ry

ra
Li b
r e
c tu
Le r
e t ho
l i n u
University Medical Center Groningen
n a
Department Medical Microbiology and Infection Prevention
O by
Expert Center Anaerobic Infections
Linda Veloo
The Netherlands
ID ©
www.mmb-umcg.eu

C M
E S
 ry
ra
Matrix Assisted Laser Desorption/Ionization time-of-flight
Mass Spectrometry (MALDI-TOF MS) Li b
r e
c tu Time of

Le r flight tube
Target at 15-25 kV
e t ho Detector

l i n u
n a
O by
Ion source
ID ©
C M
E S - “Time of flight” of individual proteins is converted into mass
information.
- Spectrum is produced
- Database is built
Veloo et al. Anaerobe 2011; 17:211-212
 ry
Workflow
ra
Li b
Direct spotting of
r
bacteria on target e
tu
using a toothpick
c
Le r
Add HCCA

e matrix
t ho
l i n u
n a Data acquisition

O by
ID ©
C M Data analyses

E S
Log score:
<1.7 no reliable identification
1.7 – 2.0 reliable genus identification
≥ 2.0 reliable species identification
 ry
ra
i b
Obtained spectrum is unique for bacterial species
L
r e
c tu
Le r
e t ho
l i n u
n a
O by
Intensity

ID ©
CM
E S
 ry
Anaerobic culture ra
Li b
r e
Phenotypic

c tu
pure culture
primary incubation

Le r2 days
2-7 days

e t ho
l i n u
aerotolerance identification
MALDI-TOF MS
n a
1 day 2-14 days

primary incubationO by
2-7 days
ID ©
C M MALDI-TOF MS testing

E S minutes
 ry
How many anaerobic bacteria can bera
identified using MALDI-TOF MS?Li b
r e
UMCG 2011/2012 tu
c
e r
e L o
i n u t h
Species ID n l
Total no. of strains 1000
a
650 65%
Genus ID O by 149 15%
No ID ID © 201 20%

C M
E S
 Performance differs per genus ry
ra
Genus* % species ID
Li b
% genus ID
Clostridium sp. (n=149)
r 97 e 3
B. fragilis sp. (n=179)
c tu 97 3

Le r
Parabacteroides sp. (n=14) 93 7
GPAC (n=133)
e t ho 85 15

l i n
Prevotella sp.(n=83)
u 78 12
n a
Propionibacterium sp. (n=129) 64 36
O by
Actinomyces sp. (n=28) 57 43
ID ©
Fusobacterium sp. (n=16) 50 50

C M
Campylobacter sp. (n=17) 41 59

E S Bilophila sp. (n=14) 0 100

Divers (n=26) 77 13

* Only isolates which could be identified with MALDI-

TOF MS (approx. 80% of all isolates)
 ry
Example from literature
ra
Li b
Species identification of clinical Prevotella isolates by Matrix-Assisted Laser
r e
Desorption Ionization-Time of Flight mass spectrometry
tu
Wybo et al. J. Clin. Microbiol. 2012; 50:1415-1418
c
Le r
e t ho
i n
Strains: 102 sequenced clinical isolates
l u
n a
1. O by
Identification MALDI-TOF MS:
using the commercial reference database
2. ID ©
using the commercial reference database + the addition of main

C Mspectral profiles (MSP) of 23 reference strains and 7 sequenced

clinical isolates

E S
 ry
How to make a Main Spectral Profilera
(MSP)
L i b
r e
c tu
e r
Intens. [a.u.]

x10 4

e L o
n t h
Intens. [a.u.]
1.5

n l i a u 4000

O by
1.0

0.5
ID © 3000

C M 2000

0.0
2000

E S
4000 6000 8000 10000 12000 14000 16000
m/z

1000

Average of at least 20 spectra

0
5920 5930 5940 5950 5960 5970 5980
m/z
 ry
ra
Example from literature
Li b
r e
Commercial reference database:

c tu
- 63 % correct species identification
Commercial database + additions:
- 83 % correct species identification

Le r
- 11 % correct genus identification - 6 % correct genus identification
- 26 % no identification
e t ho - 11 % no identification

l i n u
n a
O by
ID ©
C
database M
14 % due to the addition of main spectra of species not yet present in

E S
6 % due to the addition of main spectra of species already present in
database
 ry
ra
Summary (I)
Li b
r e
tu
1. ± 70 % of the anaerobes can be identified using MALDI-TOF MS
c
Le r
e t ho
2. Performance differs per species/genus

l i n u
3. MALDI-TOF MS database needs optimizing for the identification of
n
anaerobic bacteria
a
O by
ID ©
- addition of spectra to existing database increases performance
- reference strains

C M - clinical isolates

E S
 ry
ra
i b
ENRIA r e L
E N R I c tu A
uropean
Le r
etwork for the apid dentification of naerobes

e t ho
l i n u
Lead:
n a
O by
Prof. A.W. Friedrich (The Netherlands)

D ©
Prof. E. Nagy (Hungary)
I
C M
Coördination:
Dr. A.C.M. Veloo (The Netherlands)

E S
Prof. A.J. van Winkelhoff (The Netherlands)

In collaboration with:
Dr. W. Pusch (Bruker, Germany)
Dr. M. Kostrzewa (Bruker Germany) EU INTERREG
 ry
ENRIA project ra
Li b
Expertise laboratories:
r e
-
c tu
UMCG, Groningen, The Netherlands
-
Le r
University of Szeged, Szeged, Hungary
-
e t ho
Odense University Hospital, Odense, Denmark
-
l i n
University Hospital Brussels, Brussels, Belgium
u
-
a
Health Protection Agency, London, England
n
-
- O by
Praticien Hospitalier, Montpellier, France
Public Health Wales, Cardiff, England
ID ©
C M
E S
 ry
ra
Goal:
ENRIA project
Li b
r e
anaerobic bacteria
c tu
To optimize the MALDI-TOF MS database for the most common encountered

Le r
e t ho
l i n u
n a
At least 5 MSP’s present of each species
O by
ID ©
Strains are sent to the UMCG by the expertise laboratories

C
UMCG: M - Culture

E S -
-
Storage
Identification by sequencing
- Ethanol suspension

Validation of the optimized database

 ry
How does this look like in ra
practice:
Li b
e
r
c tu
Le r
e t ho
l i n u
n a
O by
ID ©
C M
E S
 ry
Conditions
ra
Li
Which preanalytical factors influence
b the
r e
quality of the spectrum of anaerobic
bacteria???
c tu
Le r
e t ho
l i n
- Reproducibility of spotting
u
n a
- Incubation time
O by
ID © - Pretreatment method

C M - Direct spotting
- On target extraction

ES - Full extraction

- Exposure to oxygen
 ry
Conditions ra
Li b
Reproducibility spotting
r e
c tu
e r
Amount of bacteria is important:
L
Too little e t ho
no peaks with sufficient intensity
l i n u
Too much n a
saturation of detector and only
O by prominent peaks are measured

ID ©
C M
Optimal amount: 130 µg/µl

E S
Two examiners:
an experienced one and a less experienced one
 ry
Reproducibility spotting results
ra
Li b
r e
c tu
Le r
e t ho
l i n u
n a
O by
ID ©
CM
E S
 ry
ra
Conditions
Li b
Incubation time r e
c tu
Le r24 hours

e t ho
48 hours
72 hours
l i n u 96 hours
n a
O by
ID ©
Pre-treatment method:

C M
E S - direct spotting
- on target extraction with 70% formic acid
- full extraction
Incubation time results
ry
Incubation time (hours)

ra
Species

Gram-negative bacteria
24 48 72

Li b 96

Bacteroides thetaiotaomicron

r
Prevotella intermedia
2.23
1.95a
2.21
2.03
e
2.17
2.27
2.19
2.04

tu
Fusobacterium necrophorum
Fusobacterium nucleatumb

c
Campylobacter ureolyticus
2.27
2.33
1.90
2.18
2.19
2.07
2.31
2.22
2.01
2.29
2.17
1.92a

Le r
Veillonella parvula 2.29 2.45 2.38 2.37

e t ho
% reliable species ID direct spotting 67 100 100 83

n
% reliable species ID total 67 100 100 83

n l i a u
Gram-positive bacteria
Parvimonas micra 2.33 2.29 2.30 2.27

O by
Finegoldia magna
Peptoniphilus harei
2.51c
2.02c
2.43c
2.23
2.23c
2.19c
2.21
2.20

ID ©
Peptoniphilus ivorii
Atopobium minutum
1.78c
1.96a
1.86d
2.26
2.02d
2.29
1.88d
2.25

C M Clostridium butyricum
Clostridium hathewayii
Actinomyces israellii
2.29
2.20d
<1.7e
2.13
2.36
<1.7e
2.17
2.26
<1.7e
2.08
2.36
2.03

E S Actinomyces graevenitzii
Actinomyces meyeri
Bifidobacterium dentium
Bifidobacterium longum
2.09c
2.27
2.30
2.18c
2.36
2.15
2.12
2.08c
2.00d
2.12
2.37
2.07
2.11
2.20
2.28
2.01
Propionibacterium acnes 2.14c 2.24 2.10 2.16
Eggerthella lenta 2.21c 2.20 2.20d 2.10d

% reliable species ID direct spotting 29 71 57 86

% reliable species ID total 79 86 93 93
 ry
ra
Conditions
Li b
Exposure to oxygen r e
c tu
Le r
e t ho
0 hour

l i n u
6 hours

n a 24 hours

O by 48 hours

ID ©
C M
Pre-treatment method:

E S - direct spotting
- on target extraction with 70% formic acid
- full extraction
Exposure to oxygen results
ry
Exposure to oxygen (hours)
ra
Species

Gram-negative bacteria
0 1 6 24

Li
48
b
r
Bacteroides thetaiotaomicon 2.14 2.24 2.10 2.17
e 2.35

c tu
Bacteroides stercoris
Parabacteroides johnsonii
Fusobacterium necrophorum
2.19
2.23
2.08
2.14
2.32
2.30
2.18
2.29
2.19
2.25
2.31
<1.7a
2.27
2.28
<1.7a

Le r
Fusobacterium nucleatumb
Prevotella intermedia
2.08
1.92
2.27
1.93
2.32
1.86
2.19
1.95c
2.08
<1.7a
Prevotella oris

e t ho
Alistipes onderdonkii
2.08
2.21
2.17
2.24
2.01
2.29
2.35
2.29
2.29
2.21

l i n
Veillonella parvula

u
2.25 2.13 2.2 2.20 2.16

n a
% reliable species ID direct spotting 89 89 89 78 78

O by
% reliable species ID total 89 89 89 78 78

D ©
Gram-positive bacteria

M I
Finegoldia magna
Peptoniphilus harei
Peptoniphilus ivorii
2.24
2.15
<1.7d
2.05c
2.00
1.81e
2.34
2.13
<1.7d
2.52
2.23b
1.76e
2.50
2.19
1.80e

SC Clostridium hathewayii
Clostridium ramosum
Actinomyces graevenitzii
2.36
2.03
2.05c
2.23
2.19
2.03e
2.29
2.27
2.11
2.07
2.06
2.06e
2.19
2.17
<1.7d

E Actinomyces meyeri
Bifidobacterium longum
Propionibacterium acnes
2.03
2.00c
2.18
2.27
2.12c
2.14c
2.35
1.99c
2.12
2.19
2.15
2.13
2.25
2.17
2.13
Eggerthella lenta 2.11 2.30 2.27 2.16 2.02e
Collinsella aerofaciens 2.26c 2.16 2.23c 2.32c 2.28c

% reliable species ID direct spotting 64 55 73 64 64

% reliable species ID total 91 82 82 91 82
 ry
ra
Li b
Summary (II)
r e
c tu
The fact whether an unknown anaerobic bacterium can be
e r
identified using MALDI-TOF MS mainly depends on:
L
e t ho
l i n
- The person who is spotting
u
n a
O by
- The type of colony

ID ©
- Amount of bacteria spotted

C M
- If sufficient MSP’s are present in the MALDI-TOF MS database

E S
 ry
ra
Recommendations in order to obtain Li b
a good
quality spectrum r e
c tu
Le r
e t ho
- perform the MALDI-TOF MS measurement after 48 hours of

l i n
incubation in an anaerobic environment
u
n a
- spot the bacteria on the same day
O by
ID ©
- perform an on target extraction with 70% formic acid for

C M
gram-positive anaerobic bacteria

E S- have the spotting performed by an experienced examiner

 ry
ra
Li b
r e
c tu
Le r
Accomplished so far:
e t ho
l i n u
n a
O by Collected: ± 650 strains
ID ©
CM
E S
 ry
Preliminary results ENRIA
ra
Li b
Misidentifications:
r e
Sequence ID
c tu MALDI-TOF MS ID

L e r
Anaerococcus vaginalis
e t ho Anaerococcus hydrogenalis

l i n
Bacteroides dorei
u Bacteroides vulgatus
n a
O by
Bacteroides cellulosilyticus Bacteroides intestinalis

ID ©
Parabacteroides merdae Parabacteroides johnsonii

C M
E SClostridium rectum/
Fusobacterium mortiferum
Fusobacterium mortiferum

Moryella indoligenes Fusobacterium naviforme

No MSPs present in the database

 ry
ra
Li b
Dendrogram Anaerococcus vaginalis/hydrogenalis

r e
c tu
Le r
e t ho
l i n u
n a
O by
ID ©
C M
E S
 ry
ra
Dendrogram Bacteroides vulgatus/dorei
MSP Dendrogram

Li b
e
Bacteroides massiliensis ENR0260

r
Bacteroides massiliensis ENR0230
Bacteroides massiliensis ENR0154

c tu Bacteroides
Bacteroides
Bacteroides
massiliensis DSM 17679T DSM
vulgatus PNU 71838 PNU
vulgatus HU40347_2 PNU

Le r Bacteroides
Bacteroides
vulgatus PNU 1536_5 PNU
vulgatus DSM 3289 DSM

e t ho Bacteroides
Bacteroides
Bacteroides
vulgatus DSM 1447T DSM
dorei ENR0141
dorei ENR0140

l i n u
Bacteroides
Bacteroides
dorei ENR0139
dorei ENR0137

n a Bacteroides
Bacteroides
stercoris ENR0247
eggerthii ENR0264

O by
Bacteroides eggerthii ENR0220
Bacteroides ovatus DSM 1896T DSM
Bacteroides ovatus 53152 PNU

ID © Bacteroides
Bacteroides
Bacteroides
xylanisolvens ENR0364
ovatus HU36898_1 PNU
ovatus IBS_MS_21 IBS

C M Bacteroides
Bacteroides
Bacteroides
ovatus 32456_1 PNU
ovatus 11483_2 PNU
finegoldii ENR0155

E S Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
finegoldii DSM 17565T DSM
caccae 103 PIM
caccae DSM 19024T DSM
caccae ENR0145
caccae ENR0144
1000 900 800 700 600 500 400 300 200 100 0
Distance Level
 ry
Identification of a B. dorei strain after addition of B.adorei MSP’s

i b r
e L
tu r
c
e r
e L o
B. dorei, log score 2,565

i n u t h
n l a
O by
I D ©
C M
E S B. vulgatus, log score 1,983
 ry
Dendrogram
ra
Parabacteroides merdae/johnsonii
Bacteroides intestinalis/cellulosilyticus
Li b
MSP Dendrogram
r e
c tu Parabacteroides merdae ENR0277
Parabacteroides merdae ENR0273

Le r Parabacteroides johnsonii DSM 18315T DSM

Parabacteroides merdae ENR0283

e t ho Parabacteroides goldsteinii DSM 19448T DSM

Parabacteroides goldsteinii BB28 PNU

n
Parabacteroides goldsteinii BB02 PNU

n l i a u Parabacteroides distasonis RV_00005_09 ERL

Parabacteroides distasonis HU35718_3 PNU
Parabacteroides distasonis SW26 PNU

O by
Parabacteroides distasonis HU41816_4 PNU
Parabacteroides distasonis HU36220 PNU
Parabacteroides distasonis RV 412_0209_5 LBK

ID © Parabacteroides distasonis DSM 20701T DSM

Parabacteroides distasonis 53181 PNU

M
Bacteroides massiliensis ENR0230
Bacteroides massiliensis ENR0260

SC Bacteroides massiliensis ENR0154

Bacteroides massiliensis DSM 17679T DSM
Bacteroides intestinalis DSM 17393T DSM

E Bacteroides intestinalis 110706_E9 LUMC

Bacteroides intestinalis SW20 PNU
Bacteroides cellulosilyticus ENR0168
Bacteroides cellulosilyticus ENR0149
Bacteroides cellulosilyticus ENR0228
Bacteroides cellulosilyticus ENR0237
Bacteroides cellulosilyticus ENR0236

1000 800 600 400 200 0

Distance Level
 Phylogenetic tree of fusobacteria ry
ra
Li b
r e
c tu
Le r
e t ho
l i n u
n a
O by
ID ©
CM
E S
 ry
ra
Dendrogram
C. rectum/F. mortiferumL i b
r e
MSP Dendrogram

c tu Fusobacterium nucleatum BK635_11 ERL

Le r Fusobacterium nucleatum 100617_02 PNU

Fusobacterium nucleatum 110706_B8 LUMC

o
Fusobacterium naviforme DSM 20699 DSM
Fusobacterium naviforme DSM 20699 BRB

i ne t h Fusobacterium canifelinum DSM 15543 BRB

Fusobacterium canifelinum DSM 15542T BRB

n l a u Fusobacterium necrophorum ssp necrophorum DSM 20698 DSM

Fusobacterium necrophorum ssp necrophorum DSM 21784T DSM
Fusobacterium gonidiaformans DSM 19810T DSM
Fusobacterium gonidiaformans 110706_B5 LUMC

O by Fusobacterium varium DSM 19868T DSM

Fusobacterium varium DSM 19868T BRB
Fusobacterium mortiferum DSM 19809T BRB

ID © Clostridium rectum ENR0178

Clostridium rectum ENR0317
Clostridium rectum ENR0170

C M Clostridium perfringens RV_BA_03_D LBK

Clostridium perfringens HU51221 PNU
Clostridium perfringens DSM 798 VML

S
Clostridium difficile DSM 1296T DSM
Clostridium difficile DSM 12057 DSM

E
Clostridium clostridioforme ENR0380
Clostridium clostridioforme 110706_G8 LUMC
Clostridium clostridioforme DSM 933T DSM
Clostridium clostridioforme 1021_NCTC 11224T BOG
Bacteroides pyogenes DSM 20612 DSM
Bacteroides pyogenes DSM 20611T DSM
Bacteroides fragilis DSM 9669 DSM
Bacteroides fragilis DSM 2151T DSM
1000 900 800 700 600 500 400 300 200 100 0
Distance Level
 ry
Dendrogram
ra
i b
Campylobacter gracilis: unexpected finding
L
e
MSP Dendrogram

r
c tu Campylobacter gracilis DSM 19528T DSM

L e r Campylobacter gracilis CCUG 27720T NVU

e t ho Campylobacter gracilis ENR0074

l i n u Campylobacter gracilis 0057

n a Campylobacter gracilis 0027

O by Campylobacter gracilis ENR0036

ID © Campylobacter gracilis 0058

C M Campylobacter gracilis 0026

E S Camypylobacter gracilis ENR0078

Campylobacter gracilis ENR0073

1000 900 800 700 600 500 400 300 200 100 0
Distance Level

% sequence similarity of ENR strains with C. gracilis

and each other: >99 %
 ry
ra
Achieved so far Li b
r e
c tu
Le r
e t ho Number of species

l i n u
Species originally available with >5 MSP’s
n a 35

optimization
O by
Species available with >5 MSP’s after
113

ID ©
C
MSP’s) M
Species improved (available now with 2 to 5
94

E S
Species that are new in the database 33
 ry
Estimated end result of strainsb ra
L i
r e
t u
Total no. of strains: ± 700
c
Le r
e t h o
l i n u Number of species
n a
O by
Species originally available with >5 MSP’s 35

ID ©
Species available with >5 MSP’s after
175

C M
optimization
Species improved (available now with 2 to 5

E S
MSP’s)
198

Species that are new in the database 50

 ry
ra
Future plans
Li b
r e
tu
Validation of the optimized database:
c
Le r
- proficiency testing validating laboratories
e t ho
l i n u
- performed by several laboratories throughout Europe
n a
O by
- validation of optimized database with known clinical isolates

ID ©
- capacity building

C M
E S Initiated by “Neglected Infections”of the EurSafety Health-net, which
is financially supported by the EU, Niedersachsen,
NordrheinWestfalen and the Dutch border Provinces.
 ry
ra
Ringtest
Li b
r e
c tu
Le r
Validating laboraties

e t ho
l i n u
n a
O by 7 core
laboratories
ID ©
CM
E S
 ry
ra
Li b
e
r
c tu
Le r
e t ho
l i n u
n a
O by
ID ©
CM
E S
 ry
ra
Li b
re
c tu
Le r
e t ho
l i n u
n a
O by
ID ©
CM Thank you for your attention
E S