Cultivable bacteria in infected root canals as identified by 16S rRNA gene sequencing

Abstract

Introduction: Traditionally, cultivable bacteria isolated from infected root canals have been identified by phenotype-based methods. Because 16S ribosomal RNA (rRNA) gene sequencing has emerged as a more accurate and reliable tool for bacterial identification, the present study applied this approach to identify bacterial isolates recovered from the root canals of teeth with chronic apical periodontitis. Methods: Anaerobic techniques were used for culturing; identification of the isolates was carried out by polymerase chain reaction amplification and sequencing of the V5–V8 region of the 16S rRNA gene. Bacteria were found in all samples. The mean number of taxa per canal was 3.1, ranging from 2 to 8. The median number of cultivable bacterial cells in the root canals was 4.2 × 10⁵, ranging from 2.8 × 10³ to 3.3 × 10⁷. Eighty-seven strains belonging to 52 bacterial taxa were identified. The most prevalent taxa were Fusobacterium nucleatum, Porphyromonas gingivalis, Pseudoramibacter alactolyticus, Micromonas micros and streptococci. The following bacterial phyla were represented in this study: Firmicutes (22 taxa, 46% of the identified isolates), Actinobacteria (14 taxa, 25.3% of the isolates), Bacteroidetes (eight taxa, 13.8% of the isolates), Fusobacteria (three taxa, 9.2% of the isolates) and Proteobacteria (five taxa, 5.7% of the isolates). Some of the isolates represented unnamed species not previously cultivated and characterized. In conclusion, our findings using a combined anaerobic culture–molecular identification approach confirmed the polymicrobial nature of primary endodontic infections with dominance of anaerobic bacteria. Notably several bacteria that are difficult or impossible to identify by phenotypic means were identified, including previously uncultivated taxa, cultivated-but-not-yet-characterized taxa and newly named species.