Get Permission Rohmetra, Gupta, Gupta, Beera, and Nupur: Efficacy of commercial mouth rinses containing chlorhexidine, sodium fluoride-triclosan- xylitol against salivary bacteria, streptococcus mutans : A microscopic study


Introduction

Prevalence of dental caries in children population is increasing at an alarming rate.1 The modern concepts of cariogram demonstrate micro-organisms as one of the major etiological factor in the formation of dental caries.2

Streptococcus mutans is considered one of the most important cariogenic species of the human oral microbial flora.3 Therefore, targeting Streptococcus mutans forms is the most important measure for prevention of dental caries. To cut back their level in the oral cavity will provide an additional goad for the prevention of dental caries.2 Mouthwashes have been found to be one of the safe and effective delivery system as anti-microbial agent. These mouthwashes are capable of inhibiting bacterial adhesion, colonization and metabolic activity which ultimately affects bacterial growth.2

Chlorhexidine gluconate is known as “gold standard” of all mouth rinse. It is a cationic bis-biguanide, active against an array of micro-organisms, including gram-positive and gram-negative organisms, fungi, yeasts, and viruses. When used as a mouthwash, its mode of action is purely topical and because it is poorly absorbed systemically, it is regarded as a relatively safe drug.4 Chlorhexidine has got certain side effects like long-term use like brown discoloration of teeth, some restorative materials and dorsum of tongue; taste perturbation; oral mucosal ulcerations and paresthesia; unilateral/bilateral parotid swelling, and enhanced supra-gingival calculus formation.5

Fluorides are abundantly used in oral health products including mouth rinses. Sodium fluoride mouth rinses are effective in reducing caries and inhibit carbohydrate utilization of oral microorganisms by blocking enzymes involved in the bacterial gylcolytic pathway.6

Triclosan is a non phenolic, broad spectrum antimicrobial & anti plaque agent. It is used to increase the ability of mouthwashes to bind to oral mucosa and therefore is available for longer period of time.7

Xylitol is a naturally occurring non cariogenic sugar substitute that cannot be metabolized by oral bacteria.6 It inhibit bacterial growth through two mechanisms: direct inhibition of the glycolytic route resulting from the xylitol 5-phosphate derivative and/or indirect inhibition resulting from the competition for the HPr-P carrier between glucose and xylitol. 3

Materials and Methods

A sample of 72 subjects was selected from the single school of Jodhpur city and was equally divided into 3 groups.

Group A- Rexidiene mouthwash (Chlorhexidine)

Group B- Kidodent mouthwash (Sodium Fluoride- Xylitol- Triclosan)

Group C- Distilled water (Positive control)

Prior to the study, written consent was obtained of school. Ethical clearance was obtained from the ethical committee of the institution. The subject with at least one active white spot on smooth surfaces (facial or lingual) was considered a high caries activity subject. The presence of the active lesions on these surfaces and a high caries activity imply that the subject has a high infection of mutans streptococci which means that he/she belongs to high caries risk group.

Exclusion criteria

  1. Physical limitations, which precluded the normal tooth brushing and mouth rinsing

  2. Marked intra oral soft tissue pathology

  3. 3. Medically compromised patients and subjects with history of taking antibiotics 3 months prior to or during the course of study.

  4. Subjects undergoing orthodontic treatment or with extensive intra prosthesis

  5. Children who could not brush their teeth or rinse on their own

  6. Presence of any intraoral soft tissue pathology

Stimulated saliva was collected for microbial analysis. The subjects were asked to simulate chewing action with sterilized cotton rolls for 4 min. At the end of 4 min, the students were made to expectorate into sterile container. The stimulated saliva was then transported for analysis within 30 min in ice boxes. Standardization of the saliva collection technique was followed and the same day all samples were cultured. The teachers were educated and trained in the use of mouthwash so that the children, under the supervision of the teachers, could use the mouthwash. Each of the groups used the respective mouthwash, as a daily, supervised rinse after lunch in the afternoon. The children were advised not to eat or rinse for the next 30 min. They were instructed to carry home the mouthwash bottles on weekends.

Incubation and Microbiological Enumeration

Saliva samples were serially diluted in 6 fold steps in normal saline. The serial dilution was carried out in pre-prepared sterile water blanks of 9ml each. 20 μl of saliva sample were spread on mitis salivarius agar (Hi Media Company, India) supplemented with 0.2 U/ml bacitracin (Hi Media Company, India) and sucrose (15% w/v) using cotton swab for S. mutanscount. The preparation of media, required sterilization and pouring of plates were done one day before the plating of cultures. All the plates were kept in incubator for one hour before the saliva samples were spread on it. Care was taken that all this procedure of serial dilutions and plating was done in work station called as Laminar Air Flow work station, it provides work space for sterile transfers. Petri-dishes were then kept in candle jar at 37ºC for 48 hours, than the plates were kept in anaerobic chambers and then placed in incubators at 37ºC for 48 hours. The identification of colonies were done using morphology and characteristics observed under light microscope. The confirmation of MS was done under light microscopy after heat fixed smear slide. Additional confirmation was done using gram staining and catalase negative confirmation test. Microbial counts were further expressed as colony forming units (CFU) per ml of stimulated saliva.

  1. Score 1 < 104CFU/ml

  2. Score 2 104- 105CFU/ml

  3. Score 3 105 -106 CFU/ml

  4. Score 4 > 106 CFU/ml

Data was analyzed using SPSS software version 17. Intra group comparison was done using Wilcoxon signed rank test (non-parametric equivalent to paired‘t’ test) whereas the difference between three groups was assessed using Kruskal-Wallis Annova.

Results

This study was conducted to compare the effectiveness of commercially available mouthwashes against salivary Streptococcus mutans among 8-12 years old school children.

Table 1

Comparison of mutans streptococci counts between study groups and control group before and after rinsing

Mutans Streptococci Scores Group A Group B Group C
Pre Rinse Post Rinse Pre Rinse Post Rinse Pre Rinse Post Rinse
1 2 6 2 5 2 3
2 2 6 3 6 3 2
3 5 2 6 3 6 6
4 6 1 4 1 4 4
Table 2

The means of streptococcus mutans count of all the test groups and control group before rinsing and after rinsing

Group -A n Mean SD Median Range
Pre Rinsing 15 3 1.07 3.0000 3
Post Rinsing 15 1.87 0.92 2.0000 3
Group -B
Pre Rinsing 15 2.80 1.01 3.0000 3
Post Rinsing 15 2.00 0.96 2.0000 3
Group -D
Pre Rinsing 15 2.80 1.01 3.0000 3
Post Rinsing 15 2.64 1.08 3.0000 3

The mean streptococcus mutans counts of Group A (chlorhexidienr group) post rinsing is 1.87, Group B (Sodium Fluoride-Xylitol- Triclosan group ) is 2.00

Table 3

Comparison of Pre-rinse and Post-rinse between Group A

Group A n Mean SD p-value
Pre- Rinse 15 3 1.07 0.002*
Post –Rinse 15 1.87 0.92
Table 4

Comparison of Pre Rinse And Post Rinse Between Group B

Group B n Mean SD p value
Pre- Rinse 15 2.80 1.01 0.001*
Post –Rinse 15 2.00 0.96

It showed statistically significant reduction in Streptococusmutans count (P=0.001)

Table 5

Comparison of Pre Rinse and Post Rinse between Group C (Water)

Group D n Mean SD p value
Pre- Rinse 15 2.80 1.01 0.08
Post –Rinse 15 2.64 1.08

It showed statistically non significant results (0.08)

Discussion

This study was undertaken to evaluate the efficacy of commercially available mouthwashes on salivary Streptococcus mutans on school children aged 8-12 years. These students were divided into three groups. Group A used Rexidiene (0.2% chlorhexidiene) mouthwash, Group B Kidodent (Sodium Fluoride-Triclosan-Xylitol) mouthwash, Group C used Distilled water.

In the present study saliva samples were used in this study to assess the microbial aspect of dental caries. According to Mundroff et al and Sullivan et al detection of Streptococci in saliva was an excellent means as compared to either pooled plaque or oral swab samples, as these samples do not explain the variation in caries better than the stimulated whole saliva.8, 9

In the present study the subjects were asked to simulate chewing action with sterilized cotton rolls for 4 min. At the end of 4 min, the students were made to expectorate into sterile penicillin bottles. This was in accordance to the previous study conducted by Bajaj N and Tandon S.10

According to the review by Lemos-Junior CA and Villoria GE ingestion of large amount of alcohol in mouthwash affect normal glycogenolysis and glyconeogenesis, causing hypoglycemia.11 The extrahepatic metabolism of alcohol in oral tissue has been testified. In the human mouth, aldehyde dehydrogenase (ALDH), an enzyme that converts acetaldehyde into a nontoxic acetate compound, occurs less frequently than alcohol dehydrogenase (ADH). This imbalance allows for the accumulation in oral tissues of a toxic, reactive and irritating acetaldehyde. The continuous use of mouthrinses containing alcohol should be avoided. Due to a point mutation, aldehyde dehydrogenase 2(ALDH2) isoenzyme is deficient in 30–50% of Asians. These individuals have a genetic inability to remove acetaldehyde and consequently have very high salivary acetaldehyde levels after moderate dose of alcohol.6 Therefore all the mouthwash selected for this study been alcohol free.

Children in Group A used commercially available mouthwash Rexidiene, containing 0.2% chlorhexidiene. Ernst stated that the increase in concentration of chlorhexidine from 0.1 to 0.2% provided no clinical advantages or disadvantages.12 An another study also states that the chlorhexidine used in different concentrations (0.02%, 0.06%, 0.12%) efficiently reduced the S mutans count and also by reducing the concentration of chlorhexidine, the bitter taste sensation is also reduced, making it more acceptable to children.13 Hence, in this study 0.2% concentration was used.In the present study 0.2% chlorhexidine has shown significant reduction in the mutans streptococci count (Table 3).This observation adds to the earlier studies 14, 15, 16, 17

Children in Group B used commercially available mouthwash Kidodent containing Sodium Fluoride-Triclosan-Xylitol as main ingredients. According to a study conducted,18 the use of fluoride mouthwash seems to be effective in both large group and individual studies. 0.05% sodium fluoride mouthwash is a weak solution which can be used daily, whereas 0.2% sodium fluoride mouthwash is stronger and should be used once a week. Xylitol has been incorporated into fluoride–containing mouthwashes. In vitro studies have suggested that fluoride and xylitol exert an additive inhibitory effect on growth and acid fermentation by S.mutans. 19, 20

In the present study there was a significant reduction in the S.mutans count scores within the group after using mouthwash containing Sodium Fluoride-Triclosan-Xylitol(Table 4) The reduction in S.mutans count in Group B can be due to combined effect of sodium fluoride, xylitol and triclosan. Fluoride has direct and indirect effects on bacterial cell and is also a powerful inhibitor of acid formation by plaque microorganisms. Triclosan, possesses antimicrobial action. It has got far-out hydrophobic and lipophilic nature, it adsorbs to lipid portion of the bacterial cell membrane and in low concentrations it interferes with vital transport mechanism. xyiltol as non-fermentability and non-cariogenicity as passive effects, whereas active caries prevention effects as bacteriostatic and cariostatic. 21

However, when inter group comparison were done, they showed no statistical significant difference between the groups post rinsing (p=0.16). Our study shows that all the mouthwashes used were effective in reducing the streptococcus mutans count.

Since, we found no significant difference between all the mouthwashes, with regard to their efficacy in reducing S.mutans, the use of a low fluoride–xylitol mouth wash appears to be a suitable choice for regular use in children.

Conclusion

Within the limits of the study following conclusions were drawn from this study.

  1. Group A, using Rexidine mouthwash (0.2% Chlorhexidiene) showed significant reduction in reducing streptococcus mutans count. (p= 0.002)

  2. 2. Group B, using Kidodoent mouthwash (Sodium Fluoride-Xylitol-Triclosan) showed significant reduction in reducing streptococcus mutans count. (p=0.001)

  3. When all the mouthwashes were compared there was no significant difference in reduction of streptococcus mutans count.(p= 0.16)

Source of Funding

None.

Conflict of Interest

None.

References

1 

PP Hegde BR Ashok Kumar VA Ankola Dental caries experience and salivary levels of Streptococcus mutans and Lactobacilli in 13-15 years old children of Belgaum city, KarnatakaJ Indian Soc Pedod Prev Dent 2005231236

2 

V V Kulkarni S G Damle Comparative evaluation of efficacy of sodium fluoride, chlorhexidine and triclosan mouth rinses in reducing the mutans streptococci count in saliva: An in vivo studyJ Indian SocPedod Prev20032198104

3 

V. A. C. Paula A. Modesto K. R. N. Santos R. Gleiser Antimicrobial effects of the combination of chlorhexidine and xylitolBr Dent J 201020912E19

4 

SimranR Parwani RajkumarN Parwani PJ Chitnis HimanshuP Dadlani SakurV Sai Prasad Comparative evaluation of anti-plaque efficacy of herbal and 0.2% chlorhexidine gluconate mouthwash in a 4-day plaque re-growth studyJ Indian Soc Periodontol 2013171727

5 

L Flotra P Gjermo G Rolla J Waerhaug A 4-month study on the effect of chlorhexidine mouth washes on 50 soldiersScand J Dent Res1972801017

6 

Priya Subramaniam N Nandan Effect of xylitol, sodium fluoride and triclosan containing mouth rinse on Streptococcus mutansContemp Clin Dent 20112428790

7 

T N Phan Robert E Marquis Triclosan inhibition of membrane enzymes and glycolysis of Streptococcus mutans in suspensions and biofilmsCan J Microbiol2006521097783

8 

Sheila A. Mundorff Arthur D. Eisenberg Dennis H. Leverett Mark A. Espeland Howard M. Proskin Correlations between Numbers of Microf lora in Plaque and SalivaCaries Research19902453123170008-6568, 1421-976X10.1159/000261289S. Karger AGhttps://dx.doi.org/10.1159/000261289

9 

A. Sullivan M. K. Borgstrom L. Granath G. Nilsson Number of mutans streptococci or lactobacilli in a total dental plaque sample does not explain the variation in caries better than the numbers in stimulated whole salivaCommunity Dent Oral Epidemiol 199624315963

10 

Neeti Bajaj Shobha Tandon The effect of Triphala and Chlorhexidine mouthwash on dental plaque, gingival inflammation, and microbial growthInt J Ayurveda Res2011212936

11 

Celso Augusto Lemos-Júnior Germano Eduardo Miguel Villoria Reviewed evidence about the safety of the daily use of alcohol-based mouthrinsesBraz Oral Res20082212431

12 

C P Ernst K Prockl B Willershausen The effectiveness and side effects of 0.1% and 0.2% clorhexidinemouthrinses: A clinical studyQuintessence Int1998294438

13 

R Jayaprakash A Sharma J Moses Comparative evaluation of the efficacy of different concentrations of chlorhexidine mouth rinses in reducing the mutants streptococci in saliva: An in vivo studyJ Indian Soc Pedod Prev Dent20102831626

14 

Pooja Agarwal L Nagesh Murlikrishnan Evaluation of the antimicrobial activity of various concentrations of Tulsi (Ocimum sanctum) extract against Streptococcus mutans: An in vitro studyIndian J Dent Res 20102133579

15 

S 1Spets-Happonen H Markkanen L Pöllänen T Kauppinen H Luoma Salivary S treptococcus mutans count and gingivitis in children after rinsing with a chlorhexidine-fluoride solution with and without strontiumScand J Dent Res198593432935

16 

A F Heffi B Huber The effect on early plaque formation, gingivitis and salivary bacterial counts of mouthwashes containing hexidine/zinc, aminefluoride/tin or chlorhexidineJ Clin Periodontol1987145158

17 

N Malhotra S P Rao S Acharya B Vasudev Comparative in vitro evaluation of efficacy of mouthrinses against Streptococcus mutans, Lactobacilli and Candida albicansOral Health Preven Dent2011932618

18 

John J. Clarkson Jacinta McLoughlin Role of fluoride in oral health promotionInt Dent J200050311928

19 

H. Maehara Y. Iwami H. Mayanagi N. Takahashi Synergistic Inhibition by Combination of Fluoride and Xylitol on Glycolysis by Mutans Streptococciand Its Biochemical MechanismCaries Res 2005395218

20 

Vladislav G. Petin Jin Kyu Kim Roman O. Kritsky Ludmila N. Komarova Mathematical description, optimization and prediction of synergistic interaction of fluoride and xylitolChemosphere20087258448490045-653510.1016/j.chemosphere.2008.02.023Elsevier BVhttps://dx.doi.org/10.1016/j.chemosphere.2008.02.023

21 

LaxmiS Lakade Preetam Shah Dayanand Shirol Comparison of antimicrobial efficacy of chlorhexidine and combination mouth rinse in reducing the Mutans streptococcus count in plaqueJ Indian Soc Pedod Prev Dent2014322916



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