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Background The purpose of the study was to investigate the effect of probiotics on biofilm acidogenicity and on the number of salivary Streptococcus mutans and lactobacilli in orthodontic patients. Methods This RCT was conducted on 28 young adults who were undergoing orthodontic treatment. The short-term prospective clinical trial lasted for three weeks. The test group rinsed daily with drops containing two Lactobacillus reuteri strains diluted in water, while the placebo group used drops without probiotics. The subjects were enrolled eight months since the beginning of orthodontic treatment. Plaque-pH, saliva and dental biofilm samples were obtained at baseline, one week and three weeks post intervention. Results Twenty-seven subjects successfully completed the trial period, only one drop out in the test group. No side effects were reported. A statistically significant increase in plaque pH at three weeks post-intervention was found for the test group (p < 0.05), while insignificant changes in the pH value were found for the placebo group in comparison to baseline (p > 0.05). In addition, the AUC7.0 showed a significant difference at three weeks between the test and placebo (p = 0.00002). The three-week samples of stimulated whole saliva showed a statistically insignificant difference in the number of S. mutans and lactobacilli between the two groups (p > 0.05). The qPCR analysis showed the ability of the two strains to get colonized in the dental biofilm without a significant effect on the microbial counts. Conclusion/clinical implications A mixture of Lactobacillus reuteri has the ability to reduce the pH fall at the three-week follow-up. However, the short-term use of probiotics does not appear to have an effect on the number of salivary Streptococcus mutans and lactobacilli in saliva and on the dental biofilm. Trial registration Clinicaltrial.gov (Identifier: NCT04593017/ (19/10/2020)).

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