Comparative Antimicrobial and Antibiofilm Effects of Chlorhexidine, Lactobacillus reuteri Cell-free Supernatant, and Green Tea Extract against Major Periodontopathogens: An in vitro Study

Abstract

Background: Periodontal diseases are associated with dysbiotic biofilms, and adjunctive strategies to chlorhexidine (CHX) are being explored because repeated antiseptic use may be accompanied by undesirable effects. This controlled in vitro study compared 0.12% CHX, Lactobacillus reuteri cell-free supernatant (CFS), and catechin-rich green tea extract against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. Methods: Direct antimicrobial activity was assessed by agar diffusion and broth microdilution assays, including minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determination. Biofilm-related outcomes were evaluated by crystal violet measurement of biofilm-formation inhibition and viable-count reduction in 24-hour pre-formed biofilms after 60 minutes of exposure. Each condition included three technical replicates in three independent experimental runs. Quantitative data were analysed using two-way ANOVA followed by Tukey-adjusted pairwise comparisons. Results: CHX produced the largest inhibition zones, lowest MIC and MBC values, and greatest viable-count reductions in mature biofilms across all tested species. Green tea extract showed the strongest non-CHX anti-biofilm profile, inhibiting biofilm biomass by 67-72% and producing intermediate log10 CFU reductions of 1.8-2.2 in pre-formed biofilms. L. reuteri CFS demonstrated moderate, species-dependent activity, with the most evident response against P. gingivalis. The main effect of agent was significant for inhibition-zone and biofilm outcomes (p < 0.001), and post hoc testing confirmed the ordered pattern CHX > green tea extract > CFS > vehicle. Conclusions: Within this in vitro model, CHX remained the most potent comparator, whereas green tea extract and L. reuteri CFS showed adjunctive anti-biofilm activity. Further formulation, multispecies biofilm, and host-relevant studies are required before clinical extrapolation.