Dental caries is the most common chronic disease of children worldwide. ¹ , ² It is a chronic bacterial disease, in which host factors, diet, and oral microbial flora are involved. These factors contribute to caries through dental demineralization. ³ Streptococcus mutans is a type of Gram-positive bacterium described by Clark in 1924. This bacterium plays a major role in tooth decay. ⁴ Streptococcus bacteria are initially replaced on the surface of the tooth and by preparing the environment for acidic conditions, also allow for the presence of other microorganisms. ⁵ S. mutans metabolizes sucrose to lactic acid and provides the basis for dental caries. ⁴ , ⁵ It has been suggested that therapies that interfere with the colonization of S. mutans can have a fundamental effect on reducing the incidence of caries in humans. Certain types of lactobacilli are also associated with caries. These species play a minimal role in the onset of caries but are believed to play a role in caries development. ⁶ However, not all types of lactobacilli are cariogenic and some are even known to be protective. ⁷

S. mutans can persist in an environment including mucosal surfaces exposed to salivary flow, by forming a colony or free living in saliva and proliferating; but other bacteria must attach to the mucosal surface. ⁸ , ⁹ S. mutans can be colonized in the mouth before tooth eruption and transiently infect, but for continuous colonization in the mouth, it depends on the presence of teeth. Therefore, its initial fixation occurs during the first 4–5 years of life, and its origin is usually the mother's saliva. ¹⁰ , ¹¹

Recently, the use of probiotics has been introduced as a method to reduce the amount of salivary S. mutans and subsequently dental plaque as the initiator of the cariogenesis. Probiotics are a dietary supplement made up of bacteria or potentially useful fungi. ⁴ According to the accepted definition of the World Health Organization and the US Food and Drug Administration, probiotics are “Living microorganisms that, if consumed in sufficient quantities, may have beneficial effects on maintaining their host health.” These microorganisms, through a variety of mechanisms, create unfavorable conditions for the growth of harmful microorganisms and play a major role, especially in the prevention of gastrointestinal infections. Bifidobacteria and Lactobacillus are the most commonly used probiotics. ¹² , ¹³ , ¹⁴ , ¹⁵ Previous studies have suggested that consumption of probiotic products may reduce dental caries. These studies have shown a decrease in the levels of some bacteria that are effective in causing caries such as S. mutans. ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ Probiotics or their products can have antimicrobial activity and resist the colonization of pathogens. Immunologically, they have an adjuvant effect and possibly stimulate the phagocytic process of blood leukocytes and increase IgA secretion. In addition, probiotics affect the production and activity of enzymes. They also have antigenic effects. ²¹ An in vitro study has shown that Lactobacillus plantarum probiotic, by reducing S. mutans bacteria in biofilms isolated from active caries children, can have a limiting effect on S. mutans growth. ²² In addition, the combination of probiotics with L. plantarum, rhamnosus, and acidophilus added to chocolate showed an inhibitory effect on S. mutans growth in vitro. ²³

Since there is no study on mouthrinses with such probiotic bacteria, we conducted this study for the first time in order to assess the efficacy of probiotic mouthwashes containing L. plantarum on S. mutans in comparison to placebo and fluoride.

This study was a randomized clinical trial (IRCT20180901040922N1). The subjects were randomly selected from fixed orthodontic patients who referred to the orthodontic department in 2017.

Eligibility criteria

Inclusion criteria were systemic health; no systemic and local antibiotics as well as any antiinflammatory drugs within 4 weeks of starting the study; lack of fluoride therapy history within 4 weeks of starting the study; no use of any probiotic products and xylitol-incorporated chewing gums for 4 weeks before the study and during the study; and the habit of brushing twice a day. Absence of active carious lesions and periodontal disease; permanent dentition; fixed orthodontic applications on at least eight maxillary anterior teeth; at least 3 months passed from the start of fixed orthodontic treatment; no smoking; no sensitivity to probiotic, sodium fluoride, or placebo mouthwash (participants were asked about their sensitivity to probiotics and fluoride so far: no reports of sensitivity were reported during the study); no infectious disease; and being aged between 12 and 30 years. ²⁰ , ²⁴ , ²⁵ , ²⁶ , ²⁷

Sample size

The results of a previous study were used to determine the sample size. ²⁴ The decrease in S. mutans was 100% in the intervention group and 18% in the control group. Therefore, with 95% confidence level and 90% test power using G-Power software (Axel Buchner, Universität Düsseldorf, Düsseldorf, Germany) and formula for comparison between two ratios, sample size was determined as 16 (eight in intervention group and eight in control group). As there were three comparative groups, the sample size was adjusted to 36 (12 in each of the three groups) according to this formula:. It was also increased to 48 individuals (16 individuals in each of the three groups) with a 30% dropout in mind for the total sample size. After implementation of the design and losing a number of subjects, the final sample size was reduced to 38 (12 in the fluoride group and 13 in either the placebo and probiotic group). Patients' age ranged from 12 to 20 years. Of them, 14 were males and 24 were females.

Bacteria identification

To isolate and culture Lactobacillus, 1 ml of human saliva was dissolved in 9% sterile saline and diluted to concentrations of 1/10. 0.1 ml of appropriate concentration propagated on MRS agar plate (Merck, Germany). It was incubated in aerobic conditions for 48–72 h at 30°C. Catalase − Gr + colonies were identified as Lactobacilli. These colonies were stored in 15% MRS broth (v/v) glycerol stocks for subsequent study. In order to evaluate the probiotic effect of Lactobacilli, tests such as polymerase chain reaction, acid resistance, bile resistance, antibacterial properties, Bile-salt hydrolytic activity, NaCl resistance, lysozyme resistance, antibiotic resistance, and molecular detection of Lactobacillus species were performed. After diagnostic tests, it was determined that L. plantarum is our probiotic bacterium. ²⁸

Probiotic mouthwash

The mouthwash was initially made as powder and liquid in separate containers. Each powder container contained approximately 10 ⁸colony-forming unit (CFU) bacteria lyophilized and mixed with malt and dextrin, weighing 30 mg. The liquid container was handed to subjects as a 20 ml Falcon tube containing 5 ml phosphate-buffer saline (PBS). Each patient was told to mix the powder with the liquid for 30 s before use.

Placebo

Patients were given 30 mg of dextrose powder with 5 ml of distilled water to be mixed for placebo mouthwash.

Intervention

First, patients were examined and interviewed. Individuals were included in the study after completing the informed consent form and being properly briefed by the researchers. All patients were trained according to the standard protocol of hygiene compliance during orthodontic treatment.

These subjects, who were at least 3 months into their fixed orthodontic treatment, were randomly divided into three groups according to a random number table: group 1 (control) included 13 subjects receiving placebo mouthwash; Group 2 included 12 subjects receiving sodium fluoride mouthwash; and group 3 comprised 13 patients receiving probiotic mouthwash.

From all three randomized groups, plaque sampling was performed in two stages: The 1 ^stday of study and before the intervention (T0) and 2 weeks after the mouthwash start (T1). During the mouthwash period, the patients brushed as usual and used the mouthwash twice a day after lunch and before bedtime. Each probiotic mouthwash served as a container containing powder (lyophilized and dextrose bacteria) and a container containing PBS to be mixed and homogenized before use. The placebo group used powder and liquid mouthwashes similar to the probiotic group, whereas in the liquid-containing container, distilled water, and in the powder container, only dextrose was free of lyophilized bacteria. The fluoride group was given only a 0.05% fluoride-containing container similar to that of the other groups, but the powdered container did not exist in this group. There was a leaflet in the patient package containing information on the patient's requirements including inclusion criteria and how to contact the project manager as well as checkboxes to be marked after each mouthrinsing. Each week, subjects were encouraged by a phone call to continue using mouthwash. All participants were able to contact the project manager during the project and inform him of any potential adverse effects. They also asked questions from the person in charge.

Plaque sampling

Plaque sampling was performed using the 4-pass technique recommended by Pellegrini et al. ²⁹ In this method, a senior dental student collected plaque using a sterile plaque scaler on the labial surface of the maxillary lateral incisor, adjacent to the bracket sides, in four directions of mesial, distal, occlusal, and gingival.

The dental plaque was dissolved in 5 cc of PBS and stored in a refrigerator (4°C) and sent to the Microbiology Laboratory of the Medical School on the following day. The samples were diluted serially and cultured in Mitis salivarius sucrose bacitracin agar medium under anaerobic conditions at 37°C for 24–48 h. ³⁰ Then, the number of bacteria in the dental plaque was counted based on the number of colonies grown on agar medium, using the “CFU × Dilution factor × 1/aliquot” formula and with the CFU/4-pass technique.

Statistical analysis

Descriptive statistics and 95% confidence intervals (CIs) were calculated for different variables. Data normality was assessed using Kolmogorov–Smirnov test which indicated nonnormal data. The Wilcoxon test was used to assess before-after changes of S. mutans in each group. Groups were compared using Kruskal–Wallis test of SPSS 22 (IBM, Armonk, NY, USA).

There were 4, 4, and 6 men in the groups probiotic, placebo, and fluoride (Fisher P = 0.538). The Kruskal–Wallis test showed that the three groups were similar in terms of age, brushing frequency, and the duration of brackets placed [P > 0.05, Table 1]. Furthermore, compliance with the protocol was similar between the three groups Table 2.{Table 1}{Table 2}

In the probiotic group, nine individuals showed an increase in colony counts, while 3 and 1 showed reduction and no changes, respectively. In the placebo group, 11 patients showed increases in colony counts, and two people showed a reduction. In the fluoride group, only two people showed an increase in colony counts, and the rest (10 patients) showed a reduction. According to the Wilcoxon test, in the placebo group, the colony count increased after 2 weeks (P = 0.005). In the fluoride group, the colony count reduced significantly (P = 0.025). In the probiotic group, however, there was no significant change over time [P = 0.158, Table 3and Figure 1]. There was not a significant difference among three groups in terms of either pretreatment colony counts or after-treatment colony counts Table 3. Figure 1

Estimated marginal means for colony counts.

The findings of this study suggest that, unlike fluoride mouthrinse that can have antimicrobial effects, this particular type of probiotic bacteria could not help reduce S. mutans, when used in the form of mouthrinse. There have been earlier studies on the efficacy of various forms of probiotic products in reducing S. mutans and caries, and their controversial results indicate a great ambiguity in this area. In the present study, it was found that, in practice, the probiotic mouthwash could not have a significant effect on the amount of S. mutans bacteria and had almost the same effect as did the placebo group. This was in line with some studies (Montalto and Chuang) and in contrast with most others showing a decrease in the amount of S. mutans after consumption of probiotic-containing compounds. ¹⁸ , ²⁰ , ³⁰ , ³¹ , ³² , ³³ These studies differ in the type of probiotic used: in the Nase and Caglar study, lactobacillus rhamnosus was evaluated. ¹⁸ , ³¹ The Nikawa et al. study showed the inhibitory effect of Lactobacillus reuteri on the growth of S. mutans. ³⁴ In a study by Chuang et al., results showed that consumption of pills-containing Lactobacillus paracasei had no effect on reducing salivary S. mutans. ³⁰ Caglar et al. studied the effect of probiotic-containing ice cream in a group that had high levels of S. mutans at the beginning of the study. ¹⁸ However, in the present study, the variation in the baseline colony count was high (minimum: 10,000 maximum: 7,500,000). This scattering probably originated from a 4-pass technique. In the Näse et al. study, the effect of probiotic-containing milk in preschool children with a 7-month intervention was different from the age group in the present study, and the samples received a longer probiotic duration. ³¹ In a study of thirty children between 8 and 15 years old, Megha et al. studied the effect of probiotic yogurt on orthodontic children and found that the use of these probiotics can reduce oral microorganisms and subsequently reduce caries, which differed with our findings. The conditions and methodologies of the two studies differed and they had used a much higher but inaccurate concentration (>10 ⁹). ³⁵ Studies such as Iwasakir et al., Harini and Anegundi, and Kawai et al. have shown the effect of L. plantarum in reducing gingival disease. ³⁶ , ³⁷ , ³⁸ There also appears to be a relationship between the reduction of periodontal microorganisms and the increase in S. mutans. ³⁹ , ⁴⁰ Consequently, it can be hypothesized that L. plantarum may provide the basis for increased S. mutans by removing periodontal pathogen bacteria.

There have been numerous in vitro studies on the efficacy of L. plantarum in inhibiting S. mutans. Ahn et al. reported that L. plantarum inhibits the formation of S. mutans biofilms. ⁴¹ An in vitro study also showed that L. plantarum, by reducing S. mutans bacteria in biofilms isolated from children with active caries, could have a restrictive effect on S. mutans growth. ²² Furthermore, the results of Khanafari et al. show that L. plantarum probiotic is effective in inhibiting growth of S. mutans and it may be useful to reduce the prevalence of caries. ²³ The results of an in vitro study showed that the cell-free solution containing components of L. plantarum and L. acidophilus had significant inhibitory activity on the biofilm formation of cariogenic organisms. In addition, the interference (80% decrease) in glucose synthesis by S. mutans by these lactobacilli indicates their potential role in inhibiting glucosyltransferase (Gtf-I). As a result, L. plantarum can reduce the biofilm of this carcinogenic bacterium by suppressing S. mutans virulence genes. ⁴² Therefore, the result of the present study might root in methodological strategies. In the placebo group of the current study, there was an increase in the number of S. mutans after the intervention. One of the factors that can be effective in increasing the number of bacteria is the presence of dextrose in the placebo mouthwash formulation. This sugar can be used as a substrate for the metabolism of S. mutans present in plaque and increase the number of bacteria. On the other hand, probiotic mouthwash formulation also had dextrose and increased bacterial counts (not statistically significant), but this increase was lower than the placebo group, which may reflect the inhibitory effect of L. plantarum on S. mutans. The presence of dextrose is important for the growth and activation of lyophilized bacteria, and a higher percentage of lyophilized bacteria survive after activation in dextrose-containing medium. ⁴³ Since not using dextrose may reduce the viability of probiotic bacteria, perhaps doses above 10 ⁸CFU, without dextrose, may work better in reducing S. mutans. For maximum efficacy, L. plantarum must be colonized in the oral environment. This colonization requires more time for bacterial activation as well as greater presence of bacteria in the mouth. Dissolving the lyophilized bacterial powder for 30 s is not enough to activate it. However, taking extra time of the patient at home naturally reduces the patient's cooperation and overshadows the clinical application of this mouthwash. The duration of the mouthwash usage is also important to provide the probiotic bacteria with the opportunity to colonize. The duration of mouthwash use cannot be exceeded by a certain limit, but other methods may help keep the probiotic bacteria in the mouth for longer periods. The use of probiotic varnishes may help to resolve this problem.

Much research has been done on the use of fluoride-containing mouthwashes. In addition to its protective effect on enamel, fluoride also affects the process of decay by altering the invasion of bacteria and does so in two ways: 1 – change the ability of organisms to produce acid and 2 – by facilitating the growth of some of bacteria. Fluoride inhibits glucose transport due to its effect on enolase since phosphoenolpyruvate is essential for the phosphoenolpyruvate transferase system that is capable of forming glucose-1-phosphate. Increasing the concentration of hydrogen ions inside the cell by inhibiting glucose-hydrogen ion transfer prevents glucose movement. Fluoride also inhibits the membrane's ATPase action and removes hydrogen ions from bacterial cells by preventing glycolysis and reducing the hydrogen ion gradient on the other side of the cell wall. Therefore, the overall effect of fluoride will be to inhibit acid production and to disrupt cellular energy metabolism. It is important to note that the sensitivity of the bacteria to the effects of fluoride varies. ⁴⁴ Most studies recommend a 0.2% sodium fluoride mouthwash once a week or a 0.05% mouthwash once a day. According to these studies, there is no doubt about the benefits of fluoride-containing mouthwashes in preventing tooth decay when used properly. There are other substances that have been tested for oral hygiene maintenance during orthodontic treatment. For example, Øgaard et al. ⁴⁵ compared the effect of chlorhexidine-fluoride varnish with fluoride alone and with chlorhexidine in these patients. Chlorhexidine reduced the amount of S. mutans but had no effect on the white spots, compared with fluoride, but chlorhexidine and fluoride had the best results. ⁴⁵ As a result, the simultaneous use of these two substances can have a favorable effect against dental caries.

The results of this study indicated that, compared to placebo, the experimental probiotic mouthrinse in use did not show any advantage in terms of controlling S. mutans. Therefore, our preliminary findings do not support the use of this particular dosage and type of probiotic as mouthrinse, for controlling S. mutans in the plaque around orthodontic brackets. However, fluoride mouthrinse proved effective against S. mutans in the plaque around the orthodontic bracket and is therefore recommended.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.