Background. Metagenomics is the sequencing of DNA from whole communities of organisms taken directly from the environment. As opposed to culturing methods which focus on individual species, metagenomics allows for the characterization of intact community genomes, including the identification of noncultivable bacterial species. The objective of this pilot study was to test the feasibility of a metagenomic approach in combination with a novel design of primers to analyze the pre- and post-treatment of subgingival plaque in two aggressive periodontitis patients.

Methods. Periodontal examinations and microbial sampling were performed before and after non-surgical initial phase therapy. DNA was extracted from the subgingival plaque samples and subjected to PCR amplification using primers that amplified the c2-c4 regions of the 16S rDNA. Specific bar codes were included in the primers to identify individual samples. The PCR products were pooled and sequenced for the v4 region of the 16S rDNA using the pyrosequencing 454 FLX standard platform. The results were analyzed for species identification in the Human Oral Microbiome Database (HOMD) and Ribosomal Database Project (RDP) database. The observed proportional changes of the subgingival species after therapy were verified by testing the pre- and post-therapy levels of Porphyromonas gingivalis by real-time PCR.

Results. The sequencing of the amplicons resulted in 24,673 reads and identified 208 species/phylotypes. Of those, 129 species/phylotypes were identified in both patients but their proportions varied. While >120 species/phylotypes were identified in all samples, 85–90% of the species/phylotypes were represented by ≤100 sequencing reads (≤0.2% of the total reads). Similar levels of subgingival P. gingivalis were found based on the numbers of sequencing reads or quantitative Real-time PCR analyses. Pre- and post-therapy samples showed significant quantitative and distributive species changes along with clinical improvements. Quantitatively, many known pathogenic species went from easily detectable to zero counts while the opposite occurred for many known commensal bacteria. Additionally, several novel, pathogenic and health-associated species were found.

Conclusion. High throughput metagenomic analysis is applicable to assess the changes in subgingival microbiota after nonsurgical periodontal therapy in aggressive periodontitis. The examined subgingival microbiota are characterized by high species richness (>120 species/phylotypes) dominated by a few species/phylotypes. The composition of the subgingival microbiota appears to be distinct between individuals before and after the treatment. The considerable variety of known and novel species/phylotypes detected supports further research to better understand the microbial etiopathogenesis of periodontitis.