Plaque bacteria that ferment sucrose produce acids that lower the pH level to below 5.0 in vitro and cause enamel demineralization. However, only Streptococcus mutans of all these species significantly cause caries in germ-free animals with a high-sucrose diet. This shows that microbial acid production is not the sole determinant of caries, and S. mutans must have other characteristics that are responsible for its severity and make it the main cause of caries.^[1] S. mutans forms several complex glucans such as fructans, dextrans, and mutans. In vitro experiments have shown that these glucans enable S. mutans to adhere firmly to surfaces and cause tooth decay.^[1] Adding fluoride to drinking water, producing different mouthwashes, and using pit and fissure sealants, topical products such as fluoride gels and varnishes, xylitol emulsifiers, and probiotics are among the procedures to prevent caries.

According to the definition of the World Health Organization, probiotics are living microorganisms that are prescribed in sufficient amounts and provide health benefits to the host.^[2] Streptococcus salivarius M18 and K12 (S. S. K12, S. S. M18) are probiotic bacteria used in various food products. It has been shown that these species of S. salivarius are capable of producing bacteriocin and have a narrow range of effects on preventing the growth of some other bacteria.^[3]

S. S. M18 is able to reduce the number of S. mutans but has no specific effect on the health of the gingiva and periodontal tissues.^[4] This caries prevention mechanism is attributed to the ability of S. S. M18 to produce urease and dextranase enzymes. These enzymes are able to deal with the formation of dental plaque and prevent caries.^[5,6] It has also been shown that S. S. K12 is effective in preventing and treating diseases such as halitosis, acute otitis media, pharyngotonsillitis, and oral candidiasis by producing bacteriocin.^[7-9] A study on the effect of S. S. M18 on the risk of caries and oral health showed a reduction in the amount of S. mutans and dental plaque.^[10] In another study, the comparison of saliva sample cultures at the beginning and end of the intervention showed a slight difference in the number of S. mutans between the two groups and between each group compared to the baseline. However, 9 children who had higher M18 salivary bacteria in their mouth showed a significant decrease in the number of S. mutans. Moreover, 87.5% of M18 salivary bacteria users who had a large amount of plaque at the beginning of the intervention showed a significant decrease in plaque formation, while those with a large plaque in the placebo group showed 44% decrease in plaque formation.^[4] Most of the studies conducted on probiotics have shown that their oral and dental effects are highly dependent on the type of probiotic bacteria used, and few studies have been done in this field, especially on S. S. K12 and S. S. M18 probiotic bacteria. Furthermore, very few academic studies have been done on Iranian probiotic products as well as their effects on the prevention of bacterial plaque, which requires further studies of this type.

This double-blind randomized clinical trial was conducted on 40 students of medicine and pharmacy, with the age range of 19–35 years, at Isfahan University of Medical Sciences. A checklist of subjects was prepared at the beginning and end of this study. The exclusion criteria were students with orthodontic appliances or 6-month orthodontic treatments, students who had taken antibiotics in the last month, students undergoing incomplete dental treatments, and students with immune system diseases. Further, students who needed dental treatment during the intervention, used <90% of the consumables, needed to take antibiotics during the intervention, got bacterial and viral diseases during the intervention, and changed the number of times they brushed their teeth and the type of toothpaste and mouthwash, or used a new oral hygiene method were excluded after the study. As it is shown on the diagram in figure 1, 47 volunteers enrolled for this study and 7 of them were excluded due to the study exclusion criteria. Four underwent dental treatment and 3 had orthodontic appliances. The participants’ flow diagram is represented in Figure 1.

According to Table 1, based on the results of the Kolmogorov–Smirnov test, the data were normally distributed (P > 0.05). For inferential analysis, parametric paired and independent t-tests were used.

Table 2 presents the comparison of mean scores of research variables in two periods for each group. This comparison was done using the paired t-test.

In the control group, the mean plaque score was 0.426 ± 0.259 before the intervention and 0.420 ± 0.273 after the intervention (P > 0.05). Therefore, in the control group, the number of plaques before and after the intervention did not change. In the intervention group, the mean plaque score was 0.411 ± 0.334 before the intervention and 0.759 ± 0.406 after the intervention (P < 0.05). Therefore, in the intervention group, the amount of plaque increased significantly after the intervention.

The mean plaque of the first period was compared between the control and intervention groups, indicating no significant difference between the two groups (P > 0.05). The mean plaque of the second period was compared between the control and intervention groups, which revealed a significant difference between the two groups (P < 0.05) Table 3.

The present study is one of the first studies on probiotic products made in Iran and their effectiveness in preventing dental caries. The results showed that the dental plaque index in the intervention group that received probiotic tablets (S. salivarius bacteria M18 and K12) were significantly increased. Di Pierro et al. reported that the 90-day consumption of the probiotic S. salivarius M18 could prevent dental disease,^[10] which is completely different from the results of the present study. It should be noted that the products used in the present study are made in Iran and the manufacturer of the products is different from that of the study mentioned above.

Moreover, Söderling et al. demonstrated that four types of probiotic products could effectively reduce the number of Streptococcus bacteria,^[11] which is not in line with the results of the present study. Further, they reported that the in vitro antibacterial activity of probiotic products against S. mutans is highly dependent on the pH of the environment, and the increase in the plaque in the present study can be because this product may have changed the pH of the environment in favor of S. mutans.

Contrary to the results of the current study, Burton et al. found probiotic products to be effective in reducing caries.^[4] In the present study, the probiotic bacteria used may make food more easily accessible to harmful bacteria in the mouth and teeth, thereby increasing their population. In a systematic review conducted by Poorni et al. in 2019, the quality of the articles on this subject was very poor, so they recommended further studies in this regard.^[12]

Babina et al. indicated that the probiotic product had a positive effect on reducing microbial plaque contamination, which is again different from the results of the present study.^[13] In the present study, the probiotic bacteria used may have changed the pH of the environment, thereby balancing the normal flora of the area (Echo logic Nich change) and ultimately increasing harmful oral bacteria.

The mean plaque index in the group using these Iranian probiotic tablets was significantly increased compared to the control group. Accordingly, further research is suggested to evaluate these Iranian products.