Efeito do tirosol sobre biofilmes de Streptococcus mutans
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Data
2016-02-29
Autores
Arias, Laís Salomão [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O tirosol é uma molécula de quorum-sensing (QS) que participa do controle da morfogênese em Candida albicans. Contudo, seu efeito como agente antimicrobiano sobre biofilmes de Streptococcus mutans permanece desconhecido. Assim, o objetivo deste estudo foi avaliar o efeito do tirosol em diferentes concentrações sobre a produção de ácido e formação de biofilmes por S. mutans ATCC 25175, bem como quantificar biofilmes pré-formados desta espécie desenvolvidos sobre espécimes de hidroxiapatita (HA) e tratados com tirosol. A concentração inibitória mínima (CIM) de tirosol sobre células no estado planctônico foi determinada de acordo com o método da microdiluição. Na sequência, biofilmes de S. mutans foram formados durante 48 horas sobre espécimes de HA na presença de diferentes concentrações de tirosol (11,25, 22,5, 50, 100 e 200 mmol l-1), usando saliva artificial como meio de cultura. Após, a produção de ácido foi avaliada através da mensuração do pH do meio, enquanto a formação de biofilmes foi determinada através da quantificação da biomassa total (BT), atividade metabólica (AM) e número de unidades formadoras de colônias (UFCs). Ainda, biofilmes pré-formados (24 horas) de S. mutans foram tratados com tirosol a 100 e 200 mmol l-1 por 1 minuto, duas vezes ao dia, durante três dias, totalizando biofilmes de 96 horas. Em seguida, a atividade antimicrobiana foi avaliada por meio da quantificação da BT, AM, número de UFCs e composição da matriz extracelular (proteínas e carboidratos). Gluconato de clorexidina (490 μmol l-1) foi usado como controle positivo. Os dados foram analisados por ANOVA a um critério, seguido pelos testes de Tukey e Holm-Sidak (α = 0,05). Microscopia eletrônica de varredura (MEV) foi utilizada na análise da estrutura dos biofilmes. A CIM de tirosol foi 90 mmol l-1. Tirosol em concentrações subinibitórias (11,25 e 22,5 mmol l-1) não reduziu a produção de ácido dos biofilmes de S. mutans. Entretanto, biofilmes formados na presença de tirosol a 50, 100 e 200 mmol l-1 mostraram reduções significativas na AM e no número de UFCs, variando de 23,4 a 85,5 % e 1,19 a 4,54-log10, respectivamente. Para os biofilmes pré-formados, os tratamentos com tirosol não promoveram reduções significativas, exceto para a AM do biofilme tratado com tirosol a 200 mmol l-1, a qual mostrou uma redução de 40 % (p = 0.015) quando comparada ao controle negativo. Ainda, o tratamento com tirosol a 200 mmol l-1 resultou em aumento significativo no conteúdo proteico da matriz extracelular do biofilme pré-formado de S. mutans. As imagens de MEV confirmaram os resultados de quantificação das UFCs. Portanto, foi possível concluir que o tirosol mostrou melhores efeitos sobre a formação de biofilmes do que sobre biofilmes pré-formados, e esta molécula de QS não foi capaz de reduzir a produção de ácido dos biofilmes de S. mutans. Esses resultados podem ser úteis no desenvolvimento de terapias tópicas voltadas para a prevenção de doenças orais associadas aos biofilmes, como a cárie dentária.
Tyrosol is a quorum-sensing molecule (QS) that participates in the control of Candida albicans morphogenesis. However, its effect as an antimicrobial agent on Streptococcus mutans biofilms remains unknown. Thus, the aim of this study was to evaluate the effect of tyrosol at different concentrations on the acid production and biofilm formation by S. mutans ATCC 25175, as well as to quantify pre-formed biofilms of this species developed on hydroxyapatite (HA) specimens and treated with tyrosol. Minimum inhibitory concentration (MIC) of tyrosol against planktonic cells was determined in accordance with the microdilution method. Subsequently, S. mutans biofilms were formed during 48 hours on HA specimens in the presence of different concentrations of tyrosol (11.25, 22.5, 50, 100 and 200 mmol l-1), using artificial saliva as culture medium. Next, the acid production was assessed by pH determination of the medium, while the biofilm formation was determined through quantification of total biomass (TB), metabolic activity (MA) and number of colony-forming units (CFUs). Further, S. mutans pre-formed biofilms (24 h) were treated with tyrosol at 100 and 200 mmol l-1 twice a day for 1 min, during 3 days, totaling 96-h biofilms. Then, the antimicrobial activity was evaluated through quantification of TB, MA, number of CFUs and composition of biofilms’ extracellular matrix (proteins and carbohydrates). Chlorhexidine gluconate (490 μmol l-1) was used as positive control. Data were analyzed by one-way ANOVA, followed by Tukey’s and Holm-Sidak's tests (α = 0.05). Scanning electron microscopy (SEM) observations were performed in order to analyze biofilms’ structure. MIC of tyrosol was 90 mmol l-1. Tyrosol at sub-inhibitory concentrations (11.25 and 22.5 mmol l-1) was not able to significantly reduce acid production by S. mutans biofilms. However, biofilms formed in the presence of tyrosol at 50, 100 and 200 mmol l-1 showed significant decreases in MA and number of CFUs, ranging from 23.4 to 85.5 % and 1.19 to 4.54-log10, respectively. For pre-formed biofilms, the treatments with tyrosol did not promote significant reductions, except for MA of biofilm treated with 200 mmol l-1 tyrosol, which showed a 40 % reduction (p = 0.015) compared to the negative control. Moreover, treatment with 200 mmol l-1 tyrosol resulted in a significant increase in the protein content of the extracellular matrix of S. mutans pre-formed biofilm. SEM observations confirmed the results of CFU enumeration. In conclusion, tyrosol showed better effects on biofilm formation than on pre-formed biofilm, and this QS molecule was not able to reduce acid production by S. mutans biofilms. These results may be useful in the development of topical therapies focused on preventing biofilm-associated oral diseases, such as dental caries.
Tyrosol is a quorum-sensing molecule (QS) that participates in the control of Candida albicans morphogenesis. However, its effect as an antimicrobial agent on Streptococcus mutans biofilms remains unknown. Thus, the aim of this study was to evaluate the effect of tyrosol at different concentrations on the acid production and biofilm formation by S. mutans ATCC 25175, as well as to quantify pre-formed biofilms of this species developed on hydroxyapatite (HA) specimens and treated with tyrosol. Minimum inhibitory concentration (MIC) of tyrosol against planktonic cells was determined in accordance with the microdilution method. Subsequently, S. mutans biofilms were formed during 48 hours on HA specimens in the presence of different concentrations of tyrosol (11.25, 22.5, 50, 100 and 200 mmol l-1), using artificial saliva as culture medium. Next, the acid production was assessed by pH determination of the medium, while the biofilm formation was determined through quantification of total biomass (TB), metabolic activity (MA) and number of colony-forming units (CFUs). Further, S. mutans pre-formed biofilms (24 h) were treated with tyrosol at 100 and 200 mmol l-1 twice a day for 1 min, during 3 days, totaling 96-h biofilms. Then, the antimicrobial activity was evaluated through quantification of TB, MA, number of CFUs and composition of biofilms’ extracellular matrix (proteins and carbohydrates). Chlorhexidine gluconate (490 μmol l-1) was used as positive control. Data were analyzed by one-way ANOVA, followed by Tukey’s and Holm-Sidak's tests (α = 0.05). Scanning electron microscopy (SEM) observations were performed in order to analyze biofilms’ structure. MIC of tyrosol was 90 mmol l-1. Tyrosol at sub-inhibitory concentrations (11.25 and 22.5 mmol l-1) was not able to significantly reduce acid production by S. mutans biofilms. However, biofilms formed in the presence of tyrosol at 50, 100 and 200 mmol l-1 showed significant decreases in MA and number of CFUs, ranging from 23.4 to 85.5 % and 1.19 to 4.54-log10, respectively. For pre-formed biofilms, the treatments with tyrosol did not promote significant reductions, except for MA of biofilm treated with 200 mmol l-1 tyrosol, which showed a 40 % reduction (p = 0.015) compared to the negative control. Moreover, treatment with 200 mmol l-1 tyrosol resulted in a significant increase in the protein content of the extracellular matrix of S. mutans pre-formed biofilm. SEM observations confirmed the results of CFU enumeration. In conclusion, tyrosol showed better effects on biofilm formation than on pre-formed biofilm, and this QS molecule was not able to reduce acid production by S. mutans biofilms. These results may be useful in the development of topical therapies focused on preventing biofilm-associated oral diseases, such as dental caries.
Descrição
Palavras-chave
Percepção de quorum, Anti-infecciosos, Biofilmes, Streptococcus mutans, Tirosol, Quorum Sensing, Antimicrobials, Biofilms, Tyrosol