|Year : 2019 | Volume
| Issue : 1 | Page : 67-74
Evaluation of Streptococcus mutans serotypes e, f, and k in saliva samples of 6–12-year-old school children before and after a short-term daily intake of the probiotic lozenge
Muthukrishnan Kavitha1, GS Prathima1, Gurusamy Kayalvizhi1, Adimoulame Sanguida1, G Ezhumalai2, Venkatesan Ramesh3
1 Post Graduate Student, Professor and Head, Professor, Reader, Department of Paedodontics and Preventive Dentistry, Indira Gandhi Institute of Dental Sciences, SBV, Puducherry, India
2 Senior Statistician, Mahatma Gandhi Medical College and Research Institute, SBV, Puducherry, India
3 Senior Lecturer, Al Farabi Dental College, Jeddah, Saudi Arabia
|Date of Web Publication||25-Feb-2019|
Dr. Muthukrishnan Kavitha
Department of Paedodontics and Preventive Dentistry, Indira Gandhi Institute of Dental Sciences, Puducherry
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim of this study is to assess the variations in the levels of Streptococcus mutans in saliva before and after short-term daily intake of the probiotic lozenge. To estimate the prevalence of S. mutans serotypes e, f, and k and its variations in salivary levels before and after short-term daily intake of the probiotic lozenge. Materials and Methods: This double-blind randomized controlled trial was conducted among 60 children of 6–12 years old, who were given lozenge twice daily containing probiotic bacteria twice daily, one in the morning and another in the evening after brushing for 1 month. The placebo lozenge group also followed the same protocol. A pre- and post-quantitative analysis of S. mutans and serotypes e, f, and k level were done in the saliva of both experiment and control groups. Statistical Analysis: The data thus collected were statistically evaluated using repeated measures of ANOVA, followed by post hoc test and independent t-test. Results and Conclusion: When compared with baseline, a significant reduction of S. mutans was seen after 1 month intervention and 6 months follow-up. The prevalence of serotype e was found to be 5% and serotype k was found to be 12%, whereas none of the samples carried serotype f. Future long-term research with a larger sample size is required to determine the prevalence of S. mutans serotype and longer evaluation period is required to assess the preventive role against caries development.
Keywords: Caries risk, lozenge, microorganisms, probiotics, serotypes, Streptococcus mutans
|How to cite this article:|
Kavitha M, Prathima G S, Kayalvizhi G, Sanguida A, Ezhumalai G, Ramesh V. Evaluation of Streptococcus mutans serotypes e, f, and k in saliva samples of 6–12-year-old school children before and after a short-term daily intake of the probiotic lozenge. J Indian Soc Pedod Prev Dent 2019;37:67-74
|How to cite this URL:|
Kavitha M, Prathima G S, Kayalvizhi G, Sanguida A, Ezhumalai G, Ramesh V. Evaluation of Streptococcus mutans serotypes e, f, and k in saliva samples of 6–12-year-old school children before and after a short-term daily intake of the probiotic lozenge. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2019 Jul 21];37:67-74. Available from: http://www.jisppd.com/text.asp?2019/37/1/67/252854
| Introduction|| |
Dental caries is a multifactorial disease, which affects 60%–90% of children and adults, affecting the quality of life in an early stage. It results in demineralization and destruction of hard tissues, by the production of acid from bacterial fermentation of the food debris accumulated on the tooth surface. Over the years, it has been proved that Streptococcus mutans is the causative organism for dental caries. S. mutans strongly adheres to the tooth surface and this attachment is mediated mostly by the interaction of surface proteins and bacterial polysaccharides within the biofilm., S. mutans is a species of mutans streptococci. Mutans streptococci are subdivided into seven different species and eight serotypes based on the DNA homology. Four serotypes of S. mutans have been reported, namely, serotype c, e, f, and k to be responsible for dental caries., Although the oral prevalence of S. mutan s serotype c in Indian participants with or without caries is established, the prevalence of the other three serotypes e, f, and k in caries active children is yet known.
Preventive strategies are recommended to control caries risk factors mainly based on dietary changes, i.e., reduction of cariogenic diet and enhancing host resistance to stop the progression of the carious lesion development. Most frequently, antibacterial agents are administered to reduce cariogenic microflora. However, complete eradication of caries-associated microorganisms has been difficult and almost impossible to obtain. Thus, various other methods were tried to suppress the caries activity. One of the novel self-administered strategies is by manipulation of resident microorganisms by the ingestion of probiotic organism. In probiotics, with the administration of beneficial bacteria, it may compete with pathogens for adhesion sites to antagonize these pathogens and re-establish a stable and diverse microbial community in the oral biofilm. Ideally, it has been suggested that exposure to probiotics in early life may facilitate a permanent oral health promotion. Hence, probiotic vehicle should be suitable for all ages, especially for young children without any consequences. Several appropriate vehicles of administration of probiotic strains have been proposed such as ice cream, milk, curd, tablets, kefir, lozenge, yogurt, powders, capsules, cheese, and mouthwash. It is evident from the previous studies that it could cause both systemic and local effects due to the direct contact between the probiotic bacteria and the oral tissues. Specially formulated devices and pacifier with a slow release of the microbial strain have also been tried to prevent the oral diseases in children.
One of the oral supplements in which probiotic organisms can be added and supplied daily is lozenge. A lozenge is a small, generally medicated tablet intended to be dissolved slowly in the mouth, and hence the duration of contact of probiotic strains in the oral cavity will be more. The possible impact of probiotic lozenge on the S. mutans level is less explored, and till date, no studies have been conducted to determine the effect of probiotic lozenge on S. mutans serotypes e, f, and k which is considered to be the most common serotypes of S. mutan s found in saliva samples of caries active children. Therefore, the aim of the present study is to evaluate the S. mutans count in saliva samples of 6–12 years old children before and after a short-term daily intake of the probiotic lozenge.
| Materials and Methods|| |
This double-blind randomized controlled trial was conducted among 6–12-year-old children from Udhavi Karangal Samooga Thodar Kalvi Iyakkam (Orphanage), Ariyankuppam, Puducherry. Permission to conduct the study among the children was obtained from the Director of Orphanage. This study was approved by the Internal Review Board: IRB REFERENCE NO: IGIDSIRB2015 NDP019PGKMPPD, Institutional Ethical Committee, IEC APPROVAL CODE: IGIDSIEC2016NDP019PGKMPPD. Consent from the Guardian and child's assent was obtained before enrolling the child for the study.
Selection of subjects
After screening, 60 children were selected based on the inclusion criteria.
- Children between 6 to 12 years of age
- Children with moderate-to-high caries risk based on the caries-risk assessment tool for ≥6 years old–(AAPD guidelines 2014).
- Children who are already using products-containing probiotics
- Children who are under antibiotic treatment
- Medically compromised and mentally challenged children
- Children with hereditary lactose intolerance.
Blinding and randomization
Random sequence was generated by an independent investigator, and sequence allocation was done. The allocation sequence was concealed from primary investigator in sequentially named pouches. Accordingly, the primary investigator allocated the subject as per the sequentially named pouches to the test group and control group. In this study, only one investigator who was not involved in any patient contact, dental examinations, and microbiological assays, assigned the pouches to the respective groups.
Allocation of subjects
Accordingly, 60 participants both males and females were randomly divided into two groups of 30 each. Group A probiotic lozenge containing Streptococcus fecalis T-1110 (30 million), Clostridium butyricum TO-A (2 million), Bacillus mesentricus TO-A (1 million), Lactobacillus sporogenes (50 million) with noncariogenic, sweet tasting isomaltase as base with orange flavor. Group B placebo lozenge which were identical in composition, color, taste but without the addition of probiotic strain [Figure 1]. These lozenges were given twice per day for 30 days for the cases and controls. The primary investigator visited the hostel daily in the morning and evening and distributed the lozenges to the children as per the coding for 30 days [Figure 2]. The participants were instructed to let the lozenge slowly melt in the mouth after brushing. During the intervention period of about 1 month, children were motivated to brush their teeth twice daily.
Saliva samples were collected at baseline and after 30 days of intervention and after 6 months washout period [Figure 3]. Complete oral prophylaxis was done 1 week before the intervention [Figure 4]. Dietary instructions and oral hygiene instructions were given and children were restricted on taking any other products containing probiotics.
Saliva sample collection
The unstimulated saliva samples were collected from each child in a 15 ml sterile tube (cat #546021, Tarsons, Kolkata, India), which was stored at −20°C until transported to the laboratory for bacterial analysis of S. mutans serotype e, f, and k by polymerase chain reaction (PCR). The laboratory staff and clinicians evaluating the results were blinded to the subject's group assignment.
The statistical analysis was performed using Epi-Info statistical software 7.2.2 (Centre for Disease Control and Prevention (CDC) in Atlanta, US, and licensed as public domain Epi-info, CDC Atlanta version 2.1) The data were analyzed using repeated measures of ANOVA to compare the salivary S. mutans level at baseline, 1 month, and 6 months intervention for both probiotic and placebo group. Intergroup comparison (probiotic and placebo group) at baseline, 1 month after intervention, and 6 months washout period was done using independent t-test. Friedman test was used to compare the levels of serotype e and k level.
| Results|| |
Demographic parameters of the children such as mean age were found to be 10.4 years and 10.5 years in probiotic and placebo lozenge, respectively. Out of the 60 children, 39 were boys and 21 were girls. The collected saliva samples were subjected to S. mutans species-specific PCR analysis. Agarose gel analysis of the species-specific PCR amplicons identified the prevalence of salivary S. mutans serotypes e, f, and k in the study population which is 82.5%. Among the detected serotypes, serotype e was detected in only three samples under the probiotic group [Figure 5]. Serotype k was detected in seven samples, where three samples fall under the placebo group and 4 under probiotic group serotype f was none in the study population [Figure 6], [Figure 7] and [Graph 1].
|Figure 5: Polymerase chain reaction amplification of genomic DNA with Streptococcus mutans e serotype specific primer. Streptococcus mutans e serotype-specific polymerase chain reaction amplification of genomic DNA extracted from whole saliva of patients from high caries group. Lane 1: Control Streptococcus mutans, Lanes 2 to 16: DNA samples from saliva|
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|Figure 6: Polymerase chain reaction amplification of genomic DNA with Streptococcus mutans serotype f specific primer. Streptococcus mutans f serotype specific polymerase chain reaction amplification of genomic DNA extracted from whole saliva of patients from high caries group. Lane 1: Control Streptococcus mutans, Lanes 2 to 16: DNA samples from saliva|
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|Figure 7: Polymerase chain reaction amplification of genomic DNA with Streptococcus mutans k serotype specific primer. Streptococcus mutans k serotype specific polymerase chain reaction amplification of genomic DNA extracted from whole saliva of patients from high caries group. Lane 1: Control Streptococcus mutans, Lanes 2 to 16: DNA samples from saliva|
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All children were positive for salivary S. mutans levels at baseline. The groups at baseline were matched and had no statistically significant difference (P = 0.073).
The mean salivary S. mutans level at baseline was found to be 4593.13 ± 1335.73, at 1 month it was 297.727 ± 799.25, and at 6 months washout period it was 1756.9 ± 827.523 in probiotic group with a statistically significant reduction in bacterial intensity 1 month post-intervention (P = 0.001), similarly a statistically significant postintervention change was observed in the placebo group also, with the mean salivary S. mutans level at baseline was found to be 4045.53 ± 952.33, at 1 month was 3471.69 ± 1048.58, and 4059.13 ± 1377.466 at 6 months washout period with (P = 0.001).
These comparisons suggest that both probiotic lozenge and placebo lozenge reduced the salivary S. mutans level in the children during the study. However, after the washout period of 6 months both the groups showed a marginal increase in S. mutans level when compared to 1-month intervention [Graph 2].
On comparing the salivary S. mutans serotype e level at baseline, 1-month intervention and 6 months washout period in the probiotic group, the mean salivary S. mutans level of serotype e at baseline was found to be 2068.9 ± 2436.6, at 1-month intervention, it was 2406.6 ± 4168.4, and 2698.5 ± 4674.6 after 6 months. Friedman test was used to find the changes over the 3 measurements and found that salivary S. mutans serotype e level was not statistically significant (P = 0.67) [Table 1] and [Figure 8].
|Table 1: Comparison of salivary Streptococcus mutans serotypes(e) level at baseline, 1 and 6 months follow-up in probiotic group sing Friedman test|
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|Figure 8: Polymerase chain reaction amplification of Streptococcus mutans with species specific primer followed by quantification of the band intensity to identify the extent of reduction -Comparison of serotype e. Group 1 (at baseline), Group 2 (1 month after intervention), Group 3 (6 months follow up)|
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The mean salivary S. mutans serotype k level in the probiotic group at baseline was 2367.9 ± 484.020, at 1-month intervention was 1410.23 ± 1147.45 at 6 months follow-up was 1439.5 ± 833.469. Repeated measures of ANOVA test shows that salivary S. mutans serotype e level was not statistically significant (P = 0.105).
Salivary S. mutans serotype k was compared between probiotic and placebo group at baseline, 1 month and 6 months using the Mann–Whitney U-test. The groups at baseline had no statistically significant difference (P ≤ 0.077). Post intervention comparison of the groups at 30 days revealed a non significant difference (P = 0.480). Post intervention comparison of the groups at 6 months revealed a non statistically significant difference (P = 0.724). Comparison of change in S. mutans serotype k intensity in the placebo group and probiotic group revealed statistically significant changes in bacterial intensity at 30 days post intervention and after a washout period of 6 months when compared to baseline. This non significant result may be due to the small sample size [Table 2] and [Figure 9].
|Table 2: Comparison of salivary Streptococcus mutans serotypes(k) level at baseline, 1 month intervention and 6 months follow-up between probiotic group and placebo group|
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|Figure 9: Polymerase chain reaction amplification of Streptococcus mutans with species specific primer followed by quantification of the band intensity to identify the extent of reduction - Comparison of serotype k. Group 1 (at baseline), Group 2 (1 month after intervention), Group 3 (6 months follow up)|
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| Discussion|| |
The present study was done in the hostel population to avoid subjective bias in the outcome, as all children had a common source of drinking water supply and same diet, thereby reducing bias by the intake of naturally available probiotics such as curd and pickle. Based on caries-risk assessment tool according to the American Academy of Paediatric Dentistry, 60 children with moderate-to-high caries risk were included, and children with open and untreated carious lesion were excluded from the study because in contrast to S. mutans, lactobacilli are implicated in caries progression rather than initiation of the carious lesion. In our study, children aged 6–12 years were selected, as the caries prevalence was considered to be high and there will be newly erupted permanent teeth. It has been suggested that early exposure to health-promoting strains (probiotics) has been found to be beneficial and any measures directed toward prevention against early colonization might be beneficial in the long-term prevention of dental disease.,,
The second reason for selecting this age group was the intellectual ability of the child and positive compliance can be obtained. According to Jean Piaget theory at the age of 7 years, a child largely corresponds to an increase in cognitive development whereby the child develops a sense of semi-logical reasoning to infer physical cause-effect relationships., It is common in India to provide dietary supplements of Vitamin A and Vitamin D to infants and young children. Probiotics in the form of lozenge can be used as an ideal vehicle for delivery as it can be dissolved in the oral cavity for the local action of probiotics. Moreover additional storage temperatures such as refrigerators are required.
Bifiliac lozenge (prebiotics and probiotics) which contains S. faecalis, C. butyricum, B. mesentericus, and L. sporogenes was used in the present study. The combination of probiotic strains used in our study was similar to those used by Dhawan and Dhawanin 2013. The subjects were directed to place lozenge on the tongue for few minutes and allowed to dissolve, thereby increasing the contact of lozenge, with the oral cavity for a longer period. The treatment dosing regimen was two lozenges per day, one in the morning and one in the night after tooth brushing as per the coding, which was well tolerated by the individuals and this indicates that it is suitable for use in 6–12-year-old children. To ensure maximum compliance and motivation during the intervention and to minimize bias and “drop out” the principal investigator visited the hostel throughout the study for intervention. The orange flavor of the lozenge, with the isomaltase sugar-free base, would also contribute to the 100% compliance of lozenge intake.
In the present study, there was a steep reduction in salivary S. mutans count at 1 month intervention which clearly states that probiotics are effective in reducing the S. mutans count when compared with placebo lozenge which is in agreement with similar studies.,,,,,,,, However, few studies have given contradictory results with no reduction in mutans streptococcus level even after the intervention period.,,,
In our study the placebo lozenge group also showed a marginal reduction in the salivary S. mutans count after 1-month intervention which might be due to the regular oral hygiene practice during the intervention period. In the present study at the end of 6 months washout period, we found that in children who consumed probiotic lozenge showed a significant reduction (P = 0.00) of salivary S. mutans levels when compared to baseline, but a marginal increase in salivary S. mutans levels when compared to 1-month intervention. This suggests that the effect of probiotics organisms was reduced after the washout period. Similar results were also seen in following studies., This indicates that for long-term reduction of salivary mutans streptococci level, regular ingestion of probiotic organisms is necessary and beneficial. Several mechanisms have been proposed to explain how probiotics work. Probiotic bacteria secrete various antimicrobial substances, such as organic acids, hydrogen peroxide, and bacteriocins. In addition, they compete with pathogenic agents for adhesion sites on mucosa and can also modify the surrounding environment by modulating the pH and/or the oxidation-reduction potential, which may compromise the ability of pathogens to become established.
PCR and gel electrophoresis were used in the present study to elucidate the prevalence of S. mutans serotype and their levels before and after intake of probiotic lozenge as it has been found to be suitable for the specific serotype detection and identification of human cariogenic bacteria, such as S. mutans and S. sobrinus. Rao and Austin in their study found a novel prevalence hierarchy of the three determined S. mutans serotypes k, e, and f in the saliva samples of caries active group (80%) relative to caries free group (43%) school children from southern regions of India, Further analysis of the S. mutans positive samples in both caries active and caries-free groups indicated a higher prevalence of serotype k and e, followed by serotype f in caries active group. To our knowledge, this is the first study to evaluate the prevalence of S. mutans serotypes e, f, and k in children of Puducherry; hence, this present study was aimed to evaluate the prevalence of serotype e, f, and k in moderate-to-high caries risk group children in Puducherry. Serotype analysis of 60 positive samples for S. mutans three samples were positive for serotype e, seven samples showed positive for serotype k, with the absence of serotype f in the study population. The results of our study were in accordance with the study conducted by Rao and Austin in 2014 in his study out of 99 mutans positive samples, 17 samples showed positive for serotype k and 8 samples for serotype e, followed by five samples for serotype f.
| Conclusion|| |
The administration of probiotics in moderate-to-high caries risk patients resulted in a significant reduction of a cariogenic microorganism, S. mutans. Oral supplements such as probiotic lozenge can be used as adjuncts, for prevention of dental caries in caries active children. The prevalence of the S. mutans serotypes e, f, and k in the study population was determined using serotype-specific PCR the association of the occurrence of serotypes with caries and reduction in the S. mutans level could not be determined due to the lesser prevalence of the serotypes e, f, and k. Future long-term research with a larger sample size is required to determine the prevalence of S. mutans serotype and longer evaluation period is required to assess the preventive role against caries development.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2]