Comparative evaluation of 0.12% chlorhexidine, Lactobacillus reuteri cell- free supernatant, and green tea extract against major periodontopathogens: a multi-endpoint in vitro research

Background Periodontal diseases are chronic inflammatory disorders driven by dysbiotic biofilms and host-microbial interactions. Although chlorhexidine (CHX) remains the benchmark antiseptic in periodontal practice, its adverse effects and lack of ecological selectivity have encouraged interest in probiotic-derived metabolites and plant polyphenols as alternative adjuncts. This study compared the antimicrobial, anti-biofilm, and bactericidal performance of 0.12% CHX, Lactobacillus reuteri cell-free supernatant (CFS), and a catechin-rich green tea extract against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. Methods A controlled in vitro design was used with four groups: 0.12% CHX, L. reuteri-derived CFS, green tea extract, and vehicle control. Direct antimicrobial activity was examined by agar diffusion and broth microdilution assays. Inhibition of biofilm development was quantified using crystal violet staining, and bactericidal action against mature biofilms was assessed by viable colony counting after validated neutralization and washing steps. All assays were performed in triplicate across three independent experimental runs. Data were summarized as mean ± standard deviation and analyzed by two-way ANOVA with Tukey-adjusted pairwise comparisons. Effect sizes (partial eta squared) and 95% confidence intervals were calculated. Results CHX produced the largest inhibition zones for all three species and demonstrated the lowest minimum inhibitory concentration and minimum bactericidal concentration values within the tested range. Green tea extract consistently showed stronger inhibition of biofilm development than CFS, with mean biomass inhibition ranging from 67% to 72% versus 56% to 61%, respectively. In mature biofilms, CHX achieved approximately 3-log reductions in recoverable bacteria, while green tea extract showed intermediate activity and CFS demonstrated selective moderate effects, particularly against P. gingivalis. The main effect of agent was highly significant for both inhibition-zone and biofilm outcomes (p < 0.001). Conclusions Within the limits of this in vitro study, CHX remained the most potent comparator. Green tea extract showed the strongest non-CHX anti-biofilm profile, while L. reuteri CFS demonstrated selective moderate activity. These findings support continued investigation of ecological adjuncts for periodontal biofilm control rather than direct replacement strategies for CHX.