Culture-independent Characterization of the Microbiome of Healthy Pulp

Abstract

Introduction: Advances in culture-independent molecular biotechnologies have driven a greater appreciation for the function of mutualistic microorganisms in the maintenance of states of health in humans. The purpose of this study was to test the long-held hypothesis that healthy pulp lack bacteria.

Methods: Strict inclusion criteria were used to identify 10 pristine teeth from 10 healthy patients that were scheduled to be electively extracted in compliance with an orthodontic treatment plan. Using a rigorous disinfection protocol to isolate the operating field, the pulp space was accessed, and pulp tissue was collected in vivo from each tooth using a barbed broach. Genomic DNA was extracted from each pulp sample and analyzed for the presence of bacterial DNA using universal 16S ribosomal RNA polymerase chain reaction primers and MiSeq sequencing (Illumina, San Diego, CA) of community amplicons.

Results: One hundred percent (10/10) of the tested pulp tissues demonstrated the presence of bacterial DNA, with a mean of 343 operational taxonomic units per sample (range, 191-479). These were derived from 12 genera in which Ralstonia, Actinetobacter, and Staphylococcus were predominant (43%-78% of total community). None of the negative-field controls and none of the instruments used in the study tested positive for the presence of contaminating DNA.

Conclusions: This study presents evidence to support the conclusion that the pulp spaces of pristine healthy teeth contain detectable bacterial DNA.

Keywords: Endodontic commensalism; Illumina; endodontic mutualism; pulp microbiome; pulpal symbiont.