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ORIGINAL ARTICLE
Year : 2021  |  Volume : 39  |  Issue : 3  |  Page : 279-283
 

A clinical trial on topical effect of probiotics on oral Streptococcus mutans counts in children


1 Department of Pediatric and Preventive Dentistry, Stes's Sinhgad Dental College and Hospital, Pune, Maharashtra, India
2 Department of Orthodontics and Dentofacial Orthopedics, Stes's Sinhgad Dental College and Hospital, Pune, Maharashtra, India

Date of Submission09-Dec-2020
Date of Decision10-May-2021
Date of Acceptance27-Sep-2021
Date of Web Publication22-Nov-2021

Correspondence Address:
Dr. Raju Umaji Patil
Department of Pediatric and Preventive Dentistry, Stes's Sinhgad Dental College and Hospital, S. No: 44/1 Vadgaon Bk 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_519_20

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   Abstract 


Background: Probiotics compete with the cariogenic oral bacteria like Streptococcus mutans (S. mutans) for carbohydrates resulting in substrate depletion and reduction in the number of S. mutans. Aim: The evaluation of topical effect of short-term application of Probiotic formulation on S. mutans counts in plaque of 7-12 years old children with decayed, missing, filled teeth (dmft/DMFT) score 2-5. Materials and Methods: A registered randomized clinical trial (Clinical Trials Registry of India/ICMR - CTRI/2019/09/021205) was conducted on 7-12 years medium risk 10 children with dmft/DMFT score 2-5, where an innovative probiotic formulation (prepared from capsule) was applied for 6 consecutive days, on teeth and pre/post plaque samples were evaluated for S. mutans count on mitis salivarius agar. Results: There was statistically significant and drastic reduction from 608,200 to 6600 (>90%) in the S. mutans count in plaque after the application of probiotic solution for a short period of time (6 days). Conclusion: Short-term application of probiotics can be used as an alternate topical preventive therapy to reduce the count of S. mutans effectively in oral cavity/plaque. Hence, probiotic can be used as topical caries preventive agent.


Keywords: Caries, probiotics, Streptococcus mutans, topical


How to cite this article:
Patil RU, Nachan VP, Patil SS, Mhaske RV. A clinical trial on topical effect of probiotics on oral Streptococcus mutans counts in children. J Indian Soc Pedod Prev Dent 2021;39:279-83

How to cite this URL:
Patil RU, Nachan VP, Patil SS, Mhaske RV. A clinical trial on topical effect of probiotics on oral Streptococcus mutans counts in children. J Indian Soc Pedod Prev Dent [serial online] 2021 [cited 2021 Dec 5];39:279-83. Available from: https://www.jisppd.com/text.asp?2021/39/3/279/330717





   Introduction Top


It is a well-known fact that dental caries is the main oral health problem throughout the world in both adults as well as children.[1] In addition, this dental problem places a large financial, health, and time burden on the family.[2] Over the past few decades, there have been remarkable changes in the understanding of dental caries. Traditional approaches for the management of dental caries include restoration, root canal treatment, and extraction, which have resulted in the removal of healthy tooth structure along with the carious part, while modern methods include minimal invasive dentistry, fluorides, and many newer technologies such as remineralizing agents and probiotics, which focus more on the preservation and remineralization of the tooth structure.[1]

Although the role of bacteria in the development of a carious lesion is crucial, dental caries is considered a multifactorial, plaque-associated disease. Streptococcus mutans (S. mutans) is one of the chief causative organisms of dental caries in humans.[1] S. mutans was first isolated in 1924 by Clarke. It belongs to the genus of Mutans streptococci; the other organisms in this genus are also associated with the caries process and include Streptococcus sobrinus, Streptococcus cricetus, Streptococcus rattus, Streptococcus downeii, Streptococcus macacae, and Streptococcus ferus, as given by Whiley and Beighton, in 1998.[3] S. mutans are acidogenic and aciduric bacteria that ferment sucrose and other carbohydrate substrates to produce acids and insoluble polysaccharides (dextran) that result in dental caries.[4]

According to the WHO, probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host.[5] Probiotics are considered as novel innovative agents that can be used for the prevention of dental caries with minimal adverse effects.[6] Probiotics in the oral cavity compete with the cariogenic bacteria like S. mutans for the carbohydrate substrate resulting in substrate depletion without substantial acid production and thus a reduction in the number of S. mutans.[1] Although probiotics are delivered in various forms and varieties for systemic use, there is an ultimate need to deliver probiotics with proper vehicle and form for oral effects. There are no specific formulations available for professional delivery of probiotics in oral topical form except for some self-applied tooth pastes. Very few studies are available to check the beneficial effect of these probiotic organisms in reducing the S. mutans counts, and these studies are limited mainly to adults.[1] Therefore, this in vivo experimental study is an innovative attempt to deliver probiotics in oral topical form.

The aim of the study was to evaluate the effectiveness of short-term topical probiotic formulation on S. mutans counts in plaque samples of children between 7 and 12 years of age. The objectives include evaluating S. mutans count in plaque at baseline and after application of probiotic solution; and also to compare the change in S. mutans count before and after topical application of the probiotic solution.


   Materials and Methods Top


This clinical trial was explained to the institutional research board, ethical committee, and parents to obtain legal permissions to conduct the study and was also registered in the clinical trials registry (ctri.nic.in), ICMR/National Institute of Medical Science with reference no. CTRI/2019/09/021205. Multiphase sampling technique was followed in which initially, many children's screening was done for oral health and treatment needs. 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 probiotic were excluded from the study. In this study, 10 children from the outpatient department of the Department of Paediatric and Preventive Dentistry of a Dental Institute were selected between the age group of 7–12 years with decayed, missing, filled teeth (dmft/DMFT) score 2-5 (medium risk). Children/parents not willing for the treatment and those individuals with low and high caries risk were excluded from the study.

The purpose of this study was to evaluate the short-term topical effect of unique probiotic formulation (prepared from capsule) on S. mutans counts in plaque, and after screening, only 10 individuals with dmft/DMFT 2-5 were selected for the study. Their plaque samples were collected with the help of graduated curette after applying plaque disclosing solution. These samples were stored in 1 ml saline in a sterile vial and sent for microbiological laboratory for the evaluation of S. mutans count. A fresh Probiotic formulation was prepared daily using “2 ml glycerine, 2 drops of peppermint oil and a single capsule of probiotics” [Pre Pro IBS, [Figure 1]] and was mixed cautiously till a uniform paste-like solution was formed [Figure 2]. This solution was applied topically on all the buccal and lingual surfaces of teeth for 6 consecutive days for 2 min [Figure 3]. Children were advised not to consume food and water for ½ h after topical application of the probiotic solution. After 6 days, the plaque samples were collected again and tested for the S. mutans count post intervention.
Figure 1: Composition of probiotic formulation (pre pro IBS)

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Figure 2: Preparation of probiotic formulation

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Figure 3: Topical application of probiotics

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Microbiological test

Mitis salivarius agar with bacitracin was chosen as a culture medium for the S. mutans culture. It was prepared by suspending 90.07 g of M259 mitis salivarius agar base in 1000 ml distilled water, followed by boiling to dissolve the medium completely following which it was dispensed and sterilized by autoclaving at 15 lbs. pressure (121°C) for 15 min followed by cooling to 50°C–55°C and adding 1 ml of sterile 1% potassium tellurite solution (FD052) and 0.2 units/ml of bacitracin. The medium should not be reheated after the addition of tellurite solution. Finally, the contents were mixed well and poured into sterile petri plates.

The S. mutans were isolated from the plaque sample by serial dilution method. 1 ml sample was taken into phosphate buffer, and serial dilution up to 104 was done and plated on selective agar (mitis agar) (Serial dilution method – pour plate method was used for culture).[7] The plates were incubated at 37°C for 24 h and again for 37°C for 48 h. The results were observed on the incubated plates by morphological identification/observation, i.e. small round blue color colonies were present on the plates [Figure 4]a and [Figure 4]b. Total numbers of colonies of S. mutans on mitis salivarius agar were then counted manually on a colony counter.
Figure 4: (a and b) Colonies of Streptococcus mutans on mitis salivarius agar pre- and post-probiotic application

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   Results Top


Statistical analysis was done using Statistical Package for Social Sciences (IBM SPSS Inc., Chicago II, USA, version 21). The data obtained were entered into Microsoft Office Excel Sheet. Descriptive statistics was done, and paired t-test was applied (P < 0.05 was considered as statistically significant).

The pre and post intervention microbial values of the group, along with mean values and standard deviation of the group are tabulated and calculated as shown in [Table 1], with paired t-test and P < 0.05 (statistically significant), followed by comparison between mean reduction pre and post application of topical probiotic solution. The mean S. mutans count was reduced drastically from 608,200 to 6600 (>90%).
Table 1: Colonies count of Streptococcus mutans in plaque before and after probiotic application

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   Discussion Top


Conventionally, probiotics have been associated with gut health, and most clinical interest has been focused on their use for prevention or treatment of gastrointestinal diseases; however, during the last decade, several investigators have also suggested their use for oral/dental health purposes.[5],[6] Probiotics exert their systemic action through a variety of discrete, somewhat overlapping mechanisms. These include the regulation of intestinal microbial homeostasis, interference with the ability of pathogens to colonize and infect the mucosa, modulation of local and systemic immune responses, the stabilization or maintenance of the gastrointestinal barrier function, the inhibition of pro-carcinogenic enzymatic activity and the induction of enzymatic activity that favors good nutrition.[8] Topical probiotics adopt a biological approach of caries prevention by altering oral environment. They interfere with the oral colonization of cariogenic pathogens and compete with oral bacteria like S. mutans for the carbohydrate resulting in substrate depletion.[6] Probiotics are considered as upcoming anti-microbial agents usually delivered through curds, which help in improving oral health.[8] Nase et al.[9] examined the effect of milk containing Lactobacillus rhamnosus GG, ATCC (LGG) which showed less dental caries and lower mutans streptococcus count after consumption. The effectiveness of probiotic-containing cheese on S. mutans count for short-term intervention was studied by Ahola et al., wherein there was a significant reduction in the count.[10] Probiotic chewing gum was found to reduce plaque and gingival indices along with salivary microorganisms.[11] In a short-term clinical study, ice creams containing probiotics also revealed decrease in S. mutans count significantly.[12] In all the above-mentioned literatures and studies, it is observed that there are various vehicles through which probiotics can be delivered for a short period of 1–2 weeks in the form of cheese, yogurt, milk, ice cream, mouth rinse, toothpaste, chewing gums, and many more.

The vehicle by which probiotics are ingested or delivered in the oral cavity can, however, influence the cariogenic potential and the oral colonization of a probiotic. Fortunately, the most commonly used dietary Lactobacilli are consumed in milk products (e.g. fermented milk drink, yoghurt, or cheese). When lactic acid bacteria are being consumed in milk products, the buffer capacity of the milk will decrease the production of acid.[13],[14] The presence of calcium, calcium lactate, and other organic and inorganic compounds in milk are anticariogenic and reduce the colonization of pathogens.[15] Lactobacilli, in general, are weakly adhesive to surfaces, although they are frequently isolated from retention sites in the oral cavity.[16] As the daily consumption of Lactobacilli might lead to a transient colonization of these bacteria, there might be a potential dental health risk with the daily consumption of probiotic Lactobacilli. Sookkhee et al. isolated 3790 lactic acid bacteria from orally healthy volunteers. Only five oral Lactobacillus isolates were good antimicrobial producers that could inhibit a number of oral pathogens. The strains with the omnipotent antimicrobial activity were Lactobacillus paracasei Scientific Name Search  and L. rhamnosus.[17]

However, all these formulations are delivered through some medium, but there is no formulation available for professional application till date. In addition, the consumption of these products like ice cream over the long period is not suitable for children. Whereas according to Jose et al. probiotic tooth-paste is more effective than systemic consumption, in reducing S. mutans levels.[18] It is necessary that the vehicle through which the probiotics are delivered are chosen wisely. In the present study, glycerine was chosen as the vehicle because apart from acting as a vehicle, it helps in formulating a homogenous mixture, prevents the solution from drying out, prevents microbial deterioration during storage, distribution and use, helps in stability of the solution, and provides means for translucent formula. This unique formulation was successful in reducing the S. mutans load from the study subjects. Even though this study was carried out on a small number of subjects for a short period of time, but the effectiveness can be further evaluated by carrying out similar studies on a large number of population for longer period of time and also it is important to check for how long duration of time, the effects are persistent.

As rightly said an ounce of prevention is worth a pound of cure, let's ”go PRO” and say no to bad bacteria. Due to the varied deleterious side effects of chemotherapeutics, researchers in various fields of healthcare are sorting to develop alternative strategies like the application of health-promoting bacteria (probiotics) for therapeutic purposes. With the world moving toward non-invasive and preventive dentistry, we need to look for newer and safer preventive options. This unique formulation of topical probiotics acts as a potent anti-microbial agent along with the added advantages provided through the use of glycerine as a vehicle. With further long-term studies and modifications in formulations, this innovative approach of topical probiotics might lead to the development of new promising preventive agent in dentistry for caries.


   Conclusion Top


Dental caries is the main oral health problem throughout the world in children and Probiotics are considered as novel innovative agents that can be used for prevention of dental caries with minimal adverse effects. Topical probiotics are effective in reducing the count of S. mutans.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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2.
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Van Winkelhoff AJ, Herrera GD, Winkel EG, Dellemijn-Kippuw N, Vandenbroucke-Grauls CM, Sanz M. Antimicrobial resistance in the sub gingival microflora in patients with adult periodontitis. A comparison between the Netherlands and Spain. J Clin Periodontal 2000;27:79-86.  Back to cited text no. 3
    
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Patil RU, Patil SS, Dastoor PP, Unde MP, Sahu AK. Probiotics For Life – Part II Oral health perspectives. J Dent Allied Sci 2019;8:32-8.  Back to cited text no. 6
    
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Sidhu GK, Mantha S, Murthi S, Sura H, Kadaru P, Jangra JK. 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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Näse L, Hatakka K, Savilahti E, Saxelin M, Pönkä A, Poussa T, et al. Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental caries and caries risk in children. Caries Res 2001;35:412-20.  Back to cited text no. 9
    
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Ahola AJ, Yli-Knuuttila H, Suomalainen T, Poussa T, Ahlström A, Meurman JH, et al. Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch Oral Biol 2002;47:799-804.  Back to cited text no. 10
    
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Bronkhorst A. Effect of probiotic chewing gum on caries susceptibility in orthodontic patients: A Randomised Controlled Trial. Doctoral dissertation, Faculty of Health Sciences, University of the Witwatersrand; 2011.  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
[PUBMED]  [Full text]  
13.
Gedalia I, Dakuar A, Shapira L, Lewinstein I, Goultschin J, Rahamim E. Enamel softening with Coca-Cola and rehardening with milk or saliva. Am J Dent 1991;4:120-2.  Back to cited text no. 13
    
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Kashket S, Yaskell T. Effectiveness of calcium lactate added to food in reducing intraoral demineralization of enamel. Caries Res 1997;31:429-33.  Back to cited text no. 14
    
15.
Schüpbach P, Neeser JR, Golliard M, Rouvet M, Guggenheim B. Incorporation of caseinoglycomacropeptide and caseinophosphopeptide into the salivary pellicle inhibits adherence of mutans streptococci. J Dent Res 1996;75:1779-88.  Back to cited text no. 15
    
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Van Houte J. Role of microorganisms in caries etiology. J Dent Res 1994;73:672-81.  Back to cited text no. 16
    
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Sookkhee S, Chulasiri M, Prachyabrued W. Lactic acid bacteria from healthy oral cavity of Thai volunteers: Inhibition of oral pathogens. J Appl Microbiol 2001;90:172-9.  Back to cited text no. 17
    
18.
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. 18
    


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