Exploring internal features of 16S rRNA gene for identification of clinically relevant species of the genus Streptococcus.

Abstract

Streptococcus is an economically important genus as a number of species belonging to this genus are human and animal pathogens. The genus has been divided into different groups based on 16S rRNA gene sequence similarity. The variability observed among the members of these groups is low and it is difficult to distinguish them. The present study was taken up to explore 16S rRNA gene sequence to develop methods that can be used for preliminary identification and can supplement the existing methods for identification of clinically-relevant isolates of the genus Streptococcus.
16S rRNA gene sequences belonging to the isolates of S. dysgalactiae, S. equi, S. pyogenes, S. agalactiae, S. bovis, S. gallolyticus, S. mutans, S. sobrinus, S. mitis, S. pneumoniae, S. thermophilus and S. anginosus were analyzed with the purpose to define genetic variability within each species to generate a phylogenetic framework, to identify species-specific signatures and in-silico restriction enzyme analysis.
The framework based analysis was used to segregate Streptococcus spp. previously identified upto genus level. This segregation was validated using species-specific signatures and in-silico restriction enzyme analysis. 43 uncharacterized Streptococcus spp. could be identified using this approach.
The markers generated exploring 16S rRNA gene sequences provided useful tool that can be further used for identification of different species of the genus Streptococcus.

Full text

Page 1

RESEARCHOpen Access
Exploring internal features of 16S rRNA gene
for identification of clinically relevant species of
the genus Streptococcus
Devi Lal, Mansi Verma and Rup Lal*
Abstract
Background: Streptococcus is an economically important genus as a number of species belonging to this genus
are human and animal pathogens. The genus has been divided into different groups based on 16S rRNA gene
sequence similarity. The variability observed among the members of these groups is low and it is difficult to
distinguish them. The present study was taken up to explore 16S rRNA gene sequence to develop methods that
can be used for preliminary identification and can supplement the existing methods for identification of clinically-
relevant isolates of the genus Streptococcus.
Methods: 16S rRNA gene sequences belonging to the isolates of S. dysgalactiae, S. equi, S. pyogenes, S. agalactiae,
S. bovis, S. gallolyticus, S. mutans, S. sobrinus, S. mitis, S. pneumoniae, S. thermophilus and S. anginosus were analyzed
with the purpose to define genetic variability within each species to generate a phylogenetic framework, to
identify species-specific signatures and in-silico restriction enzyme analysis.
Results: The framework based analysis was used to segregate Streptococcus spp. previously identified upto genus
level. This segregation was validated using species-specific signatures and in-silico restriction enzyme analysis. 43
uncharacterized Streptococcus spp. could be identified using this approach.
Conclusions: The markers generated exploring 16S rRNA gene sequences provided useful tool that can be further
used for identification of different species of the genus Streptococcus.
Keywords: phylogenetic framework, signature sequences, genetic heterogeneity
Background
The genus Streptococcus consists of spherical Gram
positive bacteria belonging to the class Bacilli and the
order Lactobacillales [1]. The group is large and com-
prises of numerous clinically significant species which
are responsible for wide variety of infections in human
and animals. Streptococcus of different groups are
known to cause human diseases, some species being
highly virulent and responsible for major diseases. Spe-
cies like S. pyogenes, S. agalactiae and S. pneumoniae
are important as they cause serious acute infections in
man, but several other species are also involved in a
number of diseases like infective endocarditis, abscesses
and other pathological conditions [2]. Various species of
Streptococcus are known to be associated with infections
of cattles, pigs, horses, sheeps, birds, aquatic mammals
and fishes [3]. The genus has undergone considerable
taxonomic revisions and has been divided into different
groups (pyogenic, anginosus, mitis, mutans, salivarius,
bovis) based on 16S rRNA gene sequence similarity [4].
Since many species belonging to the genus Streptococ-
cus are associated with various pathological conditions,
different protocols have been used for their identifica-
tion. Still precise identification of these species is labor-
ious. Clinical laboratories use serological grouping by
Lancefield, haemolytic reactions and phenotypic tests for
identification of various Streptococcus isolates. However,
these Lancefield groups are not species-specific [5,6]
and haemolytic activity differs within species and
depends on incubation procedures. Strains within a
given species may differ for a common trait [7,8] and
* Correspondence: ruplal@gmail.com
Molecular Biology Laboratory, Department of Zoology, University of Delhi,
Delhi-110007, India
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
© 2011 Lal et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

Page 2

even the same strain may exhibit biochemical variability
[9,10].
Various alternatives have been employed for identifi-
cation of Streptococcus isolates. These include DNA
hybridization [11-14]; rDNA restriction analysis [15-17];
use of 16S-23S rRNA interspacer region [18-21], D-ala-
nyl-D-alanine ligase gene (ddl gene) [22]; autolysin gene
(lytA) [23]; dextranase (dex) [24]; heat shock protein
(groESL) [25,26]; RNA subunit of endoribonuclease P
(rnpB) [27]; the elongation factor Tu (tuf) [28]; gyrase A
(gyrA) and topoisomerase subunit C (parC) [29];
sequence analysis of small subunit rRNA [30]; manga-
nese-dependent superoxide dismutase gene (sodA)
[31,32]; recombination and repair protein (recN) [33];
tDNA PCR fragment length polymorphism [34,35] and
use of multilocus sequencing typing loci [36,37].
16S rRNA gene sequencing has proved to be one of
the most powerful tools for the classification of microor-
ganisms [38] and has been used for identification of
clinically relevant microbes [39,40]. Therefore, molecular
tools based on 16S rRNA gene can be developed and
used for identification. However it is also true that the
correct identification of bacterial species may not be
based on the nucleotide sequence of a single gene. Mul-
tilocus Sequence Analysis (MLSA) of several house-
keeping genes has to be performed. But from practical
standpoint there is need for a simplified approach for
preliminary identification of a species, particularly under
the conditions if the amount of isolated DNA is not
enough for MLSA or it does not react with a complete
set of typing primers. The current work considers the
possibility to use 16S rRNA sequences for this purpose
and is useful for practical applications.Thus the present
study aims to explore internal features of 16S rRNA
gene for preliminary identification of a species that can
supplement the existing methods for identification.
These methods include construction of phylogenetic fra-
mework, identification of species-specific signatures and
restriction enzyme analysis.
Methods
Sequence data
16S rRNA gene sequences belonging to the genus Strep-
tococcus from RDP database http://rdp.cme.msu.edu/[41]
were analysed in the present study. These included the
sequences with relatively high number of identified
organisms (86 sequences belonging to isolates of S. dys-
galactiae, 61 to S. equi, 61 to S. pyogenes, 29 to S.agalac-
tiae, 31 S. bovis-equinus (S. bovis and S. equinus are
considered to be a single species [42]), 76 to S. gallolyti-
cus, 102 to S. mutans, 23 to S.sobrinus, 28 to S. mitis, 41
to S. pneumoniae, 73 to S. thermophilus, 32 to S. angino-
sus) and 63 sequences of uncharacterized species identi-
fied only upto genus level. The sequences belonging to
twelve sets of Streptococcus species occurring with higher
frequency were used as the master species set for gener-
ating a phylogenetic framework, species-specific signa-
tures and restriction enzyme analysis.
Phylogenetic Analyses
For phylogenetic analyses, the sequences were aligned
using multiple alignment program CLUSTAL_X [43].
Evolutionary distances between all the sequences were
calculated with DNADIST of the PHYLIP 3.6 package
[44]. The program NEIGHBOR was used to draw neigh-
bor joining [45] tree with Jukes and Cantor correction
[46]. Statistical testing of the trees was done using SEQ-
BOOT by resampling the dataset 1000 times. The trees
were viewed through TreeView Version 1.6.6 [47]. For
each of these 12 Streptococcus species data sets,
sequences that formed a single cluster were aligned and
a consensus was obtained by using JALVIEW sequence
editor [48]. The sequence close to consensus from each
group was chosen as a representative for that particular
group. Based on this, a reference set of 63 sequences
was selected to define the range of genetic variability
present in each of the Streptococcus species.
Specific Signatures
Signatures were identified in each of the species data set
using online MEME program [49]. Sequences of 12
Streptococcus species data sets were submitted group-
wise in MEME program Version 4.6.1 http://meme.sdsc.
edu/meme4_6_1/cgi-bin/meme.cgi. In order to obtain
maximum number of motifs the default setting was
modified from 3 motifs to 20 motifs. The default value
of motif widths was also modified and re-set between 25
and 50. Each of the 20 signatures was checked for its
frequency of occurrence among a particular Streptococ-
cus sp. The signatures which did not appear in other
Streptococcus spp. were considered as unique. BLAST
search against NCBI database http://www.ncbi.nlm.nih.
gov/ was carried out for these signatures to check their
uniqueness.
Restriction enzyme analysis
Eleven Type II Restriction enzymes (Table 1) were con-
sidered for these analyses. Restriction Mapper Version 3
http://restrictionmapper.org/ was used to obtain the
restriction pattern of the 12 Streptococcus species data
sets employed for construction of phylogenetic frame-
work. These restriction patterns were analyzed and a
consensus pattern was determined for each species.
Cluster analysis for restriction profile
For cluster analyses MVSP (Multi Variate Statistical
Package, Kovach Computing Services) version 3.13p was
used. Dendrograms were constructed using the
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 2 of 11

Page 3

restriction patterns generated by different restriction
enzymes for 12 framework species. The dendrograms
show the utility of these enzymes in distinguishing dif-
ferent strains.
Results
In the present study, 16S rRNA gene sequences belong-
ing to 12 different species from the genus Streptococcus
were analyzed with the aim to construct phylogenetic
framework, identification of species-specific signatures
and restriction enzyme analysis.
Phylogenetic framework
Phylogenetic tree (Additional file 1: Fig. S1) based on 61
sequences of S. pyogenes revealed 8 clusters. 8 sequences
representing these 8 clusters were chosen. These
sequences could represent genetic heterogeneity present
within this species. Similarly, sequences from other
Streptococcus spp. were analysed for genetic heterogene-
ity present within them (Additional file 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12: Fig. S2-S12). Different representative
sequences were choosen from each species that could
provide information regarding the range of genetic
variability present within each species (Table 2). 63 such
representative sequences were selected for framework
construction (Figure 1). Strains of all the species were
clearly segregated except for S. pneumoniae and S. mitis
suggesting a high level of similarity between strains of
these two species, making their identification difficult
solely on the basis of 16S rRNA gene.
The framework generated was then used to check if
uncharacterized Streptococcus spp. can be classified
among the framework species (Figure 2). Out of 63
sequences previously identified upto genus level, 43
were found to segregate with 7 Streptococcus framework
species, supported by high bootstrap values. Among
these 43 sequences, 3 segregated with S. anginosus, 21
with S. mitis, 6 with S. pneumoniae and S. gallolyticus
each, 5 with S. bovis-equinus, 1 with S. dysgalactiae and
S. thermophilus each (Figure 2). No strains could be seg-
regated with S. mutans, S. pyogenes, S. equi, S. agalac-
tiae and S. sobrinus. The framework based segregation
was further validated by checking for the presence of
species-specific signatures and restriction analysis.
Signature sequences
Out of 20 signatures identified for each of the 12 Strep-
tococcus framework species, only 1-5 unique signatures
were found (Table 3) in framework species. The unique
signatures were found in: S. mutans: 5; S. dysgalactiae:
3; S. equi, S. sobrinus and S. thermophilus: 2 each; S. gal-
lolyticus, S. agalactiae, S. pyogenes, S. bovis-equinus, S.
anginosus and S. pneumoniae: 1 each. These signatures
were found to occur with high frequency. Moreover,
these signatures were also found to be highly conserved
across a particular species showing 98-100% sequence
identity but were found to be fragmented in other spe-
cies. No unique signature could be identified for S.
mitis. But S. mitis can still be distinguished from S.
pneumoniae using the signature found unique to S.
pneumoniae. In S. mitis the signature was found to be
substituted at specific positions and thus can distinguish
these two species (Table 3). The signature found for S.
bovis-equinus was effective in distinguishing it from very
closely related species like S. lutetiensis and S. gallolyti-
cus. These signatures were further used to validate the
segregation of 43 sequences among 7 different frame-
work species. All 43 sequences were found to contain
the signature unique to the particular species thus vali-
dating the affiliation of these sequences to a particular
species.
Restriction enzyme analysis
In-silico restriction enzyme analysis using eleven type II
enzymes revealed different patterns. Restriction sites for
AluI, BfaI, HaeIII, MspI, RsaI and Sau3AI occurred with
frequency of 3-10 resulting in 4-11 fragments. The sites
for enzymes EcoRI, SmaI and HhaI were found in
majority of sequences studied but they were found to be
less frequent cutters producing single, single and double
cuts respectively. These enzymes thus are less informa-
tive and serve no purpose. Inspite of low frequency,
BamHI and HindIII can still be used for distinguishing
different Streptococcus spp. BamHI produced single but
unique cut in S. thermophilus and can be used to distin-
guish S. thermophilus isolates. HindIII produces single
but unique cut in S. sobrinus and can be used to distin-
guish S. sobrinus isolates. As can be seen from the den-
drograms (Additional file 13, 14, 15, 16, 17, 18: Fig. S13-
S18) different restriction enzymes can be used for iden-
tification and distinguishing different isolates. While
AluI was found to distinguish majority of Streptococcus
Table 1 Restriction enzymes used in the present study
S.No.Restriction enzymeCut site
AG▼CT
G▼GATCC
C▼TAG
G▼AATTC
GG▼CC
GCG▼C
A▼AGCTT
C▼CGG
GT▼AC
▼GATC
CCC▼GGG
1.
AluI
2.
BamHI
3.
BfaI
4.
EcoRI
5.
HaeIII
6.
HhaI
7.
HindIII
8.
MspI
9.
RsaI
10.
Sau3AI
11.
SmaI
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 3 of 11

Page 4

framework species, MspI produced maximum numbers
of cuts and thus proved informative for such analysis.
Closely related species like S. dysgalactiae and S. agalac-
tiae can be distinguished using BfaI, MspI and HaeIII.
Similarly, S. gallolyticus and S. bovis can be distin-
guished using BfaI and HaeIII. Therefore a combination
of AluI, BfaI, MspI or HaeIII can be used for distin-
guishing closely related organisms. The sequences segre-
gated with framework species were further validated
using in-silicorestriction enzyme analysis. The identified
sequences showed unique restriction enzyme pattern
close to the nearby framework species (Table 4) again
validating the framework based segregation. Thus com-
bining the information from framework, signature
sequences and restriction enzyme analysis it was possi-
ble to identify 43 sequences (out of 63) upto species
level which were previously designated as Streptococcus
sp. (Table 4).
Discussion
Streptococcus is a clinically important genus as a num-
ber of species belonging to this genus are human and
animal pathogens. This genus has undergone consider-
able taxonomic revisions and has been divided into dif-
ferent groups based on 16S rRNA gene sequence
similarity.
The present study aims to explore internal features of
16S rRNA gene sequences of different Streptococcus spp.
to develop methods for their identification. A phyloge-
netic framework was constructed using different repre-
sentative sequences followed by identification of
signature sequences and restriction enzymes analysis.
The framework based analysis suggests a high level of
genetic heterogeneity present within different Streptococ-
cus spp. Signature sequences specific for each Strepto-
coccus framework sp. were identified. These signature
motifs would be simple to use as a supplement to the
automated identification process. Restriction analysis has
proved to be an important tool to identify newly isolated
strains [50-52] and can be exploited for describing new
species [53]. Multiple restriction enzyme usage is
recommended for better resolution. Although it has
been documented that closely related species cannot be
distinguished solely on the basis of 16S rRNA gene, but
exploring the internal features of this gene can be of
definite use. Therefore researchers are now looking to
explore the unique features of 16S rRNA gene that have
not been explored yet [54,55]. As already described the
genus Streptococcus has been divided into different
groups based on 16S rRNA gene similarity [4]. The fra-
mework species used for these analyses belong to these
different groups.
Four framework species- S. pyogenes, S. agalactiae, S.
equi and S. dysgalactiae belong to pyogenic group
which is the largest group of the genus Streptococcus. S.
pyogenes, S. agalactiae, S. equi and S. dysgalactiae can
Table 2 Sequences used for generating phylogenetic framework
SpeciesNo. of
sequences
used
No. of clusters
obtained
No. of representative
sequences
Accession number of representative sequences
S.
dysgalactiae
8655AB002487, EU660339, AB002511, AB159678, EU075068
S. equi
6155EF406002, FM204883, EF406019, AB002516T, AJ605748T
S. pyogenes
61 88FJ798740, FJ662832, FJ662840, AB002521, AE004092, FJ662845,
CP000003, EU660342
AF459432, AB023574, AF015927, AB175037T, X59032
S. agalactiae 2945
S. bovis-
equinus
3133 AF104109, AJ305257, DQ148956
S.
gallolyticus
7655AF104114, EU163502, EU163482, EU163499, AF459431
S. mutans
10266 DQ677759, DQ677739, DQ677777, AE014133, AF139600, DQ677743,
S. sobrinus
2344 AB294731, DQ677789, DQ677790, DQ677798
AF003929T, AJ295853, EU200182, AM157440, DQ232533, AM157420,
AY281076, AB002520, AY281078
S. mitis
2879
S.
pneumoniae
4134AE008386, CP001015, AJ617796, AY525795
S.
thermophilus
7334EF990662, X68418T, EU419603, FJ749326
S. anginosus
3255AY986762, AF145239, AF104678T, AY986764, DQ232517
Total6435963
Type strains are denoted by “T” as superscript.
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 4 of 11

Page 5

100
S. agalactiaeT(AB175037)
S. agalactiae (AB023574)
S. agalactiae(AF015927)
S. agalactiae (AF459432)
S. agalactiae (X59032)
S. dysgalactiae subsp. dysgalactiae (AB159678)
S. dysgalactiae subsp. equisimilis (EU075068)
S. dysgalactiae subsp. dysgalactiae (AB002511)
S. dysgalactiae subsp. dysgalactiae (EU660339)
S. dysgalactiae subsp. dysgalactiae (AB002487)
348
423
1000
1000
735
1000
983
1000
951
S. pyogenes (FJ662845)
S. pyogenes (CP000003)
S. pyogenes (FJ662832)
S. pyogenes (EU660342)
S. pyogenes (FJ662840)
S. pyogenes (FJ798740)
S. pyogenes (AE004092)
S. pyogenes (AB002521)
S. equi subsp. equi (EF406002)
S. equi subsp. equi (FM204883)
S. equi subsp. zooepidemicusT(AB002516)
S. equi subsp. ruminatorum T(AJ605748)
S. equi subsp. zooepidemicus (EF406019)
S. gallolyticus subsp. gallolyticus (EU163482)
S. gallolyticus subsp. gallolyticus (AF104114)
S. gallolyticus subsp. gallolyticus (EU163499)
S. gallolyticus subsp. pasteurianus (EU163502)
S. gallolyticus subsp. macedonicus (AF459431)
S. equinus (AJ305257)
S. equinus (DQ148956)
S. bovis(AF104109)
S. mutans (AE014133)
S. mutans (DQ677777)
S. mutans (DQ677739)
S. mutans (DQ677759)
S. mutans (AF139600)
S. mutans (DQ677743)
S. sobrinus (DQ677790)
S. sobrinus (DQ677789)
S. sobrinus (DQ677798)
S. sobrinus (AB294731)
S. thermophilus(FJ749326)
S. thermophilus (EU419603)
S. thermophilus (EF990662)
S. thermophilus T(X68418 )
S. anginosus(AY986762)
S. anginosusT(AF104678)
S. anginosus (AF145239)
S. anginosus (AY986764)
S. anginosus (DQ232517)
S. pneumoniae(CP001015)
S. pneumoniae (AE008386)
S. mitis (AJ295853)
S. pneumoniae (AJ617796)
S. mitis (DQ232533)
S. mitis T(AF003929)
S. mitis (AM157420)
S. mitis (AM157440)
891
S. mitis (EU200182)
S. pneumoniae (AY525795)
S. mitis (AY281076)
S. mitis (AY281078)
S. mitis (AB002520)
E. coli (X80725)
481
759
472
867
797
997
1000
821
860
1000
998
1000
560
647
629
906
883
517
1000
1000
210
495
354
344
359
1000
532
858
1000
974
309
838
636
1000
304
898
992
1000
979
311
765
572
351
307
346
633
959
727
996
458
493
1000
Figure 1 Phylogenetic tree based on 63 representative 16S rRNA gene sequences from 12 Streptococcus species. The tree was
constructed by neighbour-joining method with Jukes and Cantor correction. The numbers at node represent bootstrap values (based on 1000
resampling). The accession numbers are shown in parenthesis.
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 5 of 11

Page 6

100
Streptococcus sp. (AF316591)
S. pneumoniae (CP001015)
S. pneumoniae (AE008386)
Streptococcus sp. (AF316596)
Streptococcus sp. (AF316593)
Streptococcus sp. (AF316594)
S. pneumoniae (AJ617796)
S. mitis (AJ295853)
Streptococcus sp.(AF316592)
S. mitis (DQ232533)
Streptococcus sp. (AF316595)
S. mitis (AM157440)
S. mitisT(AF003929)
Streptococcus sp. (EF151147)
S. mitis (AM157420)
S. pneumoniae (AY525795)
S. mitis (EU200182)
Streptococcus sp. (EF151145)
Streptococcus sp. (AF385523)
Streptococcus sp. (FJ405281)
Streptococcus sp. (AY880050)
Streptococcus sp. (AY880051)
468
Streptococcus sp. (AF385526)
Streptococcus sp. (AB038371)
Streptococcus sp. (EF151150)
Streptococcus sp. (AY005041)
S. mitis (AY281076)
953
Streptococcus sp. (AF479579)
Streptococcus sp. (AY005040)
Streptococcus sp. (AY134908)
Streptococcus sp. (AY741062)
Streptococcus sp. (AY741061)
1000
S. mitis (AY281078)
Streptococcus sp. (AJ871182)
Streptococcus sp. (AF385525)
Streptococcus sp. (AB174792)
Streptococcus sp. (AB174791)
Streptococcus sp. (AY005047)
S. mitis (AB002520)
Streptococcus sp. (AF543289)
Streptococcus sp. (X78826)
S. anginosus (AY986764)
Streptococcus sp. (X78825)
S. anginosus (AY986762)
849
S. anginosus (AF145239)
S. anginosus (DQ232517)
Streptococcus sp. (AY049738)
994
546
537
541
836
376
145
86
133
493
160
333
108
506
493
299
127
477
160
183
998
162
682
228
110
157
828
758
465
533
909
625
427
477
977
581
264
402
611
805
632
S. anginosusT(AF104678)
996
1000
938
709
271
Streptococcus sp. (AF221604)
S. gallolyticus subsp. gallolyticus (EU163499)
Streptococcus sp. (DQ471794)
S. gallolyticus subsp. pasteurianus (EU163502)
S. gallolyticus subsp. gallolyticus (EU163482)
S. gallolyticus subsp. gallolyticus (AF104114)
S. gallolyticus subsp. macedonicus (AF459431)
Streptococcus sp. (AF298198)
Streptococcus sp. (AB295598)
Streptococcus sp. (AF084834)
Streptococcus sp. (AF298197)
Streptococcus sp. (AJ937757)
S. equinus (AJ305257)
Streptococcus sp. (GQ139522)
Streptococcus sp. (AF298199)
Streptococcus sp. (FJ611789)
433
Streptococcus sp. (FJ611790)
S. bovis (AF104109)
S. equinus (DQ148956)
Streptococcus sp. (AY098489)
Streptococcus sp. (AB448942)
Streptococcus sp. (AJ871184)
S. thermophilus (EU419603)
Streptococcus sp. (AF349932)
664
S. thermophilus (FJ749326)
S. thermophilus (EF990662)
S. thermophilusT(X68418)
Streptococcus sp. (EF015474)
S. dysgalactiae subsp. equisimilis (EU075068)
S. dysgalactiae subsp. dysgalactiae (AB159678)
S. dysgalactiae subsp. dysgalactiae (AB002511)
S. dysgalactiae subsp. dysgalactiae (AB002487)
S. dysgalactiae subsp. dysgalactiae (EU660339)
979
S. agalactiae (AB023574)
S. agalactiae (AF459432)
S. agalactiae (X59032)
898
881
389
717
864
591
952
515
609
395
834
665
996
1000
1000
817
241
512
534
1000
105
36
365
647
997
995
1000
S. agalactiaeT(AB175037)
S. agalactiae (AF015927)
976
1000
1000
983
S. pyogenes (FJ662832)
S. pyogenes (FJ662845)
836
S. pyogenes (FJ662840)
S. pyogenes (EU660342)
S. pyogenes (FJ798740)
S. pyogenes (CP000003)
S. pyogenes (AE004092)
S. pyogenes (AB002521)
Streptococcus sp. (AB002517)
Streptococcus sp. (AJ131965)
Streptococcus sp. (AB469559)
Streptococcus sp. (AM941161)
Streptococcus sp. (AM941160)
Streptococcus sp. (AJ871181)
Streptococcus sp. (FJ405354)
Streptococcus sp. (AY442818)
Streptococcus sp. (EU483249)
Streptococcus sp. (EU483250)
Streptococcus sp. (EU483248)
Streptococcus sp. (EU483251)
S. equi subsp. equi (EF406002)
S. equi subsp. equi (FM204883)
S. equi subsp. ruminatorum T(AJ605748)
S. equi subsp. zooepidemicusT(AB002516)
S. equi subsp. zooepidemicus (EF406019)
Streptococcus sp. (AB469560)
S. mutans (DQ677777)
S. mutans (AE014133)
450
S. mutans (DQ677739)
S. mutans (DQ677759)
S. mutans (AF139600)
S. mutans (DQ677743)
S. sobrinus (DQ677790)
S. sobrinus (DQ677789)
630
S. sobrinus(DQ677798)
S. sobrinus (AB294731)
Streptococcus sp. (AJ871191)
Streptococcus sp. (Y07601)
Streptococcus sp. (FN377818)
Streptococcus sp. (FN377819)
501
857
493
566
970
1000
921
825
566
112
1000
1000
263
335
1000
999
1000
176
35
114
774
1000
998
999
635
378
320
238
1000
244
841
1000
415
480
1000
1000
962
1000
E. coli (X80725)
Figure 2 Phylogenetic tree of 63 framework sequences (bold values) and uncharacterized Streptococcus spp. The tree was constructed
by neighbour-joining method with Jukes and Cantor correction. The numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis.
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 6 of 11

Page 7

be distinguished on the basis of specific signatures
(Table 3) as well as using different restriction enzymes
(Additional file 13, 14, 15, 16, 17, 18: Figures S13-S18).
Two framework species, S. pneumoniae and S. mitis
belong to mitis group. Identification of members of mitis
group, particularly S. pneumoniae is problematic. Identi-
fication of S. pneumoniae isolates is usually done using
serological [56,57] and molecular techniques [58-60]. S.
pneumoniae isolates can be easily identified using the sig-
nature sequence as given in Table 3. We could easily dis-
tinguish S. pneumoniae isolates from S. mitis using this
signature sequence. Other members of this group (S.
mitis and S. oralis) that are almost indistinguishable on
the basis of complete 16S rRNA gene sequence can be
differentiated using different restriction enzymes. S. mitis
can be distinguished from other two species of this group
by using enzyme Sau3AI. S. pneumoniae, S.mitis and S.
oralis can be distinguished from each other by exploiting
AluI and MspI (data not shown for S. oralis).
Framework species S. anginosus belongs to anginosus
group. This group consists of only 3 species (S. angino-
sus, S. intermedius and S. constellatus). Members of
anginosus group are also difficult to identify and distin-
guish. An identification scheme for differentiation of
these 3 strains was proposed by Whiley et al. [61] and
Whiley and Beighton [62]. Commercial identification
systems [63,64] and molecular methods have been used
for identifying and distinguishing these three species
[65,14]. S. anginosus can be easily identified using the
signature sequence (Table 3) and use of restriction
enzymes. Restriction enzymes AluI, BfaI, RsaI and
HaeIII can be used for distinguishing members of angi-
nosus group efficiently (data not shown for S. interme-
dius and S. constellatus).
Framework species, S. thermophilus belongs to salivar-
ius group which is closely related to bovis group [66]
and consists of only 3 species (S. salivarius, S. vestibu-
laris, S. thermophilus). S. thermophilus can be identified
by using unique signature sequence (Table 3).
Two framework species, S. bovis and S. gallolyticus
belong to bovis group. Members of bovis group, S. bovis
and S. gallolyticus are difficult to identify. Isolates of
these two species can be distinguished using the signa-
ture specific for S. gallolyticus and S. bovis. Moreover,
the use of restriction enzymes AluI, BfaI and HaeIII can
be instrumental in distinguishing them. The signature
found for S. bovis was found to be efficient in distin-
guishing S. bovis from closely related species-S. lutetien-
sis. These two species are difficult to distinguish solely
on the basis of 16S rRNA gene.
Framework species S. mutans and S. sobrinus belong
to mutans group. These two species are also difficult to
Table 3 Unique signature sequences identified for 12 Streptococcus framework spp
Streptococcus sp. Unique Signature (Nucleotides)Frequency of occurance
S. dysgalactiae
AATACA(G)TGCAAGTAGAACGCTGAGGACTGGTGCTTGCACCGGTCCAAGGA (52-101)
TGCATCACTATGAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAA (223-272)
CATTTAAAAGGTGCAATTGCATCACTATGAGATGGACCTGCG (207-246)
77/86
86/86
86/86
S. equi
AAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAACAGTGATG (420-469)
AAAGTCCATCATGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGT (476-525)
61/61
61/61
S. pyogenes
AAACGATAGCTAATACCGCATAAGAGAGACTAACGCATGTTAGTAATTTA (172-222) 56/61
S.agalactiae
GCAGTGGCTTAACCATTGTACGCTTTGGAAACTGGAGGACTTGAGTGC (613-660) 29/29
S. bovis
GCNTTTAACNCATGTTAGPuNGCTTGAAPuGPuAGCAA (178-212)31/31
S. gallolyticus
TCTTGACATCCCGATGCTATTTCTAGAGATAGAAAGTTTCTTCGGAACAT (991-1040)76/76
S. mutans
AGTAAAAGGCTATGGCTCAACCATAGTGTGCTCTGGAAACTGTCTGACTT (611-660)
ACCTGGGCTACACACGTGCTACAATGGTCGGTACAACGAGTTGCGAGCCG (1222-1271)
ATGATAATTGATTGAAAGATGCAAGCGCATCACTAGTAGATGGACCTGCG (197-246)
ACTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTAAGGTAAGAGCTT (228-277)
AACACACTGTGCTTGCACACCGTGTTTTCTTGAGTCGCGAACGGGTGAGT (70-119)
102/102
102/102
102/102
102/102
102/102
S.sobrinus
TCACACCACGAGAGTTTGTAACACCCAAAGTCGGTGAGGTAACCATTTAT (1417-1466)
AAGTGGAACGCATTGGTAACACCGGACTTGCTCCAGTGTTACTAATGAGT (54-103)
23//23
23/23
S. mitis
TPuPyGCATGACPyANPyTNNNTTPuAAAGGTGCANTTGCAPyCACTANNAGATGGA (228-277)28/28
S. pneumoniae
TGTTGCATGACATTTPuCTTAAAAGGTGCANNTGCATCACTACCAGATGGA (179-228)41/41
S. Thermophilus
ACAATGGTTGGTACAACGAGTTGCGAGTCGGTGACGGCGAGCTAATCTCT (1246-1295)
AAGATGGACCTGCGTTGTATTAGCTAGTAGGTGAGGTAATGGCTCACCTA (234-283)
73/73
73/73
S. anginosus
ATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTAGAAAAGTCTGAAGT (581-530)32/32
The signatures were found to 98-100% identical and conserved in a particular species. Py denotes pyrimidine, Pu denoted purine and N denotes any nucleotide.
Overlapping signatures are shown in bold.The number in brackets indicate the corresponding position of the signature in 16S rRNA gene.
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 7 of 11

Page 8

distinguish. Beighton et al. (1991) [8] provided a scheme
for identification of S. mutans and S. sobrinus strains. In
the present investigations these two can be easily distin-
guished using species-specific signature and use of
restriction enzymes- AluI, BfaI, HaeIII, MspI, RsaI and
Sau3AI.
Conclusions
The species that are difficult to distinguish solely on the
basis of 16S rRNA gene sequence can be identified
using inner secrets of 16S rRNA gene, the signatures.
These signatures can be exploited for quick identifica-
tion. The aim of phylogenetic framework construction is
Table 4 Streptococcus spp. identified upto species level:
S. No. Acccession no. Close framework speciesPresence of species-specific signaturesUnique RE pattern
1
EF015474
S. dysgalactiae
Y
S. dysgalactiae (MspI)
2
AF221604
DQ471794
AF298198
AB295598
AF084834
AF298197
S. gallolyticus
Y
S. gallolyticus (BfaI)
3
AF349932
S. thermophilus
Y
S. thermophilus (AluI, HaeIII Sau3AI,BamHI)
4
X78826
X78825
AY049738
S. anginosus
Y
S. anginosus (AluI, BfaI, HaeIII)
5
AF316591
AF316596
AF316593
AF316594
AF316595
EF151147
S. pneumonaie
Y
S. pneumonaie (AluI, MspI)
6
AF316592
EF151145
AF385523
FJ405281
AY880050
AY880051
AF385526
AB038371
EF151150
AY005041
AF479579
AY005040
AY134908
AY741062
AY741061
AJ871182
AF385525
AB174792
AB174791
AY005047
AF543289
S. mitis
Y
S. mitis (AluI, MspI, Sau3AI)
7
AJ937757
GQ139522
AF298199
FJ611789
FJ611790
S. bovis-equinus
Y
S. bovis-equinus (BfaI)
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 8 of 11

Page 9

to define a range of genetic variability within the species
and later exploiting this variability for identification of
different isolates. Similarly, use of restriction enzymes
help in generating markers that can distinguish closely
related species. Present study reveals that the frame-
work, use of specific signatures in 16S rRNA gene and
pattern generated by different restriction enzymes can
be exploited for identification of isolates belonging to
the genus Streptococcus. The markers generated in the
present study are based on 16S rRNA gene sequence
which is conserved and neither subjected to changes
due to culture conditions nor exhibit biochemical varia-
bility. Thus the scheme proposed can be applied to any
isolate. The approach is cost effective and rapid way for
identification of various isolates and thus can be used to
differentiate isolates that are difficult to distinguish due
to very close traits and biochemical features. Addition-
ally the approach is simple for preliminary identification
of a species and can supplement existing automated
identification processes. But we should keep in mind
that this is a simplified procedure and thus it is also
important to know the limitations of such simplified
approach.
Additional material
Additional file 1: Phylogenetic tree based on 61, 16S rRNA gene
sequences of Streptococcus pyogenes. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 2: Phylogenetic tree based on 86, 16S rRNA gene
sequences of Streptococcus dysgalactiae. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 3: Phylogenetic tree based on 29, 16S rRNA gene
sequences of Streptococcus agalactiae. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 4: Phylogenetic tree based on 61, 16S rRNA gene
sequences of Streptococcus equi. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 5: Phylogenetic tree based on 31, 16S rRNA gene
sequences of Streptococcus bovis-equinus. The tree was constructed
by neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 6: Phylogenetic tree based on 76, 16S rRNA gene
sequences of Streptococcus gallolyticus. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 7: Phylogenetic tree based on 102, 16S rRNA gene
sequences of Streptococcus mutans. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 8: Phylogenetic tree based on 23, 16S rRNA gene
sequences of Streptococcus sobrinus. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 9: Phylogenetic tree based on 28, 16S rRNA gene
sequences of Streptococcus mitis. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 10: Phylogenetic tree based on 41, 16S rRNA gene
sequences of Streptococcus pneumoniae. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 11: Phylogenetic tree based on 32, 16S rRNA gene
sequences of Streptococcus anginosus. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 12: Phylogenetic tree based on 73, 16S rRNA gene
sequences of Streptococcus thermophilus. The tree was constructed by
neighbour-joining method with Jukes and Cantor correction. The
numbers at node represent bootstrap values (based on 1000 resampling).
The accession numbers are shown in parenthesis. Bold sequences
indicate those which are used for final framework construction.
Additional file 13: Dendrogram based on restriction digestion of 12
Streptococcus framework spp. with AluI.
Additional file 14: Dendrogram based on restriction digestion of 12
Streptococcus framework spp. with BfaI.
Additional file 15: Dendrogram based on restriction digestion of 12
Streptococcus framework spp. with HaeIII.
Additional file 16: Dendrogram based on restriction digestion of 12
Streptococcus framework spp. with MspI.
Additional file 17: Dendrogram based on restriction digestion of 12
Streptococcus framework spp. with RsaI.
Additional file 18: Dendrogram based on restriction digestion of 12
Streptococcus framework spp. with Sau3AI.
Acknowledgements
DL and MV acknowledge research fellowship provided by CSIR.
Authors’ contributions
DL, MV and RL conceived and designed the experiments. DL and MV
performed the experiments. DL, MV and RL analyzed the data. DL, MV and
RL wrote the manuscript. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 30 March 2011 Accepted: 25 June 2011
Published: 25 June 2011
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 9 of 11

Page 10

References
1.Ryan KJ, Ray CG: Sherris Medical Microbiology. McGraw Hill;, 4 2004.
2.Hardie JM, Whiley RA: The genus Streptococcus. In The Genera of Lactic
Acid Bacteria. Volume 2. Edited by: Wood BJB, Holz-apfel WH. Blackie
Academic and Professional; 1995:55-124.
3.Wilson CD, Salt GFH: Streptococci in animal disease. In Streptococci. Society
for Applied Bacteriology Symposium Series No. 7. Edited by: Skinner FA,
Quesnel LB. London, New York, San Francisco: Academic Press;
1978:143-156.
4.Kawamura Y, Hou X-G, Sultana F, Miura H, Ezaki T: Determination of 16S
rRNA sequences of Streptococcus mitis and Streptococcus gordonii and
phylogenetic relationships among members of the genus Streptococcus.
Int J Syst Bacteriol 1995, 45:406-408.
5.Farrow JAE, Collins MD: Taxonomic studies on streptococci of serological
groups C, G and L and possibly related taxa. Syst Appl Microbiol 1984,
5:483-493.
6.Lawrence J, Yajko DM, Hadley WK: Incidence and characterization of beta-
hemolytic Streptococcus milleri and differentiation from S. pyogenes
(group A), S. equisimilis (group C), and large-colony group G
streptococci. J Clin Microbiol 1985, 22:772-777.
7.Kilian M, Mikkelsen L, Henrichsen J: Taxonomic studies of viridans
streptococci: description of Streptococcus gordonii sp. nov. and emended
descriptions of Streptococcus sanguis (White and Niven 1946),
Streptococcus oralis (Bridge and Sneath 1982), and Streptococcus mitis
(Andrewes and Horder 1906). Int J Syst Bacteriol 1989, 39:471-484.
8.Beighton D, Russell RRB, Whiley RA: A simple biochemical scheme for the
differentiation of Streptococcus mutans and Streptococcus sobrinus. Caries
Res 1991, 25:174-178.
9.Hillmann JD, Andrews SW, Painter S, Stashenko P: Adaptive changes in a
strain of Streptococcus mutans during colonization of the human oral
cavity. Microb Ecol Health Dis 1989, 2:231-239.
10.Tardif G, Sulavik MC, Jones GW, Clewell DB: Spontaneous switching of the
sucrose-promoted colony phenotype in Streptococcus sanguis. Infect
Immun 1989, 57:3945-3948.
11.Schmidhuber S, Ludwig W, Schleifer KH: Construction of a DNA probe for
the specific identification of Streptococcus oralis. J Clin Microbiol 1988,
26:1042-1044.
12.Adnan S, Li N, Miura H, Hashimoto Y, Yamamoto H, Ezaki T: Covalently
immobilized DNA plate for luminometric DNA-DNA hybridization to
identify viridans streptococci in under 2 hours. FEMS Microbiol Lett 1993,
106:139-142.
13.Bentley RW, Leigh JA: Development of PCR-based hybridization protocol
for identification of streptococcal species. J Clin Microbiol 1995,
33:1296-1301.
14.Jacobs JA, Schot CS, Bunschoten AE, Schouls LM: Rapid species
identification of “Streptococcus milleri“ strains by line blot hybridization:
identification of a distinct 16S rRNA population closely related to
Streptococcus constellatus. J Clin Microbiol 1996, 34:1717-1721.
15.Jayarao BM, Dore JJ Jr, Oliver SP: Restriction fragment length
polymorphism analysis of 16S ribosomal DNA of Streptococcus and
Enterococcus species of bovine origin. J Clin Microbiol 1992, 30:2235-2240.
16.Rudney JD, Larson CJ: Use of restriction fragment polymorphism analysis
of rRNA genes to assign species to unknown clinical isolates of oral
viridans streptococci. J Clin Microbiol 1994, 32:437-443.
17.Gillespie BE, Jayarao BM, Oliver SP: Identification of Streptococcus species
by randomly amplified polymorphic deoxyribonucleic acid
fingerprinting. J Dairy Sci 1997, 80:471-476.
18.Chen CC, Teng LJ, Chang TC: Identification of clinically relevant viridians
group streptococci by sequence analysis of the 16S-23S ribosomal DNA
spacer region. J Clin Microbiol 2004, 42:2651-2657.
19.Nielsen XC, Justesen US, Dargis R, Kemp M, Christensen JJ: Identificatoin of
clinically relevant nonhemolytic streptococci on the basis of sequence
analysis of 16S-23S intergenic spacer region and partial gdh gene. J Clin
Microbiol 2009, 47:932-939.
20.Saruta K, Matsunaga T, Hoshina S, Kono M, Kitahara S, Kanemoto S, Sakai O,
Machida K: Rapid identification of Streptococcus pneumoniae by PCR
amplification of ribosomal DNA spacer region. FEMS Microbiol Lett 1995,
132:165-170.
21.Whiley RA, Duke B, Hardie JM, Hall LMC: Heterogeneity among 16S-23S
rRNA intergenic spacers of species within the ‘Streptococcus milleri
group’. Microbiol 1995, 141:1461-1467.
22.Garnier F, Gerbaud G, Courvalin P, Galimand M: Identification of clinically
relevant viridans group streptococci to the species level by PCR. J Clin
Microbiol 1997, 35:2337-2341.
Kawamura Y, Whiley RA, Shu SE, Ezaki T, Hardie JM: Genetic approaches to
the identification of the mitis group within the genus Streptococcus.
Microbiol 1999, 145:2605-2613.
Igarashi T, Ichikawa K, Yamamoto A, Goto N: Identification of mutans
streptococcal species by the PCR products of dex genes. J Microbiol
Methods 2001, 46:99-105.
Hung WC, Tsai JC, Hsueh PR, Chia JS, Teng LJ: Species identification of
mutans streptococci by groESL gene sequence. J Med Microbiol 2005,
54:857-862.
Teng LJ, Hsueh PR, Tsai JC, Chen PW, Hsu JC, Lai HC, Lee CN, Ho SW:
groESL sequence determination, phylogenetic analysis and species
differentiation for viridians group streptococci. J Clin Microbiol 2002,
40:3172-3178.
Tapp J, Thollesson M, Herrmann B: Phylogenetic relationships and
genotyping of the genus Streptococcus by sequence determination of
the RNase P RNA gene, rnpB. Int J Sys Evol Microbiol 2003, 53:1861-1871.
Picard FJ, Ke D, Boudreau DK, Boissinot M, Huletsky A, Richard D,
Ouellette M, Roy PH, Bergeron MG: Use if tuf sequences for genus specific
PCR detection and Phylogenetic analysis of 28 streptococcal species. J
Clin Microbiol 2004, 42:3686-3695.
Kawamura Y, Itoh Y, Mishima N, Ohkusu K, Kasai H, Ezaki T: High genetic
similarity of Streptococcus agalactiae and Streptococcus difficilis: S. difficilis
Eldar et al. 1995 is a later synonym of S. agalactiae Lehmann and
Neumann 1896 (Approved Lists 1980). Int J Syst Evol Microbio 2005,
55:961-965.
Bentley RW, Leigh JA, Collins MD: Intrageneric structure of Streptococcus
based on comparative analysis of smallsubunit rRNA sequences. Int J
Syst Bacteriol 1991, 41:487-494.
Poyart C, Quesne G, Coulon S, Berche P, Trieu-Cuot P: Identification of
streptococci to species level by sequencing the gene encoding the
manganese-dependent superoxide dismutase. J Clin Microbiol 1998,
36:41-47.
Poyart C, Quesne G, Trieu-Cuot P: Taxonomic dissection of the
Streptococcus bovis group by analysis of manganese-dependent
superoxide dismutase gene (sodA) sequences: reclassification of
‘Streptococcus infantarius subsp. coli’ as Streptococcus lutetiensis sp. nov.
and of Streptococcus bovis biotype II.2 as Streptococcus pasteurianus sp.
nov. Int J Syst Evol Microbiol 2002, 52:1247-1255.
Glazunova OO, Raoult D, Roux V: recN partial gene sequencing: a new
tool for identification and phylogeny within the Streptococcus genus. Int
J Syst Evol Microbiol 2010, 60:2140-2148.
De Gheldre Y, Vandamme P, Goossens H, Struelens MJ: Identification of
clinically relevant viridans streptococci and analysis of transfer DNA
intergenic spacer length polymorphism. Int J Syst Bacteriol 1999,
49:1591-1598.
Baele M, Storms V, Haesebrouck F, Devriese LA, Gillis M, Verschraegen G, de
Baere T, Vaneechoutte M: Application and evaluation of the
interlaboratory reproducibility of tRNA intergenic length polymorphism
analysis (tDNA-PCR) for identification of Streptococcus species. J Clin
Microbiol 2001, 39:1436-1442.
Bishop CJ, Aanensen DM, Jordan GE, Kilian M, Hanage WP, Spratt B:
Assigning strains to bacterial species via the internet. BMC Biology 2009,
7:3.
Hanage WP, Kaijalainen T, Herva E, Saukkoriipi A, Syrjänen R, Spratt BG:
Using multilocus sequence data to define the pneumococcus. J Bacteriol
2005, 187:6223-6230.
Woese CR: Bacterial evolution. Microbiol Rev 1987, 51:221-271.
Clarridge JE: Impact of 16S rRNA gene sequence analysis for
identification of bacteria on clinical microbiology and infectious
diseases. Clin Microbiol Rev 2004, 17:840-862.
Janda JM, Abbott SL: 16S rRNA gene sequencing for bacterial
identification in the diagnostic laboratory:pluses, perils, and pitfalls. J
Clin Microbiol 2007, 45:2761-2764.
Maidak BL, Cole JR, Lilburn TG, Parker CT, Saxman PR, Stredwick JM,
Garrity GM, Li B, Olsen GJ, Pramanik S, Schmidt TM, Tiedje JM: The RDP
(Ribosomal Database Project) continues. Nucl Acids Res 2000, 28:173-174.
Schlegel L, Grimont F, Ageron E, Grimont PAD, Bouvet A: Reappraisal of
the taxonomy of the Streptococcus bovis/Streptococcus equinus complex
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 10 of 11

Page 11

and related species: description of Streptococcus gallolyticus subsp.
gallolyticus subsp. nov., S. gallolyticus subsp. macedonicus subsp. nov.
and S. gallolyticus subsp. pasteurianus subsp. nov. Int J Syst Evol Microbiol
2003, 53:631-645.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The
CLUSTAL_X windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Res 1997,
25:4876-4882.
Felsenstein J: Phylip (Phylogeny Inference Package) version 3.57c.
Department of Genetics, University of Washington, Seattle; 1993 [http://
evolution.genetics.washington.edu/phylip.html].
Saitou N, Nei M: The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol Biol Evol 1987, 4:406-425.
Jukes TH, Cantor CR: Evolution of protein molecules. In Mammalian
Protein Metabolism. Edited by: Munro HN. New York: Academic Press;
1969:21-132.
Page RDM: TreeView: an application to display phylogenetic trees on
personal computer. Comput Appl Biosci 1996, 12:357-35.
Clamp M, Cuff J, Searle SM, Barton GJ: “The Jalview Java Alignment
Editor”. Bioinformatics 2004, 20:426-7.
Bailey TL, Williams N, Misleh C, Li WW: MEME: discovering and analyzing
DNA and protein sequence motifs. Nucleic Acids Res Web Server issue 2006,
W369-W373.
Blanc M, Marilley L, Beffa T, Aragno M: Rapid identification of
heterotrophic, thermophilic, spore-forming bacteria isolated from hot
composts. Int J Syst Bacteriol 1997, 47:1246-1248.
Heyndrickx M, Vauterin L, Vandamme P, Kersters K, De Vos P: Applicability
of combined amplified ribosomal DNA restriction analysis (ARDRA)
patterns in bacterial phylogeny and taxonomy. J Microbiol Methods 1996,
26:247-259.
Heyndrickx M, Lebbe L, Kersters K, Hoste B, De Wachter R, De Vos P,
Forsyth G, Logan NA: Proposal of Virgibacillus proomii sp. nov. and
emended description of Virgibacillus pantothenticus (Proom and Knight
1950) Heyndrickx et al. 1998. Int J Syst Bacteriol 1999, 49:1083-1090.
Bradbury JM: International committee on systematic bacteriology,
subcommittee on the taxonomy of Mollicutes. Int J Syst Evol Microbiol
2001, 51:2227-2230.
Porwal S, Lal S, Cheema S, Kalia VC: Phylogeny in aid of the present and
novel microbial lineages: Diversity in Bacillus. PLoS ONE 2009, 4:e4438.
Kalia VC, Mukherjee T, Bhushan A, Joshi J, Shankar P, Huma N: Analysis of
the unexplored features of rrs (16S rDNA) of the Genus Clostridium. BMC
Genomics 2011, 12:18.
Ortqvist A, Jonsson I, Kalin M, Krook A: Comparison of three methods for
detection of pneumococcal antigen in sputum of patients with
community-acquired pneumonia. Eur J Clin Microbiol Infect Dis 1989,
8:956-961.
Sorensen UBS: Typing of pneumococcal by using 12 pooled antisera. J
Clin Microbiol 1993, 31:2097-2100.
Garcia A, Roson B, Perez JL, Verdaguer R, Dorca J, Carratala J, Casanova A,
Manresa F, Gudiol F: Usefulness of PCR and antigen latex agglutination
tests with samples obtained by transthoracic needle aspiration for
diagnosis of pneumococcal pneumonia. J Clin Microbiol 1999, 37:709-714.
Beall B, Facklam RR, Jackson DM, Starling HH: Rapid screening for
penicillin susceptibility of systemic pneumococcal isolates by restriction
enzyme profiles of the pbp2B gene. J Clin Microbiol 1998, 36:2359-2362.
Toikka P, Nikkari S, Ruuskanen O, Leinonen M, Mertsola J: Pneumolysin
PCR-based diagnosis of invasive pneumococcal infection in children. J
Clin Microbiol 1999, 37:633-637.
Whiley RA, Fraser H, Hardie JM, Beighton D: Phenotypic differentiation of
Streptococcus intermedius, Streptococcus constellatus, and Streptococcus
anginosus strains with the “Streptococcus milleri group.”. J Clin Microbiol
1990, 28:1497-1501.
Whiley RA, Beighton D: Amended descriptions and recognition of
Streptococcus constellatus, Streptococcus intermedius, and Streptococcus
anginosus as distinct species. Int J Syst Bacteriol 1991, 41:1-5.
Facklam R: What Happened to the Streptococci: Overview of Taxonomic
and Nomenclature Changes. Clinical Microbial Reviews 2002, 15:613-630.
Von Baum H, Klemme FR, Geiss HK, Sonntag HG: Comparative evaluation
of a commercial system for identification of gram positive cocci. Eur J
Clin Microbiol Infect Dis 1998, 17:849-852.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.Bartie KL, Wilson MJ, Williams DW, Lewis AO: Macrorestriction
fingerprinting of “Streptococcus milleri“ group bacteria by pulse-field gel
electrophoresis. J Clin Microbiol 2000, 38:2141-2149.
Whiley RA, Beighton D: Current classification of the oral streptococci. Oral
Microbiol Immunol 1998, 13:195-216.
66.
doi:10.1186/1476-0711-10-28
Cite this article as: Lal et al.: Exploring internal features of 16S rRNA
gene for identification of clinically relevant species of the genus
Streptococcus. Annals of Clinical Microbiology and Antimicrobials 2011 10:28.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Lal et al. Annals of Clinical Microbiology and Antimicrobials 2011, 10:28
http://www.ann-clinmicrob.com/content/10/1/28
Page 11 of 11