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Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 150-155

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

Department of Pedodontics and Preventive Dentistry, Bharati Vidyapeeth University Dental College and Hospital, Navi Mumbai, Maharashtra, India

Date of Web Publication10-May-2017

Correspondence Address:
Shamika Kamath
Department of Pedodontics and Preventive Dentistry, Bharati Vidyapeeth University Dental College and Hospital, Navi Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JISPPD.JISPPD_13_17

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Background: Compounds present in green tea have been shown to inhibit the growth and activity of bacteria associated with oral infections. The purpose of this study was to compare the efficacy of chlorhexidine (0.12%) mouth rinse and combination (chlorhexidine and sodium fluoride) mouth rinse to that of green tea extract (0.5%) mouth rinse in reducing the salivary count of Streptococcus mutans and Lactobacillus in children. Materials and Methods: The sample for the study consisted of 75 school children aged 8–12 years with four or more (decay component) of decayed, missing, and filled teeth index. Children were divided randomly into three equal groups and were asked to rinse with the prescribed mouth rinse once daily for 2 weeks after breakfast under supervision. Nonstimulated whole salivary sample (2 ml) was collected at baseline and postrinsing and tested for the colony forming units of S. mutans and Lactobacillus. Results: The results of the study indicate that there was a statistically significant reduction in S. mutans and lactobacilli count in all the three study groups. The statistically significant reduction in the mean S. mutans and lactobacilli counts were more in 0.12% chlorhexidine group than in the combination mouth rinse and 0.5% green tea mouth rinse group. There was no statistically significant difference in the reduction of S. mutans and lactobacilli count between combination mouth rinse group and 0.5% green tea mouth rinse group. Conclusion: Green tea mouth rinse can be a promising preventive therapy worldwide for the prevention of dental caries.

Keywords: Caries prevention, green tea mouth rinse, Lactobacillus, Streptococcus mutans

How to cite this article:
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

How to cite this URL:
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 [serial online] 2017 [cited 2022 Nov 30];35:150-5. Available from: http://www.jisppd.com/text.asp?2017/35/2/150/206031

   Introduction Top

It has been estimated that the human body is made up of 1014 cells of which only 10% are mammalian. The remainders are the microorganisms that make up the resident flora of the host.[1] The mouth is extensively colonized by a broad range of microbes. One of the integral components of good general health is “good oral health.” Many children have inadequate general and oral health because of active and uncontrolled dental caries.[2] Childhood dental caries significantly contributes to the burden of pain and is associated with a marked decrease in the quality of life and general health.[3] Mutans streptococci are mainly responsible for the initial phase of the caries lesion especially in the enamel (initiation), whereas Lactobacillus is more involved with the progression of caries.[4]

The use of the antimicrobial agent to limit the growth of cariogenic microorganisms and prevent dental caries is warranted.[5] Mouthwashes have been found to be one of the safe and effective delivery systems as antimicrobial and antiplaque agent. These mouthwashes are capable of inhibiting bacterial adhesion, colonization and metabolic activity which ultimately affects bacterial growth.[6] Among the therapeutic agents used in mouthwashes, chlorhexidine is the “gold standard” or positive control for comparison with other substances due to its proven efficiency.[7] Another novel approach advocated for caries prevention is to use combination preparations of different therapeutic agents. The use of a mouth rinse containing fluoride and chlorhexidine has also shown beneficial effects in reducing the development of new caries lesions.[4]

There is an increasing demand for the use of medicinal plants having antibacterial property. Green tea is a nonfermented tea and is one of the ancient and popular therapeutic beverages consumed around the world.[8] Catechins, the major bioactive component of green tea, possess an antibacterial action and have demonstrated utility in the treatment of oral and topical infection.[9] Green tea extract mouth rinse is a safe and nontoxic mouthwash, especially for children.[10]

The previous studies have shown the ability of mouthwashes on plaque accumulation and plaque composition; either biochemical or microbiological, but the possible effect of mouthwash on bacterial load count in the mouth has received little or no attention in human studies. Hence, this study was carried out to compare the effect of chlorhexidine mouth rinse, combination mouth rinse and green tea extract mouth rinse on the colony forming units (CFU's) of salivary Streptococcus mutans and lactobacilli in children.

   Materials and Methods Top

The present study was conducted in the Department of Pedodontics and Preventive Dentistry, Bharati Vidyapeeth University Dental College and Hospital, Navi Mumbai. The sample for the study consisted of 75 school children aged 8–12 years. The research protocol was reviewed and approved by the Institutional Ethical Committee and Review Board and the study was in compliance with The Code of Ethics of the World Medication (Declaration of Helsinki). All subjects and their parents were verbally informed and written informed consent was taken from parents willing for participation in the study.

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 tooth brushing routine (using toothbrush and nonfluoridated toothpaste) and practicing no other professional or home-based oral hygiene measures were included in the study. Children with a physically challenged which might preclude the normal tooth brushing and mouth rinsing, with any other special health-care needs, with a history of taking antibiotics 1 month before and during the study, undergoing orthodontic treatment or with an intraoral prosthesis and with the presence of any intraoral pathology were excluded from the study.

The following mouth rinses were used in the study (1) Chlorhexidine (0.12%) mouth rinse: Commercially available Rexidin from Warren (Indoco Remedies Ltd., Indoco Remedies, Mumbai, India) (2) Combination mouth rinse: Commercially available Thermokind (Mankind Pharma Pvt. Ltd., Mankind Pharma Pvt. Ltd, Delhi, India) (3) Green tea extract (0.5%) mouth rinse: Freshly picked green tea leaves (Camellia sinensis) were brought from Munnar Tea Gardens, Kerala. Botanical authentication was done by plant biotechnologist. Green tea leaves were washed thoroughly with tap water, then with distilled water and air dried in shade. Once tea leaves were dry, they were finely powdered. Tea leaves extract was prepared by aqueous double distillation process by Soxhlet method. 0.5% green tea extract mouth rinse was prepared with distilled water as a solvent.

At the commencement of the study, all the children were given a demonstration of tooth brushing and mouth rinsing and were instructed to brush their teeth before breakfast in the morning and after meals in the night. The baseline nonstimulated saliva samples were collected after a week of following the brushing instructions. The baseline nonstimulated whole salivary sample (2 ml) was collected from children in the morning at least 1 h after breakfast. The subjects were instructed to sit in an upright position while drooling into the sterile graduated container. They were informed not to eat or drink anything (except water) 1 h before saliva collection to minimize possible food debris and stimulation of saliva. Salivary samples were transported in a box with ice packs to the microbiological laboratory and processed within 30 min of a collection of the samples. They were then tested for the number of CFU's for S. mutans and lactobacilli species using mitis salivarius bacitracin agar and Rogosa agar, respectively. The participants were then randomly divided into three groups by lottery method, i.e., they were asked to pick up chits of paper with a number 1, 2, or 3 written on them, each group consisting of 25 children as follows:

  • Group 1: The participants rinsed with chlorhexidine (0.12%) mouth rinse
  • Group 2: The participants rinsed with combination mouth rinse
  • Group 3: The participants rinsed with green tea extract (0.5%) mouth rinse.

The children were given prenumbered identical mouth rinse bottles and advised to rinse the mouth for 1 min using 10 ml of the respective mouth rinse daily for 2 weeks. Rinsing was carried out under the supervision of the investigator for six working days of the week in the school and on Sundays at home under parental supervision. After 2 weeks, the salivary samples were again collected at school and tested for the CFU's.

Processing of saliva samples

Mitis-Salivarius-Bacitracin agar for S. mutans and Rogosa agar for lactobacilli Species (Hi-Media Laboratories, Mumbai, India) were prepared in  Petri dish More Detailses. Both culture media and anaerobic Gas-Pak system were kept ready just before the saliva samples arrived in the laboratory. Fifty microliters (0.05 ml) of saliva samples were transferred to petri dishes using sterile micropipettes. Saliva samples were streaked onto the respective media by taking aseptic precautions. The whole inoculation procedure was done in a laminar air flow system to avoid contamination. Inoculated media were placed in Gas-Pak systems and were incubated at 37°C for 24–48 h. Saliva samples were processed in triplicates and average CFU's were taken for each sample to get best results. Once the incubation period was over, the petri dishes were taken out from the incubator. Bacterial colonies were identified by phenotypic method (by their morphology, size, and color) using a stereomicroscope and biochemical reactions such as Gram-staining and catalase reactions. The bacterial colonies were then counted as CFU's using a colony counting grid (Hi-Media Laboratories, Mumbai, India). The colony count was expressed as the number of CFU's per milliliter (CFU/ml) of saliva by adjusting for the dilution factor. The colony counting was done by the same observer under the same conditions and at the same time of the day to avoid the intraobserver variability in the results.

Statistical analysis

The data were presented using descriptive statistics such as frequency, percentage, mean, and standard deviation (SD). The mean CFU's at baseline and after 2 weeks of mouth rinsing in the three groups was compared by paired t-test and compared between the three groups using one-way ANOVA. The level of significance was set at 5%. All P < 0.05 was treated as significant. The mean change in CFU's from baseline were compared for the difference between the groups using one-way ANOVA followed by least significant difference (LSD) test for pair comparisons.

   Results Top

The mean age of the participants was 11.2 (SD: 0.93) years [Table 1].
Table 1: Descriptive statistics for age (years)

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There was no significant difference in the mean DMFT at baseline [Table 2] of the three study groups (f = 0.494, P = 0.613).
Table 2: Between-group comparison of decayed, missing, and filled teeth at baseline

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The mean pre- and post-test S. mutans [Table 3] and lactobacilli species [Table 4] in CFU/ml were compared using paired t-test and the result indicated a statistically significant difference (P < 0.0001) in all the three groups.
Table 3: Pre- and post-test comparison of Streptococcus mutans (CFU/ml) (in '1000)

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Table 4: Pre- and post-test comparison of Lactobacillus species (CFU/ml) (in '1000)

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On between group comparison against S. mutans [Table 5] and lactobacilli species [Table 6] using least significant difference test, the mean difference in CFU's in chlorhexidine 0.12% group are statistically significantly lower as compared to combination group and green tea 0.5% group. No statistically significant difference in combination and green tea 0.5% groups was observed.
Table 5: Between-group comparison of Streptococcus mutans (CFU/ml) (in '1000)

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Table 6: Between-group comparison of Lactobacillus species (CFU/ml) (in '1000)

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   Discussion Top

The development of chemotherapeutic agents like mouth rinses which are capable of inhibiting dental plaque formation has been of great interest to dental researchers.[11] The purpose of this study was to determine the efficacy of green tea extract mouth rinse in reducing the cariogenic flora like S. mutans and Lactobacillus and to compare the efficacy of chlorhexidine (0.12%) mouth rinse and combination (chlorhexidine and sodium fluoride) mouth rinse to that of green tea extract (0.5%) mouth rinse in reducing S. mutans and Lactobacillus in children.

This was a double-blind study wherein the microbiologist and study subjects were not aware to which group the subjects belonged, and coding was done for each group and each individual. No side effects or mishappenings were observed during the study. However, one subject each was lost during the study in the chlorhexidine group and green tea mouth rinse group and two subjects in the combination mouth rinse group due to the absence at the school and subsequent noncompletion of the mouth rinsing program.

Many clinical trials have shown that chlorhexidine mouth rinse which is considered to be the “gold standard” produces brown staining of teeth and affects the mucous membrane and tongue.[5] The most commonly prescribed concentration is 0.12%, which was, therefore, used in the study. The chlorhexidine mouth rinse group showed statistically significant reduction (P < 0.001) of CFU's/ml of S. mutans and lactobacilli [Table 3] and [Table 4]. According to Hennessey,[12] the bactericidal activity of chlorhexidine was presumed to be the result of adsorption of chlorhexidine to extracellular polysaccharides.

An approach to increase the efficacy of anti-plaque agent and to reduce the adverse effects is to combine one or more agents. Fluoride prevents dental caries through different processes: It inhibits adhesion of S. mutans to the tooth structure and therefore inhibits insoluble dextran production by this bacteria, it inhibits tooth demineralization and also remineralizes incipient carious lesions. Hence, a combination mouth rinse (0.05% sodium fluoride and 0.2% chlorhexidine) was used in the study. The combination mouth rinse led to statistically significant reduction (P < 0.001) of salivary S. mutans and lactobacilli [Table 3] and [Table 4]. This is in accordance with the study conducted by Jayaprakash et al. on children aged 13–16 years and they suggest that this combination along with the well-established effect of fluoride in the prevention of caries presents an important contribution to dental public health.[13]

There is always a quest for new and improved products, with the emphasis being placed on natural/nature identical products. Japanese folklore has it that “drinking green tea makes the mouth clean” and more specifically that “there is an old tradition that those who drink a large amount of green tea have less tooth decay.”[14] Tea plants are recognized as Camellia 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.[15] Tea can be considered an important dietary source of polyphenols, particularly flavonoids which are basically phenol derivatives. The main flavonoids present in tea are the polyphenolic catechins, namely, epigallocatechin-3-gallate, epigallocatechin, epicatechin-3-gallate, and epicatechin.[16] The green tea leaves used in this study were procured from the tea gardens of Munnar, Kerala and aqueous extract was made in the Biotechnology Laboratory by Soxhlet method. Since tea is consumed regularly in the aqueous form (tea infusion) this study was carried out to determine the effect of aqueous extracts of green tea on salivary S. mutans and lactobacilli. The anti-microbial, anti-inflammatory and anti-cariogenic effects of green tea extracts have been studied in many in vitro studies,[8],[16],[17],[18] rat model of experimental periodontitis [19] and clinical trials in children [5],[20],[21] as well as in adults.[9],[10],[22],[23] 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.[24] A review article by Hamilton-Miller [14] suggested that catechins present in green tea exert their anti-cariogenic activity by a direct bactericidal effect against S. mutans and Streptococcus sobrinus, prevention of bacterial adherence to teeth, inhibition of glucosyltransferase and inhibition of human and bacterial amylases. In this study, green tea mouth rinse led to statistically significant reduction (P < 0.001) of salivary S. mutans and lactobacilli [Table 3] and [Table 4]. The results of the present study strongly suggest that certain components of tea could exert a significant anticariogenic effect by virtue of their inhibitory activity against S. mutans.

In this study, the between groups comparison of S. mutans and Lactobacillus species in pre- and post-test by applying the LSD as shown in [Table 5] and [Table 6] confirms that the 0.12% chlorhexidine group showed a slightly greater statistically significant reduction of S. mutans and lactobacilli in saliva than combination mouth rinse and 0.5% green tea extract mouth rinse. There was no statistically significant difference in the reduction of S. mutans and lactobacilli count between combination mouth rinse group and 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, no bacterial resistance and causes no allergy. Moreover, green tea is 5–6 times cost-effective, easy to prepare and can be used as a home care product.[25]

Our study was carried out on a small sample which is the limitation of this study. The design of this study was cross-sectional which probably indicates the microbial count at a certain point of time. Since dental caries develops over a considerable period during which bacterial count would perhaps fluctuate in response to the changing oral environment, it is suggested that longitudinal study design is used, wherein microbial samples are taken at regular intervals. This will in turn help in studying the variation in the count of the microorganisms, as well as the important factor “time” in the Keyes circle of dental caries formation.[26]

More extensive research with larger sample size and over varying time periods should be carried out to establish the efficacy of green tea mouth rinse in the prevention of dental caries in children.

   Conclusion Top

From the results of our study, it can be concluded that green tea mouth rinse could be a good alternative mouth rinse. The phytosome technology may not be economical at this point of time, but with the advancement of dental research, this natural-based therapy could prove both useful and cost-effective. Green tea mouth rinse can be a promising preventive therapy worldwide for the prevention of dental caries and periodontal diseases.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]

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