Analysis Of Subgingival Flora Composition In Patients With Severe Periodontitis With Type 2 Diabetes Mellitus And Its Correlation With 25-hydroxyvitamin D₃ Levels

Objective:There is a significant bidirectional relationship between diabetes and periodontitis.Vitamin D is closely associated with the occurrence and development of diabetes mellitus and periodontitis.In this study,the composition of subgingival flora samples from patients with severe periodontitis with type 2 diabetes was analyzed and its correlation with serum 25-hydroxyvitamin D₃level was detected to analyze the possible related role of vitamin D in patients with diabetic periodontitis.Methods:The control group and patients treated in the Department of Periodontology of the Stomatology Hospital of China Medical University and the Department of Endocrinology of the First Hospital of China Medical University from 2019 to 2020were selected in this study.According to the inclusion and exclusion criteria,7patients were included in H group（general periodontal health group）.There were 4patients in P group（severe periodontitis group）;3 patients in PDMA group（Hb A1c<7%with good blood glucose control and severe periodontitis group with type 2diabetes）;3 patients in PDMI group（severe periodontitis group with type 2 diabetes mellitus and poor glycemic control of Hb A1c≥7%）.According to probing depth（PD）,the patients were divided into two subgroups:1 group,PD≤3 mm（normal PD）,and 2group,PD≥4 mm（deepened PD）.After the ethical approval and informed consent of the subjects,the general conditions and clinical periodontal parameters of the subjects were recorded,the fasting blood of the patients was collected,and subgingival plaque was collected by paper points.Serum 25-hydroxyvitamin D₃was detected by ELISA,and 16S rRNA amplicon sequencing was performed for subgingival plaques.The sequencing results were quality controlled and clustering of Operational taxonomic units was performed.Biodiversity analysis,flora composition analysis,and difference comparison between groups were performed.SPSS 24.0 and Majorbio Cloud Platform were used for data processing.Conclusion:1.In H,P,PDMA and PDMI groups,the serum level of25-hydroxyvitamin D₃in H group was 41.33±2.13 ng/m L,which was significantly higher than that in P group（29.97±6.48 ng/m L）,PDMA group（26.98±6.14 ng/m L）and PDMI group（22.10±6.42 ng/m L）（P<0.05）.2.A total of 1982056 sequences were detected in this study,including 25 phylums,62classes,131 orders,226 families,435 genera,718 species and 872 OTUs.3.There was no significant difference in Alpha diversity between H,P1,PDMA1 and PDMI1 groups with PD≤3 mm and P2,PDMA2 and PDMI2 groups with PD≥4 mm（P>0.05）.4.βdiversity analysis showed that there were significant differences in the species composition of subgingival flora at PD≤3 mm sites among the four groups of H,P,PDMA and PDMI（P<0.05）,the species composition of subgingival flora at PD≥4mm was significantly different between groups（P<0.05）.5.The relative abundance of Porphyromonas gingivalis,Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans in PDMI2 group was significantly lower than that in P2 group and PDMA2 group,and the relative abundance of Porphyromonas gingivalis in PDMA2 group was significantly lower than that in P2group.The relative abundance of Tannerella forsythia,Filifactor alocis and Porphyromonas endodontalis in PDMI2 group was significantly lower than that in P2group.6.The level of serum 25-hydroxyvitamin D₃was correlated with the species composition of subgingival plaque.The level of 25-hydroxyvitamin D₃in serum was positively correlated with the relative abundance of Fusobacterium,Alloprevotella and Fusobacterium nucleatum,and negatively correlated with the relative abundance of Aggregatibacter,Ammoniphilus,Sphingomonas,Acinetobacter,Stomatobaculum,W5053,Eikenella,Aggregatibacter aphrophilus,uncultured bacterium Ammoniphilus and unclassified Eikenella.Conclusion:1.The level of serum 25-hydroxyvitamin D₃decreased significantly in patients with severe periodontitis compared with healthy subjects;2.There was no significant difference in species diversity of subgingival flora between patients with severe periodontitis,patients with severe periodontitis with diabetes and healthy subjects.3.The relative abundance of specific periodontal suspected pathogenic bacteria in subgingival flora of patients with severe periodontitis with poor blood glucose control was significantly lower than that of patients with severe periodontitis alone and patients with severe periodontitis with good blood glucose control.4.The level of serum 25-hydroxyvitamin D₃was significantly correlated with the relative abundance of specific species of subgingival flora.