Identification and quantification of archaea involved in primary endodontic infections

Abstract

Members of the domain Archaea, one of the three domains of life, are a highly diverse group of prokaryotes, distinct from bacteria and eukaryotes. Despite their abundance and ubiquity on earth, including their close association with humans, animals, and plants, so far no pathogenic archaea have been described. As some archaea live in close proximity to anaerobic bacteria, for instance, in the human gut system and in periodontal pockets, the aim of our study was to assess whether archaea might possibly be involved in human endodontic infections, which are commonly polymicrobial. We analyzed 20 necrotic uniradicular teeth with radiographic evidence of apical periodontitis and with no previous endodontic treatment. Using real-time quantitative PCR based on the functional gene mcrA (encoding the methyl coenzyme M reductase, specific to methanogenic archaea) and on archaeal 16S rRNA genes, we found five cases to be positive. Direct sequencing of PCR products from both genes showed that the archaeal community was dominated by a Methanobrevibacter oralis-like phylotype. The size of the archaeal population at the diseased sites ranged from 1.3 x 10(5) to 6.8 x 10(5) 16S rRNA gene target molecule numbers and accounted for up to 2.5% of the total prokaryotic community (i.e., bacteria plus archaea). Our findings show that archaea can be intimately connected with infectious diseases and thus support the hypothesis that members of the domain Archaea may have a role as human pathogens.

FIG. 1.

Phylogenetic tree showing the positions of 16S rRNA gene types identified in infected root canals of human teeth relative to those of representative members of the four major lineages from the domain Archaea. Sequences determined in this study are shown in boldface. The scale bar corresponds to 0.1 substitution per nucleotide. The tree was calculated using 798 nucleotide positions and the neighbor-joining approach (with the Felsenstein correction), via the ARB program package (23). The statistical significance levels of interior nodes, shown as percentages, were determined by performing bootstrap analyses (1,000 replications; only values over 50% are shown).

FIG. 2.

Phylogenetic tree showing the positions of the mcrA gene types identified in infected root canals of human teeth relative to those of representative members of methanogenic archaea. Sequences determined in this study are shown in boldface. The scale bar corresponds to 0.1 substitution per nucleotide. The tree was calculated using 464 nucleotide positions and the neighbor-joining approach (with the Felsenstein correction), via the ARB program package (23). The statistical significance levels of interior nodes, shown as percentages, were determined by performing bootstrap analyses (1,000 replications; only values over 50% are shown).

FIG. 3.

Comparison of 16S rRNA gene target molecule numbers (white bars) with mcrA gene target molecule numbers (gray bars), determined by RTQ-PCR from pure cultures and from samples obtained from primary endodontic infections. Error bars indicate standard deviations from three replicate RTQ-PCR runs. Mo, M. oralis; Ms, M. smithii.

FIG. 4.

Dissociation curves of archaeal 16S rRNA gene PCR products (A) and methanogenic mcrA gene PCR products (B and C). E, endodontic samples; Mo, M. oralis; Ms, M. smithii; Mb, M. bryantii; Mm, M. maripaludis; Mh, M. hungatei; NTC, nontarget control.