ORIGINAL RESEARCH

Prevalence of Streptococcus mutans and Candida dubliniensis

in Plaque of Caries-free and Caries-active 3–6-year-old
Children by Using Polymerase Chain Reaction: A Clinical
Study
Poornima Parameshwarappa1, Yanina Singh2 , Mebin G Mathew3 , Mallikarjuna Kenchappa4,
Nagaveni Nandanhosur Basavanthappa5, Roopa Korishettar6
Received on: 29 June 2022; Accepted on: 06 August 2022; Published on: 26 December 2022

A b s t r ac t
Introduction: Oral cavity harbors numerous types of microbial flora, which change frequently with changes in the environment and which in
turn leads to the process of caries.
Aims: To evaluate the prevalence of Streptococus mutans (S. mutans) and Candida dubliniensis (C. dubliniensis) in dental plaque of caries-free
(CF) and carries-active (CA) children aged 3–6 years using polymerase chain reaction (PCR).
Material and methods: A total of 18 CA and CF children in the age group of 3–6 years were randomly selected. Plaque samples were collected
using sterile micro brushes from teeth. Specific primers were used to carry out PCR in the plaque samples. Statistical analysis was done using
Fisher’s exact test.
Results: In CA group, S. mutans were seen in 61.1% of children which is statistically significant with a p-value of 0.04 and C. dubliniensis in 27.8%
of children whereas, in CF group, S. mutans were present in 27.8% of participants and C. dubliniensis in 5.6%. These results show that both
organisms were more predominant in the CA group.
Conclusion: Though both S. mutans (61.1%) and C. dubliniensis (27.8%) were present in CA group, S. mutans was strongly associated with dental
caries. In the individuals with high scores of def and international caries detection and assessment system II, both species were more prevalent.
Keywords: Candida dubliniensis, Dental caries, Polymerase chain reaction, Streptococcus mutans.
Journal of South Asian Association of Pediatric Dentistry (2022): 10.5005/jp-journals-10077-3238

Introduction 1,4–6
Department of Pedodontics and Preventive Dentistry, College of
Caries is a dynamic process involving interactions between the Dental Sciences, Davangere, Karnataka, India
tooth structure and the microbial biofilm, along with salivary 2
Department of Pediatric and Preventive Dentistry, School of Dental
and genetic influences. The rapid alternating periods of tooth Sciences, Sharda University, Greater Noida, Uttar Pradesh, India
demineralization and remineralization result in the initiation of 3
Department of Pediatric and Preventive Dentistry, Saveetha Dental
carious lesions.1 Caries is considered a chronic childhood disease College, SIMATS, Chennai, Tamil Nadu, India
and its global prevalence rapidly increasing in children of 2–5 years Corresponding Author: Yanina Singh, Department of Pediatric and
of age, making this age group a global priority action area.2 Preventive Dentistry, School of Dental Sciences, Sharda University,
Streptococcus mutans (S. Mutans) is one of the most important Greater Noida, Uttar Pradesh, India, Phone: +91 9634557587, e-mail:
bacteria involved in the etiology and progression of the carious yaninasingh@yahoo.co.in
lesion as they can metabolize carbohydrates and produce acids, How to cite this article: Parameshwarappa P, Singh Y, Mathew MG,
tolerate extreme acidic environments, and also can synthesize et al. Prevalence of Streptococcus mutans and Candida dubliniensis in
extracellular polysaccharides which improve their adherence to Plaque of Caries-free and Caries-active 3–6-year-old Children by Using
other microorganisms and tooth surface. 3 Polymerase Chain Reaction: A Clinical Study. J South Asian Assoc
Candida species causes superficial and systemic infections in Pediatr Dent 2022;5(3):132–135.
immunocompromised patients whereas, in healthy children, the Source of support: Nil
predominant oral habitats of Candida cause carious lesions. There are Conflict of interest: None
several characteristics of Candida that are related to cariogenicity. The
fungal H+-ATPase, which actively pumps protons out of the cell causes
an extraordinarily high acid tolerance and enables rapid extracellular cultivation, cultures, direc t microscopy, enz yme tests,
acidification. Candida adheres to saliva-coated hydroxyapatite and enzyme-linked immunosorbent assays, and species-specific
binds to native or denatured collagen leading to caries.4 deoxyribonucleic acid (DNA) probes. PCR is well known for its
Many techniques are employed for the detection of sensitivity as a diagnostic tool for the detection of microbes as
microbes involved in the carious process which include: compared to other techniques. 5

© The Author(s). 2022 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.
org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to
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 Prevalence of Streptococcus mutans and Candida dubliniensis in Caries

To come up with proper treatment options, it is necessary the amplified product of size 517 base pair and 175 base pair was
to have adequate knowledge of the disease and the pathogens identified with the help of DNA ladder. Amplified product of size
involved in its process. Dental caries occurs due to the involvement 517 base pair and 175 base pair was identified as S. mutans and
of various microbes and studies for the isolation of new species C. dubliniensis, respectively.
associated with dental caries is always a topic of research.
Hence, this study will determine the presence of S. mutans and Statistical Analysis
C. dubliniensis in the plaque of CF and CA children in the age group The data were subjected to statistical analysis. The statistical test
between 3–6 years using PCR. which was used is Fisher’s exact test.

Methods R e s u lts
This research project was approved by the Institutional Ethics Table 1 shows S. mutans and C. dubliniensis in CA and CF groups.
Research Committee (CODS/181/2018-2019), and written informed In CA group, S. mutans were present in 61.1% of children which
consent was obtained from the parents or guardians. is statistically significant and C. dubliniensis in 27.8% of children
whereas, in the CF group, S. mutans were present in 27.8% of
Sample Size Determination participants and C. dubliniensis in 5.6%. These results demonstrate
Using the following formula: that both organisms were more predominant in the CA group.
When different age groups were compared, S. mutans and
• n = Z2∂2/e2
C. dubliniensis both were more prevalent in the 5-year age group,
• n = (1.96)2(1.56)2/(0.72)2
in both CA and CF groups (Table 2).
• n = 18.01 ≈ 18
Samples were collected from 36 patients (19 males and 17 females) Discussion
aged 3–6 years who visited the outpatient department for dental
Research on microbial involved in dental caries began in the
treatment. Children who were using antimicrobial mouthwashes,
1980s and the predominant species that were found are: mutans
presented with any systemic disease, or had used antibiotics within
Streptococci (MS), others include Actinomyces, Lactobacillus, Candida,
the previous 3 months were excluded from the study.
and Veillonella.6 Candida species display relevance from childhood
Sample Collection until old age, that is, thrush, caries, periodontitis, infection of
A total of 18 samples were collected from CF individuals and dental implants, and denture stomatitis.7 The fungus Candida is
18 samples from CA individuals aged 3–6 years. Before collecting recognized for its involvement in biofilm formation and it inhabits
samples, the children were asked to rinse their mouth with water around 30–40% of oral microflora in healthy individuals.8 Henriques
to remove the debris. et al.9 stated that C. dubliniensis could form a mature biofilm which
The samples were collected using sterile micro brushes from represents one of the pathogenic features of Candida species.
the buccal surface starting from the most posterior teeth toward
the anterior teeth in a sweeping motion. The collected samples Table 1: Distribution of S. mutans and C. dubliniensis in CA and CF group
were placed in reduced transport fluid (RTF) by cutting the tips with S. mutans C. dubliniensis
sterile scissors. The samples were stored in RTF at −80oC.
CA CF CA CF
Microbiological Assessment Absent 7 13 13 17
The samples of dental plaque were mechanically dispersed (vortexed 38.9% 72.2% 72.2% 94.4%
for 30 seconds) and serially diluted with phosphate-buffered saline. Present 11 5 5 1
From appropriate dilutions, aliquots of 0.05 mL were inoculated 61.1% 27.8% 27.8% 5.6%
onto the agar media, that is, Mitis Salivarius Agar for S. mutans and
Fisher’s exact test p-value = 0.04* p-value = 0.18 (NS)
Sabouraud dextrose agar for Candida. For DNA extraction, culture
colonies from agar plates were isolated. * significant; NS, not significant
Deoxyribonucleic acid (DNA) extraction was done by modified
proteinase K method using lysis buffers (Chromous Biotech, Table 2: Distribution of S. mutans and C. dubliniensis according to
different age groups
Bengaluru, India) and proteinase K enzyme (Chromous Biotech,
Bengaluru, India). The primers used were for: S. mutans C. dubliniensis
Age Absent Present Absent Present
Streptococcus mutans (S. mutans) primers
3 years 6 3 7 0
• GTFB- F 5’-ACTACACTTTCGGGTGGCTTGG-3’ 85.7% 14.3% 100.0% 0.0%
• GTFB- R 5’-CAGTATAAGCGCCAGTTTCATC-3’ 4 years 7 4 8 2
Carries dubliniensis (C. dubliniensis) primers 70.0% 30.0% 80.0% 20.0%
5 years 3 5 8 4
• GTFB- F 5’-AGTTACTCTTTCGGGGGTGGCCT-3’ 25.0% 75.0% 66.7% 33.3%
• GTFB- R 5’-AAGATCATTATGCCAACATCCTAGGTAAA-3’ 6 years 4 6 7 0
• Deoxyribonucleic acid (DNA) extraction was followed by DNA
57.1% 42.9% 100.0% 0.0%
amplification using PCR master mix (Ampliqon red) (Ampliqon,
Odense M, Denmark) followed by gel electrophoresis, in which Fisher’s exact test p-value = 0.06 (NS) p-value = 0.19 (NS)

Journal of South Asian Association of Pediatric Dentistry, Volume 5 Issue 3 (September–December 2022) 133
 Prevalence of Streptococcus mutans and Candida dubliniensis in Caries

Identification of S. mutans can be done in various ways, these Matee et al.17 reported high levels of S. mutans in some CF children.
include the methods which morphologically differentiate the All these results are suggestive that S. mutans is one of the most
bacteria using culture, biochemical tests, and PCR.10 In this study, prevalent bacterial species in CA children.
PCR was used (Figs 1 and 2), as it is more sensitive and accurate; Kneist et al.18 found the presence of C. dubliniensis more
hence it is significant in eliminating any false positives or false in plaque and carious dentin samples. In a study by Al-Ahmad
negative results; also, it is faster and less technique sensitive than et al.7 C. dubliniensis species were found in more than one quarter
traditional methods.11 (27%) of plaque samples of the CA children but were never detected
The results from the present study demonstrated the high in the control specimen, that is, CF. Lozano Moraga et al.19 found that
number of S. mutans in CA children as compared to the CF C. dubliniensis were present only in the most caries-affected group.
population. Out of 36 children, S. mutans strain was found in plaque In a study by de Jesus et al.20 authors concluded that in the pooled
samples of 16 children (11 CA children and five CF children) (Table 1). plaque mycobiome of 40 children with early childhood caries with
Fragkou et al.12 found similar results that CA children harbored more the same number of CF children, C. dubliniensis dominated the
frequently and significantly higher numbers of S. mutans that is, in mycobiome of children with caries. All these results are similar to the
15 out of 39 children, mostly with high deft. Similar results were seen results of the present study, that is the prevalence of C. dubliniensis
in a study by Fujiwara et al.13 where 39.9% of the total population was however nonsignificant, but the number of candidates having
harbored S. mutans with the majority of the population with high the species was more in the CA group than that of CF. A total of six
deft. Results showed a significant global/overall relationship children displayed the presence of C. dubliniensis in plaque samples
between MS acquisition and dental caries. A study by Hata out of which five were from the CA group and one CF (Table 1).
et al.14 and Valdez et al. 3 found counts of MS in biofilms of children Hence, C. dubliniensis is positively correlated with caries and it
having early childhood and severe childhood caries higher than increases steadily as caries severity increases.21
those found in CF children. Vacharaksa et al.15 also found a high Milgrom et al. 22 in their study found that the proportion of
count of S. mutans in his study when compared to children of the children colonized by S. mutans increased with age. Karn et al.23 also
CA group than to CF. In contrast, Loyola-Rodriguez et al.16 found found similar results and quoted that there is a trend toward an
the percentage of S. mutans isolation similar in CA and CF children. increasing percentage of children colonized with S. mutans with an
increase in age. Okada et al.5 suggested that S. mutans are generally
established in the oral cavity of children before 3 years of age.
The findings of a study conducted by Ghazal et al.24 was that the
median time without MS acquisition (50% of the children not having
positive MS test) was 2 years. Approximately 79% of the children
had positive salivary MS tests by the age of 4 years. In the present
study, we found that the subjects in the higher age group had more
caries as compared to lower age groups and proportionately higher
counts of S. mutans and C. dubliniensis were seen with an increase
in age (Table 2).
This study contributes to determining the prevalence of S.
mutans and C. dubliniensis in CA and CF children however more
such studies are required to determine new microbial species with
different age groups and among the different populations which
will benefit in understanding the pathogenesis and etiology of
the disease and will also contribute in considering new treatment
modalities. Very few studies have been reported on identifying
Candida species as a cariogenic organism and establishing the
Fig. 1: Streptococcus mutans identified under ultraviolet light
pathogenesis of C. dubliniensis in caries progression; hence more
literature is needed in this context. 25 The limitation encountered
with PCR is that it cannot differentiate dead from live bacteria. Also,
different methods for microbial isolation should be determined
as no suggested method acts as a gold standard for the isolation
and identification of microbes.

C o n c lu s i o n
The present study provided the corroboration of oral carriage
of C. dubliniensis and S. mutans in 3–6-year-old CA children. The
association of S. mutans as an active cariogenic organism has
been proved several times by various authors in the past, but very
fewer shreds of evidence have demonstrated the role of Candida
species in caries. Considering results from previous studies and the
present study, C. dubliniensis can be contemplated as one of the
associated pathogens in dental caries. However, future research
on these species has to be carried out to unearth the etiology of
Fig. 2: Candida dubliniensis identified under ultraviolet light dental caries.

134 Journal of South Asian Association of Pediatric Dentistry, Volume 5 Issue 3 (September–December 2022)
 Prevalence of Streptococcus mutans and Candida dubliniensis in Caries

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