Microorganisms and their virulence factors detection in saliva, pulp chamber and root canal of teeth associated with endodontic failure

Abstract

In the microbiota of secondary/ persistent infections predominate microorganisms resistant to endodontic procedures and able to survive in an environment with lack of space and nutrients, particularly Enterococcus faecalis. Coronal microleakage can be one of the causative agents of the failure of endodontic treatment, providing a constant source of saliva contaminants to the root canals. The aims of this study are: a) to study the saliva, pulp chamber and root canal microbiota composition of teeth with endodontic failure by checkerboard technique in order to investigate the similarity between microbiomes present in these 3 sites; b) to quantify the total bacterial load, Streptococcus spp and Enterococcus faecalis in saliva, pulp chamber and root canal, by Real Time - PCR; c) to quantify the levels of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) present in the root canals; and to correlate them with the patients clinical and radiographic aspects. Twenty patients with the presence of apical periodontitis and the need for endodontic retreatment will be selected. Samples from the endotoxic contents from saliva, pulp chamber and root canal will be collected. The extracted DNA will be submitted to the Real Time polymerase chain reaction for quantification of the bacterial load, and of the levels of Streptococcus spp. and Enterococcus faecalis. The involved microbial species will be identified by the checkerboard using probes for 40 different selected endodontic pathogens. LPS and LTA will be quantified using the Limulus Amoebocyte Lysate (LAL) and Enzyme-Linked Immunosorbent Assay (ELISA) methods, respectively. Data will be tabulated and statistically analyzed by SPSS for Windows.