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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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