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Microbiological profile analysis using nanopore technology and characterization of s. mutans isolated from primary endodontic infections

Grant number: 18/09270-4
Support type:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): September 01, 2018
Effective date (End): August 31, 2019
Field of knowledge:Health Sciences - Dentistry
Principal Investigator:Brenda Paula Figueiredo de Almeida Gomes
Grantee:Augusto Rodrigues Lima
Supervisor abroad: Jacqueline Abranches
Home Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil
Local de pesquisa : University of Florida, Gainesville (UF), United States  
Associated to the scholarship:16/23950-2 - Monitoring of infectious contents, endotoxic and gene presence of resistance to antibiotics of symptomatic and asymptomatic primary infections during endodontic therapy, BP.DR

Abstract

The knowledge of the microbiota involved in symptomatic and asymptomatic infections may clarify reasons leading to the exacerbation or chronification of an endodontic infection. Moreover, the microbial community of endodontic diseases can also be associated with systemic infections. Recently, a study found that the cariogenic Streptococcus mutans was frequently found in pulpitis. In addition to caries, S. mutans is commonly associated with extra-oral infections such as spesis and infective endocarditis. Thus, the aims of this study are: 1) to characterize the complete bacterial, fungal and viral genomes profiling with nanopore sequencing of 3 specific sites: a) root canals of necrotic teeth with symptomatology; b) abscesses associated with such canals; and c) root canals of necrotic but asymptomatic teeth and 2) to characterize strains of S. mutans isolated from root canals of primary endodontic infections with radiographic periapical lesion in relation to a) the presence of collagen adhesion proteins; b) biofilm formation capacity in the presence of sucrose and glucose; c) ability to survive extreme acid or peroxide challenges; d) systemic virulence in Galleria mellonella. Patients with a necrotic teeth with symptomatology and associated with acute apical abscess (n = 10) and patients with the presence of pulpal necrosis associated with radiographic periapical lesion (n = 10) were selected. Samples for microbiome analysis were collected from root canals [10 symptomatic and 10 asymptomatic] and and dental abscesses [10 associated with a symptomatic cases]. DNA will be extracted from the samples from 3 sites and will be performed the genomic characterization using the Nanopore sequencing. Microbiological samples were serially diluted and plated on Mitis Salivarius Agar supplemented with sucrose and bacitracin. Isolated colonies will be screened by polymerase chain reaction (PCR) using S. mutans -specific primers. The serotype of each isolated colonies will be determined as serotype c-, e-, f- or k- by PCR , using specific primers. CNM-541F and CNM-9011R primers will be used to verify the presence of cnm protein. In addition, isolated strains evaluated for biofilm formation capacity in the presence of sucrose and glucose at plaque, stress tolerance and systemic virulence in a Galleria mellonella model. The data will be tabulated and statistically analyzed.