Study of the effects of the green tea catechin epigalocatequina-3-galato no on the initial growth, maturation, and virulence of matrix-rich Streptococcus mutans biofilm

Author(s)

Maria Gerusa Brito Aragão

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC)
• Defense date: 2024-04-19

Examining board members

Silmara Aparecida Milori Corona; Carolina Steiner Oliveira Alarcon; Fernanda de Carvalho Panzeri; Sergio Luiz de Souza Salvatore

• Advisor: Silmara Aparecida Milori Corona; Carolina Patricia Aires Garbellini

Abstract

Green tea is rich in biologically active compounds such as epigallocatechin-3-gallate, which presents antimicrobial effects against Streptococcus mutans, a microorganism associated with the development of destructive caries lesions. S. mutans is an avid biofilm former that, besides being acidogenic and aciduric, in the presence of sucrose, can also synthesize insoluble polysaccharides, which are the main components of the biofilm matrix and are also highly virulent. There have been reports on the effects of EGCG on microbial viability, acidogenesis, and polysaccharide synthesis of S. mutans planktonic cultures. However, the effects of EGCG on the virulence of S. mutans biofilm have yet to be fully investigated using validated cariogenic biofilm models. Thus, the objective of this study was to evaluate the effects of EGCG on the virulence, initial growth, and maturation of S. mutans biofilm using a high-challenging validated biofilm model. In the first stage of this study, we evaluated the effects of EGCG on the virulence of S. mutans biofilm, which was cultivated in a regimen of constant exposure to sucrose and exposed twice a day to a 0.9% NaCl (negative control), EGCG 4 mg/ml, or 0.12% chlorhexidine (positive control). The culture media was changed twice a day, when its pH was also evaluated. After 67 h, the biofilm was collected for analysis of bacterial viability and quantification of soluble and insoluble polysaccharides. Morphological and 3D analyses of the biofilm were also performed using scanning electron microscopy and confocal laser scanning microscopy, respectively. In the second step of this study, the effects of chitosan nanoparticles loaded with EGCG were evaluated by using the same protocol described for experimental stage 1. The biofilm was treated with 0.9% NaCl, chitosan nanoparticles loaded with EGCG, chitosan nanoparticles, or 0.12% chlorhexidine. Acidogenesis, microbial viability and effects on the biofilm morphology were evaluated. In the third stage, an analysis of the effects of EGCG on S. mutans biofilm initial growth and maturation was performed. Hydroxyapatite discs were pre-treated with 0.9% NaCl, EGCG 4 mg/ml, or 0.12% chlorhexidine, then inoculated with S. mutans and grown for 24 or 46 h. After 24 and 46 h, the biofilm was imaged using confocal laser scanning microscopy and evaluated for changes in the 3D organization of the biofilm structure and for bacterial and EPS volume and thickness by using IMARIS® and COMSTAT, respectively. The data were evaluated by analysis of variance (ANOVA) followed by post hoc Tukey Test (α=5%). The results demonstrated that, for experimental stage 1, topical with EGCG significantly reduced the synthesis of soluble and insoluble polysaccharides, altering the 3D organization of the biofilm, which resulted in the formation of interspaced microcolony-EPS complexes that presented reduced thickness. No significant effects on microbial viability and acidogenesis were observed. Comparable results were found for stage 2 experiments, demonstrating that chitosan nanoparticles loaded with EGCG dismantle the biofilm matrix without affecting microbial viability. Lastly, stage 3 experiments demonstrated that in the 24 h protocol, EGCG led to significant reduction of biofilm biomass volume and thickness. The 46-h protocol demonstrated that the biofilm treated with EGCG did not form complex EPS-microcolony complexes and presented a 3D organization that represented the one of the biofilm treated with the negative control at 24 h. No reduction of bacterial volume was observed but important effects on the 3D structure of the biofilm were seen due to the significant reduction of biofilm matrix volume and thickness. Altogether, we demonstrated that topical treatment with EGCG reduces the virulence of S. mutans biofilm by reducing the synthesis of soluble and insoluble polysaccharides, which alters the biofilm initial growth and delays the biofilm maturation. (AU)

• FAPESP's process: 20/02658-7 - Dose-response effect of epigallocatechin-3-gallate (EGCG) on the bacterial adhesion and initial growth of Streptococcus mutans biofilm and on the progression of biofilm-induced caries lesion formed on bovine dental enamel
• Grantee: Maria Gerusa Brito Aragão
• Support Opportunities: Scholarships in Brazil - Doctorate