International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3288–3292
DOI 10.1099/ijs.0.063263-0
Streptococcus loxodontisalivarius sp. nov. and
Streptococcus saliviloxodontae sp. nov., isolated
from oral cavities of elephants
Masanori Saito, Noriko Shinozaki-Kuwahara, Masatomo Hirasawa
and Kazuko Takada
Correspondence
Masanori Saito
Department of Oral Microbiology, Nihon University School of Dentistry at Matsudo,
Chiba 271-8587, Japan
[email protected]
Four Gram-stain-positive, catalase-negative, coccoid-shaped organisms were isolated from
elephant oral cavities. The isolates were tentatively identified as streptococcal species based on
the results of biochemical tests. Comparative 16S rRNA gene sequencing studies confirmed the
organisms to be members of the genus Streptococcus. Two isolates (NUM 6304T and NUM
6312) were related most closely to Streptococcus salivarius with 96.8 % and 93.1 % similarity
based on the 16S rRNA gene and the RNA polymerase b subunit encoding gene (rpoB),
respectively, and to Streptococcus vestibularis with 83.7 % similarity based on the 60 kDa heatshock protein gene (groEL). The other two isolates (NUM 6306T and NUM 6318) were related
most closely to S. vestibularis with 97.0 % and 82.9 % similarity based on the 16S rRNA and
groEL genes, respectively, and to S. salivarius with 93.5 % similarity based on the rpoB gene.
Based on phylogenetic and phenotypic evidence, these isolates are suggested to represent novel
species of the genus Streptococcus, for which the names Streptococcus loxodontisalivarius sp.
nov. (type strain NUM 6304T5JCM 19287T5DSM 27382T) and Streptococcus saliviloxodontae
sp. nov. (type strain NUM 6306T5JCM 19288T5DSM 27513T) are proposed.
Oral streptococci (anginosus, mitis, mutans and salivarius
groups) make up a proportion of the normal flora of the oral
cavity in humans and animals (Facklam, 2002). Previously,
we reported novel species of the mutans group from the oral
cavity of various animals (Takada & Hirasawa, 2007, 2008;
Shinozaki-Kuwahara et al., 2011; Takada et al., 2010, 2013).
The mutans streptococci are known as a primary pathogen
of dental caries in humans and animals. One of the virulence
properties is the ability to produce exopolysaccharides
from sucrose (Hamada & Slade, 1980). These bacteria are
known to form characteristic colonies on Mitis Salivarius
(MS) agar demonstrating extracellular polysaccharide synthesis. During the isolation of Streptococcus mutans-like
colonies on MS agar from oral cavities of elephants, we
found four S. mutans-like isolates. After investigations,
the properties of the isolates were similar to the salivarius
group. The salivarius group includes Streptococcus salivarius,
Streptococcus vestibularis, and Streptococcus thermophilus. S.
Abbreviation: NJ, neighbour-joining.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA
gene, rpoB and groEL sequences of Streptococcus loxodontisalivarius
NUM 6304T are AB828326, AB906700 and AB906699, respectively,
and those for Streptococcus saliviloxodontae NUM 6306T are AB828327,
AB906702 and AB906701, respectively.
Four supplementary figures and a supplementary table are available with
the online version of this paper.
3288
salivarius and S. vestibularis are members of human oral
microflora (Whiley & Hardie, 1988), and S. thermophilus is
found in fermented milk products (Settachaimongkon et al.,
2014). S. salivarius and S. thermophilus produce distinctive
mucoid colonies on sucrose agar due to the production
of extracellular polysaccharides but S. vestibularis does
not. Here we describe two novel species belonging to the
salivarius group, isolated from oral cavities of elephants.
MS agar is widely used to isolate oral streptococcal species.
The oral microflora of elephants was examined by isolation
on MS agar, which contains 5 % sucrose. Streptococcal
species differentially synthesize extracellular glucan and
fructan from sucrose and form characteristic colonies on
MS agar, which facilitates their identification. The four S.
mutans-like strains (NUM 6304T, NUM 6306T, NUM 6312
and NUM 6318) obtained from samples from the oral
cavities of six elephants in a zoo (Ichihara Elephant
Kingdom, Chiba, Japan) were isolated on MS agar. These
strains formed small, raised, adherent colonies with an
irregular margin on MS agar, and were observed to grow
in extremely long chains in Brain-Heart Infusion (BHI;
BD Difco) broth at 37 uC. Growth did not occur at 45 uC
in BHI broth. The strains were grown on BHI agar supplemented with 5 % horse blood at 37 uC in a candle extinction
jar with non-haemolytic activity. Catalase activity was found
to be negative. The Lancefield grouping test was performed
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Novel streptococci from elephants
using the Streptococcal grouping kit (Oxoid). No
Lancefield carbohydrate antigens were detected from strains
NUM 6304T, NUM 6306T, NUM 6312 and NUM 6318.
Biochemical analysis was conducted using the Rapid ID
32 Strep, API 50 CH, and API ZYM systems (bioMérieux)
according to the manufacturer’s instructions. The phenotypic characteristics that differentiate the proposed novel
species from closely related species based on 16S rRNA gene
sequence similarities and type strains of species of the genus
Streptococcus are shown in Table 1. Strain NUM 6304T was
distinguished from NUM 6306T by its ability to ferment
D-mannose, raffinose and methyl b-D-glucopyranoside and
to hydrolyse urea. Strain NUM 6304T was distinguished
from S. salivarius JCM 5707T by its ability to ferment
lactose, D-mannose and raffinose. Strain NUM 6312 had the
same characteristics as strain NUM 6304T. Strain NUM
6306T was distinguished from S. vestibularis DSM 5636T by
its ability to ferment lactose, raffinose and pullulan. Strain
NUM 6318 had the same characteristics as strain NUM
6406T. The biochemical characteristics did not correspond
to any recognized species of the genus Streptococcus. The
novel isolates were subjected to further genetic studies.
DNA was extracted from bacterial cultures using the Promega
Genome kit (Promega) according to the manufacturer’s
instructions. The G+C contents of the DNA preparations
were determined by HPLC (Hirasawa & Takada, 1994)
with determinations carried out in triplicate. The value
obtained for strain NUM 6304T was 38.2±1.12 mol%
G+C (mean±standard deviation; range, 37.0–39.2 mol%);
Table 1. Characteristics differentiating Streptococcus loxodontisalivarius sp. nov. and Streptococcus saliviloxodontae sp.
nov. from the three closest related species according to 16S
rRNA gene sequence similarities
Taxa: 1, strain NUM 6304T (Streptococcus loxodontisalivarius sp.
nov.); 2, NUM 6306T (Streptococcus saliviloxodontae sp. nov.); 3, S.
salivarius JCM 5707T; 4, S. vestibularis DSM 5636T; 5, S. thermophilus
JCM 17834T; 6, S. equinus CIP 102504T. All strains produce acid
from D-glucose, D-fructose and sucrose, but not from cyclodextrin,
D-ribose, D-mannitol, D-sorbitol, melibiose, melezitose, glycogen,
D-tagatose or D-arabitol. All strains were unable to produce arginine
dihydrolase.
Characteristic
Fermentation of:
Lactose
D-Mannose
Raffinose
Pullulan
Maltose
Methyl b-D-glucopyranoside
Hydrolysis of:
Aesculin
Urea
Growth at 45 uC
http://ijs.sgmjournals.org
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the value obtained for strain NUM 6306T was 37.7±
0.81 mol% G+C (range, 37.0–38.6 mol%). The DNA
G+C content values of strains NUM 6312 and NUM
6318 showed similar results to each type strain. To assess the
phylogenetic affinity of each of the novel isolates, their 16S
rRNA genes were amplified by PCR using primer set 27f (59AGAGTTTGATCCTGGCTCAG-39) and 1525r (59-AAAGGAGGTGATCCAGCC-39), and additional primers for
determination of 16S rRNA gene sequences as described
previously (Hiraishi, 1992). Amplicons were directly
sequenced with an ABI PRISM 3130 Genetic Analyzer using
a Big Dye Terminator v1.1 Cycle Sequencing kit (Life
Technologies) and then subjected to comparative analysis.
The closest known relatives of the novel isolates were
identified by performing database searches. Identification
of the closest phylogenetic neighbours and calculation
of pairwise 16S rRNA gene sequences similarities were
performed using the EzTaxon-e server (Kim et al., 2012).
The sequence similarities based on the 16S rRNA gene
of strains NUM 6304T and NUM 6312 were in 100 %
agreement, and those of strains NUM 6306T and NUM
6318 were also in 100 % agreement. The 16S rRNA gene
sequences (1506 bp) of strains NUM 6304T and NUM 6312
showed 96.81 %, 96.75 % and 96.61 % similarity to those
of S. salivarius CIP 102503T, S. vestibularis CIP 103363T and
Streptococcus thermophilus CIP 102303T, respectively. The
16S rRNA gene sequences (1504 bp) of strains NUM 6306T
and NUM 6318 showed 97.01 %, 96.95 % and 96.81 %
similarity to those of S. vestibularis CIP 103363T, S.
salivarius CIP 102503T and Streptococcus equinus CIP
102504T, respectively. Analysis of a partial 16S rRNA gene
sequence of strains NUM 6034T and NUM 6306T showed
98.9 % similarity. Distance matrices were determined
following the assumptions described by Kimura (1980).
Using MEGA 5.2 software, trees were reconstructed by the
neighbour-joining (NJ) (Fig. 1; an extended version of this
tree is available as Fig. S1 in the online Supplementary
Material) and maximum-parsimony (Fig. S2) methods,
depicting the phylogenetic affinity of strains NUM 6304T
and NUM 6306T with members of the genus Streptococcus.
The topologies of the trees were evaluated by performing
bootstrap analysis of the sequence data using CLUSTAL W and
MEGA software (Thompson et al., 1994; Tamura et al., 2011).
Sequence similarity values were calculated manually.
Phylogenetic trees revealed the clear affiliation of strains
NUM 6304T and NUM 6306T to the genus Streptococcus,
and placed two novel species within the salivarius group.
The segments of genomic DNA of strains NUM 6304T and
NUM 6306T encoding the rpoB and groEL genes were
amplified by PCR and sequenced. The rpoB (f, 59-CGGTTATCAAATGGTTCACCTGTACGTC- 39; r, 59-CCAAACGTTGGTGAAGAAGCTCTCA-39) and groEL (f, 59-AAGCCGTCCACTCTTGATCA-39; r, 59-GCACGGAGGACAATGTTACG-39) primers were designed based on alignment
of the nucleotide sequences of each gene from S. salivarius.
The PCR mixture and procedure were as described in the
manual for KOD FX (TOYOBO). The rpoB and groEL
sequences used for phylogenetic studies were obtained from
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3289
M. Saito and others
88 S. salivarius CIP 102503T (AY188352)
100
S. thermophilus CIP 102303T (AY188354)
0.005
S. vestibularis CIP 103363T (GL831116)
S. loxodontisalivarius NUM 6304T (AB828326)
57
100
S. saliviloxodontae NUM 6306T (AB828327)
T
97 S. lutetiensis CIP 108103 (AF429763)
98
S. infantarius subsp. infantarius CIP 103233T (ABJK02000017)
S. equinus CIP 102504T (AJ301607)
99
S. gallolyticus subsp. macedonicus CIP 105683T (Z94012)
81
89
S. gallolyticus subsp. pasteurianus CIP 107122T (DQ232528)
S. gallolyticus subsp. gallolyticus CIP 105428T (X94337)
Fig. 1. Phylogenetic tree reconstructed from 16S rRNA gene sequences of the genus Streptococcus by the NJ method.
Numbers on the tree indicate bootstrap values calculated for 1000 subsets for branch points .50 %. Bar, 0.005 substitutions
per site. Dotted line indicates the proposed salivarius group. An extended version of this tree is available as Fig. S1.
the DDBJ and GenBank databases. The sequence similarities
based on rpoB and groEL genes of strains NUM 6304T and
NUM 6312 were in 100 % agreement, and those of strains
NUM 6306T and NUM 6318 were also in 100 % agreement.
Strains NUM 6304T and NUM 6312 were related most
closely to S. salivarius CIP 102503T (93.11 % similarity) and
S. vestibularis CIP 103363T (83.70 % similarity) based on
rpoB (581 bp) and groEL (600 bp) sequences, respectively.
Strains NUM 6306T and NUM 6318 were related most
closely to S. salivarius CIP 102503T (93.53 % similarity) and
S. vestibularis CIP 103363T (82.93 % similarity) based on
rpoB (526 bp) and groEL (539 bp) sequences, respectively.
Phylogenetic trees reconstructed by the NJ method with
partial sequences of rpoB (Fig. 2; an extended version of this
tree is available as Fig. S3) and groEL (Fig. S4) genes
confirmed the phylogenetic placement of representative
strains within the genus Streptococcus. The trees based on
housekeeping gene sequences, such as rpoB and groEL,
0.01
showed similar phylogenies to those based on 16SrRNA
gene sequences. Arbitrarily primed-PCR (AP-PCR) analyses
for genotyping two isolates of each novel taxon were
performed as described by Tabchoury et al. (2008). The two
strains of each novel taxon differentiated into distinct
genotypes using OPA 03 primer (data not shown).
DNA–DNA hybridization was performed in five independent experiments according to the microtitre plate method
(Ezaki et al., 1989) with minor modifications (Takada &
Hirasawa, 2007). DNA–DNA relatedness was examined
using labelled DNA from strains NUM 6304T and NUM
6306T with unlabelled single-stranded DNA from related
streptococci. DNA–DNA hybridization values of strain
NUM 6304T with NUM 6312, NUM 6306T, NUM 6318,
S. salivarius JCM 5707T, S. vestibularis DSM 5636T and
S. thermophilus JCM 17834T were 91.8 %, 29.4 %, 35.3 %,
29.8 %, 28.7 % and 27.0 %, respectively. Strain NUM 6304T
S. oligofermentans CIP 108229T (DQ232508)
S. tigurinus DSM 24864T (JQ085955)
S. pseudopneumoniae CIP 108659T (EU003819)
S. oralis CIP 102922T (AF535168)
72
S. mitis CIP 103335NT (AF535188)
98
S. pneumoniae CIP 102911T (DQ232477)
S. infantis CIP 105949T (DQ232482)
64
S. peroris CIP 105950T (DQ232483)
S. australis CIP 107167T (DQ132983)
99
S. parasanguinis CIP 104372T (DQ132985)
99 S. saliviloxodontae NUM 6306T (AB906702)
S. loxodontisalivarius NUM 6304T (AB906700)
S. salivarius CIP 102503T (AF535169)
S. thermophilus CIP 102303T (AY567833)
99
80
S. vestibularis CIP 103363T (AY687375)
53
96
Fig. 2. NJ phylogenetic tree based on rpoB gene sequences of strains NUM 6304T and NUM 6306T and related species.
Numbers on the tree indicate bootstrap values calculated for 1000 subsets for branch points .50 %. Bar, 0.01 substitutions
per site. Dotted line indicates the proposed salivarius group. An extended version of this tree is available as Fig. S3.
3290
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International Journal of Systematic and Evolutionary Microbiology 64
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Novel streptococci from elephants
showed a DNA–DNA relatedness value .70 % with strain
NUM 6312 and therefore these results confirmed that these
two isolates belonged to the same species (Wayne et al.,
1987). DNA–DNA hybridization values of strain NUM
6306T with NUM 6304T, NUM 6312, NUM 6318, S.
salivarius JCM 5707T, S. vestibularis DSM 5636T and S.
thermophilus JCM 17834T were 45.2 %, 47.3 %, 84.6 %,
45.2 %, 42.7 % and 50.1 %, respectively (Table S1). Since
strains NUM 6306T and NUM 6318 exhibited high degrees
of relatedness, their relationship at the species level was
confirmed. The DNA from other streptococci showed
low levels of DNA–DNA relatedness to strains NUM
6304T and NUM 6306T (all ,36 % and ,51 %, respectively). According to the criteria used for the delineation of
bacterial species (Wayne et al., 1987), this indicates that
strains NUM 6304T and NUM 6306T represent novel
species of the genus Streptococcus.
The results of 16S rRNA gene and housekeeping gene
sequence similarity calculations and phylogenetic analysis
(Figs 1, 2, S1, S2, S3 and S4) clearly indicated that strains
NUM 6304T, NUM 6312, NUM 6306T and NUM 6318
belong to the genus Streptococcus and the salivarius group.
Strains NUM 6304T, NUM 6312, NUM 6306T and NUM
6318 are distinguishable from recognized species of the
salivarius group based on phenotypic characteristics (Table
1). DNA–DNA relatedness values (Table S1) distinguished
strains NUM 6304T and NUM 6312, and strains NUM
6306T and NUM 6318 from recognized species of the
salivarius group. The isolates warrant classification as two
novel species of the genus Streptococcus, for which the
names Streptococcus loxodontisalivarius sp. nov. (type strain
NUM 6304T)and Streptococcushttp://dx.doi.org/10.1601/
nm.5605saliviloxodontae sp. nov. (type strain NUM 6306T)
are proposed.
Description of Streptococcus loxodontisalivarius
sp. nov.
Streptococcus loxodontisalivarius (lo.xo.don.ti.sa.li.va9ri us.
N.L n. Loxodontus scientific name for the African elephant;
L. masc. adj. salivarius of/from saliva; N.L. masc. adj.
loxodontisalivarius of saliva of an African elephant).
Cells are Gram-stain-positive, non-spore-forming cocci,
1.6–1.8 mm in diameter and occur in pairs or in long chains.
Colonies are white, non-haemolytic and 0.8–1.0 mm in
diameter after incubation on blood agar at 37 uC for 24 h.
MS agar containing sucrose makes characteristic colonies by
production of bacterial exopolysaccharide. On MS agar,
colonies appear small, dark blue and crinkled. Facultatively
anaerobic and catalase-negative. Lancefield carbohydrate
antigens are not detected. Tests for acid production by use
of the API 50 CH system show positive reactions with
D-glucose, D-fructose, sucrose, amygdalin, arbutin, salicin,
cellobiose, maltose, gentiobiose and trehalose, but not with
glycerol, erythritol, D-ribose, D-xylose, L-xylose, D-adonitol,
methyl b-D-xylopyranoside, D-galactose, D-mannose, L-sorbose, L-rhamnose, dulcitol, inositol, D-mannitol, D-sorbitol,
http://ijs.sgmjournals.org
methyl a-D-mannopyranoside, methyl a-D-glucopyranoside, N-acetylglucosamine, lactose, melibiose, inulin,
melezitose, raffinose, starch, glycogen, xylitol, turanose,
D-lyxose, D-tagatose, D-fucose, D-arabitol or gluconate.
Tests for acid production by use of the Rapid ID 32 Strep
system show positive reactions with pullulan and methyl
b-D-glucopyranoside, but not with cyclodextrin. Aesculin is
hydrolysed (API 50 CH), but hippuric acid is not (Rapid
ID 32 Strep). Voges–Proskauer test is positive. Tests for
enzyme activities by use of the API ZYM system show
positive reactions with leucine arylamidase and acid
phosphatase, but not with alkaline phosphatase, esterase
(C4), esterase lipase (C8), lipase (C14), valine arylamidase,
cystine arylamidase, trypsin, a-chymotrypsin, naphthol-ASBI-phosphohydrolase or a-fucosidase. Tests for enzyme
activities by use of the Rapid ID 32 Strep system show
positive reactions with b-glucosidase, b-galactosidase
and alanyl-phenylalanyl-proline arylamidase, but not with
arginine dihydrolase, b-glucuronidase, a-galactosidase,
pyroglutamic acid arylamidase, N-acetyl-b-glucosaminidase, glycyl-tryptophan arylamidase, b-mannosidase or
urease. In BHI broth, there is no apparent growth at 45 uC.
The type strain NUM 6304T (5JCM 19287T5DSM 27382T)
was isolated from the elephant oral cavity. The DNA G+C
content of the type strain is 38.2±1.12 mol%.
Description of Streptococcus saliviloxodontae
sp. nov.
Streptococcus saliviloxodontae [sa.li.vi.lo.xo.don9tae. L. fem.
n. saliva saliva; N.L. fem. gen. n. Loxodontae of Loxodonta
(scientific name of the African elephant); N.L. masc. adj.
saliviloxodontae of the African elephant’s saliva].
Cells are Gram-stain-positive, non-spore-forming cocci,
1.6–1.8 mm in diameter and occur in pairs or in long chains.
Colonies are white, non-haemolytic and 0.8–1.0 mm in
diameter after incubation on blood agar at 37 uC for 24 h.
MS agar containing sucrose makes characteristic colonies by
production of bacterial exopolysaccharide. On MS agar,
colonies appear small, dark blue and crinkled. Facultatively
anaerobic and catalase-negative. Lancefield carbohydrate
antigens are not detected. Tests for acid production by
use of the API 50 CH system show positive reactions
with D-glucose, D-fructose, sucrose, raffinose, D-mannose,
amygdalin, arbutin, salicin, maltose, cellobiose, gentiobiose
and trehalose, but not with glycerol, erythritol, D-ribose,
D-xylose, L-xylose, D-adonitol, methyl b-D-xylopyranoside,
D-galactose, L-sorbose, L-rhamnose, dulcitol, inositol, Dmannitol, D-sorbitol, methyl a-D-mannopyranoside, methyl
a-D-glucopyranoside, N-acetylglucosamine, lactose, melibiose, inulin, melezitose, starch, glycogen, xylitol, turanose,
D-lyxose, D-tagatose, D-fucose, D-arabitol or gluconate.
Tests for acid production by use of the Rapid ID 32 Strep
system show positive reactions with pullulan, but not with
cyclodextrin or methyl b-D-glucopyranoside. Aesculin and
urea are hydrolysed (API 50 CH), but hippuric acid is not
(Rapid ID 32 Strep). Voges–Proskauer test is positive. Tests
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3291
M. Saito and others
for enzyme activities by use of the API ZYM system
show positive reactions with leucine arylamidase and acid
phosphatase, but not with alkaline phosphatase, esterase
(C4), esterase lipase (C8), lipase (C14), valine arylamidase,
cystine arylamidase, trypsin, a-chymotrypsin, naphthol-ASBI-phosphohydrolase or a-fucosidase. Tests for enzyme
activities by use of the Rapid ID 32 Strep system show
positive reactions with b-glucosidase, alanyl-phenylalanylproline arylamidase and urease, but not with arginine
dihydrolase, b-galactosidase, b-glucuronidase, a-galactosidase, pyroglutamic acid arylamidase, N-acetyl-b-glucosaminidase, glycyl-tryptophan arylamidase or b-mannosidase.
In BHI broth, there is no apparent growth at 45 uC.
a prokaryotic 16S rRNA gene sequence database with phylotypes that
represent uncultured species. Int J Syst Evol Microbiol 62, 716–721.
The type strain NUM 6306T (5JCM 19288T5DSM 27513T)
was isolated from the elephant oral cavity. The DNA G+C
content of the type strain is 37.7±0.81 mol%.
Tabchoury, C. P. M., Sousa, M. C. K., Arthur, R. A., Mattos-Graner,
R. O., Del Bel Cury, A. A. & Cury, J. A. (2008). Evaluation of genotypic
Kimura, M. (1980). A simple method for estimating evolutionary
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M. H., Smid, E. J. & van Valenberg, H. J. F. (2014). Influence of
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profile of set-yoghurt. Int J Food Microbiol 177, 29–36.
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Acknowledgements
We are grateful to Dr Hans G. Trüper for suggesting the species name.
This work was supported in part by JSPS KAKENHI grant number
25870768.
Takada, K. & Hirasawa, M. (2008). Streptococcus dentirousetti sp. nov.,
isolated from the oral cavities of bats. Int J Syst Evol Microbiol 58,
160–163.
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