Probiotic Potential of Lactobacilli Isolated from Saliva of Periodontally Healthy Individuals

Purpose: To search for useful probiotics, we characterised antimicrobial lactic acid bacteria isolated from the oral cavities of 20 healthy volunteers from Nihon Univerisity School of Dentistry. Materials and Methods: Oral lactobacilli were isolated from the saliva of 20 periodontally healthy volunteers using the Dentocult LB dip-slide method. Primary antimicrobial screening of Lactobacillus isolates was performed using the paper disk method. Each suspected antimicrobial isolate was tested against Streptococcus mutans, S. sobrinus, and Porphyromonas gingivalis. Since P. gingivalis is considered as a keystone bacterium, we further analysed lactic acid bacteria that produced extracellular soluble antimicrobial agents that created an inhibitory zone of more than 12 mm against P. gingivalis. After two rounds of antimicrobial susceptibility testing, six isolates showing strong antibacterial effects against P. gingivalis were selected and identified as facultatively anaerobic, Gram-positive, non-spore-forming, non-capsule-forming, and catalase-negative bacilli. Selected isolates were identified to the species level through 16S rDNA sequence analyses. Results: The antimicrobial substance produced by the isolated bacilli was inactivated slightly after catalase treatment, had significantly lower activity at pH levels above 9.0, and was also reduced by heat treatment above 100°C and autoclaving. The activity of the antimicrobial substance showed some resistance to lower levels of heat, as well as pepsin and proteinase K treatments. 16S rRNA sequencing identified these isolates as Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus gasseri. Conclusion: These bacteria are antagonistic against potential periodontal pathogens and are good candidates for clinical application as probiotics. Keywords: antibacterial substance, lactobacilli, oral cavity