International Journal of Systematic and Evolutionary Microbiology (2013), 63, 260–267
DOI 10.1099/ijs.0.036749-0
Budvicia diplopodorum sp. nov. and emended
description of the genus Budvicia
Elke Lang,1 Peter Schumann,1 Brigitte Amalia Knapp,2 Ramesh Kumar,3
Cathrin Spröer1 and Heribert Insam2
Correspondence
Elke Lang
[email protected]
1
Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Inhoffenstraße 7b, 30124 Braunschweig, Germany
2
Institut für Mikrobiologie der Universität Innsbruck, Technikerstraße 25d, 6020 Innsbruck, Austria
3
Microbial Culture Collection, National Centre for Cell Science, Pune University Campus,
Ganeshkhind, Pune 411 007, India
A Gram-negative, rod-shaped, weakly motile, non-spore-forming bacterium (D9T) was isolated
from the gut of Cylindroiulus fulviceps (Diplopoda) on 1/3-strength nutrient agar plates. On the
basis of 16S rRNA gene sequence similarity, strain D9T was shown to be phylogenetically closely
related to the type strain of Budvicia aquatica, the sole species of the genus Budvicia, family
Enterobacteriaceae. The similarity of the 16S rRNA gene sequences of strain D9T and B. aquatica
DSM 5075T was 98.4 %. Other strains that showed high pairwise similarities with the isolate
belonged to the genus Yersinia: Y. frederiksenii ATCC 33641T (96.8 % 16S rRNA gene
sequence similarity), Y. massiliensis CCUG 53443T (96.8 %), Y. pestis NCTC 5923T (96.8 %), Y.
pseudotuberculosis ATCC 29833T (96.8 %), Y. similis CCUG 52882T (96.7 %) and Y. ruckeri
ATCC 29473T (96.5 % ). The similarities of sequences of the housekeeping genes rpoB, hsp60
and gyrB between strain D9T and B. aquatica DSM 5075T and other members of the
Enterobacteriaceae were less than 94 %. Phylogenetic trees based on all four gene sequences
unequivocally grouped the isolate with the type strain of B. aquatica and separately from the
genus Yersinia. Cells contained the quinones Q-8, Q-7 and MK-8. The major polar lipids were
phosphatidylglycerol and phosphatidylethanolamine. The G+C content of the DNA (48.3 mol%)
and the whole-cell fatty acid composition of strain D9T (C14 : 0, C16 : 1v7c, C16 : 0, cyclo-C17 : 0 and
C18 : 1v7c as major components) were typical for members of the Enterobacteriaceae. DNA–DNA
hybridization of strain D9T with B. aquatica DSM 5075T resulted in a relatedness of 30.4 %,
indicating that the isolate did not belong to B. aquatica. Physiological tests allowed the
phenotypic differentiation of strain D9T from B. aquatica DSM 5075T as well as from members of
the genus Yersinia. From these results, it is concluded that strain D9T represents a novel species,
for which the name Budvicia diplopodorum sp. nov. is proposed (type strain D9T 5DSM 21983T
5CCM 7845T). The description of the genus Budvicia is emended.
Millipedes (Diplopoda) act as primary decomposers in
terrestrial ecosystems and increase in abundance upon
decommission of alpine pastureland. Cylindroiulus fulviceps
was found on abandoned pastureland colonized by dwarfshrub and lichen heaths at 2000 m above sea-level in the
Alp mountains. The investigated bacterium dominated the
intestinal microflora of these millipedes independently of
the animals’ diet as shown by molecular fingerprinting of
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequence of strain D9T is HE574451 and for the gyrB, hsp60 and rpoB
sequences of strain D9T and Budvicia aquatica DSM 5075T are
JN210558–JN210563, respectively.
Four supplementary figures and two supplementary tables are available
with the online version of this paper.
260
clone libraries (Knapp et al., 2009, 2010). In this study, we
investigated the physiological and phylogenetic characteristics of strain D9T, the only strain representing this
dominating taxon in the gut of C. fulviceps that could be
isolated. Strain D9T was isolated using 1/3-strength
nutrient agar plates (Knapp et al., 2010) and turned out
to be a representative of a novel species in the genus
Budvicia. So far, the closest relative to this isolate, Budvicia
aquatica, has been isolated from freshwater (Aldová et al.,
1983) and the intestines of salmonids (SkrodenyteArbaciauskiene et al., 2006).
Gram reaction was tested by the non-staining KOH
method as described by Buck (1982) and by testing for
aminopeptidase using ready-made test strips (Merck). Cell
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Budvicia diplopodorum sp. nov.
morphology and sporulation were determined after growth
on nutrient agar (l–1: 5 g peptone, 3 g meat extract and 15 g
agar) for 2, 5 and 10 days by phase-contrast microscopy.
Motility of cells was tested by observing swarming in soft
agar (l21: 1.0 g yeast extract, 0.1 g K2HPO4 and 2.0 g agar)
incubated at 22 uC for up to 5 days. Anaerobic growth was
checked in oxygen-poor OF medium with D-glucose,
covered by paraffin (Hugh & Leifson, 1953). The colonies
of strain D9T were cream, shiny, translucent and convex and
reached a diameter of 0.5 mm after 2 days at 28 uC on R2A
agar (Reasoner & Geldreich, 1985). The isolate grew
relatively slowly and preferred media with low substrate
contents such as nutrient agar and R2A agar rather than
trypticase soy broth (TSB) agar (DSMZ medium 535; www.
dsmz.de). A slight slime production appeared upon ageing
of the cultures. The colonies consisted of Gram-negative,
non-sporulating and facultatively anaerobic rods. The cells
occurred singly and measured 0.862.5–3.0 mm. No motility
could be observed microscopically in liquid cultures
[nutrient broth (NB), TSB, R2A] incubated at 22 uC (Table
1). The type strain of B. aquatica also showed no motility
under these conditions. When inoculated into semisolid
motility agar and incubated at 22 uC, B. aquatica DSM
5075T produced a large halo of cells within 1 day whereas
strain D9T moved only a few millimeters from the
inoculation site. The following were used as reference
strains: B. aquatica DSM 5075T, Y. massiliensis DSM 21859T,
Y. frederiksenii DSM 18490T, Y. pseudotuberculosis DSM
8992T and Y. similis DSM 18211T. Y. pestis is the type species
of the genus. However, strains of the species could not be
included in the comparative laboratory work because of
safety reasons. All strains were grown routinely on R2A
medium or nutrient agar at 28 uC.
Genomic DNA extraction was carried out using the
MasterPure Gram Positive DNA Purification kit
(Epicentre Biotechnologies), according to the manufacturer’s instructions. PCR-mediated amplification of the 16S
rRNA gene and purification of the PCR product was
performed as described previously (Rainey et al., 1996).
Purified PCR products were sequenced using the
CEQTMDTCS-Quick Start kit (Beckman Coulter) as
directed in the manufacturer’s protocol. Sequence reactions
were electrophoresed using the CEQTM8000 Genetic
Analysis System. Additional 16S rRNA gene sequences used
for the phylogenetic analysis were retrieved from the EMBL,
Heidelberg, Germany. The phylogenetic tree was reconstructed using the ARB software package (version December
2007; Ludwig et al., 2004) after multiple alignment of data
with the ARB alignment tool and the SILVA SSURef 104
(release October 2010; Pruesse et al., 2007). Tree building
was performed using the ARB neighbour-joining method
(Saitou & Nei, 1987) without filters. Analysis of the 16S
rRNA gene sequence grouped the isolate within the family
Enterobacteriaceae. Phylogenetic analysis (Fig. 1) placed
strain D9T as a separate lineage adjacent to B. aquatica in a
cluster separate from the genus Yersinia. Regardless of which
other algorithm was used – maximum likelihood or
http://ijs.sgmjournals.org
maximum parsimony (data not shown) – and which taxa
were included, the genera Yersinia and Budvicia were
displayed as a group distinct from other genera of the
Enterobacteriaceae.
In addition, three housekeeping genes were analysed: RNA
polymerase B subunit (rpoB), DNA gyrase B subunit (gyrB)
and a heat-shock protein (hsp60), which have been
previously used in the differentiation of closely related
species of the family Enterobacteriaceae (Mollet et al., 1997;
Harada & Ishikawa, 1997; Dauga, 2002; Rameshkumar et al.,
2010). The amplification, sequencing and phylogenetic
analysis of the three genes were carried out as described
previously (Rameshkumar et al., 2010). The phylogenetic
trees based on each individual housekeeping gene, i.e. rpoB,
gyrB and hsp60, confirmed the tight grouping of strain D9T
with B. aquatica DSM 5075T (Figs S1–S3, available in IJSEM
Online). These groups were supported by high bootstrap
values, clearly confirming the result of 16S rRNA gene
sequence analysis that strain D9T belonged to the genus
Budvicia. Pairwise analysis of rpoB, gyrB and hsp60 sequences
showed that strain D9T had relatively low similarities (94, 88
and 93 %) with its closest relative, B. aquatica DSM 5075T.
Even lower similarities with members of other genera, e.g.
Yersinia, were found for all three genes, as shown in Figs S1–
S3, indicating a separate species status for strain D9T in the
genus Budvicia. These data revealed that rpoB, gyrB and
hsp60 sequences have a high phylogenetic resolution for
species identification in the family Enterobacteriaceae. In
contrast to the tree based on 16S rRNA gene sequences (Fig.
1), the trees based on housekeeping genes did not suggest
any other genus as a sister genus close to Budvicia.
For DNA–DNA hybridization and the determination of
G+C content, DNA was isolated using a French pressure
cell (Thermo Spectronic) and was purified by chromatography on hydroxyapatite as described by Cashion et al.
(1977). DNA–DNA hybridization was carried out in 26
SSC buffer at 69 uC as described by De Ley et al. (1970),
with the modifications described by Huß et al. (1983),
using a model Cary 100 Bio UV/VIS-spectrophotometer
equipped with a Peltier-thermostatted 666 multicell
changer and a temperature controller with in situ
temperature probe (Varian). DNA–DNA relatedness
between strain D9T and B. aquatica DSM 5075T was
30.4 % (mean of duplicate measurements: 34.7 and
25.9 %). According to the threshold value set for species
delineation (70 %; Wayne et al., 1987), this result reveals
that strain D9T does not belong to the species B. aquatica
and should be regarded as a representative of a novel
genospecies. The DNA G+C content of strain D9T, as
determined according to Tamaoka & Komagata (1984)
after degradation to nucleosides (Mesbah et al., 1989), was
48.3 mol%, which corresponds well with the value given
for B. aquatica (46±1 mol%) (Bouvet et al., 1985).
For analysis of fatty acids, cells were grown on TSA
according to the standards of the MIDI system even though
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261
E. Lang and others
Table 1. Differential physiological characters of strain D9T and its closest phylogenetic relatives
Strains: 1, Budvicia diplopodorum sp. nov. D9T; 2, B. aquatica DSM 5075T; 3, Yersinia frederiksenii DSM 18490T; 4, Y. massiliensis DSM 21859T; 5, Y.
pseudotuberculosis DSM 8992T; 6, Y. similis DSM 18211T; 7, Y. pestis [data from Brenner & Farmer (2005)]. Data were taken from this study unless
otherwise stated. +, Positive; W, weakly positive; del, delayed reaction (taking 5 days or longer unless otherwise stated); 2, negative; ND, not
determined/no data available.
Characteristic
Cultivation on MacConkey agar
Growth
Colour of colonies
Growth on Simmons citrate
Gluconate oxidation (Benedict’s
solution)
Malonate test
Starch hydrolysis
Tween 80 hydrolysis
DNase
Urease
Gas from glucose
Motility at 22 uC
Liquid media
Semisolid agar
Motility at 37 uC in liquid media
Slimy on nutrient agar
Maximum growth temperature (uC)
Growth at 4 uC
Growth in presence of 2 % NaCl
Acid from:
D-Arabinose
Lactose
D-Arabitol
Dulcitol
L-Rhamnose
2-Ketogluconate
5-Ketogluconate
Utilization of:d
N-Acetylglucosamine
Citrate
Malate
Mannose
Mannitol
Gluconate
1
2
3
4
5
6
Inhibited
Transparent
2
2
+
Red centre
2
2
+
Opaque
+
2
+
Transparent
del (4 d)
+
+
Opaque
2
+
+
Opaque
2
+
2
2
2
+
2
+
+
2
2
2
+
2
2
2
2
2
2
2
+
+
2
2
del
2
2
2
2
W,
W
2
2
+
+
ND
ND
W
ND
7
ND
ND
2
ND
2
ND
ND
2
5*
2
+
+
+
+
ND
ND
ND
ND
2D
2
+
2
2
2
2
2
+
34
+
2
+
38 (W)
+
+
2
.42
del
+
2
.42
+
+
W (NB),
2 (TSB)
2
.40
+
+
2
.42
+
+
ND
2
2
2
+
del
del
+
2
2
2
2
+
2
+
ND
ND
ND
2
2
del
2
W, del
+
2
ND
+
2
+
+
+
2
+
+
2
+
2
2
ND
ND
+
+
2
2
+
+
+
del
+
+
+
+
+
del
2
+
+
+
del
2
2
del
del
2
2
W
W
+
2
2
W
+
W
2
+
+
W
ND
+
+
2
+
+
+
2
ND
ND
ND
2
2
2
1*
ND
ND
ND
ND
ND
ND
*The percentage of strains giving a positive result is shown.
DMotility determined at 34 uC.
dFor columns 1 and 2, utilization was determined in the presence of vitamins.
the medium was suboptimal for the members of the genus
Budvicia. After incubation for 48 h at 28 uC, cells were
harvested and whole-cell fatty acid methyl esters were
obtained by methods previously described (Kämpfer &
Kroppenstedt, 1996) and separated by GC (model 5898A;
Hewlett Packard). Peaks were automatically integrated and
fatty acid names and percentages were determined using
the Microbial Identification standard software package
MIDI version 6.1 using the TSBA40 calculation method
(Sasser, 1990). Fatty acid components designated ‘summed
262
features’ by the MIDI system were identified by GC/MS
(Singlequad 320; Varian). The fatty acids of strain D9T
were dominated by C16 : 0 (25.0 %), summed feature 3
(containing C16 : 1v7c and/or C15 : 0 2-OH; 18.6 %), cycloC17 (17.9 %), C18 : 1v7c (13.2 %) and C14 : 0 (12.1 %)
according to the MIDI system (Table S1). Summed feature
3 consisted solely of C16 : 1v7c as determined by GC/MS
analysis of the methyl esters. By the same method, it was
demonstrated that summed feature 2B of the MIDI system
represented C14 : 0 3-OH and not the iso-C16 : 1 I component
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Budvicia diplopodorum sp. nov.
Yersinia aleksiciae DSM 14987T (AJ627597)
Yersinia bercovieri ATCC 43970T (AF366377)
65
Yersinia intermedia ATCC 29909T (AF366380)
Yersinia aldovae ATCC 35236T (AF366376)
66
Yersinia mollaretii ATCC 43969T (AF366382)
Yersinia kristensenii ATCC 33638T (AF366381)
64
Yersinia ruckeri ATCC 29473T (AF366385)
99 Yersinia pestis NCTC 5923T (AF366383)
Yersinia pseudotuberculosis ATCC 29833T (AF366375)
99
Yersinia similis CCUG 52882T (AM182404)
65
Yersinia frederiksenii ATCC 33641T (AF366379)
Yersinia rohdei ATCC 43380T (AF366384)
Yersinia massiliensis CCUG 53443T (EF179119)
Yersinia entomophaga DSM 22339T (DQ400782)
Yersinia enterocolitica subsp. enterocolitica ATCC 9610T (AF366378)
89
Yersinia enterocolitica subsp. palearctica DSM 13030T (FJ717344)
58
Budvicia aquatica DSM 5075T (AJ233407)
99
Budvicia diplopodorum D9T (HE574451)
T
Rahnella aquatilis DSM 4594 (AJ233426)
Hafnia paralvei ATCC 29927T (FM179943)
99
Hafnia alvei ATCC 13337T (M59155)
Serratia fonticola DSM 4576T (AJ233429)
54
0.01
66
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the nearest neighbours of strain
D9T. Bootstrap values (.50 %) based on 1000 repetitions are shown at branch nodes. The phylogenetic tree was
reconstructed using the ARB software package after multiple alignment of data with the ARB alignment tool and the SILVA
SSURef 104. Tree building was performed using the ARB neighbour-joining method without filters. The numbers shown at the
branch points indicate percentage bootstrap values from 1000 datasets. Only bootstrap values greater than 50 % are shown.
Bar, 1 % difference in nucleotide sequence.
in these taxa (Table S1). The fatty acid composition
corresponded to that determined in B. aquatica DSM
5075T, which was not described previously. The main
difference between the two strains was the ratio of
hexadecenoic acid (summed feature 3) and cyclic heptadecanoic acid. However, the conversion of the unsaturated
acids C16 : 1 and C18 : 1 into the cyclic derivatives cyclo-C17 : 0
and cyclo-C19 : 0 at the onset of the aging of cells is a known
phenomenon (Huisman et al., 1996). The high proportion
of cyclic heptadecanoic acid in the cells of strain D9T and
Y. frederiksenii DSM 18490T may reflect the fact that the
standard MIDI growth medium was suboptimal for these
organisms. As far as we are aware, all published fatty acid
compositions of members of the genus Yersinia show
percentages of cyclic C17 : 0 higher than 10 % (Jantzen &
Lassen, 1980; Whittaker et al., 2005; Tan et al., 2010;
Nagarajan et al., 2005). The compositions of the isolate and
B. aquatica DSM 5075T support the grouping of the isolate
in the family Enterobacteriaceae and suggest that the
considerable amount of C14 : 0 and the lack of C17 : 0 may
be indicative for the membership of the genus Budvicia.
The component C17 : 0 was found in three members of the
genus Yersinia tested in this study and has been reported
for the genus Yersinia and other genera within the family
Enterobacteriaceae by other authors (Jantzen & Lassen,
1980; Kämpfer et al., 2005; Whittaker et al., 2005;
Madhaiyan et al., 2010; Rameshkumar et al., 2010).
Isoprenoid quinones were extracted according to the
method of Collins et al. (1977) from cells cultivated in
NB and analysed by HPLC (Shimadzu LC 20A; Groth et al.,
1997). Cells of strain D9T contained ubiquinones Q-8 and
Q-7 (the latter in minor amounts) and menaquinone MK8. The occurrence of ubiquinone Q-8 and menaquinone
http://ijs.sgmjournals.org
MK-8 has been described for Y. similis (Sprague et al.,
2008). Dimethyl menaquinone DMK-8 and several ubiquinones have also been detected in different species of the
family Enterobacteriaceae (Collins & Jones, 1981).
Polar lipids were extracted according to the method
described by Minnikin et al. (1979) and separated by
two-dimensional TLC. To identify spots, the chromatographic behaviour of lipid components was compared with
those of authentic standard substances (Sigma) using
specific spray reagents (ninhydrin, molybdenum blue and
molybdophosphoric acid) (Embley & Wait, 1994). Strain
D9T contained phosphatidylglycerol and phosphatidylethanolamine as well as traces of unidentified aminolipids (Fig.
S4). The occurrence of phosphatidylglycerol and phosphatidylethanolamine has also been reported for members of
the genus Yersinia (Sprague et al., 2008) but diphosphatidylglycerol, reported for Yersinia species, could not
be detected in strain D9T.
Oxidase and catalase tests were performed using N,N,N9,N9tetramethyl-p-phenylenediamine dihydrochloride and 10 %
(v/v) H2O2 solutions, respectively. Tween 80 and starch
hydrolysis were tested according to Lányı́ (1987). Other
physiological tests were performed according to standard
methods (Smibert & Krieg, 1994) after incubation for up to
10 days at 28 uC. The comparison of utilization tests in
mineral medium (Stanier et al., 1966) with and without
vitamins (for vitamin composition and concentration, see
DSMZ medium 461; www.dsmz.de) revealed that, under
these conditions, growth of strain D9T was dependent on the
presence of vitamins, which could be replaced by 50 mg
yeast extract l21. We investigated which of the vitamins the
isolate depended on. However, when it was cultivated in
mineral medium with N-acetylglucosamine or glucose as the
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263
E. Lang and others
substrate and with each of the 11 individual vitamins
included in the vitamin solution, no growth was observed in
any case. In these experiments, nicotinic acid, which is
necessary for the growth of B. aquatica, was included but did
not induce growth. In conclusion, the strain seems to be
dependent on not only one but several vitamins, a situation
also described for B. aquatica (Bouvet et al., 1985).
Accordingly, the tests were carried out in vitamin-emended
mineral media where appropriate. API 20 NE, API 20 E and
API 50 CH strips were inoculated according to the
manufacturer’s instructions and read after incubation at
28 uC for 2 days. Medium E/B (for fermenters) was used in
the API 50 CH strips in order to test for acid production
from the carbohydrates.
In the API 20 E and API 50 CH strips, strain D9T and the
type strain of B. aquatica formed acid from D-glucose, Dmannitol, L-rhamnose, L-arabinose, glycerol, D-ribose, Dgalactose, D-fructose, N-acetylglucosamine and gluconate
(Table 2 and Table S2). Strain D9T showed no enzymic
reactions under these conditions whereas B. aquatica DSM
5075T exhibited b-galactosidase and urease activity and
produced hydrogen sulfide (Tables 1 and 2). The activity of
b-galactosidase in B. aquatica DSM 5075T, as well as its
absence in strain D9T, was confirmed on Brilliance E. coli
agar (Oxoid). No b-glucuronidase activity was found in
either strain on this agar. Hydrogen sulfide production by
strain D9T was observed from proteose peptone but not
from triple-sugar iron agar, while B. aquatica DSM 5075T
produced H2S under both conditions. In addition, strain
D9T could be distinguished from the type strain of B.
aquatica by its ability to acidify dulcitol and D-tagatose and
its inability to ferment D-xylose, lactose, D-arabitol and 2and 5-ketogluconate (Table S2). Overall, our results for B.
aquatica DSM 5075T were in good accordance with the
descriptions of the species (Bouvet et al., 1985; Aldová
et al., 1988; Verbarg et al., 2008). Gas production from
glucose by B. aquatica DSM 5075T, but not by strain D9T,
was observed in semisolid agar slabs containing 0.2 g yeast
extract l21 and covered by a paraffin layer. The sole
deviation from the species description of B. aquatica by
Bouvet et al. (1985) was acidification of glycerol within
48 h in the API 50 CH strips; Bouvet et al. (1985) and
Aldová et al. (1988) detected no or delayed (3 or more
days) glycerol acidification for 60 strains and Verbarg et al.
(2008) did not find acid formation from glycerol by the
type strain.
The genera Yersinia and Budvicia share a number of
characteristics. Members of both genera are biochemically
more active at 25–30 uC than at 35–37 uC, are negative for
Voges–Proskauer reaction, indole production and phenylalanine deaminase and most strains reduce nitrate to
nitrite, produce urease and acidify L-arabinose and Dxylose (Bottone et al., 2005; Bouvet et al., 1985). Hydrogen
sulfide production, the feature which was the most useful
character to delineate the members of the two genera, is no
longer suitable for that purpose according to our findings.
Among the type strains of species of the genus Yersinia
264
Table 2. API 20 E reactions of strain D9T and its closest
phylogenetic relatives
Strains: 1, Budvicia diplopodorum sp. nov. D9T; 2, B. aquatica DSM
5075T; 3, Yersinia frederiksenii DSM 18490T; 4, Y. massiliensis DSM
21859T; 5, Y. pseudotuberculosis DSM 8992T; 6, Y. similis DSM 18211T; 7,
Y. ruckeri DSM 18506T; 8, Y. pestis. Data for columns 1–7 were taken
from this study after 2 days and for column 8 from Brenner & Farmer
(2005) and Sprague et al. (2008). All strains are positive for acid
production from D-glucose and D-mannitol. All strains are negative for
arginine dihydrolase, tryptophan deaminase and gelatinase. +, Positive;
W, weakly positive; V, variable; 2, negative; ND, no data available.
Reaction
1
2
3
4
5
6
b-Galactosidase
2
2
2
2
2
2
2
2
+
2
2
2
+
+
2
2
+
2
+
+
2
W
2
+
+
+
2
2
+
+
2
+
2
2
2
2
+
2
2
2
2
+
2
+
2
2
+ 50
V 2
+ 2
2 2
2 2
2 5
2 2
2 2
2
2
+
2
2
2
+
2
2
+
2
2
2
+
+
+
+
+
2
+
+
+
+
2
+
2
+
2
2
2
2
2
+
+
2
2
2
+
2
2
2
+
2
2
2
2
2
2
2
Lysine decarboxylase
Ornithine decarboxylase
Citrate utilization
H2S production
Urease
Indole production
Acetoin production
Acid production from:
Inositol
D-Sorbitol
L-Rhamnose
Sucrose
Melibiose
Amygdalin
L-Arabinose
V
V
7
8*
2
50
1
2
20
ND
+
*Numbers are percentages of strains giving a positive result.
tested, Y. similis DSM 18211T displayed the API 20 E
reaction pattern most similar to that of strain D9T but it
differed from that of strain D9T by a positive urease
reaction (Table 2). Comparing the features of strain D9T to
those mentioned in the literature for Y. pestis, strain D9T
may be discriminated from the latter by its ability to
produce hydrogen sulfide from proteose peptone and acid
from L-rhamnose and its inability to grow at 37 uC or to
hydrolyse aesculin (Bottone et al., 2005). According to
Bottone et al. (2005), strains of Y. pestis uniformly produce
acid from trehalose, which would be another discriminating feature; however, Sprague et al. (2008) stated that only
10 % of 40 strains acidify this sugar. From the fact that
strains D9T and B. aquatica DSM 5075T did not produce
acid from D-mannose, trehalose, arbutin, aesculin or
maltose in the API 50 CH strip, whereas most or all type
strains of species of the genus Yersinia did, we conclude
that acidification of these compounds may be helpful to
distinguish members of the genus Budvicia from most
members of the genus Yersinia (Table S2).
Notably, Y. massiliensis DSM 21859T did not show arginine
dihydrolyse or lysine decarboxylase reactions (Table 2),
thus affirming the negative results found for another type
strain of this species, CCUG 53443T, and four other strains
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Budvicia diplopodorum sp. nov.
of Y. massiliensis (Souza et al., 2011) and contradicting the
original species description by Merhej et al. (2008). Citrate
utilization was stated as negative by Merhej et al. (2008)
and as positive for the type strain of Y. massiliensis by
Souza et al. (2011). In our hands, the reaction remained
negative in the API 20 E strips after 48 h, but Simmons’
citrate test turned positive after 4 days and citrate
supported growth in mineral medium after 6 days (Table
1). A different situation was observed for Y. similis DSM
18211T; this strain consistently utilized citrate in the API 20
E strip within 48 h and in mineral medium, whereas
Simmons’ citrate test remained negative for 10 days as
described for the species (Sprague et al., 2008). We
conclude that incubation time and test conditions seem
to be critical criteria when evaluating this reaction.
Y. similis DSM 18211T showed variable results in the API
20 E strip for b-galactosidase, which is described as a
negative trait for the species (Sprague et al., 2008). Y.
pseudotuberculosis DSM 8992T did not form acid from Lrhamnose in both API strips within 2 days (Table 2 and
Table S2) and showed only a weak and delayed acid
formation in semisolid agar slabs after 9 days at 28 uC
(Table 1) whereas the species descriptions report a positive
reaction (Sprague et al., 2008; Bottone et al., 2005) and had
a weak and delayed reaction (after 9 days at 28 uC) in
semisolid agar slabs, whereas the species description
reported a positive reaction (Sprague et al., 2008). The
reactions stated in this study were in good agreement with
the description of Y. frederiksenii (Ursing et al., 1980).
Chemotaxonomic and molecular properties, including
housekeeping gene (rpoB, gyrB and hsp60) sequence
similarities, indicate the membership of strain D9T in the
genus Budvicia. A low DNA–DNA relatedness with the type
strain of the only described species of the genus, B. aquatica
DSM 5075T, suggests that strain D9T represents a novel
species. In addition, biochemical traits discriminate strain
D9T from its closest relatives. Thus, a novel species,
Budvicia diplopodorum sp. nov., is proposed, with strain
D9T as the type strain.
Description of Budvicia diplopodorum sp. nov.
Budvicia diplopodorum (di.plo.po9do.rum. N.L. gen. pl. n.
diplopodorum of Diplopoda, isolated from Diplopoda).
Forms cream, shiny, translucent, convex colonies on
nutrient agar or R2A agar, reaching 0.5 mm after 2 days
at 28 uC and becoming slimy with age. Consists of Gramnegative, oxidase-negative, catalase-positive, singly occurring rods, 0.862.5–3.0 mm in size, slightly motile in soft
agar at 22 uC, but not motile in TSB, NB or R2A broth at
22 or 34 uC. No spores, gas vesicles or other cell inclusions
are detected. Grows at 4 and 34 uC, but not at 35 uC, and
with 1 % NaCl, but not with 2 % NaCl. Growth in mineral
medium with glucose or other carbohydrates or acids as
substrates depends on vitamins. Growth on McConkey
agar and Columbia-horse blood agar is inhibited; no single
colonies are formed. Negative for Simmons’ citrate and bhttp://ijs.sgmjournals.org
galactosidase, b-glucuronidase on Brilliance E. coli agar and
malonate and gluconate oxidation tests. Produces acid
from D-glucose under oxidative and fermentative conditions in Hugh’s medium. Does not produce gas during
fermentation of glucose. Produces H2S from proteose
peptone, but not in triple-sugar iron agar slants or in API
20 E strips. Hydrolyses starch weakly, but does not
hydrolyse Tween 80, DNA, aesculin, gelatin or urea. Does
not show lysine or ornithine decarboxylase, arginine
dihydrolase or tryptophan deaminase reactions, and does
not produce indole from tryptophan or produce acetoin
from pyruvate (API 20 E). Reduces nitrate to nitrite (API
20 NE). Utilizes citrate, mannitol, gluconate, N-acetylglucosamine (weakly) and malate (weakly) but does not utilize
mannose in mineral medium with vitamins. Does not
utilize mannose, maltose or phenylacetate (API 20 NE).
Produces acid from N-acetylglucosamine, L-arabinose, Dfructose, D-galactose, gluconate, D-glucose, glycerol, Dmannitol, L-rhamnose, D-ribose, D-tagatose and dulcitol
(weakly), but not from amygdalin, D-arabinose, D-arabitol,
arbutin, aesculin, inositol, 2- or 5-ketogluconate, lactose,
maltose, D-mannose, melibiose, D-sorbitol, sucrose, trehalose, D-xylose or any other substrate included in API tests
(API 50 CHE and API 20 E). The main fatty acids are
C16 : 0, C16 : 1v7c, cyclo-C17 : 0, C18 : 1v7c and C14 : 0. The
polar lipids are phosphatidylglycerol and phosphatidylethanolamine. Ubiquinone Q-8 and menaquinone MK-8
are the predominating isoprenoid quinones.
The type strain, D9T (5DSM 21983T 5CCM 7845T), was
isolated from the gut of the diplopodian Cylindroiulus
fulviceps sampled at 2000 m above sea-level. The DNA
G+C content of the type strain is 48.3 mol%.
Emended description of the genus Budvicia
Bouvet et al. 1985
The description is that given by Bouvet et al. (1985) with
the following amendments. May or may not grow at 37 uC.
May or may not produce hydrogen sulfide or acid from
glycerol. May or may not hydrolyse urea or nitrophenyl-bD-galactopyranoside. Motility may or may not be detected
in liquid media, but is detectable in semisolid agar. The
whole-cell fatty acid composition is dominated by C16 : 0,
C16 : 1v7c, cyclo-C17 : 0, C18 : 1v7c and C14 : 0.
Acknowledgements
We are grateful to G. Pötter, DSMZ, for carrying out the fatty acid
analysis. We thank P. Aumann, C. Berg, I. Brandes, N. Mrotzek, B.
Sträubler, J. Swiderski and A. Wasner for excellent technical
assistance.
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