Preliminary Analyses of the Microbiome in Rheumatoid Arthritis-associated Periodontitis

Objectives: It has been proposed that the periodontitis-associated microbiome contributes to the generation of modified host epitopes which induce pathogenic autoimmune responses that are linked with development of rheumatoid arthritis (RA). Porphyromonas gingivalis may be important in generating citrullinated proteins, but other organisms may contribute by, eg. generating carbamylated proteins. The aim of this study was to establish robust techniques to allow elucidation of the diversity and functions of the subgingival microbiomes from periodontitis in individuals with and without RA and those at risk of developing RA.
Methods: Thirty anti-citrullinated protein antibody seropositive individuals with no evidence of synovitis (CCP+), 30 healthy controls (HC) and 30 CCP+ RA patients (RA) were recruited. Subgingival plaque was collected from healthy and deep pocket sites using paper points. DNA was isolated from plaque and library preparations were made using NEBNext® Ultra™ I and II DNA Library Prep Kits for paired-end DNA sequencing on the Illumina HiSeq3000 platform.
Results: DNA yields from all samples ranged from 0 to 76 ng; 59% had <5ng DNA. Therefore, an optimised protocol for library preparations was used on low yield DNA samples (NEBNext® Ultra™ II DNA Library Prep Kit). Analyses were carried out on 14 such samples (5 CCP+, 4 RA, 5 HC) with DNA yields ranging from 0.41-3.76 ng. From these, 27.1 x 10⁶ to 114.8 x 10⁶ sequence reads were obtained. Initial taxonomic classification analysis indicated that the four most abundant phyla were Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes. The relative abundance of Porphyromonas gingivalis was high in the samples from the CCP+ group.
Conclusions: Preliminary analyses confirm the ability to carry out next generation sequencing on low yield DNA extracts of subgingival plaque. Further analyses are on-going and will help elucidate the diversity and functional potential of the subgingival microbiome in periodontitis and RA.