Bifidobacterium aesculapil sp nov., from the faeces. of the baby common marmoset (Callithrix jacchus)

Abstract

Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate phosphoketolase positive bacterial strains with a peculiar morphology were isolated from faecal samples of baby common marmoset (Callithrix jacchus). Cells of these strains showed a morphology never found in bifidobacterial species, which resembled a coiled snake, always coiled or ring shaped or forming a "Y" shape. MRM 3/1 and MRM 4/2 were chosen as representative strains and further characterized. The bacteria utilized a wide range of carbohydrates and produced urease. Glucose was fermented to acetate and lactate. The strain MRM 3/1 showed a peptidoglycan type unique among members of the genus Bifidobacterium . The DNA base composition was 64.7mol% G+C. Almost complete 16S rRNA, hsp60, clpC and rpoB gene sequences were obtained and phylogenetic relationships were determined. Comparative analysis of 16S rRNA gene sequences showed that strains MRM 3/1 and MRM 4/2 had the highest similarities to Bifidobacterium scardovii (DSM 13734T) (94.6%) and Bifidobacterium stellenboschense (DSMZ 23968T) (94.5%). Analysis of hsp60 showed that both strains were closely related to B. stellenboschense (97.5%), however, despite this high degree of similarity, our isolates could be distinguished from B. stellenboschense DSMZ 23968T also by low levels of DNA-DNA relatedness (30.4%). Therefore, strains MRM 3/1 and MRM 4/2 were located in an actinobacterial cluster and were more closely related to the genus Bifidobacterium than to other genera in Bifidobacteriaceae. On the basis of these results strains MRM 3/1T and MRM 4/2 represent a novel species within the genus Bifidobacterium, for which the name Bifidobacterium aesculapii sp. nov. is proposed; the type strain is MRM 3/1T (=DSM 26737T;= JCM 18761T).

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Bifidobacterium aesculapii sp. nov., from the faeces
of the baby common marmoset (Callithrix jacchus)
M. Modesto,
1
S. Michelini,
1
I. Stefanini,
1
A. Ferrara,
2
S. Tacconi,
2
B. Biavati
1
and P. Mattarelli
1
Correspondence
P. Mattarelli
paola.mattarelli@unibo.it
1
Department of Agricultural Sciences, University of Bologna, Italy
2
Aptuit srl, Verona, Italy
Six Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate
phosphoketolase-positive bacterial strains with a peculiar morphology were isolated from faecal
samples of baby common marmosets (Callithrix jacchus). Cells of these strains showed a
morphology not reported previously for a bifidobacterial species, which resembled a coiled snake,
always coiled or ring shaped or forming a ‘Y’ shape. Strains MRM 3/1
T
and MRM 4/2 were
chosen as representative strains and characterized further. The bacteria utilized a wide range of
carbohydrates and produced urease. Glucose was fermented to acetate and lactate. Strain MRM
3/1
T
showed a peptidoglycan type unique among members of the genus Bifidobacterium. The
DNA base composition was 64.7 mol% G+C. Almost-complete 16S rRNA, hsp60,clpC and
rpoB gene sequences were obtained and phylogenetic relationships were determined.
Comparative analysis of 16S rRNA gene sequences showed that strains MRM 3/1
T
and MRM 4/2
had the highest similarities to Bifidobacterium scardovii DSM 13734
T
(94.6 %) and
Bifidobacterium stellenboschense DSM 23968
T
(94.5 %). Analysis of hsp60 showed that both
strains were closely related to B. stellenboschense DSM 23968
T
(97.5 % similarity); however,
despite this high degree of similarity, our isolates could be distinguished from B.
stellenboschense DSM 23968
T
by low levels of DNA–DNA relatedness (30.4 % with MRM 3/1
T
).
Strains MRM 3/1
T
and MRM 4/2 were located in an actinobacterial cluster and were more closely
related to the genus Bifidobacterium than to other genera in the family Bifidobacteriaceae. On the
basis of these results, strains MRM 3/1
T
and MRM 4/2 represent a novel species within the genus
Bifidobacterium, for which the name Bifidobacterium aesculapii sp. nov. is proposed; the type
strain is MRM 3/1
T
(5DSM 26737
T
5JCM 18761
T
).
Bifidobacteria are Gram-positive, anaerobic, non-motile,
non-spore-forming bacteria and represent one of the larger
bacterial groups within the Actinobacteria. Bifidobacteria
are typically found in the gastrointestinal (GI) tracts of
humans and other mammals and the hindgut of most
social insects, such as honey bees, wasps, cockroaches and
bumblebees (Biavati & Mattarelli, 2012; Kopec
ˇny
´et al.,
2010; Killer et al., 2009). They are generally host-animal-
specific and can be separated into ‘human’ and ‘animal’
groups based on their distribution (Ventura et al., 2004).
Bifidobacteria are known to exert beneficial effects and to
play an important role in maintaining the health of their
host (Turroni et al., 2011). Hence, it is important to
understand the diversity of bifidobacteria in the GI tract
and faeces.
During the characterization of bifidobacterial distribution
in primates, six bifidobacterial strains with similar mor-
phology were isolated from fresh faecal samples of baby
subjects of the common marmoset (Callithrix jacchus),
which were individually collected from five animals kept in
animal houses at Aptuit s.r.l. Verona, in northern Italy. The
common marmoset is a small exudivore monkey from the
New World that has developed a large specialized caecum
for the digestion of the complex carbohydrates found in
tree exudates (Caton et al., 1996; Bailey & Coe, 2002). As
microbiota growth and composition are affected by GI
tract function, such as motility and nutrient availability in
the intestinal lumen, it is likely that this evolutionary
adaptation may influence the concentrations and types of
Abbreviation: F6PPK, fructose-6-phosphate phosphoketolase.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene
sequence and partial hsp60,rpoB and clpC gene sequences of strain
MRM 3/1
T
are KC807989, KC997237, KC997239 and KF164211
and those of strain MRM4/2 are KC807990, KC997238, KC997240
and KF164212, respectively. The accession number for the partial
hsp60 gene sequence of B. scardovii DSM 13734
T
is KJ689460.
Four supplementary figures are available with the online version of this
paper.
International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2819–2827 DOI 10.1099/ijs.0.056937-0
056937 G2014 IUMS Printed in Great Britain 2819

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bacteria that form part of the normal intestinal microbiota
(Bailey & Coe, 2002).
Samples of fresh rectal swabs from common marmosets
were serially diluted with peptone water (Merck) supple-
mented with cysteine hydrochloride (0.5 g l
21
); aliquots of
each dilution were inoculated onto TPY agar supplemented
with mupirocine (100 mg l
21
; Applichem), which is a
selective agent for bifidobacteria (Rada & Petr, 2000). In
each subject, we observed cells of a bacterium with a novel
and unusual morphology, resembling a coiled snake. A
total of six isolates with this morphology were obtained
from the five baby marmosets. They were namely MRM 3/
1
T
, MRM 4/2, MRM 5/13, MRM 8/7, MRM 4/6 and MRM
4/7. The isolates were subcultured on TPY agar and cells
were suspended in a 10 % (w/v) sterile skimmed milk
solution supplemented with lactose (3 %) and yeast extract
(0.3 %) and kept both freeze-dried and frozen at 2120 uC.
For all experiments, the strains were cultivated under
anaerobic conditions and maintained in TPY broth,
pH 6.9, at 37 uC, unless indicated otherwise.
In the present study, the morphological, biochemical and
molecular characterization of the isolates was carried out.
Chromosomal DNA was obtained from the isolates
according to the procedure of Rossi et al. (2000), with
slight modifications. Briefly, cells of overnight cultures
were pelleted and resuspended in 1 ml TE buffer (pH 7.6)
containing 50 mg lysozyme ml
21
and then incubated
overnight at 37 uC.
For discrimination of the isolates, molecular typing was
performed using enterobacterial repetitive intergenic con-
sensus sequences (ERIC) PCR with the primers ERIC1 (59-
ATGTAAGCTCCTGGGGATTCAC-39) and ERIC2 (59-A-
AGTAAGTGACTGGGGTGAGCG-39) (Ventura et al., 2003).
Each 20 ml reaction mixture contained 3.5 mM MgCl
2
,20 mM
Tris/HCl,50mMKCl,200mM each dNTP (HotStarTaq plus
DNA polymerase MasterMix kit; Qiagen), 30 ng DNA tem-
plate and 2 mM each primer. Amplifications were performed
using an Applied Biosystems Veriti Thermal Cycler with the
following temperature profile: 1 cycle at 94 uCfor3min;35
cycles of 94 uC for 30 s, 48 uCfor30sand72uCfor4min;
and 1 cycle at 72 uC for 6 min. Aliquots of each amplification
reaction mixture (15 ml each) were separated by electrophoresis
in 2 % (w/v) agarose gels at a voltage of 7 V cm
21
.Gelswere
stainedwithethidiumbromide(0.5
mgml
21
) and photo-
graphed under 260 nm UV light. Given that the isolates
revealed two different ERIC-PCR profiles (Fig. S1, available in
the online Supplementary Material), strains MRM 3/1
T
and
MRM 4/2 were selected as representatives and further
characterized.
The partial 16S rRNA genes of strains MRM 3/1
T
and MRM
4/2 were amplified by PCR using the primers Bif285 (59-
GAGGGTTCGATTCTGGCTCAG-39) and Bif261 (59-AAG-
GAGGTGATCCAGCCGCA-39) (Kim et al., 2010). Partial
hsp60,rpoB and clpC gene sequences were also obtained
using the primer pairs HspF3 (59-ATCGCCAAGGAGA-
TCGAGCT-39) and HspR4 (59-AAGGTGCCGCGGAT-
CTTGTT-39), BifF (59-TCGATCGGGCACATACGG-39)and
BifR2 (59-CGACCACTTCGGCAACCG-39)(Kimet al., 2010)
and BClpC-F (59–ATCGCSGARACBATYGAGA-39)and
BClpC-R (59-ATRATGCGCTTGTGCARYT-39)(Watanabe
et al., 2009), respectively. Each PCR mixture (20 ml) contained
1.5 mM MgCl
2
, 20 mM Tris/HCl, 50 mM KCl, 200 mMeach
dNTP (HotStarTaq plus DNA polymerase MasterMix kit;
Qiagen), 0.1 mM each primer and 30 or 200 ng DNA template
for the 16S rRNA gene and for each housekeeping gene, re-
spectively. Amplifications were performed using a TGradient
thermal cycler (Biometra). A touchdown PCR was used to
amplify the 16S rRNA gene and the other phylogenetic
markers as follows: initial denaturation (95 uC, 5 min) for
HotStarTaq plus activation; four cycles of denaturation at
94 uC for 60 s, annealing at 62 uC for 60 s and extension at
72 uC for 90 s; 21 cycles of denaturation at 94 uCfor60s,
annealing at 60 uC for 60 s and extension at 72 uCfor90s;
and 15 cycles of denaturation at 94 uC for 60 s, annealing at
58 uC for 60 s and extension at 72 uC for 90 s. The PCR was
completed with a single elongation step (10 min at 72 uC).
The resulting amplicons were separated on 2 % agarose gels,
followed by ethidium bromide staining. PCR fragments were
purified using the NucleoSpin extract II kit (Macherey-
Nagel) following the manufacturer’s instructions.
16S rRNA genes were directly sequenced whereas hsp60,
clpC and rpoB gene sequences were cloned using an
InsTAclone PCR Cloning kit (Fermentas). All sequencing
reactions were performed by Eurofins MWG Operon.
Almost-complete 16S rRNA gene sequence assembly was
performed using CAP (contig assembly program; Huang,
1992) in BioEdit (Hall, 1999). After editing, the closest
known relatives of the novel strains were determined by
comparison with database entries and the sequences of
closely related strains were retrieved from the EMBL and
GenBank nucleotide databases. Pairwise nucleotide sequence
similarity values were calculated using the EzTaxon server
(http://www.eztaxon.org/), which provides a web-based tool
(Kim et al.,2012).
The 16S rRNA gene sequences (about 1421 bp) of strains
MRM 3/1
T
and MRM 4/2 and of those of their closest
relatives retrieved from the DDBJ/GenBank/EMBL data-
bases were aligned by using the CLUSTAL_X2 program
(version 1.82) (Thompson et al., 1997). A phylogenetic tree
based on a total of 43 partial 16S rRNA gene sequences,
including those of members of the genus Bifidobacterium
and of related genera, was reconstructed with the neighbour-
joining method (Saitou & Nei, 1987) and evolutionary
distances were computed using Kimura’s two-parameter
method (Kimura, 1980) by using the MEGA 5.05 program
(Tamura et al., 2011). The tree was rooted using Micrococcus
luteus DSM 20030
T
(Fig. 1). The statistical reliability of the
tree was evaluated by bootstrap analysis of 1000 replicates
(Felsenstein, 1985) and the tree topology was also confirmed
with the maximum-likelihood (Cavalli-Sforza & Edwards,
1967), maximum-parsimony (Fitch, 1971) and least-squares
(Fitch & Margoliash, 1967) methods, by using MEGA 5.05
M. Modesto and others
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(Tamura et al., 2011). The 16S rRNA gene sequence
similarity between strains MRM 3/1
T
and MRM 4/2 was
about 99.6 %. They showed low sequence similarity to
known bifidobacteria; the highest similarities were found to
the type strains of Bifidobacterium scardovii and Bifido-
bacterium stellenboschense (94.6 and 94.5 %, respectively), a
recently described species from a red-handed tamarin (Sagui-
nus midas)(Endoet al., 2012). Based on the neighbour-
joining analysis, the novel strains are related phylogenetically
to B. scardovii (Fig. 1). Similar tree topologies were obtained
by using the maximum-likelihood (Fig. S2), maximum-
parsimony and least-squares methods (not shown).
Multilocus sequence analysis is a reliable and robust
technique for the identification and classification of bac-
terial isolates to the species level as an alternative to 16S
rRNA gene sequence analysis (Martens et al., 2008). For
this reason, the phylogenetic location of the novel strains
was verified by analysis of three additional phylogenetic
markers, hsp60,clpC and rpoB, which have proven to be
discriminative for classification of the genus Bifidobac-
terium (Jian et al., 2001; Ventura et al., 2006; Kim et al.,
2010).
For hsp60,clpC and rpoB genes, the sequences of strains MRM
3/1
T
and MRM 4/2 and of those of their closest relatives
retrieved from the DDBJ/GenBank/EMBL databases were
aligned by using the MAFFT program, at CBRC (http://mafft.
cbrc.jp/alignment/software/) (Katoh & Standley, 2013).
The Gblocks program (version 0.91b) as server tool at
the Castresana Lab (http://molevol.cmima.csic.es/castresana/
Gblocks.html) was then used to eliminate poorly aligned
positions and divergent regions of DNA alignments, so that
they became more suitable for phylogenetic analysis (Talavera
& Castresana, 2007).
To complete our phylogenetic determination, the partial
hsp60 gene was amplified, purified and directly sequenced
from B. scardovii DSM 13734
T
as described above, whereas,
for B. stellenboschense DSM 23968
T
, we used the partial
gene sequence obtained by Stenico et al. (2014) and
retrieved from GenBank.
Three phylogenetic trees were then reconstructed using the
neighbour-joining method. Approximately 645 bp of the
hsp60 gene, 500 bp of the clpC gene and 524 bp of the rpoB
gene sequence of the isolates and related strains were used
in the analyses.
The level of similarity for the partial hsp60 gene sequences of
strains MRM 3/1
T
and MRM 4/2 was 99.5 % and, in relation
to the type strains of their closest relatives, the levels of
similarity were about 97.5 % with B. stellenboschense, 96.2 %
with Bifidobacterium saeculare, 96 % with Bifidobacterium
pullorum and Bifidobacterium gallinarum, 94.4 % with Bifi-
dobacterium biavatii, 94 % with Bifidobacterium callitrichos
and 90.8 % with B. scardovii. Strains MRM 3/1
T
and MRM
4/2 formed a subcluster in the B. pullorum group (Fig. 2).
The sequence similarity between the clpC genes of strains
MRM 3/1
T
and MRM 4/2 was 99.2 %. The highest
sequence similarities were found to the type strains of B.
scardovii and Bifidobacterium bifidum (about 86.7 and
86.5 %, respectively). Strains MRM 3/1
T
and MRM 4/2
produced a subcluster in the B. scardovii group.
The clpC phylogenetic tree is shown in Fig. S3.
The level of similarity for the partial rpoB gene sequences of
strains MRM 3/1
T
and MRM 4/2 was 99.8 %, and the levels
of similarity in relation to their closest relatives were about
95.2, 95 and 94 % to the type strains of Bifidobacterium
cuniculi,Bifidobacterium choerinum and B. pullorum, respec-
tively. Based on the partial rpoB sequences, MRM 3/1
T
and
MRM 4/2 are placed in a distinct cluster and were related to
B. cuniculi. The rpoB phylogenetic tree is shown in Fig. S4.
These findings correlated with the results of Ventura et al.
(2006) and Endo et al. (2012) and indicated that the
phylogenetic positions of species of the genus Bifido-
bacterium are highly influenced by the genes used for the
analysis.
The 16S rRNA gene sequence similarity of strains MRM 3/
1
T
and MRM 4/2 to known species was less than 97 % and
it was lower than the recommended value for species
differentiation (98.7–99 %; Tindall et al., 2010). However,
analysis of hsp60 showed that both strains were closely
related to B. stellenboschense DSM 23968
T
(97.5 % similar-
ity). Due to this high level of similarity (the cut-off value
for bifidobacterial species differentiation of hsp60 is 96 %;
Zhu et al., 2003), DNA–DNA hybridization between strain
MRM 3/1
T
and B. stellenboschense DSM 23968
T
was also
performed. Estimation of the level of relatedness between
B. stellenboschense DSM 23968
T
and strain MRM 3/1
T
was
determined by the DSMZ, Braunschweig, Germany. Cells
were disrupted by using a Constant Systems TS 0.75 kW
(IUL Instruments). DNA in the crude lysate was purified
by chromatography on hydroxyapatite as described by
Cashion et al. (1977). DNA–DNA hybridization was
carried out as described by De Ley et al. (1970) under
consideration of the modifications described by Huss et al.
(1983) using a model Cary 100 Bio UV/Vis spectropho-
tometer equipped with a Peltier-thermostatted 666
multicell changer and a temperature controller with in
situ temperature probe (Varian). Strain MRM 3/1
T
shared
30.4 % DNA–DNA relatedness with B. stellenboschense
DSM 23968
T
, unequivocally supporting the assignment of
strain MRM 3/1
T
to a novel species.
Estimation of the G+C content in bacterial chromosomal
DNA of strain MRM 3/1
T
was done by the DSMZ. DNA
was purified on hydroxyapatite according to the procedure
of Cashion et al. (1977) and enzymically hydrolysed by the
method of Mesbah et al. (1989). The resulting deoxyr-
ibonucleosides were analysed by HPLC as described by
Tamaoka & Komagata (1984). Strain MRM 3/1
T
had a
DNA G+C content of 64.7 mol%. This value was within
the range of DNA G+C content reported for the genus
Bifidobacterium, 52–67 mol% (Biavati & Mattarelli, 2012;
Killer et al., 2010), and in particular was very similar to that
Bifidobacterium aesculapii sp. nov.
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B. choerinum ATCC 27686T (D86186)
B. animalis JCM 1190T (D86185)
B. pseudolongum JCM 1205T (D86195)
B. cuniculi YIT 4093T (AB438223)
B. gallicum JCM 8224T (D86189)
B. magnum JCM 1218T (D86193)
B. coryneforme YIT 4092T (AB437358)
B. indicum JCM 1302T (D86188)
B. asteroides CCUG 24607T (EF187235)
B. tsurumiense OMB115T (AB241106)
B. thermophilum YIT 11868T (AB437364)
B. thermacidophilum YIT 11849T (AB437362)
B. boum JCM 1211T (D86190)
B. reuteri DSM 23975T (AB613259)
B. pseudocatenulatum JCM 1200T (D86187)
B. catenulatum YIT 4016T (AB437357)
B. merycicum JCM 8219T (D86192)
B. angulatum ATCC 27535T (D86182)
B. callitrichos DSM 23973T (AB674319)
B. ruminantium JCM 8222T (D86197)
B. adolescentis YIT 4011T (AB437355)
B. stercoris Eg1T (FJ611793)
B. dentium ATCC 27534T (D86183)
82
99
100
99
100
81
98
99
95
87
93
B. aesculapii MRM 3/1T(KC807989)
B. aesculapii 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)
B. longum YIT 4021T (AB437359)
B. breve ATCC 15700T (AB006658)
B. subtile DSM 20096T (D89378)
B. gallinarum JCM 6291T (D86191)
B. saeculare DSM 6531T (D89328)
B. pullorum JCM 1214T (D86196)
B. psychraerophilum YIT 11814T (AB437351)
B. mongoliense DSM 21395T (AB433856)
B. minimum YIT 4097T (AB437350)
Gardnerella vaginalis ATCC 14018T (M58744)
Scardovia inopinata DSM 10107T (D89332)
Parascardovia denticolens DSM 10105T (D89331)
Micrococcus luteus DSM 20030T (AJ536198)
100
100
99
100
82
75
81
0.02
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obtained for B. callitrichos, described recently from a
marmoset by Endo et al. (2012).
Morphological, cultural and biochemical characterization
of the isolates according to standard techniques was
performed at 37 uC unless otherwise stated. Morphology
as examined by phase-contrast microscopy is shown in Fig.
3(a, b). Morphological characteristics determined using a
scanning electron microscope (SEM) are shown in Fig.
Fig. 1. Phylogenetic relationships of the novel bifidobacteria to related species based on 16S rRNA gene sequences. The tree
was reconstructed by the neighbour-joining method and rooted with Micrococcus luteus DSM 20030
T
. Percentages of
replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to
branches. Bootstrap values above 70 % are given at branching points. Bar, 0.02 substitutions per nucleotide position.
B. breve KCTC 3220T (GU361838)
B. longum KCTC 3128T (GU361846)
B. saguini DSM 23967T (AB674320)
B. reuteri DSM 23975T (AB674318)
B. scardovii DSM 13734T (KJ689469)
B. merycicum KCTC 3369T (GU361848)
B. ruminantium KCTC 3425T (GU361854)
B. dentium KCTC 3222T (GU361842)
B. catenulatum KCTC 3221T (GU361839)
B. pseudocatenulatum KCTC 3223T (GU361850)
B. bifidum KCTC 3202T (GU361836)
B. callitrichos DSM 23973T (AB674319)
B. aesculapii MRM 3/1T (KC997237)
B. aesculapii MRM 4/2 (KC997238)
B. stellenboschense DSM 23968T (KF294527)
B. biavatii DSM 23969T (AB674321)
B. pullorum KCTC 3274T (GU361853)
96
90
99
85
93
100
76
97
98
83
B. gallinarum KCTC 3235T (GU361844)
B. saeculare KCTC 3427T (GU361855)
B. boum KCTC 3227T (GU361837)
B. thermophilum KCTC 3225T (GU361857)
B. subtile KCTC 3272T (GU361856)
B. cuniculi KCTC 3276T (GU361841)
B. pseudolongum KCTC 3234T (GU361851)
B. pseudolongum subsp. pseudolongum KCTC 3224T (GU361852)
B. choerinum KCTC 3275T (GU361840)
B. animalis KCTC 3219T (GU361835)
B. gallicum KCTC 3277T (GU361843)
B. magnum KCTC 3228T (GU361847)
B. indicum KCTC 3230T (GU361845)
B. minimum KCTC 3273T (GU361849)
M. tubercolosis H37RvT (NC000962)
100
99
100
100
87
0.05
Fig. 2. Phylogenetic tree based on hsp60 gene sequences showing the relationships of the novel strains isolated from baby
marmosets to closely related species. The tree was reconstructed by the neighbour-joining method on the basis of a
comparison of 559 positions, and the sequence of Mycobacterium tuberculosis H37Rv
T
was used as an outgroup.
Percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown
next to branches. Bootstrap values above 70 % are given at branching points. Bar, 0.05 substitutions per nucleotide position.
Bifidobacterium aesculapii sp. nov.
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3(c). For SEM observation, strains were cultured on TPY
agar at 37 uC for 48 h under anaerobic conditions. After
culturing, a slice of agar was excised and dehydrated with a
series of increasing ethanol concentrations (50, 70, 80, 90,
95 and 100 % for 15 min each). The prepared cells were
subsequently critical-point-dried in a critical point dryer
apparatus (CPD Emitech K850) using liquid CO
2
as a
transitional fluid. Dried samples were mounted on
aluminium stubs with silver glue, coated with gold
palladium film using an ion-sputtering unit (Emitech
K500) and observed in a Philips 515 SEM at 7–10.0 kV.
The temperature range for growth of the strains was tested
using an anaerobic TPY broth at 20, 25, 30, 35, 37, 40, 42,
45 and 47 uC for 48 h. The sensitivity of the strains to low
pH was determined at 37 uC in anaerobic TPY broth
(pH 3.5, 4.0, 4.5, 5.0 and 5.5) for 48 h. The ability of the
strains to grow under aerobic and microaerophilic
conditions (CampyGen; Oxoid) was tested using TPY
agar, TPY soft agar (0.6 %), TPY broth, skimmed milk and
UHT whole milk at 37 uC for 48 h.
Haemolytic activity was determined in Columbia blood
agar (Biolife) at 37 uC under anaerobic conditions for 48 h
(Pineiro & Stanton, 2007).
Spore staining was performed using malachite green dye.
Phase-contrast microscopy (Zeiss) was used to observe the
morphology of individual cells as well as spore staining.
Gram staining and catalase and oxidase activities were
respectively determined from cells grown on TPY agar at
37 uC for 48 h under anaerobic conditions using Gram
staining individual reagents (Merck Millipore), a 3 % (v/v)
hydrogen peroxide solution and cotton swabs impregnated
with N,N,N9,N9-tetramethyl p-phenylenediamine dihy-
drochloride and dried (Oxibioswab; Biolife). The motility
of strains was determined by stabbing into TPY medium
containing 0.4 % agar, knowing that motile strains
show diffuse growth spreading from the line of inocula-
tion. Fermentation products (short-chain fatty acids) were
analysed according to the method described by Holdeman
et al. (1977). Briefly, after growth in TPY broth with 1 %
glucose, volatile acids were extracted with diethyl ether. A
Carlo Erba 5300 gas chromatograph, with a Nukol capillary
column (30 cm) at 170 uC, flame-ionization detector and
hydrogen carrier gas, was used for the analysis. All strains
tested fermented glucose to acetate and lactate in a variable
ratio ranging from 2 : 1 to 1.5 : 1.
Biochemical characterization was carried out by using the
API 20A, API 20E and API 50CHL systems (bioMe
´rieux)
following the manufacturer’s instructions. The results are
summarized in Table 1.
Bifidobacteria and members of related genera degrade
hexoses via the fructose-6-phosphate phosphoketolase
(F6PPK) pathway. F6PPK is the key enzyme in this
pathway and is considered a taxonomic marker for
identification of species of Bifidobacterium and related
genera (Biavati & Mattarelli, 2012). F6PPK activity was
determined according to the method described by Scardovi
(1986) and modified by Orban & Patterson (2000). All the
isolates possessed F6PPK activity.
The cell-wall murein composition of strain MRM 3/1
T
was
examined by the DSMZ. Analysis of partial acid hydro-
lysates revealed the presence of A4a-type, L-Lys–D-Ser–D-
Asp. This murein type is unique among members of the
genus Bifidobacterium and related genera, confirming the
novelty of this species.
According to our phylogenetic analyses based on 16S rRNA
gene and partial hsp60,clpC and rpoB sequences and the
other data obtained, strains MRM 3/1
T
, MRM 4/2, MRM
5/13, MRM 4/6, MRM 4/7 and MRM 8/7 are genetically
and phenotypically distinguishable from currently recog-
nized species of bifidobacteria and thus represent a novel
species, for which we suggest the name Bifidobacterium
aesculapii sp. nov.
Description of Bifidobacterium aesculapii
sp. nov.
Bifidobacterium aesculapii (aes.cu.la9pi.i. L. gen. masc. n.
aesculapii of Aesculapius, from the snake-like appearance
of the bacterium, resembling the serpent-entwined rod
wielded by the Roman god Aesculapius).
Cells grown in TPY broth are rods of various shapes,
occasionally swollen, always coiled or ring shaped or
(a) (b) (c)
Fig. 3. Cellular morphology of cells grown in TPY broth. (a, b) Phase-contrast photomicrographs of strains MRM 3/1
T
(a) and
MRM 4/2 (b). Bar, 10 mm (b). (c) Scanning electron photomicrograph of a cell of strain MRM 3/1
T
. Bar, 10 mm.
M. Modesto and others
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forming a ‘Y’ shape at both ends. They are Gram-posi-
tive-staining, non-motile, asporogenous, non-haemolytic,
F6PPK-positive, catalase- and oxidase-negative, indole-
negative and microaerophilic. There is no difference in
growth under either anaerobic or microaerophilic condi-
tions. Negative for arginine dihydrolase, lysine decarbox-
ylase, ornithine decarboxylase, citrate utilization and
H
2
S production. Does not reduce nitrate or nitrite.
Well-isolated colonies on the surface of TPY agar under
anaerobic conditions are white, opaque, smooth and
circular with entire edges, while imbedded colonies are
lens-shaped or elliptical. Colonies reach 1.7–2.5 mm in
diameter after 3 days of incubation. The temperature range
for growth is 25–42 uC; no growth occurs at 20 or 47 uC.
The optimum temperature for growth is 35–37 uC. Grows
at pH 4.5–7.0 with an optimum at pH 6.5–7.0. Can grow
in milk, under aerobic, microaerophilic and anaerobic
conditions. Acid is produced from D-glucose, lactose,
maltose, salicin, D-xylose, L-arabinose, melezitose, D-sorbi-
tol, D-ribose, D-galactose, gentiobiose, D-turanose, arbutin,
melibiose and potassium gluconate. Acid production from
D-mannitol, sucrose, glycerol, cellobiose, D-mannose, raffi-
nose, L-rhamnose, trehalose, D-fructose, starch, inulin and
glycogen is strain dependent. Acid is not produced from
xylitol, amygdalin, methyl a-D-glucopyranoside, N-acetyl-
glucosamine or potassium gluconate. Lactic and acetic acids
are produced as end products of glucose fermentation in
a variable ratio ranging from 1 : 2 to 1 : 1.5. Aesculin is
hydrolysed and urease is produced. The peptidoglycan type
is A4aL-Lys–D-Ser–D-Asp. Phylogenetic analysis of the 16S
rRNA gene sequence places the species in the B. scardovii
subgroup of the genus Bifidobacterium.
Table 1. Differential characteristics between the novel bifidobacteria and their closest phylogenetic relatives
Strains: 1, MRM 3/1
T
; 2, MRM 4/2; 3, MRM 8/7; 4, MRM 5/13; 5, MRM 4/6; 6, MRM 4/7; 7, B. stellenboschense DSM 23968
T
;8,B. biavatii DSM
23967
T
;9,B. scardovii DSM 13734
T
; 10, B. bifidum DSM 20456
T
. All data were obtained in this study unless indicated. +, Positive, w, weakly
positive; 2, negative; ND, not determined.
Characteristic 1 2 3 4 5 6 7 8 9 10
Utilization of:
D-Mannitol +22W2+WW22
Sucrose +2++2++ + ++
Maltose +++
W++ + + + 2
Salicin +++2++ + + 22
D-Xylose + +++++ + + 22
L-Arabinose + +++++ W+22
Glycerol +2222++ W2+
Cellobiose +2222+2+22
D-Mannose +2222+2W22
Melezitose +WWWW+W+22
Raffinose +W++2++ + +2
D-Sorbitol +W++++ WW22
L-Rhamnose +2222+2W22
Trehalose +2222+2+22
D-Ribose W++ W++ + + 22
D-Galactose W+++++ + + 2+
D-Fructose 2+++++ + + + 2
Starch +222W22 2 +2
Gentiobiose +W++++ W++2
D-Turanose W+++++ + + W2
Arbutin + +++++ + + 22
Melibiose WW+++ W++ W2
Inulin W2WWWW 2222
Potassium gluconate WW+2W+WW22
Glycogen +22222 2 W+2
Xylitol W22222 2 +22
Amygdalin 2 22222 ++ 22
Methyl a-D-glucopyranoside 2 22222 +222
N-Acetylglucosamine 2 22222 +222
DNA G+C content (mol%) 64.7 ND ND ND ND ND 66.3* 60.1* 63.1* 58*
Urease activity + +++++ 2222
*Data from Endo et al., 2012.
Bifidobacterium aesculapii sp. nov.
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The type strain, MRM 3/1
T
(5JCM 18761
T
5DSM 26737
T
),
and the reference strain MRM 4/2 (5JCM 187625DSM
26738) were isolated from fresh faecal samples of infant
common marmosets (Callithrix jacchus) that were indi-
vidually collected from animals kept in animal houses in
Aptuit s.r.l. Verona, northern Italy, in 2012. The DNA G+C
content of the type strain is 64.7 mol%.
Acknowledgements
We thank Dr Pisi Annamaria and Dr Filippini Gianfranco from
Department of Agricultural Sciences, University of Bologna, for their
technical assistance with scanning electron microscope observation.
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