IJSEM Papers in Press. Published November 10, 2014 as doi:10.1099/ijs.0.067116-0
1
Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22, and 26:
2
Streptococcus parasuis sp. nov.
3
4
R. Nomoto1*, F. Maruyama2, S. Ishida3, M. Tohya3, T. Sekizaki3 and Ro Osawa4
5
6
7
8
9
10
11
12
13
14
1
Organization for Advanced Science and Technology, Kobe University, Rokko-dai 1-1,
Nada-ku, Kobe, Hyogo 657-8501, Japan 2
Graduate School of Medical and Dental Sciences, Section of Bacterial Phathogenesis,
Tokyo Medical and Dental University, Yushima 45-5-1, Bunkyo-ku ,Tokyo 113-8510, Japan 3
Research Center for Food Safety, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan 4
Department of Bioresource Sciences, Graduate School of Agricultural Sciences, Kobe
University, Rokko-dai 1-1, Nada-ku, Kobe, Hyogo 657-8501, Japan
15
16
* Corresponding author. Tel and Fax: +81-78-803-5981
17
E-mail address: [email protected]
18
1
19
Running title: Streptococcus parasuis sp. nov.
20
Contents Category: New taxa, “Firmicutes and Related Organism”; “Streptococcaceae”
21
22
The GenBank/EMBL/DDBJ accession numbers for the recN gene sequences of isolates
23
SUT-7, SUT-286T, SUT319, SUT328 and SUT380 are AB936268−AB936272,
24
respectively; those for the 16S rRNA gene sequence of isolate SUT-286T is AB936273.
25
The GenBank/EMBL/DDBJ BioProject ID for the draft genome sequences of strains
26
86-5192, 88-1861 and 89-4109-1, and isolates SUT-7, SUT-286T, SUT319, SUT328 and
27
SUT380 is PRJDB2302.
2
28
Abstract
29
In order to clarify taxonomic position of serotypes 20, 22 and 26 of Streptococcus suis,
30
biochemical and molecular genetic studies were performed on isolates (SUT-7,
31
SUT-286T, SUT-319, SUT-328 and SUT 380) reacted with specific antisera of either
32
serotypes 20, 22 or 26 from saliva of healthy pigs as well as reference strains of
33
serotypes 20, 22 and 26. Comparative recN gene sequencing showed high genetic
34
relatedness among our isolates but markedly different from those of the type strain
35
NCTC 10234T S. suis, i.e., 74.8–75.7% sequence similarities. The genomic relatedness
36
between our strains and other streptococcal strains including S. suis were calculated
37
using average nucleotide identity values of whole genome sequences, which indicates
38
that serotypes 20, 22 and 26 should be taxonomically removed from S. suis as a novel
39
genomic species. Comparative sequence analysis revealed 99.0–100% sequence
40
similarities for 16S rRNA gene between the reference strains of serotypes 20, 22 and 26,
41
and our isolates. Isolate STU-286T had relatively high 16S rRNA gene sequence
42
similarities with S. suis NCTC 10234T (98.8%). SUT-286T could be distinguished from
43
S. suis and other closely related Streptococcus species using biochemical tests. We
44
propose to name the new species Streptococcus parasuis sp. nov., with the type strain
45
SUT-286T (=JCM 30273T, =DSM 29126T), because of its phylogenetic and phenotypic
3
46
similarity to S. suis .
47
4
48
Streptococcus suis is a major pathogen of swine and is also isolated from a variety of
49
animals such as ruminants, cats, dogs, deer, and horses (Staats et al., 1997). Moreover,
50
human S. suis infection is considered to be one of the most important emerging zoonotic
51
diseases in Asian countries (Gottschalk et al., 2010). On the basis of their
52
polysaccharide capsular antigens, a total of 33 serotypes (types 1–31, 33 and type 1/2)
53
of S. suis were described elsewhere (Gottschalk et al., 1989, 1991; Higgins et al., 1995),
54
in which serotype 2 was most frequently associated with diseases in both pigs and
55
humans (Gottschalk et al., 2007). Genetic relatedness among S. suis serotypes has been
56
described by several investigators previously. However, a phylogenetic analysis based
57
on 16S rRNA gene and chaperonin-60 gene sequences comparison (Chatellier et al.,
58
1998; Brousseau et al., 2001) indicated a marked genetic discrepancy between serotypes
59
20, 22, 26 and 33 and the rest of the serotypes of S. suis. Moreover, our previous study
60
(Le et al. 2013) presented that the reference strains of serotypes 20, 22, 26 and 33
61
should be taxonomically removed from S. suis based on DNA-DNA relatedness with the
62
type strain S. suis NCTC 10234T and phylogenetic analyses of sequences of genes
63
encoding manganese-dependent superoxide dismutase (sodA) and recombination/repair
64
protein (recN). Additionally, the reference strains of serotypes 20, 22 and 26 were found
65
to share more than 70% DNA-DNA reassociation values with each other, suggesting
5
66
that they form a novel streptococcal species. In order to clarify their taxonomic position,
67
we herein examined isolates (SUT-7, SUT-286T, SUT-319, SUT-328 and SUT-380)
68
reacted with specific antisera of either serotypes 20, 22 or 26 from saliva of 4 clinically
69
healthy pigs as well as reference strains of serotypes 20, 22 and 26. Using
70
whole-genome sequence identities and sequences of recN and 16Sr RNA gene, we
71
demonstrate that S. suis serotypes 20, 22 and 26 belong to a single novel species. On the
72
basis of the phenotypic and phylogenetic results, a novel species of the genus
73
Streptococcus, Streptococcus parasuis sp. nov., is proposed.
74
A total of 5 isolates and reference strains of serotypes 20, 22 and 26 of S. suis used in
75
this study are listed in Table 1. Isolates SUT-7, SUT-286T, SUT-319, SUT-328 and
76
SUT-380 were isolated from saliva of 4 clinically healthy pigs. Bacterial strains and
77
isolates were cultured in Todd-Hewitt agar (Becton Dickinson, Sparks, MD., USA) at
78
37°C for 24 h and grown under 5% CO2. They were stored in Luria-Bertani broth
79
(Becton Dickinson) supplemented with 30% (v/v) glycerol at −80oC until use.
80
Serotyping of these isolates was performed by capsular reaction test, as described
81
previously (Higgins and Gottschalk, 1990), using commercial antisera (Statens Serum
82
Institute, Copenhagen, Denmark). Isolates SUT-286T and SUT-380 were reacted with
83
the antisera of serotypes 20 and 22, respectively, while isolates SUT-7, SUT-319 and
6
84
SUT-328 were cross-reacted with the antisera 22/26, 20/22 and 20/22, respectively
85
(Table 1).
86
As for the streptococcal species, phylogenetic analysis based on a recN gene was
87
found to be useful taxonomic tools for identification at the species level (Glazunova et
88
al., 2010). Therefore, in order to determine the phylogenetic relations of the partial
89
sequence of recN (1056 bp) genes of our isolates were amplified and sequenced
90
following subjected comparative analysis as described previously (Le et al., 2013). The
91
obtained sequences of recN were aligned by ClustalW method using MEGA5 software
92
package (Tamura et al., 2011). Phylogenetic trees were conducted using the
93
neighbor-joining method (NJ; Saitou & Nei, 1987) with MEGA5. Other
94
phylogenetically related Streptococcus sequences of recN retrieved from GenBank were
95
also included. The stability of the groupings was estimated by bootstrap analysis with
96
1,000 replications. The percentage of similarity between nucleotide sequences was
97
calculated using BioEdit software (Hall, 1999). Comparative sequence analysis revealed
98
97.7–100% sequence similarities for recN between the reference strains of serotypes 20,
99
22 and 26, which were previously determined (Le et al., 2013), and our isolates, thereby
100
demonstrating their high genetic relatedness, but markedly different from those of the
101
type strain NCTC 10234T, i.e., 74.8–75.7% sequence similarities. Moreover, these
7
102
isolates and strains formed a branch separate from other Streptococcus species in
103
phylogenetic trees inferred from recN gene sequence comparisons (Supplementary Fig.
104
S1); the highest sequence similarity was between strain SUT-286T and S. acidominimus
105
CIP 82.4 (74.1 % similality).
106
To further elucidate the differentiation of serotypes 20, 22 and 26 from recognized
107
members of the S. suis taxon, draft genome sequences were generated. Whole genome
108
sequences of reference strains 86-5192, 88-1861 and 89-4109-1, and of isolates SUT-7,
109
SUT-286T, SUT-319, SUT-328 and SUT 380 were determined using the Illumina/Solexa
110
technology. An average of 0.75–3.38 million paired-end reads with length of 262.1 bp
111
were generated per strain by MiSeq system (Illumina, CA, USA). All the generated
112
reads were assembled into contigs using CLC Genomics Workbench v. 6.0 (CLCbio,
113
Denmark), which was also used to determine the nucleotide sequence statistics such as
114
the approximate draft genome size and DNA G+C content. The resulting draft genomes
115
of the 3 strains and 5 isolates had 54-319 contigs with 82-479 fold coverage and the
116
genome size ranged 2.12–2.55 Mb with DNA G+C contents of 38.3–40.0 mol% (Table
117
1). The DNA G+C contents of our isolates and strains are very similar to the DNA G+C
118
contents reported for whole genome sequenced strain P1/7 of S. suis (40.0 mol%)
119
(Holden et al., 2009). The degree of pairwise genome-based relatedness was calculated
8
120
as an average nucleotide identity (ANI) value following the BLAST-based ANI
121
calculation method by using JSpecies software (Richter & Rosselló-Móra, 2009). ANI
122
values for the Streptococcus strains including those designated to S. suis whose genome
123
sequences were available in GenBank database were calculated and used to generate a
124
dendrogram based on UPGMA algorithm by using TreeView program (Page, 1996) (Fig.
125
1). The ANI values among reference strains 86-5192, 88-1861 and 89-4109-1, and our
126
isolates ranged 95.3–99.9% (Table S1), this level of ANI values are higher than the 95%
127
cut-off ANI value for bacterial species proposed by Goris et al. (2007). On the other
128
hand, the ANI values between our isolates and the strains of recognized as S. suis are
129
well below the proposed cut-off ANI value for bacterial species (88.1–89.0%)
130
(Supplementary Table S1). By the combination of recN gene phylogeny and genome
131
sequence comparison, it is concluded that reference strains serotype 20, 22 and 26, and
132
our isolates represent a novel genomic species.
133
The 16S rRNA gene sequences of our isolates and reference strains 86-5192, 88-1861
134
and 89-4109-1 were obtained from the draft genome sequences. Alignment of the
135
obtained 16S rRNA gene sequences with 16S rRNA gene sequences of type strain S.
136
suis NCTC 10234T and other streptococcal species, and NJ tree was constructed using
137
MEGA5 software (Tamura et al., 2011) (Fig. 2). Statistical reliability of the
9
138
phylogenetic tree was evaluated by bootstrap analysis of 1000 replicates. Accession
139
numbers of the reference sequences used in the phylogenetic analysis are shown as part
140
of Fig. 2. Comparative sequence analysis revealed 99.0–100% sequence similarities for
141
16S rRNA gene between the reference strains of serotypes 20, 22 and 26, and our
142
isolates. Isolate STU-286T had relatively high 16S rRNA gene sequence similarities
143
with S. suis NCTC 10234T (98.8%). The next most closely related type strain was
144
Streptococcus porcorum 682-03T (97.9% similarity).
145
The 5 new isolates were Gram-stained and assessed for the presence of catalase. The
146
haemolytic reaction was determined on Columbia agar containing 5% defibrinated
147
sheep blood incubated aerobically at 37°C for 24 and 48 h. Determination of growth at
148
10, 22, 30, 37 and 42°C and with 6.5% added NaCl in brain heart infusion broth (Difco,
149
NJ, USA) at pH 7.5 was performed as recommended by Facklam & Elliott (1995). The
150
isolates were characterized biochemically using the Rapid ID 32 Strep and API ZYM
151
system (bioMérieux, Lyon, France) according to the manufacturer’s instructions. The
152
isolates exhibited almost identical biochemical characteristics. The phenotypic
153
characteristics that differentiate the proposed species from closely related species are
154
shown in Table 2.
155
Phylogenetic analyses based on the recN and 16S rRNA gene sequences and
10
156
comparative genomics show that reference strains of “S. suis” serotypes 20, 22 and 26
157
should be separated from taxon S. suis, and isolate SUT-286T represents a novel species.
158
We propose to name the new species S. parasuis because of its phylogenetic and
159
phenotypic similarity to S. suis.
160
Description of Streptococcus parasuis sp. nov.
161
Streptococcus parasuis (pa.ra.su'is. Gr. prep. para, resembling; L. gen. n. suis,
162
specific epithet of Streptococcus suis; N.L. gen. n. parasuis, resembling Streptococcus
163
suis).
164
Cells are Gram-stain-positive, non-spore-forming cocci, 0.5–1 μm in diameter,
165
occurring in pairs or in chains, commonly over 10 cells long. Colonies on blood agar are
166
small, circular and non-pigmented, 0.5–1.0 mm in diameter and α-haemolytic at 37°C.
167
Cells are indifferent bacteria, catalase-negative and non-motile. Cells are able to grow at
168
10, 22, 30, 37 and 42°C but do not grow in the presence of 6.5% NaCl. With the Rapid
169
ID32 Strep kits, acid is produced from D-trehalose, maltose, pullulan, glycogen and
170
sucrose but not from D-melibiose, L-arabinose, D-ribose, D-arabitol, D-mannitol,
171
D-sorbitol, melezitose, cyclodextrin or tagatose. Most strains produce acid from
172
D-raffinose and lactose (Rapid ID32 Strep; type strain positive). Leucine arylamidase,
173
esterase (C4), esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase (API ZYM)
11
174
are detected. Most strains do not produce β -galactosidase (Rapid ID32 Strep; type strain
175
negative). No activity is detected for N-acetyl-β -glucosaminidase, α-mannosidase,
176
α-fucosidase, lipase (C14), acid phosphatase, valine arylamidase, α-glucosidase, cystine
177
arylamidase, trypsin, α-chymotrypsin (API ZYM), alkaline phosphatase, α-galactosidase,
178
β-glucosidase, β -glucuronidase (API ZYM and Rapid ID32 Strep), glycyl-tryptophan
179
arylamidase, β -mannosidase or pyroglutamic acid arylamidase (Rapid ID32 Strep).
180
Voges–Proskauer test was negative (Rapid ID32 Strep). Arginine, hippurate and urea are
181
not hydrolysed (Rapid ID32 Strep). The type strain, SUT-286T (=JCM 30273T, =DSM
182
29126T), was isolated from saliva of a clinically healthy pig. The DNA G+C content of
183
the type strain is 39.8 mol%.
184
185
Acknowledgements
186
The S. suis serotype reference strains used in the present study were generously gifted
187
from the National Institute of Animal Health, Japan. This work was supported by a
188
Grant-in-Aid from Kieikai Research Foundation, Japan.
189
12
190
References
191
Brousseau, R., Hill, J. E., Prefontaine, G., Goh, S. H., Harel, J., and S. M.
192
Hemmingsen. 2001. Streptococcus suis serotypes characterized by analysis of
193
chaperonin 60 gene sequences. Appl Environ Microbiol 67, 4828–4833.
194
Chatellier, S., Hare, l. J., Zhang, Y., Gottschalk, M., Higgins, R., Devriese, L. A.
195
and R. Brousseau. 1998. Phylogenetic diversity of Streptococcus suis strains of various
196
serotypes as revealed by 16S rRNA gene sequence comparison. Int J Syst Bacteriol 48,
197
581–589.
198
Collins, M. D., Hutson, R. A., Hoyles, L., Falsen, E., Nikolaitchouk, N., & Foster, G.
199
(2001). Streptococcus ovis sp. nov., isolated from sheep. Int J Syst Evol Microbiol 51,
200
1147–1150.
201
Facklam, R. R. & Elliott, J. A. (1995). Identification, classification, and clinical
202
relevance of catalase-negative, gram-positive cocci, excluding the streptococci and
203
enterococci. Clin Microbiol Rev 8, 479–495.
204
Garvie, E. I. & Bramley A. J. (1979). Streptococcus uberis: an Approach to its
205
Classification. J Appl Bacteriol 46, 295–304.
206
Glazunova, O. O, Raoult, D., & Roux, V. (2010). Partial recN gene sequencing: a new
207
tool for identification and phylogeny within the genus Streptococcus. Int J Syst Evol
13
208
Microbiol 60, 2140–2148.
209
Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. &
210
Tiedje, J. M. (2007). DNA–DNA hybridization values and their relationship to
211
whole-genome sequence similarities. Int J Syst Evol Microbiol 57, 81–91.
212
Gottschalk, M., Higgins, R., Jacques, M., Mittal, K. R., and J. Henrichsen. 1989.
213
Description of 14 new capsular types of Streptococcus suis. J Clin Microbiol 27,
214
2633–2636.
215
Gottschalk, M., Higgins, R., Jacques, M., Beaudoin, M., and Henrichsen, J. (1991).
216
Characterization of six new capsular types (23 through 28) of Streptococcus suis. J Clin
217
Microbiol 29, 2590–2594.
218
Gottschalk, M., Segura, M. & Xu, J. (2007). Streptococcus suis infections in humans:
219
The Chinese experience and the situation in North America. Animal Health Res Rev 8,
220
29–45.
221
Gottschalk, M., Xu, J., Lecours, M., Grenier, D., Fittipaldi, N. & Segura. M. (2010).
222
Streptococcus suis Infections in Humans: What is the prognosis for Western countries?
223
(Part I). Clin Microbiol News 32, 89-96.
224
Hall T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and
225
analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41, 95-98.
14
226
Higgins, R. & Gottschalk, M. (1990). An update on Streptococcus suis identification. J
227
Vet Diagn Invest 2, 249–252.
228
Higgins, R., Gottschalk, M., Boudreau, M., Lebrun, A. & J. Henrichsen. (1995).
229
Description of six new capsular types (29–34) of Streptococcus suis. J Vet Diagn Invest
230
7, 405–406.
231
Holden, M. T., Hauser, H., Sanders, M., Ngo, T. H., Cherevach, I., Cronin, A.,
232
Goodhead, I., Mungall, K., Quail, M. A. & other authors. (2009). Rapid evolution of
233
virulence and drug resistance in the emerging zoonotic pathogen Streptococcus suis.
234
Pros One 4, e6072. doi: 10.1371/journal.pone.0006072.
235
Kilpper-Bälz, R., & Schleifer, K. H. (1987). Streptococcus suis sp. nov., nom. rev. Int
236
J Syst Bacteriol 37, 160–162.
237
Le, Tien, H.T., Nishibori, T., Nishitani, Y., Nomoto, R. & Osawa, R. (2013).
238
Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22, 26, and 33 based
239
on DNA-DNA homology and sodA and recN phylogenies. Vet Microbiol 162, 842-9.
240
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on
241
personal computers. Comput Appl Biosci 12, 357–358.
242
Richter, M. & Rosselló-Móra, R. (2009). Shifting the genomic gold standard for the
243
prokaryotic species definition. Proc Natl Acad Sci USA 106, 19126-19131.
15
244
Saitou, N. & Nei, M. (1987). The neighbor-joining method, a new method for
245
reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
246
Staats, J. J., Feder, I., Okwumabua, O. & Chengappa. M. M. (1997). Streptococcus
247
suis: past and present. Vet Res Commun 21, 381–407.
248
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
249
MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood,
250
Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 28,
251
2731-2739.
252
Vela, A. I., Mentaberre, G., Marco, I., Velarde, R., Lavín, S., Domínguez, L. &
253
Fernández-Garayzábal, J. F. (2011–A). Streptococcus rupicaprae sp. nov., isolated
254
from a Pyrenean chamois (Rupicapra pyrenaica). Int J Syst Evol Microbiol 61,
255
1989–1993.
256
Vela, A. I., Sánchez, V., Mentaberre, G., Lavín, S., Domínguez, L. &
257
Fernández-Garayzábal, J. F. (2011–B). Streptococcus porcorum sp. nov., isolated
258
from domestic and wild pigs. Int J Syst Evol Microbiol 61, 1585–1589.
259
260
261
16
262
Figure legends
263
Fig. 1. A dendrogram describing the UPGMA clustering of genome-sequenced
264
Streptococcus strains based on their ANI values. The accession numbers of draft
265
genome sequences of our 5 isolates (SUT-7, SUT-286T, SUT-319, SUT-328 and
266
SUT-380)
267
DRX016751−DRX016758, respectively.
and
reference
strains
of
serotypes
20,
22
and
26
were
268
269
Fig. 2. The 16S rRNA gene sequence-based phylogenetic tree of strain SUT-286T and
270
the selected type strains of the genus Streptococcus. NJ tree is shown here with
271
bootstrap support values; only values >50% are shown.
272
17
Table 1. Strains and isolates used in this study, and its assembly information of NGS sequencing. Strain
Serotype
Source
GC content N50 contig size (%) (base)
Contig count Total length (base) Accession no.
1. 86-5192
20
Diseased pig
40.0
89,116
54
2,122,005
DRX016756
2. 88-1861
22
Diseased pig
39.4
36,856
319
2,552,497
DRX016757
3. 89-4109-1
26
Diseased pig
39.8
75,931
121
2,186,636
DRX016758
4. SUT-7
22/26
Healthy porcine saliva
39.8
74,455
78
2,122,164
DRX016751
20
Healthy porcine saliva
39.8
80,669
92
2,154,045
DRX016752
6. SUT-319
20/22
Healthy porcine saliva
39.3
123,706
113
2,198,789
DRX016753
7. SUT-328
20/22
Healthy porcine saliva
38.3
105,931
204
2,122,702
DRX016754
8. SUT-380
22
Healthy porcine saliva
39.7
85,628
68
2,178,782
DRX016755
5. SUT-286
T
273
18
274
Table 2. Characteristics useful in differentiating strain SUT-286T from closely related species of the genus Streptococcus Strains: 1, SUT-286T; 2, S. suis species (including S. suis NCTC 10234T, refer to Kilpper-Bälz, & Schleifer, 1987); 3, S. porcorum 682-03T (data from Vela et al., 2011–B) ; 4, S. uberis JCM 5709T (data from Garvie et al., 1979) ; 5, S. gallinaceus CCUG 42692T (data from Vela et al., 2011–A); 6, S. ovis CCUG 39485T (data from Collins et al., 2001). Phenotypic data for strains 1 and S. suis NCTC 10234T from this study. +, Positive reaction; -, negative reaction; v, variable reaction 1
2
3
4
5
6
− + − + + − − − − + − −
Hydrolysis of arginine Voges–Proskauer test Production of: β-Glucosidase β-Galactosidase β-Glucuronidase α-Galactosidase Pyroglutamic acid arylamidase N-Acetyl-β-glucosaminidase Glycyl-tryptophan arylamidase
− − − − − − −
+ v + + + + +
+ − − − − − −
+ − + − + + −
+ + − + − − +
+ − − + − − +
− − − + + + + −
− − − + v + + +
− − − + + − − +
− + + + − − − +
+ + − + + − − +
+ + + + + + − −
Production of acid from: Ribose Mannitol Sorbitol Lactose Raffinose Glycogen Pullulan Methyl β-D-glucopyranoside 275
19
90
80
70
60
% similarity
100
Fig. 1 SUT-328 SUT-319 SUT-380 SUT-286 T 89-4109-1 (serotype 26) 86-5192 (serotype 20) SUT-7 88-1681 (serotype 22) Streptococcus gallolyticus ATCC 43143 (AP012053) Streptococcus anginosus C1051 (CP003860) Streptococcus mutans UA159 (AE014133) Streptococcus mitis B6 (FN568063) Streptococcus pneumoniae R6 (AE007317) Streptococcus agalactiae 2603V/R (AE009948) Streptococcus suis T15 (CP006246) Streptococcus suis D12 (CP002644) Streptococcus suis ST1 (CP002651) Streptococcus suis GZ1 (CP000837) Streptococcus suis P1/7 (AM946016) Streptococcus suis A7 (CP002570) Streptococcus suis SS12 (CP002640) Streptococcus suis JS14 (CP002465) Streptococcus suis 05ZYH33 (CP000407)
Fig. 2 84 73 91 99 75
Streptococcus pseudopneumoniae ATCC BAA-960T (AY612844) Streptococcus pneumoniae ATCC33400T (AF003930) Streptococcus mitis ATCC 49456T (AF003929) Streptococcus oralis ATCC35037T (AY485602) Streptococcus dentisani DSM 27088T (HG315101)
Streptococcus parasanguinis ATCC15912T (DQ303191) Streptococcus australis ATCC 700641T (AY485604)
52
Streptococcus gordonii ATCC 10558T (AF003931)
86
Streptococcus sanguinis JCM 5708T (AB596946) Streptococcus cristatus ATCC 51100T (AY584476) Streptococcus oligofermentans LMG 21585T (AY099095)
77 99
Streptococcus sinensis HKU4T (AF432856) Streptococcus intermedius KCTC 3268T (GU045403) Streptococcus gallinaceus CCUG 42692T (AJ307888) Streptococcus acidominimus LMG 17755T (JX986969) Streptococcus entericus CECT 5353T (AJ409287)
Streptococcus suis NCTC 10234T (AB002525)
98
Streptococcus parasuis SUT-286T (AB936273) Streptococcus porcorum 682-03T (FN643224) Streptococcus porci 2923-03T (AM941160) Streptococcus uberis JCM 5709T (AB023573)
75
Streptococcus porcinus ATCC 43138T (AB002523) Streptococcus pyogenes JCM5674T (AB023575)
83
Streptococcus agalactiae JCM 5671T (AB023574)
70
Streptococcus dysgalactiae subsp. equisimilis CIP 105120T (DQ232540)
98
Streptococcus ovis CCUG 39485T (Y17358) Streptococcus pluranimalium 14177T (JX986968) 75 100
Streptococcus salivarius ATCC 7073T (AY188352) Streptococcus thermophilus ATCC 19258T (AY188354) Streptococcus vestibularis ATCC49124T (AY188353) Streptococcus anginosus ATCC 33397T (AF104678)
70 98
60
Streptococcus bovis ATCC 33317T (AB002482) Streptococcus lutetiensis CIP 106849T (DQ232532) Streptococcus equinus NBRC 12553T (AB680295) Streptococcus macedonicus ACA-DC 206T (Z94012)
99
Streptococcus gallolyticus ACM 3611T (X94337)
90 85
0.005
Streptococcus pasteurianus CIP107122T (DQ232528)
1
Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22, and 26:
2
Streptococcus parasuis sp. nov.
3
4
R. Nomoto1*, F. Maruyama2, S. Ishida3, M. Tohya3, T. Sekizaki3 and Ro Osawa4
5
6
7
8
9
10
11
12
13
14
1
Organization for Advanced Science and Technology, Kobe University, Rokko-dai 1-1,
Nada-ku, Kobe, Hyogo 657-8501, Japan 2
Graduate School of Medical and Dental Sciences, Section of Bacterial Phathogenesis,
Tokyo Medical and Dental University, Yushima 45-5-1, Bunkyo-ku ,Tokyo 113-8510, Japan 3
Research Center for Food Safety, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan 4
Department of Bioresource Sciences, Graduate School of Agricultural Sciences, Kobe
University, Rokko-dai 1-1, Nada-ku, Kobe, Hyogo 657-8501, Japan
15
16
* Corresponding author. Tel and Fax: +81-78-803-5981
17
E-mail address: [email protected]
18
1
19
Running title: Streptococcus parasuis sp. nov.
20
Contents Category: New taxa, “Firmicutes and Related Organism”; “Streptococcaceae”
21
22
The GenBank/EMBL/DDBJ accession numbers for the recN gene sequences of isolates
23
SUT-7, SUT-286T, SUT319, SUT328 and SUT380 are AB936268−AB936272,
24
respectively; those for the 16S rRNA gene sequence of isolate SUT-286T is AB936273.
25
The GenBank/EMBL/DDBJ BioProject ID for the draft genome sequences of strains
26
86-5192, 88-1861 and 89-4109-1, and isolates SUT-7, SUT-286T, SUT319, SUT328 and
27
SUT380 is PRJDB2302.
2
28
Abstract
29
In order to clarify taxonomic position of serotypes 20, 22 and 26 of Streptococcus suis,
30
biochemical and molecular genetic studies were performed on isolates (SUT-7,
31
SUT-286T, SUT-319, SUT-328 and SUT 380) reacted with specific antisera of either
32
serotypes 20, 22 or 26 from saliva of healthy pigs as well as reference strains of
33
serotypes 20, 22 and 26. Comparative recN gene sequencing showed high genetic
34
relatedness among our isolates but markedly different from those of the type strain
35
NCTC 10234T S. suis, i.e., 74.8–75.7% sequence similarities. The genomic relatedness
36
between our strains and other streptococcal strains including S. suis were calculated
37
using average nucleotide identity values of whole genome sequences, which indicates
38
that serotypes 20, 22 and 26 should be taxonomically removed from S. suis as a novel
39
genomic species. Comparative sequence analysis revealed 99.0–100% sequence
40
similarities for 16S rRNA gene between the reference strains of serotypes 20, 22 and 26,
41
and our isolates. Isolate STU-286T had relatively high 16S rRNA gene sequence
42
similarities with S. suis NCTC 10234T (98.8%). SUT-286T could be distinguished from
43
S. suis and other closely related Streptococcus species using biochemical tests. We
44
propose to name the new species Streptococcus parasuis sp. nov., with the type strain
45
SUT-286T (=JCM 30273T, =DSM 29126T), because of its phylogenetic and phenotypic
3
46
similarity to S. suis .
47
4
48
Streptococcus suis is a major pathogen of swine and is also isolated from a variety of
49
animals such as ruminants, cats, dogs, deer, and horses (Staats et al., 1997). Moreover,
50
human S. suis infection is considered to be one of the most important emerging zoonotic
51
diseases in Asian countries (Gottschalk et al., 2010). On the basis of their
52
polysaccharide capsular antigens, a total of 33 serotypes (types 1–31, 33 and type 1/2)
53
of S. suis were described elsewhere (Gottschalk et al., 1989, 1991; Higgins et al., 1995),
54
in which serotype 2 was most frequently associated with diseases in both pigs and
55
humans (Gottschalk et al., 2007). Genetic relatedness among S. suis serotypes has been
56
described by several investigators previously. However, a phylogenetic analysis based
57
on 16S rRNA gene and chaperonin-60 gene sequences comparison (Chatellier et al.,
58
1998; Brousseau et al., 2001) indicated a marked genetic discrepancy between serotypes
59
20, 22, 26 and 33 and the rest of the serotypes of S. suis. Moreover, our previous study
60
(Le et al. 2013) presented that the reference strains of serotypes 20, 22, 26 and 33
61
should be taxonomically removed from S. suis based on DNA-DNA relatedness with the
62
type strain S. suis NCTC 10234T and phylogenetic analyses of sequences of genes
63
encoding manganese-dependent superoxide dismutase (sodA) and recombination/repair
64
protein (recN). Additionally, the reference strains of serotypes 20, 22 and 26 were found
65
to share more than 70% DNA-DNA reassociation values with each other, suggesting
5
66
that they form a novel streptococcal species. In order to clarify their taxonomic position,
67
we herein examined isolates (SUT-7, SUT-286T, SUT-319, SUT-328 and SUT-380)
68
reacted with specific antisera of either serotypes 20, 22 or 26 from saliva of 4 clinically
69
healthy pigs as well as reference strains of serotypes 20, 22 and 26. Using
70
whole-genome sequence identities and sequences of recN and 16Sr RNA gene, we
71
demonstrate that S. suis serotypes 20, 22 and 26 belong to a single novel species. On the
72
basis of the phenotypic and phylogenetic results, a novel species of the genus
73
Streptococcus, Streptococcus parasuis sp. nov., is proposed.
74
A total of 5 isolates and reference strains of serotypes 20, 22 and 26 of S. suis used in
75
this study are listed in Table 1. Isolates SUT-7, SUT-286T, SUT-319, SUT-328 and
76
SUT-380 were isolated from saliva of 4 clinically healthy pigs. Bacterial strains and
77
isolates were cultured in Todd-Hewitt agar (Becton Dickinson, Sparks, MD., USA) at
78
37°C for 24 h and grown under 5% CO2. They were stored in Luria-Bertani broth
79
(Becton Dickinson) supplemented with 30% (v/v) glycerol at −80oC until use.
80
Serotyping of these isolates was performed by capsular reaction test, as described
81
previously (Higgins and Gottschalk, 1990), using commercial antisera (Statens Serum
82
Institute, Copenhagen, Denmark). Isolates SUT-286T and SUT-380 were reacted with
83
the antisera of serotypes 20 and 22, respectively, while isolates SUT-7, SUT-319 and
6
84
SUT-328 were cross-reacted with the antisera 22/26, 20/22 and 20/22, respectively
85
(Table 1).
86
As for the streptococcal species, phylogenetic analysis based on a recN gene was
87
found to be useful taxonomic tools for identification at the species level (Glazunova et
88
al., 2010). Therefore, in order to determine the phylogenetic relations of the partial
89
sequence of recN (1056 bp) genes of our isolates were amplified and sequenced
90
following subjected comparative analysis as described previously (Le et al., 2013). The
91
obtained sequences of recN were aligned by ClustalW method using MEGA5 software
92
package (Tamura et al., 2011). Phylogenetic trees were conducted using the
93
neighbor-joining method (NJ; Saitou & Nei, 1987) with MEGA5. Other
94
phylogenetically related Streptococcus sequences of recN retrieved from GenBank were
95
also included. The stability of the groupings was estimated by bootstrap analysis with
96
1,000 replications. The percentage of similarity between nucleotide sequences was
97
calculated using BioEdit software (Hall, 1999). Comparative sequence analysis revealed
98
97.7–100% sequence similarities for recN between the reference strains of serotypes 20,
99
22 and 26, which were previously determined (Le et al., 2013), and our isolates, thereby
100
demonstrating their high genetic relatedness, but markedly different from those of the
101
type strain NCTC 10234T, i.e., 74.8–75.7% sequence similarities. Moreover, these
7
102
isolates and strains formed a branch separate from other Streptococcus species in
103
phylogenetic trees inferred from recN gene sequence comparisons (Supplementary Fig.
104
S1); the highest sequence similarity was between strain SUT-286T and S. acidominimus
105
CIP 82.4 (74.1 % similality).
106
To further elucidate the differentiation of serotypes 20, 22 and 26 from recognized
107
members of the S. suis taxon, draft genome sequences were generated. Whole genome
108
sequences of reference strains 86-5192, 88-1861 and 89-4109-1, and of isolates SUT-7,
109
SUT-286T, SUT-319, SUT-328 and SUT 380 were determined using the Illumina/Solexa
110
technology. An average of 0.75–3.38 million paired-end reads with length of 262.1 bp
111
were generated per strain by MiSeq system (Illumina, CA, USA). All the generated
112
reads were assembled into contigs using CLC Genomics Workbench v. 6.0 (CLCbio,
113
Denmark), which was also used to determine the nucleotide sequence statistics such as
114
the approximate draft genome size and DNA G+C content. The resulting draft genomes
115
of the 3 strains and 5 isolates had 54-319 contigs with 82-479 fold coverage and the
116
genome size ranged 2.12–2.55 Mb with DNA G+C contents of 38.3–40.0 mol% (Table
117
1). The DNA G+C contents of our isolates and strains are very similar to the DNA G+C
118
contents reported for whole genome sequenced strain P1/7 of S. suis (40.0 mol%)
119
(Holden et al., 2009). The degree of pairwise genome-based relatedness was calculated
8
120
as an average nucleotide identity (ANI) value following the BLAST-based ANI
121
calculation method by using JSpecies software (Richter & Rosselló-Móra, 2009). ANI
122
values for the Streptococcus strains including those designated to S. suis whose genome
123
sequences were available in GenBank database were calculated and used to generate a
124
dendrogram based on UPGMA algorithm by using TreeView program (Page, 1996) (Fig.
125
1). The ANI values among reference strains 86-5192, 88-1861 and 89-4109-1, and our
126
isolates ranged 95.3–99.9% (Table S1), this level of ANI values are higher than the 95%
127
cut-off ANI value for bacterial species proposed by Goris et al. (2007). On the other
128
hand, the ANI values between our isolates and the strains of recognized as S. suis are
129
well below the proposed cut-off ANI value for bacterial species (88.1–89.0%)
130
(Supplementary Table S1). By the combination of recN gene phylogeny and genome
131
sequence comparison, it is concluded that reference strains serotype 20, 22 and 26, and
132
our isolates represent a novel genomic species.
133
The 16S rRNA gene sequences of our isolates and reference strains 86-5192, 88-1861
134
and 89-4109-1 were obtained from the draft genome sequences. Alignment of the
135
obtained 16S rRNA gene sequences with 16S rRNA gene sequences of type strain S.
136
suis NCTC 10234T and other streptococcal species, and NJ tree was constructed using
137
MEGA5 software (Tamura et al., 2011) (Fig. 2). Statistical reliability of the
9
138
phylogenetic tree was evaluated by bootstrap analysis of 1000 replicates. Accession
139
numbers of the reference sequences used in the phylogenetic analysis are shown as part
140
of Fig. 2. Comparative sequence analysis revealed 99.0–100% sequence similarities for
141
16S rRNA gene between the reference strains of serotypes 20, 22 and 26, and our
142
isolates. Isolate STU-286T had relatively high 16S rRNA gene sequence similarities
143
with S. suis NCTC 10234T (98.8%). The next most closely related type strain was
144
Streptococcus porcorum 682-03T (97.9% similarity).
145
The 5 new isolates were Gram-stained and assessed for the presence of catalase. The
146
haemolytic reaction was determined on Columbia agar containing 5% defibrinated
147
sheep blood incubated aerobically at 37°C for 24 and 48 h. Determination of growth at
148
10, 22, 30, 37 and 42°C and with 6.5% added NaCl in brain heart infusion broth (Difco,
149
NJ, USA) at pH 7.5 was performed as recommended by Facklam & Elliott (1995). The
150
isolates were characterized biochemically using the Rapid ID 32 Strep and API ZYM
151
system (bioMérieux, Lyon, France) according to the manufacturer’s instructions. The
152
isolates exhibited almost identical biochemical characteristics. The phenotypic
153
characteristics that differentiate the proposed species from closely related species are
154
shown in Table 2.
155
Phylogenetic analyses based on the recN and 16S rRNA gene sequences and
10
156
comparative genomics show that reference strains of “S. suis” serotypes 20, 22 and 26
157
should be separated from taxon S. suis, and isolate SUT-286T represents a novel species.
158
We propose to name the new species S. parasuis because of its phylogenetic and
159
phenotypic similarity to S. suis.
160
Description of Streptococcus parasuis sp. nov.
161
Streptococcus parasuis (pa.ra.su'is. Gr. prep. para, resembling; L. gen. n. suis,
162
specific epithet of Streptococcus suis; N.L. gen. n. parasuis, resembling Streptococcus
163
suis).
164
Cells are Gram-stain-positive, non-spore-forming cocci, 0.5–1 μm in diameter,
165
occurring in pairs or in chains, commonly over 10 cells long. Colonies on blood agar are
166
small, circular and non-pigmented, 0.5–1.0 mm in diameter and α-haemolytic at 37°C.
167
Cells are indifferent bacteria, catalase-negative and non-motile. Cells are able to grow at
168
10, 22, 30, 37 and 42°C but do not grow in the presence of 6.5% NaCl. With the Rapid
169
ID32 Strep kits, acid is produced from D-trehalose, maltose, pullulan, glycogen and
170
sucrose but not from D-melibiose, L-arabinose, D-ribose, D-arabitol, D-mannitol,
171
D-sorbitol, melezitose, cyclodextrin or tagatose. Most strains produce acid from
172
D-raffinose and lactose (Rapid ID32 Strep; type strain positive). Leucine arylamidase,
173
esterase (C4), esterase lipase (C8) and naphthol-AS-BI-phosphohydrolase (API ZYM)
11
174
are detected. Most strains do not produce β -galactosidase (Rapid ID32 Strep; type strain
175
negative). No activity is detected for N-acetyl-β -glucosaminidase, α-mannosidase,
176
α-fucosidase, lipase (C14), acid phosphatase, valine arylamidase, α-glucosidase, cystine
177
arylamidase, trypsin, α-chymotrypsin (API ZYM), alkaline phosphatase, α-galactosidase,
178
β-glucosidase, β -glucuronidase (API ZYM and Rapid ID32 Strep), glycyl-tryptophan
179
arylamidase, β -mannosidase or pyroglutamic acid arylamidase (Rapid ID32 Strep).
180
Voges–Proskauer test was negative (Rapid ID32 Strep). Arginine, hippurate and urea are
181
not hydrolysed (Rapid ID32 Strep). The type strain, SUT-286T (=JCM 30273T, =DSM
182
29126T), was isolated from saliva of a clinically healthy pig. The DNA G+C content of
183
the type strain is 39.8 mol%.
184
185
Acknowledgements
186
The S. suis serotype reference strains used in the present study were generously gifted
187
from the National Institute of Animal Health, Japan. This work was supported by a
188
Grant-in-Aid from Kieikai Research Foundation, Japan.
189
12
190
References
191
Brousseau, R., Hill, J. E., Prefontaine, G., Goh, S. H., Harel, J., and S. M.
192
Hemmingsen. 2001. Streptococcus suis serotypes characterized by analysis of
193
chaperonin 60 gene sequences. Appl Environ Microbiol 67, 4828–4833.
194
Chatellier, S., Hare, l. J., Zhang, Y., Gottschalk, M., Higgins, R., Devriese, L. A.
195
and R. Brousseau. 1998. Phylogenetic diversity of Streptococcus suis strains of various
196
serotypes as revealed by 16S rRNA gene sequence comparison. Int J Syst Bacteriol 48,
197
581–589.
198
Collins, M. D., Hutson, R. A., Hoyles, L., Falsen, E., Nikolaitchouk, N., & Foster, G.
199
(2001). Streptococcus ovis sp. nov., isolated from sheep. Int J Syst Evol Microbiol 51,
200
1147–1150.
201
Facklam, R. R. & Elliott, J. A. (1995). Identification, classification, and clinical
202
relevance of catalase-negative, gram-positive cocci, excluding the streptococci and
203
enterococci. Clin Microbiol Rev 8, 479–495.
204
Garvie, E. I. & Bramley A. J. (1979). Streptococcus uberis: an Approach to its
205
Classification. J Appl Bacteriol 46, 295–304.
206
Glazunova, O. O, Raoult, D., & Roux, V. (2010). Partial recN gene sequencing: a new
207
tool for identification and phylogeny within the genus Streptococcus. Int J Syst Evol
13
208
Microbiol 60, 2140–2148.
209
Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. &
210
Tiedje, J. M. (2007). DNA–DNA hybridization values and their relationship to
211
whole-genome sequence similarities. Int J Syst Evol Microbiol 57, 81–91.
212
Gottschalk, M., Higgins, R., Jacques, M., Mittal, K. R., and J. Henrichsen. 1989.
213
Description of 14 new capsular types of Streptococcus suis. J Clin Microbiol 27,
214
2633–2636.
215
Gottschalk, M., Higgins, R., Jacques, M., Beaudoin, M., and Henrichsen, J. (1991).
216
Characterization of six new capsular types (23 through 28) of Streptococcus suis. J Clin
217
Microbiol 29, 2590–2594.
218
Gottschalk, M., Segura, M. & Xu, J. (2007). Streptococcus suis infections in humans:
219
The Chinese experience and the situation in North America. Animal Health Res Rev 8,
220
29–45.
221
Gottschalk, M., Xu, J., Lecours, M., Grenier, D., Fittipaldi, N. & Segura. M. (2010).
222
Streptococcus suis Infections in Humans: What is the prognosis for Western countries?
223
(Part I). Clin Microbiol News 32, 89-96.
224
Hall T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and
225
analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41, 95-98.
14
226
Higgins, R. & Gottschalk, M. (1990). An update on Streptococcus suis identification. J
227
Vet Diagn Invest 2, 249–252.
228
Higgins, R., Gottschalk, M., Boudreau, M., Lebrun, A. & J. Henrichsen. (1995).
229
Description of six new capsular types (29–34) of Streptococcus suis. J Vet Diagn Invest
230
7, 405–406.
231
Holden, M. T., Hauser, H., Sanders, M., Ngo, T. H., Cherevach, I., Cronin, A.,
232
Goodhead, I., Mungall, K., Quail, M. A. & other authors. (2009). Rapid evolution of
233
virulence and drug resistance in the emerging zoonotic pathogen Streptococcus suis.
234
Pros One 4, e6072. doi: 10.1371/journal.pone.0006072.
235
Kilpper-Bälz, R., & Schleifer, K. H. (1987). Streptococcus suis sp. nov., nom. rev. Int
236
J Syst Bacteriol 37, 160–162.
237
Le, Tien, H.T., Nishibori, T., Nishitani, Y., Nomoto, R. & Osawa, R. (2013).
238
Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22, 26, and 33 based
239
on DNA-DNA homology and sodA and recN phylogenies. Vet Microbiol 162, 842-9.
240
Page, R. D. M. (1996). TreeView: an application to display phylogenetic trees on
241
personal computers. Comput Appl Biosci 12, 357–358.
242
Richter, M. & Rosselló-Móra, R. (2009). Shifting the genomic gold standard for the
243
prokaryotic species definition. Proc Natl Acad Sci USA 106, 19126-19131.
15
244
Saitou, N. & Nei, M. (1987). The neighbor-joining method, a new method for
245
reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
246
Staats, J. J., Feder, I., Okwumabua, O. & Chengappa. M. M. (1997). Streptococcus
247
suis: past and present. Vet Res Commun 21, 381–407.
248
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011).
249
MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood,
250
Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 28,
251
2731-2739.
252
Vela, A. I., Mentaberre, G., Marco, I., Velarde, R., Lavín, S., Domínguez, L. &
253
Fernández-Garayzábal, J. F. (2011–A). Streptococcus rupicaprae sp. nov., isolated
254
from a Pyrenean chamois (Rupicapra pyrenaica). Int J Syst Evol Microbiol 61,
255
1989–1993.
256
Vela, A. I., Sánchez, V., Mentaberre, G., Lavín, S., Domínguez, L. &
257
Fernández-Garayzábal, J. F. (2011–B). Streptococcus porcorum sp. nov., isolated
258
from domestic and wild pigs. Int J Syst Evol Microbiol 61, 1585–1589.
259
260
261
16
262
Figure legends
263
Fig. 1. A dendrogram describing the UPGMA clustering of genome-sequenced
264
Streptococcus strains based on their ANI values. The accession numbers of draft
265
genome sequences of our 5 isolates (SUT-7, SUT-286T, SUT-319, SUT-328 and
266
SUT-380)
267
DRX016751−DRX016758, respectively.
and
reference
strains
of
serotypes
20,
22
and
26
were
268
269
Fig. 2. The 16S rRNA gene sequence-based phylogenetic tree of strain SUT-286T and
270
the selected type strains of the genus Streptococcus. NJ tree is shown here with
271
bootstrap support values; only values >50% are shown.
272
17
Table 1. Strains and isolates used in this study, and its assembly information of NGS sequencing. Strain
Serotype
Source
GC content N50 contig size (%) (base)
Contig count Total length (base) Accession no.
1. 86-5192
20
Diseased pig
40.0
89,116
54
2,122,005
DRX016756
2. 88-1861
22
Diseased pig
39.4
36,856
319
2,552,497
DRX016757
3. 89-4109-1
26
Diseased pig
39.8
75,931
121
2,186,636
DRX016758
4. SUT-7
22/26
Healthy porcine saliva
39.8
74,455
78
2,122,164
DRX016751
20
Healthy porcine saliva
39.8
80,669
92
2,154,045
DRX016752
6. SUT-319
20/22
Healthy porcine saliva
39.3
123,706
113
2,198,789
DRX016753
7. SUT-328
20/22
Healthy porcine saliva
38.3
105,931
204
2,122,702
DRX016754
8. SUT-380
22
Healthy porcine saliva
39.7
85,628
68
2,178,782
DRX016755
5. SUT-286
T
273
18
274
Table 2. Characteristics useful in differentiating strain SUT-286T from closely related species of the genus Streptococcus Strains: 1, SUT-286T; 2, S. suis species (including S. suis NCTC 10234T, refer to Kilpper-Bälz, & Schleifer, 1987); 3, S. porcorum 682-03T (data from Vela et al., 2011–B) ; 4, S. uberis JCM 5709T (data from Garvie et al., 1979) ; 5, S. gallinaceus CCUG 42692T (data from Vela et al., 2011–A); 6, S. ovis CCUG 39485T (data from Collins et al., 2001). Phenotypic data for strains 1 and S. suis NCTC 10234T from this study. +, Positive reaction; -, negative reaction; v, variable reaction 1
2
3
4
5
6
− + − + + − − − − + − −
Hydrolysis of arginine Voges–Proskauer test Production of: β-Glucosidase β-Galactosidase β-Glucuronidase α-Galactosidase Pyroglutamic acid arylamidase N-Acetyl-β-glucosaminidase Glycyl-tryptophan arylamidase
− − − − − − −
+ v + + + + +
+ − − − − − −
+ − + − + + −
+ + − + − − +
+ − − + − − +
− − − + + + + −
− − − + v + + +
− − − + + − − +
− + + + − − − +
+ + − + + − − +
+ + + + + + − −
Production of acid from: Ribose Mannitol Sorbitol Lactose Raffinose Glycogen Pullulan Methyl β-D-glucopyranoside 275
19
90
80
70
60
% similarity
100
Fig. 1 SUT-328 SUT-319 SUT-380 SUT-286 T 89-4109-1 (serotype 26) 86-5192 (serotype 20) SUT-7 88-1681 (serotype 22) Streptococcus gallolyticus ATCC 43143 (AP012053) Streptococcus anginosus C1051 (CP003860) Streptococcus mutans UA159 (AE014133) Streptococcus mitis B6 (FN568063) Streptococcus pneumoniae R6 (AE007317) Streptococcus agalactiae 2603V/R (AE009948) Streptococcus suis T15 (CP006246) Streptococcus suis D12 (CP002644) Streptococcus suis ST1 (CP002651) Streptococcus suis GZ1 (CP000837) Streptococcus suis P1/7 (AM946016) Streptococcus suis A7 (CP002570) Streptococcus suis SS12 (CP002640) Streptococcus suis JS14 (CP002465) Streptococcus suis 05ZYH33 (CP000407)
Fig. 2 84 73 91 99 75
Streptococcus pseudopneumoniae ATCC BAA-960T (AY612844) Streptococcus pneumoniae ATCC33400T (AF003930) Streptococcus mitis ATCC 49456T (AF003929) Streptococcus oralis ATCC35037T (AY485602) Streptococcus dentisani DSM 27088T (HG315101)
Streptococcus parasanguinis ATCC15912T (DQ303191) Streptococcus australis ATCC 700641T (AY485604)
52
Streptococcus gordonii ATCC 10558T (AF003931)
86
Streptococcus sanguinis JCM 5708T (AB596946) Streptococcus cristatus ATCC 51100T (AY584476) Streptococcus oligofermentans LMG 21585T (AY099095)
77 99
Streptococcus sinensis HKU4T (AF432856) Streptococcus intermedius KCTC 3268T (GU045403) Streptococcus gallinaceus CCUG 42692T (AJ307888) Streptococcus acidominimus LMG 17755T (JX986969) Streptococcus entericus CECT 5353T (AJ409287)
Streptococcus suis NCTC 10234T (AB002525)
98
Streptococcus parasuis SUT-286T (AB936273) Streptococcus porcorum 682-03T (FN643224) Streptococcus porci 2923-03T (AM941160) Streptococcus uberis JCM 5709T (AB023573)
75
Streptococcus porcinus ATCC 43138T (AB002523) Streptococcus pyogenes JCM5674T (AB023575)
83
Streptococcus agalactiae JCM 5671T (AB023574)
70
Streptococcus dysgalactiae subsp. equisimilis CIP 105120T (DQ232540)
98
Streptococcus ovis CCUG 39485T (Y17358) Streptococcus pluranimalium 14177T (JX986968) 75 100
Streptococcus salivarius ATCC 7073T (AY188352) Streptococcus thermophilus ATCC 19258T (AY188354) Streptococcus vestibularis ATCC49124T (AY188353) Streptococcus anginosus ATCC 33397T (AF104678)
70 98
60
Streptococcus bovis ATCC 33317T (AB002482) Streptococcus lutetiensis CIP 106849T (DQ232532) Streptococcus equinus NBRC 12553T (AB680295) Streptococcus macedonicus ACA-DC 206T (Z94012)
99
Streptococcus gallolyticus ACM 3611T (X94337)
90 85
0.005
Streptococcus pasteurianus CIP107122T (DQ232528)
