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

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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

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