German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity

Abstract

Background
Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis.

Results
Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level.

Conclusions
F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.

Full text

R E S E A R C H A R T I C L E Open Access
German Francisella tularensis isolates from
European brown hares (Lepus europaeus) reveal
genetic and phenotypic diversity
Wolfgang Müller
1
, Helmut Hotzel
1
, Peter Otto
1
, Axel Karger
2
, Barbara Bettin
2
, Herbert Bocklisch
3
, Silke Braune
4
,
Ulrich Eskens
5
, Stefan Hörmansdorfer
6
, Regina Konrad
6
, Anne Nesseler
5
, Martin Peters
7
, Martin Runge
4
,
Gernot Schmoock
1
, Bernd-Andreas Schwarz
8
, Reinhard Sting
9
, Kerstin Myrtennäs
10
, Edvin Karlsson
10
,
Mats Forsman
10
and Herbert Tomaso
1*
Abstract
Background: Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different
vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus
europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and
genotypic characteristics of F. tularensis.
Results: Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares
that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were
resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs
(B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this
set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive
isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was
useful to discriminate strains to the subspecies level.
Conclusions: F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified
using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific
PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study
indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within
eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the
distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs
are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA
sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area.
In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be
tracked to their genetic and geographical sources.
* Correspondence: herbert.tomaso@fli.bund.de
1
Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut
(Federal Research Institute for Animal Health), Naumburger Str. 96A, Jena
D-07743, Germany
Full list of author information is available at the end of the article
© 2013 Müller et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Müller et al. BMC Microbiology 2013, 13:61
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Background
Tularemia is a rare zoonotic disease caused by Francisella
tularensis, a Gram negative, facultative intracellular, fas-
tidious bacterium [1]. Most infections in animals and
humans are caused by two F. tularensis subspecies, F.
tularensis subsp. tularensis (Jellison type A) and F.
tularensis subsp. holarctica (Jellison type B). F. tularensis
type A is endemic in North America and type B is located
in Europe, Asia, and North America [2-4]. Three biotypes
of the less virulent type B have been described: biovar I
(erythromycin sensitive), biovar II (erythromycin resist-
ant), and biovar japonica which can ferment glycerol [4].
In Germany, human infections are usually caused by
skinning, preparing or eating infected hares or drinking
contaminated water. F. tularensis was sporadically diag-
nosed in humans in the first half of the 20th century in
Germany but almost disappeared in the following decades
[5,6]. Between 1983 and 1992 only four sporadic cases of
tularemia were notified in hares or rabbits from Lower
Saxony, Rhineland-Palatinate, North Rhine-Westphalia and
Baden-Württemberg, respectively [6]. After years without
reported cases in animals the re-emergence of tularemia
started in 2004 with an outbreak of tularemia in a semi-free
living group of marmosets (Callithrix jacchus)inLower
Saxony [7], and in December 2005 an outbreak with 15 hu-
man cases due to contact with infected hares was reported
from Hesse [8]. The detection of F. tularensis subsp.
holarctica in organ samples of these hares using PCR assays
was the beginning of our investigations of tularemia in
European brown hares (Lepus europaeus)inGermany.
A variety of PCR methods has been established for the
detection of F. tularensis DNA in both clinical and envir-
onmental specimens [9-11]. Farlow et al. developed a typ-
ing assay based on the variable-number of tandem repeats
(VNTRs) [12] and Johansson et al. also described a
twenty-five VNTR marker typing system that was used to
determine the worldwide genetic relationship among F.
tularensis isolates [1]. Byström et al. selected six of these
25 markers that were highly discriminatory in a study of
tularemia in Denmark [13]. Vogler et al. [14] investigated
the phylogeography of F. tularensis in an extensive study
based on whole-genome single nucleotide polymorphism
(SNP) analysis. From almost 30,000 SNPs identified
among 13 whole genomes 23 clade- and subclade-specific
canonical SNPs were identified and used to genotype 496
isolates. This study was expanded upon in another study
that used a combination of insertion/deletions (INDELs)
and single nucleotide polymorphism analysis [15].
The aim of this study was to elucidate the molecular epi-
demiology of F. tularensis in European brown hares in
Germany between 2005 and 2010. Several previously pub-
lished typing markers were selected and combined in a
pragmatic approach to test whether they are suitable to elu-
cidate the spread of tularemia in Germany. This included
cultivation, susceptibility testing to erythromycin, a PCR
assay for subspecies differentiation detecting a 30 bp dele-
tion in the Ft-M19 locus, VNTR typing, INDEL, SNP, and
MALDI-TOF analysis. This is important because it im-
proves our understanding of the spread of tularemia and
may help to recognize outbreaks that are not of natural
origin.
Results
Cultivation and identification of isolates
Cultivation of bacteria from organ specimens was suc-
cessful in 31 of 52 hares which had a positive PCR result
targeting the locus Ft-M19 that was also used to differ-
entiate F. tularensis subsp. holarctica from other F.
tularensis subsp. [11]. F. tularensis subsp. holarctica was
identified in all 52 cases.
Biovars
Seventeen isolates were susceptible to erythromycin cor-
responding to biovar I, whereas fourteen were resistant
(biovar II). The geographic distribution is given in
Table 1, Figure 1 and the susceptibility of the isolates in
Additional file 1: Table S2.
VNTR typing
In a pilot study, six loci (Ft-M3, Ft-M6, Ft-M20, Ft-M21,
Ft-M22, and Ft-M24) were amplified and sequenced, but
only the loci Ft-M3, Ft-M6, and Ft-M24 were discrimin-
atory. The strains tested in the pilot study are indicated
in Additional file 1: Table S2(*). The following identical
results were obtained for all these strains: Ft-M20: 255
bp; Ft-M21: 403 bp; Ft-M22: 241 bp. The loci Ft-M3 and
Ft-M6 (repeat: TTG GTG AAC TTT CTT GCT CTT)
were further used to analyse DNA samples extracted
from cultivated bacteria. Sequencing of Ft-M3 identified
two different repetitive elements, Ft-M3a (ATC CTT
ATT), and Ft-M3b (GTC TTT GTT), respectively. The
number of these repeats was determined separately. The
size obtained for Ft-M24 (repeat: ATA AAT TAT TTA
TTT TGA TTA) correlated with the size observed previ-
ously for the B.IV (B.18) clade. All VNTR results are
given in Additional file 1: Table S2.
INDEL analysis
Conventional PCR assays with subsequent gel electro-
phoretic size determination of the amplicon allowed clear
discrimination between amplicons with or without the re-
spective deletions which was confirmed by sequencing in
some cases (data not shown). The 31 isolates showed four
different INDEL patterns (Additional file 1: Table S2).
Based on INDELs and SNPs (see below) 14 isolates were
assigned to clade B.I (B.20), one to B.II (B.17), and 16 to
B.IV (B.18), according to the nomenclature in Karlsson
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et al. 2013 [16], where the B.I clade was re-defined to in-
clude both B1 and B3 in Svensson et al. [15].
SNP typing
The results of SNP typing are given in Additional
file 1: Table S2. All strains used in a pilot study
showed ancestral states of the following SNPs: B.16:
G; B.21:G; B.22:G; B.24:T. These strains are indicated
(*) in Additional file 1: Table S2. Therefore, only the
SNPs B.17, B.18, B.19, and B.20 were further investi-
gated for all isolates.
MALDI-TOF MS analysis
All isolates (n=31) yielded high quality spectra. MALDI-
TOF was found to be useful for rapid identification of
isolates to subspecies level within one hour. However,
the obtained clusters (Figure 2) did not conform to the
genetic clusters (Additional file 1: Table S2).
Geographical clustering
Cases of tularemia in hares were identified in eight of six-
teen federal states of Germany reaching from islands in the
North Sea to regions at Lake Constance in the southern
Table 1 Original and geographic data of Francisella tularensis subsp. holarctica isolates (BW –Baden-Württemberg,
BY - Bavaria, NRW –North Rhine-Westphalia, LS –Lower Saxony, SN –Saxony, TH - Thuringia)
Year Strain number Site Federal state Latitude [°NORTH] Longitude [°EAST] Altitude [m]
2006 06T0001 Moorgrund TH 50,838005 10,291767 279
2007 08T0001 Dingelstädt TH 51,315205 10319329 335
2007 08T0008 Allersberg BY 49,251389 11,234261 388
2007 08T0010 Sehnde LS 52,31262 9,967105 71
2007 08T0013 Ehingen BY 49,300734 10,571476 415
2008 08T0014 Weissach-Flacht BW 48,833991 8,91309 406
2008 08T0015 Leonberg-Höfingen BW 48,816676 9,016877 379
2007 08T0070 Einbeck-Kohnsen LS 51,707717 10,000538 121
2008 08T0071 Brake LS 53,326329 8,478167 2
2008 08T0072 Göttingen-Roringen LS 51,532638 9,92816 153
2008 08T0073 Twülpstedt-Rümmer LS 52,224403 11,01102 127
2008 08T0075 Würzburg BY 49,794256 9,927489 173
2009 09T0105 Geseke NRW 51,639416 8,509738 105
2009 09T0108 Geseke NRW 51,639416 8,509738 105
2009 09T0109 Geseke NRW 47,724358 9,406025 518
2009 09T0114 Markdorf BW 51,639416 8,509738 105
2009 09T0116 Geseke NRW 51,444502 12,169177 115
2009 09T0146 Wiedemar SN 48,864962 9,024819 337
2008 09T0163 Hemmingen BW 53,770141 7,693722 1
2008 09T0164 Spiekeroog LS 53,770141 7,693722 1
2008 09T0165 Spiekeroog LS 53,770141 7,693722 1
2008 09T0166 Spiekeroog LS 53,770141 7,693722 1
2008 09T0167 Spiekeroog LS 53,770141 7,693722 1
2008 09T0169 Spiekeroog LS 53,745892 7,480842 4
2008 09T0171 Langeoog LS 53,745892 7,480842 4
2009 09T0179 Langeoog LS 53,745892 7,480842 4
2010 10T0014 Hemmingen BW 52,314054 9,722783 56
2010 10T0115 Waltrop NRW 51,624087 7,39465 69
2010 10T0125 Geseke NRW 51,639223 8,469223 103
2010 10T0128 Empfingen BW 48,391957 8,708282 491
2010 10T0131 Oppenweiler BW 48,985678 9,460219 270
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part of Germany. All cases were found below 500 m above
sea level. Isolates belonging to biovar I could be found in
the western part of Germany whereas biovar II occurred in
the eastern region (Table 1 and Additional file 1: Table S2,
Figure 1). Molecular typing resulted in further discrimin-
ation of clusters within the biovars. Isolates resistant to
erythromycin and genetically assigned to clade B.I were
found only in Lower Saxony, Thuringia, Bavaria and Sax-
ony. Strains that were sensitive to erythromycin could be
assigned to clade B.II (Ftind38) and B.IV (B.18) as given in
Additional file 1: Table S2.
Stability testing
The investigated markers for two Francisella isolates
(06T0001 from hare and 10T0191 from fox) were stable
even after 20 passages in cell culture and had identical
results for the markers Ft-M3 (297 bp), Ft-M6 (311 bp),
Ftind33 (deletion), Ftind38 (insertion), and Ftind49
(insertion).
Discussion
In Thuringia the first case of tularemia in a hare was
reported in 2006 [17]. In Lower Saxony 2,162 European
brown hares and European rabbits (Oryctolagus cuniculus)
were screened for tularemia between 2006 and 2009 using
cultivation and PCR assays. Francisella specific PCR as-
says were positive in 23 hares and 1 rabbit which were fur-
ther confirmed by cultivation of F. tularensis subsp.
holarctica in 12 hares [18]. In the present study, cases of
tularemia in hares in Germany from 2005 to 2010 were in-
vestigated. During this period a total of 52 hares were
found positive in PCR assays for F. tularensis subsp.
holarctica DNA and from 31 of these cases Francisella
strains could be isolated. MALDI-TOF analysis was also
Figure 1 Germany: areas where Francisella tularensis subsp. holarctica isolates were found in hares. Erythromycin sensitive strains occur in
regions marked with blue dots, erythromycin resistant strains occur in regions marked with red dots.
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used to rapidly identify Francisella to the subspecies level
as was previously shown by Seibold et al. [19].
Several positive specimens were found on the North Sea
islands Langeoog and Spiekeroog (LS), around Soest (NR),
Darmstadt (H), and Böblingen (BW). These natural foci
and also sporadic cases in other regions of Germany were
found below 500 m above sea level. In the Czech Republic
typical natural foci of tularemia occurred in alluvial forests
and field biotopes below 200 m sea level with mean an-
nual air temperature between 8.1-10.0°C and mean annual
precipitation of 450–700 mm [20]. In Germany, an out-
break of tularemia in a colony of semi-free living marmo-
sets was located in a region with geographic and
ecological conditions similar to the hare habitats in the
Czech Republic: field biotopes 175 m above sea level
(<200 m) with 9.2°C mean annual air temperature and
642 mm mean annual precipitation [8]. In Germany, tular-
emia of hares occurs in regions with rather humid soil like
in alluvial forests and alongside rivers, but this obviously
corresponds with the natural habitat of hares.
Specimens were screened using a PCR assay targeting
Ft-M19 described by Johansson et al. [11] which allows
the simultaneous identification of the species F. tularensis
and the differentiation of the subspecies holarctica from
other (sub-) species. All samples could be attributed to
F. tularensis subsp. holarctica.
We found a clear segregation of clade B.I and clade B.IV
in Germany, B.I strains dominate in eastern Germany and
B.IV within western Germany (Figure 1). Clade B.I is
known to dominate in Europe between Scandinavia and
the Black Sea [15,16,21-23]. The other dominating
European clade is B.IV (B.18) which can be found over a
large area of western and central Europe, and, based upon
this study, western Germany [21,23-26]. We found only
one strain of the B.II clade isolated in Bavaria. Strains of
the B.II clade are most frequently isolated in the USA, but
are found sporadically in Europe as well [16,21].
The phylogeographical pattern of clade B.I and B.IV, co-
incide with the geographical distribution of biovar II and
biovar I strains, respectively. Previously, biovar I strains
(erythromycin sensitive) have been reported from Western
Europe (France, Germany, Spain and Switzerland), North-
America, Eastern Siberia and the Far East while biovar II is
present in the European part of Russia as well as Northern,
Central and Eastern Europe (Austria, Germany, Sweden
and Turkey) [27-31]. A mixture of both biotypes has been
reported in Sweden, Norway, Bulgaria, Russia and
Kazakhstan [27,28,32]. Isolation of both biovars from ro-
dents in a single settlement in Moscow as well as from
water samples collected in the Novgorod region [27] indi-
cate coexistence of the biovars in the same epidemio-
logical foci. Taken together, a geographical separation of
F. tularensis strains seems to exist in Germany. The
phenotypically defined biovar I (erythromycin sensitive)
and phylogenetically defined clade B.IV strains are con-
fined in western Germany, whereas biovar II (erythro-
mycin resistance) and clade B.I strains cluster in eastern
Germany. This is interesting and may reflect a competi-
tion between the two subpopulations or unknown
underlying ecological or epidemiological differences.
A deletion in the genome of F. tularensis subsp.
holarctica in RD
23
is typical for strains of F. tularensis
F. tul. ssp. tularensis_FSC 237
F. tul. ssp. mediasiatica_F063
F. tul. ssp. mediasiatica_F064
F. tul. ssp. novicida_F048
F. tul. ssp. novicida_F059
F. tul. ssp. holarctica_09T0171
F. tul. ssp. holarctica_08T0071
F. tul. ssp. holarctica_08T0073
F. tul. ssp. holarctica_09T0163
F. tul. ssp. holarctica_08T0072
F. tul. ssp. holarctica_09T0169
F. tul. ssp. holarctica_09T0167
F. tul. ssp. holarctica_09T0164
F. tul. ssp. holarctica_09T0165
F. tul. ssp. holarctica_09T0166
F. tul. ssp. holarctica_09T0109
F. tul. ssp. holarctica_08T0010
F. tul. ssp. holarctica_09T0114
F. tul. ssp. holarctica_08T0001
F. tul. ssp. holarctica_08T0070
F. tul. ssp. holarctica_08T0008
F. tul. ssp. holarctica_10T0131
F. tul. ssp. holarctica_09T0105
F. tul. ssp. holarctica_08T0015
F. tul. ssp. holarctica_09T0179
F. tul. ssp. holarctica_08T0013
F. tul. ssp. holarctica_08T0014
F. tul. ssp. holarctica_09T0108
F. tul. ssp. holarctica_06T0001
F. tul. ssp. holarctica_09T0116
F. tul. ssp. holarctica_08T0075
F. tul. ssp. holarctica_10T0115
F. tul. ssp. holarctica_10T0125
F. tul. ssp. holarctica_10T0128
F. tul. ssp. holarctica_09T0146
F. tul. ssp. holarctica_10T0014
05
10 15
Figure 2 Dendrogram constructed from MALDI-TOF mass spectrometry spectra of 31 Francisella tularensis ssp. holarctica strains and
representatives of ssp. tularensis,mediasiatica, and novicida.
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subsp. holarctica in France, the Iberian Peninsula and also
Switzerland, where biovar I predominates [24,25,27].
However, in one erythromycin susceptible isolate from
Bavaria (08T0013), classified in this study as belonging to
the B.II clade, RD
23
was not deleted, thus showing that de-
letion of RD
23
is not correlated with sensitivity to erythro-
mycin. The molecular mechanisms of resistance to
erythromycin have not been functionally established, but
mutations identified in domain V of the 23S rRNA of
biovar II strains, could provide a likely explanation [33].
Although 25 VNTR markers have been described for
the typing of Francisella, it is pragmatic to investigate only
loci of interest depending on the prevalent subspecies of
F. tularensis, the efficiency of PCR assays for single loci,
and existing data [1,13,34]. Sequence analysis of the locus
Ft-M3 resulted in two different repeats denominated here
as Ft-M3a corresponding with SSTR9E and Ft-M3b corre-
sponding with SSTR9A as described previously by
Johansson et al. [35]. Johansson et al. and Byström et al.
also found that locus Ft-M3 is the most variable marker
[1,13]. In the Francisella genome variations of DNA se-
quences in spite of identical repeat length have been de-
scribed for short-sequence tandem repeats [35,36]. Locus
Ft-M6 showed less variability with only three PCR frag-
ment sizes being observed among the strains. We obtained
the same amplicon sizes that were described in previous
studies for locus Ft-M3 (Additional file 1: Table S2)
[14,37] and for locus Ft-M6 (Additional file 1: Table S2)
[14,37]. Svensson et al. developed a sophisticated real-time
PCR array for hierarchical identification of Francisella iso-
lates [15]. Only three (Ftind33, Ftind38, Ftind49) out of
five INDEL loci were discriminatory among our set of
F. tularensis subsp. holarctica isolates. Ftind48 is a marker
for B.I to B.IV clades (non-japonica/non-california) and is
not expected to vary for these isolates, and Ftind50 is
targeting a specific deletion that so far only has been
found in LVS. It was possible to simplify these assays to
conventional PCR assays that allowed a simple read out
based on gel electrophoresis. We identified clusters of
strains that had the same INDELs and SNPs as strains de-
scribed by Svensson et al. [15]. In our study the analysis of
VNTR and INDELs of two F. tularensis subsp. holarctica
strains (06T0001, 10T0191) that were passaged twenty
times in Ma-104 cells showed that these genomic ele-
ments were stable. Johansson et al. demonstrated for two
VNTR loci (SSTR9 and SSTR16) that they were actually
stable over 55 passages [35]. The VNTR pattern for strains
belonging to clade B.I was more variable compared with
the pattern obtained for clade B. IV (Additional file 1:
Table S2), as was observed previously [21,23-25]. This
might indicate that clade B.IV is more recently introduced
in Germany than clade B.I.
We have applied several typing tools in a polyphasic ap-
proach in order to determine their value for identifying
groups of Francisella strains in Germany. We found
strains belonging to biovars I and II of F. tularensis subsp.
holarctica. Although SNP loci are the most informative
markers for typing of Francisella this method may have to
be adapted to local strains [37,38].
Conclusions
F. tularensis seems to be a re-emerging pathogen in
Germany that infects hares in many regions and causes
a potential risk for exposed humans such as hunters and
others who process hares. The pathogen can easily be
identified using PCR assays directly on DNA extracted
from organ specimens or cultivated strains. Isolates can
also be identified rapidly using MALDI-TOF MS in
routine laboratories where specific PCR assays for
F. tularensis are not established. To identify differences
and genetic relatedness of Francisella strains, analysis of
VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs
(Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18,
B.19, and B.20) was shown to be useful in this set of
strains. When time and costs are limiting parameters
isolates can be analysed using simplified PCR assays with
a focus on genetic loci that are most likely discrimin-
atory among strains found in a specific area. For the fu-
ture whole genome sequencing using next generation
sequencing is desirable and should provide more genetic
information of Francisella strains. Based on these data a
more detailed view on the epidemiology of tularemia will
become possible [39].
Methods
Samples
Organ specimens (e.g. spleen, liver, lung, and/or kidney)
of European brown hares that were suspicious of tular-
emia were collected by local veterinary authorities in
Germany since 2005 and sent for confirmatory testing to
the National Reference Laboratory for Tularemia of the
Friedrich-Loeffler-Institut in Jena. Francisella strains
were cultivated on cysteine heart agar (Becton Dickinson
GmbH, Heidelberg, Germany) supplemented with 10%
chocolatized sheep blood and antibiotics in order to sup-
press the growth of contaminants. One litre of culture
medium contained 100 mg ampicillin (Sigma-Aldrich
Chemie, Taufkirchen, Germany) and 600 000 U poly-
myxin B (Sigma-Aldrich Chemie). Plates were incubated
at 37°C with 5% CO
2
for up to 10 days. Typical colonies
are grey-green, mostly confluent, glossy, and opaque.
Gram staining was performed routinely and showed
Gram negative coccoid bacteria.
The reference strains F. tularensis subsp. tularensis
(FSC 237), mediasiatica (FSC 147), and F. novicida
(ATCC 15482) were obtained from the Bundeswehr
Institute of Microbiology, Munich, Germany, and F.
philomiragia (DSMZ 7535) was obtained from the
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German Collection of Microorganisms and Cell Cul-
tures, Braunschweig, Germany, respectively.
Erythromycin susceptibility
All F. tularensis subsp. holarctica isolates were tested for
their erythromycin susceptibility using Erythromycin discs
[30 μg] and M.I.C.Evaluator
™
(Oxoid, Wesel, Germany) in
order to discriminate the susceptible biovar I from the re-
sistant biovar II as described previously [40].
DNA extraction
50 mg of organ material or 200 μl of cell culture super-
natant were lysed and the DNA was extracted using the
High Pure PCR Template Preparation Kit (Roche Diag-
nostics, Mannheim, Germany) according to the manufac-
turer`s instructions. If cultivation was successful some
colonies were resuspended in 200 μl phosphate-buffered
saline, boiled at 90°C for 10 minutes and DNA was pre-
pared as described above. Finally, DNA was eluted in 200
μl elution buffer. 5 μl were applied in each PCR assay.
Diagnostic PCR assay
F. tularensis subsp. holarctica was identified using a
PCR assay with primer pair C1/C4 targeting the locus
Ft-M19 that distinguishes the two major subspecies F.
tularensis subsp. holarctica and F. tularensis subsp.
tularensis which was carried out as described by
Johansson et al. [11].
VNTR typing
In pilot experiments 6 VNTR loci (Ft-M3, Ft-M6, Ft-M20,
Ft-M21, Ft-M22, and Ft-M24) were investigated as de-
scribed by Byström et al. [13]. The loci found discrimin-
atory were then subsequently analysed in all 31 isolates.
The amplification of the VNTR loci was carried out under
the same cycling conditions as the diagnostic PCR assay
except for the annealing temperature of 56°C. The frag-
ments were cut out of the agarose gel and DNA was puri-
fied using the innuPrep Gel Extraction Kit (Analytik Jena
AG, Jena, Germany) according to the manufacturer’s
instructions. Subsequently, DNA amplificates were se-
quenced as described below.
INDEL analysis
Five INDELs (Ftind33, Ftind38, Ftind48, Ftind49, and
Ftind50) that are discriminatory among F. tularensis
subsp. holarctica were selected from the loci described
by Svensson et al. [15]. The real-time PCR assays with
melting curve analyses were simplified by using conven-
tional PCR assays. The primers “CP”and “OUT”for the
respective loci were used as described by Svensson et al.
The reaction mixture consisted of 5 μl 10 x PCR buffer
with 1.5 mM MgCl
2
(Genaxxon, Stafflangen, Germany),
2μl of dNTP mix (each 2 mM, Carl Roth GmbH,
Karlsruhe, Germany), 1 μl of each primer, 0.2 μlofTa q
DNA polymerase (5 U/μl, Genaxxon), 5 μl of DNA ex-
tract and deionised water to a final volume of 50 μl.
After denaturation at 95°C for 5 min, 35 cycles of ampli-
fication were performed with denaturation at 95°C for
30 s, primer annealing at 60°C for 60 s, and primer ex-
tension at 72°C for 30 s. After a final extension step at
72°C for 30 s amplicons were separated using 2.5% agar-
ose gel electrophoresis and visualized using ethidium
bromide staining under UV light.
SNP typing
Four of ten SNPs (B.17, B.18, B.19, and B.20) that have been
found to be useful for the typing of F. tularensis subsp.
holarctica strains were selected from the loci described by
Svensson et al. [15]. The primers “C”and “D”for the
respective loci described by Svensson et al. were used, but
the primers “D”were shortened by removing the SNP spe-
cific last nucleotide and the non-binding GC-rich tails that
were originally added to the allele-specific primer (i.e.
gcgggcagggcggc). SNPs were detected by sequence analysis
of the PCR products. The nomenclature used for the desig-
nation of clades is according to Karlsson et al. 2013 [16].
DNA sequencing
Purified DNA fragments were subjected to cycle sequen-
cing with BigDye
™
Terminator Cycle Sequencing Ready
Reaction Kit (Applied Biosystems, Darmstadt, Germany).
Amplification primers were also used as sequencing
primers. Nucleotide sequences were determined on an
ABI Prism 310 Genetic Analyzer (Applied Biosystems).
Analysis of sequence data
VNTR sequence data were aligned using BioEdit
(Biological sequence alignment editor, Ibis Therapeutics,
Carlsbad, CA, USA).
Stability testing
The stability of the markers Ft-M3, Ft-M6, Ftind33,
Ftind38, and Ftind49 was assessed for two F. tularensis
subsp. holarctica strains that were isolated from a hare
(06T0001) and a red fox (Vulpes vulpes) (10T0191), re-
spectively. The isolates were passaged twenty times on
MA-104 cells in 12.5 ml cell culture flasks (Becton
Dickinson GmbH, Heidelberg, Germany). Confluent mono-
layers of MA-104 cells were washed with phosphate- buff-
ered saline, pH 7.4. The bacterial suspensions or cell
culture samples were inoculated on the cells at 37°C for
1 h. The inoculum was replaced with Dulbecco’sModified
Eagle’s Medium (DMEM) and incubated at 37°C in a hu-
midified air atmosphere with 5% CO
2
. After incubation for
3 to 5 days when the cells detached from the surface, the
bacteria were harvested by two freeze-thaw cycles. The bac-
teria/cell suspensions were used for preparation of DNA.
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MALDI-TOF typing
Samples were taken from single colonies, ethanol-
precipitated and extracted with 70% formic acid as de-
scribed by Sauer et al. [41]. The extract was diluted with
one volume acetonitrile and 1.5 μL of the mixture was
spotted to a steel MALDI target. The dried extract was
overlaid with 1.5 μL of a saturated solution of
α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/
2.5% trifluoroacetic acid as matrix and was again allowed
to dry. A custom-made database of reference spectra, or
main spectra (MSP), was constructed using the BioTyper
software (version 1.1, Bruker Daltonics, Bremen,
Germany) following the guidelines of the manufacturer.
Each sample was spotted six-fold and four single spectra
with 500 laser pulses each were acquired from each spot
with an Ultraflex I instrument (Bruker Daltonics) in the
linear positive mode in the range of 2,000 to 15,000 Da.
Acceleration voltage was 25 kV and the instrument was
calibrated in the range of 4,364 to 10,299 Da with refer-
ence masses of an extract of an Escherichia coli DH5-
.alpha; strain prepared according to Sauer et al. [41].
MSP were generated within the mass range of 2,500 to
15,000 Da with the following default parameters: com-
pression of the spectrum data by a factor of 10, baseline
smoothing by the Savitsky-Golay algorithm (25 Da frame
size), baseline correction by 2 runs of the multi-polygon
algorithm, and peak search by spectra differentiation.
The number of peaks was limited to 100 per MSP and
all peaks were normalized to the most intense peak with
an intensity of 1.0. The minimum frequency of occur-
rence within the 24 single spectra was set to 50% for
every mass. Peak lists of MSP were exported for further
evaluation.
Additional file
Additional file 1: Table S2. Results of VNTR, SNP, INDEL analysis and
erythromycin sensitivity testing of Francisella tularensis subsp. holarctica
isolates. The number of repeats is given for Ft-M3a, Ft-M3b, and Ft-M6.
The number of base-pairs is given for Ft-M24. Derived state of SNPs and
INDELs is in boldface. Nomenclature is according to Karlsson et al. (2013)
[16], where the B.I clade was re-defined to include both B1 and B3 [15]
(DEL, deletion; IN, insertion; bp, basepairs; BW –Baden-Württemberg, BY -
Bavaria, NRW –North Rhine-Westphalia, LS –Lower Saxony, SN –Saxony,
TH –Thuringia; n.d., not done).
Competing interests
The authors declare that they have no competing interests.
Authors’contributions
WM participated in the design of the study, evaluated VNTR data and
drafted the manuscript. HH performed PCR assays and DNA sequencing and
critically revised the manuscript. PO performed cultivation on nutrient agar
and cell culture, erythromycin susceptibility testing, and critically revised the
manuscript. AK performed MALDI-TOF MS experiments, data analysis and
drafted the respective sections in the manuscript. BB performed MALDI-TOF
MS experiments and data analysis. HB isolated and cultivated strains and
critically revised the manuscript. SB performed post mortem examination and
bacterial culture and revised the manuscript. UE performed post mortem
examination and bacterial culture and revised the manuscript. SH provided
sample specimens and strains and critically revised the manuscript. RK
provided sample specimens and strains and critically revised the manuscript.
AN performed post mortem examination and bacterial culture and revised
the manuscript. MP contributed tissues of hares with tularemia from the
region of Soest (NRW). MR did PCR assays to identify Francisella tularensis in
samples and bacterial cultures and revised the manuscript. GS participated in
the data analysis and critically revised the manuscript. BAS isolated and
cultivated a Francisella tularensis strain from European brown hare in Saxony
and critically revised the manuscript. RS isolated and cultivated a Francisella
tularensis strain from European brown hare in Bavaria and critically revised
the manuscript. KM participated in the data analysis of typing data and
critically revised the manuscript. EK typed strains and critically revised the
manuscript. MF participated in the data analysis and critically revised the
manuscript. HT participated in the design of the study, coordinated the
experiments, analysed the data, and finalized the manuscript. All authors
read and approved the final manuscript.
Acknowledgements
We are grateful to Kerstin Cerncic, Renate Danner, Byrgit Hofmann, and
Karola Zmuda for their excellent technical assistance.
Author details
1
Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut
(Federal Research Institute for Animal Health), Naumburger Str. 96A, Jena
D-07743, Germany.
2
Institute of Molecular Biology, Friedrich-Loeffler-Institut
(Federal Research Institute for Animal Health), Südufer 10, Greifswald-Insel
Riems D-17493, Germany.
3
Thüringer Landesamt für Lebensmittelsicherheit
und Verbraucherschutz, Tennstedter Str. 9, Bad Langensalza D-99947,
Germany.
4
Lower Saxony State Office for Consumer Protection and Food
Safety, Eintrachtweg 17, Hannover D-30173, Germany.
5
Landesbetrieb
Hessisches Landeslabor, Schubertstr. 60, Gießen D-35393, Germany.
6
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit,
Veterinärstr. 2, Oberschleißheim D-85764, Germany.
7
Staatliches
Veterinäruntersuchungsamt Arnsberg, Zur Taubeneiche 10-12, Arnsberg
D-59821, Germany.
8
Official Laboratory for Public and Veterinary Health
Saxony, Leipzig, Bahnhofstr. 58-60, Leipzig D-04158, Germany.
9
Chemisches
und Veterinäruntersuchungsamt Stuttgart, Schaflandstr. 3/3, Fellbach
D-70763, Germany.
10
CBRN Defence and Security, Swedish Defence Research
Agency (FOI), Umeå SE-90182, Sweden.
Received: 16 February 2012 Accepted: 15 March 2013
Published: 21 March 2013
References
1. Johansson A, Farlow J, Larsson P, Dukerich M, Chambers E, Byström M,
Fox J, Chu M, Forsman M, Sjöstedt A, Keim P: Worldwide genetic
relationships among Francisella tularensis isolates determined by
multiple-locus variable-number tandem repeat analysis. JBacteriol
2004, 186:5808–5818.
2. Keim P, Johansson A, Wagner DM: Molecular epidemiology, evolution,
and ecology of Francisella.Ann N Y Acad Sci 2007, 1105:30–66.
3. Petersen JM, Schriefer ME: Tularemia: emergence/re-emergence. Vet Res
2005, 36:455–467.
4. Ellis J, Oyston PC, Green M, Titball RW: Tularemia. Clin Microbiol Rev 2002,
15:631–646.
5. Knothe H: Epidemiology of tularemia. Beitr Hyg Epidemiol 1955, 7:1–122.
6. Grunow R, Priebe H: Tularämie - zum Vorkommen in Deutschland.
Epidemiol Bull 2007, 7:51–56.
7. Splettstoesser WD, Mätz-Rensing K, Seibold E, Tomaso H, Al Dahouk S, Grunow
R, Essbauer S, Buckendahl A, Finke EJ, Neubauer H: Re-emergence of
Francisella tularensis in Germany: fatal tularaemia in a colony of semi-free-
living marmosets (Callithrix jacchus). Epidemiol Infect 2007, 135:1256–1265.
8. Hofstetter I, Eckert J, Splettstoesser W, Hauri AM: Tularaemia outbreak in
hare hunters in the Darmstadt-Dieburg district, Germany. Euro Surveill
2006, 11, E060119.3.
9. Splettstoesser WD, Tomaso H, Al Dahouk S, Neubauer H, Schuff-Werner P:
Diagnostic procedures in tularaemia with special focus on molecular and
immunological techniques. J Vet Med B Infect Dis Vet Public Health 2005,
52:249–261.
Müller et al. BMC Microbiology 2013, 13:61 Page 8 of 9
http://www.biomedcentral.com/1471-2180/13/61

10. Johansson A, Berglund L, Eriksson U, Göransson I, Wollin R, Forsman M,
Tärnvik A, Sjöstedt A: Comparative analysis of PCR versus culture for
diagnosis of ulceroglandular tularemia. J Clin Microbiol 2000, 38:22–26.
11. Johansson A, Ibrahim A, Göransson I, Eriksson U, Gurycova D, Clarridge JE
3rd, Sjöstedt A: Evaluation of PCR-based methods for discrimination of
Francisella species and subspecies and development of a specific PCR
that distinguishes the two major subspecies of Francisella tularensis.
J Clin Microbiol 2000, 38:4180–4185.
12. Farlow J, Smith KL, Wong J, Abrams M, Lytle M, Keim P: Francisella
tularensis strain typing using multiple-locus, variable-number tandem
repeat analysis. J Clin Microbiol 2001, 39:3186–3192.
13. Byström M, Böcher S, Magnusson A, Prag J, Johansson A: Tularemia in
Denmark: identification of a Francisella tularensis subsp. holarctica strain
by real-time PCR and high-resolution typing by multiple-locus variable-
number tandem repeat analysis. J Clin Microbiol 2005, 43:5355–5358.
14. Vogler AJ, Birdsell D, Price LB, Bowers JR, Beckstrom-Sternberg SM,
Auerbach RK, Beckstrom-Sternberg JS, Johansson A, Clare A, Buchhagen JL,
Petersen JM, Pearson T, Vaissaire J, Dempsey MP, Foxall P, Engelthaler DM,
Wagner DM, Keim P: Phylogeography of Francisella tularensis: global
expansion of a highly fit clone. J Bacteriol 2009, 191:2474–2484.
15. Svensson K, Granberg M, Karlsson L, Neubauerova V, Forsman M, Johansson
A: A real-time PCR array for hierarchical identification of Francisella
isolates. PLoS One 2009, 4:e8360.
16. Karlsson E, Svensson K, Lindgren P, Byström M, Sjödin A, Forsman M,
Johansson A: The phylogeographic pattern of Francisella tularensis in
Sweden indicates a Scandinavian origin of Eurosiberian tularaemia.
Environ Microbiol 2012. doi:10.1111/1462-2920.12052. n/a–n/a.
17. Müller W, Bocklisch H, Schüler G, Hotzel H, Neubauer H, Otto P: Detection
of Francisella tularensis subsp. holarctica in a European brown hare
(Lepus europaeus) in Thuringia, Germany. Vet Microbiol 2007, 123:225–229.
18. Runge M, Von Keyserlingk M, Braune S, Voigt S, Grauer S, Pohlmeyer K,
Wedekind M, Splettstoesser WD, Seibold E, Otto P, Müller W: Prevalence of
Francisella tularensis in brown hare (Lepus europaeus) populations in
Lower Saxony, Germany. Eur J Wildlife Res 2011, 57:1085–1089.
19. Seibold E, Maier T, Kostrzewa M, Zeman E, Splettstoesser W: Identification
of Francisella tularensis by whole-cell matrix-assisted laser desorption
ionization-time of flight mass spectrometry: fast, reliable, robust, and
cost-effective differentiation on species and subspecies levels. J Clin
Microbiol 2010, 48:1061–1069.
20. Pikula J, Treml F, Beklova M, Holesovska Z, Pikulova J: Ecological conditions
of natural foci of tularaemia in the Czech Republic. Eur J Epidemiol 2003,
18:1091–1095.
21. Vogler AJ, Birdsell DN, Lee J, Vaissaire J, Doujet CL, Lapalus M, Wagner DM,
Keim P: Phylogeography of Francisella tularensis ssp. holarctica in France.
Lett Appl Microbiol 2011, 52:177–180.
22. Chanturia G, Birdsell DN, Kekelidze M, Zhgenti E, Babuadze G, Tsertsvadze N,
Tsanava S, Imnadze P, Beckstrom-Sternberg SM, Beckstrom-Sternberg JS,
Champion MD, Sinari S, Gyuranecz M, Farlow J, Pettus AH, Kaufman EL,
Busch JD, Pearson T, Foster JT, Vogler AJ, Wagner DM, Keim P:
Phylogeography of Francisella tularensis subspecies holarctica from the
country of Georgia. BMC Microbiol 2011, 11:139.
23. Gyuranecz M, Birdsell DN, Splettstoesser W, Seibold E, Beckstrom-Sternberg
SM, Makrai L, Fodor L, Fabbi M, Vicari N, Johansson A, Busch JD, Vogler AJ,
Keim P, Wagner DM: Phylogeography of Francisella tularensis subsp.
holarctica, Europe. Emerg Infect Dis 2012, 18:290–293.
24. Dempsey MP, Dobson M, Zhang C, Zhang M, Lion C, Gutiérrez-Martín CB,
Iwen PC, Fey PD, Olson ME, Niemeyer D, Francesconi S, Crawford R, Stanley
M, Rhodes J, Wagner DM, Vogler AJ, Birdsell D, Keim P, Johansson A,
Hinrichs SH, Benson AK: Genomic deletion marking an emerging
subclone of Francisella tularensis subsp. holarctica in France and the
Iberian Peninsula. Appl Environ Microbiol 2007, 73:7465–7470.
25. Pilo P, Johansson A, Frey J: Identification of Francisella tularensis cluster in
central and western Europe. Emerg Infect Dis 2009, 15:2049–2051.
26. Gehringer H, Schacht E, Maylaender N, Zeman E, Kaysser P, Oehme R, Pluta
S: Presence of an emerging subclone of Francisella tularensis holarctica in
Ixodes ricinus ticks from south-western Germany. Ticks Tick-borne Dis
2012:1–8. doi:10.1016/j.ttbdis.2012.09.001.
27. Kudelina RI, Olsufiev NG: Sensitivity to macrolide antibiotics and
lincomycin in Francisella tularensis holarctica.J Hyg Epidemiol Microbiol
Immunol 1980, 24:84–91.
28. Petersen J, Molins C: Subpopulations of Francisella tularensis ssp.
tularensis and holarctica: identification and associated epidemiology.
Future Microbiol 2010, 5:649–661.
29. Georgi E, Schacht E, Scholz HC, Splettstoesser WD: Standardized broth
microdilution antimicrobial susceptibility testing of Francisella tularensis
subsp. holarctica strains from Europe and rare Francisella species.
J Antimicrob Chemother 2012, 67:2429–33.
30. Kreizinger Z, Makrai L, Helyes G, Magyar T, Erdélyi K, Gyuranecz M:
Antimicrobial susceptibility of Francisella tularensis subsp. holarctica
strains from Hungary, Central Europe. J Antimicrob Chemother 2012.
doi:10.1093/jac/dks399.
31. Yesilyurt M, Kiliç S, Celebi B, Celik M, Gül S, Erdogan F, Ozel G: Antimicrobial
susceptibilities of Francisella tularensis subsp. holarctica strains isolated
from humans in the Central Anatolia region of Turkey. J Antimicrob
Chemother 2011, 66:2588–92.
32. Kunitsa TN, Meka-Mechenko UV, Izbanova UA, Abdirasilova AA,
Belonozhkina LB: Properties of the tularemia microbe strains isolated from
natural tularemia foci in Kazakhstan. CO, USA: Presented at 7th International
Conference on Tularemia, Breckenridge; 2012:70. S4-32.
33. Biswas S, Raoult D, Rolain J: A bioinformatic approach to understanding
antibiotic resistance in intracellular bacteria through whole genome
analysis. Int J Antimicrob Agents 2008, 32:207–220.
34. Goethert HK, Telford SR 3rd: Nonrandom distribution of vector ticks
(Dermacentor variabilis) infected by Francisella tularensis.PLoS Pathog
2009, 5:e1000319.
35. Johansson A, Göransson I, Larsson P, Sjöstedt A: Extensive allelic variation
among Francisella tularensis strains in a short-sequence tandem repeat
region. J Clin Microbiol 2001, 39:3140–3146.
36. Vodop’ianov AS, Vodop’ianov SO, Pavlovich NV, Mishan’kin BN: [Multilocus
VNTR-typing of Francisella tularensis strains]. Zh Mikrobiol Epidemiol
Immunobiol 2004, 2:21–25.
37. Svensson K, Bäck E, Eliasson H, Berglund L, Granberg M, Karlsson L, Larsson
P, Forsman M, Johansson A: Landscape epidemiology of tularemia
outbreaks in Sweden. Emerg Infect Dis 2009, 15:1937–1947.
38. Pandya GA, Holmes MH, Petersen JM, Pradhan S, Karamycheva SA, Wolcott
MJ, Molins C, Jones M, Schriefer ME, Fleischmann RD, Peterson SN: Whole
genome single nucleotide polymorphism based phylogeny of Francisella
tularensis and its application to the development of a strain typing
assay. BMC Microbiol 2009, 9:213.
39. La Scola B, Elkarkouri K, Li W, Wahab T, Fournous G, Rolain JM, Biswas S,
Drancourt M, Robert C, Audic S, Löfdahl S, Raoult D: Rapid comparative
genomic analysis for clinical microbiology: the Francisella tularensis
paradigm. Genome Res 2008, 18:742–750.
40. Tomaso H, Al Dahouk S, Hofer E, Splettstoesser WD, Treu TM, Dierich MP,
Neubauer H: Antimicrobial susceptibilities of Austrian Francisella tularensis
holarctica biovar II strains. Int J Antimicrob Agents 2005, 26:279–284.
41. Sauer S, Freiwald A, Maier T, Kube M, Reinhardt R, Kostrzewa M, Geider K:
Classification and identification of bacteria by mass spectrometry and
computational analysis. PLoS One 2008, 3:e2843.181.
doi:10.1186/1471-2180-13-61
Cite this article as: Müller et al.:German Francisella tularensis isolates
from European brown hares (Lepus europaeus) reveal genetic and
phenotypic diversity. BMC Microbiology 2013 13:61.
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