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

Comparative evaluation of 0.12% chlorhexidine, Lactobacillus reuteri cell- free supernatant, and green tea extract against major periodontopathogens: a multi-endpoint in vitro research | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparative evaluation of 0.12% chlorhexidine, Lactobacillus reuteri cell- free supernatant, and green tea extract against major periodontopathogens: a multi-endpoint in vitro research Mehmet Murat Taskan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9355651/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract 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. Biological sciences/Biotechnology Biological sciences/Microbiology chlorhexidine Lactobacillus reuteri green tea extract Porphyromonas gingivalis Aggregatibacter actinomycetemcomitans Fusobacterium nucleatum Figures Figure 1 Introduction Periodontal diseases are among the most prevalent chronic inflammatory conditions in humans and arise from a complex interaction between a dysbiotic microbial biofilm and a susceptible host response [ 1 – 4 ]. Contemporary periodontal biology no longer frames disease as a simple infection caused by a single pathogen. Instead, current concepts emphasize ecological imbalance, altered interspecies interactions, and dysregulated host-microbe homeostasis within the subgingival environment [ 2 – 7 ]. Within this study, Porphyromonas gingivalis is frequently cited as a keystone pathogen because it can modulate host defense pathways, reshape the community structure of the biofilm, and amplify pathogenicity out of proportion to its abundance [ 6 , 7 ]. Aggregatibacter actinomycetemcomitans remains clinically relevant due to its historical association with aggressive forms of periodontal destruction and its array of leukotoxic and invasive virulence traits [ 8 , 9 ]. Fusobacterium nucleatum serves as an important bridging organism that facilitates co-aggregation and maturation of multispecies biofilms, thereby increasing ecological complexity and resilience [ 10 , 11 ]. The use of these organisms together therefore provides a biologically coherent model for screening candidate adjunctive agents in periodontology. Chlorhexidine gluconate remains the best established antiseptic comparator in clinical dentistry because of its broad antimicrobial spectrum and substantivity [ 12 – 16 ]. Nevertheless, its long-term or repeated use is limited by adverse effects such as taste alteration, tooth and tongue staining, calculus promotion, and mucosal irritation [ 12 , 14 – 16 ]. There is also increasing conceptual concern that broad-spectrum suppression may not represent the most desirable long-term ecological strategy in a microbiome-driven disease. These limitations have encouraged investigation of adjuncts that can attenuate pathogenic biofilm behavior while potentially exerting less collateral disruption. Probiotic-based approaches have received substantial attention in periodontal research, particularly those involving Lactobacillus reuteri [ 17 – 24 ]. Clinical trials and systematic reviews suggest that probiotic adjuncts may improve periodontal parameters when combined with conventional therapy, although findings remain heterogeneous [ 17 – 20 , 22 , 24 ]. Proposed mechanisms include competitive exclusion, production of antimicrobial metabolites such as reuterin, pH modification, signaling interference, and immune modulation [ 21 , 23 ]. However, the biological activity of a cell-free supernatant must be distinguished from that of a live probiotic formulation. A supernatant acts through soluble metabolites present at the time of administration and lacks active colonization, metabolic renewal, and interspecies competition after delivery. Plant-derived polyphenols represent a second major ecological strategy of interest. Green tea contains catechins, especially epigallocatechin gallate, epicatechin gallate, and epigallocatechin, which have been associated with antimicrobial, anti-adhesive, anti-inflammatory, and anti-biofilm effects in oral models [ 25 – 31 ]. In relation to periodontal pathogens, catechins may reduce adhesion, influence extracellular matrix development, attenuate virulence-associated proteolytic activity, and increase susceptibility to conventional antimicrobials [ 25 – 28 , 31 ]. Importantly, the therapeutic appeal of such compounds may lie less in absolute bactericidal potency and more in biofilm modulation. Many laboratory studies assess candidate agents using only a single antimicrobial assay, which can oversimplify interpretation. Agar diffusion depends in part on molecular diffusion through the medium, broth microdilution reflects planktonic growth suppression, and biofilm assays capture a different dimension of microbial behavior altogether. A multi-endpoint design provides a more balanced appraisal by distinguishing direct growth inhibition from interference with biofilm formation and from killing within established biofilms. Accordingly, the aim of the present study was to compare 0.12% CHX, L. reuteri-derived cell-free supernatant, and a catechin-rich green tea extract in a in vitro design incorporating direct antimicrobial, anti-biofilm, and mature-biofilm viability outcomes against P. gingivalis, A. actinomycetemcomitans, and F. nucleatum. The null hypothesis was that no significant difference would be observed among the tested agents across the evaluated endpoints. Materials and Methods Study design This study was designed as a controlled, comparative in vitro experiment with four groups: 0.12% CHX, L. reuteri-derived CFS, standardized green tea extract, and vehicle control. Four outcome domains were assessed: agar diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), inhibition of biofilm development, and viable-count reduction in pre-formed biofilms. Each assay was performed in technical triplicate and repeated in three independent experimental runs (total n = 9 observations per condition for descriptive analysis). Microorganisms and culture conditions Reference strains were selected to represent functionally distinct components of the periodontal biofilm: P. gingivalis ATCC 33277, A. actinomycetemcomitans ATCC 29523, and F. nucleatum ATCC 25586. P. gingivalis and F. nucleatum were cultured anaerobically at 37 C in an atmosphere of approximately 85% N 2 , 10% H 2 , and 5% CO 2 . A. actinomycetemcomitans was maintained under capnophilic conditions. Organisms were revived from stock cultures, subcultured on strain-appropriate agar media, and transferred into broth for preparation of standardized inocula. Before testing, suspensions were adjusted to 0.5 McFarland standard, corresponding approximately to 1 x 10^8 CFU/mL, and then diluted according to assay requirements. ( Table 1 ) Table 1 Microorganisms and principal culture conditions used in the experiment. Species Reference strain Atmosphere Primary role in periodontal biofilm model Porphyromonas gingivalis ATCC 33277 Anaerobic Keystone pathogen and dysbiosis amplifier Aggregatibacter actinomycetemcomitans ATCC 29523 Capnophilic Aggressive-periodontitis-associated pathogen Fusobacterium nucleatum ATCC 25586 Anaerobic Bridging organism in multispecies biofilm maturation Preparation and standardization of test agents The CHX group consisted of a commercially available 0.12% chlorhexidine gluconate solution used as supplied. For the probiotic-metabolite group, L. reuteri was grown in de Man, Rogosa and Sharpe broth for 24 hours at 37 C. Cultures were centrifuged at 4000 rpm for 10 minutes, and the supernatant was sterilized through a 0.22-micrometer membrane filter to remove intact bacterial cells. The resulting CFS was standardized before use by pH adjustment to 6.5 ± 0.2 and normalization of optical density. Protein content was estimated by a Bradford-based assay to improve batch consistency. Although detailed metabolomic profiling was beyond the scope of the present study, these standardization steps were included to reduce variability between batches. The green tea group consisted of a catechin-rich extract prepared in sterile distilled water. The extract was selected to reflect a polyphenol-rich composition and was standardized according to supplier data for high catechin content. Serial two-fold dilutions were prepared freshly for MIC and MBC testing. The vehicle control contained the corresponding solvent system without active ingredient. ( Table 2 ) Table 2 Standardization characteristics of the tested agents. Agent Preparation Standardization / notes 0.12% chlorhexidine Commercial mouthrinse-grade solution Used as supplied; reference antiseptic comparator L. reuteri CFS 24-h broth culture, centrifuged and 0.22-micrometer filtered pH adjusted to 6.5 ± 0.2; optical density normalized; protein estimate recorded Green tea extract Catechin-rich extract in sterile distilled water Freshly prepared; serial dilutions used for MIC and MBC testing Vehicle control Corresponding solvent system without active ingredient Included in all assays Agar diffusion assay Uniform bacterial lawns were created on species-appropriate agar surfaces using standardized inocula. Wells with equal diameter (6 mm) were created aseptically, and 50 microliters of each test solution were dispensed into each well. Plates were incubated under the relevant atmospheric conditions for each species. Following incubation, inhibition zones were measured in millimeters using a digital caliper by two independent blinded observers. The mean of the two readings was used for analysis. Vehicle control was expected to yield no measurable inhibition. MIC and MBC determination Broth microdilution testing was performed in sterile 96-well microplates using two-fold serial dilutions of each agent. MIC was defined as the lowest concentration demonstrating no visible turbidity after incubation under species-specific conditions. For MBC determination, aliquots from non-turbid wells were plated onto agar and incubated. The lowest concentration yielding no colony growth was recorded as the MBC. When a bactericidal threshold was not reached within the tested range, the result was recorded as not reached (NR). Biofilm inhibition assay To evaluate inhibition of early biofilm development, bacterial suspensions were seeded into polystyrene microplates and exposed to test agents during the initial adhesion and growth phase. After 24 hours of incubation, wells were gently washed with phosphate-buffered saline to remove non-adherent cells, fixed with methanol, stained with 0.1% crystal violet, and destained before optical density measurement at 570 nm. The percentage inhibition of biofilm biomass was calculated relative to untreated control wells using the formula: ((OD_control - OD_test) / OD_control) x 100. Viability assay in pre-formed biofilms For mature-biofilm testing, biofilms were allowed to establish for 24 hours before exposure. After maturation, wells were treated with the respective agents for a standardized contact period of 60 minutes. Residual test solution was then removed, and a validated washing and neutralization sequence was applied to minimize antimicrobial carryover. Neutralization solution consisted of a lecithin and Tween 80-containing buffer used in line with common antiseptic-neutralization practice. Neutralization efficacy was verified in a control procedure confirming that residual antimicrobial activity did not distort post-treatment colony counts. Biofilms were then mechanically disrupted, serially diluted, plated, and incubated. Outcomes were expressed as log10 CFU reduction relative to untreated controls. Controls, quality assurance, and blinding Vehicle controls were included in all assays. Neutralization controls were incorporated in the mature-biofilm experiment to verify that differences in colony counts reflected treatment effects rather than residual carryover activity. Measurements of inhibition zones and spectrophotometric outputs were performed by investigator who was blinded to agent identity at the time of reading. (M.M.T.) Media sterility and growth controls were included in each run, and all experiments were repeated independently three times to improve internal consistency. Statistical analysis All quantitative results were expressed as mean ± standard deviation. The primary inferential framework used two-way analysis of variance with agent and species as fixed factors. Where relevant, Tukey-adjusted pairwise comparisons were applied to compare group means while controlling for multiple testing. Assumptions were checked using Shapiro-Wilk testing for normality of residuals and Levene testing for homogeneity of variance. In addition to p values, effect sizes were expressed as partial eta squared (partial eta^2), and 95% confidence intervals were calculated for selected contrasts. Statistical significance was set at p < 0.05. Results Vehicle control yielded no inhibition zone, no relevant inhibition of biofilm formation, and no measurable bactericidal reduction in the mature-biofilm assay. Because the control findings were consistently negligible, the principal emphasis below is on the comparative performance of the three active interventions. Agar diffusion findings CHX produced the largest inhibition zones against all three organisms. The highest mean zone was observed against P. gingivalis, followed by F. nucleatum and A. actinomycetemcomitans. Green tea extract showed a clear but smaller inhibitory halo in all species, while CFS displayed modest inhibition that remained consistently greater against P. gingivalis than against the other two species. The ranking CHX > green tea extract > CFS was preserved across species. ( Table 3 , Fig. 1 ) Table 3 Mean inhibition zone diameters in the agar diffusion assay (mm). Agent P. gingivalis A. actinomycetemcomitans F. nucleatum 0.12% CHX 23.1 ± 1.1 19.8 ± 1.5 21.4 ± 1.3 L. reuteri CFS 12.8 ± 0.9 10.9 ± 1.0 11.8 ± 1.1 Green tea extract 16.9 ± 1.2 14.6 ± 1.3 15.8 ± 1.2 Vehicle 0 0 0 MIC and MBC findings Broth microdilution results reinforced the superiority of CHX as the direct antimicrobial comparator. CHX achieved the lowest MIC and MBC values for all three species. Green tea extract showed measurable inhibitory and bactericidal thresholds but required substantially higher concentrations than CHX. CFS displayed selective activity, with better inhibitory and bactericidal performance against P. gingivalis than against A. actinomycetemcomitans and F. nucleatum. In the latter organisms, bactericidal thresholds were more difficult to achieve within the tested concentration range. ( Table 4 ) Table 4 MIC and MBC findings for the tested agents. MIC and MBC values are given as percent concentration equivalents within the tested range. NR, not reached within tested range. Agent Outcome P. gingivalis A. actinomycetemcomitans F. nucleatum 0.12% CHX MIC 0.0005 0.0010 0.0005 0.12% CHX MBC 0.0010 0.0020 0.0010 L. reuteri CFS MIC 12.5 25.0 25.0 L. reuteri CFS MBC 25.0 NR 50.0 Green tea extract MIC 1.56 3.12 3.12 Green tea extract MBC 3.12 6.25 6.25 Inhibition of biofilm development When agents were present during early biofilm formation, CHX again produced the strongest suppression of biomass accumulation, with all means exceeding 80% inhibition. Notably, green tea extract consistently outperformed CFS in all species. The magnitude of the green tea advantage over CFS ranged from approximately 10 to 12 percentage points, suggesting that catechin-rich preparations may be especially relevant for modulating the developmental phase of periodontal biofilms. ( Table 5 ) Table 5 Percentage inhibition of biofilm formation relative to untreated control (mean ± SD). Agent P. gingivalis A. actinomycetemcomitans F. nucleatum 0.12% CHX 84 ± 4 81 ± 5 83 ± 4 L. reuteri CFS 61 ± 5 56 ± 6 58 ± 5 Green tea extract 72 ± 4 67 ± 5 69 ± 4 Vehicle 3 ± 2 2 ± 2 2 ± 1 Viability in pre-formed biofilms The mature-biofilm assay revealed the expected attenuation of antimicrobial effect compared with planktonic testing, yet meaningful differences remained. CHX produced approximately 3-log reductions across all species, reflecting substantial bactericidal action despite the protective architecture of established biofilm. Green tea extract generated intermediate reductions, generally between 1.8 and 2.2 log10 CFU. CFS showed lower but measurable activity, again with the largest response observed against P. gingivalis. Neutralization controls confirmed that these viable-count results were not attributable to residual antiseptic carryover. ( Table 6 ) Table 6 Log10 CFU reduction in pre-formed biofilms relative to untreated control (mean ± SD). Agent P. gingivalis A. actinomycetemcomitans F. nucleatum 0.12% CHX 3.1 ± 0.3 2.8 ± 0.4 3.0 ± 0.3 L. reuteri CFS 1.5 ± 0.2 1.0 ± 0.2 1.2 ± 0.3 Green tea extract 2.2 ± 0.3 1.8 ± 0.3 2.0 ± 0.3 Vehicle 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 Inferential analysis For inhibition-zone outcomes, two-way ANOVA showed a highly significant main effect of agent (p < 0.001), a smaller but significant main effect of species (p = 0.011), and a significant agent-species interaction (p = 0.018). For inhibition of biofilm development, the main effect of agent remained highly significant (p green tea extract > CFS > vehicle. The difference between green tea extract and CFS was statistically significant for the biofilm endpoint (p = 0.032). Effect sizes indicated that the choice of agent explained a substantial proportion of the observed variance. (Table 7 ) These findings should be interpreted within the constraints of this controlled in vitro model. Table 7 Summary of inferential analysis for key endpoints. Endpoint Source F value p value Partial eta^2 Interpretation Agar diffusion Agent 148.6 < 0.001 0.82 Very large agent effect Agar diffusion Species 5.4 0.011 0.19 Small to moderate species effect Agar diffusion Agent × species 3.2 0.018 0.14 Significant interaction Biofilm inhibition Agent 126.9 < 0.001 0.79 Very large agent effect Biofilm inhibition Species 3.7 0.037 0.12 Modest species effect Biofilm inhibition Agent × species 2.4 0.061 0.09 Trend without strong interaction Discussion The present study compared three conceptually different antimicrobial strategies for periodontal biofilm control: a conventional broad-spectrum antiseptic, a probiotic-derived metabolite preparation, and a plant polyphenol-rich extract. Across all principal endpoints, 0.12% CHX remained the most potent comparator. This overall pattern is consistent with its longstanding role as the benchmark antiplaque and antiseptic agent in periodontal care [ 12 – 16 , 32 ]. However, the present data also underscore that antimicrobial superiority does not answer the entire translational question. Biofilm-related periodontal therapy is not only about maximal killing; it is also about how a compound modulates adhesion, maturation, ecological structure, and the balance between suppression and selectivity. The green tea extract findings were particularly notable because this agent did not approach CHX in direct planktonic potency but showed the strongest non-CHX performance in the anti-biofilm assay. This observation is biologically plausible and is in line with prior reports describing catechin-mediated interference with P. gingivalis biofilm formation, adherence, and virulence behavior [ 25 – 31 , 33 ]. Catechins, especially epigallocatechin gallate, may alter membrane function, reduce extracellular matrix development, attenuate proteolytic activity, and influence the expression of virulence-associated genes. In practical periodontal terms, this suggests that green tea-derived preparations may hold more promise as ecological biofilm modulators than as direct replacements for high-potency antiseptics. The CFS preparation displayed moderate and species-dependent effects, again with the most favorable response observed against P. gingivalis. This selective pattern may reflect differential sensitivity of target organisms to soluble metabolites generated by L. reuteri culture supernatants. Previous probiotic literature has emphasized mechanisms such as reuterin production, competition for nutrients and adhesion sites, pH changes, and immune signaling modulation [ 17 – 24 , 34 , 35 ]. Yet the distinction between a live probiotic product and a cell-free metabolite fraction should be emphasized. A live organism can potentially colonize transiently, dynamically produce bioactive compounds, and participate in interspecies competition, whereas a supernatant represents a finite biochemical mixture delivered at a single time point. The present findings should therefore be interpreted specifically as evidence about probiotic-derived metabolites, not as a surrogate for the full probiotic effect. An important strength of this study is the multi-endpoint design. Agar diffusion is easy to perform and useful for initial screening, but it is influenced by diffusion characteristics and therefore does not provide a complete measure of antimicrobial performance. Broth microdilution offers a more direct assessment of growth inhibition in planktonic culture, whereas crystal violet assays evaluate biomass accumulation during biofilm development. Mature-biofilm viable counting then adds a bactericidal outcome under more resistant conditions. By integrating these assays, the study captures complementary dimensions of microbial behavior instead of relying on a single simplified indicator. The inferential findings further strengthen interpretation. Agent choice exerted a very large effect size for both inhibition-zone and biofilm endpoints, indicating that the differences observed were not merely statistically detectable but also practically substantial within the experimental context. Species differences were smaller, although still meaningful. The significant interaction observed in the inhibition-zone analysis suggests that the relative performance of the agents was not perfectly uniform across organisms, which is consistent with species-specific cell-envelope properties, growth behavior, and susceptibility patterns. The more limited interaction in the biofilm model may indicate that the rank order among interventions was more stable once organisms were assessed under the common challenge of surface-associated growth. From a translational perspective, the observed hierarchy suggests that CHX is still the most plausible comparator when strong short-term antimicrobial suppression is desired, for example after periodontal instrumentation or during acute plaque-control phases [ 12 , 14 – 16 ]. Green tea extract appears particularly attractive when the therapeutic goal is to interfere with pathogenic biofilm development while potentially reducing reliance on an antiseptic with known adverse effects. CFS may be best conceptualized as a biologically derived adjunct with selective moderate activity, potentially useful in combined or stepwise ecological strategies. The data do not support direct replacement of CHX by either ecological comparator, but they do support further work on adjunctive and formulation-based applications. Several limitations should be acknowledged. First, the study used reference strains rather than patient-derived multispecies subgingival communities, and therefore cannot reproduce the structural and metabolic complexity of clinical plaque [ 36 – 38 ]. Second, the study was entirely in vitro and could not replicate salivary flow, host proteins, gingival crevicular fluid, repeated mechanical disruption, oxygen gradients, or immune interactions. Third, although the CFS was standardized by pH, optical density, and protein estimation, comprehensive metabolomic characterization was not performed. Because probiotic-derived activity depends on the precise composition of the metabolite pool, future work would benefit from quantifying relevant soluble factors and testing batch stability. Fourth, while the green tea extract was catechin-rich, botanical products can vary considerably between preparations, and more detailed analytical standardization would strengthen reproducibility. Additionally, although efforts were made to standardize experimental conditions, variations in laboratory preparation and extract composition may influence reproducibility across different settings. Additionally, although experiments were performed in triplicate across independent runs, the replicate structure remains more suitable for controlled laboratory comparison than for direct clinical generalization. Future work should move toward multispecies biofilm models containing keystone and accessory organisms in shared architecture, ideally combined with confocal microscopy, host-cell compatibility assays, inflammatory readouts, and formulation stability testing [ 36 , 39 , 40 ]. Time-kill studies, repeated-dose exposure models, and synergy testing with mechanical debridement or low-dose antiseptics could clarify whether ecological adjuncts are best used as stand-alone short-term rinses, local-delivery systems, or supporting components within a broader periodontal management strategy. Taken together, the present findings support a nuanced view of adjunctive periodontal antimicrobial therapy. CHX remains the most effective direct antimicrobial comparator in this model, but non-CHX ecological agents demonstrated meaningful and biologically interesting effects that may be relevant for future biofilm-directed strategies. The most important message is not that ecological adjuncts match CHX in raw potency, but that they may offer different and potentially complementary therapeutic functions within personalized periodontal care. Conclusion Within the limits of this in vitro study, 0.12% CHX remained the most potent antimicrobial and bactericidal comparator against the tested periodontopathogens. Green tea extract showed the strongest non-CHX anti-biofilm activity, while L. reuteri CFS demonstrated selective moderate inhibition, particularly against P. gingivalis. These findings suggest that plant-derived and probiotic-derived preparations may be better positioned as ecological adjuncts rather than direct replacements for CHX. All raw and statistical datasets corresponding to the reported results are provided in Supplementary File 1 . Further work using analytically characterized formulations, multispecies biofilms, and host-relevant models is needed before translational conclusions can be drawn for clinical periodontal care. However, direct clinical extrapolation of these findings should be interpreted with caution due to the in vitro nature of the study. Abbreviations CHX: Chlorhexidine CFS: Cell-free supernatant MIC: Minimum inhibitory concentration MBC: Minimum bactericidal concentration CFU: Colony-forming unit ATCC: American Type Culture Collection Declarations Ethics approval and consent to participate: Not applicable. This manuscript presents an in vitro laboratory study and did not involve human participants, human tissue, or animal experimentation. Consent for publication: Not applicable. Competing interests: No competing interests. Funding: No external financial support was received for the preparation of this manuscript draft. Author Contribution Conceptualization, methodology, investigation, data interpretation, drafting, critical revision, and final approval should be specified by the submitting author. Acknowledgement I would like to thank Specialist Pharmacist Elif Sirma Taskan for mentoring me during the laboratory stages of this study. Data Availability All data generated and/or analysed during this study are included in this published article and its supplementary information file. (Supplementary File 1) References Caton, J. G. et al. A new classification scheme for periodontal and peri-implant diseases and conditions - Introduction and key changes from the 1999 classification. J. 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Periodontol . 36 (10), 850–856 (2009). Sanz, M. et al. Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal diseases. J. Clin. Periodontol . 44 (Suppl 18), S5–S11 (2017). Cekici, A., Kantarci, A., Hasturk, H. & Van Dyke, T. E. Inflammatory and immune pathways in the pathogenesis of periodontal disease. Periodontol 2000 . 64 (1), 57–80 (2014). Lamont, R. J., Koo, H. & Hajishengallis, G. The oral microbiota: dynamic communities and host interactions. Nat. Rev. Microbiol. 16 (12), 745–759 (2018). Chapple, I. L. C. et al. Periodontal health and gingival diseases and conditions on an intact and a reduced periodontium. J. Clin. Periodontol . 45 (Suppl 20), S68–S77 (2018). Herrera, D., Retamal-Valdes, B., Alonso, B. & Feres, M. Acute periodontal lesions (periodontal abscesses and necrotizing periodontal diseases) and endo-periodontal lesions. J. Clin. Periodontol . 45 (Suppl 20), S78–S94 (2018). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 10 May, 2026 Reviewers agreed at journal 30 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers invited by journal 16 Apr, 2026 Editor assigned by journal 16 Apr, 2026 Editor invited by journal 15 Apr, 2026 Submission checks completed at journal 14 Apr, 2026 First submitted to journal 14 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9355651","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":628048557,"identity":"a5c26c14-f4f9-4d40-8e7f-90611fd8050b","order_by":0,"name":"Mehmet Murat Taskan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYNCCAwwM/CA6oYAo5cwQLZINIC0GpGgxOADiEKNFd9r5gx9/nLHLMz6/OvHDAwMGeX6xA/i1mN1OZpbmuZFcbHbj7WYJoMMMZ85OIKiFQZrhA3PithtnN4C0JBjcJqyF+eePD/WJm2ec3fyDWC1sEjw3Didu4O/dRrQtZtY8Z44nzrjBu80iwUCCGL8kPr7541h1Yn//2c03f1TYyPNLE9CCABJglRLEKgcB/gOkqB4Fo2AUjIKRBADM/EoS0/fVqQAAAABJRU5ErkJggg==","orcid":"","institution":"Tokat Gaziosmanpaşa University","correspondingAuthor":true,"prefix":"","firstName":"Mehmet","middleName":"Murat","lastName":"Taskan","suffix":""}],"badges":[],"createdAt":"2026-04-08 10:44:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9355651/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9355651/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107723055,"identity":"06a0440f-ef95-4538-bb92-d94981e26c37","added_by":"auto","created_at":"2026-04-24 11:26:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118243,"visible":true,"origin":"","legend":"\u003cp\u003eComparative antimicrobial activity of test agents.\u003c/p\u003e\n\u003cp\u003eThe antimicrobial effects of chlorhexidine (CHX), green tea extract, and Lactobacillus reuteri cell-free supernatant (CFS) against Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were evaluated using an agar diffusion assay. Bars represent mean inhibition zone diameters (mm) ± standard deviation (SD) obtained from three independent experiments. CHX demonstrated the highest antimicrobial activity across all tested species, followed by green tea extract and CFS.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9355651/v1/d69483106baf7653b7e612c1.png"},{"id":107723371,"identity":"06268c8d-8142-4fc4-be3e-fbc84a1f0ba6","added_by":"auto","created_at":"2026-04-24 11:27:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":524105,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9355651/v1/10a66b1e-64d8-441e-9bac-6167f236b41d.pdf"},{"id":107723114,"identity":"855a2b4a-23aa-4f2a-83fc-4b3c294f58d1","added_by":"auto","created_at":"2026-04-24 11:26:52","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19364,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9355651/v1/49a591f122a35e7ed3db1b78.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative evaluation of 0.12% chlorhexidine, Lactobacillus reuteri cell- free supernatant, and green tea extract against major periodontopathogens: a multi-endpoint in vitro research","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeriodontal diseases are among the most prevalent chronic inflammatory conditions in humans and arise from a complex interaction between a dysbiotic microbial biofilm and a susceptible host response [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Contemporary periodontal biology no longer frames disease as a simple infection caused by a single pathogen. Instead, current concepts emphasize ecological imbalance, altered interspecies interactions, and dysregulated host-microbe homeostasis within the subgingival environment [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWithin this study, Porphyromonas gingivalis is frequently cited as a keystone pathogen because it can modulate host defense pathways, reshape the community structure of the biofilm, and amplify pathogenicity out of proportion to its abundance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Aggregatibacter actinomycetemcomitans remains clinically relevant due to its historical association with aggressive forms of periodontal destruction and its array of leukotoxic and invasive virulence traits [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Fusobacterium nucleatum serves as an important bridging organism that facilitates co-aggregation and maturation of multispecies biofilms, thereby increasing ecological complexity and resilience [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The use of these organisms together therefore provides a biologically coherent model for screening candidate adjunctive agents in periodontology.\u003c/p\u003e \u003cp\u003eChlorhexidine gluconate remains the best established antiseptic comparator in clinical dentistry because of its broad antimicrobial spectrum and substantivity [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nevertheless, its long-term or repeated use is limited by adverse effects such as taste alteration, tooth and tongue staining, calculus promotion, and mucosal irritation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. There is also increasing conceptual concern that broad-spectrum suppression may not represent the most desirable long-term ecological strategy in a microbiome-driven disease. These limitations have encouraged investigation of adjuncts that can attenuate pathogenic biofilm behavior while potentially exerting less collateral disruption.\u003c/p\u003e \u003cp\u003eProbiotic-based approaches have received substantial attention in periodontal research, particularly those involving Lactobacillus reuteri [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Clinical trials and systematic reviews suggest that probiotic adjuncts may improve periodontal parameters when combined with conventional therapy, although findings remain heterogeneous [\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Proposed mechanisms include competitive exclusion, production of antimicrobial metabolites such as reuterin, pH modification, signaling interference, and immune modulation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the biological activity of a cell-free supernatant must be distinguished from that of a live probiotic formulation. A supernatant acts through soluble metabolites present at the time of administration and lacks active colonization, metabolic renewal, and interspecies competition after delivery.\u003c/p\u003e \u003cp\u003ePlant-derived polyphenols represent a second major ecological strategy of interest. Green tea contains catechins, especially epigallocatechin gallate, epicatechin gallate, and epigallocatechin, which have been associated with antimicrobial, anti-adhesive, anti-inflammatory, and anti-biofilm effects in oral models [\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29 CR30\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In relation to periodontal pathogens, catechins may reduce adhesion, influence extracellular matrix development, attenuate virulence-associated proteolytic activity, and increase susceptibility to conventional antimicrobials [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Importantly, the therapeutic appeal of such compounds may lie less in absolute bactericidal potency and more in biofilm modulation.\u003c/p\u003e \u003cp\u003eMany laboratory studies assess candidate agents using only a single antimicrobial assay, which can oversimplify interpretation. Agar diffusion depends in part on molecular diffusion through the medium, broth microdilution reflects planktonic growth suppression, and biofilm assays capture a different dimension of microbial behavior altogether. A multi-endpoint design provides a more balanced appraisal by distinguishing direct growth inhibition from interference with biofilm formation and from killing within established biofilms.\u003c/p\u003e \u003cp\u003eAccordingly, the aim of the present study was to compare 0.12% CHX, L. reuteri-derived cell-free supernatant, and a catechin-rich green tea extract in a in vitro design incorporating direct antimicrobial, anti-biofilm, and mature-biofilm viability outcomes against P. gingivalis, A. actinomycetemcomitans, and F. nucleatum. The null hypothesis was that no significant difference would be observed among the tested agents across the evaluated endpoints.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis study was designed as a controlled, comparative in vitro experiment with four groups: 0.12% CHX, L. reuteri-derived CFS, standardized green tea extract, and vehicle control. Four outcome domains were assessed: agar diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), inhibition of biofilm development, and viable-count reduction in pre-formed biofilms. Each assay was performed in technical triplicate and repeated in three independent experimental runs (total n\u0026thinsp;=\u0026thinsp;9 observations per condition for descriptive analysis).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicroorganisms and culture conditions\u003c/h3\u003e\n\u003cp\u003eReference strains were selected to represent functionally distinct components of the periodontal biofilm: P. gingivalis ATCC 33277, A. actinomycetemcomitans ATCC 29523, and F. nucleatum ATCC 25586. P. gingivalis and F. nucleatum were cultured anaerobically at 37 C in an atmosphere of approximately 85% N\u003csub\u003e2\u003c/sub\u003e, 10% H\u003csub\u003e2\u003c/sub\u003e, and 5% CO\u003csub\u003e2\u003c/sub\u003e. A. actinomycetemcomitans was maintained under capnophilic conditions. Organisms were revived from stock cultures, subcultured on strain-appropriate agar media, and transferred into broth for preparation of standardized inocula. Before testing, suspensions were adjusted to 0.5 McFarland standard, corresponding approximately to 1 x 10^8 CFU/mL, and then diluted according to assay requirements. \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMicroorganisms and principal culture conditions used in the experiment.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReference strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAtmosphere\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePrimary role in periodontal biofilm model\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePorphyromonas gingivalis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC 33277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnaerobic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKeystone pathogen and dysbiosis amplifier\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAggregatibacter actinomycetemcomitans\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC 29523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCapnophilic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAggressive-periodontitis-associated pathogen\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFusobacterium nucleatum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCC 25586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnaerobic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBridging organism in multispecies biofilm maturation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePreparation and standardization of test agents\u003c/h3\u003e\n\u003cp\u003eThe CHX group consisted of a commercially available 0.12% chlorhexidine gluconate solution used as supplied. For the probiotic-metabolite group, L. reuteri was grown in de Man, Rogosa and Sharpe broth for 24 hours at 37 C. Cultures were centrifuged at 4000 rpm for 10 minutes, and the supernatant was sterilized through a 0.22-micrometer membrane filter to remove intact bacterial cells. The resulting CFS was standardized before use by pH adjustment to 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 and normalization of optical density. Protein content was estimated by a Bradford-based assay to improve batch consistency. Although detailed metabolomic profiling was beyond the scope of the present study, these standardization steps were included to reduce variability between batches. The green tea group consisted of a catechin-rich extract prepared in sterile distilled water. The extract was selected to reflect a polyphenol-rich composition and was standardized according to supplier data for high catechin content. Serial two-fold dilutions were prepared freshly for MIC and MBC testing. The vehicle control contained the corresponding solvent system without active ingredient. \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStandardization characteristics of the tested agents.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePreparation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandardization / notes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.12% chlorhexidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCommercial mouthrinse-grade solution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUsed as supplied; reference antiseptic comparator\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL. reuteri CFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24-h broth culture, centrifuged and 0.22-micrometer filtered\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epH adjusted to 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2; optical density normalized; protein estimate recorded\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen tea extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatechin-rich extract in sterile distilled water\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFreshly prepared; serial dilutions used for MIC and MBC testing\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVehicle control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorresponding solvent system without active ingredient\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIncluded in all assays\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eAgar diffusion assay\u003c/h3\u003e\n\u003cp\u003eUniform bacterial lawns were created on species-appropriate agar surfaces using standardized inocula. Wells with equal diameter (6 mm) were created aseptically, and 50 microliters of each test solution were dispensed into each well. Plates were incubated under the relevant atmospheric conditions for each species. Following incubation, inhibition zones were measured in millimeters using a digital caliper by two independent blinded observers. The mean of the two readings was used for analysis. Vehicle control was expected to yield no measurable inhibition.\u003c/p\u003e\n\u003ch3\u003eMIC and MBC determination\u003c/h3\u003e\n\u003cp\u003eBroth microdilution testing was performed in sterile 96-well microplates using two-fold serial dilutions of each agent. MIC was defined as the lowest concentration demonstrating no visible turbidity after incubation under species-specific conditions. For MBC determination, aliquots from non-turbid wells were plated onto agar and incubated. The lowest concentration yielding no colony growth was recorded as the MBC. When a bactericidal threshold was not reached within the tested range, the result was recorded as not reached (NR).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm inhibition assay\u003c/h2\u003e \u003cp\u003eTo evaluate inhibition of early biofilm development, bacterial suspensions were seeded into polystyrene microplates and exposed to test agents during the initial adhesion and growth phase. After 24 hours of incubation, wells were gently washed with phosphate-buffered saline to remove non-adherent cells, fixed with methanol, stained with 0.1% crystal violet, and destained before optical density measurement at 570 nm. The percentage inhibition of biofilm biomass was calculated relative to untreated control wells using the formula: ((OD_control - OD_test) / OD_control) x 100.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eViability assay in pre-formed biofilms\u003c/h3\u003e\n\u003cp\u003eFor mature-biofilm testing, biofilms were allowed to establish for 24 hours before exposure. After maturation, wells were treated with the respective agents for a standardized contact period of 60 minutes. Residual test solution was then removed, and a validated washing and neutralization sequence was applied to minimize antimicrobial carryover. Neutralization solution consisted of a lecithin and Tween 80-containing buffer used in line with common antiseptic-neutralization practice. Neutralization efficacy was verified in a control procedure confirming that residual antimicrobial activity did not distort post-treatment colony counts. Biofilms were then mechanically disrupted, serially diluted, plated, and incubated. Outcomes were expressed as log10 CFU reduction relative to untreated controls.\u003c/p\u003e\n\u003ch3\u003eControls, quality assurance, and blinding\u003c/h3\u003e\n\u003cp\u003eVehicle controls were included in all assays. Neutralization controls were incorporated in the mature-biofilm experiment to verify that differences in colony counts reflected treatment effects rather than residual carryover activity. Measurements of inhibition zones and spectrophotometric outputs were performed by investigator who was blinded to agent identity at the time of reading. (M.M.T.) Media sterility and growth controls were included in each run, and all experiments were repeated independently three times to improve internal consistency.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll quantitative results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The primary inferential framework used two-way analysis of variance with agent and species as fixed factors. Where relevant, Tukey-adjusted pairwise comparisons were applied to compare group means while controlling for multiple testing. Assumptions were checked using Shapiro-Wilk testing for normality of residuals and Levene testing for homogeneity of variance. In addition to p values, effect sizes were expressed as partial eta squared (partial eta^2), and 95% confidence intervals were calculated for selected contrasts. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eVehicle control yielded no inhibition zone, no relevant inhibition of biofilm formation, and no measurable bactericidal reduction in the mature-biofilm assay. Because the control findings were consistently negligible, the principal emphasis below is on the comparative performance of the three active interventions.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAgar diffusion findings\u003c/h2\u003e \u003cp\u003eCHX produced the largest inhibition zones against all three organisms. The highest mean zone was observed against P. gingivalis, followed by F. nucleatum and A. actinomycetemcomitans. Green tea extract showed a clear but smaller inhibitory halo in all species, while CFS displayed modest inhibition that remained consistently greater against P. gingivalis than against the other two species. The ranking CHX\u0026thinsp;\u0026gt;\u0026thinsp;green tea extract\u0026thinsp;\u0026gt;\u0026thinsp;CFS was preserved across species. \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean inhibition zone diameters in the agar diffusion assay (mm).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP. gingivalis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA. actinomycetemcomitans\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF. nucleatum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.12% CHX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL. reuteri CFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen tea extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMIC and MBC findings\u003c/h2\u003e \u003cp\u003eBroth microdilution results reinforced the superiority of CHX as the direct antimicrobial comparator. CHX achieved the lowest MIC and MBC values for all three species. Green tea extract showed measurable inhibitory and bactericidal thresholds but required substantially higher concentrations than CHX. CFS displayed selective activity, with better inhibitory and bactericidal performance against P. gingivalis than against A. actinomycetemcomitans and F. nucleatum. In the latter organisms, bactericidal thresholds were more difficult to achieve within the tested concentration range. \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMIC and MBC findings for the tested agents. MIC and MBC values are given as percent concentration equivalents within the tested range. NR, not reached within tested range.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eP. gingivalis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA. actinomycetemcomitans\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF. nucleatum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.12% CHX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.12% CHX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.0010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL. reuteri CFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL. reuteri CFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen tea extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen tea extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of biofilm development\u003c/h2\u003e \u003cp\u003eWhen agents were present during early biofilm formation, CHX again produced the strongest suppression of biomass accumulation, with all means exceeding 80% inhibition. Notably, green tea extract consistently outperformed CFS in all species. The magnitude of the green tea advantage over CFS ranged from approximately 10 to 12 percentage points, suggesting that catechin-rich preparations may be especially relevant for modulating the developmental phase of periodontal biofilms. \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage inhibition of biofilm formation relative to untreated control (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP. gingivalis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA. actinomycetemcomitans\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF. nucleatum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.12% CHX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e84\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e81\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e83\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL. reuteri CFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e61\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e56\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e58\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen tea extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e72\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e67\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e69\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eViability in pre-formed biofilms\u003c/h2\u003e \u003cp\u003eThe mature-biofilm assay revealed the expected attenuation of antimicrobial effect compared with planktonic testing, yet meaningful differences remained. CHX produced approximately 3-log reductions across all species, reflecting substantial bactericidal action despite the protective architecture of established biofilm. Green tea extract generated intermediate reductions, generally between 1.8 and 2.2 log10 CFU. CFS showed lower but measurable activity, again with the largest response observed against P. gingivalis. Neutralization controls confirmed that these viable-count results were not attributable to residual antiseptic carryover. \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLog10 CFU reduction in pre-formed biofilms relative to untreated control (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP. gingivalis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA. actinomycetemcomitans\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF. nucleatum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.12% CHX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL. reuteri CFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreen tea extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInferential analysis\u003c/h2\u003e \u003cp\u003eFor inhibition-zone outcomes, two-way ANOVA showed a highly significant main effect of agent (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), a smaller but significant main effect of species (p\u0026thinsp;=\u0026thinsp;0.011), and a significant agent-species interaction (p\u0026thinsp;=\u0026thinsp;0.018). For inhibition of biofilm development, the main effect of agent remained highly significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while species had a modest but statistically significant influence (p\u0026thinsp;=\u0026thinsp;0.037). Post hoc analysis confirmed the ordered pattern CHX\u0026thinsp;\u0026gt;\u0026thinsp;green tea extract\u0026thinsp;\u0026gt;\u0026thinsp;CFS\u0026thinsp;\u0026gt;\u0026thinsp;vehicle. The difference between green tea extract and CFS was statistically significant for the biofilm endpoint (p\u0026thinsp;=\u0026thinsp;0.032). Effect sizes indicated that the choice of agent explained a substantial proportion of the observed variance. (Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) These findings should be interpreted within the constraints of this controlled in vitro model.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of inferential analysis for key endpoints.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndpoint\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePartial eta^2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInterpretation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgar diffusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e148.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVery large agent effect\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgar diffusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSmall to moderate species effect\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgar diffusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgent \u0026times; species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSignificant interaction\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiofilm inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e126.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVery large agent effect\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiofilm inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModest species effect\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiofilm inhibition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgent \u0026times; species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTrend without strong interaction\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study compared three conceptually different antimicrobial strategies for periodontal biofilm control: a conventional broad-spectrum antiseptic, a probiotic-derived metabolite preparation, and a plant polyphenol-rich extract. Across all principal endpoints, 0.12% CHX remained the most potent comparator. This overall pattern is consistent with its longstanding role as the benchmark antiplaque and antiseptic agent in periodontal care [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, the present data also underscore that antimicrobial superiority does not answer the entire translational question. Biofilm-related periodontal therapy is not only about maximal killing; it is also about how a compound modulates adhesion, maturation, ecological structure, and the balance between suppression and selectivity.\u003c/p\u003e \u003cp\u003eThe green tea extract findings were particularly notable because this agent did not approach CHX in direct planktonic potency but showed the strongest non-CHX performance in the anti-biofilm assay. This observation is biologically plausible and is in line with prior reports describing catechin-mediated interference with P. gingivalis biofilm formation, adherence, and virulence behavior [\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29 CR30\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Catechins, especially epigallocatechin gallate, may alter membrane function, reduce extracellular matrix development, attenuate proteolytic activity, and influence the expression of virulence-associated genes. In practical periodontal terms, this suggests that green tea-derived preparations may hold more promise as ecological biofilm modulators than as direct replacements for high-potency antiseptics.\u003c/p\u003e \u003cp\u003eThe CFS preparation displayed moderate and species-dependent effects, again with the most favorable response observed against P. gingivalis. This selective pattern may reflect differential sensitivity of target organisms to soluble metabolites generated by L. reuteri culture supernatants. Previous probiotic literature has emphasized mechanisms such as reuterin production, competition for nutrients and adhesion sites, pH changes, and immune signaling modulation [\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21 CR22 CR23\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Yet the distinction between a live probiotic product and a cell-free metabolite fraction should be emphasized. A live organism can potentially colonize transiently, dynamically produce bioactive compounds, and participate in interspecies competition, whereas a supernatant represents a finite biochemical mixture delivered at a single time point. The present findings should therefore be interpreted specifically as evidence about probiotic-derived metabolites, not as a surrogate for the full probiotic effect.\u003c/p\u003e \u003cp\u003eAn important strength of this study is the multi-endpoint design. Agar diffusion is easy to perform and useful for initial screening, but it is influenced by diffusion characteristics and therefore does not provide a complete measure of antimicrobial performance. Broth microdilution offers a more direct assessment of growth inhibition in planktonic culture, whereas crystal violet assays evaluate biomass accumulation during biofilm development. Mature-biofilm viable counting then adds a bactericidal outcome under more resistant conditions. By integrating these assays, the study captures complementary dimensions of microbial behavior instead of relying on a single simplified indicator.\u003c/p\u003e \u003cp\u003eThe inferential findings further strengthen interpretation. Agent choice exerted a very large effect size for both inhibition-zone and biofilm endpoints, indicating that the differences observed were not merely statistically detectable but also practically substantial within the experimental context. Species differences were smaller, although still meaningful. The significant interaction observed in the inhibition-zone analysis suggests that the relative performance of the agents was not perfectly uniform across organisms, which is consistent with species-specific cell-envelope properties, growth behavior, and susceptibility patterns. The more limited interaction in the biofilm model may indicate that the rank order among interventions was more stable once organisms were assessed under the common challenge of surface-associated growth.\u003c/p\u003e \u003cp\u003eFrom a translational perspective, the observed hierarchy suggests that CHX is still the most plausible comparator when strong short-term antimicrobial suppression is desired, for example after periodontal instrumentation or during acute plaque-control phases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Green tea extract appears particularly attractive when the therapeutic goal is to interfere with pathogenic biofilm development while potentially reducing reliance on an antiseptic with known adverse effects. CFS may be best conceptualized as a biologically derived adjunct with selective moderate activity, potentially useful in combined or stepwise ecological strategies. The data do not support direct replacement of CHX by either ecological comparator, but they do support further work on adjunctive and formulation-based applications.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. First, the study used reference strains rather than patient-derived multispecies subgingival communities, and therefore cannot reproduce the structural and metabolic complexity of clinical plaque [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Second, the study was entirely in vitro and could not replicate salivary flow, host proteins, gingival crevicular fluid, repeated mechanical disruption, oxygen gradients, or immune interactions. Third, although the CFS was standardized by pH, optical density, and protein estimation, comprehensive metabolomic characterization was not performed. Because probiotic-derived activity depends on the precise composition of the metabolite pool, future work would benefit from quantifying relevant soluble factors and testing batch stability. Fourth, while the green tea extract was catechin-rich, botanical products can vary considerably between preparations, and more detailed analytical standardization would strengthen reproducibility. Additionally, although efforts were made to standardize experimental conditions, variations in laboratory preparation and extract composition may influence reproducibility across different settings. Additionally, although experiments were performed in triplicate across independent runs, the replicate structure remains more suitable for controlled laboratory comparison than for direct clinical generalization.\u003c/p\u003e \u003cp\u003eFuture work should move toward multispecies biofilm models containing keystone and accessory organisms in shared architecture, ideally combined with confocal microscopy, host-cell compatibility assays, inflammatory readouts, and formulation stability testing [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Time-kill studies, repeated-dose exposure models, and synergy testing with mechanical debridement or low-dose antiseptics could clarify whether ecological adjuncts are best used as stand-alone short-term rinses, local-delivery systems, or supporting components within a broader periodontal management strategy.\u003c/p\u003e \u003cp\u003eTaken together, the present findings support a nuanced view of adjunctive periodontal antimicrobial therapy. CHX remains the most effective direct antimicrobial comparator in this model, but non-CHX ecological agents demonstrated meaningful and biologically interesting effects that may be relevant for future biofilm-directed strategies. The most important message is not that ecological adjuncts match CHX in raw potency, but that they may offer different and potentially complementary therapeutic functions within personalized periodontal care.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWithin the limits of this in vitro study, 0.12% CHX remained the most potent antimicrobial and bactericidal comparator against the tested periodontopathogens. Green tea extract showed the strongest non-CHX anti-biofilm activity, while L. reuteri CFS demonstrated selective moderate inhibition, particularly against P. gingivalis.\u003c/p\u003e \u003cp\u003eThese findings suggest that plant-derived and probiotic-derived preparations may be better positioned as ecological adjuncts rather than direct replacements for CHX. All raw and statistical datasets corresponding to the reported results are provided in \u003cb\u003eSupplementary File 1\u003c/b\u003e. Further work using analytically characterized formulations, multispecies biofilms, and host-relevant models is needed before translational conclusions can be drawn for clinical periodontal care. However, direct clinical extrapolation of these findings should be interpreted with caution due to the in vitro nature of the study.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cul\u003e\n \u003cli\u003eCHX: Chlorhexidine\u003c/li\u003e\n \u003cli\u003eCFS: Cell-free supernatant\u003c/li\u003e\n \u003cli\u003eMIC: Minimum inhibitory concentration\u003c/li\u003e\n \u003cli\u003eMBC: Minimum bactericidal concentration\u003c/li\u003e\n \u003cli\u003eCFU: Colony-forming unit\u003c/li\u003e\n \u003cli\u003eATCC: American Type Culture Collection\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e \u003cp\u003eNot applicable. This manuscript presents an in vitro laboratory study and did not involve human participants, human tissue, or animal experimentation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication:\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eNo competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eNo external financial support was received for the preparation of this manuscript draft.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, methodology, investigation, data interpretation, drafting, critical revision, and final approval should be specified by the submitting author.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eI would like to thank Specialist Pharmacist Elif Sirma Taskan for mentoring me during the laboratory stages of this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated and/or analysed during this study are included in this published article and its supplementary information file. (Supplementary File 1)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCaton, J. G. et al. A new classification scheme for periodontal and peri-implant diseases and conditions - Introduction and key changes from the 1999 classification. \u003cem\u003eJ. Periodontol\u003c/em\u003e. \u003cb\u003e89\u003c/b\u003e (Suppl 1), S1\u0026ndash;S8 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarsh, P. D. Dental plaque as a microbial biofilm. \u003cem\u003eCaries Res.\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e (3), 204\u0026ndash;211 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarsh, P. D. Dental plaque as a biofilm and a microbial community - implications for health and disease. \u003cem\u003eBMC Oral Health\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e (Suppl 1), S14 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarsh, P. D. 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Acute periodontal lesions (periodontal abscesses and necrotizing periodontal diseases) and endo-periodontal lesions. \u003cem\u003eJ. Clin. Periodontol\u003c/em\u003e. \u003cb\u003e45\u003c/b\u003e (Suppl 20), S78\u0026ndash;S94 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"chlorhexidine, Lactobacillus reuteri, green tea extract, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum","lastPublishedDoi":"10.21203/rs.3.rs-9355651/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9355651/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePeriodontal 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.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA 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\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and analyzed by two-way ANOVA with Tukey-adjusted pairwise comparisons. Effect sizes (partial eta squared) and 95% confidence intervals were calculated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCHX 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\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWithin 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.\u003c/p\u003e","manuscriptTitle":"Comparative evaluation of 0.12% chlorhexidine, Lactobacillus reuteri cell- free supernatant, and green tea extract against major periodontopathogens: a multi-endpoint in vitro research","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 11:24:28","doi":"10.21203/rs.3.rs-9355651/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-10T12:21:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164724762326647388036815390142135789426","date":"2026-04-30T10:01:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283024352557690457736779534313716033637","date":"2026-04-17T05:46:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T00:58:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T00:46:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-15T13:12:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-14T17:29:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-14T17:16:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9ba9631c-0cda-4bc9-ab50-731b37e031e5","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-10T12:21:43+00:00","index":44,"fulltext":""},{"type":"reviewerAgreed","content":"164724762326647388036815390142135789426","date":"2026-04-30T10:01:39+00:00","index":42,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66848061,"name":"Biological sciences/Biotechnology"},{"id":66848062,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-24T11:24:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 11:24:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9355651","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9355651","identity":"rs-9355651","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}