Multispecies biofilm models to study new therapeutic strategies against secondary/persistent endodontic infections

Abstract

Post-treatment apical periodontitis is a polymicrobial disease. Identifying the active bacterial composition in endodontic infections is essential to direct effective treatment strategies. Recently, new approaches have been suggested for removal of polymicrobial biofilm-associated infections, including the use of quorum-sensing inhibitors, enzymes, and antimicrobial peptides or amino acids. Interestingly, promising results were shown in inhibiting biofilm formation and/or decreasing the viability of mono-species biofilms formed by oral or non-oral bacterial species. However, to date, there is little information on the effectiveness of the aforementioned approaches on multispecies biofilms. Objectives: The purposes of this study will be as follows: (a) to investigate the active bacterial community of teeth with post-treatment apical periodontitis; (b) to examine the bacterial colonization patterns/ spatial arrangement of bacteria on gutta-percha cones; and (c) to explore the antimicrobial properties of an antimicrobial peptides mixture consisting of lantibiotic nisin and D-amino acids (DDAs) on endodontic multispecies biofilms. Methods: Next Generation Sequence (NGS) approaches based on ribosomal RNA (rRNA) and rDNA genes will be used for analysis of root canal samples of teeth with post-treatment apical periodontitis. DNA and RNA will be extracted from samples, and complementary DNA (cDNA) will be synthetized using reverse transcription reaction. DNA and cDNA will be subject to PCR with primers that amplify V1-V2 region of 16S rRNA gene followed NGS analysis. The relationship between 16S rRNA and rDNA frequency for each operational taxonomic unit (OTU) will be used as an index of activity of specific taxa in the root canal samples. In order to provide additional information on the bacterial colonization of the obturation materials, gutta-percha samples from root canals will be analyzed using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Additionally, a modified subgingival biofilm model, which consists of 11 species, will be exposed to a mixture of the antimicrobial peptides lantibiotic nisin and DDAs alone or combined with NaOCl 1%, and biofilm viability will be assessed by colony forming units (CFUs) and bacterial viability staining solution/CLSM image analysis. (AU)