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Lactobacillus, KBL13T, were, from, salivarius, species, using, strain, subsp., rRNA, isolated, gene, phylogenetic, that, aviarius, hayakitensis, with, Lys–Asp, sequence, content, Syst, which, type, Japan, DNA–DNA, sequences, strains, Microbiol, al.,, GBL13

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Lactobacillus hayakitensis sp. nov., isolated from
intestines of healthy thoroughbreds
Hidetoshi Morita,1 Chiharu Shiratori,1 Masaru Murakami,1 Hideto Takami,2
Yukio Kato,1 Akihito Endo,3 Fumihiko Nakajima,4 Misako Takagi,5
Hiroaki Akita,4 Sanae Okada3 and Toshio Masaoka1
Correspondence
Hidetoshi Morita
morita@azabu-u.ac.jp
1School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa
229-8501, Japan
2Microbial Genome Research Group, Japan Agency of Marine-Earth Science and Technology,
2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan
3Nodai Culture Collection Center, Department of Brewing, Tokyo University of Agriculture,
1-1-1 Sakuragaoka, Setagaya, Tokyo 156-8502, Japan
4Northern Farm, 275 Hayakita-genbu, Abira-cho, Yufutsu-gun, Hokkaido 059-1432, Japan
5Clossfield-Bio Inc., 1-1-20, Higashi Nihonbashi, Chuo, Tokyo 103-0004, Japan
Two strains, KBL13T and GBL13, were isolated as one of intestinal lactobacilli from the faecal
specimens from different thoroughbreds of the same farm where they were born in Hokkaido,
Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming
and non-motile rods. KBL13T and GBL13 homofermentatively metabolize glucose, and produce
lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA–DNA
hybridization, DNA G+C content and biochemical characterization indicated that these two
strains, KBL13T and GBL13, belong to the same species. In the representative strain, KBL13T,
the DNA G+C content was 34.3 mol%. Lactobacillus salivarius JCM 1231T (5ATCC 11741T;
AF089108) is the type strain most closely related to the strain KBL13T as shown in the
phylogenetic tree, and the 16S rRNA gene sequence identity showed 96.0 % (1425/1484 bp).
Comparative 16S rRNA gene sequence analysis of this strain indicated that the two isolated
strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest
relatives, L. salivarius, Lactobacillus aviarius, Lactobacillus saerimneri and Lactobacillus
acidipiscis. DNA–DNA reassociation experiments with L. salivarius and L. aviarius confirmed that
KBL13T represents a novel species, for which the name Lactobacillus hayakitensis sp. nov. is
proposed. The type strain is KBL13T (5JCM 14209T5DSM 18933T).
Lactobacilli are important members of healthy gastrointestinal tracts of mammals and humans, and some of
them are frequently administered as probiotics for their
beneficial roles in mammalian and human health.
However, there have only been a few studies on the
Lactobacillus flora of thoroughbred gastrointestinal contents by using the culturing method (Mitsuoka &
Kaneuchi, 1977; Morotomi et al., 2002). In our study, we
isolated the following lactobacilli present in the intestinal
tract of healthy thoroughbreds: Lactobacillus gasseri,
Lactobacillus johnsonii, Lactobacillus ruminis, Lactobacillus
reuteri, Lactobacillus salivarius, Lactobacillus crispatus and
Lactobacillus agilis. These species are well-known
species isolated from mammalian gastrointestinal tracts.
Lactobacillus equi was also found during our study, which
is a dominant and indigenous species in equine gastrointestinal tracts (Morotomi et al., 2002). As part of a study
on the intestinal microbiota in thoroughbreds, two strains,
KBL13T and GBL13, were isolated from different thoroughbreds. A polyphasic taxonomic study of these strains was
performed using phenotypic characterization and phylogenetic as well as genetic methods; the results obtained by
using these methods consistently revealed the isolates,
KBL13T and GBL13, to represent a novel Lactobacillus
species, from intestines of thoroughbreds, for which the
name Lactobacillus hayakitensis sp. nov. is proposed.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain KBL13T is AB267406.
Trees showing the phylogenetic relationship, based on the 16S rRNA
gene sequence, of the isolate in comparison to species of the
Lactobacillus salivarius phylogenetic group constructed by the maximum-parsimony and maximum-likelihood methods are available with the
online version of this paper.
International Journal of Systematic and Evolutionary Microbiology (2007), 57, 2836–2839 DOI 10.1099/ijs.0.65135-0
2836 65135 G 2007 Crown Copyright Printed in Great Britain
Bacterial strains, KBL13T and GBL13, were isolated from
fresh faeces of different healthy thoroughbreds of the same
farm where they were born in Hokkaido, Japan. The fresh
faeces of each thoroughbred were transferred under
anaerobic conditions by AnaeroPack (Mitsubishi Gas
Chemical) at 4 uC to our laboratory within 24 h. The
initial processing and subsequent weighing and dilution of
the specimens were carried out under anaerobic conditions. Each dilution was then spread on to BL agar plates
(Eiken Chemical) and incubated anaerobically at 37 uC for
2 days. All further cultivation was performed at 37 uC in
ABCM broth (Eiken Chemical). The 16S rRNA gene
sequences of the isolates were determined as described
previously (Endo & Okada, 2005). The 1484 bp of the 16S
rRNA gene sequence of KBL13T was consistent with those
of GBL13. DNA–DNA hybridization was carried out by
using the microdilution-well technique, with photobiotin
for labelling of the DNA (Ezaki et al., 1989). KBL13T and
GBL13 shared high levels of DNA–DNA relatedness (99.5–
100.0 %). The closest known relatives of the isolates were
determined by performing database searches, and the
sequences of closely related species were retrieved from the
DDBJ database. Multiple alignments of the sequences were
carried out with the CLUSTAL_X program, version 1.18
(Thompson et al., 1997). Distance matrices for the aligned
sequences were calculated by using the two-parameter
method of Kimura (1980). The neighbour-joining method
was used to construct a phylogenetic tree (Saitou & Nei,
1987). The robustness of individual branches was estimated
by using bootstrapping with 1000 replicates (Felsenstein,
1985). Phylogenetic trees were also constructed by using
the maximum-likelihood (Cavalli-Sforza & Edwards, 1967)
and maximum-parsimony (Kluge & Farris, 1969) methods
with PHYLIP version 3.65 (Felsenstein, 2005).
In a neighbour-joining dendrogram created based on the
sequence of KBL13T and sequences from the GenBank
database, the phylogenetic position of KBL13T was
determined. KBL13T was placed within the L. salivarius
phylogenetic group (Canchaya et al., 2006) and was most
closely related to L. salivarius, Lactobacillus aviarius,
Lactobacillus saerimneri and Lactobacillus acidipiscis as
shown in Fig. 1. Recently, on the basis of a polyphasic
analysis, Li et al. (2006) indicated that L. salivarius subsp.
salivarius and L. salivarius subsp. salicinicus did not merit
separate subspecies status. As the information of the
physiological characteristics of L. salivarius JCM 1150 is
available to us (previously described as L. salivarius subsp.
salicinicus JCM 1150), the physiological characteristics of
KBL13T and GBL13 were compared with those of L.
salivarius JCM 1231T (5ATCC 11741T; AF089108) and
JCM 1150 as shown in Table 1. L. salivarius JCM 1231T and
JCM 1150, L. aviarius subsp. aviarius JCM 5666T and L.
aviarius subsp. araffinosus JCM 5667T used in the study
were obtained from the Japan Collection of
Microorganisms. A high similarity of 96.0 % (1425/
1484 bp) was observed in the 16S rRNA gene sequences
of KBL13T and L. salivarius JCM 1231T. Identical tree
topologies were obtained by using the maximum-likelihood and maximum-parsimony methods (see
Supplementary Figs S1 and S2 available in IJSEM Online).
The DNA G+C content was determined by hydrolysing the
DNA enzymically and quantifying the nucleosides by HPLC
according to the method of Ezaki et al. (1990). The DNA
G+C content of KBL13T and GBL13 were 34.3 and
34.8 mol%, respectively. The DNA G+C content of their
closest relatives, L. salivarius JCM 1231T and L. aviarius
subsp. araffinosus JCM 5667T was 34.7 and 41.3 mol%,
respectively. The DNA G+C content of KBL13T was found
to be within the range of 32.0–55.0 mol%, which is the range
reported for Lactobacillus species (Kandler & Weiss, 1986).
The sugar fermentation patterns were determined using
the API 50CH system (bioMérieux) according to the
Fig. 1. Phylogenetic relationship of the isolate
to the species of the L. salivarius phylogenetic
group based on the 16S rRNA gene
sequences is shown. The tree was constructed by the neighbour-joining method. L.
delbrueckii ATCC 9649T was used as an
outgroup. Bootstrap percentages above
70.0 % are given at the branching points.
Lactobacillus hayakitensis sp. nov.
http://ijs.sgmjournals.org 2837
manufacturer’s instructions. The results were recorded
after 48 h at 37 uC. The isomer of lactic acid produced
from glucose was determined by using an F-kit (D-lactic
acid/L-lactic acid; Roche Diagnostics Corporation). Other
biochemical tests, such as those on motility, growth at a
fixed temperature and gas production from glucose, were
performed by using the methods described by Mitsuoka
(1969). Table 1 shows the characteristics most useful in
distinguishing the strains studied from closely related
lactobacilli. Since KBL13T and GBL13 were found to be the
same species, KBL13T was used as a representative strain in
the experiments described below.
DNA–DNA hybridization analyses (Ezaki et al., 1989) were
performed, including those for the two most closely related
species, L. salivarius JCM 1231T and JCM 1150, and L.
aviarius subsp. araffinosus JCM 5667T, based on the 16S
rRNA gene sequence analysis. DNA–DNA relatedness
values between KBL13T and L. salivarius JCM 1231T and
JCM 1150, and L. aviarius subsp. araffinosus JCM 5667T
were 14.2, 12.1 and 7.9 %, respectively. These values are
well below the threshold of 70.0 % that is suggested for
species delineation (Stackebrandt & Goebel, 1994), indicating that strain KBL13T represents a separate genomic
species. Analysis by high-performance thin-layer chromatography showed that meso-diaminopimelic acid was
not contained in the peptidoglycan of the strain KBL13T,
and an analysis, by ultraperformance liquid chromatography according to the methods described by Komagata &
Suzuki (1987), of the cell wall composition revealed the
Lys–Asp peptydoglycan type in the presence of Lys, Glu,
Ala and Asp.
DNA–DNA relatedness showed a clear separation of strain
KBL13T from its phylogenetic relatives, it is considered that
the strain studied represents a novel species belonging to
the genus Lactobacillus, for which the name Lactobacillus
hayakitensis sp. nov. is proposed.
Description of Lactobacillus hayakitensis sp. nov.
Lactobacillus hayakitensis (ha.ya.ki.ten9sis. N.L. masc. adj.
hayakitensis of Hayakita, which is the name of the area
where the bacterium was originally isolated).
Cells are Gram-positive, 3.0–5.0 mm long and 1.0–1.5 mm
wide, non-motile and non-spore-forming rods. They occur
singly or in pairs. Colonies are small (1.5 mm), circular to
slightly irregular, convex, with a smooth to rough surface,
and white when grown on MRS agar. The optimum growth
temperature is 37 uC. Strain KBL13T is not able to grow in
4.5 % NaCl and at 15 uC, but grows in 3.0 % NaCl and at
45 uC. Cells are catalase-negative. Glucose is metabolized
homofermentatively and lactate is the sole final product.
Strain KBL13T produces L(+)-lactic acid. Acid is produced
from glucose, fructose, mannose, mannitol, N-acetyl-Dglucosamine, arbutin, aesculin, salicin, cellobiose, maltose,
sucrose and gentiobiose. Amygdalin and raffinose are
weakly fermented. In this species, some strains cannot
ferment N-acetyl-D-glucosamine, arbutin and raffinose.
The DNA G+C content of the type strain is 34.3 %, and
Table 1. Physiological characteristics of strains KBL13T and GBL13 and type strains of the closely related Lactobacillus species
Strains: 1, KBL13T; 2, GBL13; 3, L. salivarius JCM 1231T; 4, L. salivarius JCM 1150; 5, L. aviarius subsp. aviarius JCM 5666T (Fujisawa et al. 1984); 6,
L. aviarius subsp. araffinosus JCM 5667T (Fujisawa et al. 1984). +, Positive; –, negative; W, weakly positive; ND, no data available. All strains were
positive for the following characteristics: fermentation of glucose, fructose, mannose, maltose, sucrose; growth in MRS broth at 37 uC and no
growth in MRS broth at 15 uC. The DNA G+C contents were determined by HPLC.
Characteristic 1 2 3 4 5 6
Lactic acid isomers L L Mainly L Mainly L DL DL
Fermentation of:
Galactose – – + + + –
Rhamnose – – + – ND ND
Sorbitol – – + + ND ND
N-Acetyl-D-glucosamine + – + + ND ND
Amygdalin W W – – – –
Arbutin + – – + ND ND
Aesculin + + – + + –
Salicin + + – + + –
Cellobiose + + – – + –
Lactose – – + + – –
Melibiose – – + + + –
Trehalose – – + + + +
Raffinose W – + + + –
Gentiobiose + + – – ND ND
Growth on MRS medium at 45 uC + + + + ND ND
DNA G+C content (mol%) 34.3 34.8 34.7 ND 38.7 41.3
Peptidoglycan type Lys–Asp Lys–Asp Lys–Asp Lys–Asp Lys–Asp Lys–Asp
H. Morita and others
2838 International Journal of Systematic and Evolutionary Microbiology 57
the cell wall composition of the strain exhibits the Lys–Asp
peptydoglycan type.
The type strain, KBL13T (5JCM 14209T5DSM 18933T),
was isolated from the faeces of a thoroughbred.
Acknowledgements
We would like to thank Ms Sayuri Nagata of the Laboratory of Food
Science, Azabu University, for her technical assistance. We also wish to
thank the Private University Scientific Foundation for financial support.
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Lactobacillus hayakitensis sp. nov.
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