Original Article

Antibacterial effects of children’s and adults’ toothpastes containing different amounts of fluoride: An in vitro study

Introduction

Dental caries is the most common oral disease affecting children, adolescents, and adults.^1,2 In a 2017 global survey, the World Health Organization reported 532 million cases of dental caries in primary dentition and about 2.3 billion for caries in permanent dentition.³ According to data collected during 1995‒2019, the prevalence of dental caries in Asian children is about 52%‒58%.¹ This chronic infectious disease is mainly caused by a group of streptococcal species, with multiple contributing factors helping to increase or reduce the risk of caries progression.⁴

Even though dental caries is prevalent, the disease is preventable. One of the most applicable and common oral health care preventive methods is brushing the teeth with fluoride toothpaste. Brushing teeth causes mechanical removal of dental plaque and food debris, reducing the number of cariogenic bacteria and helping to maintain the balance of normal flora in the oral cavity.⁵ Meanwhile, the ingredients in fluoride toothpaste contribute to caries prevention via two main avenues. The first is via antimicrobial agents added to the toothpaste formula, such as triclosan, sodium lauryl sulfate (SLS), and herbal extracts.⁶ The second is the promotion of remineralization by fluoride.^7,8 Evidence has shown that tooth brushing with fluoride toothpaste significantly reduces caries increments in both primary and permanent dentition; moreover, the higher the fluoride concentration, the better the remineralization and caries preventive effects.⁹ Additionally, several studies have suggested fluoride itself has antimicrobial properties by inhibiting the activity of bacterial enzymes.^6,10

In recent years, the recommended fluoride concentration in toothpaste for caries prevention has shifted from 500‒1000 ppm to 1000‒1500 ppm, which is better to increase preventive efficiency.^11-13 Consequently, many new commercially available toothpaste products for children and adults have been marketed.^14,15 However, while many reports have been published on the remineralization effect of 1450‒1500 ppm fluoride,^8,16,17 few studies have investigated and compared the antibacterial activity of commercially available 1450‒1500 ppm fluoride toothpastes with those having lower fluoride concentrations, and the findings in this regard are still inconclusive.^18,19 Furthermore, the antibacterial activities of children’s and adults’ toothpastes containing 1450‒1500 ppm fluoride have never been compared. Obtaining this information would further guide dental professionals on the recommendation and selection of toothpastes. Therefore, we aimed to investigate the antibacterial activity of commercially available toothpastes containing 500, 1000‒1100, and 1450‒1500 ppm fluoride and compare the antibacterial activity of children’s and adults’ toothpastes.

Methods

Toothpastes

The toothpastes were classified into three groups according to fluoride concentration: (i) 500 ppm fluoride, (ii) 1000‒1100 ppm fluoride, and iii) 1450‒1500 ppm fluoride. The necessary sample size was calculated using G*Power 3.1²⁰ with a power of 80% and significance level of 5%, where means and standard deviations were based on previous reports from Randall et al¹⁸ and Evans et al.¹⁹ This yielded an estimate of ten samples per group. Accordingly, we selected 30 fluoride toothpastes in each group for this study: 500 ppm, 1000‒1100 ppm, and 1450‒1500 ppm fluoride, which were commercially available (both online and off-the-shelf) in the market in Thailand. For the 500-ppm fluoride group, only toothpastes for children were available, and consequently, only children’s toothpastes were selected. For the 1000‒1100-ppm and 1450‒1500-ppm groups, five children’s products and five adult products were selected. Table 1 presents the details of the toothpastes, including product name, manufacturer, ingredients, and fluoride concentration.

Microorganisms and in vitro growth conditions

The three strains of bacteria used for this investigation were obtained from the American Type Culture Collection (ATCC). The strains consisted of Streptococcus mutans (ATCC 25175), Streptococcus salivarius (ATCC 13419), and Lactobacillus casei (ATCC 334), which were revived and cultured on brain heart infusion agar (BD Difco^TM Spark, MD, USA) under anaerobic condition (5% CO₂) at 37 °C.

In vitro investigation of bacterial growth inhibition using agar well diffusion method

The antimicrobial activity of the toothpastes was investigated using the agar well diffusion assay and measurement of the zone of inhibition. Each bacterial strain was first inoculated in brain heart infusion broth (BD Difco^TM, USA) and incubated at 37 °C under 5% CO₂ for 24 hours, after which the inoculums were prepared and adjusted to a turbidity of 0.5 McFarland. Next, 100 µL of each bacterial suspension containing 1.5×10⁸ CFUs/mL was uniformly spread on plated brain heart infusion agar using a sterile cotton swab. A sterile cork borer with a diameter of 6 mm was used to cut four wells into the agar. These wells were seeded with 40 μL of each toothpaste, or 0.12% chlorhexidine and sterile distilled water, respectively, for the positive and negative controls, as shown in Figure 1. Afterward, the agar plates were incubated at 37 °C under 5% CO₂ for 24 hours, and the diameters of the inhibition zones were measured in millimeters. The test was repeated five times for each tested agent, and the data were presented as mean ± SD.

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Figure 1.

Inhibition zone of toothpastes and controls against Streptococcus mutans on an agar plate. a) Negative control (sterile distilled water). b) positive control (0.12% chlorhexidine). c) toothpaste No. 1 (Fluocaril^® Original). d) toothpaste No. 2 (Dentiste’ Kids).

Figure 1.

Inhibition zone of toothpastes and controls against Streptococcus mutans on an agar plate. a) Negative control (sterile distilled water). b) positive control (0.12% chlorhexidine). c) toothpaste No. 1 (Fluocaril^® Original). d) toothpaste No. 2 (Dentiste’ Kids).

Statistical analysis

Data were analyzed using SPSS 18 (IBM, Armonk, NY, USA). The Kruskal-Wallis test, followed by pairwise comparisons, was used to compare the three groups of toothpaste with different fluoride concentrations. The Mann-Whitney U test was used to compare children’s and adults’ toothpastes. The chi-squared test was used to examine the association of bacterial growth inhibition with the presence of SLS, an antimicrobial agent, in the toothpaste. A P value of <0.05 was considered statistically significant.

Results

Table 2 presents the inhibition zone sizes obtained for each of the three strains of bacteria: S. mutans, S. salivarius, and L. casei. Comparisons of the three different fluoride concentrations revealed no significant differences in the inhibition zones for any bacterial strain (P>0.05), as shown in Figure 2. However, significant differences were observed when the inhibition zones of children’s and adults’ toothpastes were compared, with children’s toothpastes exhibiting significantly lower inhibition zones for all the three strains (P=0.000002, P=0.000006, and P=0.00003 for S. mutans, S. salivarius, and L. casei, respectively). Further comparisons of children’s and adults’ toothpastes having the same fluoride concentration revealed the toothpastes for children consistently had significantly smaller inhibition zones, as illustrated in Figures 3A and 3B. The presence of SLS in the toothpaste ingredients was significantly associated with antibacterial activity (P<0.001).

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Figure 2.

Bacterial growth inhibition of fluoride toothpastes containing 500, 1000‒1100, and 1450‒1500 ppm fluoride. Straight horizontal lines (⎯) represent medians and crosses (×) indicate means. No differences were observed (P>0.05).

Figure 2.

Bacterial growth inhibition of fluoride toothpastes containing 500, 1000‒1100, and 1450‒1500 ppm fluoride. Straight horizontal lines (⎯) represent medians and crosses (×) indicate means. No differences were observed (P>0.05).

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Figure 3.

Bacterial growth inhibition of children’s and adult’s fluoride toothpastes. A) Toothpastes containing 1000‒1100 ppm fluoride. B) Toothpastes containing 1450‒1500 ppm fluoride. Straight horizontal lines (⎯) represent medians and crosses (×) indicate means (**P<0.01, *P<0.05).

Figure 3.

Bacterial growth inhibition of children’s and adult’s fluoride toothpastes. A) Toothpastes containing 1000‒1100 ppm fluoride. B) Toothpastes containing 1450‒1500 ppm fluoride. Straight horizontal lines (⎯) represent medians and crosses (×) indicate means (**P<0.01, *P<0.05).

Discussion

This study investigated the antibacterial activity of commercial fluoride toothpastes in terms of their ability to inhibit the growth of S. mutans, S. salivarius, and L. casei, bacterial strains that play roles in biofilm formation and the initiation and progression of dental caries.^21-24 We found that toothpastes containing 500 ppm fluoride had the smallest inhibition zones, with no significant differences in zone sizes between fluoride concentrations. This finding is consistent with previous reports that fluoride concentration is not correlated with toothpaste antibacterial activity.^18,25 Some studies have suggested that the antimicrobial function of fluoride requires an acidic environment, which occurs in the oral cavity when the critical pH is reached.^26,27 Our in vitro investigation maintained a neutral pH for bacterial growth, which might explain our not observing an antibacterial action of fluoride in this study. In contrast, Evans et al¹⁹ reported commercial toothpastes containing 1450 ppm fluoride to exhibit significantly greater growth inhibition of S. mutans and S. sanguinis than toothpastes containing 500 ppm fluoride. However, this difference might be attributable to the fact that the 1450 ppm fluoride toothpastes tested in their study also contained antimicrobial agents such as triclosan and sodium bicarbonate, which were not present in the 500 ppm fluoride toothpastes.^6,19

We also investigated and compared the antibacterial activity of fluoride toothpastes for children and adults. This study is the first to compare the antibacterial effects of commercially available fluoride toothpastes for children and adults with the same fluoride concentration. Our results showed that the toothpastes for adults resulted in significantly greater inhibition of bacterial growth than those for children, possibly because many commercial toothpaste products for adults contain SLS, a surfactant with antimicrobial properties that interferes with microorganism biological processes and membrane integrity.²⁸ Our findings concerning an association between SLS presence and toothpaste’s capacity to inhibit bacterial growth are consistent with previous publications that found toothpastes containing SLS exhibited greater bacterial growth inhibition than those without SLS.^18,29 SLS creates foam during brushing, leading to the impression of cleanliness^28,30; however, it also alters taste perception, contributing to a bitter taste after exposure^30,31 and has been reported to cause some tissue irritation.^28,30 Distinct from adults’ toothpastes, many toothpastes for children have no SLS due to this taste alteration and chance of irritation. Consequently, fluoride toothpastes for adults demonstrated significantly greater antibacterial activity than those for children.

Our study had some limitations regarding other factors that could impact bacterial growth. For example, microbial growth and activity are affected by other microorganisms in plaque biofilm and by salivary pH in the oral cavity^26,27,32; however, this study was an in vitro investigation and inherently limited the influences of such environmental factors. Therefore, absolute data on bacterial growth inhibition in the oral cavity could not be provided.

Fluoride toothpastes that are branded and marketed for children usually have attractive flavors, smells, colors, and packaging to motivate them to brush their teeth. Children have been reported to prefer toothpastes with a fruity smell and sweet flavor.³³ However, for those in late childhood and early teens, toothpaste flavor and smell may not significantly affect their brushing decisions and cooperation. Therefore, a recommendation for these groups to use commercially available adults’ fluoride toothpastes may help them gain both remineralization and antimicrobial benefits.

Conclusion

Fluoride concentrations ranging from 500 to 1500 ppm did not affect the ability of commercially available toothpastes to prevent bacterial growth. On the other hand, whether a toothpaste is formulated for children or adults was found to influence its effect on bacterial growth, with adults’ toothpastes exhibiting greater antibacterial activity. This inhibitory effect is mainly due to SLS, an antimicrobial agent widely added to adult formulations. Consumers and dental health professionals should be aware of this differential effect and consider it when selecting a toothpaste.

Competing Interests

Authors have no conflicts of interest to declare.

Ethical Approval

The study was performed according to the Declaration of Helsinki. Neither humans nor animals were used in this study. We used well-known bacterial strains supplied by a business (ATCC) for our research. We do not have the ethical approval code because, according to what we understand, ethical approval was not necessary for this study.

Funding

None.

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