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Original research
From a social standpoint, extrinsic environmental factors
such as socioeconomic situation and access to dental care also
contribute to the development and composition of the oral
In t r o d u c t I o n
Understanding the relationship between the oral microbiome
and oral health has evolved. Biomarkers that participate in the
states of eubiosis and dysbiosis have been identified, establishing
a predictive association between microbial communities and oral
diseases such as dental caries.
From birth to adulthood, the or al microbiome is modulated by
different factors. These factors include intrinsic factors (biological
characteristics inherent to the host) and extrinsic factors (external
stimuli with the potential to influence host characteristics), which
can be divided into those modulated by the host (lifestyle and
behavior) and those not intentionally modulated by the host
(environmental factors).1
Within the host-modulated extrinsic factors, diet and oral
hygiene habits have signific ant effects on the oral micro biome and
may or may not favor an imbalance of the oral medium.
Frequent intake of fermentable carbohydrates is a necessary,
though not sufficient, condition for developing dental caries.
Fermentation of carbohydrates in the diet by microorganisms
present in the microbiota produces organic acids that lower oral
pH. When acid produ ction exceeds salivary buf fer capacity, the pH
descends, favoring an increase in acid-producing and acid-tolerant
bacterial species. When this condition persists over time, the oral
microbiome reaches a state of dysbiosis.
1,3,5Universidad de Buenos Aires, Facultad de Odontología, Cátedra de
Odontología Preventiva y Comunitaria, Instituto de Investigaciones en
Salud Publica, Buenos Aires, Argentina
2,4Universidad de Buenos Aires, Facultad de Odontología, Cátedra de
Microbiología y Parasitología, Laboratorio de Diagnostico Microbiológico
y Molecular, Buenos Aires, Argentina
Corresponding Author: Celina F Cornejo, Cátedra de Odontología
Preventiva y Comunitaria, Facultad de Odontología, Universidad de
Buenos Aires, Buenos Aires, Argentina, Phone: +541152876254, e-mail:
celinafcornejo3001@gmail.com
How to cite this article: Cornejo CF, Soken LJ, Salgado PA, etal. Detection
of Streptococcus mutans and Streptococcus sobrinus and Their Association
with Oral Microbiome Stressors in 6–18-month-old Infants. Int J Clin
Pediatr Dent 2023;16(1):68–73.
Source of support: Nil
Conflict of interest: None
Patient consent statement: The author(s) have obtained written
informed consent from the patient’s parents/legal guardians for
publication of the case report details and related images.
Detection of Streptococcus mutans and Streptococcus sobrinus
and Their Association with Oral Microbiome Stressors
in 6–18-month-old Infants
Celina F Cornejo1, Luciana J Soken2, Pablo A Salgado3, Laura A Gliosca4, Aldo F Squassi5
Ab s t r A c t
Aim: To determine the presence of Streptococcus mutans (S. mutans) and Streptococcus sobrinus (S. sobrinus) and their association with extrinsic
and intrinsic variables in 6–18-month-old infants.
Methods: This was an analytical, cross-sectional study of 65 6–18-month-old infants who visited the Centers for Early Childhood in Buenos
Aires City. Three groups were established according to the presence of teeth—group I (GI)—edentulous infants, group II (GII)—infants
with 1–8 teeth, and group III (GIII)—infants with 9–16 teeth. Data on the variables, diet, use of artificial teats, and oral hygiene were gathered
using a self-administered questionnaire. An oral examination was performed according to the International Caries Detection and Assessment
System (ICDAS II) criterion. A saliva sample was taken by aspiration with a sterile plastic syringe. Cariogenic Streptococci (CS) were counted
using the adherence test in modified gold broth (AT-MGB). Molecular detection and quantification were performed by quantitative polymerase
chain reaction (qPCR) (gtfB, gtfT, and tuf).
Results: A total of 12% of infants received oral hygiene, 38% used bottles, 30% used pacifiers, and 55% had sugar intake. S. sobrinus and S. mutans
were detected in 57.1 and 28.6% of the children with caries, respectively. Groups I, II, and III had CS counts of log 2, 3.4, and 3.7, respectively.
S. sobrinus was detected in 26.7% of GI, 52.9% of GII, and 85.7% of GIII, while S. mutans was detected in 13.3%, 35.3%, and 57.7%, respectively.
Conclusion: The prevalence of S. sobrinus was higher than S. mutans in all groups. The presence of CS was significantly associated with sugar
intake. No association was found between S. mutans and S. sobrinus and the presence of caries, hygiene habits, or use of artificial teats.
Clinical Significance: This study supports the role of diet in developing a cariogenic biofilm in children under 2 years of age.
Keywords: Colony forming units, Early childhood caries, Quantitative polymerase chain reaction, Streptococcus mutans, Streptococcus sobrinus,
Sugar intake.
International Journal of Clinical Pediatric Dentistry (2023): 10.5005/jp-journals-10005-2489
Cariogenic Streptococci in Early Childhood Caries
International Journal of Clinical Pediatric Dentistry, Volume 16 Issue 1 (January–February 2023) 69
underprivileged children from 45 days to 3 years of age. Both these
CPIs are located in District 4, w hich has one of the highest percentages
of households with UBN in Buenos Aires City, and where people’s
only access to healthcare is via the government-managed system.13
The eligible population for this study comprised 6–18-month-old
infants who attended the CPIs. Inclusion criteria for the
sample were 6–18 months of age, attending the selected
early childhood centers, and signed informed consent from
legal guardians (approved by CUPAD Ethics Committee—
EXP-UBA—0072332/201 7 Nº O12/2018 CETICA FOUBA). Exclusion
criteria were preexisting s ystemic diseases that could affect the o ral
microbiome or having received antibi otic therapy within the month
prior to being included in the study. Elimination criteria were any
infant not accompanied by parents or legal guardians at the time
of sampling and infants who we re edentulous at 12 months of age.
The study sampl e consisted of 65 infants who met the legal and
ethical principles and the inclusion and exclusion criteria.
The presence of teeth was the criterion used to form three
study groups—GI n = 30 edentulous infants, GII n = 20 infants
with 1–8 teeth, and GIII n = 15 infants with 9–16 teeth. Demographics,
diet, use of artificial teats, and oral hygiene of each participating
infant were surveyed through a questionnaire containing closed
questions (yes/no), self-administered by infants’ parents and/or
legal guardians. The item ”diet” or sugar intake was considered as
the intake of sucrose in the form of refined sugars added to the
infant’s usual diet. ”Artificial teat s” included the use of bottles and/or
pacifiers. ”Oral hygiene” was recorded as the presence or absence
of hygiene practices at some time of the day.
Dental clinical diagnosis was performed by a calibrated
professional (κ > 0.7), following ICDAS II criteria.14
For the microbiological and molecular s tudy, saliva samples were
taken from the floor of the mouth by aspiration with a sterile plastic
syringe. Samples were sent to the laboratory (Microbiological and
Molecular Diagnosis Laboratory of the School of Dentistry, Buenos
Aires University, Buenos Aires, Argentina), where they were vortexed
(”Velp Scientifica” ZX-3, Analogical 3000 rpm, Italy) and divided into
equal portions, of which one portion was preserved at −20°C for
molecular processing and another used for microbiological culture.
Cariogenic Streptococci (CS) were counted using the
AT-MG B.15 Cultures were incubated under anaerobic conditions
(Jarra 2,5 L, AnaeroJar OXOID™, AnaeroGen™ AN0035 kit, OXOID™)
for 48 hours at 36° ± 1°C, after which th ey were assessed by calibrated
personnel (κ > 0.7) under a stereoscopic microscope (binocular
stereomicroscope ”Arcano” ST30-L, China) at 50× magnification.
Molecular detection of S. mutans and S. sobrinus was done
by qPCR (real-time PCR). The genomic material was obtained
using a commercial kit (PrestoTM Mini gDNA Bacteria Kit, Geneaid
Biotecj Ltd., New Taipei city, Taiwan), following the manufacturer’s
instructions.
Deoxyribonucleic acid (DNA) integrity was quantified and
evaluated by spectrom etry (CYTATION 3 Cell Imaging reader, Biotek,
Winooski, Vermont, United States of Ame rica). The original ex tracts
were normalized at 20 ng/mL for use in the qPCR reactions.
Presence and quantification of S. mutans and S. sobrinus were
performed using the qPCR technique in a CFX96TM Real-Time
System thermocycler (Bio-Rad Laboratories, Inc., Hercules, California,
United States of America). Species-specif ic primers were used, and
the target was the gene encodin g the glycosyltransferase enzymes
gtfB for S . mutans and gtf T for S. sobrinus.16 Total Streptococci were
quantified by detection of the gene tuf.17 The reactions were
performed in duplicate, using SsoAdvanced Universal SYBR Green
microbiome.2,3 Like other countries in Latin America, Argentina
uses a direct multidimensional method to assess poverty, which
measures economic poverty and the living conditions of groups
and communities known as the Unmet B asic Needs (UBN) method;
it seeks to measure deficiencies in a series of needs considered
essential and generally discriminated into two main dimensions.
The first is asso ciated with minimum requirements related to private
household consumption—food, housing, and clothing. The second
is associated with public services provided to the community—
potable water, sanitation, public transport, healthcare, education,
and cultural facilities. Based on a standardized definition, a given
condition can be compared, need by need, or satisfier by satisfier,
thereby providing direct observation of unmet needs.4
The process of tooth eruption is o ne of the intrinsic factors that
modulate the oral microbiome because it creates a new habitat
in the ecosystem. The formation of the gingival sulcus at the
beginning of the eruptive period develops a new ecosystem that
favors colonization by anaerobi c microorganisms. At the same time,
the emergence of teeth provides surfaces to which biofilm-forming
microorganisms such as viridans group Streptococcus, in particular
S. mutans, can adhere.5
Some authors consider that colonization by CS (S. mutans-S.
sobrinus) occurs only after the dental eruption, while others have
detected the presence of microorganisms in predentate infants. It
has been reported that at least 20% of 2–3-year-old have been
colonized by S. mutans before tooth eruption, with colonization
increasing to 80% after tooth eruption.6,7
Recent studies on caries at early ages show that it is associated
with various cariogenic microorganisms. However, in agreement
with classical studies, new rese arch based on molecular techniques
continues to highlight the presence of S. mutans (serotypes
c, e, f, and k) and S. sobrinus (serotypes d and g) as cariogenic
microorganisms causing dental caries where the coexistence of
S. mutans and S. sobrinus would have a synergetic effect.8,9 Some
pathogenicity factors that could explain the cariogenic activity
of these microorganisms are related to the synthesis and use of
different glycosyltransferase-type enzymes and the presence of
glucan-binding proteins, which are essential during the process
of colonization of the enamel surface. Rupf et al.10 reported the
presence and acidogenic capacity of S. sobrinus, relating it to a
higher probability of developing dental caries on smooth surfaces
even though it is present in a lower proportion than S. mutans.
From birth to 2–3 years of age, children may be fed (exclusive or
mixed feeding) using artificial teats and bottles and maybe given
pacifiers to calm anxiety or pain and regulate feeding or sleeping
schedules. Studies have analyzed how the use of artificial teats
and pacifiers, the quality of their components, the frequency with
which they are used, and the addition of sugar may impact the
development of dental caries in infants.11,12
AI m
The aim of this study was to deter mine the presence of S. muta ns and
S. sobrinus and their association with intrinsic and extrinsic factors
in the establishment of dental caries in 6–18-month-old infants.
methods
This was an analytical, observational, and cross-sectional study
conducted at two early childhood centers [Certificate of Parental
Improvement (CPI), according to the acronym in Spanish], which are
facilities that aim to ensure the healthy growth and development of
Cariogenic Streptococci in Early Childhood Caries
International Journal of Clinical Pediatric Dentistry, Volume 16 Issue 1 (January–February 2023)
70
affected by incipient lesions (code 1-2-3 ICDAS II) and two
cavitated surfaces (code 4 -5-6 I CDAS II). Infants in GIII had a 66.7%
prevalence of dental caries, with a maximum of three surfaces
affected by incipient lesions and eight cavitated surfaces. In
samples corresponding to patients with carious lesions in GII, the
presence of CS was detected in 18.20% of S. mutans and 36.4%
of S. sobrinus, while in GIII, it was 40% for S. mutans and 80% for
S. sobrinus.
Cariogenic Streptococci (CS) counts by AT-MGB expressed in
colony-forming units pe r mL of saliva (CFU/mL) were 1 × 102 (Log10 2)
in G1; 2.5 × 103 (Log10 3.4) in G2 and 5.01 × 103 (Log10 3.7) in
GIII, with significant difference between GI and GIII, p < 0.01.
Molecular detection showed the presence of CS in edentulous
infants, and significant increases were observed in groups with
teeth (p < 0.01).
The relative abundance of Streptococcus spp (total Streptococci—
gene tuf) was 0.04% ± 0.048 for S. mutans and 0.017% ± 0.018 for
S. sobrinus, resulting in a mean relative abundance of 0.03%.
The extrinsic var iable ”sugar intake” was significantly associated
with the presence of both S . mutans and S. sobrinus, with p = 0.007
[odds ratio (OR) 5.93 (95% CI, 1.48–23.750] and p = 0.014 [OR = 3.70
(95% CI, 1.27–10.72)], respectively.
Detection of S. mutans and S. sobrinus with relation to the
extrinsic and intrinsic variables studied in each group showed
greater exponential growth of S. sobrinus than S. mutans (Fig. 3).
dI s c u s s I o n
The results of our study s howed that the detection of CS increased in
direct relation to infant ag e and the number of teeth, in agreement
with data published by Avasare etal.,20 who consider the presence
of more than five teeth to be a risk factor for acquiring CS. This
linear increase in detection in relation to the number of teeth may
Supermix (Bio-Rad Laboratories, Inc., Hercules, California, United
States of America) in a final volume of 10 mL, following the cycling
conditions recommended by the manufacturer.
The calibration curves for the genes gtfB, gtfT, and tuf were
based on a series of dilutions of DNA from strains of S. mutans
(ATCC 2517) and S. sobrinus (Ss001—culture collection of the
Department of M icrobiology at the School of Dentistr y, Universidad
de Buenos Aires, Buenos Aires, Argentina) and by sequencing two
gtfT genes) (ABI 3130xl Genetic Analyzer, Applied Biosystems,
Waltham, Massachusetts, United States of America). The number
of copies per dilution was calculated considering the genome size
for S. mutans as 2.6 M b18 and for S. sobrinus as 2.20 Mb.19 The targets
employed correspond to single-copy genes. The quantifiable range
for both species was 1 × 101–1 × 108 c/µL (copies/µL). The curves
employed were accepted with an ef ficiency of 84. 3% for S. sobrinus
and 93.6% for S. mutans.
Statistical Treatment
Qualitative variables were described by calculating the frequency
distribution, percentages, and 95% confidence intervals (CIs). For
quantitative variables, median, minimum, and maximum were
calculated. To compare the association between qualit ative variables,
the independence test (Chi-square) was applied, and for expected
frequencies lower than five, exact tests were used. To compare
proportions between groups, independent proportion comparison
tests with Bonferroni correction were used, and 95% CIs were
calculated for the difference in proportions. To evaluate risk, the OR
with 95% CIs was applied.
Statistical tests for indep endent samples were applied in all cases ,
with a significance level l ower than 5%, to reject the nu ll hypothesis.
Data were processed using the sof tware Statistical Package for
the Social Sciences (version 27), Statistics and data 14, MS Excel 2019,
MedCalc; Epidat 4.2.
results
Demographics are shown in Tabl e1, with distribution according to
sex and age for each study group.
Three infants from G2 and one from G3 were not included in
the microbiological/mol ecular analysis because it was not possible
to obtain a large enough sample. The presence of mutans group
Streptococcus per study group was as follows—GI, S. sobrinus
in 26.7% (95% CI, 13.5–4 4.1) and S. mu tans in 6.7% (95% CI, 1.4–19.7);
GII, S. sobrinus in 52.9% (95% CI, 30.3–74.6) and S. mutans in 35.3%
(95% CI, 16.3–58.9); and GIII, S. sobrinus in 85.7% (95% CI, 61.5–96.9)
and S. muta ns in 57.1% (95% CI, 31.9–79.7) (Fig. 1).
The data from the self-administered questionnaire showed
that 22.2% of the infants received oral hygiene, 38.5% used a
bottle, 30.8% used a pacifier, and 55.4% had refined sugar intake.
Figure 2 shows the detection of S . mutans and S. sobrinus and their
association with the extrinsic factors studied.
Clinical diagnosis showed that infants in G2 had a 55.0%
prevalence of dental caries, with a maximum of four surfaces
Table1: Demographic characteristics of the infants
Variables Total (n = 65) GI (n = 30; 46.2%) GII (n = 20; 30.8%) GIII (n = 15; 23.1%)
Sex Male 28 (43.1%) 11 (36.7%) 8 (40.0%) 9 (60.0%)
Female 37 (56.9%) 19 (63.3%) 12 (60.0%) 6 (40.0%)
The median age in months
(minimum-maximum)
66–22 66,796–20 1812–22
Fig. 1: Distribution of cariogenic Streptococci by groups
Cariogenic Streptococci in Early Childhood Caries
International Journal of Clinical Pediatric Dentistry, Volume 16 Issue 1 (January–February 2023) 71
the presence of CS. Our data are consistent with those reported
by Li etal.,5 establishing the relationship between the presence
of incipient lesions and bacterial species in children with and
without lesions.
The use of saliva samples may be insufficient for establishing
an association between the presence of carious lesions and CS
in this age group. Thus, future research should use alternative
sampling techniques and new sequencing techni ques for sample
processing.
The presence of carious lesions in infants in our study had
values similar to those reported in Ecuador by Valarezo-Bravo
and Marino-Solis,25 who used the same diagnostic criterion and
reported a prevalence of 49.3%. The systematic review by Ganesh
etal.26 on early childhood caries in India reported a prevalence
higher than 40%. However, studies by other authors such as Un
Lam etal.27 in Singapore and He etal.28 in China found v alues lower
than 35% for caries prevalence in infants up to 2 years old. These
differences may respond to socioeconomic and cultural differences
between countries. The role of CS as primary pathogens in dental
caries may depend on the study population, with their weight
being lower in populations with access to prevention programs
and higher in populations lacking access to prevention strategies
and treatment of caries.29
result from the dynamism in microbial communities associated
with tooth eruption.21
S. mutans and S. sobrinus were detected in the current study
from the group of 6-month-old infants. This finding agrees with
the data reported by Plonka etal.22 for a study on S. mutans
acquisition time and age of caries onset in a cohort of children
from birth to 36 months of age, which established that 6% of
the children who had developed caries at 30 months of age had
been colonized by S. mutans at the age of 6 months. Thus, early
detection of these cariogenic species could have a predictive
effect and enable preventive interventions to modulate oral
microbiota.
In contrast to other studies that report only the presence of
S. mutans, the techn iques used in our study found a more signif icant
presence of S. sobrinus among total participating infants as well as
within each study group. Thes e results are similar to those reported
by Gizani etal.23 Recent studies have repor ted the high acidogenic
capacity of S. sob rinus and established that when it is detec ted, there
is a greater probability of dental caries on smooth surfaces even if
it is present in a lower proportion.24
Through the data obtained fro m clinical diagnosis, the current
study showed the presence of incipient and cavitated carious
lesions but was unable to establish a significant association with
Fig. 2: Association of S. sobrinus and S. mutans with extrinsic variables
Fig. 3: Distribution of intrinsic and extrinsic variables according to the quantity of teeth
Cariogenic Streptococci in Early Childhood Caries
International Journal of Clinical Pediatric Dentistry, Volume 16 Issue 1 (January–February 2023)
72
clInIcAl sI g n I f I c A n c e
This study supports the role of diet in developing a cariogenic
biofilm in children under 2 years of age. This study supports the
role of diet in developing a cariogenic biofilm in children under
2 years of age and the need to imp lement interventions to educate
parents or caregiver s on feeding practices that may avoid dysbiosis.
Acknowledgment
This study was suppor ted by the University of Buenos Aires Grant–
UBACYT 20720190100007BA.
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14. Pitts N. ”ICDAS”—an international system for caries detection and
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15. Gliosca LA, Stoppani N, Lamas NS, etal. Validation of an adherence
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16. Yoshida A, Suzuki N, Nakano Y, et al. Development of a 5’
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The CFU/mL count is another method used to establish
the relationship between cariogenic microorganisms and the
presence or progression of carious lesions. Various longitudinal
studies have demonstrated the predictive value of the microbial
count and established that high CS counts are associated with
the presence and progression of dental caries.30,31 The results of
the counts in our study are similar to those reported by Edelstein
etal. 2016.32 However, we were unable to establish an association
between CS count and carious lesions. Fontana etal.33 found that
the S. mutans count and its association with total Streptococci
did not behave as significant predictors for caries progression
in 2–5-year-olds.
Streptococcus mutans (S. mutans) and S. sobrinus relative
abundance found by qPCR in the current study was similar to that
reported by Teng etal.34 and Dzidic etal.35 for 6–24-month-old
infants. Those studies also found no significant association
between the levels of thes e microbial species and the development
of carious lesions. However, other authors36,37 had found
significant associations when the relative abundance of CS
(S. mutans-S. sobrinus) reached 0.73%, establishing that values
higher than this may be good estimators for future caries
development. It is worth highlighting that even though the
frequency of detection of S. sobrinus in our study was higher
than the detection of S. mutans, the quantification and relative
abundance for S. mutans compared to Streptococcus spp. were
higher than for S. sobrinus. It would therefore be interesting to
increase the number of samples a nd perform a longitudinal study
to evaluate the behavior of the intrinsic and ex trinsic variables with
regard to the onset of carious l esions and how they are associated
with the proportions of CS in infants up to the age of 24 months.
Our study found that more than half the population had
refined sugar intake, which acted as a stress factor and favored
the presence of CS, as reported by Wan etal.38 Studies of infant
nutritional transition from birth to 5 years of age have claimed
that this is the period when infants learn what, when, and how
much to eat, based on the transmission of beliefs, attitudes, and
cultural practices regarding nutrition. It has been reported that
some of the influencing factors involved in an early introduction
to sugary foods and beverages are related to affordability of and
greater access to foods rich in refined sugars, low socioeconomic
level, and parents who study or work outside the home for long
periods.39,40
In the current study, no association was found b etween the use
of artificial teats and the presence of carious lesions or CS levels.
Other studies claim that the use of ar tificial teats is associated with a
higher frequenc y of dental caries in infants and the early acquisitio n
of CS, and a lower level of dental hygiene habits ,11,12 although these
findings were also linked to the addition of refined sugars to those
artificial teats.
co n c lu s I o n
It is relevant to highlight that S. mutans and S. sobrinus presented
low relative abundance in relation to total Streptococci detected
in the saliva samples analyzed. However, there was a significant
predominance of S. sobrinus to S. mutans in all three study groups.
Both species were obser ved to increase in direct relation to infant
age and the number of teeth according to dental chronology.
Analysis of the relationship between microbiology and the
different extrinsic factors showed a significant association with
refined carbohydrate intake.
Cariogenic Streptococci in Early Childhood Caries
International Journal of Clinical Pediatric Dentistry, Volume 16 Issue 1 (January–February 2023) 73
29. Johansson I, Witkowska E, Kaveh B, etal. The microbiome in
populations with a low and high prevalence of caries. J Dent
Res 2016;95(1):80–86. DOI: 10.1177/0022034515609554
30. Piva F, Pereira JT, Luz PB, etal. A longitudinal study of ear ly childhood
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