_Hadar_VAB
12. 754_Agona_VAB
Freiburg, 12.06.2007
Microorganism databases
Acetobacter aceti subsp. aceti Acetobacter pasteurianus subsp.lovaniensis Acetobacter pasteurianus subsp.pasteurianus Actinomadura aurantiaca Actinomadura libanotica Actinomadura livida Agrobaterium tumefaciens Arthrobacter globiformis Arthrobacter oxydans Arthrobacter pyridinolis Arthrobacter sulfureus Bacillus alcalophilus Bacillus cohnii Bacillus sphaericus Brevibacillus brevis Brevibacterium linens Cellulomonas flavigena Cellulomonas turbata Corynebacterium glutamicum Comamonas testosteronii Gluconobacter oxydans subsp. oxydans Gluconobacter oxydans subsp.oxydans Gordonia amarae Gordonia rubropertincta Gordonia terrae Halomonas denitrificans Halomonas elongata Halomonas elongata Halomonas halmophila Halomonas halmophila Hydrogenophaga flava Hydrogenophaga pseudoflava
Methylobacterium mesophilicum Methylobacterium organophilum Methylobacterium radiotolerans Methylobacterium rhodesianum Paracoccus versutus Paracoccus versutus Pseudomonas balearica Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas oleovorans Pseudomonas putida Pseudomonas stutzeri Pseudonocardia hydrocarbonoxydans Rhizobium leguminosarum Rhodococcus coprophilus Rhodococcus fascians Rhodococcus globerulus Rhodococcus rhodnii Rhodococcus rhodochrous Rhodococcus ruber Sinorhizobium meliloti Starkaya novella Starkaya novella Streptomyces albus Streptomyces avidinii Streptomyces azureus Streptomyces badius Streptomyces griseus Streptomyces hirsutus Streptomyces lavendulae Streptomyces phaeochromogenes Streptomyces violaceoruber Streptomyces viridisporus
Libraries:
• Generation of reference pattern for new
microorganisms by users
• Ready-to-use libraries with microbial strains for direct identification (BioTyper 2.0)
Freiburg, 12.06.2007
MALDI BioTyper – database development
DSMZ, Braunschweig, Germany
German Culture Collection
IEB/Czech Culture Collection
Czech Culture Collection
Robert Koch Institute, Berlin, Germany
Infectious disease control centre
Friedrich Loeffler Institute, Riems, Germany
Veterinary infectious disease control centre
Hans Knoell Institute, Jena, Germany
Streptomycetes strain collection
Institute of Environmental Sciences, Leipzig, Germany
Environmental biology
Institute for Bacteriology and Mycology, Leipzig, Germany
Veterinary microbiology
Max-Planck-Istitute für Molecular Biology
Plant Pathogens
LBCM, Lorient, France
Marine microbiology
Partners in database assembly (1)
Freiburg, 12.06.2007
University of Lausanne, Switzerland
U.S. Department of Agriculture, Agricultural Research Service, USA
VetOMICS centre, Vienna, Austria
Food microbiology
Institute for Laboratory Medicine, University Hospital Leipzig, Germany
Institute for Clinical Chemistry, Mannheim; Germany
Institute for Medical Microbiology, Immunology and Parasitology, U. of Bonn, Germany
Institute for Microbiology and Hygiene, Charité, Berlin
Institute for Medical Microbiology, University Hospital, Giessen
Institute for Paradontology + Institute for Hygiene, Münster, Germany
Institute of Physico Chemical Medicine, Moscow, Russia
Hopital Pitié-Salpêtrière, Paris, France
University hospital, Inst. Of Bacteriology, Strassbourgh, France
Institute of Clinical Microbiology, Szeged, Hungary
Children’s Hospital of the King/EVMS, Norfolk, USA
Clinical microbiology
Partners in database assembly (2)
MALDI BioTyper – database development
Freiburg, 12.06.2007
MALDI BioTyper – database development
Status of the Bruker microorganism database, June 2007:
>2500 database entries
>1420 different species
>220 different genera
Steadily growing!
Very high proportion of reference and type strains!
Freiburg, 12.06.2007
Application examples
Freiburg, 12.06.2007
42
63
.38
51
64
.55
64
60
.15
72
45
.48
44
94
.22
75
13
.30
61
76
.31
56
73
.61
86
00
.46
89
89
.05
96
31
.46
82
39
.00
69
29
.01
* Arthrob_sulfureus_B571\0_F8\1\1SLin
0
2000
4000
6000
8000
Inte
ns.
[a.u
.]
42
63
.21
51
64
.04
44
93
.98
72
53
.89
64
60
.00
61
75
.92
56
73
.45
89
89
.24
86
00
.54
96
31
.12
75
13
.41
69
28
.55
82
38
.88
* DSM 20167T\0_G4\1\1SLin, Smoothed, "Baseline subt."
0
1000
2000
3000
4000
5000
6000
Inte
ns.
[a.u
.]
4000 5000 6000 7000 8000 9000m/z
DSMZ / microflex / 2006 / source: DSMZ quality
control
Bruker / autoflex / 2005 / source: UFZ Leipzig
Arthrobacter sulfureus DSM 20157 from different sources
High reproducibility and applicability
Freiburg, 12.06.2007
High reproducibility and applicability
Measurement of one sample using three different Bruker MALDI TOF instruments
0
1
2
3
4
4 x10
5000 6000 7000 8000 9000 10000 11000 m/z
autoflex II TOF/TOF
microflex LT
ultraflex III TOF/TOF
• Analysis of 24 non-fermenting bacteria with microflex LT/MALDI BioTyper
• Measurement of the same extracts again after 2 months, on three different MALDI TOF instruments with adopted, standardized parameter files
• Very reproducible spectra could be generated on the three instruments
• Same identification results over time and regardless the used instrument
Freiburg, 12.06.2007
Taxonomic application
Dendrogram for Clostridia in coop. with Prof. Krüger, Dr. Grosse-Herrenthey, Prof. Böhnel
Very high similarity with 16S rDNA results Presentation in the ClinProt Forum Publication in prep.
Freiburg, 12.06.2007
Industrial application
• Microbial contamination in a facial creme produced by a large health&beauty
product manufacturing facility in North America
• Source of the contaminant – water supply?
• Cultivation of both water supply biofilm bacteria and of contaminant
• Inactivation and shipment to Bruker lab in Billerica
• BioTyper analysis (matching of profile spectra against Bruker db, PCA, MSP
score based dendrogram)
• Parallel analysis by API and Vitek 2 (both Biomerieux) and RiboPrinter
(DuPont Qualicon)
Freiburg, 12.06.2007
Industrial application
Contaminant of facial creme Herbaspirillum huttiensis DSM 10281T
Water supply biofilm:
Burkholderia cenocepacia LMG 12614
API failed in ID, Vitek 2 only identified creme contaminant!
Riboprinter confirmed BioTyper result!
MALDI BioTyper result:
Freiburg, 12.06.2007
Biochemical test: Enteroccocus species, motility negative
MALDI BioTyper: Mixed culture
Staphylococcus epidermidis and Enterococcus faecalis
MALDI BioTyper Identification Results:
Detected Species log(Score)
--------------------------------------------------------------------------------------
Staphylococcus epidermidis 10547 CHB 2.236
Staphylococcus epidermidis CHB 2.175
Enterococcus faecalis 11756 1_CHB 2.158
Enterococcus faecalis ATCC 7080 THL 2.118
Enterococcus faecalis 20247_4 CHB 2.050
Enterococcus faecalis ATCC 29212_CHB 2.047
Enterococcus faecalis ATCC 29212 THL 2.024
Staphylococcus epidermidis DSM 3269 2.004
Staphylococcus epidermidis ATCC 14990 THL 1.973
Staphylococcus epidermidis 6b s ESL 1.900
Clinical application
Freiburg, 12.06.2007
Re-examination “Enterococcus“
Freiburg, 12.06.2007
Clinical application
MALDI BioTyper analysis of 35 biochemically characterized clinical Klebsiella isolates from Russia (Prof. Govorun, IPCM)
Six BioTyper results were deviating from the original identification,
Further analysis by 16S rDNA sequencing:
biochemical tests BioTyper 16S RNA sequencing
Klebsiella ornithinolytica 306 Klebsiella pneumoniae Klebsiella pneumoniae
Klebsiella oxytoca 201 Klebsiella pneumoniae Klebsiella pneumoniae
Klebsiella oxytoca 234 Escherichia coli Escherichia coli
Klebsiella oxytoca 250 Morganella morganii Morganella morganii
Klebsiella ozaenae 36 Klebsiella pneumoniae Klebsiella pneumoniae
Klebsiella rhinoscleromatis 72 Stenotrophomonas maltophilia
Stenotrophomonas sp.
MALDI BioTyper: All deviating results were confirmed by sequencing!
Freiburg, 12.06.2007
• Minimal sample preparation
• Powerful bioinformatic approaches
• Species to strain resolution, mixture detection
• High throughput at low costs per analysis
• Non-expert identification possible
• Dedicated databases of high quality
MALDI BioTyper
MALDI BioTyper:
Highest quality in microorganism ID at low costs
Freiburg, 12.06.2007
• How about virus detection?
• What to do, if the microorganism cannot be cultivated?
• How to handle mixed populations?
• How to handle new emerging agents?
But…
Freiburg, 12.06.2007
Automated Microorganism
Detection System
IBIS Biosciences / Bruker Daltonics
For research use only, not for use in diagnostic procedures
Freiburg, 12.06.2007
• Over 1,000 infectious microbes known to cause disease in
humans*
• 217 viral species
• 538 bacterial species
• 307 fungi
• 66 parasitic protozoa
• Numerous strain variations of each species (i.e., >100 strains of
Streptococcus pyogenes)
• Emergence of multi-drug resistant and highly pathogenic strain
types
• Unknown and uncultivable pathogens
Problems Addressed by Ibis T5000
* Taylor et al., Phil. Trans. R. Soc. Lond. B (2001) 356, 983-989.
Freiburg, 12.06.2007
Objectives of the system
• Broad identification of all clinical microbes
Bacteria, Viruses, Fungi, Protozoa
• No culture
• Mixed populations of microbes (single method for simultaneous wide range identification)
• Quantitative
• High resolution genotyping and strain identification
• Identify emerging agents not previously seen
• Costs effective, high throughput
Freiburg, 12.06.2007
1. Identify genomic regions for identification
Informative variable DNA sequences flanked by conserved sequences
How does it work?
2. Amplify variable
genomic regions
Using broad-range
PCR-primers
3. Measure nucleic acid mass
ESI-TOF mass spectrometry
4. Identify micro organisms
Base-composition fingerprints
Freiburg, 12.06.2007
• Exact mass measurement is used to back-calculate base
compositions consistent with the measured molecular weight.
• The four letter code and their exact masses:
A = 313.0576 T = 329.0526
G = 289.0464 C = 304.0461
Base calculations I
Exact Mass of both strands facilitates unambiguous
base composition determination
Freiburg, 12.06.2007
ppm
0-25
50
100
250
500
# comp pairs
1
13
66
378
1447
AwGxCyTz
AzGyCxTw
Base calculations II
Freiburg, 12.06.2007
(M-30H+)30-
MW = 32,588.90
A28 G29 C25 T24
800 900 1000 1100 1200 1300 m/z
33- 31-
29-
27-
25-
35-
37-
39-
41-
1089 1093 m/z
Electrospray Ionization does not break DNA
Freiburg, 12.06.2007
Animal Reservoirs
of Infectious
Agents
Environmental
Surveillance of
Public Places
Clinical
Diagnostics/
Biosurveillance
Water Quality
Biosurveillance
What kind of samples?
Freiburg, 12.06.2007
Bruker micrOTOF™
mass spectrometer
Magnetic bead-based
desalting station for PCR
plates AND autosampler
Signal analysis and sample tracking software
Instrument Overview
For research use only, not for use in diagnostic procedures
Robotics Arm
And
Plate Hotel
Amplicon
desalting
Module
Infusion
Module
micrOTOF
Sample tracking software and extensive microorganism database
Freiburg, 12.06.2007
Example 1
Broad Bacterial Identification
Pneumonia Epidemic in Marine Recruits
Collaborator: CMDR Kevin Russell M.D.
Naval Health Research Center
Outbreaks of Severe Pneumonia at a recruit training camp,
San Diego 2002/2003
- Hundreds ill
- 160 hospitalized
- 1 death
Identify the pathogens and co-pathogens
Determine the pathogen strain genotype
Freiburg, 12.06.2007
Example of a Broad-Range Test
Primary pathogen:
• Streptococcus pyogenes (GAS)
• emm-type 3
known virulent strain
Secondary pathogens:
• Haemophilus influenzae
• Neisseria meningitidis
5 other military facilities
• Determined these sites had a mixture of strain types
Throughput
• >200/samples per day
*Ecker et al. (2005) Proc.
Natl. Acad. Sci. Vol. 102:
p8012-8017
T5000 in routine use at Naval Health Research Center
S. Pyogenes
[A38 G31 C29 T23]
PCR Calibrant
[A34 G29 C27 T26]
Primer 356: rplB
S. Pyogenes
[A24 G37 C30 T25]
H influenzae
[A23 G37 C26 T27]
Neisseria meningitidis
[A27 G34 C27 T27]
Primer 347: 16S rDNA
Organism
Cumulative
Estimate of
Genomes/Swab
Relative Abundances
Haemophilus influenzae 7.38E+05 1.00
Neisseria meningitidis 3.77E+05 0.51
Streptococcus pyogenes 1.89E+05 0.26
Freiburg, 12.06.2007
Sampath R, Russell KL, Massire C, Eshoo MW, Harpin V, Blyn LB, Melton R, Ivy C, Pennella T, Li F, Levene H, Hall TA, Libby
B, Fan N, Walcott DJ, Ranken R, Pear M, Schink A, Gutierrez J, Drader J, Moore D, Metzgar D, Addington L, Rothman R,
Gaydos CA, Yang S, St George K, Fuschino ME, Dean AB, Stallknecht DE, Goekjian G, Yingst S, Monteville M, Saad MD,
Whitehouse CA, Baldwin C, Rudnick KH, Hofstadler SA, Lemon SM, Ecker DJ.: Global surveillance of emerging influenza virus
genotypes by mass spectrometry. PLoS ONE. 2007 May 30;2:e489.
.
Ecker JA, Massire C, Hall TA, Ranken R, Pennella TT, Agasino Ivy C, Blyn LB, Hofstadler SA, Endy TP, Scott PT, Lindler L,
Hamilton T, Gaddy C, Snow K, Pe M, Fishbain J, Craft D, Deye G, Riddell S, Milstrey E, Petruccelli B, Brisse S, Harpin V, Schink
A, Ecker DJ, Sampath R, Eshoo MW.: Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus
PCR and mass spectrometry. J Clin Microbiol. 2006 Aug;44(8):2921-32.
Hofstadler SA, Drader JJ, Schink A.: Selective Ion Filtering by Digital Thresholding: A Method To Unwind Complex ESI-Mass Spectra
and Eliminate Signals from Low Molecular Weight Chemical Noise. Anal Chem. 78(2): 372-378. January 15, 2006.
Hall TA, Budowle B, Jiang Y, Blyn L, Eshoo M, Sannes-Lowery KA, Sampath R, Drader JJ, Hannis JC, Harrell P, Samant V,
White N, Ecker DJ, Hofstadler SA: Base composition analysis of human mitochondrial DNA using electrospray ionization mass
spectrometry: a novel tool for the identification and differentiation of humans. Anal Biochem. 2005 Sep 1;344(1):53-69.
Ecker DJ, Sampath R, Blyn LB, Eshoo MW, Ivy C, Ecker JA, Libby B, Samant V, Sannes-Lowery KA, Melton RE, Russell K,
Freed N, Barrozo C, Wu J, Rudnick K, Desai A, Moradi E, Knize DJ, Robbins DW, Hannis JC, Harrell PM, Massire C, Hall TA,
Jiang Y, Ranken R, Drader JJ, White N, McNeil JA, Crooke ST, Hofstadler SA.: Rapid identification and strain-typing of respiratory
pathogens for epidemic surveillance.Proc Natl Acad Sci U S A. 2005 May 31;102(22):8012-7. Epub 2005 May 23.
Sampath R, Hofstadler SA, Blyn LB, Eshoo MW, Hall TA, Massire C, Levene HM, Hannis JC, Harrell PM, Neuman B, Buchmeier
MJ, Jiang Y, Ranken R, Drader JJ, Samant V, Griffey RH, McNeil JA, Crooke ST, Ecker DJ.: Rapid identification of emerging
pathogens: coronavirus.Emerg Infect Dis. 2005 Mar;11(3):373-9.
http://www.ibisbiosciences.com/pubs.html
List of Selected References
Freiburg, 12.06.2007
• Minimal sample preparation
• Proteomic Profile
• Open System
• Bacteria, Yeast, Fungi
• Requires cultivation step
• Applications:
• Strain quality control
• Food contaminations
• Medical microbiology
• Water control
• Environmental
• PCR
• DNA Analysis
• Closed system
• Viruses included
• No cultivation step
• Main applications:
• Hospital-associated infections
• Infection control
• Influenza infections
• Surveillance studies
MALDI BioTyper T5000
Two systems for complementary applications in
microorganism identifications!
Comparison
Freiburg, 12.06.2007
Acknowledgement
www.ibisbiosciences.com
www.bdal.de and www.bdal.com
Dr. Wolfgang Pusch
Dr. Gerold Schwarz
Bruker Daltonik GmbH
Shaun Lonergan
David J. Ecker, Ph.D.
Steven A. Hofstadler, Ph.D.
Vanessa Harpin
Ibis Biosciences
Thomas Maier
Kristina Schlosser
Thomas Wenzel
Thorsten Mieruch
Stefan Klepel
Uwe Renner
Jan-Henner Wurmbach
Karl-Otto Kräuter
Alexander Rueegg
Bruker Daltonik GmbH
BioTyper-team: Ibis-Bruker-team:
Freiburg, 12.06.2007
Questions?
Thank you!!