Evaluation of virulence factors, antimicrobial activity and cariogenic bacteria cell viability in the presence of Terpinen-4-ol: in vitro study

Author(s)

Ester Alves Ferreira Bordini

• Document type: Master's Dissertation
• Press: Araraquara. 2016-05-31.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Defense date: 2016-03-23
• Advisor: Denise Madalena Palomari Spolidorio

Abstract

The use of essential oils has been widespread in the treatment of infectious diseases that affect the oral cavity to be effective against pathogens in biofilms. Among these natural products, there is the Terpinen-4-ol a biocide membrane-active, with broad spectrum of action against gram-positive bacteria, gram-negative and multi-resistant bacteria. In this study, we investigated the antimicrobial effect of Terpinen-4-ol on planktonic and biofilm cultures of Streptococcus mutans (S. mutans) and gene expression gbpA (glucan binding protein A) involved in the adhesion of biofilm. The antimicrobial activity of Terpinen-4-ol (0.059 % to 0.95 %) was assessed by the broth microdilution test for the determination of MIC and MBC for the microorganism in planktonic form. The biofilm formed in cell culture plate were treated with different concentrations of Terpinen-4-ol and cell metabolism resulting plaque was assessed using the hidróxido de 2,3-bis (2-metoxi-4-nitro-5-sulfo-fenil) -2H-tetrazólio-5-caboxanilide (XTT). The analysis of the biofilm formed on enamel blocks and dentin treated with Terpinen-4-ol (0.24 % and 0.95 %) and chlorhexidine (CHX 0.12 %) for 60 seconds was performed by XTT assay and spectral Ratio (Green/Red) of images obtained by microscopy Laser Scanning Confocal (CLSM). The gbpA gene expression was investigated by quantitative RT-PCR after S. mutans have been exposed to Terpinen-4-ol and CHX for 15 and 30 minutes. The data were normally distributed and therefore parametric tests of ANOVA One-way and Tukey were used. All statistical tests were considered at a significance level of 5 % (p<0.05). Terpinen-4-ol showed antimicrobial activity at concentrations 0.24% for both planktonic microorganisms in culture and to biofilm formation in cell culture plate and enamel with microbial reduction near 100 %. There was no statistically significant difference between Terpinen-4-ol and CHX. For biofilm on dentin, the concentration of Terpinen-4-ol 0.95 % showed antimicrobial activity both by analysis by XTT and by CLSM. There was a reduction in gbpA expression when in contact with Terpinen-4-ol for 15 minutes, however the gene overexpression occurred after 30 minutes of exposure. CHX caused overexpression of the gbpA gene in all tested times. These findings demonstrate that Terpinen-4-ol shows a significant antimicrobial activity and ability to modulate the expression of genes involved in cariogenic biofilm formation, can be used in treating oral infectious diseases. (AU)

• FAPESP's process: 14/23241-6 - Evaluation of virulence factors and cell viability of cariogenic bacteria in the presence of Terpinen-4-ol: in vitro study
• Grantee: Ester Alves Ferreira Bordini Galvani
• Support Opportunities: Scholarships in Brazil - Master