Infection, Genetics and Evolution 33 (2015) 381–392

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Genomics of Streptococcus salivarius, a major human commensal

Christine Delorme, Anne-Laure Abraham, Pierre Renault, Eric Guédon ⇑
INRA, UMR 1319 Micalis, Domaine de Vilvert, F-78352 Jouy-en-Josas, France
AgroParisTech, UMR MICALIS, Jouy-en-Josas, France

a r t i c l e i n f o a b s t r a c t

Article history: The salivarius group of streptococci is of particular importance for humans. This group consists of three
Received 10 July 2014 genetically similar species, Streptococcus salivarius, Streptococcus vestibularis and Streptococcus thermophilus.
Received in revised form 30 September 2014 S. salivarius and S. vestibularis are commensal organisms that may occasionally cause opportunistic
Accepted 2 October 2014
infections in humans, whereas S. thermophilus is a food bacterium widely used in dairy production. We
Available online 13 October 2014
developed Multilocus sequence typing (MLST) and comparative genomic analysis to confirm the clear
separation of these three species. These analyses also identified a subgroup of four strains, with a core
Keywords:
genome diverging by about 10%, in terms of its nucleotide sequence, from that of S. salivarius sensu stricto.
MLST
Comparative genomic
S. thermophilus species displays a low level of nucleotide variability, due to its recent emergence with the
Streptococci development of agriculture. By contrast, nucleotide variability is high in the other two species of the
HGT salivarius group, reflecting their long-standing association with humans. The species of the salivarius
Diversity group have genome sizes ranging from the smallest (1.7 Mb for S. thermophilus) to the largest
Adaptation (2.3 Mb for S. salivarius) among streptococci, reflecting genome reduction linked to a narrow, nutrition-
ally rich environment for S. thermophilus, and natural, more competitive niches for the other two species.
Analyses of genomic content have indicated that the core genes of S. salivarius account for about two
thirds of the genome, indicating considerable variability of gene content and differences in potential
adaptive features. Furthermore, we showed that the genome of this species is exceptionally rich in genes
encoding surface factors, glycosyltransferases and response regulators. Evidence of widespread genetic
exchanges was obtained, probably involving a natural competence system and the presence of diverse
mobile elements. However, although the S. salivarius strains studied were isolated from several human
body-related sites (all levels of the digestive tract, skin, breast milk, and body fluids) and included clinical
strains, no genetic or genomic niche-specific features could be identified to discriminate specific group.
Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction
The salivarius group consists of three genetically similar species
(Kawamura et al., 1995; Poyart et al., 1998): Streptococcus salivarius
and Streptococcus vestibularis, two commensal bacteria, and Strep-
tococcus thermophilus, a nonpathogenic dairy species belonging to
the viridans group of streptococci. The viridans group consists of
26 streptococcal species with similar phenotypic characteristics.
These bacteria are mostly isolated from the human oral microbial
ecosystem, and can be classified into five subgroups (anginosus,
mitis, salivarius, sanguinis and mutans) (Facklam, 2002). The sali-
varius group of the genus Streptococcus is a cluster with significant
support from sequence analysis using the 16S (Bentley et al., 1991;
Kawamura et al., 1995), sodA (Poyart et al., 1998) and rnpB genes
⇑ Corresponding author at: INRA, UMR 1319 Micalis, Domaine de Vilvert, F-78352
Jouy-en-Josas, France. Tel.: +33 1 34 65 25 25; fax: +33 1 34 65 25 21.
E-mail address: eric.guedon@jouy.inra.fr (E. Guédon).
(Tapp et al., 2003) and, more recently, 136 genes from the core
streptococcal genome (Richards et al., 2014). The most recent of
these studies showed that S. downei and S. criceti, which are usually
included in the mutans group, actually cluster tightly with the sal-
ivarius group on the phylogenetic tree (Richards et al., 2014).
S. salivarius, which can be isolated from human saliva and the
tongue dorsum, is one of the first bacteria to colonize the mucosa
in the first few days after birth (Pearce et al., 1995; Rotimi et al.,
1985). This key streptococcal species is maintained as a dominant
population in the human oral cavity and the upper airways,
throughout the life of its human carrier (Nakajima et al., 2013;
Tappuni and Challacombe, 1993). S. salivarius is also one of the pri-
mary inhabitants of the intestinal microbiota. A metagenomic
analysis of the feces revealed its presence in more than 90% of
the individuals analyzed to generate the first human gut microbial
gene catalog (it was formerly described as S. thermophilus due to a
lack of appropriate genomic references) (Qin et al., 2010). In the
first few days after birth, S. salivarius has been recovered from fecal

http://dx.doi.org/10.1016/j.meegid.2014.10.001
1567-1348/Ó 2014 Elsevier B.V. All rights reserved.
382 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392
samples collected from preterm infants and newborns (Favier
et al., 2002; Millar et al., 1996; Park et al., 2005). The proportions
of streptococci in the bacterial community differ along the length
of the human intestinal tract, with these species predominating
in the jejunum, present in the distal ileum and absent from the
ascending colon and rectum (Booijink et al., 2010; Wang et al.,
2005). S. salivarius was isolated from jejunum biopsy specimens
and from samples collected from subjects undergoing ileostomy
(van den Bogert et al., 2013b; Wang et al., 2005). S. salivarius was
also detected in microbiota from the human stomach, cecum and
rectosigmoidal colon (Hakalehto et al., 2011; Hayashi et al.,
2005). The abundance of streptococci among which S. salivarius
represents a significant part in intestinal metagenomic samples
indicates that these bacteria achieve a true development rather
than being washed down with saliva flow (Qin et al., 2010; Qin
et al., 2014; van den Bogert et al., 2013b; Zoetendal et al., 2012).
No S. salivarius strain has yet been unambiguously characterized
from non-human sources, suggesting that this bacterium may be
considered specific to humans.
Several reports have indicated that S. salivarius plays a positive
role in oral and digestive tract ecology. S. salivarius may exert its
impact on human health through effects on the stability of micro-
biota composition, bacterial interference and interaction with the
host. Streptococcus is the dominant genus in the tonsillar crypts
of children aged two to four years with tonsillar hyperplasia, and
a decrease in the abundance of S. salivarius/S. vestibularis has been
reported in children with recurrent tonsillitis (Jensen et al., 2013).
The predominance of S. salivarius in microbial profiles is associated
with stabilization, as opposed to acute cystic fibrosis (Filkins et al.,
2012). A low abundance of S. salivarius in patients with halitosis
has been reported, but not in all studies (Kazor et al., 2003;
Riggio et al., 2008). Negative bacterial interference plays a
fundamental role in surface colonization and in the limitation of
pathogen emergence. The antagonistic effects of S. salivarius have
been described in competition with S. mutans and Streptococcus
sobrinus for tooth sites during initial oral colonization (Tanzer
et al., 1985), and during the adhesion of Streptococcus pyogenes to
the human pharyngeal cell layer (Guglielmetti et al., 2010). S. sali-
varius has also been shown to inhibit epithelial colonization by the
periodontal pathogen Aggregatibacter actinomycetemcomitans
(Sliepen et al., 2009b; Teughels et al., 2007). This predominant col-
onizer of the oral cavity is also the main producer of bacteriocins,
such as lantibiotics in particular (Hyink et al., 2007; Ross et al.,
1993; Wescombe et al., 2012a; Wescombe et al., 2011). These com-
pounds inhibit the growth of Streptococcus pneumoniae (Santagati
et al., 2012), S. pyogenes (Dempster and Tagg, 1982) and other spe-
cies (Wescombe et al., 2011). Antimicrobial peptides with broad-
spectrum activity, including those from the streptococci cited
above, have also been characterized from strains recovered from
fecal samples (Birri et al., 2012; O’Shea et al., 2009). S. salivarius
K12, a bacteriocin-producing strain, is used as an oral probiotic,
initially in New Zealand and now widely in the world, for the pre-
vention of streptococcal pharyngitis (Burton et al., 2011; Tagg and
Dierksen, 2003). Diverse colonization outcomes and potential
applications of this strain in the control of respiratory infections
have been reported (Dierksen et al., 2007; Power et al., 2008;
Wescombe et al., 2012b). A lower incidence of bacterial throat
and ear infections was recently reported in a preliminary trial of
S. salivarius K12 administration in children (Di Pierro et al.,
2012). This strain, which inhibits the growth of the salivary bacte-
ria responsible for halitosis, can modify the factors underlying bad
breath and the composition of the oral microbiota in subjects with
halitosis (Burton et al., 2006). In addition, S. salivarius K12 has been
shown to protect against Candida albicans invasion, by inhibiting
adhesion through mechanisms independent of its antimicrobial
activity (Ishijima et al., 2012). S. salivarius has also been shown
to affect the immune response, by inhibiting the inflammatory
pathways activated by different pathogens. S. salivarius inhibited
the IL-8 production and NF-jB activation induced by the enteric
pathogens Pseudomonas aeruginosa and Salmonella enterica serovar
Typhimurium and a periodontal pathogen (Cosseau et al., 2008;
Frick et al., 2007; Sliepen et al., 2009a). An immunomodulatory
response to S. salivarius was established in several oral cell lines
but also in intestinal epithelial cells (Frick et al., 2007; Kaci et al.,
2011). The downregulation of other molecular functions was dem-
onstrated in global analyses of human bronchial epithelial cells
(Cosseau et al., 2008). The anti-inflammatory potential of S. saliva-
rius was recently confirmed, in studies demonstrating an inhibition
of inflammation in mouse models of severe and moderate colitis.
The in vitro and in vivo anti-inflammatory responses were observed
with live bacteria but not with heat-killed bacteria. These
responses are, therefore, linked to the metabolic activity of S. sali-
varius (Kaci et al., 2014; Kaci et al., 2011).
The bacteria of the viridans group of streptococci are
commensal organisms, but they may cause both local and systemic
infections (Doern and Burnham, 2010; Maeda et al., 2011). S. sali-
varius displays only weakly virulent but, in opportunistic infec-
tions, it can cause invasive disease, such as bacteremia in
immunocompromised patients (Corredoira et al., 2005; Han
et al., 2006), endocarditis (Kitten et al., 2012) and meningitis after
brain abscess, cranial trauma, and cerebrospinal fluid (CSF) fistula
(Wilson et al., 2012). S. salivarius is one species of the viridans
group most frequently recovered from patients with meningitis
following spinal procedures because of contamination of the pro-
cedure site (Rubin et al., 2007; Trautmann et al., 2002). In several
recent cases of iatrogenic meningitis, the strains recovered from
the CSF of patients and the saliva of the physician concerned were
identical (Chitnis et al., 2012; Shewmaker et al., 2010; Srinivasan
et al., 2012), showing a direct contamination of the patient via a
respiratory droplet from the physician who was not wearing a face
mask. In this review, we summarize the available and new data for
the phylogeny, ecology, genomics and metabolism of the S. saliva-
rius species.
2. Materials and methods
2.1. Phylogenetic analyses
The nucleotide sequences of internal fragments from the five
selected genes of the strains identified in Fig. 1 were concatenated
(2.359-bp): ddlA (encoding D-alanine ligase), pyrE (encoding oro-
tate phosphoribosyltransferase), thrS (encoding threonyl-tRNA
synthetase), dnaE (encoding DNA polymerase III) and sodA (super-
oxide-dismutase). Phylogenetic trees of the nucleotide sequences
for each housekeeping locus were constructed by the neighbor-
joining (NJ) method using MEGA version 4 software (http://
www.megasoftware.net) (Tamura et al., 2007). A model of the
Kimura 2-parameter distance was used to estimate distances for
nucleotide sequences. The significance of the observed clusters in
the trees constructed by the NJ method was assessed by bootstrap
analysis with 1000 replicates.
2.2. Sequence data and genome mining
Details of the genome sequences used in our analyses are pre-
sented in Tables S1 and S2. All genome sequences were obtained
directly from National Center for Biotechnology Information
(NCBI). RAST facilities were used for whole-genome comparisons
(Overbeek et al., 2014). Genomes were screened for urease genes
by dedicated Blast searches using urease genes from S. salivarius
JIM8777. Carbohydrate and amino-acid biosynthesis pathways
 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392 383

Strain Species Source Location Year

100
JIM8774 JIM8774 S. sal. oral cavity 2 France 2004
JIM8776 JIM8776 S. sal. oral cavity 1 France 2004
99
SR4 (sp.) SR4 sp. human oral NA
CAG:79 (sp.) CAG:79 sp. NA NA
100 CIP53158 CIP53.158 S. sal. NA Ithaca, USA 1954
CIP55126 CIP55.126 S. sal. NA France 1955
JIM8223 JIM8223 S. sal. oral cavity 3 Germany 2000
JIM8775 JIM8775 S. sal. oral cavity 1 France 2004
SK126 SK126 S. sal. Human skin NA
CIP102503T CIP102503 T S. sal. Human blood New York, USA 1950
LMG13104 LMG13104 S. sal. NA Munchen, Germany 1983
100 HSISS1 (sp.) HSISS1 sp. ileostomy effluent Netherlands 2010
100
HSISS4 (sp.) HSISS4 sp. ileostomy effluent Netherlands 2010
JIM8777 JIM8777 S. sal. oral cavity 1 France 2004
LMG14652 LMG14652 S. sal. Human blood Ostersund, Sweden 1993
57.I 57.I S. sal. NA NA
CIP102505 CIP102505 S. sal. NA Rockfeller, USA 1971
CIP104994 CIP104994 S. sal. Human blood Tourcoing, France 1996
M18 M18 S. sal. oral swab from healthy volunteer Dunedin, New Zealand
99
JIM8221 JIM8221 S. sal. oral cavity 4 Germany 2000
CCHSS4 CCHSS4 S. sal. Human blood Cochin hosp,, France 2001
JIM8421 JIM 8421 S. sal. Breast milk Finland 2001
CCHSS7 CCHSS7 S. sal. Human blood Cochin hosp,, France 2004
JIM8772 JIM8772 S. sal. oral cavity 2 France 2004
94
LMG13108 LMG13108 S. sal. NA London hosp., UK 1983
LMG13106 LMG13106 S. sal. NA London hosp., UK 1983
100 CCHSS3 CCHSS3 S. sal. Human blood Cochin hosp,, France 2002
JIM8771 JIM8771 S. sal. oral cavity 2 France 2004
JIM8773 JIM8773 S. sal. oral cavity 2 France 2004
85 JIM8222 JIM8222 S. sal. oral cavity 4 Germany 2000
CCHSS1 CCHSS1 S. sal. Human blood Cochin hosp., France 2003
LMG13109 LMG13109 S. sal. NA London hosp., UK 1983
CCHSS2 CCHSS2 S. sal. Human blood Cochin hosp., France 2001
JIM8224 JIM8224 S. sal. oral cavity 3 Germany 2000
K12 K12 S. sal. saliva from healthy child NA
ACS2(sp.) ACS2 sp. NA NA
HSISS2 (sp.) HSISS2 sp. ileostomy effluent Netherlands 2010
100 PS4 PS4 sp. milk from a healthy woman Spain 2010
100 HSISS3 (sp.) HSISS3 sp. ileostomy effluent Netherlands 2010
98
C150 (sp.) C150 sp. human airway samples NA
100 ATCC49124 ATCC49124 S. vest. oral cavity England 1988
76 CIP103363T CIP103363 T S. vest. oral cavity London hosp., UK 1987
CCHSV6 CCHSV6 S. vest. Human blood Cochin hosp,, France 2004
LMG14647 LMG14647 S. vest. vesbular mucosa Malmö, Sweden 1966
100 LMG17855 LMG17855 S. vest. vesbular mucosa Malmö, Sweden 1966
81
LMG17856 LMG17856 S. vest. Human dental plaque Malmö, Sweden 1966
FO396 F0396 S. vest. human oral cavity England 1988
CCHSV5 CCHSV5 S. vest. Human blood Cochin hosp,, France 2002
98 LMG14645 LMG14645 S. vest. vesbular mucosa Malmö, Sweden 1966
LMG14646 LMG14646 S. vest. vesbular mucosa Malmö, Sweden 1966
97 100
LMG17854 LMG17854 S. vest. vesbular mucosa Malmö, Sweden 1966
100 MTCC5460 MTCC5460 S. th. fermented milk product (curd) India 1984
MTCC5461 MTCC5461 S. th. fermented milk product (curd) India 1984
CAG236 CAG236 S. th. NA NA
100 IF8CT IF8CT S. th. curd of “Grana Padano” cheese Veneto, Italy 2012
DGCC7710 DGCC7710 S. th. Commercial starter culture NA
TH982 TH982 S. th. Buffalo moarella curd Campania, Italy 2003
TH1435 TH1435 S. th. Arsanal goat cheese from raw milk Friuli Venezia Giulia, Italy 2011
LMD-9 LMD-9 S. th. starter for yogurt and mozzarella cheese USA
JIM8232 JIM8232 S. th. milk France 2002
87
M17PTZA946 M17PTZA496 S. th. Fonna cheese Valle d'Aosta, Italy 1996
TH1477 TH1477 S. th. Cow milk Veneto, Italy 2012
CNRZ1066 CNRZ1066 S. th. yogurt starter France 1986
LMG18311 LMG18311 S. th. yogurt UK 1974
MTH17CL396 MTH17CL396 S. th. Fonna cheese Valle d'Aosta, Italy 1996
TH1436 TH1436 S. th. Arsanal goat cheese from raw milk Friuli Venezia Giulia, Italy 2011
MN-ZLW-002 MN-ZLW-002 S. th. yogurt block Gannan region, China
NDO3 ND03 S. th. naturally fermented yak milk Qinghai, China
TH985 TH985 S. th. Buffalo mozzarella whey Campania, Italy 2003
NA, not available
0,01

Fig. 1. Phylogenetic tree of 9 Streptococcus sp. (sp.), 18 S. thermophilus (STH), 11 S. vestibularis (SVE) and 31 S. salivarius (SSAL) strains, based on the concatenated sequences of
five housekeeping genes: ddlA, dnaE, thrS, pyrE and sodA.
384 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392
were constructed for S. salivarius genomes from the information
provided by the PATRIC platform (http://patricbrc.vbi.vt.edu),
using the comparative pathway tool (Wattam et al., 2014). Mobile
elements and transposase genes were identified with the protein
family sorter tools of the PATRIC platform. The subcellular distribu-
tions of proteins in streptococci were obtained from the PSORTdb
database (http://db.psort.org), with PSORTb version 3.0 prediction
results (Yu et al., 2011). CAZy data from the Carbohydrate Active
Enzymes database (http://www.cazy.org) were used to compare
the distribution of glycosyltransferase and glycosylhydrolase genes
in streptococcal genomes (Lombard et al., 2014). Bacterial genomes
were mined for genes encoding proteins responsible for sensing
and responding to environmental signals, using the Microbial Sig-
nal Transduction database, MiST2.2 (http://mistdb.com) (Ulrich
and Zhulin, 2010). Percentage of identity between pairs of genomes
was computed from the genomic regions that were aligned with
MUMmer (version 3.07) (Kurtz et al., 2004). Regions overlapping
on more than 20 nucleotides were discarded. Hierarchal clustering
was performed for S. salivarius genomes, based on the percentage
of identity or the presence/absence of genes, with the heatmap.2
function from the R package gplots.
3. Results and discussion
3.1. Phylogeny and population structure of the salivarius group
The salivarius group contains three recognized species, S. ther-
mophilus, S. vestibularis and S. salivarius, which are genetically very
similar. S. vestibularis was recently described as a new species
present in the human oral cavity (Whiley and Hardie, 1988). It
has frequently been shown to be associated with human infections
since its identification (Doyuk et al., 2002). S. thermophilus is the
only nonpathogenic streptococcus originating from dairy products.
It was accorded full species status species in 1991, having previ-
ously been classified as a subspecies of S. salivarius (S. salivarius
ssp. thermophilus) (Schleifer et al., 1991). S. thermophilus genomes
are characterized by a high proportion of pseudogenes, indicating
that strains of this species have undergone major reductive evolu-
tion in relation to their lifestyle, which is simpler than that of other
streptococci (Bolotin et al., 2004; Goh et al., 2011).
Multilocus sequence typing (MLST) has been carried out on
bacteria from the salivarius group, to decipher the phylogenetic
relationship between the three closely related species and to assess
the genetic diversity of 26 S. thermophilus, 9 S. vestibularis and 27 S.
salivarius strains (Delorme et al., 2010; Delorme et al., 2007). We
present here a new phylogenetic tree constructed with the
sequences of 69 strains, including all the available sequenced
strains, and using the concatenated sequences of five housekeeping
genes (ddlA, thrS, pyrE, sodA and dnaE), excluding genes probably
subject to inter-species recombination in S. salivarius species
(Fig. 1) (Delorme et al., 2010). This tree confirmed our previous
analysis, separating the strains belonging to S. vestibularis and S.
thermophilus species from those of S. salivarius. It also revealed
the existence of an additional cluster of four strains, supported
by significant bootstrap values in S. salivarius species. Two of these
strains, HSISS2 and HSISS3, were isolated from ileostoma effluent,
whereas the other two, PS4 and C150, were isolated from the
breast milk of a healthy woman and a human airway sample. This
result confirms the observations based on a tree constructed by the
alignment of multiple protein sequences from 450 orthologous
streptococcal groups, in which HSISS2 and HSISS3 clustered sepa-
rately from other S. salivarius strains isolated from the same set
of samples (Van den Bogert et al., 2013a).
We looked for associations, by inferring the MLST tree and
strain origin. The lack of clustering of clinical isolates and commen-
sal organisms into separate groups indicated that the clinical
strains did not belong to a distinct S. salivarius population (Fig. 1)
(Delorme et al., 2010, 2007). Strains from the same ecotype, such
as ileostoma effluent (4) or breast milk (2), were clearly separated
from each other and associated with oral strains in the MLST
scheme. The clear lack of association between strains and the
sources from which they were isolated (oral cavity, small intestine,
blood or breast milk), suggests that it is not possible to define a
group of S. salivarius isolates as specific to a particular environ-
ment. Finally, S. salivarius isolates from the same individual display
a high level of genetic diversity, as demonstrated by the four
S. salivarius strains recovered from the same oral cavity and
ileostoma effluent sample (Fig. 1) (Delorme et al., 2007; Van den
Bogert et al., 2013a). Similar observations have been reported for
several other mucosal streptococci from the oral cavity, such as
S. mitis (Hohwy et al., 2001), S. mutans (Kulkarni et al., 1989) and
Streptococcus oralis (Alam et al., 2000). Between three and 10
distinct genotypes of S. mitis, S. oralis and Streptococcus infantis
have been identified in the upper respiratory of an individual
(Bek-Thomsen et al., 2008). Population genetic analyses indicated
that the genetic diversity of human streptococci was due to a
succession of clones rather than the establishment of stable strains
(Hohwy et al., 2001). No information about the dynamics of
S. salivarius populations, through analyses of the proportion of clones
and changes in this proportion over time, is currently available.
S. vestibularis strains group together into a single MLST cluster
and form a separate species (Fig. 1) (Delorme et al., 2007). The
members of this species are mostly commensal organisms of the
oral cavity, but they are significantly less frequently isolated than
S. salivarius. Little is known about the genomics of this species,
although two genome sequences were recently made available. S.
thermophilus is clearly separated from the two commensal species
and has a low genetic diversity, suggesting recent emergence
within the salivarius group (Fig. 1) (Delorme et al., 2010). This spe-
cies is thought to have a recent origin, emerging with the start of
human dairy activity, about 7000 years ago, with its ancestor
exploiting milk as a new ecological niche (Bolotin et al., 2004;
Fox, 1993). MLST clustering, the specific common tkt locus
(Delorme et al., 2007), and the presence of the same chromosomal
inversion between S. thermophilus and S. vestibularis as in S. saliva-
rius genomes (see below), indicate that this species probably
diverged from a strain related to S. vestibularis.
3.2. Dynamics of S. salivarius genomes
The genomes of 12 S. salivarius strains from both MLST groups,
have been determined, and three of these genome sequences are
complete (JIM8777, CCHSS3, 57.I) (Table S1). The corresponding
strains were isolated from diverse human sites, including the oral
cavity and saliva (JIM8777, K12, M18), dental plaque (57.I), the
upper respiratory tract (C150), the small intestine (HSISS1, HSISS2,
HSISS3, HSISS4) skin (SK126), human blood (CCHSS3) and breast
milk (PS4). The diversity of isolate origins suggests that S. salivarius
can adapt and colonize various niches in its human host. This abil-
ity may be reflected in genome size, which ranges from 2.05 Mb to
2.42 Mb (mean of 2.24 Mb + 0.12), one of the largest genomes
among streptococci (Tables S1 and S2). Two of the strains with
the largest genomes, K12 and M18, contain a megaplasmid
(approximately 0.18 Mb) (Barretto et al., 2012; Heng et al., 2011;
Hyink et al., 2007). These megaplasmids harbor gene clusters with
specific ecological functions, such as bacteriocin production, which
may interfere with pathogen proliferation and stabilize the oral
microbiota (Hyink et al., 2007; Wescombe et al., 2006;
Wescombe et al., 2011), and surface fimbriae, which are involved
in co-aggregation reactions with other microbial species and
adhesion to human epithelial cells (Burton et al., 2013; Levesque
et al., 2004). Several contigs from the HSISS3 strain display striking
 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392 385

similarity to regions of these two megaplasmids, suggesting that
the assembled genome may include extrachromosomal sequences
(data not shown). The core genome defined for all S. salivarius
available genomes is estimated to consist of 1350 orthologous
genes (Table S3). However, pairwise comparisons of S. salivarius
strains indicate differences between genomes of more than 500
genes, suggesting significant differences in the adaptive features
of different strains (Fig. S1). As a representative example, the first
two strains to be sequenced, JIM8777 and CCHSS3, have 1691
orthologous genes in common and contain 252 and 295 specific
genes, respectively. Interestingly, a pairwise comparison of strains
based on the determination of an overall percentage of identity
across their genome sequences indicated that the group of four
strains previously identified by MLST displayed more similarity
to each other than to other S. salivarius strains (Fig. 2). The mean
level of nucleotide divergence ranged from 4% to 7% within clus-
ters, but reached 10% between clusters. This analysis also pointed
out the genomic proximity between strains 57.I, M18 and CCHSS3.
A comparison of S. salivarius (12), S. vestibularis (2) and S. ther-
mophilus (15) genome sequences revealed several inversion events.
An inversion was detected in S. thermophilus species with respect
to S. salivarius, as illustrated in Fig. S2, for S. salivarius JIM8777
and S. thermophilus CNRZ1060. Interestingly, the regions surround-
ing the inversion points in the two S. vestibularis draft genomes
presented complete synteny with the S. thermophilus genome. Con-
sistent with our MLST analysis, the simplest hypothesis would be
that S. thermophilus and S. vestibularis are descended from a com-
mon ancestor presenting this inversion. A pairwise comparison
between circularized S. salivarius genomes also identified inversion
events in this species (Fig. S2). The first such event was highlighted
by a dotplot comparison of JIM8777, CCHSS3 and M18. This event
was also detected in S. salivarius 57.I, but coupled with a second
inversion. This second inversion occurred between two copies of
an IS element located close to the inverted chromosomal region,
and resulted in a realignment of the genome with that of
JIM8777 (Fig. S2).
Horizontal gene transfer (HGT) is increasingly frequently being
identified as an evolutionary mechanism facilitating environmen-
tal adaptation through the acquisition of new genes in streptococci.
Recent explorations of the phylogenetic relationships between
Streptococcus species have revealed the strong impact of HGT on
the evolution of the genome in this genus, particularly in the

Color Key

0.9 0.94 0.98

Value

M18

57.I

CCHSS3

JIM8777

SK126

HSISS4

HSISS1

K12

HSISS3

HSISS2

C150

PS4
57.I
PS4

K12

SK126

M18
C150

HSISS2

HSISS3

HSISS1

HSISS4

JIM8777

CCHSS3

Fig. 2. Genomic relationship between S. salivarius strains. Hierarchical clustering analysis of S. salivarius strains was performed using pairwaise percentage identity of the
genome sequences.
386 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392
salivarius group (Richards et al., 2014). HGT provides new genes
encoding proteins with additional physiological properties of use
for adaptation and survival in a particular niche, as documented
for the dairy S. thermophilus species (Delorme et al., 2010; Eng
et al., 2011). Intraspecific recombination events have occurred fre-
quently in the S. salivarius population, generating sequence diver-
sity in the core genome (Delorme et al., 2007). Moreover, several
regions of the K12 and M18 megaplasmids are almost identical
to chromosomal genes found in S. salivarius strains 57.I and
CCHSS3, indicating possible genetic exchanges between chromo-
somal regions and plasmids (Heng et al., 2011). Gene exchanges
also seem to have taken place between S. salivarius and other spe-
cies. HGTs with other streptococci present in the human oral envi-
ronment, such as S. pneumoniae and Streptococcus parasanguinis,
have been characterized (Delorme et al., 2007) and recombination
events have been detected in genes encoding glycosyltransferases
(Giffard et al., 1993). We identified a 17-kb genomic region of S.
salivarius K12 located between RSSL_01392 and RSSL_01412 and
constituting a mosaic of three fragments, two of which are more
than 97% identical to DNA sequences from distantly related species
(Fig. 3A and Table S4). The RSSL_01397-RSSL_01405 region is
almost identical to a plasmid-encoded region from Lactococcus
lactis and Enterococcus faecium, and a mobile element region from
Lactobacillus sakei. This region bears the lacRABDEFGX genes encod-
ing the lactose PTS system and the lactose assimilation pathway
(Fig. 3B) (see Section 3.4.). The downstream region from
RSSL_01406 to RSSL_01412 is 99% and 97% identical to a genomic
region of S. mutans LJ23 and two S. pneumoniae strains (Table S4),
respectively. This region, which encodes a thioredoxin
(RSSL_01407), a thioredoxin reductase (RSSL_01408) and a disul-
fide oxidoreductase (DSBA homolog, RSSL_01411) in particular, is
also present in strains HSISS1 and HSISS4, indicating that gene
transfer is not confined to the K12 strain. The sources of the trans-
ferred genes and the mechanisms of transfer are unknown, but the
mosaic structure of the region is consistent with the occurrence of
multiple transfer events. These transfers probably took place
recently, given the high level of identity between the homologous
regions of these distantly related species.
3.3. Functions enriched in S. salivarius genomes
A previous analysis showed that the salivarius group differed
from other groups of streptococci in terms of Gene Ontology
(GO) term enrichment for urea metabolism (urea activity, urea
metabolic process, nickel cation binding, cobalt and calcium
transport) and transposition (Richards et al., 2014). However, this
analysis was performed with only one representative genome for
each species. For confirmation of these findings, a search was car-
ried out for the complete urease cluster and mobile elements in all
available genomes from the salivarius group. All these genomes,
except for that of S. salivarius SK126, were found to contain a com-
plete urease operon (Table S5). By contrast, a high mobile-element
content seems to be a feature more specific to S. thermophilus than
to the rest of the salivarius group (Table S6).
We performed additional genomic comparisons, to decipher
other features. An analysis of the subcellular distribution of pro-
teins predicted by PSORTdb 2.0 indicated that S. salivarius was
among the streptococcal species with the largest number of extra-
cellular components (cell wall and extracellular proteins), whereas
S. thermophilus was among the streptococcal species with the
smallest number of extracellular components (Fig. 4A and
Table S7) (Rey et al., 2005; Yu et al., 2011). Extracellular compo-
nents, such as adhesins, play a determinant role in the interactions
of streptococci with their environments, including colonization
and escape from the host immune system (Nobbs et al., 2009).
The physiological and ecological role of this class of proteins may
thus be a very important feature of the lifestyle of this bacterium
with respect to the large number of potential extracellular compo-
nents predicted. S. salivarius extracellular components have been
shown to be involved in adhesion to buccal epithelial cells
(Handley et al., 1987) and human salivary proteins (Schenkels
et al., 1993), in co-aggregation reactions with pathogens
(Levesque et al., 2003) or commensal organisms in oral cavities
(Handley et al., 1987; Weerkamp and Jacobs, 1982), in the synthe-
sis of glucans (Rathsam and Jacques, 1998; Simpson et al., 1995)
and in biofilm formation (Ogawa et al., 2011). Conversely, it has
been suggested that these features have largely disappeared from
S. thermophilus, following its adaptation to the milk niche
(Bolotin et al., 2004).
A similar search of the CAZy server revealed that S. salivarius
had a high glycosyltransferase (GTF) content (approximately twice
the number of glycosyltransferase genes found in most strepto-
cocci), but a similar number of glycosylhydrolase (GH) genes to
other streptococcal genomes (Fig. 4B and Table S8). GTFs catalyze
the formation of glycoside bonds between a saccharide from a
donor substrate (e.g. nucleotide sugars, sugar phosphates, sugar
lipids, saccharides) and an acceptor (e.g. saccharides, proteins, lip-
ids, nucleic acids). They are thus involved in the biosynthesis of
various compounds, including glucans (Komatsu et al., 2011;
Simpson et al., 1995), EPS/CPS (Han et al., 2012; Lakkitjaroen
et al., 2014; Minic et al., 2007), glycolipids (Meiers et al., 2014),
polyketides and peptidoglycan (Di Guilmi et al., 1998; Helassa
et al., 2012), as well as glycoproteins (Weerkamp and Jacobs,
1982; Zhou and Wu, 2009; Zhou et al., 2010). Their activities are
associated with bacterial fitness and resistance, adhesion, invasion,
biofilm formation, colonization and virulence in numerous strepto-
coccal species (Aviles-Reyes et al., 2014; Bowen and Koo, 2011;
Chaudhuri et al., 2007; Forquin et al., 2007; Gregoire et al., 2011;
Koo et al., 2010; Meiers et al., 2014; Taniguchi et al., 2010; Yang
et al., 2014; Yeh et al., 2006). Little is known about the role of GTFs
in S. salivarius, but it seems likely, given the large number of these
enzymes present in this species and their great potential for chem-
ical modifications, that they play a particular role in the interaction
with the host.
The adaptive capacity of S. salivarius was assessed by generating
an inventory of potential regulatory proteins. S. salivarius genomes
were predicted to contain about six regulatory components per
100 kilobase pairs, corresponding to less than 1% of all the proteins
encoded by the genome (Table S9). This frequency is similar to the
mean values obtained for other streptococci. However, we noted
that the S. salivarius and Streptococcus agalactiae genomes differed
from the other genomes of streptococci in containing a much larger
number of genes encoding response regulators (RR) (Fig. 4C). The
RR counts were normalized by dividing by the total number of
regulatory proteins identified by MiST 2.2 (Ulrich and Zhulin,
2010). The mean number of RR per streptococcal genome
corresponds to about 10% of all regulatory components, for a value
similar to those obtained for Escherichia coli K-12 (9%),
Pseudomonas aeruginosa PAO1 (10%), Enterococcus faecalis D32
(8%), Bacillus subtilis 168 (13%) and Streptomyces coelicolor A3
(9%). In S. salivarius, this proportion is even higher, at almost 15%
(Fig. 4D). RR and TCSs (two-component systems) are usually
involved in controlling stress and adaptive responses, bacteriocin
production, natural competence and biofilm formation (for a
detailed description of TCSs in several S. salivarius strains, see
(Van den Bogert et al., 2013a)). Their role in the physiological
and ecological lifestyle of S. salivarius remains to be elucidated.
3.4. Metabolic capacity of S. salivarius on the basis of genomic data
The metabolic potential of strains isolated from different sites
was investigated by exploration of the genome, to determine
 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392 387

Fig. 3. Horizontal gene transfer in S. salivarius. (A) Schematic diagram of a 17-kb mosaic region of horizontal gene transfer in the genome of S. salivarius K12, and the
corresponding region in JIM8777. Two large fragments of the K12 region display more than 97% DNA/DNA identity with Lactococcus lactis (red) and Streptococcus mutans
(blue) sequences. (B) Routes of lactose utilization in S. salivarius. Black, genes present in all S. salivarius strains. Red, K12-specific genes present in the previous 17-kb mosaic
region. Green, genes absent from K12 but present in most S. salivarius strains. Locus tags correspond to those of S. salivarius K12.

whether strains from different sites had specific features. We over-
came possible bias due to genome annotation, by collecting infor-
mation from the PATRIC database, which is based on the de novo
gene detection and annotation of genomic sequences by RAST,
before metabolic reconstruction (Overbeek et al., 2014; Wattam
et al., 2014). An analysis of about 200 re-annotated metabolic
genes revealed that some strains presented a striking level of gene
decay (up to 15% pseudogenes) (Fig. 5A, Tables S10 and S11). The in
silico reconstruction of metabolic pathways showed that S. salivari-
us strains were almost identical (Fig. 5B, Tables S10 and S11),
except for several substantial divergences (see below). Hierarchical
clustering based on the presence/absence of these metabolic genes
identified three groups of strains. One of these groups contained
PS4, C150, HSISS2 and HSISS4 and was distinguished by having a
 388
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tr co tr c s pn co S c c u us t o c
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c c id e b s o c s s p s C C s 1 3 s c o co er e c cu g i G S
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ep
t e G p id i s h a g i 3 1 to S s r m r m L M 1. 1 /7
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t u S1 mi bs n g C 2 4 cc tr t s i h 1 89
ep o p lu il 8
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t c c y A ep c s c u c a J
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g p c cc ta n 0 S e p p r m h a s u is 5
tr to y
S in o t o c o c c u s n s s G 5 0 ep c o o p r y n s C ST
tr s o u u N S o g
to c e hi gi 1 3
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t s u m e S S t oc u s es us C 5 1
S o c s u s u r i s 20 tr re c i s J 1 0
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tr o b a n ta 0 2 5
S e c to t o s a t er . M 8 0
tr S p t c u s p . g i n s 14 c o c o ga m a 2
ep tr o s w n U 0 e 3
to S e p co g h o s A J S c c c c u l ac di u nf r 2
tr to c a il u 1 t r u s s ti s e d
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cc tr to o c s l y i M C 2 9 tr e to i nt p y o e I C 2
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S r d l o l y s p n T it is 8 tr a l t r co e p s A 5 SF
us on ti a n e u C B ep
S tr c to a c e p cc t o p y C A Z Y 1
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st s n o n 5 S Str
l lo ep to A o 3 tr e e cc e c o a cc e n C 3 3
l t c S r. T s ia 7 pt pto us S su c c g a u s e s 19
S y t i o c o o cc t r e C h C u s e tr bs us l a o S 8
t r cu c u p a C C R S oco coc dy S t ep p c r
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ep c u s ga p
re t o . e in te t i a e al i s SI -
S t o s u u s t h c o is s A A 0 5 co q r N U 1
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ep o c s e r m c b 2 co g a ct o
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co s g o p h o . 9 u s o ly l y t s s e n B 6
c c m a l lo p h i l u r a l C H d tic icu Str ub pa pto eu AC 19
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tr a s u t o e s a cc n 7

10
15
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0
5
S s o c e d y tic u s J IM U
tr li o u L o ep la ub b c q n u i a 1 3
s o s
S ep g n s M 8 5 t o ct sp p c u i g u m e
c ia . c s R
tr t o o fe i c u AT G 2 3 S o c e . g a g a u s i m i n i s i ti s 6
ep c o rm s C 1 8 2 t r c s u l l l o a i li
to c e A C 3 e p u s b lo l n s F B
co c n C 1 t o g sp l y t y t i g i G G W 2 6
cc S us ta A 4 3 1 c o o r . ic c u n o S 1
tr p ns - D 1 4
S us e S c c do eq us s A su _ 1 3
tr u n u A T s 2
tr p S pt ne A C 3 e p s ii is T C C 4
ep a tr oc um S 1
e 1 9 t p s im C
S t o a p oc o . 3 8r S S o c o n e t r . i li C C 4 2 3 8
t r c o s a to c n 0 tr tr c u C s B 3
e p c n c u ia 8 ep ep c m h A A 1
S to t o u s o n a ll i T C A -2 4 3
t o c u g u o c s in e D 9 c i s
C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392

c o s i n c u ia 3 tr
ep c o c S t r o c e q u a e su C 1 07 0
S cc t h i s s s e 9 to c u ep c u i s AT bs 2 3
tr u er A u S co S
e s F cc
s to s g u b C t r . 9 4
tr S p a c o a s C C
S pt o p m o T C i s P 1
tr c n e p C 1 us e p t t r e r a cc u l l ol p . 7 0 H 1
ep o u h 1 /
S eq o c pt s a s y t eq 0 6
S
S tre t o c c m il u s 5 9 7 ui o c oc n g a g i c u u i 6 9
tr p co u s o n L 1 c u oc ui al s 40
ep t S c s ia M 2 tr e su c

number of regulatory proteins identified by MiST2. Red dots, S. salivarius; orange dots, S. thermophilus; green dots, S. vestibularis.
t o o c o t re cu u i e D pt bs S t s p u s n i s a c t U C 4 7
S c o c p t s s TI -9
oc p . r e p y o p A ia e N 3
tr oc S z to g ne T C
t A 4
S ep c c o lu 0 G cu r o
e p o c o e n u m C 90
s
tr t o S c u u s co t e t 5 H R
tr s t h c c i e A 4
e e t ep c e s o 1 9
ep c
o S o c id cu
M n 59
to c S p t o p a er m us n s i 6 8 S qu i St Str o c e m s s G i a e 1 2
c o c u tr e c r a o s u s tr s r e e cu i u A D
e p u p p t s cu is S 3
c c s p o c s a p is 0 3 to b s t oc oc s s 9 1 0 9
us ag toc c u ng h il S 3 c o p. o oc al M 8 H 7 5
a u S S
a l o s u s 1 tr c c z o c c c u i va G C A 0
S ga a ct cc u a g i n i s N 2 ep us oe us s r i u S H3
t r l a ia s a l D to s
p n p id a g a n s J 1 0 5 3
ep c e s a F 0
t o tia N a n cti W 2 3 co e e a g I 6
c e E a c c S t u m m l a c u in M 8 5
i
S S o c 2 M 3 gu i e A 1 3 S u s r e p o n c u t ia e is 7 8 0
t r t c 60 1 n 9 tr p t o i s
e p re u 3 6 is 0 e s a A 2 SK
t o p t s s V / se S 9 S pto eu c oc e H T C 60 3 6
t r c d c u C 3V
c o o c a l R r K3 ep o o u n
cc o c iv a s e o va 6 to cc u p n s s g a 3 5 /R
u s c u r iu ro r c o s e u al r y 2 4
I c c p n m iv a 1 9 6
s a s s s J ty p II us e o n ri A
li v a l i I M e V u u -
s a m i ae s 5 6
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s s 7 iu T 49
C 5 s IG 3
C 7 JI R
H .I M 4
S
S 87
3 77

glycosyltransferases (GTF) identified by CAZy. (C) Number of response regulators (RR) identified by MiST2. (D) Percentage of response regulators (RR%) among the total
Fig. 4. Functions enriched in S. salivarius genomes. (A) Percentage of extracellular components (cell wall and extracellular proteins) identified by PSORTdb. (B) Number of
 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392 389

A
100

Metabolic genes (%)

Present
Absent
Pseudogene
50

18

SI 2
SI 1
SI 4
3
77
C 50

.I
M 6

4
SI 2
SS

H SS
H SS
H SS
SS
PS
JI 1 2

H K1
57
M
87
C 1C
H
K
S

B
HSISS4

HSISS1

JIM8777

57.I

M18

CCHCSS3

SK126

K12

C150

PS4

HSISS3

HSISS2

Fig. 5. Genome-based metabolic features of S. salivarius. (A) Proportion of the 200 metabolic genes present, absent or present as pseudogenes, by S. salivarius strain. (B)
Hierarchical clustering analysis was performed on the strains on the basis of the absence (white)/presence (grey) of the 200 metabolic genes studied. Genes present as
pseudogenes were considered to be present. The set of 200 metabolic genes used in these analyses is described in Table S12.

high rate of metabolic gene absences (Fig. 5). Strains did not cluster
together according to their site of isolation. For example, despite
their isolation from the same site and the same individual, HSISS2
and HSISS3, which belong to a particular MLST cluster, appear to be
metabolically different from HSISS1 and HSISS4, which are more
closely related to the oral isolate JIM8777.
The analysis of S. salivarius genomes predicted a large capacity
to synthesize most amino acids (Table S10). However, none of
the strains encoded a diaminopimelate epimerase (EC 5.1.1.7),
the enzyme required to synthesize lysine from aspartate. The his-
tidine biosynthetic pathway of HSISS2 and HSISS3 was found to
be incomplete. Six his genes were not detected in the genomes of
these strains and one of the genes identified was a pseudogene).
The alanine dehydrogenase gene (EC 1.4.1.1) was missing from
PS4 and HSISS3. Moreover, several amino-acid biosynthetic genes
in strains M18, PS4, HSISS1, HSISS2, HSISS3 and HSSISS4 appear
to be pseudogenes, suggesting that these strains may have addi-
tional amino-acid requirements.
All the strains were predicted to have complete PTS systems
involved in the import of mannose, maltose/glucose, sucrose and
fructose, and most of the enzymes involved in the catabolism of
these sugars (Table S11). Interestingly, HSISS2, HSISS3, PS4 and
C150, which belong to the same phylogenetic cluster, are probably
unable to use trehalose and beta-glucosides, because they lack the
corresponding PTS. Consistent with this hypothesis, the gene
encoding a,a-phosphotrehalase (EC 3.2.1.93), which is involved
in the conversion of PTS-transported trehalose into D-glucose-6P,
appears to be a pseudogene in these four strains. Conversely, these
strains and M18 each have a gene encoding a-galactosidase (EC
3.2.1.22), indicating that they may be able to metabolize other sug-
ars, such as raffinose and melibiose. Only strains M18 and CCHSS3
appear to have a complete cellobiose PTS. The substantial func-
tional diversity and evolution of S. salivarius genomes are illus-
trated particularly well by lactose metabolism. Strains SK126 and
K12 lack the lactose-galactose antiporter and several genes
involved in lactose assimilation via the galactose pathway
(Fig. 3B). Furthermore, the gene encoding b-galactosidase (EC
3.2.1.23) appears to be a pseudogene in strains HSISS1, HSISS2
and HSISS4, suggesting that these strains may also be unable to
metabolize lactose. However, as mentioned above, the K12 strain
is nevertheless able to assimilate this disaccharide through a lac-
tose PTS and the tagatose-6P pathway, a route involving proteins
acquired by horizontal gene transfer (Fig. 3). PTS excel in condi-
tions of low substrate concentration, whereas lactose antiporters
are more typically present in species living in lactose-rich environ-
ments (El Kafsi et al., 2014). This suggests that the lactose–galact-
ose antiporter may fulfill an important ancestral function that may
be dispensable in the environment occupied by S. salivarius, given
390 C. Delorme et al. / Infection, Genetics and Evolution 33 (2015) 381–392
its elimination from two of the 12 strains studied. Consistent with
this hypothesis, the acquisition of a dedicated PTS by the K12 strain
may confer a selective advantage in environments with a low lac-
tose concentration, such as the oral cavity. Further investigations
are required to obtain a full understanding of the physiological
and ecological origins of the functional diversity of S. salivarius
strains.
Finally, most S. salivarius genomes are predicted to encode
enzymes for the production of several fermentation end-products,
such as L-lactate, acetaldehyde, formate, ethanol, acetate, propio-
nate, acetoin and 2,3-butanediol from pyruvate, some of which
interfere with human signaling pathways and immunomodulation
(Bari et al., 2011; Harty and Handley, 1988; Maslowski and
Mackay, 2011; Vinolo et al., 2011).
4. Conclusion
We carried out a phylogenetic and comparative genomic
analysis of S. salivarius strains isolated from diverse human sites.
S. salivarius is a highly diverse species, which is clearly phylogenet-
ically separate from the other species of the salivarius group. In
addition, phylogenetic analyses showed that S. thermophilus and
S. vestibularis were descended from a common ancestor. As in most
streptococcal species, sequence diversity is maintained by the
extensive horizontal transfer of core genome genes within and
between species, and by the acquisition of new genes. A cluster
of four S. salivarius strains was identified by MLST analysis. Support
for the existence of this cluster was obtained from comparative
genomics studies and genome-based metabolic capacity analysis.
More detailed analysis is now required to determine whether this
group corresponds to a new species or a subspecies of S. salivarius.
Our findings also support distinct genomic and evolutionary pat-
terns within the S. salivarius species sensu stricto including large
chromosomal inversion and similar gene content. We have shown
that S. salivarius is one of the streptococcal species with the largest
numbers of extracellular proteins, glycosyltransferases and
response regulators, three functional categories of proteins with
major physiological and ecological functions, such as adhesion to
surfaces and host-commensal interactions. S. salivarius was iso-
lated from diverse sites in human, suggesting it is able to adapt
and colonize a variety of niches in its host as well as to resist in
the bloodstream. However, we found no phylogenetic, genomic
or metabolic relationship between strains as a function of their
isolation site, so as yet unidentified factors probably contribute
to the adaptation of S. salivarius. Subtle differences between strains
such as gene expression level and regulatory pattern may be
involved in the niche adaptation. Moreover, this study, mainly
based on core genome assessment, revealed that HGT is a driver
for S. salivarius genomic evolution and environmental adaptation.
For this reason, a particular interest would be paid on specific gene
contents to identify niche specific factors through physiological
and genomic analysis performed on a wider strain sampling.
S. salivarius appears thus to be an outstanding model to depict
lifestyles of commensal bacteria.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.meegid.2014.10.
001.
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