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ORIGINAL ARTICLE
Year : 2021  |  Volume : 39  |  Issue : 3  |  Page : 310-315
 

Comparison of the Streptococcus mutans colony count changes in plaque following chlorhexidine (0.12%) mouth rinse and green tea extract (0.5%) mouth rinse in 8–12-year-old children


1 Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Navi Mumbai, Maharashtra, India
2 Director, D Y Patil Dental College, Pune, Maharashtra, India
3 Department of Microbiology, Namo Medical Education and Research, Dadra and Nagar Haveli, India

Date of Submission10-Jun-2021
Date of Acceptance14-Sep-2021
Date of Web Publication22-Nov-2021

Correspondence Address:
Dr. Shamika Kamath
Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Navi Mumbai - 400 614, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jisppd.jisppd_208_21

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   Abstract 


Background: Mouth rinses are solutions or liquids used to rinse the mouth to have a therapeutic effect by relieving infection or preventing dental caries due to their antimicrobial properties. Aims: This study was carried out to compare the effect of 0.12% chlorhexidine mouth rinse and 0.5% green tea extract mouth rinse on the colony-forming units (CFUs) of Streptococcus mutans in plaque in children. Materials and Methods: The sample for the study consisted of fifty schoolchildren aged 8–12 years with four or more (decay component) of decayed, missing, and filled teeth index. Children were divided randomly into two groups and were asked to rinse with the prescribed mouth rinse twice daily for 2 weeks under supervision. The plaque samples were collected at baseline (prerinsing) and postrinsing and tested for the CFUs of S. mutans. Results: The results of the study indicate that there was a statistically significant reduction (pre- and postrinsing) in S. mutans colony count in both the study groups. There was no statistically significant difference in the reduction of S. mutans colony count between 0.12% chlorhexidine mouth rinse group and 0.5% green tea mouth rinse group. Conclusion: Green tea mouth rinse is effective against S. mutans – one of the major dental plaque microorganisms.


Keywords: Caries prevention, chlorhexidine mouth rinse, green tea mouth rinse


How to cite this article:
Kamath S, Hegde R, Kamath N. Comparison of the Streptococcus mutans colony count changes in plaque following chlorhexidine (0.12%) mouth rinse and green tea extract (0.5%) mouth rinse in 8–12-year-old children. J Indian Soc Pedod Prev Dent 2021;39:310-5

How to cite this URL:
Kamath S, Hegde R, Kamath N. Comparison of the Streptococcus mutans colony count changes in plaque following chlorhexidine (0.12%) mouth rinse and green tea extract (0.5%) mouth rinse in 8–12-year-old children. J Indian Soc Pedod Prev Dent [serial online] 2021 [cited 2021 Nov 29];39:310-5. Available from: https://www.jisppd.com/text.asp?2021/39/3/310/330704





   Introduction Top


Dental plaque is the community of microorganisms found on a tooth surface as a biofilm, embedded in a matrix of polymers of host and bacterial origin. It is a structurally and functionally organized biofilm, has a diverse microbial composition, and forms in an ordered way.[1] The microbial composition is different in the healthy and diseased plaque. Oral bacterial plaque is the most common cause of dental caries and periodontal disease which can lead to destruction of tooth structure and early loss of periodontal supporting tissues.[2] Dental caries is initiated by the process of fermentation of carbohydrates by the microorganisms, in which the production of strong organic acids such as lactate, formate, and pyruvate cause demineralization of the tooth surface.[3] Among the microorganisms, a major cause of the disease is believed to be commensal bacteria which exist in dental plaque, particularly oral streptococci.[4] There is a shift toward microbial community dominance by acidogenic and acid-tolerating species such as mutans streptococci group in plaque in the pathogenesis of dental caries. Streptococcus mutans (a subgroup of mutans streptococci group) is well known as the initiator of dental caries.[5]

Mouth rinses are solutions or liquids used to rinse the mouth to have a therapeutic effect by relieving infection or preventing dental caries due to their antimicrobial properties. The daily use of antimicrobial mouth rinse shown to have significant antiplaque activity would be a meaningful, cost-effective addition to mechanical oral hygiene methods.[6] Among the mouth rinses used, chlorhexidine, a cationic biguanide, is considered the “gold standard” based on its bactericidal and bacteriostatic activities. Research on herbal mouth rinses has been on the rise due to their wide range of biological and medicinal activities, higher safety margins, low cost, and combating the limitations of drawbacks of chemicals such as teeth discoloration, mucosal erosions, burning sensation, and alteration of taste.[7] Among the herbs, Camellia sinensis or tea plant has a unique phytochemical and pharmacological profile, which makes it a perfect candidate for use as an active constituent in oral mouthwashes.[8] During producing the harvested green tea leaves, catechin, the key to tea's health benefits, should be precluded from oxidation. Compared to black tea, green tea has a higher concentration of polyphenol (the most important antioxidant in green tea leaves) and thus more therapeutic benefits.[9] The inhibition of acid production from dental plaque and mutans streptococci by epigallocatechin gallate (EGCG), one of the green tea catechins, was examined and the results suggested that EGCG is effective in reducing acid production in dental plaque.[10]

Previous dental studies reveal the use of herbal mouthwashes mainly in adult populations, while there is a lack of research regarding the pediatric population. Hence, this study was carried out to compare the effect of 0.12% chlorhexidine mouth rinse (positive control) and 0.5% green tea extract mouth rinse on the colony-forming units (CFUs) of S. mutans in plaque in children.


   Materials and Methods Top


A randomized, double-blind, two-group parallel clinical trial was conducted in the Department of Paediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Navi Mumbai, for 2 weeks. A sample size of fifty children was selected out of screened 307 children by simple random sampling between the age group of 8 and 12 years [Figure 1]. The research protocol was reviewed and approved by the Institutional Ethical Review Committee. Participants were informed about the purpose and design of the investigation, and written informed consent was obtained from parents willing for participation in the study.
Figure 1: CONSORT flowchart

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Inclusion and exclusion criteria

Children with at least four decayed and/or missing due to caries or filled teeth (decayed, missing, and filled teeth ≥4) and adhering to an at least once daily toothbrushing routine (using toothbrush and nonfluoridated toothpaste) and practicing no other professional or home-based oral hygiene measures were included in the study. Children with physical disability which might preclude the normal toothbrushing and mouth rinsing, with any other special health-care needs, with a history of taking antibiotics 1 month prior to and during the study period, undergoing orthodontic treatment, or with an intraoral prosthesis and with the presence of any intraoral pathology were excluded from the study. The sample size was determined by using the sample size formula: n = 4qp/L2 where q is 95% confidence level, p is prevalence, and L is allowable error.

The selected fifty children were given demonstration of mouth rinsing. The baseline plaque (prerinse) samples were collected from all the fifty subjects with a sterile probe tip from the buccal surface of the right maxillary first molar and the labial surface of the maxillary incisor. They were informed not to eat or drink anything (except water) 1 h before plaque collection to minimize possible food debris. The plaque samples were transferred to labeled sterile vials immediately after collection and transported to the microbiological laboratory and processed within 30 min of collection of the samples.

The following mouth rinses were used in the study: Group A (25 subjects): chlorhexidine (0.12%) mouth rinse: commercially available Rexidin from Warren (Indoco Remedies Ltd.) and Group B (25 subjects): green tea extract (0.5%) mouth rinse.

Preparation of green tea extract (0.5%) mouth rinse

Freshly picked green tea leaves (C. sinensis) were brought from Munnar Tea Gardens, Kerala. Botanical authentication was done by a plant biotechnologist. Green tea leaves were washed thoroughly with tap water and then with distilled water and air-dried in shade. Once tea leaves were dry, they were finely powdered. Tea leave extract was prepared by aqueous double distillation process by Soxhlet method in the biotechnology laboratory. 0.5% green tea extract mouth rinse was prepared with distilled water as a solvent.[11]

The subjects were given prenumbered identical mouth rinse bottles and advised to rinse the mouth for 1 min using 10 ml of the mouth rinse twice daily for 2 weeks under parental supervision. Subjects were asked to report immediately if they felt any discomfort/irritation while using the mouthwashes during the course of the study. After 2 weeks, the plaque (postrinse) samples were collected.

Microbiological processing of samples

All prerinse and postrinse plaque samples from labeled sterile vials were sonicated for 20 s. 0.5 ml of plaque samples was inoculated on petri plates of mitis salivarius-bacitracin agar (HiMedia, Mumbai). The whole inoculation procedure was done in a laminar airflow system to avoid contamination. Inoculated media were placed in GasPak anaerobic jar and were incubated at 37°C for 48 h. After 48 h of incubation, all the Petri plates were removed from the incubator and CFUs/ml were counted after identifying and confirming as S. mutans by phenotypic methods. Colony counts were made using Digital Colony Counter and verified by manual count. All the plaque culture plates were inoculated in duplicate and average count was taken for final calculations which were recorded in MS Excel sheet.

Statistical analysis

The data were presented using descriptive statistics such as mean, median, and standard deviation. The mean CFU at baseline and after 2 weeks of mouth rinsing in the two groups was compared by paired t-test. Shapiro–Wilk test of normality was used to determine if the data deviated significantly from a normal distribution. The level of significance was set at 5%. All P < 0.05 were treated as significant. The homogeneity of variances of differences in the two groups was assessed by Levene's test. The mean change in CFUs from baseline was compared for difference between the groups using unpaired (independent) t-test for pair comparisons.


   Results Top


[Table 1] shows descriptive statistics for prerinse and postrinse plaque S. mutans in two groups – chlorhexidine mouth rinse and green tea mouth rinse. The mean prerinse plaque S. mutans in the chlorhexidine group was 21.77 (±2.062). The postrinse S. mutans in the chlorhexidine group was 8.45 (±8.68). The mean prerinse plaque S. mutans in the green tea group was 23.06 (±1.520). The postrinse S. mutans in the green tea group was 8.79 (±8.71). [Table 2] shows the normality of data was tested using Shapiro–Wilk test. The result of Shapiro–Wilk test suggests no significant deviation from the normal distribution in both the groups (P > 0.05).
Table 1: Descriptive statistics for plaque Streptococcus mutans colony-forming units/ml (105/ml) – within-group comparison

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Table 2: Normality test (Shapiro–Wilk)

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[Table 3] shows that in the chlorhexidine group, the mean difference between pre- and postrinse plaque S. mutans was 13.3 with SE of 0.45. The result of paired t-test indicates that there is a significant decrease in the postrinse plaque S. mutans (t = 29.6, P < 0.001). In the green tea group, the mean difference between pre- and postrinse plaque S. mutans was 14.3 with SE of 0.365. The result of paired t-test indicates that there is a significant decrease in the postrinse plaque S. mutans (t = 39, P < 0.001).
Table 3: Paired samples t-test

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[Table 4] shows the descriptive statistics of between-group comparison. The mean difference in prerinse and postrinse plaque S. mutans in chlorhexidine was 13.3 (±2.25) and the mean difference in prerinse and postrinse plaque S. mutans in green tea was 14.3 (±1.83). The results are also shown in the box plot [Figure 2]. [Table 5] indicates the test of homogeneity of variances test using Levene's test. The result indicates that variances of differences in the two groups were not significantly different from each other (P > 0.05).
Table 4: Group descriptive – between-group comparison

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Figure 2: Box plot depicting the mean difference in prerinse and postrinse plaque Streptococcus mutans in chlorhexidine and green tea mouth rinse group

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Table 5: Homogeneity of variances test (Levene's test)

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[The difference between prerinse and postrinse difference between the two groups was compared using unpaired t-test. [Table 6]] shows the result of independent t-test which indicates that there is no significant difference in the prerinse and postrinse differences between the two groups (t = −1.63, P = 0.110). This means that no statistically significant difference in the chlorhexidine 0.12% mouth rinse and green tea 0.5% mouth rinse groups was observed.
Table 6: Independent samples t-test

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   Discussion Top


According to Sturdevant, dental caries is an infectious microbiological disease of the teeth that results in localized dissolution and destruction of the calcified tissue.[12] Basically, dental caries results from the interplay of three main factors over time: dietary carbohydrates, cariogenic bacteria within dental plaque, and susceptible hard tooth surfaces.[13] These cariogenic bacteria adhere to the tooth surface and produce a sticky glycocalyx film composed of glucan resulting from the action of glucosyltransferase on dietary sucrose. Accumulation of bacteria causes dental plaque formation, within which there is continuing acid production by the bacterial plaque.[14] S. mutans is the main cariogenic bacteria. The streptococci are acidogenic and can ferment glucose, lactose, sucrose, and maltose. The major end product of fermentation is lactic acid which causes demineralization of tooth structure.[15]

Mechanical cleaning methods alone, such as brushing, are not enough to eliminate bacteria in the biofilm, and antimicrobial agents are often used in addition to prevent the adhesion of microorganisms in the mouth, reproduction, and biofilm formation. Among the various antimicrobial delivery systems, mouthwashes have been found to be one of the safest and most effective vehicles, especially in young children, as they have the ability to deliver therapeutic ingredients to all accessible surfaces in the mouth including the interproximal surfaces. Chlorhexidine is the most frequently used antimicrobial agent and is accepted as the gold standard, but chlorhexidine also has negative effects, which can include staining of teeth and peripheral tissues, oral mucosal erosion, taste disturbances, paresthesia, and toxicity. Thus, it is not recommended for pediatric patients.[16]

Natural products have shown to be good alternative to synthetic chemical substances for caries prevention.[17] During the last two decades, green tea has received much attention globally due to its diverse pharmacological properties. “Better to be deprived of food for 3 days, than tea for one” an ancient Chinese proverb elucidates the significance of tea. Although native of China, tea is widely used in the world for over 2000 years.[18]

Tea plants are recognized as C. sinensis by botanists. They are small bushy plants about 3–4 feet high. Green tea is produced from leaves that are picked and heated quickly, either in a pan or with hot steam, to stop enzymatic action and to prevent fermentation, thereby retaining the polyphenols.[19] There are four main types of catechins in green tea: epigallocatechin-3-gallate (EGCG), epigallocatechin, epicatechin-3-gallate, and epicatechin.[20] The polyphenol contents of green tea have been reported to inhibit varieties of pathogenic bacterial growth such as Helicobacter pylori, methicillin-resistant Staphylococcus aureus, S. mutans, Streptococcus sobrinus,  Salmonella More Details typhi, Shigella dysenteriae, Shigella flexneri, and Vibrio cholerae.[14] An in vitro study has confirmed that green tea has antibacterial activity against predominant cariogenic bacteria, namely S. mutans.[18] The cariogenic activity of catechins present in green tea was found to be related to its role in the depletion of thiol group which in turn exerted bactericidal effect.[21] A study showed that Tea polyphenols (TPP) are effective against adherent cells of S. mutans. Thus, TTP would be useful for development as an antimicrobial agent against oral microorganisms and has great potential for use in mouthwash solutions to prevent dental caries.[22] Hence, this study was done to assess the anticariogenic, oral health benefits of green tea. No side effects or mishappenings were observed during the study.

There was no statistically significant difference in the reduction of S. mutans count between the chlorhexidine mouth rinse group and the 0.5% green tea mouth rinse group. However, green tea has certain advantages over chlorhexidine such as it does not stain, has no lingering after taste, has no bacterial resistance, and causes no allergy. Moreover, green tea is 5–6 times cost-effective, is easy to prepare, and can be used as a home care product.[6] Another study showed that green tea extract showed good efficacy in controlling bacterial growth, and is recommended as a better-tasting alternative for daily oral hygiene due to a lack of known side effects.[16]

In our previous study,[11] 0.12% chlorhexidine mouth rinse had shown a slightly greater statistically significant reduction of S. mutans in saliva than 0.5% green tea extract mouth rinse. In the present study, results have shown that 0.5% green tea extract mouth rinse is as effective as 0.12% chlorhexidine mouth rinse against dental plaque microorganisms in decreasing the CFU of S. mutans. This is in accordance with an in vivo study which had shown that green tea catechin is effective as a mouthwash against S. mutans and having better action in plaque as compared to saliva.[23] A systematic review on C. sinensis mouthwashes in oral care recommends that “considering the main trend in clinical evidence and favourable safety profile, Camellia sinensis products are able to act as antiseptic, anti-plaque, and anti-inflammatory agents and can be used as useful mouthwashes in the future clinical studies and practice.”[24]

Green tea extract mouth rinse proved to be effective in reduction of S. mutans when it was used over the period of this study, however, in order to see its long-term effects, longitudinal studies need to be conducted. Although it did not show any side effects of its usage during the course of the study, further clinical trials are required to check for its minimum inhibitory concentration and its effects on systemic health also. Keeping this in mind, this herbal product can have clinical implication of being an effective alternative to chlorhexidine and could be advocated and recommended by dental professionals as an antimicrobial agent in the prevention of dental caries.


   Conclusion Top


Within the limits of the present study, it is concluded that the green tea extract mouth rinse is effective against one of the major dental plaque microorganisms: S. mutans when compared to 0.12% chlorhexidine mouth rinse. Further research is needed to identify the real benefits of green tea as a therapeutic and preventive agent for dental plaque microorganisms and to identify the specific active constituents in green tea that could be useful as anticaries/anti-plaque agents.

Acknowledgments

The authors would like to express gratitude to the participants and their parents in the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Ardakani MR, Golmohammadi S, Ayremlou S, Taheri S, Daneshvar S, Meimandi M. Antibacterial effect of Iranian green-tea-containing mouthrinse vs. chlorhexidine 0.2%: An in vitro study. Oral Health Prev Dent 2014;12:157-62.  Back to cited text no. 2
    
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Hegde RJ, Kamath S. Comparison of the Streptococcus mutans and Lactobacillus colony count changes in saliva following chlorhexidine (0.12%) mouth rinse, combination mouth rinse, and green tea extract (0.5%) mouth rinse in children. J Indian Soc Pedod Prev Dent 2017;35:150-5.  Back to cited text no. 11
    
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Roberson TM. Cariology: The lesion, etiology, prevention and control. In: Sturdevant's Art and Science of Operative Dentistry. 5th ed. Elsevier: ISBN 9788131204771; 2008.  Back to cited text no. 12
    
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Abdolmehdi A, Jamshid K, Mohammad MF. Inhibitory activity of green tea (Camellia sinensis) extract on some clinically isolated cariogenic and periodontopathic bacteria. Med Princ Pract 2013;22:368-72.  Back to cited text no. 14
    
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Tafazoli A, Tafazoli Moghadam E. Camellia sinensis mouthwashes in oral care: A systematic review. J Dent (Shiraz) 2020;21:249-62.  Back to cited text no. 24
    


    Figures

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