Influencia de concentrações subinibitórias de agentes antimicrobianos sobre a fisiologia e genética de S.mutans
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Data
2016-12-16
Autores
Gazola Filho, Jaime [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Streptococcus mutans é conhecido por sua capacidade de aderir, colonizar e formar biofilme nas superfícies dentárias. Para sobreviver na cavidade bucal, S. mutans coordena a expressão de vários genes em resposta aos fatores ambientais, como na presença de agentes antimicrobianos. Os objetivos deste estudo foram: 1) avaliar a influência de concentrações subinibitórias (sub-MIC) de fluoreto de sódio (NaF) e digluconato de clorexidina (Chx) sobre o crescimento planctônico; 2) avaliar a influência desses antimicrobianos em concentrações sub-MIC na formação de biofilme das cepas de S. mutans; 3) avaliar a expressão dos genes covR e vicR de cepas de S. mutans expostas aos sub-MIC de Chx e NaF. Cepas de S. mutans padrão (ATCC 25175 e UA159) e isolada de criança com cárie precoce da infância (3FV2) foram reativadas em BHI caldo e expostas a Chx e NaF para determinação da MIC e MBC. A partir desses valores, curvas de crescimento foram obtidas para as cepas de S. mutans expostas a concentrações sub-MIC de Chx (0,25 x MIC a 0,75 x MIC) e NaF (0,125 x MIC a 0,75 x MIC). Ensaios de biofilme foram realizados para as cepas de S. mutans expostas ou não aos sub-MIC dos agentes antimicrobianos (0,25 x MIC; 0,5 x MIC e 0,75 x MIC). A expressão dos genes covR e vicR foram avaliadas por PCR quantitativo para as cepas de S. mutans em condições planctônicas expostas a 0,125/0,25/0,5 x MIC de NaF e Chx. Os dados obtidos foram submetidos à análise estatística considerando p≤0,05. Os resultados mostraram que sub-MIC de Chx e de NaF afetaram o crescimento planctônico das cepas de S. mutans, de maneira dose-dependente. Sub-MIC de Chx afetou somente a formação de biofilme de 3VF2 e valores de NaF acima de 0,5 x MIC causaram redução do crescimento do biofilme para todas as cepas testadas, de maneira dose-dependente, com exceção da cepa ATCC 25175, que foi afetada igualmente por todas as concentrações avaliadas. Concentrações sub-MIC gradativas de Chx induziram a expressão crescente do gene vicR das cepas avaliadas. Quando NaF e Chx foram associados, o gene covR teve sua expressão reduzida quando expostas a 0,5 x MIC. Conclui-se que concentrações sub-MICs de NaF acima de 0,5 x MIC causaram redução do crescimento planctônico e em biofilme das cepas de S. mutans. Concentrações sub-MIC de Chx induzem a expressão de vicR podendo favorecer a formação de biofilme pelas cepas de S. mutans.
Streptococcus mutans is known for its ability to adhere, colonize and form biofilm on dental surfaces. To survive in the oral cavity, S. mutans coordinates the expression of several genes in response to environmental factors, such as in the presence of antimicrobial agents. The objectives of this study were: 1); to evaluate the influence of subinhibitory concentrations (sub-MIC) of chlorhexidine (Chx) and sodium fluoride (NaF) on planktonic growth 2) to evaluate the influence of sub-MIC of these agents on biofilm formation of S. mutans strains; 3) to evaluate the expression of the covR and vicR genes of S. mutans strains exposed to sub-MIC of Chx and NaF. Standard strains of S. mutans (ATCC 25175 or UA159) and clinical strain isolated from child with caries (3FV2) were reactivated in BHI broth and exposed to Chx and NaF for determination of MIC and MBC. From these values, growth curves were obtained for S. mutans strains exposed to sub-MIC concentrations of Chx (0.25 x MIC to 0.75 x MIC) and NaF (0.125 x MIC to 0.75 x MIC). Biofilm assays were performed for S. mutans strains exposed or not to the sub-MIC of antimicrobial agents (0.25, 0.5 and 0.75 x MIC). Expression of covR and vicR genes were evaluated by quantitative PCR for strains of S. mutans under plankton conditions exposed to 0.125 / 0.25 / 0.5 x MIC of NaF and Chx. The data were submitted to statistical analysis considering p≤0.05. The results showed that Chx and NaF sub-MIC affected planktonic growth of S. mutans strains in a dose-dependent manner. Sub-MIC of Chx only affect the biofilm formation for ATCC 25175, and, NaF values above 0.5 x MIC caused reduction of the biofilm growth for all tested strains, in a dose-dependent manner, except for ATCC 25175, which was also affected by all concentrations evaluated. Gradual sub-MIC concentrations of Chx induced increasing expression of the vicR gene of the strains evaluated. When NaF and Chx were related, the covR gene had its expression reduced when exposed to 0.5xMIC. It is concluded that sub-MICs concentrations of NaF above 0.5 x MIC caused reduction of planktonic and biofilm growth of S. mutans strains. Sub-MICs concentrations of Chx induce vicR expression and may favor biofilm formation by S. mutans strains.
Streptococcus mutans is known for its ability to adhere, colonize and form biofilm on dental surfaces. To survive in the oral cavity, S. mutans coordinates the expression of several genes in response to environmental factors, such as in the presence of antimicrobial agents. The objectives of this study were: 1); to evaluate the influence of subinhibitory concentrations (sub-MIC) of chlorhexidine (Chx) and sodium fluoride (NaF) on planktonic growth 2) to evaluate the influence of sub-MIC of these agents on biofilm formation of S. mutans strains; 3) to evaluate the expression of the covR and vicR genes of S. mutans strains exposed to sub-MIC of Chx and NaF. Standard strains of S. mutans (ATCC 25175 or UA159) and clinical strain isolated from child with caries (3FV2) were reactivated in BHI broth and exposed to Chx and NaF for determination of MIC and MBC. From these values, growth curves were obtained for S. mutans strains exposed to sub-MIC concentrations of Chx (0.25 x MIC to 0.75 x MIC) and NaF (0.125 x MIC to 0.75 x MIC). Biofilm assays were performed for S. mutans strains exposed or not to the sub-MIC of antimicrobial agents (0.25, 0.5 and 0.75 x MIC). Expression of covR and vicR genes were evaluated by quantitative PCR for strains of S. mutans under plankton conditions exposed to 0.125 / 0.25 / 0.5 x MIC of NaF and Chx. The data were submitted to statistical analysis considering p≤0.05. The results showed that Chx and NaF sub-MIC affected planktonic growth of S. mutans strains in a dose-dependent manner. Sub-MIC of Chx only affect the biofilm formation for ATCC 25175, and, NaF values above 0.5 x MIC caused reduction of the biofilm growth for all tested strains, in a dose-dependent manner, except for ATCC 25175, which was also affected by all concentrations evaluated. Gradual sub-MIC concentrations of Chx induced increasing expression of the vicR gene of the strains evaluated. When NaF and Chx were related, the covR gene had its expression reduced when exposed to 0.5xMIC. It is concluded that sub-MICs concentrations of NaF above 0.5 x MIC caused reduction of planktonic and biofilm growth of S. mutans strains. Sub-MICs concentrations of Chx induce vicR expression and may favor biofilm formation by S. mutans strains.
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Palavras-chave
Streptococcus mutans, Cárie dentária, Teste de sensibilidade microbiana, Fluoreto de sódio, Clorexidina