Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies / Extração e purificação de RNA proveniente de dentina humana cariada: uma abordagem para viabilizar estudos de expressão gênica

Background: RNA isolation from bacteria within dentine caries lesions could be difficult due to reduced amount of collectable biomass and high mRNA instability. Attempting to overcome this challenge we describe one protocol developed to extract and purify total RNA from dentine lesions. Objective: customize a bacterial RNA extraction and purification method from human carious dentine. Methods: quantity and purity of extracted RNA were measured with a microvolume UV-VIS spectrophotometer, RNA integrity was assessed by standard denaturing agarose gel electrophoresis and images were captured under ultraviolet light with camera and analyzed. DNase treatment removed genomic DNA and an additional step of purification was carried out in silica spin column. Results: final yield (ng/µl) was 67.01 ± 22.33, absorbance ratio A260/A280 = 2.0 ± 0.07 and RNA integrity were obtained. The purified samples were reversely transcribed and the expression of atpD and fabM gene from Streptococcus mutans analyzed by quantitative real-time PCR. Conclusion: the extraction methodology developed produced high-quality RNA from dentine microbiota for transcriptional analysis.

Introdução: o isolamento de RNA de bactérias dentro de lesões de dentina cariada pode ser difícil devido à quantidade reduzida de biomassa e alta instabilidade de mRNA. Na tentativa de superar esse desafio, descrevemos um protocolo desenvolvido para extrair e purificar o RNA total das lesões dentinárias. Objetivo: personalizar um método de extração e purificação de RNA bacteriano a partir da dentina cariada humana. Métodos: a quantidade e a pureza do RNA extraído foram medidas com um espectrofotômetro UV-VIS de microvolume, a integridade do RNA foi avaliada por eletroforese em gel de agarose desnaturante padrão e as imagens foram capturadas sob luz ultravioleta e analisadas. O tratamento com DNase removeu o DNA genômico e uma etapa adicional de purificação foi realizada em coluna de spin de sílica. Resultados: o rendimento final (ng / µl) foi de 67,01 ± 22,33, a razão de absorbância A260 / A280 = 2,0 ± 0,07 e a integridade do RNA foram obtidas. As amostras purificadas foram transcritas reversamente e a expressão do gene atpD e fabM de Streptococcus mutans analisadas por PCR quantitativo em tempo real (RT-qPCR). Conclusão: a metodologia de extração desenvolvida produziu RNA de alta qualidade da microbiota dentinária para análises transcricionais.

Antimicrobial activity of mouth rinses against bacteria that initially colonizes dental's surface / Atividade antimicrobiana de enxaguatórios bucais sobre bactérias que iniciam a colonização das superfícies dentais

Abstract Introduction Much advertising in mouthwash is conveyed in all media appealing to the anti-plaque effect and rendering a disservice to the community. Mouth rinses are available over-the-count and differ on their compositions and antimicrobial effectiveness. Objective In this study, we evaluated the antimicrobial activity of 35 widely available mouth rinses against bacterial species involved in initiation of dental biofilm - Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius, and Streptococcus sanguinis. Material and method The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the evaluated mouth rinses were determined according to the Clinical & Laboratory Standards Institute protocols. Data were submitted to Kruskal-Wallis test and Mann-Whitney post hoc (α=0.05). Result About 70% of the mouth rinses achieved high antibacterial activity and 30%, a low antibacterial activity against all the species tested. The most ineffective mouth rinse showed antibacterial activity (MIC) at 1:1 dilution, while the most effective showed activity even at 1:2048 dilution, which may imply prolonged effect in the mouth. About 51% of mouth rinses showed bactericidal activity, and it was verified that cetylpyridinium chloride or chlorhexidine digluconate containing in the formulation were associated with the highest activity. Conclusion Most - but not all - mouth rinses commercially available are effective in inhibiting in vitro initial colonizers of dental surfaces.

Resumo Introdução Muita publicidade sobre enxaguatórios bucais é veiculada em todos os meios de comunicação apelando para o efeito anti-placa e prestando um desserviço à comunidade. Grande quantidade de enxaguatórios bucais está disponível no mercado e estes diferem em suas composições e eficácia antimicrobiana. Objetivo Neste estudo, avaliamos a atividade antimicrobiana de 35 enxaguatórios bucais amplamente disponíveis contra espécies bacterianas envolvidas na iniciação do biofilme dental - Streptococcus gordonii, Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius e Streptococcus sanguinis. Material e método A Concentração Inibitória Mínima (CIM) e a Concentração Bactericida Mínima (CBM) dos enxaguatórios avaliados foram determinadas de acordo com os protocolos do Clinical & Laboratory Standards Institute. Os dados foram submetidos ao teste Kruskal-Wallis e Mann-Whitney post hoc (α=0,05). Resultado Aproximadamente 70% dos enxaguatórios bucais alcançaram alta atividade antibacteriana e 30%, baixa atividade antibacteriana contra todas as espécies testadas. O enxaguatório bucal mais ineficaz mostrou atividade antibacteriana (CIM) na diluição de 1:1, enquanto a mais eficaz mostrou atividade mesmo na diluição de 1:2048, o que pode implicar em efeito prolongado na boca. Cerca de 51% dos enxaguatórios bucais apresentaram atividade bactericida, e verificou-se que formulações contendo cloreto de cetilpiridíneo ou digluconato de clorexidina estavam associados à maior atividade. Conclusão A maior parte - mas não todos - dos enxaguatórios bucais comercialmente disponíveis são eficazes na inibição de colonizadores iniciais de superfícies dentárias in vitro.

Effect of iodonium salt and chitosan on the physical and antibacterial properties of experimental infiltrants

Abstract Resinous infiltrants are indicated in the treatment of incipient carious lesions, and further development of these materials may contribute to greater control of these lesions. The aim of this study was to analyze the physical and antibacterial properties of experimental infiltrants containing iodonium salt and chitosan. Nine experimental infiltrants were formulated by varying the concentration of the diphenyliodonium salt (DPI) at 0, 0.5 and 1 mol%; and chitosan at 0, 0.12 and 0.25 g%. The infiltrants contained the monomeric base of triethylene glycol dimethacrylate and bisphenol-A dimethacrylate ethoxylate in a 75 and 25% proportion by weight, respectively; 0.5 mol% camphorquinone and 1 mol% ethyl 4-dimethylaminobenzoate. The degree of conversion was evaluated using Fourier transformer infrared spectroscopy, and the flexural strength and elastic modulus using the three-point bending test. Sorption and solubility in water, and antibacterial analysis (minimum inhibitory concentration and minimum bactericidal concentration) were also analyzed. Data was analyzed statistically by two-way ANOVA and Tukey's test (p<0.05), with the exception of the antibacterial test, which was evaluated by visual inspection. In general, the infiltrant group containing 0.5% DPI and 0.12% chitosan showed high values of degree of conversion, higher values of elastic modulus and flexural strength, and lower sorption values in relation to the other groups. Antibacterial activity was observed in all the groups with DPI, regardless of the concentration of chitosan. The addition of DPI and chitosan to experimental infiltrants represents a valid option for producing infiltrants with desirable physical and antibacterial characteristics.