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MALDI BiotyperMicrobial Identificationfor the 21st century… and beyo d
Dr. Francesc Márquez
beyond
Bruker Española S.A.
• MALDI TOF para identificar microorganismos • MALDI-TOF para identificar microorganismos
• La solución MALDI Biotyper
• Estudios de evaluación
ó• Ejemplos de identificación
• Micobacterias y Hongosy g
Microbiology- How is (was) ID DoneMethods for microorganism identificationMethods for microorganism identification
• Ribosomal DNA sequencing (16S rDNA)q g ( )• PCR based detection (several)• Microarrays/FISH hybridization/fluorescence microscopy• RiboPrinter/Pulsed field gel electrophoresis (PFGE)• FT-IR
FAME (f id h l ) GC MS• FAME (fatty acid methyl ester) GC-MS• Biochemistry: phenotypical tests
automated liquid culturing or manual plating
Biochemical methods for microorganism identification
Biolog system
microorganism identification
BD Phoenix system
Biomerieux API-stripsSiemens MicroScan system
Established in routine labsbut based on
“Ancient Technology“Biomerieux Vitek system
“Ancient Technology“
Microbiology- How is (was) ID Done
Biochemical methods Take 24-48 hours for results to develop after culture $4 $10 per sample $4-$10 per sample Automated Culture media dependent
d l l h d Sequencing and molecular methods 4-6 hours after culture Will ID even non-viable organisms (+/- aspects) Can be automated, $30-$50 per sample Other specific detection methods based on primers
available Riboprinter for strain typing FAME
Wouldn´t it be great, ...
• if you had a technology that can analyze gram+and gram- bacteria without prior knowledge?
• if this technology could also analyze yeasts,fungi and mycobacteria?
if thi th d ld b l l h• if this method would be a molecular approachand not a phenotypical one?
• if this method would give the IDif this method would give the IDin less than a minute?
• if this method had a much better taxonomicall ti th bi h i l t ti ?resolution than biochemical testing?
• if this would be available at avery acceptable price per sample?y p p p p
MALDI BiotyperMALDI BiotyperMicrobial Identificationf th 21st t dfor the 21st century… and beyond
• MALDI TOF para identificar microorganismos • MALDI-TOF para identificar microorganismos
• La solución MALDI Biotyper
• Estudios de evaluación
ó• Ejemplos de identificación
• Micobacterias y Hongosy g
Qué es el MALDI-TOF?
MALDI-TOF-MS
Matrix Assisted Laser Matrix Assisted Laser Desorbtion Ionization - Time Of Flight - Mass Spectrometry
b l lé lbalanza para moléculas
Pesando moléculas
¿ ó d ¿cómo podemos poner un microorganismo en un espectrómetro de masas y transformarlo en moléculas ?
Preparación de muestra
M est aMuestra
DisoluciónDisoluciónde matriz
Mezclamos ambas l l d MALDI
Muestra preparada
en la placa de MALDI
p p
Las moléculas se pueden generarpor evaporaciónpor evaporación
En los espectrómetros de masas MALDI usamos un láserpara evaporar la muestra.p p
La matriz protege la muestra
MALDI:
UV-Laser (355 or 337nm)
MALDI:Thermo labilemolecules aremolecules are embedded into host lattice of small matrix
H+
D i I i i i molecules in order to protect them.
MALDI Matrix Sample
Desorption Ionisiation
MALDI-target
Matrix Sample
MALDI-Ionisation
Principio delMALDI-TOF MS.
Vacuumlock
Vacuum system
Sampleplate
Analyte molecules i t i b t
Accelerationgrids
Drift tube Ion detector
Mass spectrumin matrix substance
Mass spectrum essentially reflects analyte molecules
• MALDI TOF para identificar microorganismos • MALDI-TOF para identificar microorganismos
• La solución MALDI Biotyper
• Estudios de evaluación
ó• Ejemplos de identificación
• Micobacterias y Hongosy g
Identificación de microorganismos por MALDI TOF MSMALDI TOF MSFlujo de trabajo con MALDI Biotyper:
IdentificaciónS l ió d l i? IdentificaciónSelección de una colonia?
Microorganismodesconocido
MALDI-TOF MS Interpretación de los datosPreparación en la placa de MALDI (disponibles placasreutilizables o de un solo uso).
Tiempo para obtener el resultado de una muestra: ~ 5 min
• Taxonomical Resolution comparable to sequencing
• Faster than conventional techniques
• Cost-effective
Each microbial species has a unique proteomic fingerprint
06a.u.
]
proteomic fingerprint.
E.coli43
64.0
0.64
.64
5000
Inte
ns. [
a
ribosomal Protein m/zRL36 4364,33RS32 5095,82RL34 5380 39
5380
6254
.31
5.49
4000
RL34 5380,39RL33meth. 6255,39RL32 6315,19RL30 6410,60RL35 7157,74
63
5096
.01
7157
.65
73.8
7
90
3000RL29 7273,45RL31 7871,06RS21 8368,76
772
7
6410
.
70.6
2
68.9
9
2000
787
836
0
1000
04000 4500 5000 5500 6000 6500 7000 7500 8000
m/z
MALDI BiotyperIdentifica bacterias gram+ y gram levaduras y hongosIdentifica bacterias gram+ y gram-, levaduras y hongosmediante su “huella dactilar“ proteómica
Bacillus subtilis
Escherichia coli
1 min
Candida albicans
Aspergillus fumigatus
MALDI Biotyper Features
• 12-24 hour culture recommended• Sensitive, can work with 100,000 cells, small colonies Sensitive, can work with 100,000 cells, small colonies
preferred• Results in minutes after culture, not hours or days
bl l $ 3 / l• Consumables cost approximately $0.30/sample• Automated, up to 96 samples can be done in a single
runrun• Robust, reproducible and easy to use• No gram stain required, works for gram positives, gram
i d f inegatives, yeast and fungi• Library compiled with collaborators from many fields
Unknown Organism Specifications…
Classifications of organisms that can be analyzed: Bacteria, yeast, molds, mycobacteria
Cultivation Media: studies have demonstrated that different Cultivation Media: studies have demonstrated that different media types do not affect results.
Cultivation Temperatures: studies have demonstrated that different air temperatures, composition, and humidity do not p , p , yaffect results. Cultures that have been refrigerated after growth do not generate acceptable spectra
Age of Organism: cultures should be fresh, for example 12-24 h th f t ti b t i d 24 48 h f t hour growth for most routine bacteria and 24-48 hours for yeast. Older cultures can be analyzed, however, the scores may be lower (repeat using a fresh isolate)
ghk@bdal.com02/2009
Preparación de muestrap
Transferencia directa:
Tocar una colonia con un dispositivo para transferirlo a la placa de MALDI como un mondadientes.
Transferir una pequeña cantidad a un pocillo y extender. Dejar secar al aire. Cubrir con 1 µL de matriz HCCA dejar secar al aire. Analizar.
Identificación Rutinaria
Transferencia Directa90 – 95 %
Ex DT Ex DT ~8%
Extracc.~3 %
Seq.Seq.
Preparación de muestra
Extracción con Etanol y ácido Fórmico
Transferirmaterial
Aguadesionizada
Preparación de muestra
Extracción con Etanol y ácido Fórmico
Etanol
Inactivaciónde patógenos
Preparación de muestra
Extracción con Etanol y ácido Fórmico
Etanol
Preparación de muestra
Extracción con Etanol y ácido Fórmico
Ácido fórmicoAcetonitrilo
Preparación de muestra
Extracción con Etanol y ácido Fórmico
Analizarsobrenadante
10 min10 min
Preparación de muestra
MALDI Biotyper – Puntuación basada en los patrones de coincidenciapatrones de coincidencia
El microorganismo desconocido se compara contra la librería de referencia El microorganismo desconocido se compara contra la librería de referencia.
Resultado de identifación mediante código de colores.
Scoring Algorithm
• Score increased for each peak matched between measured and library spectrummeasured and library spectrum
• Score decreased for peaks that don’t match• Overall match of intensity pattern added to score• Results in a number from 0 to 1000, final score is the
logarithm of that number (0-3)• Above 2 0 is considered a reliable species match• Above 2.0 is considered a reliable species match• Between 1.7 and 2.0 is considered a genus level match• Below 1.7 is considered no match • Robust for species level ID not for strain typing
Results - Color Coded Identification & Consistency CategoriesConsistency Categories
MALDI Biotyper – Result tableyp
MALDI Biotyper – Ranking Listyp g
MALDI Biotyper OC
Unknown samples
Match to references
Identification results
MALDI BiotyperOpen Microbiology ConceptOpen Microbiology Concept
• Open Interfaces to complementary Open Interfaces to complementary technologies
• Customers can generate own library entries
• Customer-generated entries can be exchanged between cooperating labs
• More than 100 partnering labs for the library generation
Involve microbiology experience of our customers in • Involve microbiology experience of our customers in consortia for certain microbial groups:
FungiMycobacteriayAnaerobes (ENRIA)Yeasts (CBS ring test)
• Collaboration with other market leading • Collaboration with other market leading companies (BD, Siemens)
Open Library Concept C ió t l b t iCooperación entre laboratorios
MSPExport MSPExport
Own strainsof interest
Generate own Library Entries
(MSPs)
Partnering Labsor BionumericsSoftware(MSPs) Software
MALDI BiotyperBase de datos de referenciaAustriaBelgium
Base de datos de referencia
BelgiumBrazilCzech Republic Current Bruker database:FranceGermanyHungaria
> 5.989 Strains (more than 80.000 Spectra)> 2.400 different species
450 diff t HungariaItalyNetherlands
> 450 different genera
Strains from > 100 partners/ 18 RussiaSwedenSwitzerland
different countries
Very high proportion of sequenced, SpainSouth AfricaUK
y g p p q ,reference and type strains
UKUSA
• MALDI TOF para identificar microorganismos • MALDI-TOF para identificar microorganismos
• La solución MALDI Biotyper
• Estudios de evaluación
ó• Ejemplos de identificación
• Micobacterias y Hongosy g
Baja influencia de las condiciones de cultivoEstudios de Evaluación
Pseudomonas oleovorans crecido en medios diferentes
j
Psdm. oleovorans B396_Medium 360
01000
Psdm. oleovorans B396_Medium 464
01000
Psdm. oleovorans B396_Medium 53
1000
0 Psdm. oleovorans B396_Medium 65
01000
Psdm. oleovorans B396 Medium 98Psdm. oleovorans B396_Medium 98
0500
1000
Psdm. oleovorans B396_MRS10
2000
01000
Psdm. oleovorans B396_YPD
010002000
0 4000 5000 6000 7000 8000 9000 10000 11000m/z
Estudios Multi-CentroEstudio de Evaluación de Cumplimiento
Test de est és
Estudio de Evaluación de Cumplimiento
Test de estrésIdentificación de 20 especies de cultivos de colección complicadasde identificar, medidas múltiples en días consecutivos, p(reproducibilidad inter- e intra-dia)
Objetivo: Exactitud en la identificación > 90%
Test de perfil de trabajo diario en laboratorioIdentificación de aislados clínicos en el trabajo diarío de un l b t i d ét d bi í i ( i ió )laboratorio, comparado con métodos bioquímicos (o secuenciación)
Objetivo: Exactitud en la identificación > 90%
Manual for Clinical Microbiology, Murray, Patrick R., ed. in chief; Baron, Ellen Jo, ... [et al.], 9th ed. ASM
Miller, J.M. 1991, Evaluating biochemical identification systems. J. Clin. Microbiol. 29:1559-2101
Manual for Clinical Microbiology, Murray, Patrick R., ed. in chief; Baron, Ellen Jo, ... [et al.], 9th ed. ASM Press, 2007, page 202
Estudios Multi-CentroEstudio de Evaluación de Cumplimiento
Bacterias Gram-positivas:
Estudio de Evaluación de Cumplimiento
Kocuria kristinae DSMZ 20321Streptococcuspyogenes DSMZ 2071
Test de estrésBacterias Gram positivas:
pyogenes DSMZ 2071Staphylococcuslugdunensis DSMZ 4804
Enterobacteriaceae:
Staphylococcussciuri DSMZ 20345Streptococcus equi DSMZ 20727
Leclercia adecarboxylata DSMZ 5077Proteus vulgaris DSMZ 13387Enterobacter cloacae DSMZ 30062 Streptococcus equi DSMZ 20727
Bacterias Gram-negativas no fermentadoras:
Enterobacter cloacae DSMZ 30062
Acinetobacter baumannii DSMZ 30007Stenotrophomonasmaltophilia DSMZ 50170maltophilia DSMZ 50170
Estudios Multi-CentroEstudio de Evaluación de CumplimientoEstudio de Evaluación de Cumplimiento
Otras Bacterias Gram-negativasLevaduras:
Test de estrés
Otras Bacterias Gram negativas
Ochrobactrumanthropi DSMZ 6882
Saccharomyces cerevisiae DSMZ 70449Yarrowia lipolytica DSMZ 1345C did t i li DSMZ 1346 Brevundimonas
diminuta DSMZ 7234S hi b t i
Candida tropicalis DSMZ 1346Candida albicans DSMZ 1386Candida parapsilosis DSMZ 5784 Sphingobacterium
spiritivorum DSMZ 11722
Candida parapsilosis DSMZ 5784Candida glabrata DSMZ 11950Clavispora lusitaniae DSMZ 70102
Estudios Multi-CentroEstudio de Evaluación de CumplimientoEstudio de Evaluación de Cumplimiento
Resultados del test de estrés
Valores obtenidos para los estudios intra-día
Día 1: exactitud en la identificación= 96%Día 2: exactitud en la identificación = 93%Día 3 e actit d en la identificación 93%Día 3: exactitud en la identificación = 93%
Exactitud en la identificación inter-día= 94%Exactitud en la identificación inter día= 94%
*) Basado en espectros generados con el sistema Microflex LT) Basado en espectros generados con el sistema Microflex LT
Estudios Multi-Centro
f”Weighted Laboratory Profile Test”
ID of clinical isolates compared to biochemical methods (or sequencing)
Microorganism Group N MALDI BiotyperIdentification
Biochemical ID
p ( q g)
Non-Fermenting Gram-Negative Bacteria
274 95% 88%
Enterobacteriaceae 439 100% 98%
Other Gram-Negative Bacteria 251 95% 89%
Gram-Positive Bacteria 497 97% 94%
Yeasts 348 97% 98%Yeasts 348 97% 98%
Sum / Overall for ID Acc. 1809 97% 94%
0.39% Identification only on genus level (Biochemical: 0.77%)
1.77% No identification (Biochemical: 2.16%)
0.88% False-Positive Identification (Biochemical: 2.93%)
• MALDI TOF para identificar microorganismos • MALDI-TOF para identificar microorganismos
• La solución MALDI Biotyper
• Estudios de evaluación
ó• Ejemplos de identificación
• Micobacterias y Hongosy g
MALDI Biotyper - nonclinical
Food• Trace-back of contaminations• Dairy products
Pharma• Trace-back of contaminations• Control of raw materials
2 C S• Beer production• Rapid Salmonella detection• Fish and meat species• Product authenticity testing
• 21 CFR Part 11 Support
Product authenticity testing
A i lt /Animal Health
Agriculture/Environmental• Ballast water analysis
• Mastitis• Difficult organisms• Subtyping to differentiate
d i f ti fy
• Taxonomy• Dereplication of microbial
communities
dangerous infections fromopportunistic pathogens
• Tracking of microbes specificfor certain poultry flocks.for certain poultry flocks.
Identification of lactic acid bacteria
www.bruker.com
Mass spectra of different Lactobacillus species. MS fil h t b t L t b illi i ith
© Copyright Bruker Corporation. All rights reserved.
MS profiles were heterogeneous between Lactobacilli species with slight differences within each group.
Beverage Spoiling Microorganisms
Pectinatus sp S cerevisiae
Pediococcus ssp.Debaryomyces hansenii
Pectinatus sp. S. cerevisiae
Wickerhamomyces anomalusJürgen Behr
L. brevisCarola KernJulia UsbeckMathias HutzlerMathias HutzlerRudi F. Vogel
CO2, Ethanol, Off-flavour
State-of-the-art differentiation of beverage spoilers by MALDI-TOF MS biotyping
100%
90 69.4 50%
75%
90.6
25%
0%1.100 1.1205 1.1326 1.1370 1.315
correct falsely classified as: 1.100 1.1205 1.841 1.317
Correct
False
strain level identification
St i l l id tifi ti hi d i d 90% f t t l f th Strain level identification was achieved in around 90% of a total of more than 200 samples
Product Authenticity Testing
May 22, 2015, WPU@bdal.de
MALDI BiotyperTrace back of contaminations in productionTrace-back of contaminations in production
• Establish Library Entries from swabs of different production places.
• Build a taxonomy tree
• In case of contamination match isolate against this tree.
• Decrease production downtime in case ofcontamination by rapid identification of the sourcecontamination by rapid identification of the source.
• Prof. Pierre Venter, Fonterra, New Zealand
May 22, 2015, WPU@bdal.de 51
Diferenciación de Listeria spp
Espect os de dife entes especies de Liste iaEspectros de diferentes especies de Listeria:L.monocytogenes, L.ivanovii ssp ivanovii, L.ivanovii ssp londoniensis,L.innocua, L.seeligeri, and L.welshimeri.
Diferenciación de Listeria spp
Identificacion de Listeria: Patrón de cotejo standard“ and weighted“ Identificacion de Listeria: Patrón de cotejo „standard and „weighted La introducción de criterios de peso específico incrementa la distancia entre el patrón correcto (L. monocytogenes) y el cercano mejor relacionado (L. ivanovii).
• MALDI TOF para identificar microorganismos• MALDI-TOF para identificar microorganismos
• La solución MALDI Biotyper
• Estudios de evaluación
• Ejemplos de identificación
• Micobacterias y Hongosy g
Fungi Library & Mycobacteria Libraryg y y y
> 850 References>100 References > 850 References
MALDI Biotyper - Resumen
• Sensitive, can work with 100,000 cells, small colonies preferred
• Results in minutes after culture, not hours or days• Automated, up to 96 samples can be done in a single
runrun• Robust, reproducible and easy to use• No gram stain required, works for gram positives, gram
negatives, yeast and fungi• Library compiled with collaborators from many fields,
not only clinicalnot only clinical.
MALDI Biotyper - Resumen
• Not a proteomics experiment! We see a “fingerprint” of small soluble proteinssmall soluble proteins
• Makes the technique independent of culture conditions.• Includes choice of culture media, temperature , p• Very robust, in use in over >1800 labs worldwide• Excellent lab to lab reproducibility• Suitable for operation by bench level technologists, does
not require expert operator• Open libraryOpen library
www.bruker.com
© Copyright Bruker Corporation. All rights reserved.
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