Open Journal of Stomatology, 2011, 1, 18-24 OJST
doi:10.4236/ojst 2011.12004 Published Online June 2011 (
Published Online June 2011 in SciRes.
The prevalence of virulent clonal strains of mutans
streptococci in vivo and co-culture succession of the strains
in vitro
Virulence potential of mutans streptococci
Mingyu Li*, Guang-yun Lai, Jun Wang
Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Ninth People’s Hospital, Medical College,
Shanghai Jiao Tong University, Shanghai, China.
E-mail: *
Received 8 March 2011; revised 20 April 2011, accepted 3 May 2011.
Purpose:To examine selected putative virulent prop-
erties of mutans streptococci in two groups with dif-
ferent caries activity and to examine co-culture hy-
bridization of the strains in vitro. Methods: A set of
strains from caries-free subjects (115) and another set
from caries-active subjects (165) were isolated. Each
strain was characterized for three virulence determi-
nants. The clinical bacteria were then cocultured by
three strains exhibiting the highest levels of virulence.
Isolate colonies of last filial generation bacteria were
enrichment-incubated and estimated for virulence
again. RAPD-PCR and MLEE analyses were proc-
essed for parental bacteria and last filial generation
one. Results: No difference associated with caries ac-
tivity of the subjects from whom the isolates originated.
Virulent properties of a filial generation strains was
not different in the same generation, but was very
different from their parent strains. Conclusion: The
coexist properties of virulent polyclonal strain of MS
may hold in a very general conditional sense in a
dental plaque ecosystem in vivo, however, one of the
co-culture strains may became dominant and displa-
ced the others as the result of continuous ecological
succession in vitro.
Keywords: Ecological Phenomenon; Mutans
Streptococci; Co-Culture; Virulent; in Vitr o
Dental caries is a transmissible and poly-microbial eco-
logical disease, in which mutans streptococci (MS),
mainly Streptococcus mutans and Streptococcus sobri-
nus play the major role. The main virulent factors of MS
include adhesion, acidogenicity and aciduricity [1-3].
Primary mechanism for adherence of S. mutans is the
production of glucan from sucrose via glucosyltrans-
ferases (GTFs) [4,5]. Acid production and aciduricity are
other important caries-inducing virulent factors [6-8].
MS is generally considered to be the principal aetiologi-
cal agent of dental caries [9,10]; however, they are widely
distributed not only in populations with moderate or high
caries prevalence [11,12] but also in populations having
no or low caries experience [13,14]. Caries can also de-
velop in the absence of MS species [15].
So Takahashi and Nyvad present a new view of caries,
the Extended Caries Etiological Hypothesis, that it is no
longer just a consideration of which specific bacteria are
present, but rather what those bacteria are doing. They
are behaving as acidogenic/aciduric bacteria contributing
to the dental caries disease process [16]. The virulence
properties of MS have the coexist properties of a poly-
clonal epidemic [17]. The three highest virulent charac-
ters (GTFs activity, acid production and aciduricity) were
not assembled together on a same strain [18]. Kuramitsu
and Wang suggested that S. gordonii, and perhaps addi-
tional nonmutans streptococci, can attenuate some of the
virulence properties of S. mutans [19].
Despite its importance, we have only a limited under-
standing of the biological nature of MS virulent strains
colonization. How the prevalence of virulent factors of
MS from strain to strain in a dental plaque ecosystem in
vivo. How do polyclonal strains replace one another in
ecological succession in vitro? To attempt to determine
what happens to ecological sites where more than one S.
mutans strain is present, co-culture of this study was
performed by the 3 clinical strains which exhibited the
highest level of virulence properties in glycosyltrans-
ferase activity, acid production, and acid tolerance, re-
M. Y. Li et al. / Open Journal of Stomatology 1 (2011) 18-24
Copyright © 2011 SciRes. OJST
spectively. The last filial generation bacteria on an agar
plate were picked up and estimated for virulence proper-
ties again. Genetic studies were processed to understand
how parent bacteria and last filial one variations.
2.1. Subjects
The experimental procedures were approved by the Eth-
ics Committee of Ninth People’s Hospital, Medical Col-
lege, Shanghai Jiao Tong University.
The study consisted of 100 subjects aged between 5
and 25 years (13.5 ± 4.7). Groups of caries-free and car-
ies-active groups were 50 and 50, with the DMF (de-
cayed, missing, filled) in caries-free (dmft = 0) and car-
ies-active (dmft > 4), respectively. A total of 280 MS
(both sobrinus and mutans species) was obtained from
dental biofilm of the subjects. MS form Groups of car-
ies-free and caries-active groups were 115 and 165.
2.2. Bacterial Isolates and Species Confirmation
Plaque biofilm samples were plated on Mitis Salivarius
Bacitracin (MSB) agar plates (Difco Laboratories, De-
troit, MI). They were cultured in an Anaerobic Work
Station (BUG BOX DUAL, Ruskinn, England) with
85% of N2, 15% of CO2 at 37˚C for 2 days. In order to
detect MS, preliminary identification was done by Gram’s
staining method. Further classification was done to ex-
amine their sensitivity to antibiotics and fermentation of
carbohydrates etc, by using a biochemical appraisal and
verification system, API 20 Strep test kits according to
the manufacturer’s instructions (bioMerieux Vitek Inc.).
S. mutans UA159 and S. sobrinus 6715 were used as
standard laboratory strains.
2.3. GTFs Activity, Acid Production and
Aciduricity Assay
Identified MS were incubated anaerobically in Trypti-
case soy broth (TSB) in an incubator at 37˚C for 18 h.
The samples of bacterial supernatants were obtained by
means of centrifugation. Extracellular GTFs activity was
determined by means of direct enzyme assay [20]. Each
supernatant was exposed to 14C glucosyl-sucrose (Shang-
hai Institute of Nuclear Research of Chinese Academy of
Science, Shanghai, China, final concentration = 100
mmol/L sucrose, 40 μmol/L dextran 9,000 in buffered-
KCl, pH 6.5) for 4 h at 37˚C, to form glucans. GTFs ac-
tivity by direct enzyme assay was expressed as micromol
glucans formed per milliliter supernatant.
The acid sensitivity (tolerance) was assayed by using
broth micro-dilution method on 96-microwell plates.
Bacteria were incubated anaerobically in 5 ml of WIL-
KINS-CHALGREN anaerobe broth (Oxoid, England) in
the incubator at 37˚C for 24 hours to a density equal to
that of a no. 1 McFarland standard. For the evaluation of
acid sensitivity, strains were sub-cultured into fresh an-
aerobe medium at pH of 5.5, 5.0, 4.5, 4.0, 3.5, 3.0 and
2.5 (medium pH was adjusted with HCl). The control
was treated in the same way except the medium at pH
7.5. After 24 hours incubation, bacterial growth was
observed by a stereoscope and recorded. At the same
time, from every microwell, 50 μl of suspension was
removed and smeared on an agar plate. Bacterial colony
was counted correspondingly after 48 hours incubation.
The pH value at which there was no bacterial growth
was considered as acid sensitivity.
For the evaluation of acid production, the strains were
cultured in the anaerobe broth with glucose in microwell
plate at pH of 7.5. After 24 hours incubation in an an-
aerobic chamber, acid production of bacterial cultures
was measured by an Orion pH meter (Model 501, Orion
Research, Inc., Cambridge, MA). Un-inoculated broth
(control) was also cultured under the same conditions.
The experiments of virulent assay were performed in
quadruplicate and the result was presented by means.
2.4. Co-Culture in Vitro
Hybridization co-culture was performed by 3 strains with
ratio of 1:1:1 (v/v/v) anaerobically in Trypticase soy
broth (TSB) in an incubator at 37˚C, which exhibited the
highest levels of aciduricity, acidogenicity and GTFs
activity, respectively. Each respective strain was cultured
alone under the same condition as control. After one
week of co-culture, during which the growth medium
was replenished daily, the cultures was plated out, indi-
vidual colonies were enrichment incubated and assayed
for aciduricity, acidogenicity and GTFs activity again.
2.5. RAPD-PCR Analysis
A total of 26 of 96 strain colonies from last filial genera-
tion were next subjected to RAPD-PCR analysis. The
protocol for DNA amplification was based on Pereira
[21], with a little modification. For DNA extraction,
cells were washed in 0.1 M Tris buffer, lysed with ly-
sozyme + 10% SDS and extracted with phenol-chloro-
form. DNA was precipitated with sodium acetate and
ethanol, and its concentration was calculated by mea-
suring A260. Quality was estimated from A260/A280 ratio.
RAPD working primers [21] were OPA-3 (5’-AGTCA-
GCCAC-3’), OPA-5 (5’-AGGGGTCTTG-3’), and OPA-
18 (5’-AGGTGACCGT-3’) from Shanghai Sangon Bio-
logical Engineering Technology & Services Co., Ltd
(Shanghai, China). The RAPD reaction was performed
in a total volume of 50 μl consisting of 5 μl template
DNA, 10 × PCR buffer (750 mM Tris-HCl, 200 mM
(NH4)2SO4, 0.1% Tween 20), 3.5 mM MgCl2, 200 μM
deoxynucleoside triphosphates, 1.25 U of Taq DNA po-
M. Y. Li et al. / Open Journal of Stomatology 1 (2011) 18-24
Copyright © 2011 SciRes. OJST
lymerase (Fermentas, Lithuania), and 1 μM of primer.
The amplification was done in a Perkin Elmer 9600
Thermal Cycler. PCR cycling procedures were as fol-
lows; 50 cycles of 94˚C for 1 min, 37˚C for 1 min and
72˚C for 1 min. A final step of extension was applied at
72˚C for 10 min. Fifteen microliters of PCR products were
analysed by electrophoresis on 4% denaturing PAGE
(polyacrylamide gel electrophoresis) and displayed by
silver staining.
2.6. Multilocus Enzyme Electrophoresis Analysis
Soluble proteins were extracted by grinding 100 mg freeze-
dried bacteria with pestle and mortar in liquid nitrogen
and 5 ml buffer solution (0.1M Tris-HCl, pH 6.8). The
mixture was centrifuged for 20 min at 17.000 rpm and
the supernatant collected. The protein content in super-
natant was estimated. Protein content was adjusted to 1
mg/ml per sample.
PAGE was performed by the method described by
Laemmli [22]. Proteins were analyzed on a dual vertical
slab mini-gel system (Bio-Rad Mini-Protean, Hercules,.
CA, USA). From each sample, 20 μl of extract was loaded
on a polyacrylamide gel. The separation gel (10%) and
staking gel (3.5%) were prepared from an acrylamide
monomer solution (Roth, Karlsruhe, Germany). Elec-
trophoresis was carried out at a constant current of 35
mA through the stacking gel, and at 90 mA through the
separation gel at 4˚C. After the running time, gels were
revealed for enzyme active band detection of glucose
dehydrogenase (GDH), glucose-6-phosphate dehydroge-
nase, glucosyltransferase (GTF), lactate dehydrogenase
(LDH), leucine aminopeptidase (LAP), malate dehydro-
genase (MDH), mannitol-1-phosphate dehydrogenase
(M1P), mannose-phosphate isomerase (MPI), nucleoside
phosphorylase (NSP), phenylalanyl-leucine peptidase
(PLP), and glutamic-oxalacetic transaminase (GOT).
After appearing, the bands were scored according to
their respective relative mobilities [23].
2.7. Statistical Analysis
The GTFs activity and acidogenicity of strains between
groups from caries-free and caries-active subjects were
compared by means of student-t tests.
The distribution of MS in dental plaque of different
carious experience persons was different. The detection
rate of MS isolated from caries-active (61%) were higher
than that isolated from caries-free patients (35%) (P <
0.005), however, the distribution of virulence potential
strains of MS was almost the same in caries-free subjects
and caries-active subjects (Figure 1). The three principal
virulence factors were not put together in one strain
(data not shown). There was no significant difference
between the two groups (P > 0.05) of caries-free and
Figure 1. Acidogenicity, aciduricity and GTFs activity distribution of mutans streptococcus strains of clinical isolates
from caries-active and caries-free subjects.
M. Y. Li et al. / Open Journal of Stomatology 1 (2011) 18-24
Copyright © 2011 SciRes. OJST
caries-active in GTFs activity and acidogenicity (Table
In the test of co-culture, a total of 96 individual colo-
nies were isolated and characterized biochemically.
Virulent properties of a filial generation strains was not
different in the same generation, but was very different
from their parent strains (Table 2). The virulent poten-
tial of the control strains did not change after one week
culture alone (data not shown). A total of 26 of 96 strain
colonies from last filial generation were next subjected
to RAPD-PCR analysis. RAPD-PCR data showed that
the band profiles of filial generation bacteria were not
significantly different from each other but different from
any ones of the parent strains (Figure 2). It may suggest
that one strain (strain A) of last filial generation may
become dominant and displaced the others (Table 2,
Figures 2 and 3). In the results of MLEE, only GOT,
NSP, PLP, MPI, LAP as well as M1P showed activity
for S. mutans strain (Figure 3). The results of MLEE
were similar to that of RAPD-PCR. RAPD was proved
to be more discriminatory than MLEE.
This work described an analysis of acidogenicity, acidu-
ricity and GTFs activity amongst 280 clinical strains of
MS. It was found that the distribution of virulence po-
tential of MS was nearly the same in strains from caries-
free subjects and caries-active subjects. Three highest
virulent characters (GTFs activity, aciduricity, and aci-
dogenicity) were not fixed together on a same strain. The
result is consistent with the study by Guo [18]. So we
may proposed a hypothesis that MS strains with varying
degree of virulence potential may play different func-
tions in vivo (for example one strain plays a function of
GTFs activity, other strain plays a function of acid pro-
duction and another strain plays a function of aciduric-
ity), which makes them a good collective group to obtain
the best potential of cariogenic.
Co-culture of MS strains was performed by the 3
clinical strains which exhibited the highest level of dif-
ferent virulent potential. The results of filial generation
obtained neither a combination group of MS strains nor
one strain with the highest virulence potential. Virulent
properties of a filial generation strains was not different
in the same generation, but was very different from their
parent strains. RAPD-PCR and MLEE analysis revealed
that the band profiles of filial generation bacteria were
not significantly different from each other but different
from any ones of the parent strains. In the micro-eco-
logical environmental condition in vitro, one strain may
become dominant and displace the others. This cannot
exclude simple adaptation; however, the data indicated
the different ecological succession outcomes of poly-
clonal virulent strains between in vitro and in vivo.
The niche concept was defined by G. E. Hutchinson. A
species can only live within a certain environment, or
within a certain environment a species can live. Accord-
ing to the competitive exclusion principle, no two spe-
cies can occupy the same niche in the same environment
for a long time [24]. Different strains of the same species
are more likely to occupy the same ecological niche, and
hence undergo more intense competition than different
species [25]. In the study of Rukayadi, if two different
bacterial strains occupy the same niche on the leaf sur-
face, then the antagonism between them can occur only
when they use the same nutrient source and occupy the
same niche [26]. The nontoxigenic strains applied to soil
occupy the same niches as the natural occurring toxi-
Table 1. GTFs activity and acidogenicity of clinical isolates of mutans streptococci.
Caries-free (115 Strains)Caries-active (165 Strains)P-value (Student-t test)
GTFs activity (per milliliter supernatant) 0.9611 ± 0.6630 1.2218 ± 0.6799 0.0790
Acidogenicity (pH) 4.8624 ± 0.5857 4.5657 ± 0.4543 0.0893
The result of aciduricity is not an absolute term and state at Figure 1.
Table 2. The actual virulence properties of each strain of last filial generation and parental bacteria of 3 strains which
exhibited the highest levels of aciduricity, acidogenicity and GTF activity.
GTFs activity (per
milliliter supernatant) Aciduricity pH Acidogenicity pH
Strain of the most potential acidogenic capacity 0.27 5.0 3.9
Strain of the most potential aciduricity capacity 1.67 3.0 5.59
Strain of the most potential GTFs activity capacity2.2 4.5 4.28
Strain colonies of last filial generation (mean ± SD, n = 96) 1.36 ± 0.08 4.0 ± 0.04 4.2 ± 0.3
M. Y. Li et al. / Open Journal of Stomatology 1 (2011) 18-24
Copyright © 2011 SciRes. OJST
Figure 2. RAPD-PCR analysis of the gene transformation of three parental generation and last filial generation mutans
streptococci strains. M: marker, A, B and C: parental generation strains, A: acidogenicity strain, B: aciduricity strain, C:
GTFs activity strain. 1 - 26: mutans streptococci strains of last filial generation. Bands spectrum from filial generation mu-
tans streptococci were almost same but different from their three parental generation strains.
Figure 3. Multilocus enzyme electrophoresis analysis of three parental generation and filial generation mutans strep-
tococcus strains. A, B and C: parental generation strains, A: acidogenicity strain, B: aciduricity strain, C: GTFs activ-
ity strain. 1 - 7: mutans streptococci strains of last filial generation. GOT: glutamic-oxalacetic transaminase, NSP: nu-
cleoside phosphorylase, PLP: phenylalanyl-leucine peptidase, MPI: mannose-phosphate isomerase, LAP: leucine
aminopeptidase, M1P: mannitol-1-phosphate dehydrogenase. Enzyme loci from filial generation mutans streptococci
were almost same.
genic strains. They are capable of competing and dis-
placing toxigenic strains [27]. These data are more in
agreement with our results which indicate that different
strains of the same species undergo more intense compe-
tition if they need protection from the surrounding harsh
ecological niche. A group of different strains of the same
specie can not occupy the same ecological niche in vitro.
In conclusion, the coexist properties of virulent poly-
clonal strain of MS may hold in a very general condi-
tional sense in a dental plaque ecosystem in vivo, how-
M. Y. Li et al. / Open Journal of Stomatology 1 (2011) 18-24
Copyright © 2011 SciRes. OJST
ever, one of the co-culture strains may became dominant
and displaced the others as the result of continuous eco-
logical succession in vitro.
This work was supported by Science and Technology Commission of
Shanghai (08DZ2271100) and Shanghai Leading Academic Discipline
Project (Project Number: S30206). Authors of this manuscript do not
have any financial conflicts of interest and the funder (s) has/have not
played any decision-making role in the research.
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GTF: glucosyltransferases; RAPD-PCR: Random amplified polymorphic DNA - Polymerase Chain Reaction.