(53) Influence of Lactobacillus reuteri, Bifidobacterium animalis subsp. lactis, and prebiotic inulin on dysbiotic dental biofilm composition ex vivo

Introduction: Probiotic bacterial supplementation has shown promising results in enhancing periodontitis treatment and supporting periodontal health. However, oral health benefits associated with probiotics appear transient without repeated dosing. Prebiotic oligosaccharides may increase the sustainability of probiotic bacterial strains. The purpose of this investigation was to evaluate the influence of Lactobacillus reuteri and Bifidobacterium animalis subsp. lactis supplementation with and without prebiotic inulin using an ex vivo dental biofilm model.

Methods: Subgingival plaque specimens from three periodontitis patients were used to establish biofilms on hydroxyapatite disks under anaerobic conditions. Biofilms were cultured in a tryptic soy broth/hemin/menadione medium, with or without initial supplementation with varying dilutions of Lactobacillus reuteri or Bifidobacterium animalis subsp. lactis cultures and continuous supplementation with prebiotic inulin concentrations of 0, 5, or 20 mg/mL. Biofilms were grown to maturity over eight days and then used to establish subsequent generations. Ten generations (80 days of aggregate bacterial growth) were completed. Relative abundances of the bacterial genera present in each generation were evaluated using 16S rRNA next generation sequencing. Hierarchal multiple regression was utilized to compare relative abundances across experimental groups, and community diversity analysis was completed using QIIME 2.

Results: Inulin supplementation lead to statistically significant dose-dependent increases in the relative abundances of Lactobacillus and Bifidobacterium species (p < 0.001) and concomitant statistically significant decreases in the relative abundances of Streptococcus, Veillonella, Fusobacterium, Parvimonas, and Prevotella species (p < 0.001). Inoculation with L. reuteri or B. animalis subsp. lactis resulted in a statistically significant increase in the relative abundance of only the supplemented probiotic genera (p < 0.05), and introduction of probiotic bacteria did not significantly affect the relative abundance of any other genera assessed in this study. Supplemental inulin lead to a statistically significant decrease in the alpha diversity of the biofilms (p < 0.001). Community profiles of biofilms grown with or without inulin appeared indistinguishable by weighted UniFrac distance throughout the ten generations.

Conclusions: This ex vivo biofilm model appears suitable for investigating the effects of probiotic and prebiotic combinations. Initial inoculation of dysbiotic biofilms with probiotic L. reuteri or B. animalis subsp. lactis did not induce significant compositional changes within the microbiota. In contrast, inulin supplementation significantly influenced biofilm diversity favoring probiotic species, even in the absence of probiotic supplementation. Research investigating the potential correlation between periodontal health and the described prebiotic effects appears warranted.

Lead Author’s Contributions to the Research Project: The Lead Author was responsible for study conception and design, data collection, analysis and interpretation of the data, and the composition of the abstract.