“Análise da viabilidade e expressão de genes de virulência de Streptococcus mutans em lesões dentinárias de crianças com cárie precoce da infância”

Abstract

Early childhood caries (ECC) is characterized by the presence of one or more lesions on dental surface of children under 6 years old, being considered a public health issue. Streptococcus mutans (SM) is considered the main etiologic agent of the disease and has several virulence features that allow its survival in the oral cavity and, therefore, its selection under environmental stress situations. In this context, this research had as objectives; (a) to describe a method of total RNA extraction and purification to conduct studies on bacterial gene expression from in vivo dentine caries lesions (Chapter 01); (b) to identify and quantify the metabolically active SM and analyze the expression of its virulence genes atpD, fabM, nox, pdhA and aguD on active and arrested dentin lesions (Chapter 02). Twenty-nine samples of active dentin caries and 16 samples of arrested dentin lesions were collected from children with ECC. The samples were submitted to extraction and purification of total RNA through a customized method, based on the use of a mechanical lysis and a commercial extraction kit and submitted to reverse transcriptase (RT) reaction to obtain complementary DNA (cDNA). Next, to demonstrate the primers specificity for the virulence genes on in vivo samples, 06 cDNA samples were submitted to Sanger’s sequencing for each primer. After verifying the specificity, RT-qPCR (quantitative real-time reverse transcription polymerase chain reaction) were executed for all the samples. The results showed that the bacterial RNA could be extracted in appropriate quantity and quality through the customized protocol, making its use on gene expression studies on dentin affected by caries (Chapter 01). The RT-qPCR results showed that, despite the low quantification, if compared to total streptococci and total bacteria, SM was detected as metabolically active with similar quantification on active and arrested lesions (p>0.05). Despite that, a higher expression of the virulence genes pdhA and aguD was detected on arrested lesions (p≤0,05) (Chapter 02). It was concluded that after standardization of an effective RNA extraction technique, reactions of RT-qPCR were efficiently executed for SM identification, quantification, and gene expression on samples of human dentin affected by caries. This bacterium proved to be viable, however, only inactive lesions showed larger expression of the genes pdhA and aguD, probably to make its metabolic activity viable, even in unfavorable survival conditions.