| Abstract|| |
Background and Objectives: The aciduric mutans streptococci (MS) group, including Streptococcus mutans (SM) and Streptococcus sobrinus (SS), is highly cariogenic. Relationship between increments in dental caries and the presence of these species is not yet clarified. It is of great importance to separately determine the presence of these two species for understanding their role in dental caries, accurate prediction, and effective prevention. Hence, this study was undertaken to detect the presence of SM and SS in plaque samples of caries-free (CF), early childhood caries (ECC), and severe early childhood caries (S-ECC) and also to compare the possible relationship between these species with the occurrence of ECC. Materials and Method: In all, 120 healthy children between 3 and 6 years of age were randomly allocated into three groups: Group A (n = 40) CF, Group B (n = 40) ECC, and Group C (n = 40) S-ECC. Plaque samples were collected from all approximal surface and gingivobuccal surfaces of teeth using a sterile universal scaler and dmft/dmfs scores were taken. The presence of SM and SS was evaluated using real-time polymerase chain reaction. Results: The presence of SM in CF, ECC, and S-ECC was 10.0%, 27.5%, and 42.5%, respectively, and SS was 5.0%, 40.0%, and 47.5% in CF, ECC, and S-ECC, respectively. Comparison of mean relative quantification (RQ) values of SM between three study groups showed significant results between CF and S-ECC at P value 0.003. Comparison of mean RQ values of SS showed significant results between CF to ECC and CF to S-ECC at P value <0.001. This study showed significant results between the mean dmft/dmfs scores in three study groups and the presence of high levels of SM and SS in ECC and S-ECC groups. However, the study showed weak positive correlation between dmfs scores and both SM and SS species in ECC and S-ECC. Interpretations and Conclusion: Higher levels of SS were more closely related to the caries-active lesions leading to severity of dental disease, that is, both in ECC and S-ECC. There was significant difference between mean dmft/dmfs scores in three study groups and it also showed positive correlation between dmfs scores and SM and SS colonization in ECC and S-ECC status.
Keywords: Caries experience, dental plaque, preschool children, real-time PCR, Streptococcus mutans,Streptococcus sobrinus
|How to cite this article:|
Veena R L, Nagarathna C. Correlation of streptococcus mutans and streptococcus sobrinus colonization with and without caries experience in preschool children. Indian J Dent Res 2020;31:73-9
|How to cite this URL:|
Veena R L, Nagarathna C. Correlation of streptococcus mutans and streptococcus sobrinus colonization with and without caries experience in preschool children. Indian J Dent Res [serial online] 2020 [cited 2022 Jan 20];31:73-9. Available from: https://www.ijdr.in/text.asp?2020/31/1/73/281811
| Introduction|| |
Early childhood caries is recognized as a serious public health problem due to its high prevalence, impact on quality of life, potential for increasing risk of caries in the permanent dentition, and its role in general health. Early childhood disease (ECC) is defined as the presence of one or more decayed (noncavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces in any primary tooth in a child 71 months of age or younger. S-ECC is defined as one or more cavitated, missing (due to caries), or filled smooth surfaces in primary maxillary anterior teeth or a decayed, missing, or filled score of ≥4 (age 3), ≥5 (age 4), or ≥6 (age 5) surfaces from ages 3 to 5. The AAPD guidelines differentiate S-ECC from ECC as a consequence of a more destructive disease process based on age and number of carious, filled, or missing tooth surfaces.
The onset and progression of dental caries are determined by a wide array of risk factors, including genetic, dietary, environmental, socioeconomical, and behavioral determinants. The changes in the acidity and metabolic status of the oral cavity may induce alterations in the microbial population of the oral biofilms. Dental plaque or biofilm are complex microbial communities found on tooth and mucosal surfaces. The aciduric MS group, including SM and SS, is highly cariogenic microorganisms identified using saliva and plaque samples. SM has been identified as the principal cariogenic bacterium for caries initiation, whereas SS is thought to enhance caries initiation progression and development.
The presence of MS in plaque or saliva of caries-free (CF) preschool children is associated with significant increase in caries risk. The plaque samples were processed using microbiological culture and polymerase chain reaction methods (PCR) and acceptable results were obtained. Quantitative real-time PCR with species-specific primers can provide an accurate and sensitive method for detecting and quantification of individual species and bacterial population. There is also a growing recognition in the literature that the cariogenic potential may be determined by complex interactions in dental plaque biofilms rather than solely the virulence properties of a single organism. Hence, in our study PCR was used to detect and compare the presence of SM and SS in dental plaque sample of CF, ECC, and S-ECC conditions in preschool children.
Currently, it is of great importance to separately determine the presence of these two species in children for understanding its role in dental caries for creating a complete model of caries etiology and accurate prediction and effective prevention of dental caries.,
Thus, the purpose of this cross-sectional study was to detect the presence of Streptococcus mutans (SM) and Streptococcus sobrinus (SS) in plaque samples of children, without caries experience and with caries experience, that is, ECC and S-ECC conditions. And also to compare the possible relationship between SMand SSand dental caries.
| Materials and Methods|| |
This study was conducted in the Department of Pedodontics and Preventive Dentistry and was approved by the Institutional Review Board (RRDC and H/115/2015-2016). The time period of the study was from November 2015 to October 2017. The sample size has been estimated using the GPower software (version. 188.8.131.52., released 2014; Kiel University, Germany). Considering the effect size to be measured (dz) at 25%, power of the study at 80%, and the margin of the error at 10%, the total sample size needed is 120. The selection criteria for teeth included in the study were as follows: (1) children with only primary dentition having normal occlusal relationship, (2) children with ECC [presence of one or more decayed (noncavitated, white lesion, or cavitated lesions), missing tooth due to caries, or filled tooth surfaces in any primary tooth in a child 71 months of age or younger], (3) children with S-ECC [children with one or more cavitated, missing (due to caries), or filled smooth surfaces in primary maxillary anterior teeth or a decayed, missing, or filled score of ≥4 (age 3), ≥5 (age 4), or ≥6 (age 5) surfaces], and (4) CF children (had no caries including white-spot lesions or restorations). Children who were on antibiotics within the past 3 months, children with existing restorations and undergone any kind of orthodontic treatment, and those who require special healthcare needs were excluded from the study.
Totally 120 preschool children were included in the study. The study design was explained to the child's parent or guardian, from whom informed consent was obtained before the start of the procedure. The study design, protocol, and informed consent were approved by the Institutional Review Board. Routine case history was taken and dental examination was carried out to identify CF, ECC, and S-ECC conditions using plain dental mirror and explorer on the dental chair with optimal artificial light. All examinations and dental treatments were completed in standard dental operatories under ideal conditions. The World Health Organization caries diagnostic criteria were used for determining the dmft [decayed, missing, filled, teeth] and dmfs [decayed, missing, filled, surfaces] index. Based on the scores, the children were allocated into the following three groups: Group A (n = 40) – CF children, Group B (n = 40) – children with early childhood caries, and Group C (n = 40) – children with severe early childhood caries.
All the subjects were instructed to refrain from tooth brushing and eating for two hours before sample collection. An aseptic environment was maintained throughout the sample collection. All the teeth were cleaned with water and then isolated with cotton rolls and gently dried with compressed air before plaque samples were collected. Supragingival plaque samples were collected from all approximal surfaces and gingivobuccal surfaces of teeth from each subjects using a sterile universal scaler [Figure 1]. Plaque samples were placed into marked sterile Eppendorf tubes which contained transport media, 200 μL of 10 × TE buffer (Tris–HCl EDTA). Samples were then transferred immediately to the laboratory and stored at −70°C until processing in the laboratory (New Brunswick Scientific ultra-low-temperature freezer).
PCR procedure was carried out in laboratory for molecular biology. Analysis of SM and SS was done using real-time PCR method.
Processing of samples
Analysis of SM and SS was done using qRT-PCR. DNA extraction of the samples was done using highly purified Invitrogen DNA isolation kit (Purelink™ DNA extraction kit; Applied BioSystems, India). The standard “Proteinase K” method was followed for DNA isolation. The extracted DNA was purified using a purification procedure designed for purifying genomic DNA using a spin column-based centrifugation procedure on a total time of 10–15 min. Custom SYBR® Green assay reagents for SM and SS (Applied Biosystems) were used in this study. The primer sequence specific to SM and SS selected for the study was as follows:
Forward: 5′-TGC TAT CTT TCC CTA GCA TG-3′
Reverse: 5′-GGT ATT CGG TTT GAC TGC-3′
Forward primer: 3′-TCCTACGGGAGGCAGCAGT-5′
Reverse primer: 5′-GGACTACCAGGGTATCTAATCCTGTT-3′
Each set of PCR analyses included a negative control (water blank). The conditions for real-time PCR were as follows: holding stage at 95°C for 10 s followed by 40 cycles of shuttle heating at 95°C for 15 s and at 60°C for 1 min. The melt curve stage was at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. 16S rRNA was used as an endogenous control (SYBR® Green assay reagents). Relative quantification (RQ) for SM and SS was based on Ct (the number of PCR cycles necessary to obtain the threshold signal of fluorescence) values. All the calculations were done using Applied Biosystems Software.
Data obtained from Applied Biosystem were sent for statistical analysis. Statistical Package for Social Sciences for Windows (version 22.0, released 2013; IBM Corp, Armonk, NY) was used to perform statistical analyses. Descriptive analysis of all the explanatory and outcome parameters was done using mean and standard deviation for quantitative variables, frequency, and proportions for categorical variables. One-way analysis of variance test followed by Tukey's post hoc analysis was used to compare the mean dmft and dmfs scores between different study groups. Kruskal–Wallis test followed by Mann–Whitney U-test was used to compare the mean SM and SS count between different study groups. Spearman's correlation test was used to estimate the relationship between dmfs and microbial count in ECC and S-ECC groups. The level of significance (P-value) was set at <0.05.
| Results|| |
Demographic details of our study showed that 57.5% were males and 42.5% were females in CF group and 65% were males and 35% were females in ECC and S-ECC groups. The mean age was 3–6 years in CF and S-ECC and 4–6 years in ECC children [Table 1]. In all, 120 samples were subjected for PCR analysis, and SM was detected in 10.0% of CF group, 27.5% of ECC group, and 42.5% of the S-ECC group. SS was detected in 5.0%, 40.0%, and 47.5% in the CF, ECC, and S-ECC, respectively [Table 2]. PCR yielded 26.7% SM and 30.8% SS from the plaque samples.
|Table 2: Comparison of presence of S. mutans and S. sobrinus microorganisms in three different study groups using Chi-square test|
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Comparison of mean RQ values of SM between three study groups showed significant results between CF (Group A; RQ = 0.076 ± 0.267) and S-ECC (Group C; RQ = 0.162 ± 0.325) at P value 0.003. Comparison of mean RQ value of SS between three study groups also showed significant results between CF (Group A; RQ = 0.071 ± 0.326) and ECC (Group B; RQ = 4.239 ± 13.543) and S-ECC (Group C; RQ = 7.198 ± 18.966) at P value <0.001 [Table 3]. This study showed significant results between the mean dmft/dmfs scores in three study groups and the presence of higher levels of SM and SS in ECC and S-ECC groups [Table 4]. However, the study showed weak positive correlation between dmfs scores and both SM and SS species in ECC and S-ECC [Table 5].
|Table 3: Comparison of mean S. mutans and S. Sobrinus RQ value between different study groups using Kruskal-Wallis test followed by Mann-Whitney U post hoc analysis|
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|Table 4: Comparison of mean dmft and dmfs scores between different study groups using one-way ANOVA test followed by Tukey's post hoc analysis|
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|Table 5: Spearman's correlation test to estimate the relationship between dmfs scores, S. mutans, and S. sobrinus in ECC and S-ECC groups|
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| Discussion|| |
Dental caries is an infectious disease in which members of the MS group have been implicated as major etiological agents. Most humans harbor MS, but not all humans manifest caries. The levels of SM in the oral cavity have been shown to have a correlation with both past caries experience and future caries activity. The presence of SS may be correlated with the development of caries but possibly when the disease process has already started and colonization is favored by a low environmental pH.
In our study, higher level of both microorganisms quantified in caries prevalence groups. Higher scores indicate early colonization in the primary teeth, and the severity of the carious activity was consistent with other reports. The virulence of MS is directly related to properties that enable these organisms to colonize and thrive on the tooth surfaces during acidic conditions. In our present study, comparison of mean RQ values of SM between three study groups showed significant results between CF and S-ECC. These results were similar with other previous studies done in preschool children; they found that SM in caries active group was significantly higher than the CF group.,,,, Contrary to the above findings, studies reported high levels of MS in some CF children., Comparison of mean RQ value of SS between three study groups also showed significant results between CF to ECC and S-ECC, and this suggests that SS was associated with active dental caries and children with caries experience. These results were similar to other previous studies, and they found that more S-ECC children were SS-positive compared with CF.,,,, Thus, the presence of SS was a risk factor for high caries activity in severe early childhood caries. Contrary to the above findings, a high incidence of SS-positive subjects in Kenya who did not have active dental caries was found.
Infants with caries had higher isolation frequencies and higher counts of SM, lactobacilli, and yeasts (but not S. sobrinus) compared with those who were clinically CF. Sucrose intake is one of the factors that correlate with the level of infant colonization. The ability to synthesize water-insoluble glucans from sucrose is an important virulence factor in initial caries development, in that it increases mutans streptococcal adherence and accumulation in the plaque of young children. The mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonization by SM and this mutacin exhibits different degrees of inhibition against other bacteria.,, SM can be so dominant that the plaque above the carious lesions consists almost entirely of this microorganism in relation to other pathogenic bacteria. Caries-active subjects tend to carry more genotypes of SM and multiple strains with stronger cariogenic ability and adherence ability, compared with CF subjects. Reasons for early colonization of microorganisms could be most of the children receive the organisms from their mother through vertical transmission which occurs in two ways, that is, direct transmission involves the comingling of the parent's and child's saliva, as during kissing, indirect transmission involves the placement of objects into the parent's mouth and then into the infant's mouth. In addition, studies have shown that acquisition of MS has been found to be most common during a discrete window period that occurs at around 7–31 months in primary dentition. Some have suggested that there is a window of infectivity for MS at an early age, after which colonization is not likely to occur, whereas others have reported its predentate presence in infants as young as 3 months of age.,,, Previous studies, reviewed by Gibbons and Van Houte, have demonstrated that MS have a feeble capacity to become attached to epithelial surfaces. Therefore, it seemed unlikely that these organisms could colonize in the mouth of a normal infant before the teeth erupt.
ECC is associated not only with increased levels of SM but also with elevated levels of total streptococci in the mouth., Hence, we also see an increase in SS level. Bacterial levels were significantly increased at each of the caries sites including white spot, cavitated lesions, and excavated carious dentin, compared to those found on intact enamel surfaces. The initial attachment of SS to the pellicle is minimal and in a less specific manner but, once attached, it can, in the presence of sucrose, accumulate by massive glucan formation. Any Glucosyltransferase(GTF) s (and especially those secreted from SM) present in the pellicle promote the initial attachment of SS explaining why SS is rarely found without SM. It was, however, recently noted that SS becomes established later than SM in the oral cavities of children over the age of 3 years. It is possible that SS is an indicator microorganism of an already diseased environment: when SS is present in the oral environment, it is unlikely that this individual is CF. Caries can also be found in an environment in which SS is below detection levels. However, the presence of these species in healthy children can represent a marker of ECC risk facilitating the detection of this disease in healthy individuals.
This study showed significant results between the mean dmft/dmfs scores in three study groups and the presence of high levels of SM and SS in ECC and S-ECC groups.,, However, the study showed weak positive correlation between dmfs scores and both SM and SS species in ECC and S-ECC. In ECC, it is common to see caries extending in almost all the surfaces of teeth including labial and lingual aspects. Higher dmfs does not necessarily mean higher caries activity or risk. There were children who had high dmfs because of high filled score, or high decayed score. The former might represent high caries history, and the latter high caries activity. Dmft/dmfs scores are the crucial indicators of caries in young children. Weak positive correlation between dmfs scores and two microorganisms studied could be due to the small sample size, comprising preschool children. It is assumed as reported earlier that the carious teeth significantly increased by ages because as age increases, more exposure to cariogenic factors will occur. Second, dental caries is a multifactorial disease including microorganisms; other factors such as genetic, dietary, environment, socioeconomical, and behavioral determinants also influence the severity of disease which we did not consider clinically in our study.
In this study, supragingival dental plaque was used as a source for detection of cariogenic bacteria.,,,, When the microorganism count of a specific surface such as molar fissures in dental caries or restoration is required, the most useful method is dental plaque collection. In our study, we used universal scalers as they have tips on both ends and designed to work in anterior/posterior or buccal/lingual and all the approximal surfaces of oral cavity. MS are more often associated with more acidic response of caries-affected plaque when compared with saliva and also shows 70% sensitivity for estimating MS., The intention to relate the presence of cariogenic bacteria and dental caries using saliva as a source of the bacteria does not permit establishing an effective association. Even though the presence of SM is high in saliva, it is lower on the surface of enamel where the bacteria actually manifests its capacity to produce acid leading to subsequent demineralization.
In this study, we chose qRT-PCR with SYBR Green reagents for detection of SM and SS. PCR is a powerful and reliable tool for the detection of bacterial concentrations in clinical samples. It cannot detect the presence or absence and also the amount of bacteria that can be co-related to clinical conditions. The use of PCR presents with advantages such as easy sample handling, ability to detect low numbers of bacterial species with a detection limit of as few as 25–100 cells, stability of samples on freezing over long periods of time, 10–100 times more sensitive than the other identification method, and also it reduces the time to 6– 7 h for processing. qRT-PCR monitors the levels of cariogenic bacteria in dental plaque; however, quantitative analysis for RQ determines the changes in steady-state mRNA levels of a gene across multiple samples and expresses it relative to the levels of an endogenous control RNA (i.e. 16sRNA) which is known as RQ. However, the disadvantage of these direct PCR methods is the potentially low sensitivity for directly detecting bacteria from clinical samples.
As ECC and S-ECC are a bacterial-dependent disease, it is probable that the pathogenic effects of these two microorganisms may be far greater than that of either SM or SS acting alone. In addition to SM and SS, recent discoveries have shown that other microorganisms in saliva and dental plaque may also contribute to the pathogenicity of S-ECC and ECC. Besides microorganisms, other risk determinants of cariogenic factor should be considered including older school children. More research is warranted to elucidate the synergistic mechanisms of these two microorganisms particularly in a mixed microbial environment and to determine the reliability and accuracy of the use of relative levels in predicting caries formation and also the mechanism by which these two microorganisms compete with each other to result in disease or health of the oral cavity for determining disease progression and prevention.
| Conclusion|| |
In this study, qRT-PCR detected the presence of SM and SS in caries-experienced children. Higher levels of SS were more closely related to active caries lesions which also showed involvement of multisurface lesions leading to the severity of dental diseases in children which is a characteristic feature of early childhood caries.
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Conflicts of interest
There are no conflicts of interest.
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Department of Pedodontics and Preventive Dentistry, Rajarajeswari Dental College and Hospital, Bengaluru - 560 074, Karnataka
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]