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Exploring microorganisms in subgingival plaque of periodontitis through 16S rRNA gene high-throughput sequencing and culturomics

genomics
microbiology
dentistry

Abstract

Background. Periodontitis, a chronic inflammatory disease affecting oral and general health globally, is primarily caused by oral microbiome dysbiosis, yet nearly a quarter of oral bacteria remain uncultivated.

Methods. The culturomics of dental plaque from periodontitis patients were executed with SHI and NEMM media with vancomycin (vSHI and vNEMM). High-throughput 16S rRNA gene sequencing analyzed the difference between healthy and periodontitis groups and different culture conditions. Isolates were cultivated, expanded, and identified with Sanger sequencing of the full-length 16S rRNA gene and BLAST searches through EzBioCloud and NCBI BLAST websites.

Results. High-throughput results indicated insufficient growth of Gram-negative bacteria in both NEMM and SHI media; thus, vancomycin was added to enrich Gram-negative bacteria. We found that Kingella , Aggregatibacter, Capnocytophaga, and Veillonella were better enriched in vSHI and vNEMM, respectively. 532 and 690 strains were isolated, respectively, through culturomics with vNEMM and vSHI. Of these, 4 strains were found as potential new species/subspecies, providing bacterial strain materials for further research in periodontitis.

Conclusions. This study provides a method to isolate periodontal pathogens via culturomics. A total of 1,222 strains of bacteria were isolated, including 4 potential novel subspecies ( Kingella oralis , Capnocytophaga ochracea , Veillonella rodentium, and Rothia dentocariosa) . These clinical isolates will support future periodontitis studies.

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