Bifidobacterium kashiwanohense sp. nov., isolated
from healthy infant faeces
Hidetoshi Morita,1Akiyo Nakano,1Hiromi Onoda,1Hidehiro Toh,2
Kenshiro Oshima,3Hideto Takami,4Masaru Murakami,1Shinji Fukuda,5, 6
Tatsuya Takizawa,1Tomomi Kuwahara,7Hiroshi Ohno,5, 6Soichi Tanabe8
and Masahira Hattori3
1School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara,
Kanagawa 229-8501, Japan
2Advanced Science Institute, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045,
3Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa,
Chiba 277-8562, Japan
4Microbial Genome Research Group, Japan Agency of Marine-Earth Science and Technology,
2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan
5Laboratory for Epithelial Immunobiology, RIKEN Research Center for Allergy and Immunology,
1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan
6Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro, Tsurumi,
Yokohama, Kanagawa 230-0045, Japan
7Department of Molecular Bacteriology, Institute of Health Biosciences, University of Tokushima,
Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan
8Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama,
Higashi-Hiroshima, Hiroshima 739-8528, Japan
Strains HM2-1 and HM2-2Twere isolated from the faeces of a healthy infant and were
characterized by determining their phenotypic and biochemical features and phylogenetic
positions based on partial 16S rRNA gene sequence analysis. They were Gram-positive,
obligately anaerobic, non-spore-forming, non-gas-producing, and catalase-negative non-motile
rods. They did not grow at 15 or 45 6C in anaerobic bacterial culture medium, and their DNA
G+C content was in the range 56–59 mol%. In enzyme activity tests, strains HM2-1 and HM2-2T
were positive for a/b-galactosidases and a/b-glucosidases but negative for b-glucuronidase and
cystinearylamidase.Ananalysisof thecell-wallcompositionof strainsHM2-1andHM2-2Trevealed
the presence of glutamic acid, alanine and lysine. The presence of fructose-6-phosphate
phosphoketolase shows that isolates HM2-1 and HM2-2Tare members of the genus
Bifidobacterium. These two isolates belong to the same species of the genus Bifidobacterium.
Strain HM2-2Twas found to be related to Bifidobacterium catenulatum JCM 1194T(97.4% 16S
rRNA gene sequence identity: 1480/1520 bp), Bifidobacterium pseudocatenulatum JCM 1200T
(97.2%: 1472/1514 bp), Bifidobacterium dentium ATCC 27534T(96.7%: 1459/1509 bp) and
Bifidobacterium angulatum ATCC 27535T(96.5%: 1462/1515 bp). The predominant cellular fatty
acids of strains HM2-1 and HM2-2Twere 16:0 and 18:1v9c, with proportions greater than 18%
of the total. Phylogenetic analyses involving phenotypic characterization, DNA–DNA hybridization
and partial 16S rRNA gene sequencing proves that the strains represent a novel species of the
genus Bifidobacterium, for which the name Bifidobacterium kashiwanohense sp. nov. is proposed.
The type strain is HM2-2T(5JCM 15439T5DSM 21854T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and partial hsp60 gene sequences of strain HM2-1 are AB491757 and AB578933,
respectively. Those for strain HM2-2Tare AB425276.2 and AB491759.2, respectively.
Four supplementary figures are available with the online version of this paper.
International Journal of Systematic and Evolutionary Microbiology (2011), 61, 2610–2615
2610024521G2011 IUMSPrinted in Great Britain
The genus Bifidobacterium represents one of the bacterial
groups within the class Actinobacteria and is prevalent
within the gastrointestinal tracts of humans and animals
(Mikkelsen et al., 2003). At the time of writing, the genus
Bifidobacterium is represented by over 30 species and
subspecies, and the type strains of Bifidobacterium adoles-
centis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B.
dentium,B. longumsubsp.infantis,B. longumsubsp.longum,
B. pseudocatenulatum and B. scardovii were isolated from
human specimens (Scardovi, 1986; Hoyles et al., 2002;
Sakata et al., 2002). The bifidobacteria have not been found
to cause human diseases; instead, they are considered to be
probiotic micro-organisms (Fuller, 1991; Ventura et al.,
2009; Whorwell et al., 2006; Brenner et al., 2009).
During our studies, two isolates, HM2-1 and HM2-2T,
from faeces of a healthy infant were not identified to the
species level. Our results show that these isolates represent
a novel Bifidobacterium species, for which the name
Bifidobacterium kashiwanohense sp. nov. is proposed.
We isolated 51 strains of putative bifidobacteria inhabiting
the faeces of a healthy infant (male; 1.5 years old) in 2008.
Blood liver (BL) agar plates (Eiken Chemical) and anaerobic
bacterial culture medium (ABCM broth; Eiken Chemical)
were used for cell cultures. All 51 isolates were anaerobically
cultured at 37 uC in ABCM broth and on BL agar plates for
24 and 72 h, respectively. Of the 51 isolates, Bifidobacterium
longum subsp. longum (20 strains), B. longum subsp. infantis
(2 strains), B. bifidum (6 strains) and B. breve (3 strains)
were identified, whereas the other isolates (20 strains) were
not identified to the species level. Of the 20 other isolates,
the unidentified strains HM2-1 and HM2-2Tformed a
subcluster in the genus Bifidobacterium in the taxonomic
study. Strains HM2-1 and HM2-2Twere found to be Gram-
positive, obligately anaerobic, non-spore-forming, non-gas-
producing and catalase-negative non-motile rods. Table 1
shows the major characteristics of isolates HM2-1 and
HM2-2T, and the related strains B. catenulatum JCM 1194T,
B. pseudocatenulatum JCM 1200T, B. dentium JCM 1195T
and B. angulatum JCM 7096T. The results were recorded
after 48 h at 37 uC. The isomer of lactic acid produced from
D-glucose was determined by using an F-kit [D(2)-lactic
acid/L(+)-lactic acid; Roche]. Other biochemical tests on
motility, growth at a fixed temperature and gas produc-
tion from D-glucose were performed using the methods
described by Mitsuoka (1969). The optimal temperature for
growth of strains HM2-1, HM2-2T, JCM 7096Tand JCM
1194Twas 37 uC; no growth was observed at 15 or 45 uC in
ABCM broth. Although all strains grew at pH 7.2 and
pH 8.0, no growth was observed at pH 4.5. B. pseudocate-
nulatum JCM 1200Tgrew weakly at 45 uC and pH 4.5. B.
dentium JCM 1195Tgrew at pH 4.5. Acid production from
carbohydrates in ABCM broth is shown in Table 1. For the
determination of phenotypic characteristics, API ZYM
tests (bioMe ´rieux) were performed in duplicate using the
methods recommended by the manufacturer. Cells were
collected from each ABCM culture by centrifugation at
12000 r.p.m. for 5 min, inoculated onto the test strips and
incubated for 4 h at 37 uC. Nineteen enzymic activities were
estimated by the API ZYM tests, and strains HM2-1 and
HM2-2Tshowed 7 enzymic activities (Table 1). Enzyme
activity tests were positive for a/b-galactosidases and a/b-
glucosidases but negative for b-glucuronidase and cystine
arylamidase. Isolates HM2-1 and HM2-2Thad fructose-6-
phosphate phosphoketolase activity like the reference strains
(Biavati & Mattarelli, 1991; Gavini et al., 1991). An analysis
of the cell-wall composition revealed the presence of
glutamic acid, alanine and lysine by ultraperformance liquid
chromatography according to the methods described by
Komagata & Suzuki (1987). The ACQUITY UPLC System
(Waters) wasused in thisstudy.Whole-cellfatty acid methyl
Table 1. Physiological characteristics of isolates HM2-1 and
HM2-2Tand the type strains of closely related species of the
Strains: 1, HM2-1; 2, HM2-2T; 3, B. catenulatum JCM 1194T; 4, B.
pseudocatenulatum JCM 1200T; 5, B. dentium JCM 1195T; 6, B.
angulatum JCM 7096T. +, Positive; –, negative; W, weakly positive.
All strains were positive for the following characteristics: fermentation
of glucose, fructose, mannose, maltose and sucrose; growth in ABCM
broth at 37uC, at intial pH 7.2 and 8.0, and under anaerobic
conditions; no growth in ABCM broth at 15uC under anaerobic
conditions. All strains produced L-lactic acid and did not produce gas
from D-glucose. All strains produced acid from D-xylose, D-ribose, L-
arabinose, D-glucose, D-galactose, sucrose, maltose, lactose, melibiose,
raffinose and salicin. None of the strains produced acid from glycerol
or rhamnose. All strains possessed leucine arylamidase, a-galactosi-
dase, b-galactosidase, a-glucosidase and b-glucosidase; none pos-
sessed alkaline phosphatase, esterase (C4), lipase (C14), valine
arylamidase, trypsin, a-chymotrypsin, naphthol-AS-BI-phosphohy-
drolase, N-acetyl-b-glucosaminidase, a-mannosidase or a-fucosidase.
DNA G+C contents were determined by HPLC.
Initial pH 4.5
Esterase lipase (C8)
DNA G+C content
Bifidobacterium kashiwanohense sp. nov.
esters were obtained by saponification, methylation and
extraction followed by analysis using a standardized
Microbial Identification System (Microbial ID) (Tan et al.,
2010). The Sherlock standard library MOORE5 5.00 made it
possible to standardize the bacterial fatty acid analysis
(http://www.midi-inc.com) in Table 2.
The DNA G+C contents of strains HM2-1 and HM2-2T
were determined in three separate trials by high-perform-
ance liquid chromatography (Okamoto et al., 2008). The
DNA G+C contents of strains HM2-1 and HM2-2Twere
58.6 and 56.3 mol%, respectively. These differ from the
values obtained for B. catenulatum JCM 1194Tand B.
dentium JCM 1195T, whose DNA G+C contents were 54.7
and 61.2 mol%, respectively (Scardovi & Crociani, 1974).
However, the DNA G+C content of strains HM2-1 and
HM2-2Twere similar to those of B. pseudocatenulatum
JCM 1200T(57.5 mol%) and B. angulatum JCM 7096T
(59.0 mol%) (Scardovi & Crociani, 1974; Scardovi et al.,
The 16S rRNA genes of strains HM2-1 and HM2-2Twere
amplified by PCR using the universal primers 27F and
1492R (Weisburg et al., 1991). The closest known relatives
of the isolate were determined by performing database
searches, and the sequences of closely related species were
retrieved from public databases. Multiple alignments of the
sequences were carried out using CLUSTAL W (Thompson
et al., 1994). We used the neighbour-joining method to
reconstruct a phylogenetic tree (Saitou & Nei, 1987) and
estimated the robustness of the individual branches by
performing bootstrapping analysis with 1000 replicates
(Felsenstein, 1985). Phylogenetic trees were also recon-
structed using the maximum-likelihood (Cavalli-Sforza &
Edwards, 1967) and maximum-parsimony (Kluge & Farris,
1969) methods with PHYLIP version 3.66. The partial 16S
rRNA gene sequence of strain HM2-2Twas consistent with
that of strain HM2-1. DNA–DNA hybridization was carried
out using the microdilution-well technique, with photo-
biotin for DNA labelling (Ezaki et al., 1989). Immobilized
DNA in microdilution wells was incubated with a photo-
biotin-labelled DNA probe prepared from each strain at
48 uC for 3 h in hybridization buffer containing 50%
formamide, after 30 min of incubation at 37 uC in
prehybridization buffer. The fluorescence intensity was
measured with a MicroPlate reader (PerkinElmer) at a
wavelength of 360 nm for excitation and 450 nm for
emission. Values (%) of DNA–DNA hybridization were
the mean of the two trials in this study. Strains HM2-1 and
HM2-2Tshared high levels of DNA–DNA relatedness
polymorphic DNA (RAPD) PCR performed using the
random primers 103 (59-GTGACGCCGC-39), 127 (59-
ATCTGGCAGC-39) and 173
(University of British Columbia) to compare strains HM2-
1 and HM2-2T. Their RAPD profiles (Supplementary Fig. S1
available in IJSEM Online) showed that they were different
strains of the same species, supported by phenotypic and
biochemical features and their phylogenetic position based
on partial 16S rRNA gene sequence and DNA–DNA
Two organisms are considered to represent the same
species if their purified DNA exhibits greater than 70%
hybridization (Wayne et al., 1987). DNA–DNA hybridiza-
tion analysis was carried out comparing isolates HM2-1
and HM2-2Tand other related strains listed in Table 1
according to the method previously by Ezaki et al. (1989).
DNA–DNA relatedness values between strain HM2-2T
and B. catenulatum JCM 1194Twere 8.1%±1.6% and
14.6%±2.4% using the probes for HM2-2Tand B.
catenulatum JCM 1194T, respectively. DNA–DNA related-
ness values using the reference strains as DNA probes for
strain HM2-2Twere as follows: HM2-2Tand B. pseudoca-
tenulatum JCM 1200T(6.5%), HM2-2Tand B. dentium
JCM 1195T(5.7%), and HM2-2Tand B. angulatum JCM
We reconstructed a phylogenetic tree based on a total of 44
partial 16S rRNA gene sequences, including those of
members of the genus Bifidobacterium and related genera.
The tree was rooted using Actinomyces bovis (Fig. 1).
Strains HM2-1 and HM2-2Thad 16S rRNA gene sequences
that were similar to those of B. catenulatum, B. angulatum
and B. dentium. Levels of similarity for the partial 16S
rRNA gene sequence of HM2-2Tin relation to B.
catenulatum JCM 1194T, B. pseudocatenulatum JCM
1200T, B. dentium ATCC 27534Tand B. angulatum
Table 2. Cellular fatty acid contents (%) of isolates HM2-1 and
HM2-2Tand the type strains of closely related species of the
pseudocatenulatum; 5, B. dentium; 6, B. angulatum. Data for reference
strains were obtained from the standard Library MOORE5 5.00 of the
Sherlock Microbial Identification System.
1,HM2-1; 2,HM2-2T; 3,B.catenulatum;4,B.
H. Morita and others
2612International Journal of Systematic and Evolutionary Microbiology 61
ATCC 27535Twere 97.4% (1480/1520 bp), 97.2% (1472/
1514 bp), 96.7% (1459/1509 bp) and 96.5% (1462/1515
maximum-parsimony and maximum-likelihood analyses
(Supplementary Figs S2 and S3).
Heat-shock protein (hsp60; also known as groEL, cpn60,
groES or dnaK) genes have also been adopted for taxonomic
studies of bifidobacterial species (Jian et al., 2001; Zhu et al.,
2003; Simpson et al., 2004; Delcenserie et al., 2005; Ventura
et al., 2005). The partial hsp60 gene sequence of strain HM2-
2Twas obtained by using the conditions and primers for
PCR amplification described by Jian et al. (2001) and
Okamoto et al. (2008). We reconstructed a phylogenetic tree
based on a total of 40 sequences of hsp60 genes, including
those of members of the genus Bifidobacterium and related
genera (Supplementary Fig. S4). Levels of similarity for the
partial hsp60 gene sequence of HM2-2Tin relation to B.
catenulatum JCM 1194T, B. pseudocatenulatum JCM 1200T,
B. dentium ATCC 27534Tand B. angulatum ATCC 27535T
were 96.1% (567/590 bp), 90.8% (536/590 bp), 92.0%
(544/591 bp) and 89.8% (531/591 bp), respectively. The
16S rRNA and hsp60 gene sequences were well related and
useful for determining the phylogeny of strain HM2-2T.
Detailed characteristics of the novel isolates are provided in
the species description and in Table 1, and these character-
istics were compared with those of the phylogenetic relatives
Fig. 1. Phylogenetic relationship between isolates HM2-1 and HM2-2T, species of the genus Bifidobacterium and related
genera, determined by 16S rRNA gene sequencing. The tree was reconstructed using the neighbour-joining method.
Actinomyces bovis NCTC 11535Twas used as an outgroup. Bootstrap percentages (based on 1000 replicates) are shown at
nodes; values lower than 50% are not indicated. Accession numbers are given in parentheses. Bar, 10% difference in
Bifidobacterium kashiwanohense sp. nov.
B. catenulatum JCM 1194T, B. pseudocatenulatum JCM
1200T, B. angulatum JCM 7096Tand B. dentium JCM 1195T.
The isolates were able to produce L(+)-lactic acid from D-
glucose. Anaerobic conditions were essential for the growth
of strains HM2-1 and HM2-2T. These two isolates could be
distinguished from B. pseudocatenulatum JCM 1200T, B.
angulatum JCM 7096Tand B. dentium JCM 1195Tby the
pattern of acid production from D-mannose, cellobiose,
trehalose, melezitose, dextrin, starch, inulin, D-mannitol, D-
sorbitol and D-fructose. The cellular fatty acid profiles of
strains HM2-1, HM2-2Tand the related genera B. catenu-
latum, B. pseudocatenulatum, B. dentium and B. angulatum
ofstrainsHM2-1 and HM2-2Twere16:0 and 18:1v9c, with
proportions greater than 18% of the total.
Experimental data suggest that isolates HM2-1 and HM2-
2Tcan be assigned to the genus Bifidobacterium on the basis
of their phylogenetic position; it can be concluded that these
two isolates might be considered as representing a single
novel species. Thus, these two isolates represent a novel
species, for which we propose the name Bifidobacterium
kashiwanohense sp. nov.
Description of Bifidobacterium kashiwanohense
Bifidobacterium kashiwanohense (ka.shi.wa.no.hen9se. N.L.
neut. adj. kashiwanohense of Kashiwanoha in Japan, which
is the name of the area, University of Tokyo, where this
bacterium was originally isolated).
Cells are Gram-positive, non-motile, non-spore-forming
rods measuring 1.061.3 mm. Colonies on BL agar after
incubation under anaerobic conditions for 2 days at 37 uC
are beige, smooth and approximately 1.0 mm in diameter.
No growth is observed at 15 or 45 uC. Produces L(+)-lactic
acid from D-glucose. Produces acid from D-xylose, D-ribose,
D-mannose, cellobiose, D-sorbitol, L-arabinose, D-glucose,
D-fructose, D-galactose, sucrose, maltose, lactose, melibiose,
raffinose and salicin, and, to a weaker extent, from dextrin.
Exhibits esterase lipase (C8), leucine arylamidase, acid
phosphatase, a-galactosidase, b-galactosidase, a-glucosidase,
b-glucosidase, and fructose-6-phosphate phosphoketolase
activities. The cell wall contains glutamic acid, alanine and
lysine. The DNA G+C content is 56–59 mol%.
The type strain is HM2-2T(5JCM 15439T5DSM 21854T),
isolated from faeces of a healthy infant (1.5 years old).
We wish to thank the Matching Fund Subsidy for financial support.
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Bifidobacterium kashiwanohense sp. nov.