A methodological study on microbial in vivo sampling methods of root canal microbiota for next generation gene sequencing analysis.

Introduction: The aim was to evaluate the suitability of paper points or endodontic nickel-titanium files to sample microorganisms for in vivo investigation of endodontic microbiota by 16S rDNA sequencing.

Methods: 45 patients presenting clinical and radiological sings of apical periodontitis were recruited for sampling, giving their written informed consent. Glide paths were assessed using C-Pilot Files and K-Files under electronic root canal length control under aseptic conditions. Microbial samples were taken from 84 root canals in duplicates, the first sample with a sterile paper point (size 15), the second with a sterile file (size 20/.06). After DNA extraction, the hypervariable region V4 of the bacterial 16 S rRNA gene was amplified and sequenced (Illumina MiSeq). Sequencing data were trimmed with Cutadapt and exact amplicon sequence variants generated by DADA2. Taxonomy was assigned based on the Human Oral Microbiome Database (eHOMD). Statistical analysis of diversity parameters comprised Wilcoxon signed-rank tests and PERMANOVA. Compositional differences were evaluated by differential abundance analysis (DESeq2). Microbial contamination during the sampling process and analysis were evaluated.

Results: Concerning alpha diversity, richness and dissimilarity differed non-significantly between paper point and instrument samples (P>.05) while a significant difference was observed in Shannon Index (P<.05). Regarding beta diversity, paper point and instrument samples presented with similar microbial community compositions (P=1.0,PERMANOVA). Paper point controls contained significantly higher proportions of Pseudomonadales (P<.05).

Conclusions: Paper point and endodontic instrument sampling generate valid specimens for 16S rDNA community profiling. Endodontic instrument sampling is easier to execute and therefore, could be the technique of choice.