Introduction

White spot lesions are caused by S. Mutans and are common on teeth during and after orthodontic treatment. The complex design of orthodontic bands and brackets can create an ecological environment that promotes colonization and growth of cariogenic S. Mutans. They are most commonly found in the cavities and crevices of teeth. They abundantly colonize plaque biofilms in the oral cavity. One of the virulence factors of S. Mutans in caries development is its ability to adhere to tooth surfaces and form biofilms.^[1] They grow and synthesize dextran capsules that bind to tooth enamel and form biofilms. It demineralizes tooth enamel, causing tooth decay and bacterial infection of the teeth. ^[2] These bacteria are more common in orthodontic patients than in non-orthodontic patients. They are most common around the gingival region of bands of the posterior teeth, but anteriorly, the lateral incisors, then the canines, of both arches are most commonly affected. The maxillary lateral incisors and canines accumulated the highest level of dental biofilm, which may be because these teeth are located at the corners of the mouth and receive relatively less tooth brushing strokes during oral hygiene. ^[3]To inhibit or reverse enamel demineralization, various methods have been proposed such as fluoride delivery systems, casein phosphopeptides, amorphous calcium phosphate, and argon laser enamel surface attenuation. Continuous fluoride release from fluoride-containing sealants, elastomeric strands, primers, and adhesives has also been found useful.

Often the application of fluoride not only requires frequent visits to the dentist but also causes discoloration of the teeth, leading to decreased esthetics. Biological methods such as antibiotics, antimicrobial therapy with chlorhexidine, povidone iodine, fluoride, and penicillin have gained importance in recent years. The application of broad spectrum antibiotics and antimicrobial therapy can suppress the caries infection but never totally eliminate it.^[4] This disadvantage led to the idea of using probiotics as an alternative. The idea of probiotics dates back to the first decade of 1900s when the Ukranian bacteriologist and Nobel Laureate Metchnikof (1908) studying the flora of the human intestine developed a theory that senility in humans is caused by poisoning of body by the products of some of these bacteria. To prevent the multiplication of these organisms he proposed a diet containing milk fermented by lactobacilli, which produce large amounts of lactic acid that could increase the life span of humans. ^[5]

Several studies have concluded that there are positive effects of probiotics on gingivitis, periodontitis, halitosis, and candidiasis as well along with their anticaries effect. Studies have established that the level of S. Mutans is reduced after the use of probiotics. However, few studies have been conducted examining the effects of topical administration of probiotic agents such as mouthwashes, lozenges, cheese, milk chewing gum, and yogurt.^[6], ^[7] The aim of this study was to evaluate the effect of probiotic toothpaste in reducing S. Mutans and improving periodontal health in patients undergoing fixed orthodontic treatment.

Materials and Methods

This prospective clinical trial was conducted at the Department of Orthodontics and Orthodontics, Swami Devi Dyal Hospital and Dental College, Barwala, Panchkula. Microbiological analysis were performed at the Microbiology Department of Swami Devi Dyal Hospital and Dental College Barwala Panchkula. Ethical approval for this study was obtained from the Institutional Review Board of Swami Devi Dyal Dental College, Barwala. ([Figure 1])

Results

Evaluation of S mutans colony counts

The method was performed manually by quantitative differential culture at the beginning (T0) and at the end of 30 days (T1).

Sextant	Description
First/S1	Maxillary molar and premolar area; first quadrant (17-14)
Second/S2	Maxillary canine and incisor area; first/second quadrant (13-23)
Third/S3	Maxillary premolar and molar area: second quadrant (24-27)
Fourth/S4	Mandibular molar and premolar area; third quadrant (37-34)
Fifth/S5	Mandibular canine and incisor area; third/fourth quadrant (33-43)
Sixth/S6	Mandibular premolar and molar area; fourth quadrant (44-47)

Table 1 Orthodontic plaque index

Time period	N	Mean ± SD CFU/ml (10-2)	Standard error mean	Mean difference	Z value	P value
T0 Baseline	60	63.3±24.23	3.51	33.61	-6.73	<0.001
T1 after 1 month	60	29.70±15.41	1.98

Table 2 Comparison of streptococcal colony count.

Time period	N	Mean ± SD CFU/ml (10-2)	Standard error mean	Mean difference	Z value	P value
T0 Baseline	60	1.23±0.53	0.06875	1.11667	-7.386	<0.001
T1 after 1 month	60	0.1±0.32	0.04179

Table 3 Orthodontic plaque index (OPI)

[Table 2] shows a comparison of S. Mutans colony counts before and after probiotic administration for 30 days. The mean S. Mutans colony count at baseline (T0) was 63.31 ±27.23 x 10^-2 CFU/mL and after applying probiotic toothpaste for one month (T1) was found to be 29.70 ± 15.41 x 10^-2 CFU/mL. The mean standard error at T0 was 3.51 and at T1 was 1.98.

We found the Z-score to be -6.73. This indicates that the raw score is 6.73 standard deviations below the mean. P-value was observed to be <0.001, which is highly significant.

Discussion

The present study was conducted to evaluate the effect of probiotic toothpaste on S. Mutans level in patients undergoing fixed orthodontic treatment. The study was initiated 3 months after orthodontic procedure to rule out a precipitous decline in bacterial counts after bracket application. After applying the probiotic toothpaste twice daily for 1 month, the number of S. Mutans colonies was significantly reduced. At T0, the mean S. Mutans colony count was found to be 63.31 ± 27.23 x 10 -2 CFU/ml. A mean streptococcal colony count of 29.70 ± 15.41 x 10-2 CFU/ml was found in T1. The mean plaque index at T0 was found to be 1.23 ±0.53. After using the probiotic toothpaste twice daily for one month (T1), a mean plaque index of 0.11 ± 0.32 was recorded.

To overcome the limitations of traditional disease control strategies, many researchers are developing 'probiotics' to treat infections that cause tooth decay. Probiotics are defined by FAO/WHO (Food and Agriculture Organization/World Health Organization) as live microorganisms that, when administered in adequate amounts (either in food or as dietary supplements), confer a health benefit on the host. (improved microbiological balance in intestine tract). Such non-pathogenic organisms (yeasts or bacteria, especially lactobacilli) are present in food and can have beneficial effects on the health of the host. Although probiotics have been used in fermented products for decades, the potential use of probiotics as a medical nutritional therapy has not been officially recognized. ^[9]

Metchnikoff was the first one to state that consumption of fermented milk with viable bacteria could enhance the longevity of Bulgarian people. The term "probiotic" rather than "antibiotic" was first proposed by Lilley and Stillwell in 1965. ^[10] The use of probiotics has come a long way since the 20th century. It has been used to modify the microbial flora of the stomach and intestines. Probiotics can create a biofilm that acts as a protective layer of oral tissue against oral disease by keeping bacterial pathogens away from the oral tissue by filling the space for pathogenic bacteria to enter.

Some probiotic species also secrete antibacterial compounds called bacteriocins for example, Reuterin. ^[11], ^[12] A major drawback of using saliva to estimate the amount of S. Mutans is that S. mutans in saliva represents the total number of microbes in the oral cavity from previous carious lesions, the tongue, and other sites that harbor microbes, and is also unique to the tooth surface. Saliva clearance in anterior teeth is much less when compared to posterior teeth. ^[13]

SK Cildir et al in 2009 ^[14] had shown that daily consumption of fruit yogurt containing Bifidobacterium animalis subsp lactis DN-173010 could reduce salivary levels of S. Mutans in orthodontic patients using fixation devices. Similar results regarding the use of probiotic yogurt were reported by Jose, Padma Nabhan, and Chithranjan in 2013, ^[15] Bafna et al in 2017 ^[16] and Megha et al ^[17] in 2018. Bifidobacterium bacteria and lactobacillus duo is naturally heterofermentative, producing both lactic and acetic acid. Bifidobacteria also have the ability to bind to Fusobacterium nucleatum-coated hydroxyapatite, and lactobacilli have been shown to transiently colonize the oral cavity. These properties of both Bifidobacterium and Lactobacillus confers the ability to adhere to oral mucosa and dental tissues as part of a biofilm whereby inhibiting the growth of dental pathogens to be effective against oral infections. conflict and also explain the observed reduction by S.Mutans count in saliva in the previous study. ^[17]

Chuang et al, ^[18] found that the use of probiotic containing tablets did not create a statistical difference in terms of lactobacillus quantities compared with the control group. However the probiotic intervention tended to increase the amount of lactobacillus. They suggested that this increase in lactobacillus level could be because the oral tablet used in their study contained L.paracasei extract which was not considered cariogenic. Studies on use of probiotic mouth rinse by Shah et al in 2018^[19] and Goyal et al in 2019^[2] showed that daily use of probiotic mouth rinse could reduce the levels of S. Mutan in the saliva in orthodontic patients with fixed appliance which was in concordance to our study.

The antiplaque activity of probiotic mouth rinse may be achieved in several ways such as reducing bacterial adhesion to the tooth surfaces, inhibiting growth and proliferation of microorganisms on the tooth surface inhibiting formation of intracellular plaque matrix, modifying plaque biochemistry to reduce the formation of cytotoxic products and modifying plaque ecology to a less pathogenic flora. Probiotic mouth rinses utilize the natural commensal bacteria to provide a natural defense system against harmful bacteria. Goyal et al in 2018 ^[20] found that the level of P.gingivalis were significantly decreased with probiotic mouthwash hence probiotic mouthwash can be used as a adjunctive measure along with regular brushing to improve periodontal status during fixed orthodontic treatment Amflor mouthwash can effectively be used for white spot lesions with gingival and periodontal problems. Previous studies on the use of probiotic toothpaste by Jose et al ^[15] in 2013, Majstorvoic ^[21] in 2013 and Baka ^[22] in 2018 reported a significant decrease in S.mutans level in the plaque around in orthodontic brackets.

This reduction can be attributed to the formation of a biofilm that prevents adhesion of pathogens, the competitive agents produced by the bacteria.

The topical application of probiotic tooth paste was found to be more efficient in reducing S.mutans levels than probiotic curd but this difference was not statistically significant. ^[15], ^[16]

Conclusion

After applying the probiotic toothpaste twice daily for 1 month, the number of S. Mutans colonies was significantly reduced.

A statistically significant difference in plaque index was observed after using the probiotic toothpaste (P-value was observed to be <0.001). Orthodontists should probably work towards introducing an oral hygiene regimen involving a probiotic dentrifice for routine orthodontic treatment. Since this was a short term study longer period of evaluation would establish the long term advantages of probiotics in orthodontic patients and relative merits of local probiotic therapy.

Conflict of Interest

None.

Source of Funding

None.