Oral Microbiota Shift After 12-week Supplementation With Lactobacillus Reuteri DSM 17938 and PTA 5289

Ethics statements The study was approved by the Regional Ethical Review Board in Umeå, Sweden (Dnr 2011-380-31M) and was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants.

Subjects and study design Healthy adult volunteers, aged 20-66 years, among students and employees at the Faculty of Medicine, Umeå University, Sweden were recruited to a double-blind, randomized controlled trial (RCT) through advertisements. Inclusion criteria were a self-reported healthy status and no use of antibiotics or probiotic products during 3 months prior to the study. Based on previous studies regarding the persistence of probiotic strains [31,32], the recruitment goal was at least 15 people per study group.

Forty-four volunteers, none of whom used tobacco products, were recruited and randomly allocated to either a test (n = 22) or placebo group (n = 22; Figure S1). Participants were asked to allow 2 lozenges per day to slowly melt in the mouth and to circulate the dissolved tablet contents around their mouths. One lozenge was taken in the morning and 1 in the evening for 12 weeks. The test lozenges contained L. reuteri (DSM 17938 and PTA 5289; 108 CFU per strain; BioGaia AB, Stockholm, Sweden), isomalt, hydrogenated palm oil, peppermint and menthol flavoring, peppermint oil, and sucralose (http://www.biogaia.com/product/biogaia-prodentis-oral-lozenges). The placebo lozenges were identical to the test lozenges in appearance, taste, and composition except the lactobacilli. Compliance was monitored as the percentage of lozenges consumed of the total assigned number. The remaining lozenges were counted when the containers were returned for monthly refills to assess this factor. Compliance was considered acceptable if ≤15% of the lozenges remained. Participants were asked to abstain from oral hygiene for 48 h and to not consume any food for at least 4 h before sampling. Participants were also instructed to not eat probiotic products throughout the study period.

Saliva and biofilm sampling Saliva and tooth biofilm samples were obtained immediately before (baseline) and after 4, 8, and 12 weeks of L. reuteri supplementation (Figure S1). Follow-up samples were collected 1 and 6 months after supplementation was terminated. Furthermore, whole stimulated saliva (~5 mL) was generated by chewing 1 g of paraffin and collected into ice-chilled sterile test tubes. One milliliter of saliva was used for cultural analysis, and the remaining saliva was centrifuged at 3,500 × g for 10 min at 4°C. The pellets were stored at -80°C until DNA extraction for strain-specific PCR reactions. For the pyrosequencing analysis, pooled supragingival plaque was collected with sterilized toothpicks and transferred to Eppendorf tubes (Sarstedt, Nümbrecht, Germany) containing 200 µL of TE-buffer (10 mM Tris, 1 mM EDTA, pH 7.6). The samples were stored at -80°C until DNA extraction.

Identification of lactobacilli by culture and PCR Aliquots of saliva were plated onto Rogosa agar (Merck, Darmstadt, Germany) to obtain Lactobacillus counts and on selective agar for tentative identification of the L. reuteri (DSM 17938 and PTA 5289) strains [33]. All plates were anaerobically incubated at 37°C for 48-72 h, except L. reuteri PTA 5289, which was anaerobically incubated at 40°C for 72 h.