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ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 4  |  Page : 378-382
 

Comparative evaluation of antimicrobial effectiveness of probiotic milk and fluoride mouthrinse on salivary Streptococcus mutans counts and plaque scores in children – An in vivo experimental study


Department of Pedodontics and Preventive Dentistry, STES'S Sinhgad Dental College and Hospital, Pune, Maharashtra, India

Date of Web Publication7-Nov-2019

Correspondence Address:
Dr. Raju Umaji Patil
Department of Pedodontics and Preventive Dentistry, STES'S Sinhgad Dental College and Hospital, S. No. 44/1, Vadgaon Budruk, Off., Sinhgad Road, Pune - 411 041, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_45_19

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   Abstract 


Aim: The aim of the study was to compare the effectiveness of probiotic milk and fluoride mouthwash on salivary Streptococcus mutans (SM) counts and plaque index (PI) scores in children. Materials and Methods: Thirty children of 8–13 years with zero decayed, missing, and filled teeth (dmft)/DMFT were randomly divided into two equal groups of 15 each; baseline saliva samples were collected, and plaque scores were recorded. Group A received probiotic milk and Group B received fluoride mouthwash for 7 days, and then, saliva samples were collected for colony counting, and plaque scores were recorded on the 8th day. Study Design: This was a double-blind experimental in vivo study and randomized sample selection. Results: The results showed that there was statistically significant reduction in both salivary SM counts and plaque scores in both the groups after 7 days period. Intergroup comparison showed that there was no statistically significant difference between the two groups (i.e., both groups are equally effective in caries reduction). Conclusion: Probiotic milk was as effective as fluoride mouthwash in reducing SM counts and PI scores.


Keywords: Dental caries, fluoride mouthwash, Lactobacillus casei, probiotics, Streptococcus mutans


How to cite this article:
Patil RU, Dastoor PP, Unde MP. Comparative evaluation of antimicrobial effectiveness of probiotic milk and fluoride mouthrinse on salivary Streptococcus mutans counts and plaque scores in children – An in vivo experimental study. J Indian Soc Pedod Prev Dent 2019;37:378-82

How to cite this URL:
Patil RU, Dastoor PP, Unde MP. Comparative evaluation of antimicrobial effectiveness of probiotic milk and fluoride mouthrinse on salivary Streptococcus mutans counts and plaque scores in children – An in vivo experimental study. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2019 Nov 17];37:378-82. Available from: http://www.jisppd.com/text.asp?2019/37/4/378/270483





   Introduction Top


It is a well-known fact that dental caries is the major oral health issue throughout the world both in children and adults.[1],[2] Over the past century, there have been remarkable changes in our understanding of caries etiology. Caries management by both traditional approaches such as extraction/restorations and modern methods such as fluorides, minimal invasive dentistry, and remineralizing agents has been tried.[1],[3] Although the role of bacteria in the development of a caries lesion is crucial and Streptococcus mutans (SM) is one of the chief causative organisms, dental caries is considered as a multifactorial plaque-associated disease. Mechanical plaque control by toothbrushing is the most dependable and commonly practiced oral hygiene measure; also, numerous anti-plaque agents have been tried for improving oral health such as the use of fluorides and newer advances such as xylitol, triclosan, and probiotics.[4] Fluoride is one of the most important and effective components of preventive dental programs in children.[2] Fluorides are abundantly used in oral health products including mouthrinses. Sodium fluoride mouthrinses are effective in reducing caries by constantly providing ionic fluoride during acid challenge and formation of a more resistant layer on enamel which prevent acid dissolution and inhibit carbohydrate utilization of oral microorganisms by blocking enzymes involved in the bacterial glycolytic pathway.[4],[5],[6],[7]

Probiotics are living microorganisms, principally bacteria, which are safe for human consumption and have beneficial effects on human health, beyond basic nutrition. Their ability to survive in an acidic environment, which is present during a carious attack on the tooth, makes probiotics a choice to interact with these organisms. There are increasing experimental and clinical data to support their use in medicine.[1],[8] Probiotics are mainly delivered through dairy products which are easily available and easily accepted, especially by children. Among most of the studies, proved beneficial probiotic organisms are Lactobacilli and Bifidobacteria.[1],[8] Various studies have compared the usage of probiotics in dairy products such as cheese, yogurt, ice cream, and milk. Probiotics cause minimal or no systemic toxicity if ingested and has beneficial systemic affects unlike fluorides which if ingested in large amounts can be fatal in children.[1],[8],[9]

Studies in children are limited comparing the beneficial effect of probiotics to fluorides in reducing salivary SM counts and plaque index (PI) scores.[1] It is known that the use of 0.05% fluoride mouthrinse for a week is effective clinically in caries prevention,[1],[10] as it increases the fluoride content of the outer enamel layer by 15.9%. Similarly, probiotic agents have been used for a week to prevent dental diseases by reduction of the microbial count.[1],[10] This study was done to see the immediate short-term effects of probiotic milk and fluoride mouthwash in 8–13 years age group children with the aim to compare the salivary levels of SM and plaque scores before and after administration.


   Materials and Methods Top


This randomized controlled trial/experimental in vivo study design was explained to the institutional research board, school headmasters, and parents to obtain legal permissions to conduct the study. Multiphase sampling technique was followed, in which initially, many urban schoolchildren's screening was done for oral health and treatment needs, following which 30 caries-free children with zero decayed, missing, and filled teeth (DMFT)/dmft index and using nonfluoridated toothpaste between the ages of 8 and 13 years were selected. Children with any recent history of dental treatment, systemic antibiotics or topical fluoride treatments (1 month prior to baseline), habitual use of dairy probiotics, xylitol chewing gums, severe medical conditions, and those who are allergic to dairy products were excluded from the study. These children were randomized into two groups of 15 each. Diet instructions and oral hygiene maintenance were told to continue during the study period. On the next day, 2 ml of unstimulated saliva was taken from each of the participants in 20 ml disposable sterile container during 10–11 am of school hours. The saliva samples collected were transported to the laboratory for culture within 1 h of collection in a vaccine icebox and were subjected to microbial analysis using serial dilution–pour plate method on freshly prepared mitis-salivarius agar culture plates and were inoculated for 48 h. Colonies were identified by Gram-staining and were counted using manual colony counter. Colonies were expressed as the number of colony-forming units per ml of saliva. PI scores were also recorded for all the subjects using Silness and Loe PI. The readings obtained were tabulated and considered as baseline data.

The double-blinding procedure was followed, in which the subjects as well as the individuals examining the subjects and collecting the data were blinded. For the next 7 days, Group A was given 10 ml of probiotic milk (Yakult) and Group B was given 10 ml of sodium fluoride (NaF) mouthwash (Fluoritop) once daily in the morning time continuously for 7 days. The probiotic milk used had 6.5 billion Lactobacillus casei strain Shirota, manufactured by Yakult Danone India Pvt. Ltd. (New Delhi). The fluoride mouthwash contained NaF IP – 0.044% w/v, with available fluoride – 200 ppm, in an aqueous base of potassium sorbate (NF) – 0.10%, with the commercial name Fluoritop.

Unstimulated saliva was collected on the next day after the study period of 7 days (i.e., on the 8th day) and was inoculated/incubated, and colonies were counted. Similarly, PI scores were also recorded on the 8th day using Silness and Loe PI. Data were recorded and tabulated [Table 1] and [Table 2]. This data was compiled on a MS Office Excel Sheet (v 2010 © Microsoft, USA) and were subjected to statistical analysis using the Statistical Package for the Social Sciences. (IBM SPSS Inc., Chicago II, USA, version 22.0)
Table 1: Inter- and intragroup comparison of Streptococcus mutans counts

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Table 2: Inter- and intragroup comparison of plaque scores

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   Results Top


Since the data of SM counts did not follow a normal curve, nonparametric tests were used for statistical significance. Intergroup comparison of differences in means of SM counts (between the two groups) was done using Mann–Whitney U-test for pairwise comparisons. Since the data of PI counts followed a normal curve, parametric tests were used for statistical significance. Intergroup comparison of differences in means of PI (between the 2 groups) was done using independent t-test (unpaired t-test).

Intragroup comparison of differences in means of SM counts in both the groups (at baseline and time interval 8 days) was done using the Wilcoxon signed-rank test. Intragroup comparison of differences in means of PI counts in both the groups (at baseline and time interval 8 days) was done using the paired t-test.

Data obtained from the baseline and after 7 days were arranged and tabulated [Table 1] and [Table 2]. The study revealed that there was a significant reduction of both the salivary SM levels and PI scores in both the groups after the study period (P < 0.001). There was no significant difference between the groups over the study period [Graph 1], [Graph 2], [Graph 3], [Graph 4]. These comparisons suggest that both probiotic milk and fluoride mouthwash reduced the salivary SM level and plaque scores in the children during the study period.




   Discussion Top


Chemotherapeutics are widely used to prevent and treat infection caused by indigenous and exogenous microbes. The availability of cheap and effective antibiotics in the latter half of the 20th century has revolutionized the treatment of infectious diseases and reduced the death rate. The Nobel Laureate in immunology Macfarlane Burnet had stated in 1962 that by the late 20th century, we can anticipate the virtual elimination of infectious diseases as a significant factor in social life. However, the development of resistance to a wide range of antibiotics by some important pathogens has raised a possibility of a return to the preantibiotic dark ages. The developments have encouraged researchers in various fields of health care to develop alternative antimicrobial approaches. The application of health-promoting bacteria for therapeutic purposes is one of the strongest emerging fields in this regard.[1],[8] In an effort to reduce the use of antibiotics in the face of increasing development of antimicrobial-resistant bacteria, the WHO has advocated a policy of Microbial interference therapy, i e., the use of nonpathogens to eliminate pathogens. Probiotics basically replace the pathogenic bacteria by nonpathogenic. Hence, this is also called as replacement therapy.[1],[8]

Toothbrushing alone cleans just the buccal and lingual tooth surfaces and is not effective in removing interproximal and subgingival plaque, especially around malposed teeth and also during the mixed dentition period in children.[1] Hence, numerous anti-plaque agents have been tried for improving oral health such as the use of local and systemic fluorides, pit and fissure sealants, and the newer advances which include xylitol, triclosan, and probiotics.[4] An ideal anti-plaque agent for regular use in children should not interfere with biologic processes occurring in the mouth, be harmless to the oral mucosa, should have low toxicity if swallowed, and should be both sugar- and alcohol-free.[4] Fluoride is considered the gold standard in the prevention of dental caries.[3],[5],[6],[7]

The most commonly used probiotic organisms are from the Bifidobacterium and Lactobacillus groups.[8],[9] They act by competitively inhibiting the pathogenic bacteria because they have greater adhesion to the tissues. They inhibit pathogens but do not inhibit friendly bacteria. Studies have shown that once the pathogenic organisms are replaced, the reintroduction of the pathogen does not occur easily. The benefits of probiotics on oral health in preventing gingivitis, halitosis, and caries have been recognized, and thus, probiotics have been incorporated into mouthwashes and dentifrices. They cause no systemic toxicity if ingested and have beneficial systemic effects. Hence, probiotics are truly novel newer agents which can be used for the prevention of dental caries with minimal adverse effects.

In this in vivo study, comparative evaluation of antimicrobial effectiveness of probiotic milk and fluoride mouthrinse was done on salivary SM counts and plaque scores for a period of 1 week to check the effectiveness as anti-cariogenic agents in 8–13 years age group children. Although plaque microbial count is a better measure than salivary microbial count, it is difficult to collect adequate quantities of plaque for microbial culture, especially from the oral cavities of children. Furthermore, according to Bowden, determination of the numbers of a given species in nonstimulated saliva may indicate whether it is actively growing in plaque.[11] The values obtained showed that there was highly statistically significant difference (P < 0.001) between the mean value obtained for both salivary SM counts and PI scores at the baseline and on the 8th day in both the groups. While intergroup comparison showed a statistically nonsignificant difference between the two groups, both the groups are equally effective in caries reduction. This is in agreement with the previous studies where the Mutans streptococci (MS) levels in saliva were compared after the consumption of probiotic ice cream and curd.[10] Thus, probiotics could be an alternative strategy of displacing pathogenic microorganisms by beneficial bacteria in the oral cavity.[12]

A similar study on systemic consumption of probiotic curd and the use of probiotic toothpaste to reduce SM in plaque around orthodontic brackets concluded that there is significant reduction in the SM levels, although the probiotic toothpaste was more effective than systemic consumption.[13] In another cross-over study,[10] the effect of yogurt milk and NaF mouthrinse on the salivary MS levels was done and found that both can reduce the salivary MS levels to significant levels. Hence, probiotics containing dairy products have anti-cariogenic properties and can be used for caries prevention.[10]

Hedayati-Hajikand et al.[14] in a randomized control trial to evaluate the effect of probiotic chewing tablets on early childhood caries development in preschoolchildren concluded that early childhood caries development could be reduced through the administration of these probiotic chewing tablets as adjunct to daily use of fluoride toothpaste. Chinnappa et al.[12] in a study compared the MS levels in saliva, before and after consumption of probiotic ice cream and curds. The study concluded that after 7 days, probiotic ice cream and curd showed a statistically significant reduction in MS counts as compared to the control ice cream and curd. Hence, the use of probiotic products could be an alternative strategy of displacing pathogenic microorganisms by probiotic bacteria and can thus be exploited for the prevention of enamel demineralization.

Soderling [15] stated that theoretically probiotic organisms such as Lactobacillus and Bifidobacterium have been implicated as causative organisms for dental caries, as they survive in an acidic environment and are often seen associated with deep carious lesions. However, several studies have shown that probiotics are beneficial in causing a reduction in the number of cariogenic organisms and carious lesions. But these studies were conducted for short term only. Hence, long-term clinical studies with the disease occurrence as the primary outcome measure are needed to establish beneficial versus adverse effects of probiotics on oral health. The reason for this being that although probiotics can affect the main cariogenic microorganisms, namely SM, Lactobacillus themselves may be associated with the caries progression. However, Lactobacilli have poor initial adhesion to the tooth surface, and they cannot establish themselves sufficiently to cause dental caries; however, in the presence of an existing carious lesion, the Lactobacilli may aid in the progression of the carious process and may further worsen the caries. Hence, caries-free individuals were chosen to reduce the risk of progression of the existing carious lesions. Furthermore, since the new age concept of minimally invasion dentistry focuses on the reduction of caries risk rather than surgical treatment of already established carious lesions, this study focuses on the measurement of reduction in caries risk in healthy mouths before the lesion has already occurred rather than measuring the reduction in incidence in individuals with already established carious lesions.[16],[17],[18] In addition, a shorter duration was chosen to assess the immediate effects of probiotics on SM and caries' risks; however, further long-term studies are required to study the long-term effects of these agents including the ability to establish themselves permanently in the oral cavity without causing caries.[17],[18]


   Conclusion Top


Probiotic milk was found to be as effective as fluoride mouthwash in the reduction of both salivary SM counts and plaque scores, and since it can be ingested by the individual and has minimal systemic toxic effects, it can be used, especially in children (above 2 years), as an alternative to fluoride mouthwash as a preventive measure for dental caries. However, the prolonged use of such agents and their effects on caries is still to be determined.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Ashwin D, Ke V, Taranath M, Ramagoni NK, Nara A, Sarpangala M. Effect of probiotic containing ice-cream on Salivary Mutans Streptococci (SMS) levels in children of 6-12 years of age: A randomized controlled double blind study with six-months follow up. J Clin Diagn Res 2015;9:ZC06-9.  Back to cited text no. 1
    
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Patil RU, Sahu A, Kambalimath HV, Panchakshari BK, Jain M. Knowledge, attitude and practice among dental practitioners pertaining to preventive measures in paediatric patients. J Clin Diagn Res 2016;10:ZC71-5.  Back to cited text no. 2
    
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Shashikiran ND, Subba Reddy VV, Patil RU. Minimal intervention – Part I a review of new concept in caries management. J Int Coll Dent 2004;50:15-21.  Back to cited text no. 3
    
4.
Subramaniam P, Nandan N. Effect of xylitol, sodium fluoride and triclosan containing mouth rinse on Streptococcus mutans. Contemp Clin Dent 2011;2:287-90.  Back to cited text no. 4
[PUBMED]  [Full text]  
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Bergstrand F, Twetman S. A review on prevention and treatment of post-orthodontic white spot lesions – Evidence-based methods and emerging technologies. Open Dent J 2011;5:158-62.  Back to cited text no. 5
    
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Shashikiran ND, Subba Reddy VV, Patil RU. Fluoride update. Karnataka State Dent J 2005;24:26-7.  Back to cited text no. 6
    
7.
Shashikiran ND, Subba Reddy VV, Patil R. Evaluation of fluoride release from teeth after topical application of NaF, SnF2 and APF and antimicrobial activity on mutans streptococci. J Clin Pediatr Dent 2006;30:239-45.  Back to cited text no. 7
    
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Sidhu GK, Mantha S, Murthi S, Sura H, Kadaru P, Jangra JK, et al. Evaluation of lactobacillus and Streptococcus mutans by addition of probiotics in the form of curd in the diet. J Int Oral Health 2015;7:85-9.  Back to cited text no. 8
    
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Bhalla M, Ingle NA, Kaur N, Yadav P. Mutans streptococci estimation in saliva before and after consumption of probiotic curd among school children. J Int Soc Prev Community Dent 2015;5:31-4.  Back to cited text no. 9
    
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Ghasempour M, Sefdgar SA, Moghadamnia AA, Ghadimi R, Gharekhani S, Shirkhani L. Comparative study of kefir yogurt-drink and sodium fluoride mouth rinse on salivary mutans streptococci. J Contemp Dent Pract 2014;15:214-7.  Back to cited text no. 10
    
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Bowden GH. Does assessment of microbial composition of plaque/saliva allow for diagnosis of disease activity of individuals? Community Dent Oral Epidemiol 1997;25:76-81.  Back to cited text no. 11
    
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Chinnappa A, Konde H, Konde S, Raj S, Beena JP. Probiotics for future caries control: A short-term clinical study. Indian J Dent Res 2013;24:547-9.  Back to cited text no. 12
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Jose JE, Padmanabhan S, Chitharanjan AB. Systemic consumption of probiotic curd and use of probiotic toothpaste to reduce Streptococcus mutans in plaque around orthodontic brackets. Am J Orthod Dentofacial Orthop 2013;144:67-72.  Back to cited text no. 13
    
14.
Hedayati-Hajikand T, Lundberg U, Eldh C, Twetman S. Effect of probiotic chewing tablets on early childhood caries – A randomized controlled trial. BMC Oral Health 2015;15:112.  Back to cited text no. 14
    
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Soderling E. Probiotics and dental caries. Microb Ecol Health Dis 2012;23:55-6.  Back to cited text no. 15
    
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Stamatova I, Meurman JH. Probiotics: Health benefits in the mouth. Am J Dent 2009;22:329-38.  Back to cited text no. 16
    
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Dastoor PP, Patil RU, Unde MP, Patil SS. Probiotics for life – Part I general health perspectives. J Dent Allied Sci 2018;7:75-80.  Back to cited text no. 17
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Patil RU, Patil SS, Dastoor PP, Unde MP. Probiotics for life – Part II oral health perspectives. J Dent Allied Sci 2019:8. [In Press].  Back to cited text no. 18
    



 
 
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