Methodological approaches for the bacteriome analysis of the root canal system of a tooth affected by apical periodontitis
Abstract
Introduction, aim: Apical periodontitis (AP) is an inflammatory disease of the dental periradicular tissues caused by a bacterial infection. Various methodological approaches are used to determine the bacteria inhabiting the root canals, however, the analysis of the entire root system of a tooth affected by AP still remains a challenge.
The aim of our study was to perform a literature search focused on sample collection procedures and methodologies for bacteriome analysis, and then propose a suitable methodological approach for the purpose of studying the etiopathogenesis of this disease. Methods: After searching the PubMed database, we selected only publications of the original work type in which the bacterial DNA of human teeth was analyzed, for the search.
Results: Methodologically, the studies differ greatly, in terms of sample collection, DNA isolation, and bacterial DNA analysis itself. A common method of sample collection is the use of sterile endodontic paper points.
Although this method of sampling is suitable in clinical practice, it is considered insufficient for a comprehensive analysis of the environment of the root canal system, due to the morphology of the tooth itself and the presence of ramifications. Another method of sampling is resection of the root tip using sterile burs and subsequent grinding of the apex or smearing with sterile endodontic paper pins.
Only the apical part of the tooth is used to determine the bacteriome, therefore bacteria that colonize the coronal part of the tooth and participate in the etiopathogenesis of the disease cannot be analyzed. In recent studies, a method is used in which the entire extracted tooth affected by AP is ground into a fine homogeneous powder using cryogenic grinding.
It is possible to determine the complex bacteriome of the root canal system and the pulp chamber from the dust of a crushed tooth, and therefore this method seems optimal for the sample preparation from an experimental study point of view. Most often, an effective column method with various purification kits is used for DNA isolation, and for subsequent DNA analysis, methodologies based on the principle of the polymerase chain reaction are mostly used.
Sequencing the variable regions of the gene for 16S rRNA is nowadays already the gold standard for categorizing bacteria and characterizing bacterial communities. Conclusion: To study the AP bacteriome, it seems most appropriate to use extracted tooth samples and immediate freezing of the sample without further preanalytical steps.
The crushed tooth is a suitable matrix for the isolation of microbial DNA with commercially available kits, provided that sterile conditions are maintained during cryogenic grinding. Currently, next-generation sequencing is the best choice for determining the bacteriome and obtaining information about the relative abundance of bacterial genera, both analytically and economically.