Effects of probiotic bacterium Weissella cibaria CMU on periodontal health and microbiota: a randomised, double-blind, placebo-controlled trial

This study was conducted in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. Approval for the study was obtained from the Kangwon National University (KNU) Institutional Review Board (KWNUIRB-2018-05-003-005, Chuncheon, Korea). The purpose and procedure of the study were explained to all participants. Participants were also informed that refusal to participate would not disadvantage them in any way, and they were free to withdraw from the study at any time. Written informed consent was obtained from all participants prior to enrolment.

Participants were recruited through an offline poster and an online public notice using social network services aimed at undergraduate students, graduate students, and school personnel at KNU (Chuncheon, Korea). The sample size was calculated using the G * Power 3.1 programme. The number of participants needed for the independent t-test with significance level α = 0.05 bilateral test, power = 0.8, and effect size = 0.7 was 68. The initial sample size was planned at 96, considering a dropout rate of 40%; 100 participants were enrolled in the current study. The effect size was set to medium or high based on previous studies that reported the effects of W. cibaria CMU administration. The dropout rate was set at high as the subjects were college students or working adults. Random allocation sequence for test group or placebo group was generated via Microsoft Excel [18] by a research assistant not participated in this study intervention. The formula was following: If (Rank (B2, $B$2: $B$101) > 50, 1, 0). Sequential numbered opaque sealed envelopes was used until assignment and opened sequentially at screening. Other 3 research assistants enrolled and assigned participants to intervention and there was not any restriction in random allocation. A total of 100 subjects were screened, and 92 were randomly assigned to the probiotic test group (n = 49) or placebo control group (n = 43), after excluding eight subjects who did not meet the inclusion criteria or refused to participate during the two-week run-in period. Twenty-four additional subjects were excluded from the eight-week intervention phase, and data were finally analysed for 68 subjects. The inclusion criteria were as follows: subjects who were able to comply with the protocol, over 20 years of age with more than 20 natural teeth, no tongue problems, no gum diseases, and with oral VSC concentration of 1.5 ng/10 mL or more. The exclusion criteria were as follows: subjects who had received antibiotic treatment within the previous month; those currently visiting their dentists for treatment; with adverse reactions to lactose or fermented milk products; consistently using probiotic supplements; with a dry mouth; with systemic diseases that would cause halitosis; who could not see or hear sufficiently; with mental illness; and those who had participated in another clinical trial within the previous month.

The 800 mg probiotic tablet contained 1.0 × 10⁸ colony forming units (CFU)/g of W. cibaria CMU (oraCMU) provided by OraPharm Inc. (Seoul, Korea). Other ingredients included isomalt, sucralose, peppermint flavour, maltodextrin, and magnesium stearate. The placebo was a tablet from the same manufacturer with the same taste, texture, and appearance lacking oraCMU. Subjects were instructed to melt and suck one tablet in their mouth every night before bedtime after brushing their teeth. Water or food was prohibited after the treatment. The study treatment was conducted for 8 weeks.

A randomised double-blind, placebo-controlled trial was performed. To secure the homogeneity of the oral condition of the subjects, they were required to visit M Dental Clinic in Chuncheon 1 week prior to starting the test treatment, received an oral examination by a dentist, and underwent dental scaling and root planing (SRP). After SRP, they had a recovery period of 1 week for the regeneration of the gums. One week after the recovery period, the probiotic tablet was administered to the experimental group, and a placebo tablet of the same shape was administered to the control group. The typical maxillary and mandibular teeth, including the maxillary right first molar (#16), maxillary left central incisor (#21), maxillary left first premolar (#24), mandibular left first molar (# 36), mandibular right central incisor (#41), and mandibular right first premolar (#44) were selected for clinical examination. Clinical examination including probing depth (PD), bleeding on probing (BOP), plaque index (PI), and gingival index (GI) was performed at three visits (baseline, at four, and 8 weeks). The measurement of bacteria in subgingival plaque was also performed in the three visits.

During the experimental period, all subjects were provided with the same type of toothpaste and toothbrush and were instructed to use them throughout the study. All the interventions were performed in double-blind state: participants were distinguished by only registration number, intervention providers (research assistants) neither know who was test group or placebo group as well. The study was conducted in the period of July to November 2018.

PD was used to evaluate the probing pocket depth by measuring the distance from the marginal gingiva to the epithelial attachment region using a probe, depending on the observed changes of the gingival sulcus [19].

The criteria used for GI evaluation were applied to teeth and tooth surfaces to assess BOP. The presence of bleeding from the base of the gingival sulcus was denoted as (+), and its absence was denoted as (−). The incidence of bleeding (%BOP) was calculated [20].

Using Loe and Silness PI technique [21], each tooth surface was coloured with a red colourant, and the tooth surface was divided into two parts (occlusal and gingival surfaces) to measure plaque accumulation and thickness of the gingival margin. The evaluation criteria were: 0 = no plaque; 1 = thinly attached to the gingival margin and apparent after lightly scraping with a probe or applying a tooth colourant; 2 = moderate plaque that can be visually recognised along the gingival margin; and 3 = thick plaque accumulation in the gingival pockets, as well as gingival margin and tooth surface. The total PI score per subject was calculated with the average value of each tooth surface.

GI was evaluated at the proximal, distal, buccal, and lingual sites and each site was assigned 0–3 points [22]: 0 = healthy gingiva; 1 = gingivitis with a slight colour change and slight swelling, but without bleeding by mild irritation; 2 = gingivitis with redness, swelling, and bleeding by mild irritation; and 3 = advanced inflammation with marked redness and swelling and the possibility of ulceration and natural bleeding. The total PI score per subject was calculated with the average value of each tooth surface.

#15 paper points were inserted to the gingival sulcus of four sites of two maxillary teeth (anterior and posterior) and two mandibular teeth (anterior and posterior) of subjects with PD less than 4 mm for 10 s and were then placed in a 1.5 mL tube. They were immediately stored at − 20 °C until just before analysis. DNA was extracted from the collected paper points using the AccuPrep Universal RNA Extraction Kit (Bioneer, Daejeon, Korea). The extraction was performed according to the manufacturer’s instructions. OligoMix (YD Global Life Science Co., Ltd., Seongnam, Korea) and three oligonucleotides (forward primer, reverse primer, and probe) (Table 1) that react specifically to each bacterium were used [23]. To prepare the polymerase chain reaction (PCR) reaction sample, 9 μL of OligoMix, 10 μL of 2x probe qPCR mix (Takara Bio Inc., Shiga, Japan), and 1 μL of template DNA were combined. A 96-well plate with the PCR reaction sample was placed in the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, USA) to amplify the DNA. The conditions of PCR were as follows: initial denaturation at 95 °C for 30 s, denaturation at 95 °C for 10 s, and annealing for 30 s at 62 °C with 40 repeated cycles. The cycle threshold (Ct) value was calculated using the Bio-Rad CFX Manager Software program, and the number of copies was derived by plotting the Ct value in the standard curve of each bacterium.

All study results were evaluated according to the per protocol analysis. All data were analysed using SPSS 21.0 for Windows (IBM Corp., Armonk, USA). An independent t-test, x² test, and Fisher’s exact test were used for confirmation of homogeneity between the two groups at baseline. The Kolmogorov-Smirnov test and the Shapiro-Wilk test were used to check the normality of the data. For the clinical variables and the concentrations of bacteria, an independent t-test allowed a comparison between probiotic and placebo groups. Otherwise, the Mann-Whitney U test was used. A value of P ≤ 0.05 was considered statistically significant.

CONSORT flow diagram of this study is shown in Fig. 1. The baseline characteristics of the study subjects are shown in Table 2. No significant differences were observed between the groups (P > 0.05).

As shown in Table 3, both groups had similar mean BOP at baseline. Treatment resulted in a significant reduction in BOP from baseline (P < 0.05). There was a statistically significant difference in the BOP mean values between groups in the maxillary buccal site at week 8 and in the maxillary lingual site at week 4 (P < 0.05). However, there were no statistically significant inter-group differences in BOP reduction as well as the mean BOP in all the observed teeth. As shown in Fig. 2, no significant inter-group differences were observed at baseline, 4 weeks, and 8 weeks for PD, GI, and PI (P > 0.05).

The Gram-negative and -positive oral bacterial data in subgingival plaques are presented in Tables 4 and 5, respectively. As shown in Table 4, similar findings on Gram-negative oral bacteria were observed in both groups at baseline (P > 0.05). After four and 8 weeks, significant differences in the number of F. nucleatum were observed in the groups (P < 0.05). Most Gram-negative oral bacteria decreased in the probiotic group at four and 8 weeks. Among the Gram-positive oral bacteria, Actinomyces viscosus, and a group of Streptococcus (GS) were higher in the probiotic group than those in the control group (P < 0.05). However, GS significantly decreased between baseline and 4 weeks in the probiotic group (P < 0.05), while there was a significant increase in A. viscosus in the placebo group (P < 0.05). Both bacteria showed statistically significant differences among the groups over 4 weeks (P < 0.05). The number of Staphylococcus aureus in the placebo group significantly increased compared to the baseline at week 8, and this was significantly higher than that of the probiotic group (P < 0.05; Table 5).