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MALDI Biotyper

Microbial Identification
for the 21st century… and
beyo d
beyond

Dr. Francesc Márquez


Bruker Española S.A.
• MALDI-TOF
MALDI TOF para identificar microorganismos

• La solución MALDI Biotyper

• Estudios de evaluación

• Ejemplos de identificación
ó

• Micobacterias y Hongos
g
Microbiology- How is (was) ID Done
Methods for microorganism identification

• Ribosomal DNA sequencing


q g ((16S rDNA))
• PCR based detection (several)
• Microarrays/FISH hybridization/fluorescence microscopy
• RiboPrinter/Pulsed field gel electrophoresis (PFGE)
• FT-IR
• FAME (f
(fatty acid
id methyl
h l ester)) GC-MS
GC MS
• Biochemistry: phenotypical tests
automated liquid culturing or manual plating
Biochemical methods for
microorganism identification

Biolog system

BD Phoenix system

Siemens MicroScan system


Biomerieux API-strips

Established in routine labs


but based on
“Ancient Technology“
Biomerieux Vitek system
Microbiology- How is (was) ID Done

 Biochemical methods
 Take 24-48 hours for results to develop after culture
 $4-$10
$4 $10 per sample
 Automated
 Culture media dependent
 Sequencing and d molecular
l l methods
h d
 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 this
thi method
th d would
ld be
b a molecular
l l approach
h
and not a phenotypical one?

• if this method would give the ID


in less than a minute?

• if this method had a much better taxonomical


resolution
l ti than
th biochemical
bi h i l testing?
t ti ?

• if this would be available at a


veryy acceptable
p p
price p
per sample?
p
MALDI Biotyper
Microbial Identification
f
for th
the 21st century…
t and
d
beyond
• MALDI-TOF
MALDI TOF para identificar microorganismos

• La solución MALDI Biotyper

• Estudios de evaluación

• Ejemplos de identificación
ó

• Micobacterias y Hongos
g
Qué es el MALDI-TOF?

MALDI-TOF-MS

Matrix Assisted Laser


Desorbtion Ionization - Time Of
Flight - Mass Spectrometry

b l
balanza para moléculas
lé l
Pesando moléculas

¿cómo
¿ ó podemos
d poner un
microorganismo en un espectrómetro
de masas y transformarlo en moléculas ?
Preparación de muestra

M est a
Muestra

Disolución
de matriz

Mezclamos ambas
en la
l placa
l de
d MALDI

Muestra p
preparada
p
Las moléculas se pueden generar
por evaporación

En los espectrómetros de masas MALDI usamos un láser


para evaporar
p p la muestra.
La matriz protege la muestra

UV-Laser (355 or 337nm)

MALDI:
Thermo labile
H+
molecules are
embedded into host
lattice of small matrix
D
Desorption
i I i i i
Ionisiation
molecules in order to
protect them.
MALDI-
MALDI Matrix Sample
target

MALDI-Ionisation
Vacuum
Principio del
lock MALDI-TOF MS.

Vacuum system

Sample Acceleration Drift tube Ion detector


plate grids
Analyte molecules Mass spectrum
i matrix
in t i substance
bt

Mass spectrum essentially reflects


analyte molecules
• MALDI-TOF
MALDI TOF para identificar microorganismos

• La solución MALDI Biotyper

• Estudios de evaluación

• Ejemplos de identificación
ó

• Micobacterias y Hongos
g
Identificación de microorganismos por
MALDI TOF MS
Flujo de trabajo con MALDI Biotyper:

? S l
Selección
ió de
d una colonia
l i Identificación

Microorganismo Preparación en la
MALDI-TOF MS Interpretación de los datos
desconocido placa de MALDI
(disponibles placas
reutilizables 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
fingerprint.
a.u.]

06
4364.0
Intens. [a

E.coli ribosomal Protein m/z


5000
RL36 4364,33

0.64
RS32 5095,82

6254..64
5380
RL34 5380 39
5380,39
RL33meth. 6255,39
4000
RL32 6315,19

315.49
RL30 6410,60
RL35 7157,74

63
RL29 7273,45
5096.01

3000
RL31 7871,06

7157.65
RS21 8368,76

73.87
6410.90

7
727
2000

70.62

68.99
787

836
1000

0
4000 4500 5000 5500 6000 6500 7000 7500 8000
m/z
MALDI Biotyper
Identifica bacterias gram+ y gram-,
gram levaduras y hongos
mediante 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
preferred
• Results in minutes after culture, not hours or days
• Consumables
bl cost approximately l $$0.30/sample
3 / l
• Automated, up to 96 samples can be done in a single
run
• Robust, reproducible and easy to use
• No gram stain required, works for gram positives, gram
negatives,
i yeast and
d fungi
f i
• 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
media types do not affect results.
 Cultivation Temperatures: studies have demonstrated that
different air temperatures,
p , composition,
p , and humidity
y do not
affect 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
hour growth
th for
f mostt routine
ti bacteria
b t i and d 24-48
24 48 hours
h for
f yeast.
t
Older cultures can be analyzed, however, the scores may be lower
(repeat using a fresh isolate)

02/2009
ghk@bdal.com
Preparación
p de muestra

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 Directa
90 – 95 %

Ex DT
~8%

Extracc.
~3 %

Seq.
Preparación de muestra

Extracción con Etanol y ácido Fórmico

Transferir
material

Agua
desionizada
Preparación de muestra

Extracción con Etanol y ácido Fórmico

Etanol

Inactivación
de patógenos
Preparación de muestra

Extracción con Etanol y ácido Fórmico

Etanol
Preparación de muestra

Extracción con Etanol y ácido Fórmico

Ácido fórmico
Acetonitrilo
Preparación de muestra

Extracción con Etanol y ácido Fórmico

Analizar
sobrenadante

10 min
Preparación de muestra
MALDI Biotyper – Puntuación basada en los
patrones de coincidencia

El microorganismo desconocido se compara contra la librería de referencia.


referencia

Resultado de identifación mediante código de colores.


Scoring Algorithm

• Score increased for each peak matched between


measured 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 22.0
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 Categories
MALDI Biotyper
yp – Result table
MALDI Biotyper
yp – Ranking
g List
MALDI Biotyper OC

Match to
Unknown references
samples

Identification
results
MALDI Biotyper
Open Microbiology Concept

• 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
consortia for certain microbial groups:
Fungi
Mycobacteria
y
Anaerobes (ENRIA)
Yeasts (CBS ring test)

• Collaboration with other market leading


companies (BD, Siemens)
Open Library Concept
C
Cooperación
ió entre
t llaboratorios
b t i

Export MSP

Own strains Generate own Partnering Labs


of interest Library Entries or Bionumerics
(MSPs) Software
MALDI Biotyper
Base de datos de referencia
Austria
Belgium
Brazil
Czech Republic Current Bruker database:
France
> 5.989 Strains (more than 80.000 Spectra)
Germany
> 2.400 different species
Hungaria
> 450 different
diff t genera
Italy
Netherlands Strains from > 100 partners/ 18
Russia different countries
Sweden
Switzerland Very
y high
g pproportion
p of sequenced,
q ,
Spain reference and type strains
South Africa
UK
USA
• MALDI-TOF
MALDI TOF para identificar microorganismos

• La solución MALDI Biotyper

• Estudios de evaluación

• Ejemplos de identificación
ó

• Micobacterias y Hongos
g
Estudios de Evaluación
Baja
j influencia de las condiciones de cultivo
Pseudomonas oleovorans crecido en medios diferentes

Psdm. oleovorans B396_Medium 360

1000
0 Psdm. oleovorans B396_Medium 464

1000
0 Psdm. oleovorans B396_Medium 53

1000
0 Psdm. oleovorans B396_Medium 65

1000
0 Psdm. oleovorans B396
B396_Medium
Medium 98

1000
500
0 Psdm. oleovorans B396_MRS10

2000
1000
0 Psdm. oleovorans B396_YPD
2000
1000
0 4000 5000 6000 7000 8000 9000 10000 11000
m/z
Estudios Multi-Centro
Estudio de Evaluación de Cumplimiento

Test de estrés
est és
Identificación de 20 especies de cultivos de colección complicadas
de identificar,, medidas múltiples
p en días consecutivos
(reproducibilidad inter- e intra-dia)
Objetivo: Exactitud en la identificación > 90%

Test de perfil de trabajo diario en laboratorio


Identificación de aislados clínicos en el trabajo diarío de un
l b
laboratorio,
t i comparado d con métodos
ét d bioquímicos
bi í i ( secuenciación)
(o i ió )
Objetivo: Exactitud en la identificación > 90%

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-Centro
Estudio de Evaluación de Cumplimiento

Bacterias Gram
Gram-positivas:
positivas:
Test de estrés Kocuria kristinae DSMZ 20321
Streptococcus
pyogenes DSMZ 2071

Enterobacteriaceae: Staphylococcus
lugdunensis DSMZ 4804
Leclercia adecarboxylata DSMZ 5077 Staphylococcus
Proteus vulgaris DSMZ 13387 sciuri DSMZ 20345
Enterobacter cloacae DSMZ 30062 Streptococcus equi DSMZ 20727

Bacterias Gram-negativas no fermentadoras:


Acinetobacter baumannii DSMZ 30007
Stenotrophomonas
maltophilia DSMZ 50170
Estudios Multi-Centro
Estudio de Evaluación de Cumplimiento

Test de estrés
Levaduras: Otras Bacterias Gram
Gram-negativas
negativas
Saccharomyces cerevisiae DSMZ 70449
Ochrobactrum
Yarrowia lipolytica DSMZ 1345
anthropi DSMZ 6882
C did tropicalis
Candida t i li DSMZ 1346
Brevundimonas
Candida albicans DSMZ 1386 diminuta DSMZ 7234
Candida parapsilosis DSMZ 5784
Sphingobacterium
S hi b t i
Candida glabrata DSMZ 11950 spiritivorum DSMZ 11722
Clavispora lusitaniae DSMZ 70102
Estudios Multi-Centro
Estudio 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:
3 exactitud
e actit d en la identificación = 93%

Exactitud en la identificación inter


inter-día=
día= 94%

*)) Basado en espectros generados con el sistema Microflex LT


Estudios Multi-Centro
”Weighted Laboratory Profile
f Test”
ID of clinical isolates compared
p to biochemical methods (
(or sequencing)
q g)

Microorganism Group N MALDI Biotyper Biochemical ID


Identification

Non-Fermenting Gram-Negative 274 95% 88%


Bacteria
Enterobacteriaceae 439 100% 98%

Other Gram-Negative Bacteria 251 95% 89%

Gram-Positive Bacteria 497 97% 94%

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
MALDI TOF para identificar microorganismos

• La solución MALDI Biotyper

• Estudios de evaluación

• Ejemplos de identificación
ó

• Micobacterias y Hongos
g
MALDI Biotyper - nonclinical

Food Pharma
• Trace-back of contaminations • Trace-back of contaminations
• Dairy products • Control of raw materials
• Beer production • 2 CFR
21 C Part 11 Support
S
• Rapid Salmonella detection
• Fish and meat species
• Product authenticity testing

Animal Health
Agriculture/
A i lt / • Mastitis
Environmental • Difficult organisms
• Ballast water analysis
y • Subtyping to differentiate
• Taxonomy d
dangerous iinfections
f ti ffrom
• Dereplication of microbial opportunistic pathogens
communities • Tracking of microbes specific
for certain poultry flocks.
Identification of lactic acid bacteria

www.bruker.com

Mass spectra of different Lactobacillus species.


MS profiles
fil were heterogeneous
h t between
b t Lactobacilli
L t b illi species
i with
ith
slight differences within each group.
© Copyright Bruker Corporation. All rights reserved.
Beverage Spoiling Microorganisms

Debaryomyces hansenii Pediococcus ssp.

Pectinatus sp.
sp S.
S cerevisiae

Jürgen Behr Wickerhamomyces anomalus

Carola Kern
Julia Usbeck L. brevis
Mathias Hutzler
Rudi F. Vogel

CO2, Ethanol, Off-flavour


State-of-the-art differentiation of beverage
spoilers by MALDI-TOF MS biotyping

100%

75%

50%
9.4
90 6
90.6

25%

0%
Correct 1.100 1.1205 1.1326 1.1370 1.315

False correct falsely classified as: 1.100 1.1205 1.841 1.317

strain level identification

 Strain
St i level
l l id
identification
tifi ti was achieved
hi d iin around
d 90% off a ttotal
t l off more than
th
200 samples
Product Authenticity Testing

May 22, 2015, WPU@bdal.de


MALDI Biotyper
Trace back of contaminations in production
Trace-back

• 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 of


contamination by rapid identification of the source.
source

• Prof. Pierre Venter, Fonterra, New Zealand

May 22, 2015, WPU@bdal.de 51


Diferenciación de Listeria spp

Espectros
Espect os de diferentes
dife entes especies de Listeria:
Liste ia
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


standard“ and „weighted
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
MALDI TOF para identificar microorganismos

• La solución MALDI Biotyper

• Estudios de evaluación

• Ejemplos de identificación

• Micobacterias y Hongos
g
Fungi
g Library
y & Mycobacteria
y Library
y

>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
run
• 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 clinical.
clinical
MALDI Biotyper - Resumen

• Not a proteomics experiment! We see a “fingerprint” of


small 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 library
www.bruker.com

© Copyright Bruker Corporation. All rights reserved.