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Study of bacterial diversity of infected root canal with acute apical abscess by culture, cloning and 16S rRNA sequencing

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Author(s):
Letícia Maria Menezes Nobrega
Total Authors: 1
Document type: Doctoral Thesis
Press: Piracicaba, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
Defense date:
Examining board members:
Brenda Paula Figueiredo de Almeida Gomes; Fabio Roberto Dametto; Rejane Andrade de Carvalho; Rogerio de Castilho Jacinto; Caio Cezar Randi Ferraz
Advisor: Brenda Paula Figueiredo de Almeida Gomes
Abstract

Acute apical abscesses are one of the most frequently treated conditions in endodontic emergency. It is a microbiologically heterogeneous disease presenting different bacterial profiles among the patients. Multiple bacterial combinations play a role in disease, acting synergistically and increasing their virulence, which leads to further damage to the host. Considering the microbial etiology of pulp and periapical disease, it is important to identify correctly microorganisms present in acute endodontic infection. Molecular methods of bacterial identification based on the 16S rRNA gene sequencing represent an important tool for identification and determination of the taxonomic position of microorganisms. The assessment of the endodontic microbiota by metagenomic approaches revealed that these techniques are sensitive and specific to evaluate the bacterial diversity of root canal infections, making possible the identification of some unexpected or not often associated with endodontic infection, including as-yet-uncultivated. The aim of this study was to investigate the bacterial diversity in the root canals of teeth with APA by culture, clonal analysis and 16S rRNA sequencing. Microbial samples were taken from 20 root canals using sterile paper points which were immediately placed into the VMGA III transport medium. A total of 220 isolated strains, previously identified by biochemical methods, were submitted to DNA extraction, 16S rRNA gene amplification and sequencing. Ten out of the 20 samples collected were also subjected to the clonal analysis using electrocompetent Escherichia coli DH5?. The nucleotides sequences obtained were compared with the GenBank database from National Center of Biotechnology Information through the BLAST. Thirty-four different bacteria were identified biochemically and 57 by 16S rRNA sequencing, in an average of 6 species per root canal. Sequencing allowed the identification of 97% of isolates against 70.5% identified biochemically. There was an agreement of 49% between the biochemical and 16S rRNA gene sequencing identification. Strains not identified biochemically (65/220) were characterized in 97% (63/65) by sequencing. The most frequently identified bacteria by sequencing were Prevotella spp., Pseudoramibacter alactolyticus, Parvimonas micra, Dialister invisus, Filifactor alocis and Peptostreptococcus stomatis. A total of 689 clones were analyzed and 76 phylotypes identified, of which 48 (63.15%) were different species and 28 (36.84%) were taxa reported as-yet-uncultivable or as yet-uncharacterized species. Prevotella spp., Fusobacterium nucleatum, Filifactor alocis and Peptostreptococcus stomatis, were the most frequently detected species, followed by Dialister invisus, Parvimonas micra, Phocaeicola abscessus, Porphyromonas spp. and the uncharacterized Lachnospiraceae oral clone. No specie was detected in all studied samples and some species were identified in only one case. Culture-independent methods shown that endodontic microbiota was underestimated in culture studies. Although some species predominate in acute primary endodontic infections, it was concluded that this infection is microbiologically heterogeneous, characterized by a wide diversity in which anaerobic gram-negatives are most frequently, and that the association of conventional and molecular approaches allow a better understanding of these microorganisms (AU)

FAPESP's process: 09/07760-5 - MICROBIAL INVESTIGATION OF INFECTED ROOT CANAL OF SYMPTOMATIC TEETH WITH PERIAPICAL LESION BY CULTURE, CLONING AND 16S rRAN SEQUENCING
Grantee:Letícia Maria Menezes Nóbrega
Support Opportunities: Scholarships in Brazil - Doctorate