The Effect Of Calcitriol On The Anaerobic Cultures Of Bacterial Genera From The Subgingival Plaque Of Patients With Severe Chronic Periodontitis With Amplicon Sequencing Of 16S RRNA

Objective:Vitamin D is an indispensable nutrient for human beings,and the deficiency of vitamin D is closely related to periodontitis.The purpose of this study was to explore the effect of calcitriol?1,25-?OH?₂D₃,hereinafter referred to as 1,25D?on the composition of subgingival plaque in patients with severe chronic periodontitis,and to provide a scientific basis for the clinical adjuvant treatment of periodontitis with 1,25D.Methods:Eight patients with severe chronic periodontitis were randomly selected from the periodontal department of the stomatological hospital affiliated to China medical university.Subgingival plaque was collected by paper point method.Subgingival plaque of the same patient source was inoculated in BHI liquid culture containing 0.05%anhydrous ethanol?Con group?,10^-1010 mol/L 1,25D?Low group?,10^-8mol/L 1,25D?High group?,and cultured in anaerobic environment for 48 h.Paired t-test was used to analyze the concentration of plaque in three groups.Total DNA samples from 8 patients were extracted,primers were designed and synthesized,PCR amplification and product purification were conducted,and Miseq library was constructed.Then sequencing was conducted by Illumina Miseq sequencing platform.According to the sequencing data,Wilcoxon signed rank sum test was used to analyze the Chao,Shannon and Simpson indexes of the three groups of samples,and the comparison between the two groups was carried out,so as to explain the difference of the diversity of Aphla in the subgingival plaque of the three groups of samples.Beta diversity of the three sample communities was analyzed by PCoA analysis and Adonis analysis based on btray_curtis distance matrix.At the overall level,wilcoxon signed test was used to statistically analyze the differences of all genera between Con group and Low,Con group and High group,and Low group and High group.In the analysis of individual patients,fisher's exact test was used to evaluate the significance of the differences in relative abundance between the two samples,and on this basis,statistical analysis was conducted according to the proportion of patients whose relative abundance of each genera increased?or decreased?significantly.Results:With the end of anaerobic culture,the bacterial concentrations of 0.05%anhydrous ethanol,10^-1010 mol/L 1,25D,10^-88 mol/L 1,25D were 1.03±0.19?10⁹CFU/mL??0.85±0.14?10⁹ CFU/mL??0.74±0.13?10⁹ CFU/mL?,respectively,with statistically significant differences?P<0.001?.A total of 970369 sequences were detected by high-throughput sequencing,including 5 phyla,12 classes,22 orders,31families,44 genera,56 species and 63 OTUs.On the genera level,Aphla diversity index Chao,Shannon,Simpson index showed no significant difference among the three groups?P>0.05?.There was no significant difference in Beta diversity among the three groups?P>0.05?.There was no significant difference in relative abundance among the three groups?P>0.05?.We defined the proportion of patients with significant increase?or decrease?in relative abundance of a certain bacteria?>50%?as the dominant proportion.In this study,with the treatment of 10^-10mol/L 1,25D,the relative abundance increased significantly for the dominant bacterial genera as follows,Solobacterium?75.00%?,Gemella?66.67%?and Streptococcus?62.50%?,the relative abundance decreased significantly for the dominant bacterial genera as follows,Granulicatella?85.71%?and Veillonella?85.71%?.With the treatment of 10^-88 mol/L 1,25D,the bacterial genera the relative abundance increased significantly for the dominant bacterial genera as follows,were Staphylococcus?100.00%?and Veillonella?57.14%?,the relative abundance decreased significantly for the dominant bacterial genera as follows,Gemella?66.67%?,Streptococcus?62.50%?,Granulicatella?71.43%?and Leptotrichia?60.00%?.Conclusion:Results of 1,25D treatments on the anaerobic cultures of subgingival plaque samples from patients with severe chronic periodontitis suggest that:?1?1,25D may reduce the number of bacteria.?2?As for the overall analysis,we found no significant effect of 1,25D on the diversity of Alpha diversity,Beta diversity and the relative abundance of bacterial genera.As for the individual patients,1,25D may change the community composition and relative abundance of bacterial genera of subgingival plaques.