Oral diseases are categorized in the top 100 causes of disability-adjusted life years. ¹ Among them, dental caries affects 60%–90% of children as well as majority of adults and shows serious economic, social, and functional impacts on individuals. ² , ³ According to the World Health Organization (WHO), oral diseases are the fourth-most expensive disease to be treated in industrialized countries. ²

Based on ecological plaque hypotheses, there is a dynamic relationship between the composition of dental plaque (biofilm and microbial community) and the environment. Hence, caries is the consequence of bacterial unfavorable shift from beneficial organisms (that preferentially grow at neutral pH) to acid producer (tolerant to the acidic conditions) driven by changes in the dental environment to caries-conducive environment (sugar accessibility and/or low saliva flow). Hence, dental caries can be controlled by bacterial activity inhibition and interfering with the factors that drive the microbiota shifts (i.e., reducing the amount and frequency of sugar intake). ⁴

Mutans streptococci (MS) including Streptococcus mutans and Streptococcus sobrinus as members of acid-generating and acid-tolerating bacterial community using glucosyltransferases (Gtfs) metabolize sucrose as a substrate to synthesize insoluble and soluble glucans, which change the extracellular composition of the biofilm structure and increase bacterial adhesion and biofilm accumulation on dental surface. ⁵ , ⁶ Studies have been conducted on special populations to investigate the determinative role of MS in dental caries and the association of S. mutans and S. sobrinus with caries formation. ⁷ , ⁸ , ⁹

There is also an agreement that sugars are implicated in dental caries. ¹⁰ , ¹¹ , ¹² Sugar by pH decrease can change the composition of the oral microflora toward caries-promoting one and increase the colonization and outgrowth of MS. ⁴ It is shown that people with higher level of sugar consumption have significantly higher incidence of dental caries than those with lower level of sugar consumption in their diet. ¹³ , ¹⁴ , ¹⁵ , ¹⁶

In addition, tooth brushing can modulate the number of MS ¹³ and the impact of sugar on dental caries. ¹⁴ It is shown that poor oral hygiene practices can be a risk factor for DMFT scores and preventive measurements such as fissure sealant and fluoride therapy can prevent dental caries occurrence. ¹⁵ , ¹⁶

To prevent dental caries or estimate future caries activity, caries etiology revisiting is necessary. Numerous studies have shown the relationship between mouth microflora/sugar/tooth brushing and caries in children, ⁷ , ⁸ , ¹² , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ and such estimations in adults have been largely ignored. This cross-sectional study which was conducted on 20–30-year-old adults, for the first time in Iran, evaluates the association of S. mutans, sugar consumption, and oral hygiene with DMFT scores, and is aimed to compare the influence of the aforementioned factors on DMFT score changes. In other words, the presence or absence of S. mutan s (as a member of biofilm community) and its influence on DMFT scores were compared with sugar consumption, an environmental-driven factor for microbial shift, and with tooth brushing.

According to the results of the studies, the incidence of caries is higher in the co-existence of S. mutans and S. sobrinus in mouth microflora. ⁷ , ⁹ , ¹⁷ , ²¹ It has also been shown that S. sobrinus, unlike S. mutans, is not detected in any healthy controls, and may be a more effective factor of caries than S. mutans. ²¹ As the purpose of the present study is to estimate the presence of S. mutans and its relationship with higher caries experience and to compare its effect with sugar consumption and tooth brushing factors, molecular detection of S. mutans and S. sobrinus from saliva and plaque samples was performed, and samples negative for S. sobrinus were selected as the study population. The identification of S. mutans and S. sobrinus was based on gtf B and gtf I gene detection for S. mutans and S. sobrinus, respectively. These genes express important cariogenic factors and have nucleotide sequences specific to S. mutans and S. sobrinus. ²² , ²³

Study population, dental caries, sugar consumption, and tooth brushing habit assessments

A total of 459 individuals aged 20–30 years (in Tehran province) were included in this cross-sectional study. People who were excluded were as follows: (a) those who used fluoride or taken antibiotics within the previous 3 months or with systemic diseases; (b) had <24 permanent teeth; and (c) used dental appliances (orthodontic appliances and dentures). It should be mentioned that, in Iran, water supply and food, salt, or milk fluoridation is not conducted, so in the studied population, the only exposure to fluoride is the fluoride in the toothpaste. Dental examinations are performed using a plane dental mirror and an explorer under artificial light. The DMFT value was calculated following the WHO's caries diagnostic criteria. ²⁴ The research assistants were encouraged to review the caries scoring criteria protocol. Calibration or interrater reliability analysis using the Kappa statistic was performed (with 13 individuals) by comparison with a dentist who did not participate in the fieldwork to determine consistency among raters. ²⁵

A Sugar Frequency Questionnaire (SFQ) consisting of 22 items on commonly consumed snacks in Iran was designed [Appendix A]. The internal consistency of the questionnaire was assessed by Cronbach's alpha and the items in which their removal would increase the alpha amount were reconsidered. Each participant was asked how often in a month, week, or day each item was consumed. A unit of sweet food consumption per day (unit/day) and a mean of unit per day were calculated for each participant. A score for the number of consumed sweet foods was also recorded (from 1 to 22) for each participant. Population grouping to low-, intermediate-, and high-level sugar consumption was done according to the numbers calculated for each person using the sugar consumption score (sugar consumption score: mean unit/day/score from 1 to 22) [Appendix A]. Population grouping for sugar consumption based on the yielded sugar consumption scores was as follows:

Low: 0.44/4–0.76/10

Each study participant was inquired about his/her tooth brushing frequency, and it was categorized as never or rarely, once a day (1/d), twice a day (2/d), and three times a day (3/d).

Streptococcus mutans and Streptococcus sobrinus detection

The plaque samples were collected by rubbing the vestibular face of the smooth and proximal surfaces of the central and lateral incisors, canines, premolars, and the first and second molars with a sterile swab. Sample size was calculated based on Okada et al.'s study. ²⁶ The mean concentration of DNA recovered from the saliva and plaque samples with approximately 660 μg/ml was sufficient for performing a polymerase chain reaction (PCR)-based survey. Each plaque sample was immediately placed in a sterile tube containing saliva sample of the same participant. Tubes were then vortexed for 30 s for content homogenization and processed within 2–3 h after collection.

Cells from 500-μl homogenized plaque and saliva mixture were pelleted down and washed twice with TE buffer (10 mM trisaminomethane hydrochloride [Tris-HCl], 1 mM ethylenediaminetetraacetic acid [EDTA], pH: 8), suspended in 200 μl boiling buffer (Tris-HCl 10 mM, pH: 8, EDTA 40 mM, pH: 8, sodium dodecyl sulfate [SDS] 0.2%, pH: 7.2), boiled for 10 min, and immediately transferred to ice to release DNA from cells or incubated in lysis buffer (Tris-HCl 10 mM, pH: 8; EDTA 40 mM, pH: 8; SDS 0.2%, pH: 7.2; lysozyme 2 mg/ml) for 2 h at 37°C with occasional inversion. The cell suspension was then mixed with 200-μl proteinase k solution (Tris-HCl 10 mM, pH: 8; proteinase K 2 mg/ml) and incubated at 50°C for 2 h with occasional inversion. Extra incubation at 68°C for 30 min was performed for proteinase K inactivation. Each suspension was mixed with 300-μl equilibrated phenol solution (Sigma-Aldrich, USA) by vigorous shaking for 5 min and centrifuged (12,000 × g, 15 min). The upper DNA-containing layer was mixed (by shaking) with 300 μl of equal volume of phenol: chloroform (1:1 v/v) and also treated with 30 μl 3 M sodium acetate buffer (pH 5.2). The DNA was precipitated with 2.5–3 volumes of ice-cold 95% ethanol and allowed to precipitate overnight. After wash by 70% ethanol and removal of the ethanol, the DNA dissolved in 30-μl autoclaved TE buffer (Tris-HCl 10 mM, EDTA 1 mM, pH: 8). The DNA was quantified spectrophotometrically by calculating the A 260/A 280 ratios and the A 260 values to determine protein impurities and DNA concentrations.

Standard PCR amplification of gtf B and gtf I gene for S. mutans and S. sobrinus detection, respectively, was performed using 20-μl PCR buffer containing 2 μl PCR buffer (10 mM Tris, 50 mM KCl, pH: 8), 0.2 mM of each of the deoxy nucleoside triphosphates, 2.5 mM MgCl ₂, 10 pM of primers ²² , ²³ Table 1, 0.5 U of Taq DNA polymerase, and 10 ng of target DNA. A total of 35 cycles of amplification were performed with template DNA denaturation at 94°C for 2 min, primer annealing at 60°C for 30 s, and primer extension at 72°C for 2 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gel (Sigma, Germany) containing ethidium bromide (0.5 μg/ml) (CinnaGen, Iran).{Table 1}

Statistical analysis

Data were processed for analysis using SPSS software (version 20.0/PC; Chicago, IL, USA), and statistical significance was set at P < 0.05. As mentioned in the materials and methods section, calibration or interrater reliability analysis for DMFT score recording was done using the Kappa statistic, and the internal consistency of the questionnaire was assessed by Cronbach's alpha.

The frequency of S. mutans existence in individuals with and without caries lesion was analyzed by Chi-square test. Data from the samples were recorded as DMFT score (dependent variable), and the three independent variables were as follows: S. mutans existence in saliva and plaque (two categories of positive or negative), sugar consumption (three categories of low, intermediate, and high), and tooth brushing habits (four categories of never or rarely, 1/d, 2/d, and 3/d). The significant differences of DMFT scores for the two categories of S. mutans variable were assessed using independent sample t-test. For sugar consumption and tooth brushing habits, one-way analysis of variance was performed to verify statistical differences, and Tukey's honestly significant difference test was used for multiple mean comparisons (α = 0.05). As the DMFT score distribution was normal, linear multiple regression was used to study the influence of independent variables (S. mutans, sugar consumption, and tooth brushing habits) on DMFT scores.

Ethical issues

This study was conducted with the understanding and written consent of each participant and in full accordance with the World Medical Association Declaration of Helsinki. It was independently reviewed and approved by Islamic Azad Medical University's ethics committee (ethical code: IR.IAU.TMU.REC.1396.299). Volunteers' data were anonymized prior to the analysis.

PCR reaction by gtf B and gtf I gene-specific primers produced DNA fragments of 517 bp and 712 bp from S. mutans and S. sobrinus, respectively Figure 1. Figure 1

Detection of Streptococcus mutansin saliva and dental plaque samples by polymerase chain reaction amplification. (a) Glucosyltransferase B gene (517 bp) amplification from saliva and plaque samples. Lanes 1–4: Polymerase chain reaction products from Streptococcus mutans-positive samples. Lane 5–7: Polymerase chain reaction product from Streptococcus mutans-negative samples. Lane 8: Control chromosomal DNA from Streptococcus mutans(PTCC 1683) (positive control). Lane 9: No chromosomal DNA (negative control). Lane 10: 1 kb DNA ladder (fermentase). (b) Polymerase chain reaction amplification of glucosyltransferase I gene (712 bp) from saliva and plaque samples. Lane 1: No chromosomal DNA (negative control). Lane 2 Control chromosomal DNA from Streptococcus sobrinus(PTCC 1601) (positive control). Lanes 3, 6, and 7: Polymerase chain reaction product from Streptococcus sobrinus-positive samples. Lanes 4, 5, and 8–14: Polymerase chain reaction product from Streptococcus sobrinus-negative samples. Lane 15: 1 kb DNA ladder (fermentase).

Dental caries is a multifactorial incident, and its prevalence in a population is influenced by a number of factors including microflora, sugar consumption, genetic, socioeconomic status, and oral hygiene habits. ¹⁸ , ²⁷ Microbiota of the dental plaque, being exposed by sugars, makes plaque environment to be acidogenic and initiates dental caries. ⁴ Moreover, diets rich in sucrose can change the population of the oral microflora toward caries-promoting one. ⁴ In this study, in addition to the new report of S. mutan s epidemiology and DMFT scores and the evaluation of association of S. mutan s with sugar consumption and tooth brushing habits with DMFT scores in Iranian adults, the effect of the three mentioned factors in caries occurrence was compared using DMFT scores analysis, to find the most effective one.

The mean DMFT score found in the present study was 6.62. The prevalence of S. mutans, found in this study, was 77.1%, and the frequency of the presence of S. mutans in caries-active group was statistically significantly higher than the caries-free group (P < 0.001) Table 2. This study also showed that there is a significant difference in mean DMFT scores between S. mutans-positive and S. mutans-negative categories (P < 0.001) Table 3. No epidemiological studies have been conducted in Iran to investigate the frequency of S. mutans in adults. In a study by Ghasempour et al. ¹⁹ on 4–6-year-old children, 65% of the children were harboring S. mutans in their saliva, and the frequency of S. mutans in caries-active children was significantly higher than that of caries-free one (P = 0.046). Similar observations regarding the association of S. mutans with higher DMFT scores have been made in other parts of the world in different age groups. ⁸ , ¹⁰ , ¹⁸ , ²⁸

While in this study S. mutans showed a strong relationship with caries incidence and DMFT, it is not present in all individuals with caries (13.2% of the population with dental caries were not harboring S. mutans). This finding supports the “nonspecific plaque hypothesis,” in which caries is the consequence of the net metabolic activity of the biofilm, ²⁹ which can be attributed to other contributing factors in caries occurrence or other members of complex microbial community including Streptococcus salivarius, Streptococcus vestibularis, Streptococcus parasanguinis, Lactobacillus, Veillonella, Actinomyces, Propionibacterium, Atopobium, and Bifidobacterium dentium. ²¹ , ³⁰ , ³¹ , ³²

S. mutans was detected in 28.5% of individuals without caries lesion, and this may be because of the differences between S. mutans strains' capabilities in adherence to hydroxyapatite or bacteriocin production. ³³ In addition, the importance of prominent microbial community members that contribute in promoting health by ammonia production and pH homeostasis ³⁴ should be considered as an explanation.

Sugar consumption quantity as an environmental aspect of ecological plaque hypotheses was investigated in 20–30-year-old adults in this study, and the results showed a statistically significant different in mean DMFT scores between the three sugar consumption categories (low, intermediate, and high) (P < 0.000). In the SFQ, people were asked about the amount of extrinsic sugar consumed in different times, and the selection of items was based on Iranian people's nutritional habits. The observed positive relationship between sugar consumption and caries scores, apart from the use of fluoride, is in accordance with other studies. ¹¹ , ¹² , ¹⁴

Tooth brushing habits (with fluoride toothpaste) as a factor related to dental caries score were also investigated among the studied population, and there was a positive relationship (P = 0.016) between the frequency of brushing per day and decreased DMFT score. Some studies have also shown lower DMFT scores in 3–5–year-old children with earlier initiation of tooth brushing in their life and more frequency of brushing per day. ¹⁵ Tooth brushing frequency and its association with lower dental caries experience is also confirmed in adults. ¹⁰

In this study, the effect of S. mutans, sugar consumption, and tooth brushing on DMFT changes was compared using linear multiple regression test. The results showed that S. mutans has the greatest impact on DMFT score changes (β =0.474) followed by sugar consumption with remarkable different in beta value (β = 0.179). It is also shown that tooth brushing has no effect on DMFT changes (β= −0.117) Table 4. In other words, tooth brushing was not found as a more preventative factor of caries than S. mutans and sugar consumption. Acid production and pH drop by MS occurs 5–10 min following sugar consumption. ³⁵ A significant effect of the time that sugar is available to mouth microflora is seen in DMFT scores ²⁰ so, tooth brushing can be a hindrance to caries-producing factors. However, there was no possibility to record the brushing time for each individual in this study to verify tooth brushing factor on its merit. This limitation can be a justification for the ineffectiveness of brushing frequency in DMFT changes in this study.

In accordance with the results of the previous studies that consider dental caries as a multifactorial incident, ¹⁸ , ²⁷ the adjusted R ²of 0.325 in the present study showed that 32.5% of DMFT changes in the present study population could be explained by three factors that were entered into regression model including S. mutans (28.2%), sugar consumption (3.6%), and tooth brushing (0.7%), and additional factors such as other acidogenic species, genetic factors, and socioeconomic status may consist the remaining (67.5%). It should be mentioned that, in one population, different explanations may be relevant for each individual, age group, and each part of teeth.

This study provided the first report of S. mutans frequency and DMFT score in Iranian 20–30-year-old adults. More importantly, it revealed that S. mutans existence in mouth microflora of the studied population can act as a stronger factor in dental caries development than sugar consumption and tooth brushing. In other words, this study shows that, among oral bacteria, S. mutans is more powerful than sugar because in this study, there were high DMFT scores in S. mutans-positive people than the negative one with equal sugar consumption. Hence, more preventive solutions should be presented for S. mutans and its caries development mechanisms. The method used in this study is useful because it allows comparison of sugar consumption and S. mutans' effects on DMFT score. The results of the present study which can be strengthened by similar surveys, using the presented method, on different populations, have notable clinical significance for preventive and therapeutic purposes.

Acknowledgment

The authors would like to thank all individuals whose participation made this study possible.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.