IJSEM Papers in Press. Published May 27, 2014 as doi:10.1099/ijs.0.056937-0
1
Bifidobacterium aesculapii sp. nov., from the faeces of the baby common marmoset (Callithrix
2
jacchus)
3
Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1*
4
1
Department of Agricultural Sciences, University of Bologna Italy
5
2
Aptuit srl, Verona, Italy
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*Corresponding author:
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Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42,
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40127 Bologna, Italy Phone+393497620217 Fax+390512096274
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[email protected]
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Key words: New species, Bifidobacterium, Bifidobacterium aesculapii, common marmoset, Callitrix
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jacchus
21 22
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences and hsp60, rpoB
23
and clpC partial gene sequences of B. aesculapii MRM 3/1T are KC807989, KC997237, KC 997239
24
and KF 164211 and of B. aesculapii MRM4/2 are KC 807990, KC997238, KC997240 and
25
KF164212.
26
27
Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate phosphoketolase
28
positive bacterial strains with a peculiar morphology were isolated from faecal samples of baby
29
common marmoset (Callithrix jacchus). Cells of these strains showed a morphology never found in
30
bifidobacterial species, which resembled a coiled snake, always coiled or ring shaped or forming a
31
“Y” shape. MRM 3/1 and MRM 4/2 were chosen as representative strains and further characterized.
32
The bacteria utilized a wide range of carbohydrates and produced urease. Glucose was fermented to
33
acetate and lactate. The strain MRM 3/1 showed a peptidoglycan type unique among members of
34
the genus Bifidobacterium. The DNA base composition was 64.7mol% G+C. Almost complete 16S
35
rRNA, hsp60, clpC and rpoB gene sequences were obtained and phylogenetic relationships were
36
determined. Comparative analysis of 16S rRNA gene sequences showed that strains MRM 3/1 and
37
MRM 4/2 had the highest similarities to Bifidobacterium scardovii (DSM 13734T) (94.6%) and
38
Bifidobacterium stellenboschense (DSMZ 23968T) (94.5%). Analysis of hsp60 showed that both
39
strains were closely related to B. stellenboschense (97.5%), however, despite this high degree of
40
similarity, our isolates could be distinguished from B. stellenboschense DSMZ 23968T also by low
41
levels of DNA-DNA relatedness (30.4%). Therefore, strains MRM 3/1 and MRM 4/2 were located
42
in an actinobacterial cluster and were more closely related to the genus Bifidobacterium than to
43
other genera in Bifidobacteriaceae. On the basis of these results strains MRM 3/1T and MRM 4/2
44
represent a novel species within the genus Bifidobacterium, for which the name Bifidobacterium
45
aesculapii sp. nov. is proposed; the type strain is MRM 3/1T (=DSM 26737T;= JCM 18761T).
46 47
Bifidobacteria are Gram positive, anaerobic, non-motile and non-spore-forming bacteria and
48
represent one of the great bacterial groups within the Actinobacteria. Bifidobacterium spp. are
49
typically found in the gastrointestinal tracts (GIT) of humans and other mammals and hindgut of
50
most social insects, such as honey bees, wasps, cockroaches and bumblebeesbees (Biavati &
51
Mattarelli, 2012; Kopečný et al., 2010; Killer et al., 2009). They are generally host-animal-specific
52
microorganisms and can be separated into “human” and “animal” group based on their distribution
53
(Ventura et al., 2004). Bifidobacteria are well known to posses beneficial effects and to play an
54
important role in maintaining the health of their host (Turroni et al., 2011). Hence, it is the
55
importance of understanding diversity of bifidobacteria in GIT and faeces.
56
During the characterization of bifidobacterial distribution in primates, six bifidobacterial strains
57
with similar morphology were isolated from fresh faecal samples of baby subjects of the common
58
marmoset (Callithrix jacchus), which were individually collected from five animals kept in animal
59
houses in Aptuit s.r.l. Verona, North Italy. The common marmoset is a small exudivore monkey
60
from the new world, which has developed a large specialized caecum for the digestion of complex
61
carbohydrates found in tree exudates (Caton et al., 1996; Bailey & Coe, 2002). As microbiota
62
growth and composition are affected by GIT functioning, such as motility and nutrient availability
63
in intestinal lumen, it is likely that this evolutionary adaptation may influence the concentrations
64
and the types of bacteria that reside as part of the normal intestinal microbiota (Bailey & Coe,
65
2002).
66
Samples of fresh rectal swabs from common marmosets were serially diluted with Peptone Water
67
(Merck) supplemented with cysteine hydrochloride (0.5 g/L); aliquots of each dilution were
68
inoculated onto TPY agar supplemented with mupirocine (100 mg/L) (Applichem) which is a
69
selective agent for bifidobacteria (Rada & Petr, 2000). In each subject we observed cells of a
70
bacterium with a new and unusual morphology, resembling a coiled snake. A total of six isolates
71
with this morphology were obtained from the 5 baby marmosets. They were namely MRM 3/1,
72
MRM 4/2, MRM 5/13, MRM 8/7, MRM 4/6 and MRM 4/7. The isolates were subcultured on TPY,
73
their cells were suspended in a 10 % (w/v) sterile skim milk solution, supplemented with lactose (3
74
%) and yeast extract (0.3 %) and kept both freeze dried and frozen at – 120°C. For all experiments,
75
the strains were cultivated under anaerobic conditions and maintained in TPY broth, pH 6.9 at 37
76
°C, unless indicated otherwise.
77 78
In the present study the morphological, biochemical and molecular characterizations of the isolates
79
were carried out.
80 81
Chromosomal DNA was obtained from isolates according to the procedure of Rossi et al. (2000),
82
with slight modifications. Briefly, the cells of overnight cultures were pelletted and resuspended in
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1 ml of TE buffer (pH 7.6) containing 50 mg/ml lysozyme and then incubated overnight at 37°C.
84 85
For discrimination of the isolates, molecular typing was performed using the enterobacterial
86
repetitive intergenic consensus sequences (ERIC) PCR with the primer pairs ERIC1 (5’-
87
ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’)
88
(Ventura et al., 2003). Each 20 µl reaction mixture contained 3.5 mM of MgCl2, 20 mM of Tris-
89
HCl, 50 mM of KCl, 200 µM of each deoxynucleoside triphosphate (HotStarTaq plus DNA
90
polymerase MasterMix Kit; Qiagen), 30 ng of DNA template and 2 µM of each primer.
91
Amplifications were performed using an Applied Biosystem Veriti Thermal Cycler (Applied
92
Biosystems, Foster City, CA) with the following temperature profiles: 1 cycle at 94 °C for 3 min;
93
35 cycles at 94 °C for 30 s, at 48 °C for 30 s, and at 72 °C for 4 min; and 1 cycle at 72 °C for 6 min.
94
Aliquots of each amplification reaction mixture (15 µl each) were separated by electrophoresis in 2
95
% (w/v) agarose gels at a voltage of 7 V/cm. Gels were ethidium bromide stained (0.5 µg/ml) and
96
photographed under 260-nm UV light.
97 98
Given that isolates revealed two different ERIC-PCR profiles (see Supplementary fig. S1), strains
99
MRM 3/1T and MRM 4/2 were selected as representatives and further characterized.
100 101
The partial 16S rRNA genes of the strains MRM 3/1T and MRM 4/2 were amplified by PCR using
102
the
103
AAGGAGGTGATCCAGCCGCA-3’) (Kim et al., 2010). The partial hsp60, rpoB and clpC gene
104
sequences were also obtained using the primer pairs HspF3 (5’-ATCGCCAAGGAGATCGAGCT-
105
3’) and HspR4 (5’-AAGGTGCCGCGGATCTTGTT-3’), BifF (5’-TCGATCGGGCACATACGG-
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3’) and BifR2 (5’-CGACCACTTCGGCAACCG-3’) (Kim et al., 2010) and BClpC-F (5’–
107
ATCGCSGARACBATYGAGA-3’)
108
(Watanabe et al., 2009), respectively.
primer
pairs
Bif285
(5’-GAGGGTTCGATTCTGGCTCAG-3’)
and
BClpC-R
and
Bif261
(5’-
(5’-ATRATGCGCTTGTGCARYT-3’)
109 110
Each PCR mixture (20 µl) contained 1.5 mM of MgCl2, 20 mM of Tris-HCl, 50 mM of KCl, 200
111
µM of each deoxynucleoside triphosphate (HotStarTaq plus DNA polymerase MasterMix Kit;
112
Qiagen), 0.1 µM of each primer and 30 ng or 200 ng of DNA template for 16S rRNA gene and for
113
each housekeeping gene, respectively. Amplifications were performed using a TGradient thermal
114
cycler (Biometra). A touch down PCR was used to amplify 16S rRNA gene and for all phylogenetic
115
markers, and it was performed as follows: initial denaturation (95 °C, 5 min) for HotStarTaq plus
116
activation; 4 cycles with denaturation at 94 °C for 60 s, annealing at 62 °C for 60 s, and extension at
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72 °C for 90 s; 21 cycles with denaturation at 94 °C for 60 s, annealing at 60°C for 60 s, and
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extension at 72 °C for 90 s; 15 cycles with denaturation at 94 °C for 60 s, annealing at 58°C for 60
119
s, and extension at 72 °C for 90 s; the PCR was completed with a single elongation step (10 min at
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72 °C).
121 122
All the resulting amplicons were separated on 2 % agarose gels, followed by ethidium bromide
123
staining. PCR fragments were purified using the NucleoSpin extract II kit (Macherey-Nagel, Duren,
124
Germany) following manufacturer’s instructions.
125
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The 16S rRNA gene sequences were directly sequenced whereas, for inferring a correct
127
phylogenetic study hsp60, clpC and rpoB gene sequences were cloned using InsTAclone PCR
128
Cloning Kit (Fermentas). All sequencing reactions were performed by Eurofins MWG Operon.
129 130
Almost-complete 16S rRNA gene sequence assembly was performed using
131
program (Huang, 1992), in BIOEDIT (Hall, 1999).
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After editing, the closest known relatives of the novel strains were determined by comparison with
133
DataBase entries and sequences of the closely related species were retrieved from the EMBL and
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GenBank nucleotide databases. Pairwise nucleotide sequence similarity values were calculated
135
using the EzTaxon server (www.eztaxon.org) which provides a web-based tool (Kim et al., 2012).
CAP,
contig assembly
136 137
The 16S rRNA gene sequences (about 1421 bp) of strains MRM 3/1T and MRM 4/2 and of those of
138
their closest relatives retrieved from the DDBJ/GenBank/EMBL databases were aligned by using
139
the
140
total of 43 partial 16S rRNA gene sequences including those of members of the genus
141
Bifidobacterium and of related genera, was constructed with the neighbor-joining method (Saitou &
142
Nei, 1987) and the evolutionary distances were computed using the Kimura 2-parameter method
143
(Kimura, 1980) by using the
CLUSTAL_X2
program (version 1.82) (Thompson et al., 1997). A phylogenetic tree based on a
MEGA
5.05 program (Tamura et al., 2011). The tree was rooted using
T
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Micrococcus luteus DSM 20030 (Fig. 1). The statistical reliability of the tree was evaluated by
145
bootstrap analysis of 1000 replicates (Felsenstein, 1985) and the tree topology was also confirmed
146
with the maximum-likelihood (Cavalli-Sforza & Edwards, 1967) maximum-parsimony (Fitch,
147
1971) and least squares (Fitch & Margoliash, 1967) methods, by using MEGA 5.05 program (Tamura
148
et al., 2011). The 16S rRNA gene sequences similarity between the strains MRM 3/1T and MRM
149
4/2 was about 99.6%. Furthermore they showed low sequence similarities to the known
150
bifidobacteria and the highest values were found to Bifidobacterium scardovii and Bifidobacterium
151
stellenboschence (94.6 and 94.5%, respectively) which are a recently new species described in red -
152
handed tamarin (Saguinus midas) by Endo et al. (2012). Based on a neighbor joining analysis, the
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novel strains are phylogenetically related to B. scardovii (Fig. 1). Similar tree topologies were
154
obtained by using the maximum-likelihood (see Supplementary Fig. S2), maximum-parsimony and
155
least squares methods (data not shown).
156 157
Multilocus sequence analysis is a reliable and robust technique for the identification and
158
classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence
159
analysis (Martens et al., 2008). For this reason, the phylogenetic location of the novel strains was
160
also verified by the analysis of three additional phylogenetic markers such as hsp60, clpC and rpoB
161
genes, which have proven to be discriminative for classification of the genus Bifidobacterium (Kim
162
et al., 2010; Ventura et al., 2006; Jian et al., 2001).
163 164
For hsp60, clpC and rpoB genes, the sequences of strains MRM 3/1T and MRM 4/2 and of those of
165
their closest relatives retrieved from the DDBJ/GenBank/EMBL databases were aligned by using
166
MAFFT program, at
167
Gblocks
168
(“http://molevol.cmima.csic.es/castresana/Gblocks_server.htm”) was then used to eliminate poorly
169
aligned positions and divergent regions of DNA alignments, so that they become more suitable for
170
phylogenetic analysis (Talavera & Castresana 2007).
171
For completing phylogenetic determination hsp60 partial gene sequence was amplified, purified,
172
and directly sequenced from B. scardovii DSM 13734T as above described, whereas for B.
173
stellenboschense DSM 23968T we used the partial gene sequence obtained by Stenico et al., (2014)
174
and retrieved from the GenBank nucleotide databases (Accession number KF 294527).
175
The GenBank/EMBL/DDBJ accession number for the hsp60 partial gene sequence of B. scardovii
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DSM 13734T is KJ689460.
177
Three phylogenetic trees were then constructed using the neighbor joining method. Approximately
178
645 bp of the hsp60 gene, 500 bp of the clpC and 524 bp of the rpoB gene sequences of the isolates
179
and related species were used in the analyses.
CBRC (http://mafft.cbrc.jp/alignment/software/) (Katoh, 2013).
program
(version
0,91b)
as
server
tool
at
Castresana
Lab
180 181
The level of similarity for the partial hsp60 gene sequences of the strains MRM 3/1T and MRM 4/2
182
was 99.5 % and in relation to their closest relatives the levels of similarity were about 97.5 % with
183
B. stellenboschense, 96.2 % with Bifidobacterium saeculare, 96 % with Bifidobacterium pullorum
184
and Bifidobacterium gallinarum, 94.4 % with Bifidobacterium biavatii and 94 % with
185
Bifidobacterium callitrichos and 90.8 % with B. scardovii. Strains MRM 3/1T and MRM 4/2
186
produced a subcluster in the B. pullorum group (Fig. 2).
187 188
The sequences similarity between the clpC gene of strains MRM 3/1T and MRM 4/2 was 99.2 %.
189
The highest sequence similarities were found to B. scardovii and Bifidobacterium bifidum (about
190
86.7 % and 86.5 %, respectively). Strains MRM 3/1T and MRM 4/2 produced a subcluster in the B.
191
scardovii group.
192
The clpC phylogenetic tree is shown in Supplementary Fig. S3.
193
194
The level of similarity for the partial rpoB gene sequence of the strains MRM 3/1T and MRM 4/2
195
was 99.8 % and the levels of similarity in relation to their closest relatives were about 95.2 %, 95
196
%,and 94 % to Bifidobacterium cuniculi, Bifidobacterium choerinum and B. pullorum, respectively.
197
Based on the rpoB partial sequences, MRM 3/1T and MRM 4/2 are placed in a distinct cluster and
198
were related to B. cuniculi. The rpoB phylogenetic tree is shown in Supplementary Fig. S4.
199 200
This finding correlated with the results of Ventura et al. (2006) and Endo et al. (2012) and indicated
201
that the phylogenetic position of Bifidobacterium spp. was highly influenced on the genes used for
202
the analysis.
203 204
The 16S rRNA gene sequence similarity values of strains MRM 3/1T and MRM 4/2 to known
205
species were less than 97 % and it was lower than the recommended value for species
206
differentiation (98.7- 99 %) (Tindall et al., 2010). However, analysis of hsp60 showed that both
207
strains were closely related to B. stellenboschense DSM 23968T (97.5%)
208 209
Due to this high level of similarity (cut-off value for bifidobacterial species differentiation of hsp60
210
is 96%, Zhu et al., 2003) DNA-DNA hybridization between B. aesculapii and B. stellenboschense
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has been also performed. Estimation of level of homology among B. stellenboschense DSM 23968T
212
and strain MRM 3/1T was determined by DSMZ, Braunschweig, Germany. Cells were disrupted by
213
using a Constant Systems TS 0.75 KW (IUL Instruments, Germany). DNA in the crude lysate was
214
purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA-DNA
215
hybridization was carried out as described by De Ley et al. (1970) under consideration of the
216
modifications described by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-
217
spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature
218
controller with in situ temperature probe (Varian). Type strain MRM 3/1 shared 30.4 % DNA-DNA
219
homology to B. stellenboschense DSMZ 23968T unequivocally supporting the description of B.
220
aesculapii as new species.
221 222
Estimation of the G+C content in bacterial chromosomal DNA of type strain MRM 3/1T was
223
determined by DSMZ, Braunschweig, Germany. DNA was purified on hydroxyapatit according to
224
the procedure of Cashion et al. (1977) and was enzymatically hydrolyzed by the method of Mesbah
225
et al. (1989). The resulting deoxyribonucleosides were analyzed by HPLC as described by Tamaoka
226
& Komagata (1984). Type strain MRM 3/1 had G+C content of 64.7 mol% G+C. This value was
227
within the G+C content values for the genus of Bifidobacterium which ranged from 52-67 mol %
228
(Biavati & Mattarelli, 2012; Killer et al., 2010) and in particular was very similar to that obtained
229
from the species Bifidobacterium callitrichos recently described in marmoset by Endo et al. (2012).
230
Morphological, cultural and biochemical characterization of the isolates according to standard
231
techniques were performed at 37 °C unless otherwise stated.
232
Morphology examined by phase-contrast microscopy is shown in Fig. 3(a), (b). Morphological
233
characteristics determined using a Scanning Electron Microscope (SEM) are shown in Fig. 3(c). For
234
SEM observation the strains were cultured in TPY agar at 37°C for 48 hours under anaerobic
235
conditions. After culturing, a slice of agar was excised and dehydrated with a series of increasing
236
ethanol concentrations (at 50, 70, 80, 90, 95 and 100% for 15 minutes each). The prepared cells
237
were subsequently critical-point-dried in a critical point dryer apparatus (CPD Emitech K850) using
238
liquid CO2 as a transitional fluid. Dried samples were mounted on aluminum stubs with silver glue,
239
coated with gold palladium film, using an ion-sputtering unit (Emitech K500) and observed in a
240
Philips 515 SEM at 7-10.0 Kv.
241
The temperature growth range of the strains was tested using an anaerobic TPY broth at a
242
temperature gradient of 20, 25, 30, 35, 37, 40, 42, 45 and 47 °C for 48 h. The sensitivity of the
243
strains to low values of pH was determined at 37 °C in anaerobic TPY broth (pH gradient 3.5, 4.0,
244
4.5, 5.0 and 5.5) for 48 h. The ability of the strains to grow under aerobic and microaerophilic
245
conditions (CampyGen, Oxoid) was tested using TPY agar, TPY soft agar (0.6 %), TPY broth, skim
246
milk and UHT whole milk at 37 °C for 48 h.
247
Hemolytic activity was determined in Columbia Blood Agar (Biolife) at 37 °C under anaerobic
248
conditions for 48 h (Pineiro & Stanton, 2007).
249
Spore staining was performed using malachite green dye. Phase-contrast microscopy (ZEISS) was
250
used to observe the morphology of individual cells as well as spore staining.
251 252
Gram staining, catalase and oxidase activities were determined in cells grown on TPY agar at 37 °C
253
for 48 h under anaerobic conditions using Gram staining individual reagents (Merck Millipore), a 3
254
% (v/v) hydrogen peroxide solution and cotton swabs impregnated with N,N,N’,N’- Tetramethyl-p-
255
phenylendiamine dihydrochloride and dried (Oxibioswab, Biolife), respectively. The motility of
256
strains was determined by stabbing into TPY medium containing 0.4 % agar, knowing that motile
257
strains show a diffuse growth spreading from the line of inoculation. Fermentation products (short-
258
chain fatty acids) were analyzed according to the method described by Holdeman et al. (1977).
259
Briefly after growth in TPY broth with 1% glucose, volatile acids were extracted with diethyl ether.
260
A Carlo Erba 5300 gas chromatograph, with a Nukol capillary column (30 cm) at 170 °C, flame-
261
ionization detector and hydrogen carrier gas, was used for the analysis. All strains tested fermented
262
glucose to acetate and lactate in a variable ratio ranging from 2:1 to 1.5:1.
263
Biochemical characterization was carried out by using the API20A, API20E and API 50CHL
264
systems (BioMérieux) following the manufacturer’s instructions. The results are summarized in
265
Table 1.
266
Bifidobacteria and related genera degrade hexoses via the fructose-6-phosphate phosphoketolase
267
(F6PPK) pathway. F6PPK is the key enzyme in this pathway and . is considered a taxonomic
268
marker for identification of Bifidobacterium spp. and related genera (Biavati & Mattarelli, 2012).
269
The detection of F6PPK activity was determined according to the method described by Scardovi
270
(1986) and modified by Orban & Patterson (2000). All the isolates possess F6PPK activity (Table
271
1).
272
The cell wall murein composition of strain MRM 3/1T was examined by DSMZ. Analysis of partial
273
acid hydrolysates revealed the presence of an A4alpha L-Lys-D-Ser-D-Asp. This murein type is
274
unique among members of the genus Bifidobacterium and related genera confirming the novelty of
275
this species.
276
According to the phylogenetic analyses based on 16S rRNA gene, on hsp60, clpc and rpoB partial
277
sequences, and to other data obtained, the strains MRM 3/1T, MRM 4/2, MRM 5/13, MRM 4/6,
278
MRM 4/7 and MRM 8/7 are genetically and phenotypically distinguishable from the currently
279
recognized species of bifidobacteria and thus represent a novel species, for which we suggest the
280
name Bifidobacterium aesculapii sp. nov.
281
Description of Bifidobacterium aesculapii sp. nov.
282
Bifidobacterium aesculapii (aes.cu.la'pi.i. L. gen. masc. n. aesculapii from the snake-like
283
appearance of the bacterium, resembling the serpent-entwined rod wielded by the Roman God
284
Aesculapius).
285
Cells grown in TPY broth are rods of various shapes, occasionally swollen, always coiled or ring
286
shaped or forming a “Y” shape at both ends. They are Gram-positive-staining, non-motile,
287
asporogenous, non hemolytic, F6PPK-positive, catalase- and oxidase-negative, indole negative and
288
microaerophilic. There was no difference in the growth of the strains under both anaerobic and
289
microaerophilic conditions. The strains were also negative for Arginine DiHydrolase, Lysine
290
DeCarboxylase, Ornithine DeCarboxylase, negative for citrate utilization and H2S production.
291
They did not reduce nitrate as well as nitrite. The well isolated colonies on the surface of TPY agar
292
under anaerobic conditions are white, opaque, smooth and circular with entire edges while the
293
imbedded colonies are lens-shaped or elliptical. Colonies reach 1.7-2.5 mm in diameter after 3 days
294
of incubation. The temperature range is 25-42 °C; no growth occurs at 20 or 47°C. The optimum
295
temperature for growth is 35°-37 °C. They grow at pH 4.5 -7.0 with an optimum at pH 6.5-7.0. The
296
strains can grow in milk, in aerobiosis, microaerophilic and anaerobiosis conditions. Acid is
297
produced from D-glucose, D-lactose, maltose, salicin, D-xylose, L-arabinose, melezitose, D-sorbitol,
298
D-ribose, D-galactose,
299
production from D-mannitol D-sucrose, glycerol, cellobiose, D-mannose, raffinose, L-rhamnose, D-
300
trehalose, D-fructose, starch, D-inulin and glycogen is strain dependent. Acid is not produced from
301
xylitol, amygdalin, methyl-alpha-D-glucoside, N-acetylglucosamine and gluconate. Lactic and
302
acetic acid are produced as end product of glucose fermentation in a variable ratio ranging from 1:2
303
to 1:1.5. Aesculin is hydrolysed and urease is produced. The DNA G+C content of the type strain is
304
64.7 mol%. The peptidoglycan type is A4alpha L-Lys-D-Ser-D-Asp. Phylogenetic analysis of the
305
16S rRNA gene sequence places the species in the B. scardovii subgroup of the genus
306
Bifidobacterium.
307
The type strain MRM 3/1T (=JCM 18761T =DSM 26737T) and the reference strain MRM 4/2
308
(=JCM 18762 =DSM 26738) were isolated from fresh faecal samples of infant common marmoset
309
(Callithrix jacchus), which were individually collected from 5 animals kept in animal houses in
310
Aptuit s.r.l. Verona, North Italy in 2012.
311
Acknowledgements
312
We thank Dr. Pisi Annamaria and Dr. Filippini Gianfranco from Department of Agricultural
313
Sciences, University of Bologna, for their technical assistance with scanning electron microscope
314
observation.
315
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402 403
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405 406
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409 410
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413 414
Turroni, F., van Sinderen, D. & Ventura M. (2011). Genomics and ecological overview of the
415
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416
Ventura, M., Meylan, V. & Zink R. (2003). Identification and Tracing of Bifidobacterium Species
417
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418
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419
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genetics and physiology of bifidobacteria. Antonie Van Leeuwenhoek 86, 205–223.
421
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422
Sinderen, D. (2006). Analysis of bifidobacterial evolution using a multilocus approach. Int J Syst
423
Evol Microbiol 56, 2783–2792.
424 425
Watanabe, K., Makino H., Sasamoto,M., Kudo,Y., Fujimoto, J. & Demberel, S. (2009).
426
Bifidobacterium mongoliense sp. nov., from airag, a traditional fermented mare’s milk product from
427
Mongolia. Int J Syst Evol Microbiol 59, 1535–1540.
428 429 430 431 432 433 434 435 436
437
Figure 1. Phylogenetic relationship of the two novel bifidobacteria to related species based on 16S
438
rRNA gene sequences. The tree was constructed by the neighbour-joining method and rooted with
439
Micrococcus luteus DSM 20030T. The percentage of replicate trees in which the associated taxa
440
clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap
441
percentages above 70 are given at branching points.
442 443 444
Figure 2. Phylogenetic tree based on hsp60 gene sequences showing the relationship of the novel
445
strains isolated from baby marmoset with closely related species. The tree was constructed by the
446
neighbour-joining method on the basis of a comparison of approximately 559 positions, and the
447
sequence of Mycobacterium tuberculosis H37Rv was used as an outgroup. The percentage of
448
replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates)
449
are shown next to the branches. Bootstrap percentages above 70 are given at branching points.
450 451 452
Figure 3. Cellular morphology of cells grown in TPY. Phase-contrast photomicrographs: (a),
453
MRM 3/1T, (b), MRM 4/2. Scanning electron photomicrograph: (c) MRM 3/1T.
454 455 456 457 458 459
Table 1.
460
Differential characteristics between novel bifidobacteria and their closest phylogenetic relatives: B.
461
stellenboschense DSM 23968T, B. biavatii DSM 23967T, B. scardovii DSM 13734T and B. bifidum
462
DSM 20456T.
463 464
465
Table 1:
466
Differential characteristics between novel bifidobacteria and their closest phylogenetic relatives: B. stellenboschense (DSM 23968 T) and B. biavatii
467
(DSM 23967 T) B. scardovii (DSM 13734 T) and B. bifidum (DSM 20456 T).
468
MRM
MRM
MRM
MRM
MRM
B.
B.
MRM 3/1T
4/2
8/7
5/13
4/6
4/7
biavatii
bifidum
D_Mannitol
+
-
-
w
-
+
w
w
-
-
D _Saccharose
+
-
+
+
-
+
+
+
+
+
D_Maltose
+
+
+
w
+
+
+
+
+
-
Salicine
+
+
+
-
+
+
+
+
-
-
D_Xylose
+
+
+
+
+
+
+
+
-
-
L_Arabinose
+
+
+
+
+
+
w
+
-
-
Glycerol
+
-
-
-
-
+
+
w
-
+
D_Cellobiose
+
-
-
-
-
+
-
+
-
-
D_Mannose
+
-
-
-
-
+
-
w
-
-
D_Melezitose
+
w
w
w
w
+
w
+
-
-
D_Raffinose
+
w
+
+
-
+
+
+
+
-
D_Sorbitol
+
w
+
+
+
+
w
w
-
-
L_Rhamnose
+
-
-
-
-
+
-
w
-
-
D_Trehalose
+
-
-
-
-
+
-
+
-
-
D_Ribose
W
+
+
w
+
+
+
+
-
-
Characteristics
B. stellenboschense B. scardovii
Carbohydrates:
D_Galactose
W
+
+
+
+
+
+
+
-
+
D_Fructose
-
+
+
+
+
+
+
+
+
-
Starch
+
-
-
-
w
-
-
-
+
-
Gentiobiose
+
w
+
+
+
+
w
+
+
-
D_Turanose
W
+
+
+
+
+
+
+
w
-
Aburtine
+
+
+
+
+
+
+
+
-
-
D_Melibiose
W
w
+
+
+
w
+
+
w
-
Inulin
W
-
w
W
w
w
-
-
-
-
W
w
+
-
w
+
w
w
-
-
Glycogen
+
-
-
-
-
-
-
w
+
-
Xylitol
W
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
+
-
-
-
-
-
+
+
-
-
+
+
w
-
-
-
-
-
-
-
+
-
-
-
64.7
N.D.
N.D.
N.D.
N.D.
N.D.
66.3
60.1
63.1
58
+
+
+
+
+
-
-
-
-
Potassium Gluconate
Amiygdalin α-Methyl-DGlucopyranoside D_Turanose NAcetylglucosamine
DNA GC Content (Mol%) Enzymatic Activity Urease
+
469 470
Bifidobacterium aesculapii sp. nov., from the feaces of the baby common marmoset (Callithrix jacchus) Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1* 1Department of Agricultural Sciences, University of Bologna Italy 2Aptuit srl, Verona, Italy
*Corresponding author: Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42, 40127 Bologna, Italy Phone+393497620217 Fax+390512096274 [email protected]
Figure 1. Phylogenetic relationship of the two novel bifidobacteria to related species based on 16S rRNA gene sequences. The tree was constructed by the neighbour-joining method and rooted with Micrococcus luteus DSM 20030T. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap percentages above 70 are given at branching points.
B. choerinum ATCC 27686T (D86186)
82
B. animalis subsp. animalis JCM 1190T (D86185)
B. pseudolongum subsp. pseudolongum JCM 1205T (D86195) 81
B. cuniculi YIT 4093T (AB438223)
95
B. gallicum JCM 8224T (D86189)
B. magnum JCM 1218T (D86193)
B. coryneforme YIT 4092T (AB437358)
99 98
B. indicum JCM 1302T (D86188)
B. asteroides CCUG 24607T (EF187235)
B. tsurumiense OMB115T (AB241106)
B. thermophilum YIT 11868T (AB437364) 100
B. thermacidophilum subsp. thermacidophilum YIT 11849T (AB437362)
87
B. boum JCM 1211T (D86190)
B. reuteri DSM 23975T (AB613259) 100
B. pseudocatenulatum JCM 1200T (D86187)
B. catenulatum YIT 4016T (AB437357)
B. merycicum JCM 8219T (D86192)
B. angulatum ATCC 27535T (D86182)
99
B. callitrichos DSM 23973T (AB674319)
B. ruminantium JCM 8222T (D86197) 93 99
B. adolescentis YIT 4011T (AB437355)
B. stercoris Eg1T (FJ611793)
B. dentium ATCC 27534T (D86183) T 100 MRM 3/1 (KC807989)
MRM 4/2 (KC807990)
B. stellenboschense DSM 23968T (AB559505)
B. scardovii YIT 11867T (AB437363)
B. biavatii DSM 23969T (AB559506)
B. bifidum YIT 4039T (AB437356)
B. saguini DSM 23967T (AB559504)
75 81
B. longum subsp. longum YIT 4021T (AB437359)
B. breve ATCC 15700T (AB006658)
B. subtile DSM 20096T (D89378) T 99 B. gallinarum JCM 6291 (D86191)
B. saeculare DSM 6531T (D89328) 100
B. pullorum JCM 1214T (D86196)
B. psychraerophilum YIT 11814T (AB437351)
82
B. mongoliense DSM 21395T (AB433856)
B. minimum YIT 4097T (AB437350)
Gardnerella vaginalis ATCC 14018T (M58744)
Scardovia inopinata DSM 1010T (D89332) 100
Parascardovia denticolens DSM 10105T (D89331)
Micrococcus luteus DSM 20030T (AJ536198)
0.02
Bifidobacterium aesculapii sp. nov., from the feaces of the baby common marmoset (Callithrix jacchus) Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1* 1Department of Agricultural Sciences, University of Bologna Italy 2Aptuit srl, Verona, Italy
*Corresponding author: Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42, 40127 Bologna, Italy Phone+393497620217 Fax+390512096274 [email protected]
Figure 2. Phylogenetic tree based on hsp60 gene sequences showing the relationship of the novel strains isolated from baby marmoset with closely related species. The tree was constructed by the neighbour-joining method on the basis of a comparison of approximately 559 positions, and the sequence of Mycobacterium tuberculosis H37Rv was used as an outgroup. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap percentages above 70 are given at branching points.
B. breve KCTC 3220T (GU361838)
85 93
B. longum subsp. longum KCTC 3128 T 8GU361846)
B. saguini DSM 23967T (AB674320)
97
B. reuteri DSM 23975T (AB674318)
B. scardovii DSM 13734T (KJ689469)
99
B. merycicum KCTC 3369T (GU361848)
B. ruminantium KCTC 3425T (GU361854)
83
B. dentium KCTC 3222T (GU361842)
76
B. catenulatum KCTC 3221T (GU361839)
90
B. pseudocatenulatum KCTC 3223T (GU361850)
96
B. bifidum KCTC 3202T (GU361836)
B. callitrichos DSM 239673T (AB674319) 100
MRM 3/1T (KC997237)
98
MRM 4/2 (KC997238)
B. stellenboschense DSM 23968T (KF294527)
B. biavatii DSM 23969T (AB674321)
B. pullorum KCTC 3274T (GU361853)
B. gallinarum KCTC 3235T (GU361844)
87 100
B. saeculare KCTC 3427T (GU361855)
B. boum KCTC 3227T (GU361837) 100
B. thermophilum KCTC 3225T (GU361857)
B. subtile KCTC 3272T (GU361856)
B. cuniculi KCTC 3276T (GU361841) 100
B. pseudolongum subsp. globosum KCTC 3234T (GU361851)
B. pseudolongum subsp. pseudolongum KCTC 3224T (GU361852)
B. choerinum KCTC 3275T (GU361840)
B. animalis subsp. animalis KCTC 3219T (GU361835)
B. gallicum KCTC 3277T (GU361843) 99
B. magnum KCTC 3228T (GU361847)
B. indicum KCTC 3230T 8GU361845)
B. minimum KCTC 3273T 8GU361849)
M. tubercolosis H37RvT (NC 000962) 0.05
1
Bifidobacterium aesculapii sp. nov., from the faeces of the baby common marmoset (Callithrix
2
jacchus)
3
Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1*
4
1
Department of Agricultural Sciences, University of Bologna Italy
5
2
Aptuit srl, Verona, Italy
6 7 8
*Corresponding author:
9
Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42,
10
40127 Bologna, Italy Phone+393497620217 Fax+390512096274
11
[email protected]
12 13 14 15 16 17 18 19
Key words: New species, Bifidobacterium, Bifidobacterium aesculapii, common marmoset, Callitrix
20
jacchus
21 22
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences and hsp60, rpoB
23
and clpC partial gene sequences of B. aesculapii MRM 3/1T are KC807989, KC997237, KC 997239
24
and KF 164211 and of B. aesculapii MRM4/2 are KC 807990, KC997238, KC997240 and
25
KF164212.
26
27
Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate phosphoketolase
28
positive bacterial strains with a peculiar morphology were isolated from faecal samples of baby
29
common marmoset (Callithrix jacchus). Cells of these strains showed a morphology never found in
30
bifidobacterial species, which resembled a coiled snake, always coiled or ring shaped or forming a
31
“Y” shape. MRM 3/1 and MRM 4/2 were chosen as representative strains and further characterized.
32
The bacteria utilized a wide range of carbohydrates and produced urease. Glucose was fermented to
33
acetate and lactate. The strain MRM 3/1 showed a peptidoglycan type unique among members of
34
the genus Bifidobacterium. The DNA base composition was 64.7mol% G+C. Almost complete 16S
35
rRNA, hsp60, clpC and rpoB gene sequences were obtained and phylogenetic relationships were
36
determined. Comparative analysis of 16S rRNA gene sequences showed that strains MRM 3/1 and
37
MRM 4/2 had the highest similarities to Bifidobacterium scardovii (DSM 13734T) (94.6%) and
38
Bifidobacterium stellenboschense (DSMZ 23968T) (94.5%). Analysis of hsp60 showed that both
39
strains were closely related to B. stellenboschense (97.5%), however, despite this high degree of
40
similarity, our isolates could be distinguished from B. stellenboschense DSMZ 23968T also by low
41
levels of DNA-DNA relatedness (30.4%). Therefore, strains MRM 3/1 and MRM 4/2 were located
42
in an actinobacterial cluster and were more closely related to the genus Bifidobacterium than to
43
other genera in Bifidobacteriaceae. On the basis of these results strains MRM 3/1T and MRM 4/2
44
represent a novel species within the genus Bifidobacterium, for which the name Bifidobacterium
45
aesculapii sp. nov. is proposed; the type strain is MRM 3/1T (=DSM 26737T;= JCM 18761T).
46 47
Bifidobacteria are Gram positive, anaerobic, non-motile and non-spore-forming bacteria and
48
represent one of the great bacterial groups within the Actinobacteria. Bifidobacterium spp. are
49
typically found in the gastrointestinal tracts (GIT) of humans and other mammals and hindgut of
50
most social insects, such as honey bees, wasps, cockroaches and bumblebeesbees (Biavati &
51
Mattarelli, 2012; Kopečný et al., 2010; Killer et al., 2009). They are generally host-animal-specific
52
microorganisms and can be separated into “human” and “animal” group based on their distribution
53
(Ventura et al., 2004). Bifidobacteria are well known to posses beneficial effects and to play an
54
important role in maintaining the health of their host (Turroni et al., 2011). Hence, it is the
55
importance of understanding diversity of bifidobacteria in GIT and faeces.
56
During the characterization of bifidobacterial distribution in primates, six bifidobacterial strains
57
with similar morphology were isolated from fresh faecal samples of baby subjects of the common
58
marmoset (Callithrix jacchus), which were individually collected from five animals kept in animal
59
houses in Aptuit s.r.l. Verona, North Italy. The common marmoset is a small exudivore monkey
60
from the new world, which has developed a large specialized caecum for the digestion of complex
61
carbohydrates found in tree exudates (Caton et al., 1996; Bailey & Coe, 2002). As microbiota
62
growth and composition are affected by GIT functioning, such as motility and nutrient availability
63
in intestinal lumen, it is likely that this evolutionary adaptation may influence the concentrations
64
and the types of bacteria that reside as part of the normal intestinal microbiota (Bailey & Coe,
65
2002).
66
Samples of fresh rectal swabs from common marmosets were serially diluted with Peptone Water
67
(Merck) supplemented with cysteine hydrochloride (0.5 g/L); aliquots of each dilution were
68
inoculated onto TPY agar supplemented with mupirocine (100 mg/L) (Applichem) which is a
69
selective agent for bifidobacteria (Rada & Petr, 2000). In each subject we observed cells of a
70
bacterium with a new and unusual morphology, resembling a coiled snake. A total of six isolates
71
with this morphology were obtained from the 5 baby marmosets. They were namely MRM 3/1,
72
MRM 4/2, MRM 5/13, MRM 8/7, MRM 4/6 and MRM 4/7. The isolates were subcultured on TPY,
73
their cells were suspended in a 10 % (w/v) sterile skim milk solution, supplemented with lactose (3
74
%) and yeast extract (0.3 %) and kept both freeze dried and frozen at – 120°C. For all experiments,
75
the strains were cultivated under anaerobic conditions and maintained in TPY broth, pH 6.9 at 37
76
°C, unless indicated otherwise.
77 78
In the present study the morphological, biochemical and molecular characterizations of the isolates
79
were carried out.
80 81
Chromosomal DNA was obtained from isolates according to the procedure of Rossi et al. (2000),
82
with slight modifications. Briefly, the cells of overnight cultures were pelletted and resuspended in
83
1 ml of TE buffer (pH 7.6) containing 50 mg/ml lysozyme and then incubated overnight at 37°C.
84 85
For discrimination of the isolates, molecular typing was performed using the enterobacterial
86
repetitive intergenic consensus sequences (ERIC) PCR with the primer pairs ERIC1 (5’-
87
ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’)
88
(Ventura et al., 2003). Each 20 µl reaction mixture contained 3.5 mM of MgCl2, 20 mM of Tris-
89
HCl, 50 mM of KCl, 200 µM of each deoxynucleoside triphosphate (HotStarTaq plus DNA
90
polymerase MasterMix Kit; Qiagen), 30 ng of DNA template and 2 µM of each primer.
91
Amplifications were performed using an Applied Biosystem Veriti Thermal Cycler (Applied
92
Biosystems, Foster City, CA) with the following temperature profiles: 1 cycle at 94 °C for 3 min;
93
35 cycles at 94 °C for 30 s, at 48 °C for 30 s, and at 72 °C for 4 min; and 1 cycle at 72 °C for 6 min.
94
Aliquots of each amplification reaction mixture (15 µl each) were separated by electrophoresis in 2
95
% (w/v) agarose gels at a voltage of 7 V/cm. Gels were ethidium bromide stained (0.5 µg/ml) and
96
photographed under 260-nm UV light.
97 98
Given that isolates revealed two different ERIC-PCR profiles (see Supplementary fig. S1), strains
99
MRM 3/1T and MRM 4/2 were selected as representatives and further characterized.
100 101
The partial 16S rRNA genes of the strains MRM 3/1T and MRM 4/2 were amplified by PCR using
102
the
103
AAGGAGGTGATCCAGCCGCA-3’) (Kim et al., 2010). The partial hsp60, rpoB and clpC gene
104
sequences were also obtained using the primer pairs HspF3 (5’-ATCGCCAAGGAGATCGAGCT-
105
3’) and HspR4 (5’-AAGGTGCCGCGGATCTTGTT-3’), BifF (5’-TCGATCGGGCACATACGG-
106
3’) and BifR2 (5’-CGACCACTTCGGCAACCG-3’) (Kim et al., 2010) and BClpC-F (5’–
107
ATCGCSGARACBATYGAGA-3’)
108
(Watanabe et al., 2009), respectively.
primer
pairs
Bif285
(5’-GAGGGTTCGATTCTGGCTCAG-3’)
and
BClpC-R
and
Bif261
(5’-
(5’-ATRATGCGCTTGTGCARYT-3’)
109 110
Each PCR mixture (20 µl) contained 1.5 mM of MgCl2, 20 mM of Tris-HCl, 50 mM of KCl, 200
111
µM of each deoxynucleoside triphosphate (HotStarTaq plus DNA polymerase MasterMix Kit;
112
Qiagen), 0.1 µM of each primer and 30 ng or 200 ng of DNA template for 16S rRNA gene and for
113
each housekeeping gene, respectively. Amplifications were performed using a TGradient thermal
114
cycler (Biometra). A touch down PCR was used to amplify 16S rRNA gene and for all phylogenetic
115
markers, and it was performed as follows: initial denaturation (95 °C, 5 min) for HotStarTaq plus
116
activation; 4 cycles with denaturation at 94 °C for 60 s, annealing at 62 °C for 60 s, and extension at
117
72 °C for 90 s; 21 cycles with denaturation at 94 °C for 60 s, annealing at 60°C for 60 s, and
118
extension at 72 °C for 90 s; 15 cycles with denaturation at 94 °C for 60 s, annealing at 58°C for 60
119
s, and extension at 72 °C for 90 s; the PCR was completed with a single elongation step (10 min at
120
72 °C).
121 122
All the resulting amplicons were separated on 2 % agarose gels, followed by ethidium bromide
123
staining. PCR fragments were purified using the NucleoSpin extract II kit (Macherey-Nagel, Duren,
124
Germany) following manufacturer’s instructions.
125
126
The 16S rRNA gene sequences were directly sequenced whereas, for inferring a correct
127
phylogenetic study hsp60, clpC and rpoB gene sequences were cloned using InsTAclone PCR
128
Cloning Kit (Fermentas). All sequencing reactions were performed by Eurofins MWG Operon.
129 130
Almost-complete 16S rRNA gene sequence assembly was performed using
131
program (Huang, 1992), in BIOEDIT (Hall, 1999).
132
After editing, the closest known relatives of the novel strains were determined by comparison with
133
DataBase entries and sequences of the closely related species were retrieved from the EMBL and
134
GenBank nucleotide databases. Pairwise nucleotide sequence similarity values were calculated
135
using the EzTaxon server (www.eztaxon.org) which provides a web-based tool (Kim et al., 2012).
CAP,
contig assembly
136 137
The 16S rRNA gene sequences (about 1421 bp) of strains MRM 3/1T and MRM 4/2 and of those of
138
their closest relatives retrieved from the DDBJ/GenBank/EMBL databases were aligned by using
139
the
140
total of 43 partial 16S rRNA gene sequences including those of members of the genus
141
Bifidobacterium and of related genera, was constructed with the neighbor-joining method (Saitou &
142
Nei, 1987) and the evolutionary distances were computed using the Kimura 2-parameter method
143
(Kimura, 1980) by using the
CLUSTAL_X2
program (version 1.82) (Thompson et al., 1997). A phylogenetic tree based on a
MEGA
5.05 program (Tamura et al., 2011). The tree was rooted using
T
144
Micrococcus luteus DSM 20030 (Fig. 1). The statistical reliability of the tree was evaluated by
145
bootstrap analysis of 1000 replicates (Felsenstein, 1985) and the tree topology was also confirmed
146
with the maximum-likelihood (Cavalli-Sforza & Edwards, 1967) maximum-parsimony (Fitch,
147
1971) and least squares (Fitch & Margoliash, 1967) methods, by using MEGA 5.05 program (Tamura
148
et al., 2011). The 16S rRNA gene sequences similarity between the strains MRM 3/1T and MRM
149
4/2 was about 99.6%. Furthermore they showed low sequence similarities to the known
150
bifidobacteria and the highest values were found to Bifidobacterium scardovii and Bifidobacterium
151
stellenboschence (94.6 and 94.5%, respectively) which are a recently new species described in red -
152
handed tamarin (Saguinus midas) by Endo et al. (2012). Based on a neighbor joining analysis, the
153
novel strains are phylogenetically related to B. scardovii (Fig. 1). Similar tree topologies were
154
obtained by using the maximum-likelihood (see Supplementary Fig. S2), maximum-parsimony and
155
least squares methods (data not shown).
156 157
Multilocus sequence analysis is a reliable and robust technique for the identification and
158
classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence
159
analysis (Martens et al., 2008). For this reason, the phylogenetic location of the novel strains was
160
also verified by the analysis of three additional phylogenetic markers such as hsp60, clpC and rpoB
161
genes, which have proven to be discriminative for classification of the genus Bifidobacterium (Kim
162
et al., 2010; Ventura et al., 2006; Jian et al., 2001).
163 164
For hsp60, clpC and rpoB genes, the sequences of strains MRM 3/1T and MRM 4/2 and of those of
165
their closest relatives retrieved from the DDBJ/GenBank/EMBL databases were aligned by using
166
MAFFT program, at
167
Gblocks
168
(“http://molevol.cmima.csic.es/castresana/Gblocks_server.htm”) was then used to eliminate poorly
169
aligned positions and divergent regions of DNA alignments, so that they become more suitable for
170
phylogenetic analysis (Talavera & Castresana 2007).
171
For completing phylogenetic determination hsp60 partial gene sequence was amplified, purified,
172
and directly sequenced from B. scardovii DSM 13734T as above described, whereas for B.
173
stellenboschense DSM 23968T we used the partial gene sequence obtained by Stenico et al., (2014)
174
and retrieved from the GenBank nucleotide databases (Accession number KF 294527).
175
The GenBank/EMBL/DDBJ accession number for the hsp60 partial gene sequence of B. scardovii
176
DSM 13734T is KJ689460.
177
Three phylogenetic trees were then constructed using the neighbor joining method. Approximately
178
645 bp of the hsp60 gene, 500 bp of the clpC and 524 bp of the rpoB gene sequences of the isolates
179
and related species were used in the analyses.
CBRC (http://mafft.cbrc.jp/alignment/software/) (Katoh, 2013).
program
(version
0,91b)
as
server
tool
at
Castresana
Lab
180 181
The level of similarity for the partial hsp60 gene sequences of the strains MRM 3/1T and MRM 4/2
182
was 99.5 % and in relation to their closest relatives the levels of similarity were about 97.5 % with
183
B. stellenboschense, 96.2 % with Bifidobacterium saeculare, 96 % with Bifidobacterium pullorum
184
and Bifidobacterium gallinarum, 94.4 % with Bifidobacterium biavatii and 94 % with
185
Bifidobacterium callitrichos and 90.8 % with B. scardovii. Strains MRM 3/1T and MRM 4/2
186
produced a subcluster in the B. pullorum group (Fig. 2).
187 188
The sequences similarity between the clpC gene of strains MRM 3/1T and MRM 4/2 was 99.2 %.
189
The highest sequence similarities were found to B. scardovii and Bifidobacterium bifidum (about
190
86.7 % and 86.5 %, respectively). Strains MRM 3/1T and MRM 4/2 produced a subcluster in the B.
191
scardovii group.
192
The clpC phylogenetic tree is shown in Supplementary Fig. S3.
193
194
The level of similarity for the partial rpoB gene sequence of the strains MRM 3/1T and MRM 4/2
195
was 99.8 % and the levels of similarity in relation to their closest relatives were about 95.2 %, 95
196
%,and 94 % to Bifidobacterium cuniculi, Bifidobacterium choerinum and B. pullorum, respectively.
197
Based on the rpoB partial sequences, MRM 3/1T and MRM 4/2 are placed in a distinct cluster and
198
were related to B. cuniculi. The rpoB phylogenetic tree is shown in Supplementary Fig. S4.
199 200
This finding correlated with the results of Ventura et al. (2006) and Endo et al. (2012) and indicated
201
that the phylogenetic position of Bifidobacterium spp. was highly influenced on the genes used for
202
the analysis.
203 204
The 16S rRNA gene sequence similarity values of strains MRM 3/1T and MRM 4/2 to known
205
species were less than 97 % and it was lower than the recommended value for species
206
differentiation (98.7- 99 %) (Tindall et al., 2010). However, analysis of hsp60 showed that both
207
strains were closely related to B. stellenboschense DSM 23968T (97.5%)
208 209
Due to this high level of similarity (cut-off value for bifidobacterial species differentiation of hsp60
210
is 96%, Zhu et al., 2003) DNA-DNA hybridization between B. aesculapii and B. stellenboschense
211
has been also performed. Estimation of level of homology among B. stellenboschense DSM 23968T
212
and strain MRM 3/1T was determined by DSMZ, Braunschweig, Germany. Cells were disrupted by
213
using a Constant Systems TS 0.75 KW (IUL Instruments, Germany). DNA in the crude lysate was
214
purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA-DNA
215
hybridization was carried out as described by De Ley et al. (1970) under consideration of the
216
modifications described by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-
217
spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature
218
controller with in situ temperature probe (Varian). Type strain MRM 3/1 shared 30.4 % DNA-DNA
219
homology to B. stellenboschense DSMZ 23968T unequivocally supporting the description of B.
220
aesculapii as new species.
221 222
Estimation of the G+C content in bacterial chromosomal DNA of type strain MRM 3/1T was
223
determined by DSMZ, Braunschweig, Germany. DNA was purified on hydroxyapatit according to
224
the procedure of Cashion et al. (1977) and was enzymatically hydrolyzed by the method of Mesbah
225
et al. (1989). The resulting deoxyribonucleosides were analyzed by HPLC as described by Tamaoka
226
& Komagata (1984). Type strain MRM 3/1 had G+C content of 64.7 mol% G+C. This value was
227
within the G+C content values for the genus of Bifidobacterium which ranged from 52-67 mol %
228
(Biavati & Mattarelli, 2012; Killer et al., 2010) and in particular was very similar to that obtained
229
from the species Bifidobacterium callitrichos recently described in marmoset by Endo et al. (2012).
230
Morphological, cultural and biochemical characterization of the isolates according to standard
231
techniques were performed at 37 °C unless otherwise stated.
232
Morphology examined by phase-contrast microscopy is shown in Fig. 3(a), (b). Morphological
233
characteristics determined using a Scanning Electron Microscope (SEM) are shown in Fig. 3(c). For
234
SEM observation the strains were cultured in TPY agar at 37°C for 48 hours under anaerobic
235
conditions. After culturing, a slice of agar was excised and dehydrated with a series of increasing
236
ethanol concentrations (at 50, 70, 80, 90, 95 and 100% for 15 minutes each). The prepared cells
237
were subsequently critical-point-dried in a critical point dryer apparatus (CPD Emitech K850) using
238
liquid CO2 as a transitional fluid. Dried samples were mounted on aluminum stubs with silver glue,
239
coated with gold palladium film, using an ion-sputtering unit (Emitech K500) and observed in a
240
Philips 515 SEM at 7-10.0 Kv.
241
The temperature growth range of the strains was tested using an anaerobic TPY broth at a
242
temperature gradient of 20, 25, 30, 35, 37, 40, 42, 45 and 47 °C for 48 h. The sensitivity of the
243
strains to low values of pH was determined at 37 °C in anaerobic TPY broth (pH gradient 3.5, 4.0,
244
4.5, 5.0 and 5.5) for 48 h. The ability of the strains to grow under aerobic and microaerophilic
245
conditions (CampyGen, Oxoid) was tested using TPY agar, TPY soft agar (0.6 %), TPY broth, skim
246
milk and UHT whole milk at 37 °C for 48 h.
247
Hemolytic activity was determined in Columbia Blood Agar (Biolife) at 37 °C under anaerobic
248
conditions for 48 h (Pineiro & Stanton, 2007).
249
Spore staining was performed using malachite green dye. Phase-contrast microscopy (ZEISS) was
250
used to observe the morphology of individual cells as well as spore staining.
251 252
Gram staining, catalase and oxidase activities were determined in cells grown on TPY agar at 37 °C
253
for 48 h under anaerobic conditions using Gram staining individual reagents (Merck Millipore), a 3
254
% (v/v) hydrogen peroxide solution and cotton swabs impregnated with N,N,N’,N’- Tetramethyl-p-
255
phenylendiamine dihydrochloride and dried (Oxibioswab, Biolife), respectively. The motility of
256
strains was determined by stabbing into TPY medium containing 0.4 % agar, knowing that motile
257
strains show a diffuse growth spreading from the line of inoculation. Fermentation products (short-
258
chain fatty acids) were analyzed according to the method described by Holdeman et al. (1977).
259
Briefly after growth in TPY broth with 1% glucose, volatile acids were extracted with diethyl ether.
260
A Carlo Erba 5300 gas chromatograph, with a Nukol capillary column (30 cm) at 170 °C, flame-
261
ionization detector and hydrogen carrier gas, was used for the analysis. All strains tested fermented
262
glucose to acetate and lactate in a variable ratio ranging from 2:1 to 1.5:1.
263
Biochemical characterization was carried out by using the API20A, API20E and API 50CHL
264
systems (BioMérieux) following the manufacturer’s instructions. The results are summarized in
265
Table 1.
266
Bifidobacteria and related genera degrade hexoses via the fructose-6-phosphate phosphoketolase
267
(F6PPK) pathway. F6PPK is the key enzyme in this pathway and . is considered a taxonomic
268
marker for identification of Bifidobacterium spp. and related genera (Biavati & Mattarelli, 2012).
269
The detection of F6PPK activity was determined according to the method described by Scardovi
270
(1986) and modified by Orban & Patterson (2000). All the isolates possess F6PPK activity (Table
271
1).
272
The cell wall murein composition of strain MRM 3/1T was examined by DSMZ. Analysis of partial
273
acid hydrolysates revealed the presence of an A4alpha L-Lys-D-Ser-D-Asp. This murein type is
274
unique among members of the genus Bifidobacterium and related genera confirming the novelty of
275
this species.
276
According to the phylogenetic analyses based on 16S rRNA gene, on hsp60, clpc and rpoB partial
277
sequences, and to other data obtained, the strains MRM 3/1T, MRM 4/2, MRM 5/13, MRM 4/6,
278
MRM 4/7 and MRM 8/7 are genetically and phenotypically distinguishable from the currently
279
recognized species of bifidobacteria and thus represent a novel species, for which we suggest the
280
name Bifidobacterium aesculapii sp. nov.
281
Description of Bifidobacterium aesculapii sp. nov.
282
Bifidobacterium aesculapii (aes.cu.la'pi.i. L. gen. masc. n. aesculapii from the snake-like
283
appearance of the bacterium, resembling the serpent-entwined rod wielded by the Roman God
284
Aesculapius).
285
Cells grown in TPY broth are rods of various shapes, occasionally swollen, always coiled or ring
286
shaped or forming a “Y” shape at both ends. They are Gram-positive-staining, non-motile,
287
asporogenous, non hemolytic, F6PPK-positive, catalase- and oxidase-negative, indole negative and
288
microaerophilic. There was no difference in the growth of the strains under both anaerobic and
289
microaerophilic conditions. The strains were also negative for Arginine DiHydrolase, Lysine
290
DeCarboxylase, Ornithine DeCarboxylase, negative for citrate utilization and H2S production.
291
They did not reduce nitrate as well as nitrite. The well isolated colonies on the surface of TPY agar
292
under anaerobic conditions are white, opaque, smooth and circular with entire edges while the
293
imbedded colonies are lens-shaped or elliptical. Colonies reach 1.7-2.5 mm in diameter after 3 days
294
of incubation. The temperature range is 25-42 °C; no growth occurs at 20 or 47°C. The optimum
295
temperature for growth is 35°-37 °C. They grow at pH 4.5 -7.0 with an optimum at pH 6.5-7.0. The
296
strains can grow in milk, in aerobiosis, microaerophilic and anaerobiosis conditions. Acid is
297
produced from D-glucose, D-lactose, maltose, salicin, D-xylose, L-arabinose, melezitose, D-sorbitol,
298
D-ribose, D-galactose,
299
production from D-mannitol D-sucrose, glycerol, cellobiose, D-mannose, raffinose, L-rhamnose, D-
300
trehalose, D-fructose, starch, D-inulin and glycogen is strain dependent. Acid is not produced from
301
xylitol, amygdalin, methyl-alpha-D-glucoside, N-acetylglucosamine and gluconate. Lactic and
302
acetic acid are produced as end product of glucose fermentation in a variable ratio ranging from 1:2
303
to 1:1.5. Aesculin is hydrolysed and urease is produced. The DNA G+C content of the type strain is
304
64.7 mol%. The peptidoglycan type is A4alpha L-Lys-D-Ser-D-Asp. Phylogenetic analysis of the
305
16S rRNA gene sequence places the species in the B. scardovii subgroup of the genus
306
Bifidobacterium.
307
The type strain MRM 3/1T (=JCM 18761T =DSM 26737T) and the reference strain MRM 4/2
308
(=JCM 18762 =DSM 26738) were isolated from fresh faecal samples of infant common marmoset
309
(Callithrix jacchus), which were individually collected from 5 animals kept in animal houses in
310
Aptuit s.r.l. Verona, North Italy in 2012.
311
Acknowledgements
312
We thank Dr. Pisi Annamaria and Dr. Filippini Gianfranco from Department of Agricultural
313
Sciences, University of Bologna, for their technical assistance with scanning electron microscope
314
observation.
315
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Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. (1997). The
411
CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by
412
quality analysis tools. Nucleic Acids Res 25, 4876-4882
413 414
Turroni, F., van Sinderen, D. & Ventura M. (2011). Genomics and ecological overview of the
415
genus Bifidobacterium. Int. J Food Microbiol 149), 37-44.
416
Ventura, M., Meylan, V. & Zink R. (2003). Identification and Tracing of Bifidobacterium Species
417
by Use of Enterobacterial Repetitive Intergenic Consensus Sequences. Appl Environ Microbiol 69,
418
4296–4301.
419
Ventura, M., Van Sinderen, D., Fitzgerald, G.F. & Zink, R. (2004). Insights into the taxonomy,
420
genetics and physiology of bifidobacteria. Antonie Van Leeuwenhoek 86, 205–223.
421
Ventura, M., Canchaya, C., Del Casale, A., Dellaglio, F., Neviani, E., Fitzgerald, G.F. & Van
422
Sinderen, D. (2006). Analysis of bifidobacterial evolution using a multilocus approach. Int J Syst
423
Evol Microbiol 56, 2783–2792.
424 425
Watanabe, K., Makino H., Sasamoto,M., Kudo,Y., Fujimoto, J. & Demberel, S. (2009).
426
Bifidobacterium mongoliense sp. nov., from airag, a traditional fermented mare’s milk product from
427
Mongolia. Int J Syst Evol Microbiol 59, 1535–1540.
428 429 430 431 432 433 434 435 436
437
Figure 1. Phylogenetic relationship of the two novel bifidobacteria to related species based on 16S
438
rRNA gene sequences. The tree was constructed by the neighbour-joining method and rooted with
439
Micrococcus luteus DSM 20030T. The percentage of replicate trees in which the associated taxa
440
clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap
441
percentages above 70 are given at branching points.
442 443 444
Figure 2. Phylogenetic tree based on hsp60 gene sequences showing the relationship of the novel
445
strains isolated from baby marmoset with closely related species. The tree was constructed by the
446
neighbour-joining method on the basis of a comparison of approximately 559 positions, and the
447
sequence of Mycobacterium tuberculosis H37Rv was used as an outgroup. The percentage of
448
replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates)
449
are shown next to the branches. Bootstrap percentages above 70 are given at branching points.
450 451 452
Figure 3. Cellular morphology of cells grown in TPY. Phase-contrast photomicrographs: (a),
453
MRM 3/1T, (b), MRM 4/2. Scanning electron photomicrograph: (c) MRM 3/1T.
454 455 456 457 458 459
Table 1.
460
Differential characteristics between novel bifidobacteria and their closest phylogenetic relatives: B.
461
stellenboschense DSM 23968T, B. biavatii DSM 23967T, B. scardovii DSM 13734T and B. bifidum
462
DSM 20456T.
463 464
465
Table 1:
466
Differential characteristics between novel bifidobacteria and their closest phylogenetic relatives: B. stellenboschense (DSM 23968 T) and B. biavatii
467
(DSM 23967 T) B. scardovii (DSM 13734 T) and B. bifidum (DSM 20456 T).
468
MRM
MRM
MRM
MRM
MRM
B.
B.
MRM 3/1T
4/2
8/7
5/13
4/6
4/7
biavatii
bifidum
D_Mannitol
+
-
-
w
-
+
w
w
-
-
D _Saccharose
+
-
+
+
-
+
+
+
+
+
D_Maltose
+
+
+
w
+
+
+
+
+
-
Salicine
+
+
+
-
+
+
+
+
-
-
D_Xylose
+
+
+
+
+
+
+
+
-
-
L_Arabinose
+
+
+
+
+
+
w
+
-
-
Glycerol
+
-
-
-
-
+
+
w
-
+
D_Cellobiose
+
-
-
-
-
+
-
+
-
-
D_Mannose
+
-
-
-
-
+
-
w
-
-
D_Melezitose
+
w
w
w
w
+
w
+
-
-
D_Raffinose
+
w
+
+
-
+
+
+
+
-
D_Sorbitol
+
w
+
+
+
+
w
w
-
-
L_Rhamnose
+
-
-
-
-
+
-
w
-
-
D_Trehalose
+
-
-
-
-
+
-
+
-
-
D_Ribose
W
+
+
w
+
+
+
+
-
-
Characteristics
B. stellenboschense B. scardovii
Carbohydrates:
D_Galactose
W
+
+
+
+
+
+
+
-
+
D_Fructose
-
+
+
+
+
+
+
+
+
-
Starch
+
-
-
-
w
-
-
-
+
-
Gentiobiose
+
w
+
+
+
+
w
+
+
-
D_Turanose
W
+
+
+
+
+
+
+
w
-
Aburtine
+
+
+
+
+
+
+
+
-
-
D_Melibiose
W
w
+
+
+
w
+
+
w
-
Inulin
W
-
w
W
w
w
-
-
-
-
W
w
+
-
w
+
w
w
-
-
Glycogen
+
-
-
-
-
-
-
w
+
-
Xylitol
W
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
-
+
-
-
-
-
-
+
+
-
-
+
+
w
-
-
-
-
-
-
-
+
-
-
-
64.7
N.D.
N.D.
N.D.
N.D.
N.D.
66.3
60.1
63.1
58
+
+
+
+
+
-
-
-
-
Potassium Gluconate
Amiygdalin α-Methyl-DGlucopyranoside D_Turanose NAcetylglucosamine
DNA GC Content (Mol%) Enzymatic Activity Urease
+
469 470
Bifidobacterium aesculapii sp. nov., from the feaces of the baby common marmoset (Callithrix jacchus) Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1* 1Department of Agricultural Sciences, University of Bologna Italy 2Aptuit srl, Verona, Italy
*Corresponding author: Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42, 40127 Bologna, Italy Phone+393497620217 Fax+390512096274 [email protected]
Figure 1. Phylogenetic relationship of the two novel bifidobacteria to related species based on 16S rRNA gene sequences. The tree was constructed by the neighbour-joining method and rooted with Micrococcus luteus DSM 20030T. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap percentages above 70 are given at branching points.
B. choerinum ATCC 27686T (D86186)
82
B. animalis subsp. animalis JCM 1190T (D86185)
B. pseudolongum subsp. pseudolongum JCM 1205T (D86195) 81
B. cuniculi YIT 4093T (AB438223)
95
B. gallicum JCM 8224T (D86189)
B. magnum JCM 1218T (D86193)
B. coryneforme YIT 4092T (AB437358)
99 98
B. indicum JCM 1302T (D86188)
B. asteroides CCUG 24607T (EF187235)
B. tsurumiense OMB115T (AB241106)
B. thermophilum YIT 11868T (AB437364) 100
B. thermacidophilum subsp. thermacidophilum YIT 11849T (AB437362)
87
B. boum JCM 1211T (D86190)
B. reuteri DSM 23975T (AB613259) 100
B. pseudocatenulatum JCM 1200T (D86187)
B. catenulatum YIT 4016T (AB437357)
B. merycicum JCM 8219T (D86192)
B. angulatum ATCC 27535T (D86182)
99
B. callitrichos DSM 23973T (AB674319)
B. ruminantium JCM 8222T (D86197) 93 99
B. adolescentis YIT 4011T (AB437355)
B. stercoris Eg1T (FJ611793)
B. dentium ATCC 27534T (D86183) T 100 MRM 3/1 (KC807989)
MRM 4/2 (KC807990)
B. stellenboschense DSM 23968T (AB559505)
B. scardovii YIT 11867T (AB437363)
B. biavatii DSM 23969T (AB559506)
B. bifidum YIT 4039T (AB437356)
B. saguini DSM 23967T (AB559504)
75 81
B. longum subsp. longum YIT 4021T (AB437359)
B. breve ATCC 15700T (AB006658)
B. subtile DSM 20096T (D89378) T 99 B. gallinarum JCM 6291 (D86191)
B. saeculare DSM 6531T (D89328) 100
B. pullorum JCM 1214T (D86196)
B. psychraerophilum YIT 11814T (AB437351)
82
B. mongoliense DSM 21395T (AB433856)
B. minimum YIT 4097T (AB437350)
Gardnerella vaginalis ATCC 14018T (M58744)
Scardovia inopinata DSM 1010T (D89332) 100
Parascardovia denticolens DSM 10105T (D89331)
Micrococcus luteus DSM 20030T (AJ536198)
0.02
Bifidobacterium aesculapii sp. nov., from the feaces of the baby common marmoset (Callithrix jacchus) Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1* 1Department of Agricultural Sciences, University of Bologna Italy 2Aptuit srl, Verona, Italy
*Corresponding author: Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42, 40127 Bologna, Italy Phone+393497620217 Fax+390512096274 [email protected]
Figure 2. Phylogenetic tree based on hsp60 gene sequences showing the relationship of the novel strains isolated from baby marmoset with closely related species. The tree was constructed by the neighbour-joining method on the basis of a comparison of approximately 559 positions, and the sequence of Mycobacterium tuberculosis H37Rv was used as an outgroup. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap percentages above 70 are given at branching points.
B. breve KCTC 3220T (GU361838)
85 93
B. longum subsp. longum KCTC 3128 T 8GU361846)
B. saguini DSM 23967T (AB674320)
97
B. reuteri DSM 23975T (AB674318)
B. scardovii DSM 13734T (KJ689469)
99
B. merycicum KCTC 3369T (GU361848)
B. ruminantium KCTC 3425T (GU361854)
83
B. dentium KCTC 3222T (GU361842)
76
B. catenulatum KCTC 3221T (GU361839)
90
B. pseudocatenulatum KCTC 3223T (GU361850)
96
B. bifidum KCTC 3202T (GU361836)
B. callitrichos DSM 239673T (AB674319) 100
MRM 3/1T (KC997237)
98
MRM 4/2 (KC997238)
B. stellenboschense DSM 23968T (KF294527)
B. biavatii DSM 23969T (AB674321)
B. pullorum KCTC 3274T (GU361853)
B. gallinarum KCTC 3235T (GU361844)
87 100
B. saeculare KCTC 3427T (GU361855)
B. boum KCTC 3227T (GU361837) 100
B. thermophilum KCTC 3225T (GU361857)
B. subtile KCTC 3272T (GU361856)
B. cuniculi KCTC 3276T (GU361841) 100
B. pseudolongum subsp. globosum KCTC 3234T (GU361851)
B. pseudolongum subsp. pseudolongum KCTC 3224T (GU361852)
B. choerinum KCTC 3275T (GU361840)
B. animalis subsp. animalis KCTC 3219T (GU361835)
B. gallicum KCTC 3277T (GU361843) 99
B. magnum KCTC 3228T (GU361847)
B. indicum KCTC 3230T 8GU361845)
B. minimum KCTC 3273T 8GU361849)
M. tubercolosis H37RvT (NC 000962) 0.05
