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

Author(s)

Priscila Amanda Francisco

• Document type: Master's Dissertation
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2017-02-22

Examining board members

Brenda Paula Figueiredo de Almeida Gomes; Francisco Montagner; José Flávio Affonso de Almeida

• Advisor: Brenda Paula Figueiredo de Almeida Gomes

Abstract

In the microbiota of secondary/persistent infections, microorganisms resistant to endodontic procedures predominate, especially Enterococcus faecalis. Coronary microleakage is an etiologic agent of endodontic treatment failure, promoting a constant source of saliva contamination to the root canals. The aims of the present study were: a) to study the microbiota composition of saliva (S), pulp chamber (PC) and root canals (RC) of teeth with endodontic failure, by means of the checkerboard technique, in order to investigate the simultaneity among the bacterial species present in these three sites; b) to quantify the levels of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) existing in the S, PC and RC investigated; c) to correlate the bacteria found between the sites, within the sites and with the LPS/LTA; d) to correlate the microorganisms and the LPS/LTA with the patient clinical aspects. Twenty teeth with presence of periapical lesion and need for endodontic retreatment, were selected. Samples were collected from saliva, pulp chamber and root canal. The extracted DNA was subjected to the checkerboard method using probes for 40 different species. The LPS and LTA samples were quantified by the Limulus Amoebocyte Lysate (LAL) and Enzyme-Linked Immunosorbent Assay (ELISA) methods, respectively. Bacteria were detected in all samples, both Gram-positive and Gram-negative, facultative and strict anaerobes, with an average number of species of 35, 20, and 29, for S, PC and RC, respectively. Simultaneous bacteria most found, at the 3 studied sites, were: E. faecium, P. micra, F. nucleatum sp. nucleatum, E. faecalis, E. saburreum and C. ochracea. LPS and LTA were found in 95% and 100% of the RC, with a mean of 3.26 EU/mL and 578.67 pg/mL. Highly significant positive and negative associations (p <0.01) were found between species for each site, being 2 positive in S; 66 positive and 44 negative in PC; and 7 positive and 11 negative in RC, all with ODDS values> 2 or <0.5. A positive statistical association (p <0.05) between the sites of the teeth showing signs of microleakage (11/20) was found between S. oralis from S and S. oralis from RC (p = 0.015 and ODDS = 7). In the teeth that did not show signs of microleakage (9/20) a negative association was found between C. showae of PC and C. showae from RC (p = 0.048 and ODDS = 0.250). There were four positive associations between the Gram-negative bacteria concentration and the LPS (V. parvula-PC/LPS-S, C. showae-PC/LPS-S and C. ochracea-PC/LPS-RC), and two negative (F. nucleatum sp. nucleatum-PC/LPS-PC and D. pneumosintes-RC/LPS-RC) (p <0.01). Between the Gram-positive bacteria concentration and the LTA, there were one positive association (E. hirae-RC/LTA-PC) and three negative (all at PC between LTA and E. nodatum, S. mitis and E. nodatum) (p <0.01). With regard to correlations with the clinical aspects of patients, three significant negative associations (p <0.05) were found between pain to percussion and D. pneumosintes, F. periodonticum and E. corrodens. LPS of S presented a moderate positive correlation with the fistula presence (p = 0.02). The pain to percussion and LTA of the PC had a positive positive correlation (p = 0.035). It was concluded that the microbiota of root canal associated with secondary/persistent infection is heterogeneous, with a similarity among the microbiotas in the 3 sites, with greater detection of LTA than LPS. Associations between the microorganisms present in the saliva, pulp chamber and root canal suggest a possible communication among the sites (AU)

• FAPESP's process: 15/19215-2 - Microorganisms and their virulence factors detection in saliva, pulp chamber and root canal of teeth associated with endodontic failure
• Grantee: Priscila Amanda Francisco
• Support Opportunities: Scholarships in Brazil - Master