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| Author(s): |
Priscila Amanda Francisco
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Piracicaba, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba |
| Defense date: | 2021-05-20 |
| Examining board members: |
Brenda Paula Figueiredo de Almeida Gomes;
Marcos Sergio Endo;
Daniel Rodrigo Herrera;
Caio Cezar Randi Ferraz;
José Flávio Affonso de Almeida
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| Advisor: | Brenda Paula Figueiredo de Almeida Gomes |
| Abstract | |
Microbial role in the intracanal infection can be inferred by its release of substances detected by meta-proteomic analyses. Furthermore, human proteins from the immunological response can equally be screened by proteomic technology. Considering the negative and sometimes life-threatening effects of the intracanal microbiota pathological actions, it is sometimes necessary the prescription of antibiotic supplementation in restrict caes in the treatment of endodontic infections. Thus, the aims of this study were: 1- to characterize by Mass Spectrometry the proteome of 60 root canals of teeth with different endodontic situation, as: a) vital pulp (VP); b) primary infection (PI) and c) secondary/persistent infection (SI); 2- to investigate the antimicrobial susceptibility of E. faecalis and S. mutans obtained from root canals affected by SI. Twenty patients with VP and periapical heath, 20 patients with apical periodontitis and PI and 20 patients with apical periodontitis and SI were selected. Samples from the root canal content were collected and processed by LC-ESI-MS/MS. The spectra generated were searched against human and microbial protein databases (Swiss PROT and TREMBL) using Proteome Discoverer 1.3 software, SEQUEST algorithm and UniProtKB tools. Strains of E. faecalis and S. mutans were recovered during 20 cases of endodontic retreatments through culture technics and confirmed by 16S rRNA gene sequencing. These isolates had their antimicrobial susceptibility tested through the E-test system. Diverse antimicrobial agents were tested. Minimum inhibitory concentration (MIC) breakpoints readings were done at the point of intersection between the ellipse-shaped inhibition halo and the E-test tape. Susceptibility or resistance was determined following interpretation guides. The MICs of antibiotics that inhibited 50% and 90% of the strains were calculated and expressed as MIC50 and MIC90, respectively. Through LC-ESI-MS/MS, 2950; 3871 and 2548 human and 3425; 5609 and 3544 microbial different accession numbers, were revealed from the root canal samples of vital pulp; primary infection with periapical lesion; and secondary infection with periapical lesion, respectively. Protein’s member of inflammation and bone regulations pathways as: MAPK, NF-kappa-B, MCSF-1 and RANKL, were encountered in all endodontic situations, while Calcium/calmodulin-dependent protein kinase type 1D, Tumor necrosis factor ligand superfamily member 11 and the Tumor necrosis factor receptor superfamily member 11A were found only at infections cases. Several microbial functions were present in all endodontic situations specially relating to cell adhesion, which varied between symbiosis or parasitic forms. Biofilm formation, toxin release and cell invasion molecules were also found. Thirty-seven strains of E. faecalis and seven of S. mutans were obtained. All E. faecalis isolates were susceptible to amoxicillin, amoxicillin + clavulanate, moxifloxacin and vancomycin. Higher rates (75 – 84%) of antimicrobial effectiveness have also been found for ciprofloxacin, chloramphenicol and doxycycline. Azithromycin (45.9%) and erythromycin (56.8%) showed intermediate rates of susceptibility, while 21.6 %, 24.3% and 24.3% of the E. faecalis strains expressed resistance to doxycycline, rifampicin and tetracycline, respectively. Three antimicrobial agents were totally effective against S. mutans, amoxicillin, amoxicillin + clavulanate and tetracycline. Besides, six strains were found to be susceptible to benzylpenicillin, clindamycin, erythromycin and vancomycin. Azithromycin and chloramphenicol showed the less favorable results, with susceptibility rates of 28.6% and 14.3%, respectively. Amoxicillin, amoxicillin + clavulanate acid and moxifloxacin had the lowest MIC90 for both strains. In conclusion, human and microbial proteins were identified in all endodontic conditions. PI contained a greater number of proteins than the others endodontic situations. Important inflammation and bone resorption pathways members were encountered, whilst many detected microbial proteins were related to symbiont adhesion to host tissues and pathogenic activities, such as: biofilm formation, toxin release, proteolysis, and cell invasion. E. faecalis strains were found to be totally susceptible to amoxicillin, amoxicillin + clavulanate, moxifloxacin, and vancomycin. While all S. mutans isolates were susceptible to amoxicillin, amoxicillin + clavulanate, and tetracycline. Furthermore, MIC90 showed that 1mg/ml of amoxicillin or amoxicillin + clavulanate acid, 0.75mg/ml of moxifloxacin, and 3mg/ml of vancomycin would be enough to inhibit at least 90% of both strains (AU) | |
| FAPESP's process: | 17/16516-7 - Investigation of the microbiological content, matrix metalloproteinase expression and antimicrobial susceptibility of specific bacteria in endodontic failure cases |
| Grantee: | Priscila Amanda Francisco |
| Support Opportunities: | Scholarships in Brazil - Doctorate |