Available online 6 May 2024, 105990

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EGCG treatments reduce the synthesis of EPS in mature S. mutans cariogenic biofilm

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SEM demonstrates that EGCG changes the morphology of mature cariogenic biofilm

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Confocal microscopy demonstrates that EGCG treatments dismantle the biofilm matrix

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Effects against S. mutans biofilm are achieved with short-timed treatment

ABSTRACTIntroduction

There have been reports on the effects of epigallocatechin gallate (EGCG) against Streptococcus mutans viability and acidogenesis. However, the effects of EGCG on the virulence of S. mutans biofilm development have yet to be fully investigated using validated cariogenic biofilm models.

Objective

Thus, this study aimed to evaluate the effects of EGCG on S. mutans biofilm virulence using a validated cariogenic model and clinically relevant treatment regimens, twice a day for 1.5 minutes.

Methods

The effects of EGCG on the morphology and 3D structure of the biofilms were evaluated by scanning electron (SEM) and confocal laser scanning microscopy, respectively.

Results

No significant change in S. mutans viability or culture medium pH were observed when comparing EGCG-treated and NaCl treated biofilms. EGCG significantly reduced the accumulation of soluble and insoluble polysaccharides, resulting in the formation of a biofilm with interspaced exopolysaccharide-microcolony complexes unevenly distributed on enamel. The SEM images of the biofilm treated with EGCG depict multilayers of cells arranged in short chains of microorganisms adhered to an unstructured matrix, which is not continuous and does not enmesh or protect the microorganisms entirely. Importantly, confocal images demonstrated that treatment with EGCG affected the 3D structure and organization of S. mutans biofilm, which presented a biofilm matrix more confined to the location of the microcolonies.

Conclusion

In conclusion, EGCG lowered the virulence of S. mutans matrix-rich biofilm by reducing the synthesis of biofilm matrix components, altering the biofilm matrix structure, organization, and distribution.

Section snippetsINTRODUCTION

The dental biofilm is a complex ecosystem formed by a diverse microbial community embedded in a matrix of polymers of bacterial and salivary origin (Marsh, 2006). Frequent exposure to low pH due to carbohydrate fermentation by bacteria is the driving force for dental caries development (Marsh, 2006, Marsh, 2018, Takahashi, 2015, Takahashi & Nyvad, 2010 and 2011). Severe and prolonged exposure to an acidic microenvironment leads to dysbiosis, making the dental biofilm microbial community less

Test agents and substrate

The test agents were Epigallocatechin gallate (EGCG) (E4143, Sigma-Aldrich) and chlorhexidine digluconate (CHX) (C9394, Sigma-Aldrich). This study evaluated 3 distinct treatment groups: Chlorhexidine 0.12% (CHX) (positive control), NaCl 0.9% (NaCl) (negative control), and 4 mg/mL Epigallocatechin gallate (EGCG). This EGCG concentration was chosen based on previously reported effects of EGCG 4 mg/mL in reducing S. mutans count in saliva (Vilela et al., 2020) and its inhibitory effect on the gtf

Biofilm acidogenesis and microbial viability

Regarding acidogenesis, enamel slabs were placed in a medium with a neutral pH. In the first 19 h of biofilm growth, S. mutans metabolized sucrose and acidified the milieu, generating a pH drop. We demonstrated that the pH of the medium dropped from around 7 at the initial inoculation to around 4 at 19 h. After 19 h of biofilm development, no significant control of biofilm acidogenesis was observed as shown by the pH of the culture medium of the biofilm treated with EGCG 4 mg/mL when compared to

Discussion

Although natural products and their derivatives present potential as adjunctive therapeutic agents against cariogenic bacteria, several gaps need to be filled to translate more linearly the potential of natural products into clinically relevant oral care products for dental caries prevention, control, and treatment (Philip et al., 2019, Jean et al., 2011, Freires & Rosalen, 2016). For instance, there are known effects of the green tea catechin EGCG against cariogenic bacteria grown in

CONCLUSION

In conclusion, topical treatments with EGCG lowered the virulence of S. mutans matrix- rich biofilm by reducing the synthesis of biofilm matrix components, altering the biofilm matrix structure, organization and distribution, which is desirable when developing novel therapeutic strategies for preventing, controlling and treating dental caries.

Statement of Ethics

This study did not involve human participants or animals. Therefore, there was no need for ethical approval.

Funding Sources

This study was supported by the São Paulo State Research Support Foundation (FAPESP) (grant #2022/01576-2 and #2020/02658-7. This study was financed in part by the Coordination for the Improvement of Higher Education Personnel (CAPES) – Brazil– Finance Code 001. A.C.T. We also thank the Brazilian National Council for Scientific and Technological Development (CNPq) for the research support to the author S.A.M.C (304366/2020-0). This work was also in part supported by NIH/NIDCR. R01DE023810,

Uncited references

(Hajishengallis et al., 2017, Koo et al., 2009, Newman and Cragg, 2020, Wen et al., 2021, Xiao and Koo, n.d)

CRediT authorship contribution statement

Maria Aragao: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Silmara Corona: Writing – review & editing, Writing – original draft, Validation, Resources, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization. Carolina Patricia Aires: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources,

Declaration of Generative AI and AI-assisted technologies in the writing process

AI and AI-assisted technologies were not use in the writing process of this manuscript

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We would like to express our acknowledgment to our laboratory technician A. C. P for helping preparing all materials for polysaccharide extraction experiments and for being a handful staff for the execution of the hole study.

Declaration of Competing Interest.

The authors have no conflicts of interest to declare

Author Contributions

All authors contributed to the present study. M.G.B.A assisted in designing the study and experiments, performed the experiments, analyzed the data and drafted the manuscript. X.H provided resources, supervision and

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