Topic Application of the Probiotic Streptococcus dentisani Improves Clinical and Microbiological Parameters Associated With Oral Health

Abstract

Streptococcus dentisani 7746, isolated from dental plaque of caries-free individuals, has been shown to have several beneficial effects in vitro which could contribute to promote oral health, including an antimicrobial activity against oral pathogens by the production of bacteriocins and a pH buffering capacity through ammonia production. Previous work has shown that S. dentisani was able to colonize the oral cavity for 2-4 weeks after application. The aim of the present work was to evaluate its clinical efficacy by a randomized, double-blind, placebo-controlled parallel group study. Fifty nine volunteers were enrolled in the study and randomly assigned to a treatment or placebo group. The treatment consisted of a bucco-adhesive gel application (2.5 10⁹ cfu/dose) with a dental splint for 5 min every 48 h, for a period of 1 month (i.e., 14 doses). Dental plaque and saliva samples were collected at baseline, 15 and 30 days after first application, and 15 days after the end of treatment. At baseline, there was a significant correlation between S. dentisani levels and frequency of toothbrushing. Salivary flow, a major factor influencing oral health, was significantly higher in the probiotic group at day 15 compared with the placebo (4.4 and 3.4 ml/5 min, respectively). In the probiotic group, there was a decrease in the amount of dental plaque and in gingival inflammation, but no differences were observed in the placebo group. The probiotic group showed a significant increase in the levels of salivary ammonia and calcium. Finally, Illumina sequencing of plaque samples showed a beneficial shift in bacterial composition at day 30 relative to baseline, with a reduction of several cariogenic organisms and the key players in plaque formation, probably as a result of bacteriocins production. Only 58% of the participants in the probiotic group showed increased plaque levels of S. dentisani at day 30 and 71% by day 45, indicating that the benefits of S. dentisani application could be augmented by improving colonization efficiency. In conclusion, the application of S. dentisani 7746 improved several clinical and microbiological parameters associated with oral health, supporting its use as a probiotic to prevent tooth decay.

Keywords: bacteriocin; dental caries; dental plaque; probiotic; salivary flow.

Figure 1

Diagram of clinical study experimental protocol. In this double-blind study, participants were randomly assigned to one of the two treatment groups (placebo or probiotic). On the first visit (Day 0), treatment was applied by the dentist and participants were explained how to perform the 14 successive applications at home every 48 h during a month. At each of the visits (Day 0, Day 15, Day 30, and Day 45), several clinical parameters were measured (plaque and gingival index, and salivary flow), and plaque and saliva samples were collected to determine the acidogenicity and buffer capacity of biofilm and saliva. All samples at day 0 were collected before any administration of the probiotic or placebo and therefore S. dentisani levels at day 0 correspond to the endogenous levels of this organism in the participants. The image on the right shows bucco-adhesive product administration. Lyophilized S. dentisani cells (with a total dose of 2.5 × 10⁹ CFUs) mixed with thickener and adherence excipients were resuspended with 6 mL of water, and resultant gel was transferred to the individualized mouth splint using a syringe. The placebo group product had the same excipients but no probiotic.

Figure 2

Clinical parameters. Data show average values of (A) plaque index (PI), (B) gingival index (GI), and (C) salivary flow in each group (placebo in black and probiotic group in gray) measured at the different visits. Bars, from left to right, represent mean values (SEM) at: V0, baseline visit; V15, 15 days of treatment; V30, end of treatment; and V45, 15 days after the end of treatment. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

Shifts in the relative abundance in the most abundant bacteria in dental plaque. (A–C) Boxplots show levels of different bacterial species in the probiotic group before and after treatment (probiotic V0 vs. V30). Lines join the samples before and after the treatment period (paired samples). (D–F) Levels of different bacterial species in placebo and probiotic groups at the end of treatment (placebo V30 vs. probiotic V30). The relative abundance values of Fusobacterium nucleatum shown in (B) correspond to ASV 0216 and (E) to the reads of ASV 0090, respectively, both 99% identical to F. nucleatum type strain. The results of Wilcox paired test are also shown.

Figure 4

Calcium and ammonium salivary levels. Boxplots show unstimulated saliva concentration (mg/L) of (A) calcium and (B) ammonium in the probiotic group between baseline levels (Probiotic V0) and the end of treatment (probiotic V30). Wilcox test results for the comparison are also shown.

Figure 5

Salivary and plaque pH. Bars show mean values of (A) basal saliva pH (16–18 h. after last tooth-brushing), (B) salivary pH after plaque removal and before a sugar rinse, and (C) salivary pH 10 min after a sugar rinse, measured in unstimulated saliva samples from all patients in the probiotic (gray columns) and placebo (black) groups, at the four study visits (V0, V15, V30, and V45). (D) Mean values of plaque pH measured ex vivo 10 min after a 10% sugar pulse. Columns from left to right represent mean values (SD) at: V0, baseline visit; V15, 15 days during treatment; V30, end of treatment; and V45, 15 days after the end of treatment. *P < 0.05.

Figure 6

Streptococcus dentisani salivary levels. qPCR quantification of S. dentisani in saliva (number of bacterial cells/ml) throughout the study. Columns from left to right represent mean values (SEM) at: V0, baseline visit; V15, 15 days during treatment; V30, end of treatment; and V45, 15 days after the end of treatment in each study group (placebo in black and probiotic group in gray). *P < 0.05 and **P < 0.01.