Identification of Gallibacterium species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry evaluated by multilocus sequence analysis

Abstract

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) whole-cell fingerprinting was used for characterization of 66 reference strains of Gallibacterium. The 4 recognised Gallibacterium species and Gallibacterium genomospecies 1 yielded reproducible and unique mass spectrum profiles, which were confirmed with Bruker Biotyper reference database version 3. The reproducibility of MALDI-TOF MS results were evaluated varying the age and storage of the cultures investigated. Reliable species identification was possible for up to 8 days of storage at 4°C and less reliable if the bacteria were stored at room temperature (20°C). However, if the strains were grown longer than 48h at 37°C under microaerobic atmosphere, poor identification results were obtained, due to changes in protein profile. The MALDI-TOF MS results of all 66 strains demonstrated 87.9% concordance with results based upon biochemical/physiological characterization. In addition, diversities outlined by MALDI-TOF MS were verified by sequencing the rpoB (n=43), 16S rRNA (n=28), infB (n=14), and recN (n=14) genes (multilocus sequence analysis, MLSA). In addition, discrepancies were observed between some of the genes sequenced. Results obtained demonstrated that MALDI-TOF MS fingerprinting represents a fast and reliable method for identification and differentiation of the 4 recognised Gallibacterium species and possible a fifth species Gallibacterium genomospecies 1, with applications in clinical diagnostics.

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Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
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International Journal of Medical Microbiology
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Identification of Gallibacterium species by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry evaluated by multilocus
sequence analysis
Merima Alispahica,∗, Henrik Christensenb, Claudia Hess a, Ebrahim Razzazi-Fazelic,
Magne Bisgaardb, Michael Hess a
aClinic for Avian, Reptile and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria
bDepartment of Veterinary Disease Biology, Faculty of Life Science, University of Copenhagen, Denmark
cVetOMICS Core Facility for Research/Proteomics and Metabolomics, University of Veterinary Medicine, Vienna, Austria
article info
Article history:
Received 15 December 2010
Received in revised form 4 March 2011
Accepted 5 March 2011
Keywords:
Gallibacterium
MALDI-TOF MS
MLSA
Characterization
abstract
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) whole-
cell fingerprinting was used for characterization of 66 reference strains of Gallibacterium. The 4 recognised
Gallibacterium species and Gallibacterium genomospecies 1 yielded reproducible and unique mass
spectrum profiles, which were confirmed with Bruker Biotyper reference database version 3. The
reproducibility of MALDI-TOF MS results were evaluated varying the age and storage of the cultures
investigated. Reliable species identification was possible for up to 8 days of storage at 4 ◦C and less reliable
if the bacteria were stored at room temperature (20 ◦C). However, if the strains were grown longer than
48hat37◦C under microaerobic atmosphere, poor identification results were obtained, due to changes in
protein profile. The MALDI-TOF MS results of all 66 strains demonstrated 87.9% concordance with results
based upon biochemical/physiological characterization. In addition, diversities outlined by MALDI-TOF
MS were verified by sequencing the rpoB (n= 43), 16S rRNA (n=28), infB (n= 14), and recN (n= 14) genes
(multilocus sequence analysis, MLSA). In addition, discrepancies were observed between some of the
genes sequenced. Results obtained demonstrated that MALDI-TOF MS fingerprinting represents a fast
and reliable method for identification and differentiation of the 4 recognised Gallibacterium species and
possible a fifth species Gallibacterium genomospecies 1, with applications in clinical diagnostics.
© 2011 Elsevier GmbH. All rights reserved.
Introduction
Four species of Gallibacterium,G. anatis,G. melopsittaci,G. salp-
ingitidis, and G. trehalosifermentans have been recognised so far
(Euzeby, 1997; Christensen et al., 2003; Bisgaard et al., 2009). How-
ever, 16S rRNA gene sequence data clearly indicated the existence
of a probable new species G. genomospecies 1 and G. genomo-
species 2 (Christensen et al., 2003). In addition, Bisgaard et al. (2009)
showed that 16S rRNA groups III (G. genomospecies 3) and V (G.
group V) should be classified as novel species of Gallibacterium. All
taxa of Gallibacterium reported so far seem to be associated with
birds, although isolates have been reported from cattle and pigs,
too (Gerlach, 1977; Mushin et al., 1980; Bisgaard and Dam, 1981;
Christensen et al., 2003; Jordan et al., 2005; Bisgaard et al., 2009).
G. anatis is a common organism of the upper respiratory and lower
∗Corresponding author. Tel.: +43 1 25077 4710; fax: +43 1 25077 5192.
E-mail addresses: merima.alispahic@vetmeduni.ac.at,amerima@gmail.com
(M. Alispahic).
genital tract of poultry. Disease associated with this microorganism
is related to egg peritonitis, decrease in egg production, and occa-
sionally an increase in mortality (Gerlach, 1977; Mushin et al., 1980;
Bisgaard and Dam, 1981; Jordan et al., 2005; Neubauer et al., 2009).
A novel RTX-toxin, GtxA, in G. anatis was recently demonstrated as
an important virulence factor for haemolytic and leukotoxic activity
(Kristensen et al., 2010).
Like, most other genera of the family Pasteurellaceae Pohl 1981,
the genus Gallibacterium represents a phenotypically heteroge-
neous group (Christensen et al., 2003). Phenotypic characterization
therefore constitutes a laborious and time-consuming diagnostic
method, which may also give ambiguous results due to variable
outcome of tests included. For the same reason, interpretation of
earlier studies, in which only relatively few phenotypic characters
have been investigated might be difficult (Bisgaard, 1993).
Various genotypic methods have been developed for identifica-
tion of Gallibacterium (Bojesen et al., 2003b, 2007; Christensen et al.,
2004). The specificity of these methods, however, remains to be
investigated including the recently published taxa of Gallibacterium
(Bisgaard et al., 2009).
1438-4221/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2011.03.001

Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry evaluated by multilocus sequence analysis. Int. J. Med. Microbiol. (2011), doi:10.1016/j.ijmm.2011.03.001
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Bacterial identification by matrix-assisted laser desorp-
tion/ionization time-of-flight mass spectrometry (MALDI-TOF MS)
is based on generating complex fingerprint spectra of biomarker
molecules by measuring the exact size of peptides and proteins,
which are assumed to represent high-abundant proteins with
housekeeping functions, such as ribosomal or nucleic acid-binding
proteins (Claydon et al., 1996; Suh and Limbach, 2004). The
procedure is fast, requires minimal amounts of colony material, is
suitable for high-throughput routine analysis, and therefore has
a great potential for application in routine clinical microbiology
laboratories as reviewed by Carbonnelle et al. (2010).
In this study, the MALDI Biotyper system was assessed for
the first time for identification of taxa of Gallibacterium, result-
ing in a reference database. To achieve this task, a well-defined
population of Gallibacterium species was needed. Therefore, from
all strains investigated biochemical/physiological characters were
obtained. Also, the few already known reference/type strains from
different Gallibacterium species were tested. Moreover, the repro-
ducibility of MALDI results obtained was evaluated varying the age
and storage conditions of the cultures investigated, which is rel-
evant in a diagnostic laboratory. Since taxonomic investigations
have demonstrated difficulties as to obtain correct identification
within this area, diversities outlined by MALDI-TOF MS were veri-
fied by sequencing the rpoB, 16S rRNA, infB,recN genes (multilocus
sequence analysis, MLSA).
Materials and methods
Bacterial strains and phenotypic characterization
Sixty-six Gallibacterium reference strains were included in this
study (Table 1). These strains have previously been characterized in
detail by phenotypic methods, and a number of these strains have
also been characterized by genotypic methods as shown in Table 1
(Christensen et al., 2003; Bojesen et al., 2003a,b, 2007; Bisgaard
et al., 2009). Pasteurella multocida 08/14290 field strain was iden-
tified by phenotypic methods and Biotyper reference database
library version 3. All bacteria were grown on Columbia agar (COS)
containing 5% sheep blood (BioMerieux, Vienna, Austria) and inoc-
ulated at 37 ◦C for 24h under microaerophilic conditions.
Sample preparation
Sample preparation for MALDI-TOF MS was performed as pre-
viously described in detail (Alispahic et al., 2010). Each sample
was spotted 8 times onto the MALDI target plate to test techni-
cal replication. Then, the sample was overlaid with 2 ␮l of matrix
(alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile/2.5% tri-
fluor acetic acid, according to the protocol of Bruker) and dried
again. All steps were performed at room temperature.
MALDI-TOF MS parameters
Mass spectra were collected using Ultraflex II MALDI-TOF/TOF
mass spectrometer (Bruker Daltonik GmbH, Leipzig, Germany) in
linear mode, i.e., using a mass range of 2000–20,000 Da (parameter
setting: IS1 20.0 kV, IS2 18.7 kV, lens 6.25kV, detector gain 1634 V).
Five hundred single spectra (10 times 50 shots with a 50 Hz nitrogen
laser from different positions of the target spot) were summarised,
and each spot was measured 3 times automatically. The instru-
ment was externally calibrated with Bruker bacterial test standard
(BTS, Bruker). Proteins used for calibration are as follows: riboso-
mal proteins RL36, 4364.3 m/z; RS32, 5095.8 m/z; RS34, 5380.4 m/z;
RS33meth, 6254.4 m/z; RL29, 7273.5 m/z; RS19, 10299.1 m/z; RNase
A, 13683.2 m/z; and myoglobin, 16952.3 m/z.
Creation of reference database library
Each individual spectrum was scrutinised by eye in the flex-
Analysis software 3.0 (Bruker Daltonik GmbH, Leipzig, Germany),
and atypical spectra were excluded from further analysis (e.g. flat
line spectrum, spectrum containing high matrix background sig-
nal). A reference database library was established for MALDI-TOF
MS-based species identification following the manufacturer’s rec-
ommendations for Ultraflex measurement and MALDI Biotyper 2
software package (Bruker Daltonik GmbH, Leipzig, Germany). In
brief, for each database entry, at least 20 individually measured
mass spectra fingerprints were imported into the MALDI Biotyper
2 software. After smoothing, baseline correction, and peak-picking,
the resulting peak lists (up to 70 peak masses) were used by the
program to calculate and to store a main spectrum containing the
information about average peak masses, average peak intensities,
and peak frequency.
Dendrogram construction
For strain identification, the formation of the dendrogram is
based on cross-wise minimum spanning tree (MSP) matching.
Similar MSPs result in a high matching score value. Each MSP is
compared with all MSPs of the analysed set. The list of score val-
ues is used to calculate normalised distance values between the
analysed species, resulting in a matrix of matching scores. The
visualization of the respective relationship between the MSPs is
displayed in a dendrogram using the standard settings of the MALDI
Biotyper 2.0 software. Species with distance levels <500 have been
described as reliably classified (Sauer et al., 2008). Pasteurella mul-
tocida strain 08/14290 was used as an outgroup in the dendrogram.
MALDI-TOF MS reproducibility test
To test the reproducibility of MALDI-TOF MS-based species
identification, 8 reference strains were selected randomly, and
their reproducibilities of spectra under 5 different conditions were
tested. Condition number 1: bacteria were grown on COS agar and
incubated for 3 and 8 days at 37 ◦C under microaerobic conditions.
Condition number 2: bacteria were grown on COS agar at 37 ◦C for
24 h under microaerobic conditions and then left at room tempera-
ture (∼20 ◦C) under a microaerobic atmosphere. Condition number
3: the same as condition number 2, but incubated and stored in
an aerobic atmosphere. Condition number 4: the same as condi-
tion number 2, but stored at 4 ◦C. Condition number 5: the same as
condition number 3, but with storage at 4 ◦C. For all conditions, a
small amount of biomass was used to measure 5 spots (resulting in
5 spectra for each sample) with MALDI-TOF MS after 3 and 8 days,
respectively. Resulting spectra were imported into Biotyper soft-
ware for identification using the already made MSP library used for
dendrogram creation.
Sequencing of rpoB, recN, infB, and 16S rRNA genes
The partial rpoB sequence (Table 1) was determined according
to Mollet et al. (1997) covering the region 509–680 (Escherichia
coli pos.) of the deduced protein sequence as reported previ-
ously (Angen et al., 2003; Korczak et al., 2004). Partial recN gene
sequences (Table 1) were determined as described by Kuhnert
and Korczak (2006), with 1340 bp of the gene being sequenced.
Sequencing of infB gene (Table 1) was performed according to pre-
viously described protocols (Korczak et al., 2004; Kuhnert et al.,
2004). Sequencing of the 16S rRNA gene (Table 1) was also per-
formed according to previous reports (Christensen et al., 2002;
Kuhnert et al., 2002; Angen et al., 2003). 16S rRNA, rpoB,recN,
and infB gene sequences determined in the present investigation

Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry evaluated by multilocus sequence analysis. Int. J. Med. Microbiol. (2011), doi:10.1016/j.ijmm.2011.03.001
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Table 1
Reference strains used in this study.
Strain name Source of isolation Results GenBank accession number
Biochemical/physiological MALDI-TOF MSaGenotypicb16S rRNA rpoB recN infB
2737/89 Turkey,
septicaemia
G. anatis biovar 11 G. genomospecies 1 G. anatis HQ629809 HQ629815 HQ629854 HQ629805
220/89 No data G. anatis biovar 24 G. anatis G. anatis AF228006c
34346ov Chicken, ovary G. anatis biovar 4 G. anatis
F281 Duck G. anatis biovar
anatis
G. anatis G. anatis HQ629825
F282 Duck G. anatis biovar
anatis
G. anatis
3911/90 Rosella,
septicaemia
G. anatis biovar 4 G. anatis
12656/12L Chicken,
septicaemia
G. anatis biovar 4 G. anatis
10672/9Salp Chicken, salpingitis G. anatis biovar 4 G. anatis G. anatis AF228004c
5821/88 A. militensis,
septicaemia
G. anatis biovar 4 G. anatis
10814/13Salp Chicken, salpingitis G. anatis biovar 4 G. anatis
1779/90 Guinea fowl,
septicaemia
G. anatis biovar 4 G. anatis G. anatis HQ629817
4258/90dPartridge,
septicaemia
G. anatis biovar 4 G. anatis
2125/89 Chicken,
septicaemia
G. anatis biovar 11 G. anatis G. anatis HQ629816
121/89 Pheasant,
septicaemia
G. anatis biovar 18 G. anatis
20558/3kloak Goose G. anatis biovar 19 G. anatis G. anatis AF228010c
1746/87 Chicken,
septicaemia
G. anatis biovar 18 G. anatis
CCM5995 Chicken G. anatis biovar 20 G. anatis G. anatis AF228003cHQ629824
10816/12 Salpingitis, chicken G. anatis biovar 18 G. genomospecies 1 G. anatis HQ629810 HQ629845 HQ629857 HQ629800
444/89 Budgerigar,
septicaemia
G. anatis biovar 18 G. anatis G. anatis HQ629849
10673/1Salp Chicken, salpingitis G. anatis biovar 1 G. anatis
3348/80 Goose, septicaemia G. anatis biovar 17 G. anatis G. anatis AF228005cHQ629828
3268/87 Chicken,
septicaemia
G. anatis biovar 18 G. anatis
4224/88 No data G. anatis biovar 22 G. anatis G. anatis AF228014cHQ629822
1939/89 Budgerigar,
septicaemia
G. anatis biovar 18 G. anatis
IPDH 697/78 Chicken G. anatis biovar 15 G. anatis G. anatis AF228007cHQ629827
14542/2L Chicken,
septicaemia
G. anatis biovar 12 G. anatis G. anatis HQ629826
42447/2L Chicken,
septicaemia
G. anatis biovar 12 G. anatis
442/89 Budgerigar,
septicaemia
G. anatis biovar 18 G. anatis
P6Organs Bovine G. anatis biovar
anatis
G. anatis
99066106 Duck brain G. anatis biovar
anatis
G. anatis G. anatis HQ629834
H3Lung Bovine G. anatis biovar
anatis
G. anatis G. anatis HQ629847

Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
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Table 1 (Continued)
Strain name Source of isolation Results GenBank accession number
Biochemical/physiological MALDI-TOF MSaGenotypicb16S rRNA rpoB recN infB
B96/24 Bovine brain G. anatis biovar
anatis
G. anatis G. anatis AY039598 HQ629844
HPA126 Duck G. anatis biovar
anatis
G. anatis G. anatis HQ629842
B96/21 Pig brain G. anatis biovar
anatis
G. anatis G. anatis HQ629832
2589/89 Chicken,
septicaemia
G. anatis biovar 1 G. anatis G. anatis HQ629839
B96/26 Bovine G. anatis biovar
anatis
G. anatis G. anatis HQ629833
1307/89 Budgerigar,
septicaemia
G. anatis biovar 1 G. anatis
66Liver Goose G. anatis biovar
anatis
G. anatis G. anatis HQ629843
F280 Duck G. anatis biovar
anatis
G. anatis
P. sp. 38 Chicken, yolk G. anatis biovar 12 G. anatis G. anatis HQ629821
H2Lung Bovine G. anatis biovar
anatis
G. anatis G. anatis HQ629848
6263/88 Chicken,
septicaemia
G. anatis biovar 12 G. anatis G. anatis HQ629837
HIM996-9 Duck G. anatis biovar
anatis
G. anatis
10672/6 Chicken, salpingitis G. anatis biovar 1 G. anatis G. anatis AF228008cHQ629830
12158/5 Chicken, salpingitis G. anatis biovar 3 G. anatis G. anatis AF228009cHQ629835
646/89 Chicken,
septicaemia
G. anatis biovar 1 G. anatis G. anatis HQ629823
5693/88 Chicken,
septicaemia
G. anatis biovar 12 G. anatis G. anatis HQ629814
4851/87 Budgerigar,
septicaemia
G. anatis biovar 12 G. anatis
3076/88 Duck, septicaemia G. anatis biovar 17 G. anatis G. anatis HQ629840
F279 Duck G. anatis biovar
anatis
G. anatis G. anatis AF228002c
F149T(CCUG15563T) Duck G. anatis biovar
anatis
G. anatis G. anatis AF228001cAY314032cDQ410892cAY508843c
3109/88 Budgerigar,
septicaemia
G. anatis biovar 12 G. anatis G. anatis HQ629838
CCM5974dChicken G. anatis biovar 8 G. genomospecies 1 G. genomosp. 1 by
rpoB and 16S rRNA.
G. anatis by recN
and infB
AF228015cAY314033cHQ629850 HQ629802
CCM5975dChicken G. anatis biovar 5 G. genomospecies 1 G. genomosp. 1 by
rpoB and 16S rRNA.
G. anatis by recN
and infB
AF228016cHQ629831 HQ629853 HQ629803

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Table 1 (Continued)
Strain name Source of isolation Results GenBank accession number
Biochemical/physiologicalMALDI-TOF MSaGenotypicb16S rRNA rpoB recN infB
29934Liver Chicken,
septicaemia
G. anatis biovar 24 G. genomospecies 1 G. genomosp. 1 by
rpoB and 16S rRNA.
G. anatis by recN
and infB
HQ629811 HQ629836 HQ629851 HQ629804
139/89 Chicken,
septicaemia
G. anatis biovar 23 G. genomospecies 1 G. genomosp. 1 by
rpoB and 16S rRNA.
G. anatis by recN
and infB
HQ629812 HQ629841 HQ629852 HQ629806
3191/88 Chicken,
septicaemia
G. anatis biovar 8 G. anatis G. genomosp. 1 AY038591cHQ629820
F151dDuck, salpingitis Gallibacterium
variant (G.
genomospecies 3)
Clusters with G.
salpingitidis
G. salpingitidis by
rpoB and 16S rRNA.
G. genomosp. 3 by
recN and infB
EU423996cEU424014cEU424049cEU424032c
52/S3/90Td(CCUG 55631T) Budgerigar,
septicaemia
G.
trehalosifermentans
G.
trehalosifermentans
G.
trehalosifermentans
by 16S rRNA and
recN
EU339199cEU424012cEU424048cEU424030c
F450Td(CCUG 36331T) Parakeet,
septicaemia
G. melopsittaci G. melopsittaci G. melopsittaci by
16S rRNA and recN
EU339196cEU424003cEU424039cEU424021c
F150Td(CCUG 15564T) Duck, salpingitis G. salpingitidis G. salpingitidis
(clusters with
genomospecies 3
F151 strain)
G. salpingitidis EU424000cEU424018cEU424053cEU424036c
39199/1L Pheasant,
pneumonia
Gallibacterium
group V
G. anatis Gallibacterium
group V
EU339207cEU424020cEU424055cEU424038c
CCM5976 Chicken G. anatis biovar 9
(genomospecies 2)
G. anatis G. anatis by rpoB G.
genomosp. 2 by
16S rRNA
AF228017cHQ629846
B96/41 Bovine lung G. anatis biovar
anatis
G. anatis G. genomosp. 3 by
rpoB G. anatis by
16S rRNA, recN,
and infB
HQ629813 HQ629819 HQ629855 HQ629801
B96/20dBovine lung G. anatis biovar
anatis
G. anatis G. salpingitidis rpoB
G. anatis 16S rRNA
AY038592cHQ629829
B96/27 Bovine lung G. anatis biovar
anatis
G. anatis G. salpingitidis by
rpoB G. anatis by
16S rRNA, recN,
and infB
HQ629808 HQ629818 HQ629856 HQ629807
Abbreviations: G., Gallibacterium; G. genomosp., Gallibacterium genomospecies.
aMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
bResults from sequencing one or more genes as indicated with accession number.
cGene sequence published in previous publications (9, 16, 22).
dStrains used for testing the reproducibility of MALDI-TOF MS results under different conditions.

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have been deposited with GenBank/EMBL/DDBJ under the acces-
sion numbers listed in Table 1. Pairwise comparisons for similarity
were performed by the program WATER included in EMBOSS (Rice
et al., 2000). Multiple alignment and phylogenetic trees were con-
structed by the neighbour joining method based on Jukes and
Cantor corrected similarity matrices by ClustalX (Thompson et al.,
1997) and drawn by MEGA4 (Tamura et al., 2007).
Results
A reproducible signal pattern was obtained from all 66 reference
Gallibacterium strains used for MALDI/Biotyper reference database
development (Table 1). Signal patterns obtained were compared
with data in the Bruker Biotyper reference database version 3 (con-
tained 3024 bacterial strains). The Bruker database only contained
the type strain of G. anatis (DSM 16844T) which matched to the
G. anatis type strain included in the present study with a 2.703
log(score) value. All the 4 recognised Gallibacterium species yielded
unique mass spectral profiles (Fig. 1) and therefore could be eas-
ily differentiated by score values and dendrogram analysis that
showed separate clades/clusters for each species in the Biotyper
software.
According to the dendrogram generated by the MALDI Biotyper
software based upon mass signals and intensities (Fig. 2), the type
species of the genus Gallibacterium,G. anatis (F149T), clustered with
55 other isolates (cluster 1). This cluster, however, splits into 2
groups at a distance level between 200 and 300, the majority of iso-
lates including the type strain of the type species clustering below a
200-distance level indicating a high similarity between the isolates.
Gallibacterium group V (39199/1L) and strain CCM5976 (Gallibac-
terium genomospecies 2) formed a separate cluster with strain P. sp.
38 which branched deeply with the type species of the genus Gal-
libacterium, but below the 200-distance level. Strains 29934liver,
CCM5974 (Gallibacterium genomospecies 1), 2737/89, CCM5975,
10816/12, and 139/89 formed cluster 2 demonstrating a distance
level closer to G. anatis (600–500). Moreover, the type strain of
G. salpingitidis (F150T) clustered with G. genomospecies 3 strain
(F151) (cluster 3) at a distance between 200 and 300. Both iso-
lates, however, clustered with the type strain of the type species,
G. anatis, at a distance above 600. The mass signal pattern of G.
trehalosifermentans (52/33/90T) (cluster 4) and of G. melopsittaci
(F450T) (cluster 5) differed significantly from G. anatis demon-
strating a distance level above 800 and differed to each other at
a distance level between 800 and 700. The strain relatedness doc-
umented in the dendrogram complements identification by score
values (data not shown).
Reproducibility was carried out to investigate if identification of
8 randomly chosen strains was possible under different growth and
storage conditions relevant in a clinical laboratory. Reliable species
identification [log(score) above 2.3] was possible after 3 and 8 days
of storage at 4 ◦C. Less reliable species identification [6/8 strains
correctly identified, but with a lower log(score)] was seen if the
bacteria were stored at room temperature (∼20 ◦C) for 3 days. The
reliability declined further after 8 days of storage [3/8 strains cor-
rectly identified to species level with log(score) just above 2.0] (data
not shown). However, if the strains were grown for 3 or 8 days at
37 ◦C under microaerobic atmosphere, poor identification results
were obtained [2/8 strains with log(score) just above 2.0]. No dis-
tinction was seen if the strains were stored at different atmospheres
(data not shown).
The strain relatedness documented in the MALDI dendro-
gram classified 58 strains out of 66 investigated (88%) concordant
to results based upon biochemical/physiological characteriza-
tion. Discrepancies were seen with strains 2737/89, 10816/12,
CCM5974, CCM5975, 29934 Liver, 139/89, F151, and 39199/1L.
To investigate if clusters outlined by MALDI-TOF MS (Fig. 2)
also reflect genotypic relationship, the partial rpoB sequences of
36 strains were generated and compared with 7 strains from Gen-
Bank covering the diversity outlined in Fig. 2. In general, 30 strains
out of 43 were assigned to G. anatis with rpoB sequence which was
concordant to MALDI-TOF MS results, except for strains 10816/12
and 2737/89. Hence, clustering of genomospecies 2 with G. anatis
was supported by rpoB (Fig. 3a). Sequencing of rpoB also classified
strains CCM5975, 29934 Liver, 139/89, and CCM5974 with Gal-
libacterium genomospecies 1 (Fig. 3a) concordant to MALDI-TOF
MS (cluster 2 in Fig. 2). Moreover, the clustering of G. salpingitidis
with genomospecies 3 (cluster 3) was confirmed by rpoB. Interest-
ingly, G. melopsittaci and G. trehalosifermentans clustered together
in rpoB analysis, while major differences were observed as to Gal-
libacterium group V. However, MALDI-TOF MS clearly identified G.
melopsittaci and G. trehalosifermentans as 2 different species, though
Gallibacterium group V was identified as G. anatis.
In addition, to verify the diversities in Figs. 2 and 3a, the par-
tial 16S rRNA sequences of 6 strains were generated and compared
with 22 strains from GenBank. The recN and infB sequence of 8
strains were generated and compared with 6 strains (for both
genes) from GenBank. Two bovine isolates (B96/20 and B96/27)
were classified with G. salpingitidis, while a third isolate, B96/41,
clustered with Gallibacterium genomospecies 3 with rpoB analysis.
However, all 3 isolates identified as G. anatis by MALDI-TOF MS,
biochemical/physiological characterization, and sequencing of 16S
rRNA, recN, and infB genes. Moreover, sequencing of recN and infB
did not allow separation of genomospecies 1 (CCM5974) from G.
anatis (Fig. 3c–d). Contrary to MALDI-TOF MS, strains 10816/12 and
2737/89 (cluster 2), clustered with G. anatis type strain in all genes
sequenced (Fig. 3a–d). A concatenated analysis of 16S rRNA, rpoB,
recN, and infB gene sequences confirmed the existence of the taxa
previously outlined by Bisgaard et al. (2009) (data not shown).
Discussion
The genus Gallibacterium includes a very diverse group of bacte-
ria that vary in pheno- and genotypic characteristics, independent
of hosts range, geographical location, and time of isolation explain-
ing the difficulties in classification and identification of taxa making
up the genus Gallibacterium. So far, classification and identifica-
tion of Gallibacterium species have been based upon phenotypic
and genotypic methods, including DNA–DNA hybridisation, PFGE,
AFLP, and 16S rRNA sequencing (Bisgaard, 1977, 1993; Christensen
et al., 2003).
More recently, a number of strains were characterized by infB,
recN, and rpoB gene sequencing (Bisgaard et al., 2009). However,
many of these methods are complex, costly, and often require days
to complete arguing for further techniques keeping in mind that
every method has its own limitations. In addition, inconsistent
results may be obtained with different genes as demonstrated in
the present paper. The rpoB gene sequence is strongly conserved
within the various species of the family of Pasteurellaceae, and the
resolution is generally greater than that of the 16S rRNA sequence,
but still does not allow the separation of very closely related species
(Korczak et al., 2004).
This study investigated if MALDI-TOF MS allows unambiguous
separation and identification of Gallibacterium species recognised
as mainly avian pathogens, thus creation of a MALDI-TOF MS ref-
erence database to be used as a basic tool for further clinical
diagnostics. With the exception of the strain named 39199/1L
(group V), the overall congruency between MALDI-TOF MS and
MLSA was good. This observation is in agreement with results
reported by Tanigawa et al. (2010) who stated that the MALDI-
TOF MS results were nearly identical to genotypic identification

Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry evaluated by multilocus sequence analysis. Int. J. Med. Microbiol. (2011), doi:10.1016/j.ijmm.2011.03.001
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M. Alispahic et al. / International Journal of Medical Microbiology xxx (2011) xxx–xxx 7
Fig. 1. MALDI-TOF mass spectrometric profiles obtained from the analysis of Gallibacterium type strains of species: (a) G. anatis, (b) G. melopsittaci, (c) G. salpingitidis, and (d)
G. trehalosifermentans. The relative intensities of the ions are shown on the Y-axis, and the masses (in Da) of the ions are shown on the X-axis. The m/zvalue stands for mass
to charge ratio. For a single positive charge, this value corresponds to the molecular weight of the protein.
Fig. 2. Classification of Gallibacterium reference strains investigated. Score-oriented (MSP) dendrogram of MALDI-TOF mass spectral profiles generated by the MALDI Biotyper
2. The dendrogram was generated with the following settings: distance measure was set at correlation, linkage at average, and score threshold value for a single organism
at 600. Strains clustering with distance levels lower than 500 could be classified up to species level.

Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry evaluated by multilocus sequence analysis. Int. J. Med. Microbiol. (2011), doi:10.1016/j.ijmm.2011.03.001
ARTICLE IN PRESS
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8M. Alispahic et al. / International Journal of Medical Microbiology xxx (2011) xxx–xxx
(16S rRNA and recA gene sequence and AFLP) for discriminat-
ing species and subspecies in the genus Lactococcus. According to
Christensen et al. (2003), AFLP and DNA reassociation data seem
to indicate the existence of only one additional genomospecies of
Gallibacterium (strain CCM5976). MALDI-TOF MS results did not
support that CCM5976 belong to an independent species (Fig. 2).
Neither did sequencing of rpoB (Fig. 3a), while 16S rRNA data
classified CCM5976 with genomospecies 2. Korczak et al. (2004)
noted that within the family Pasteurellaceae, discrepancies were
observed for some species between 16S rRNA and rpoB gene-
Fig. 3. Phylogenetic relationships between members of Gallibacterium as investigated based on neighbour joining analysis of partial rpoB gene sequences (a), nearly full length
16S rRNA gene sequences (b), partial recN gene sequences (c), and partial infB gene sequences (d). Supports for monophyletic groups by bootstrap analysis are indicated
as numbers out of 100. The strain numbers with DBJ/EMBL/GenBank accession numbers marked in bold have been sequenced in the present investigation. The scale bar
represents sequence variation considering the model for nucleotide substitution (Jukes and Cantor) and algorithm (Neighbour Joining) used in the analysis.

Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry evaluated by multilocus sequence analysis. Int. J. Med. Microbiol. (2011), doi:10.1016/j.ijmm.2011.03.001
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M. Alispahic et al. / International Journal of Medical Microbiology xxx (2011) xxx–xxx 9
Fig. 3. (Continued)
based phylogenies. The discrepancy that was observed between
MLSA and MALDI-TOF MS of Gallibacterium group V remains unex-
plained. However, comparison of whole-cell protein profiling and
DNA reassociation of strains classified as taxon 2 or 3 also demon-
strated deviations (Bisgaard, 1993). Apart from strains 2737/89 and
10816/12, the other strains of cluster 2 (Fig. 2) seem to represent
a new species of Gallibacterium previously outlined as genomo-
species 1 by genetic methods (Christensen et al., 2003). Clustering
of G. salpingitidis with Gallibacterium genomospecies 3 (F151) (clus-
ter 3) was also confirmed by 16S rRNA and rpoB sequencing results.
However, whole-genome similarity between these taxa calculated
from recN sequences showed that these taxa represent differ-
ent species (Bisgaard et al., 2009). Finally, 3 strains isolated from
bovine lungs (B96/41, B96/20, and B96/27) exhibited extensive
variation between their rpoB and their infB, 16S rRNA and recN
gene sequences. The reason behind this observation remains to be
investigated.
An increased number of papers comparing the use of
MALDI/Biotyper with the standard methods for identification
in clinical diagnostic laboratories have been published recently
(Carbonnelle et al., 2010; Giebel et al., 2010). Bizzini et al. (2010)
compared MALDI/Biotyper to the conventional phenotypic meth-
ods for identification of 1371 routine isolates, in which 93.2%
were identified to species level with both methods. Poor 16S rRNA
sequence similarity between the species Nocardia paucivorans and
Nocardia transvalensis was noted even though MALDI-TOF MS den-
drogram showed that their mass signal patterns are closely related
(Verroken et al., 2010). On the other hand, MALDI-TOF MS was
used to support the data from 16S rRNA and rpoB gene sequence
data to indicate that Acinetobacter bereziniae and A. guillouiae rep-
resent distinct groups within the genus Acinetobacter (Nemec et al.,
2010).
Bacteria rapidly respond to environmental changes that might
induce changes in the protein profile. Thus, the reproducibility of
MALDI-TOF MS results was investigated under different growth
and storage conditions of bacteria, relevant in a clinical laboratory.
Reliable identification was possible when bacteria were stored at
4◦C for up to 8 days. However, if bacteria were grown longer than
72 h, unreliable identification was seen. This might be explained
as a result of depletion of nutrients imposing changes in the pro-
tein profile (Valentine et al., 2005). Arnold et al. (1999) also stated
that the time of incubation should be carefully controlled if MALDI-
TOF MS is used for bacterial identification. Besides this the use
of MALDI-TOF MS can be limited by agar media used (Alispahic
et al., 2010). Most important, identification of bacteria on a rou-
tine basis can only be done using a reference database created
with well-characterized strains, hence, the main result of this
study.
In conclusion, this is the first study employing the MALDI-TOF
MS fingerprinting technique for the in-depth analysis of Gal-
libacterium species. MALDI-TOF MS clearly discriminated between
the 4 recognised Gallibacterium type strains and a possible fifth
species Gallibacterium genomospecies 1, in agreement with previ-
ous findings. However, several minor discrepancies were observed
between MALDI-TOF MS and MLSA, the causes of which remain
to be investigated. MALDI-TOF MS fingerprinting, however, rep-
resents a fast and reliable method for the identification and
differentiation of 4 recognised Gallibacterium species and the possi-
ble fifth species Gallibacterium genomospecies 1, with applications
in clinical diagnostics.

Please cite this article in press as: Alispahic, M., et al., Identification of Gallibacterium species by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry evaluated by multilocus sequence analysis. Int. J. Med. Microbiol. (2011), doi:10.1016/j.ijmm.2011.03.001
ARTICLE IN PRESS
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IJMM-50581; No. of Pages 10
10 M. Alispahic et al. / International Journal of Medical Microbiology xxx (2011) xxx–xxx
Acknowledgments
Excellent technical assistance was contributed by Katrine Mad-
sen and Isabelle Josipovic.
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