Postbiotic and Phytochemical Modulation of Multispecies Periodontal Biofilms: Comparative Effects on Biofilm Viability and Porphyromonas Gingivalis Virulence

Abstract

Background: Periodontal diseases are associated with complex multispecies biofilms in which Porphyromonas gingivalis acts as a key virulence-associated pathogen contributing to inflammation and tissue destruction. Owing to the limitations of conventional antimicrobials, postbiotics and phytochemicals are emerging as promising alternative strategies for reducing biofilm viability and attenuating bacterial virulence. Purpose: To compare the antibiofilm and antivirulence effects of 0.12% chlorhexidine, Lactobacillus reuteri cell-free supernatant, green tea extract, and their combination against a multispecies periodontal biofilm model. Materials and Methods: Artificial saliva-conditioned hydroxyapatite discs were used to establish a sequential multispecies biofilm containing Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis. Biofilms were allocated to five groups: untreated control, 0.12% chlorhexidine, L. reuteri cell-free supernatant, green tea extract, and combined L. reuteri cell-free supernatant plus green tea extract. Metabolic activity, total biomass, viable bacterial proportion, relative P. gingivalis load, and gingipain activity were quantified after standardized exposure. Data from three independent biological experiments with three technical replicates per condition were analyzed using one-way ANOVA and Tukey post hoc tests. Results: All active treatments significantly reduced biofilm-related outcomes compared with the untreated control group (P<0.001). Chlorhexidine showed the strongest bactericidal profile, reducing metabolic activity to 24.60 +/- 4.90% and viable bacterial proportion to 28.50 +/- 5.20%. The combined postbiotic-phytochemical treatment showed broad inhibition, reducing total biomass to 42.50 +/- 6.90%, relative P. gingivalis load to 33.10 +/- 6.30%, and gingipain activity to 28.40 +/- 5.50%. Green tea extract showed a marked antivirulence-dominant effect on gingipain activity. Conclusion: Chlorhexidine provided superior short-term bactericidal activity, whereas the combined postbiotic-phytochemical approach showed relevant multi-endpoint antibiofilm and antivirulence effects in this in vitro model. These findings support further mechanistic and translational evaluation of postbiotic-phytochemical adjuncts for periodontal biofilm modulation.