The characteristics of the periodontal microbiome influence clinical periodontal status. Gingivitis involves reversible gingival inflammation without alveolar bone resorption.

Ancient dental calculus, formed from dental plaque, is a rich source of ancient DNA and can provide information regarding the food and oral microbiology at that time. Genomic analysis of dental calculus from Neanderthals has revealed the difference in bacterial composition of oral microbiome between Neanderthals and modern humans. There are few reports investigating whether the pathogenic bacteria of periodontitis, a polymicrobial disease induced in response to the accumulation of dental plaque, were different between ancient and modern humans. This study aimed to compare the bacterial composition of the oral microbiome in ancient and modern human samples and to investigate whether lifestyle differences depending on the era have altered the bacterial composition of the oral microbiome and the causative bacteria of periodontitis. Additionally, we introduce a novel diagnostic approach for periodontitis in ancient skeletons using micro-computed tomography. Ancient 16S rDNA sequences were obtained from 12 samples at the Unko-in site (18th-19th century) of the Edo era (1603–1867), a characteristic period in Japan when immigrants were not accepted. Furthermore, modern 16S rDNA data from 53 samples were obtained from a database to compare the modern and ancient microbiome. The microbial co-occurrence network was analyzed based on 16S rDNA read abundance. Eubacterium species, Mollicutes species, and Treponema socranskii were the core species in the Edo co-occurrence network. The co-occurrence relationship between Actinomyces oricola and Eggerthella lenta appeared to have played a key role in causing periodontitis in the Edo era. However, Porphyromonas gingivalis, Fusobacterium nucleatum subsp. vincentii, and Prevotella pleuritidis were the core and highly abundant species in the co-occurrence network of modern samples. These results suggest the possibility of differences in the pathogens causing periodontitis during different eras in history.

Background. 16S rRNA gene amplicon sequencing analysis is widely used to investigate the diversity and complexity of bacterial communities in the environment. However, the bacterial composition estimated from the experimental data can differ from the original composition. Such a bias occurs depending on methodological stages, including trimming length, selected amplification regions, and referenced databases. The optimal condition to minimize the bias for oral bacterial analysis remain unknown. Therefore, this study aimed to evaluate the possible bias in 16S rRNA gene amplicon analysis using three bacterial DNA samples, namely mock1 community, which comprised 15 bacteria from various environments, mock2 community, which comprised 6 major oral bacteria, and dental calculus obtained from 5 patients, along with different trimming lengths, three databases, and nine primers targeting different hypervariable regions. Results. Mock1 community analysis results at the genus level showed the highest similarity between the data using 300 bp paired-end (PE), primer targeting V3 region, and SILVA ribosomal RNA database (SILVA) and the theoretical value obtained from the bacterial species. Mock2 community analysis with 300 bp PE showed one of the highest similarities between the theoretical value and data using the V3–V4 region with the Human Oral Microbiome Database (HOMD) at the genus level and data using the V1–V2 region with HOMD at the species level. In the species analysis of the dental calculus samples with 300 bp PE, the Shannon index value was higher in the V1–V2 region with HOMD than that in other combinations of primers and databases. The composition of the relative bacterial abundance was more markedly influenced by the inter-individual variability in the samples than the selected amplified region and/or database. Conclusion. The optimal conditions for analyzing oral microbiota with the most negligible bias were determined to be a combination of 300 bp PE, the primer targeting the V1–V2 region, and the HOMD database. Notably, this is the first report for such analyses of modern Japanese dental calculus. Furthermore, the methods of this study will be a guide for setting the appropriate sequence analysis conditions for each environment.

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