Analysis of Metabolically Active Bacterial Community of Persistent Endodontic Infections after Different Clinical Protocols of Disinfection: RNA- and DNA-based Molecular Study

Abstract

The success of endodontic treatment in teeth with necrotic pulp and apical periodontitis depends on the reduction of viable microorganisms of root canals by endodontic disinfection procedures. Our data has indicated an association of molecular methods based on rRNA and rDNA to allow simultaneous evaluation of metabolic activity and abundance of species/ taxa in a bacterial community. This proposal aims to evaluate by molecular methods the effect of different disinfection protocols on reduction, diversity and microbial metabolic activity. For this study, 75 patients with pulp necrosis and apical periodontitis will be selected. Microbiological sampling of root canals will be performed at the beginning of endodontic treatment (S1) and after chemical-mechanical preparation with Reciproc System and NaOCl 2.5% (S2). Next, patients will be divided into groups according to supplementary antimicrobial methods performed after chemical-mechanical preparation: XP (XP-endo Finisher); Group PIPS (irrigation activated by laser using the Photon-Induced Photoacoustic Streaming tips); and IC Group (conventional irrigation without activation). New microbiological sampling of root canals will be performed after the supplementary antimicrobial methods (S3), after intracanal medication with calcium hydroxide for 14 days (S4) and after re-instrumentation before root canal filling (S5). Root canal samples will be submitted to the total nucleic acids extraction. The effect of treatment protocols on total microbiota will be determined by rDNA-based qPCR using universal primers for Bacteria domain. RNA of S1, S3 and S4 samples from 30 patients (10 of each group) will be used to determine the diversity of metabolically active microbiota by Reverse Transcriptase Reaction (RT), followed by amplification (PCR) and high throughput sequencing of the hypervariable region V1-V2-V3 of 16S rRNA gene. The metabolic activity of the most prevalent species / taxa will be calculated by rRNA/ rDNA ratio estimated by qPCR using species-specific primers. The data will be analyzed by statistical tests, with 5% significance level. (AU)