Avaliação dos fatores de virulência, atividade antimicrobiana e viabilidade celular de bactéria cariogênica na presença do Terpinen-4-ol: estudo in vitro
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Data
2016-03-23
Autores
Bordini, Ester Alves Ferreira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A utilização de óleos essenciais tem sido amplamente difundida no tratamento de doenças infecciosas que acometem a cavidade oral por serem eficazes contra patógenos presentes em biofilme. Dentre estes produtos de origem natural, destaca-se o Terpinen-4-ol um biocida membrana-ativo, com amplo espectro de ação contra bactérias gram-positivas, gram-negativas e bactérias multi-resistentes. Neste estudo, investigou-se o efeito antimicrobiano do Terpinen-4-ol sobre culturas planctônicas e biofilmes de Streptococcus mutans (S. mutans) e a expressão gênica de glucano de ligação de proteína A (gbpA) envolvido na adesão em biofilme. A atividade antimicrobiana do Terpinen-4-ol (0.059 % a 0.95 %) foi avaliada pelo teste de microdiluição em caldo com a determinação da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM) para o microrganismo na forma planctônica. O biofilme formado em placa de cultura celular foi tratado com diferentes concentrações de Terpinen-4-ol e o metabolismo celular do biofilme resultante foi avaliado por meio de ensaio de hidróxido de 2,3-bis (2-metoxi-4-nitro-5-sulfo-fenil) -2H-tetrazólio-5-caboxanilide (XTT). A análise do biofilme formado sobre blocos de esmalte e dentina tratados com Terpinen-4-ol (0.24 % e 0.95 %) e Clorexidina (CHX 0.12 %) durante 60 segundos foi realizada por meio de ensaio de XTT e razão espectral (verde/vermelho) das imagens obtidas por Microscopia Confocal de Varredura a Laser (MCVL). A expressão gênica de gbpA foi investigada por RT-PCR quantitativo após exposição de S. mutans ao Terpinen-4-ol e CHX por 15 e 30 minutos. Os dados obtidos apresentaram distribuição normal e, portanto, foram utilizados os testes paramétricos de ANOVA One-way e de Tukey. Todos os testes estatísticos foram considerados em nível de significância de 5% (p<0.05). Terpinen-4-ol demonstrou atividade antimicrobiana a partir da concentração de 0.24 % tanto para o microrganismo em cultura planctônica quanto para biofilme formado em placa de cultura celular e esmalte, com redução microbiana próxima a 100 %. Não houve diferença estatisticamente significante entre Terpinen-4-ol e CHX. Para biofilme em dentina, a concentração de Terpinen-4-ol 0.95 % apresentou atividade antimicrobiana tanto pela análise por XTT quanto por MCVL. Houve redução na expressão de gbpA quando em contato com Terpinen-4-ol durante 15 minutos, no entanto a superexpressão gênica ocorreu a partir dos 30 minutos de exposição. CHX levou a superexpressão do gene gbpA em todos os tempos testados. Estes achados demonstram que Terpinen-4-ol apresenta importante atividade antimicrobiana e capacidade de modulação da expressão de genes envolvidos na formação de biofilme cariogênico, podendo ser utilizado no tratamento de doenças infecciosas bucais.
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.
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.
Descrição
Palavras-chave
Fatores de virulência, Cárie dentária, Medicamentos Fitoterápicos, Virulence factors, Dental caries, Phytotherapeutic frugs