Effect of Three Different Topical Fluoride Varnishes on Streptococcus mutans Count in Biofilm Samples of Children Aged 6–10 Years: A Randomized Controlled Trial

Abstract

Objective

This study aims to check how well three fluoride varnishes work. The varnishes are GC MI Varnish, Ultradent Enamelast, and Ivoclar Fluor Protector. They are tested to see if they can lower Streptococcus mutans levels in biofilm samples from children aged 6–10 years.

Materials and methods

A randomized controlled trial was conducted with 144 children divided into three groups. Samples of plaque were gathered initially and then again at intervals of 1, 3, and 6 months following the application of fluoride varnish. The levels of S. mutans were measured using culture methods and reported as colony-forming units per milliliter (CFU/mL).

Results

All three fluoride varnishes significantly reduced S. mutans counts over the 6-month period. GC MI Varnish showed the greatest reduction, particularly at 1 and 3 months (p < 0.0001 and p = 0.01, respectively). The mean CFU/mL for GC MI Varnish decreased from 7.64 ± 0.18 at baseline to 5.76 ± 2.05 at 6 months. Ultradent Enamelast and Fluor Protector S also demonstrated significant reductions but to a lesser extent than GC MI Varnish.

Conclusion

GC MI Varnish containing casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) demonstrated superior antimicrobial efficacy against S. mutans compared to Ultradent Enamelast and Fluor Protector S. All three fluoride varnishes were effective in reducing S. mutans counts, with GC MI Varnish showing the most significant and consistent reductions over time.

How to cite this article

Baidya D, Pathivada L, Garg N, et al. Effect of Three Different Topical Fluoride Varnishes on Streptococcus mutans Count in Biofilm Samples of Children Aged 6–10 Years: A Randomized Controlled Trial. Int J Clin Pediatr Dent 2026;19(3):343–349.

Introduction

Dental plaque is a soft, sticky material that forms on the surface of teeth. The oral microorganisms present in dental plaque play a crucial role in the onset and development of dental caries.¹ These microorganisms consist of mutans streptococci (Streptococcus mutans and Streptococcus sobrinus), Lactobacillus species, Actinomyces species, nonmutans streptococci, and yeast. They become pathogenic under conditions causing prolonged plaque acidification. S. mutans adapts to low pH and increases acid production, resulting in cariogenic plaque.²

There are different ways to manage tooth decay by stopping the interaction between risk factors like diet, bacteria, and weak teeth. These methods include changing eating habits, practicing good oral hygiene, and using fluoride and sealants. All these actions aim to affect the bacteria in the mouth, either directly or indirectly.^3,4

Fluoride varnishes are designed to extend the contact time between fluoride and tooth enamel. They create a thin coating that clings to the tooth's surface for a prolonged period, preventing the rapid loss of fluoride after it is applied and acting as reservoirs that gradually release fluoride.

Fluoride varnish can reduce the number of bacteria by interfering with how oral bacteria process carbohydrates.^5,6 This varnish usually has 5% sodium fluoride (NaF) and is often used to prevent cavities. Studies show that applying it twice a year can greatly lower the risk of cavities by keeping a high level of fluoride on teeth for a long time.^7,8

Fluoride alone does not offer a comprehensive solution for caries prevention, as the formation of fluoride reservoirs and the remineralization potential of saliva are constrained by the availability of calcium and phosphate ions. As a result, to boost the effectiveness of fluoride varnish in preventing cavities, calcium and phosphate ions have been added to improve the retention of both fluoride and calcium ions in the mouth.^9,10

Mankind's quest for novel materials to overcome caries led to casein phosphopeptide–amorphous calcium phosphate (CPP–ACP). An ACP form, stabilized by a phosphopeptide derived from the milk protein casein (CPP), can be concentrated in dental plaque. This helps buffer the activity of free calcium and phosphate ions, maintaining a state of supersaturation relative to tooth enamel, which prevents demineralization and aids in remineralization during acidogenic conditions.^11,12 Incorporating CPP into the salivary pellicle diminishes the adhesion of S. mutans, and this targeted inhibition results in noncariogenic plaque due to its antimicrobial characteristics. Recently, advanced fluoride varnishes enhanced with CPP–ACP have been introduced.^13,14

Xylitol, a sugar alcohol that does not undergo fermentation, prevents the growth and metabolic activities of various bacterial species, specifically targeting mutans streptococci. This inhibitory effect of xylitol on bacterial growth extends to other oral pathogens, contributing to its overall antimicrobial properties in the oral cavity. The mechanism of action involves xylitol's ability to interfere with bacterial energy production and adherence to tooth surfaces, ultimately reducing plaque formation and the risk of dental caries.

There is limited literature comparing the efficacy of different types of fluoride varnishes in reducing S. mutans counts in children, particularly with and without prophylactic procedures. In vitro data suggest that additives in fluoride varnishes can potentially enhance or diminish fluoride ion release, influencing the anticariogenicity of the product.^15,16

Understanding the differences in fluoride release patterns of varnishes with additives like CPP–ACP will help determine their potential to enhance or diminish the anticariogenicity of the varnish. This study aims to address these gaps in knowledge by evaluating and comparing the efficacy of three types of fluoride varnishes in reducing S. mutans counts in children, considering the impact of prophylactic procedures. The findings could contribute to more effective caries prevention strategies in pediatric dentistry.

Materials and Methods

This randomized controlled clinical study was conducted in the department of pediatric and preventive dentistry at Ayush University of Chhattisgarh, Raipur. The research protocol received approval from the Institutional Ethics Committee. Prior to the commencement of the study, parents or legal guardians of the selected participants were provided with comprehensive information regarding the study, including the materials used, their benefits, limitations, and potential drawbacks. Informed consent was obtained, allowing their children's participation. All participating children received instructions on oral hygiene practices and dental care. A total of 144 children, aged 6–10 years, who met the inclusion criteria for fluoride varnish application, were enrolled in the study following a screening process at the outpatient department of pediatric and preventive dentistry. The sample size was determined through a power analysis, which indicated an 80% power for this study.

Children aged 6–10 years, who are healthy and free from systemic disorders, and possess noncarious primary maxillary molar teeth with intact tooth structure, were included in the study. Exclusion criteria comprised individuals who had received artificial fluoride supplementation or antimicrobial therapy within the preceding 3 months, those undergoing orthodontic treatment, or those with dental fluorosis.

The children recruited for the study were randomly assigned to one of three groups: group I (MI Varnish), group II (Ultradent Enamelast varnish), and group III (Ivoclar Fluor Protector S varnish). A simple random sampling method was employed, wherein each child selected one of three colored paper chits from an opaque bag, each chit indicating the name of the varnish to be used on the day of treatment.

Plaque samples were carefully gathered from each child participant using a sterile dental explorer, specifically focusing on the right permanent maxillary first molar, a site known for hosting cariogenic bacteria.

On the same day, each child received ultrasonic scaling, a gentle yet comprehensive method to eliminate any remaining plaque and debris from their teeth, preparing them for the upcoming fluoride varnish treatment. Based on the group they were randomly placed in—GC MI Varnish, Ultradent Enamelast, or Ivoclar Fluor Protector S—each child had a precise and uniform layer of varnish applied using a soft-bristled applicator. The application process was carried out with great care to ensure optimal adherence and maximum fluoride retention. Children were then given clear postprocedural instructions to refrain from eating or drinking for at least 1 hour, allowing the varnish to form a protective, long-lasting layer over the enamel surface. Standardized application procedures (Fig. 1) for all varnishes were used to prevent unintentional unblinding.

Fig. 1:

Steps of fluoride application

After 1 month, the children were recalled to the department for follow-up, and another plaque sample was collected and sent to the laboratory for S. mutans count evaluation. This process was repeated at 1-month, 3-month, and 6-month intervals for further follow-up.

After meticulously gathering plaque samples, they were promptly placed into sterile tubes containing reduced transport fluid (RTF), a specially formulated medium intended to maintain the viability of anaerobic and facultative anaerobic organisms during transit. These samples were handled with the highest level of precision and transported to the microbiology laboratory within 3 hours to ensure the preservation of microbial integrity. Upon reaching the lab, the samples were initially processed using the method outlined by Westergren and Krasse, a reliable technique for measuring S. mutans.¹⁷ After incubation (Fig. 2), the characteristic colonies of S. mutans were counted using an automated colony counter, and the results were expressed as colony-forming units per milliliter (CFU/mL), offering a quantitative insight into the bacterial load present in each plaque sample

Fig. 2:

Streak plate method to isolate S. mutans on agar plates for further analysis

Statistical Analysis

Data was analyzed using the statistical package SPSS 26.0 (SPSS Inc., Chicago, IL), and the level of significance was set at p < 0.05. Descriptive statistics were performed to assess the mean and standard deviation of the respective groups. Normality of the data was assessed by the Shapiro–Wilk test. Inferential statistics to find out the difference between the groups were done by one-way analysis of variance (ANOVA) test followed by Bonferroni post hoc test. Within-group analysis was done using repeated measures of ANOVA followed by post hoc test.

Results

The study initially enrolled a total of 180 participants, who were equally divided into three groups of 60 each. Group I received GC MI Varnish, group II received Ultradent, and group III received Fluor Protector S. During the 1st-month follow-up, group I had one dropout, group II had six dropouts, and group III experienced eight dropouts. By the 3rd-month follow-up, group I had an additional 10 participants dropout and group II 2 dropouts, while groups II and III saw four and three more dropouts, respectively. At the 6th-month follow-up, each of group I and III recorded one more dropout and group II 2 dropouts. After accounting for all dropouts, each group ultimately had 48 participants who were successfully followed and analyzed (Flowchart 1).

Flowchart 1:

Flowchart according to CONSORT guidelines

Of the 144 participants, 66 (45.8%) were female, with 35.4% in GC MI Varnish, 43.8% in Ultradent, and 58.3% in Fluor Protector S. Males accounted for 78 participants (54.2%), with 64.6% in GC MI Varnish, 56.3% in Ultradent, and 41.7% in Fluor Protector S. The Chi-squared test showed a value of 5.21 and a p-value of 0.07, indicating no statistically significant differences in gender distribution across the groups.

Table 1 presents the comparison of mean CFU/mL across three different treatments (GC MI Varnish, Ultradent, Fluor Protector S) at baseline, and at 1, 3, and 6 months. At baseline, the mean CFU/mL values were relatively similar, with GC MI Varnish at 7.64 ± 0.18, Ultradent at 7.79 ± 0.18, and Fluor Protector S at 7.83 ± 0.17. However, significant differences emerged over time, particularly at the 1-month and 3-month marks. The one-way ANOVA results indicate a significant effect at 1 month (p < 0.0001) and at 3 months (p = 0.015), suggesting that treatment groups differed significantly during these periods. The 6-month mark did not yield significant differences (p = 0.55). Post hoc tests further clarify these differences. Notably, GC MI Varnish showed a significant difference from both Ultradent and Fluor Protector S at 1 month (p < 0.0001) and 3 months (p = 0.01). While comparisons between Ultradent and Fluor Protector S yielded no significant differences (p = 0.22 at 3 months and p = 0.94 at 6 months), the findings highlight the effectiveness of the treatments, particularly for GC MI Varnish in the earlier months of the study.

Table 1:

Comparison of mean CFU/mL across three different treatments (GC MI Varnish, Ultradent, Fluor Protector S) at baseline, and at 1, 3, and 6 months

		N	Baseline	1 month	3 months	6 months
GC MI Varnish		48	7.64 ± 0.18	6.82 ± 0.08	6.16 ± 1.13	5.76 ± 2.05
Ultradent		48	7.79 ± 0.18	7.46 ± 0.25	6.45 ± 1.30	6.02 ± 1.30
Fluor Protector S		48	7.83 ± 0.17	7.61 ± 0.82	6.86 ± 1.08	6.14 ± 1.85
One-way ANOVA, p-value			0.09	0.0001*	0.015*	0.55
Post hoc test	Group I	Group II	0.45	0.0001*	0.44	0.75
		Group III	0.07	0.0001*	0.01*	0.54
	Group II	Group III	0.60	0.37	0.22	0.94

*Denotes highly significant difference

Table 2 presents the within-group comparison of mean CFU/mL across various time points for the three treatments. All treatments exhibited significant reductions in CFU/mL over time, with overall p-values indicating substantial changes. For GC MI Varnish, the mean CFU/mL decreased from 7.64 ± 0.18 at baseline to 5.76 ± 2.05 at 6 months, showing significant differences at multiple intervals, particularly from baseline to 1, 3, and 6 months. Ultradent also demonstrated a decline from 7.79 ± 0.18 at baseline to 6.02 ± 1.30 at 6 months, with significant changes noted between baseline and 3 months, as well as between 1 and 6 months. Fluor Protector S decreased from 7.83 ± 0.17 at baseline to 6.14 ± 1.85 at 6 months, revealing significant reductions primarily at 3 and 6 months. These results indicate that all treatments are effective over time, with GC MI Varnish showing particularly notable reductions early on.

Table 2:

Within-group comparison of mean CFU/mL across various time points for the three treatments

	Baseline	1 month	3 months	6 months	RM ANOVA, p-value	Post hoc test
						Interval	p-value
GC MI Varnish	7.64 ± 0.18	6.82 ± 0.08	6.16 ± 1.13	5.76 ± 2.05	0.0001*	B vs 1M	0.004*
						B vs 3M	0.0001*
						B vs 6M	0.0001*
						1M vs 3M	0.03*
						1M vs 6M	0.0001*
						3M vs 6M	0.34
Ultradent	7.79 ± 0.18	7.46 ± 0.25	6.45 ± 1.30	6.02 ± 1.30	0.0001*	B vs 1M	0.30
						B vs 3M	0.0001*
						B vs 6M	0.0001*
						1M vs 3M	0.0001*
						1M vs 6M	0.0001*
						3M vs 6M	0.11
Fluor Protector S	7.83 ± 0.17	7.61 ± 0.82	6.86 ± 1.08	6.14 ± 1.85	0.0001*	B vs 1M	0.75
						B vs 3M	0.0001*
						B vs 6M	0.0001*
						1M vs 3M	0.004*
						1M vs 6M	0.0001*
						3M vs 6M	0.006*

*Denotes highly significant difference

Table 3 describes the changes in CFU/mL from baseline to 6 months for three treatments—GC MI Varnish, Ultradent, and Fluor Protector S. Each treatment reflects a decrease in CFU/mL, indicating effective antimicrobial activity over the 6-month period.

Table 3:

Changes in CFU/mL from baseline to 6 months for the three treatment groups

	Baseline	6 months	Mean difference	Change in percentage (%)
GC MI Varnish	7.64 ± 0.18	5.76 ± 2.05	1.88 ± 1.54	24.6
Ultradent	7.79 ± 0.18	6.02 ± 1.30	1.77 ± 1.26	22.7
Fluor Protector S	7.83 ± 0.17	6.14 ± 1.85	1.69 ± 1.46	21.5

Discussion

Dental caries ranks among the most prevalent infectious diseases affecting the mouth and is characterized as a biofilm-mediated condition.^18,19 Biofilms form when bacteria initially adhere to and subsequently accumulate on the tooth surface, indicating that managing biofilm development and buildup could reduce the occurrence of dental caries. Consequently, we concentrated on the effectiveness of fluoride varnishes in combating both the virulence and buildup of cariogenic biofilms, given the rising use of fluoride varnishes for dental caries prevention.⁶

Bacterial adhesion to the tooth surface is essential for biofilm formation. This initial attachment plays a role in both the development and maturation of biofilms and is influenced by various factors, including the growth environment, bacterial vitality, and the characteristics of the material surface.^20–22 Therefore, the adhesion of caries-associated bacteria to a surface coated with fluoride varnish was initially assessed. In this research, all tested fluoride varnishes, with the exception of Fluor Protector, were found to reduce the adhesion of S. mutans. S. mutans is recognized as the primary contributor to dental caries globally and is regarded as the most cariogenic among oral streptococci. It is chiefly identified as the initiator of dental caries. Due to the challenge of replicating the complex oral environment with its varied microbiota, the species most frequently associated with dental caries, namely S. mutans, were selected for this study.²³

Fluoride changes the physical and chemical characteristics of teeth, making them more resistant to being dissolved by acid through the creation of fluorapatite or fluorhydroxyapatite. Additionally, it boosts the maturation process after teeth have erupted, promotes remineralization, and prevents demineralization.²⁴ Fluoride acts as an inhibitor for several bacterial enzymes, including enolases, phosphatases, proton-extruding ATPases, and pyrophosphatases.²⁵ It also affects the bacterial population and modifies the plaque environment. The aqueous phase within the plaque, known as plaque fluid, contains a higher concentration of fluoride than any other oral fluid.^26,27

The current research found that fluoride varnish with CPP–ACP led to a decrease in S. mutans counts. CPP–ACP and calcium compete for identical binding sites on S. mutans, with CPP–ACP demonstrating double the binding affinity to bacterial cells compared to calcium. The increased calcium levels in plaque contribute to anticaries effects by promoting remineralization and inhibiting demineralization.

Moreover, it has been shown that a high level of free calcium can either kill bacteria or inhibit their growth. The combined anticariogenic effect of CPP–ACP and fluoride is also associated with the presence of ACP fluoride on the tooth surface, which is facilitated by CPP.¹¹ A study by Erdem et al. produced similar results, showing a reduction in the viability of S. mutans after applying varnish that contains CPP–ACP.²⁸

According to research conducted by Li et al.,²⁹ CPP–ACP exhibits a prolonged remineralizing impact on initial caries lesions. The decrease in S. mutans was more significant after 1 month compared to the 3rd month. Shen et al.³⁰ discovered that the fluoride ion in MI Varnish had its peak release at 24 hours. Cochrane et al.¹⁵ noted that MI Varnish's rapid ion release is due to the CPP–ACP complex's high-water solubility, compensating for brief oral cavity duration. Shen et al.³¹ found MI Varnish's fluoride ion reached peak release at 24 hours, with nearly full release by 48 hours. A randomized trial by Pukallus et al.³² found GC MI Varnish significantly reduced S. mutans in high-caries-risk pediatric patients after 6 months. Our findings align with Kitasako et al.,³³ who reported fluoride varnishes with CPP–ACP enhance fluoride uptake and provide better bacterial inhibition than fluoride-only products.¹⁴

When it comes to the release of fluoride ions from varnishes, it is important that the presence of calcium and phosphate ions does not reduce the availability of fluoride ions. Clinical trials have shown that fluoride is key to the varnish's ability to prevent caries. Consequently, it was deemed necessary to evaluate the impact of adding CPP–ACP on the fluoride-releasing properties of the fluoride varnish.

Enamelast varnish is a xylitol-sweetened product that contains 5% NaF dissolved in a resin solvent. It is flavored to be more appealing to children. The varnish has a patented formula that includes an adhesion-promoting agent, enhancing its retention and offering superior fluoride release and uptake. In our study, Ultradent varnish demonstrated moderate antimicrobial effectiveness. Baysan and Lynch³⁴ found similar results, noting that resin-based fluoride varnishes significantly but variably reduced bacterial biofilms, attributing the differences to variations in varnish viscosity and fluoride release rates. De Godoi et al.³⁵ conducted a study to assess the remineralization effect on artificial carious lesions, finding that Enamelast varnish facilitated surface remineralization and achieved higher fluoride concentration compared to other varnishes. Singh et al.³⁶ reported that Enamelast varnish (group II) released less fluoride over 3 months, with significantly (p < 0.001) higher fluoride release from Enamel Pro varnish (group III). However, after 6 months, Enamelast varnish released more fluoride than both Enamel Pro varnish and Fluor Protector varnish.

Matar et al.³⁷ applied Fluor Defender and Enamelast to the enamel surfaces of primary teeth, subsequently detecting biofilm formation spectrophotometrically and observing it via scanning electron microscope (SEM) to examine the antibiofilm activity of both products. Attiguppe et al.³⁸ showed that MI Varnish works better against S. mutans than NaF varnish. This leads to less plaque. They said that just releasing fluoride from NaF varnish might not be enough to fight bacteria that cause cavities or stop damage below the surface. The addition of CPP–ACP helps the varnish stop this damage.

Fluor Protector S exhibited the least antimicrobial effect, consistent with Seppä's³⁹ research, which found that varnishes with lower fluoride concentrations and minimal sustained release were less effective in reducing microbes. In the current study, baseline mean CFU/mL values were relatively similar, but significant differences emerged over time, particularly at the 1-month and 3-month intervals. Postapplication, the mean colony-forming units (CFUs) were lowest for the GC MI Varnish group, followed by Ultradent and Fluor Protector S. The GC MI Varnish group also showed minimal standard deviation, indicating consistent antimicrobial efficacy. In children without cavities who have their first set of teeth, the number of bacteria in their plaque was between 10,000 and 1,00,000. Their average bacterial score was around 7.64. For adults and older children, S. mutans bacteria levels in saliva that pose a cavity risk were between 1,00,000 and 10,00,000 per mL.

In the study, each topical fluoride used offers several benefits, such as being easy to apply, safe, noninvasive, and effective in preventing dental caries in children. Regular use decreases the likelihood of decay, particularly on smooth surfaces and newly erupted teeth, which are more prone to cavities. Topical fluoride can also reverse early demineralization signs, stopping the development of initial lesions. Moreover, professional fluoride treatments like gels, foams, or varnishes provide precise dosage control and targeted application, reducing the risk of fluoride ingestion. Overall, topical fluoride is a cost-effective, evidence-based preventive strategy that is essential for maintaining optimal oral health in children and lessening the need for restorative dental procedures. In this study, after assessing all groups for all interventions, we found that GC MI Varnish produced highly significant results across all three groups, indicating that it was more effective than the other two varnishes used in the study. Overall, all three treatments successfully reduced microbial counts over the 6-month period, with GC MI Varnish demonstrating the most significant mean difference and percentage change, underscoring its potential as an effective antimicrobial treatment.

Limitations

The research was conducted with a small group from a single geographic location, limiting the ability to apply the findings to larger populations. The absence of long-term follow-up hindered the evaluation of ongoing treatment effects over an extended period. Outcomes may have been affected by observer bias and differences in patient adherence, and elements such as socioeconomic status were not included in the analysis.

Clinical Implications and Future Direction

With the increasing focus on preventive dentistry, choosing fluoride varnishes that include additional bioactive ingredients could significantly enhance caries prevention strategies, particularly for high-risk groups. Our research indicates that GC MI Varnish should be regarded as a top choice in clinical settings. Moreover, microbial culture techniques were employed to measure S. mutans. Although culture-based assays are considered the gold standard, molecular techniques such as quantitative polymerase chain reaction (qPCR) may provide higher sensitivity and specificity. Future research could incorporate molecular methods to confirm these findings. Additionally, while the reduction in microbial load was examined, long-term clinical outcomes, like the decrease in caries incidence, were not assessed. Prospective longitudinal studies could determine if the observed reductions in microbial load led to significant clinical advantages over time.

Orcid

Nishita Garg https://orcid.org/0000-0001-6594-9098

Abhay K Jain https://orcid.org/0000-0002-2457-8144

Pallavi Bhyri https://orcid.org/0009-0005-9284-3914