Analysis Of Subgingival Plaque And Gut Microbiota Composition Of Patients With Moderate To Severe Periodontitis With Alzheimer’s Disease

Objective:The subgingival plaque and gut microbiota of patients with moderate to severe periodontitis with Alzheimer’sdisease（AD）were analyzed by high-throughput putsequencing（HTS）technology.To preliminarily explore the composition characteristics of subgingival plaque and gut microbe under different disease states,so as provide new ideas and basis for the related research on the pathogenesis and prevention of AD.Methods:According to the diagnosis and inclusion criteria,from June to December 2020,recruiting 15 patients with moderate and severe periodontitis with AD from the Department of geriatrics of the second people’s Hospital of Guizhou Province（AP group）,15 patients with moderate and severe periodontitis from the Department of periodontitis of the Affiliated Stomatological Hospital of Zunyi Medical University（group P）and 15 healthy volunteers from the Medical Examination Center（group H）.The basic information（name,age,height,weight,etc.）as well as the number of missing teeth,periodontal clinical indexes,mini-mental state examination（MMSE）,The Mediterranean-DASH Intervention for Neurodegenerative Delay diet score table and Bristol stool classification table were recorded.The subgingival plaque（P）and feces（f）samples of each subject were collected and DNA of all samples were extracted.The 16 S r DNA V4 region of the sample was amplified by PCR and sequenced by Illumina Nova Seq;The structural characteristics and diversity of subgingival plaque and gut microflora were analyzed by Qiime（Version 1.9.1）,R（Version 2.15.3）,mothur,SPSS 18.0 and other software,and the differences of subgingival microbe and intestinal microbe in the three groups and their relationship with AD were analyzed.Results: 1.There were no significant differences in gender,body mass index,education level,MIND diet and stool traits among H,P and AP groups（P>0.05）;The age of AP group was significantly higher than that of H group and P group（P < 0.05）;The MMSE score in AP group was significantly lower than that in H group and P group（P<0.05）;The number of teeth missing in AP group was higher than that in H group and P group,and the difference between AP group and H group was statistically significant（P<0.05）;The periodontal clinical indexes（probing bleeding rate,probing depth and clinical attachment loss）in AP group and P group were worse than H group（P<0.05）.2.OTU venn and Core-Pan diagrams of subgingival microbe and fecal microbe of patients in the three groups showed that there were 3537 OTUs in group H.p,2660 OTUs in group P.p and 2884 OTUs in group AP.p,respectively among subgingival microbe;Among fecal microbe,H.f group,P.f group and AP.f group have 1629,1841,3216 OTU respectively;H.p group and H.f group share 13.82% OTU,P.p group and P.f group share 14.14% OTU,AP.p and AP.f share 24.39% OTU.Important periodontal pathogens: T.denticola,P.nigrescens,T.forsythia,P.intermedia were found to be the core microorganisms in the H.p group,P.p group,and AP.p group,and P.gingivalis was only the core microorganism of the H.p and P.p groups,but not the core microbe microorganism of the AP.p group;The contents of P.gingivalis and P.intermedia in P.p group were significantly higher than those in H.p group（P<0.05）;P.gingivalis was found in AP.f group and P.f group,while P.intermedia was only found in AP.f group,all the important Periodontal pathogenic bacteria were not found in the fecal samples of the normal group.3.Through Alpha diversity index analysis.In subgingival plaque,P group（5.258）and AP group（5.018）was slightly higher than H group（P>0.05）;In fecal samples,AP group was slightly higher than P group,and P group was slightly higher than H group,but the differences were not statistically significant（P>0.05）;Beta diversity showed that the similarity between subgingival microbial community and fecal microbial community in AP group was higher than that in P group and H group,and the subgingival microbiome composition of AP group was more similar to that of P group.4.The dominant species of subgingival plaque and fecal microorganisms in AP group,P group and H group were statistically analyzed at phylum,genus and species levels.The results showed that:（1）Proteobacteria,Firmicutes,Actinobacteriota and Bacteroidota are common phylum of subgingival microbe and fecal microbe;The ratio of Firmicutes/Bacteroidota（F/B）in AP group and P group was decreased in subgingival plaque and fecal microbe compared with that in H group,and the decrease in subgingival plaque ratio was statistically significant（P<0.05）;The Bacteroidota,Synergistota and Spirochaetota of the AP.p group and the P.p group were significantly larger than those of the H.p group（P<0.05）,the Spirochaetota and Synergistota of the AP.p group were slightly larger than those of the P.p group,and the difference was not statistically significant（P > 0.05）;The endemic dominant phylum of P.f group was Fusobacteriota,and that of H.f group was Verrucomicrobiota;The relative average abundance of Proteobacteria in AP.f group was slightly higher than that in P.f group,and that in P.f group was higher than that in H.f group,but there was no statistical significance（P> 0.05）.（2）)At the genus level of subgingival plaque,the relative abundance of Haemophilus,Rothia,Streptococcus,Actinomyces and Leptotrichia in AP group decreased compared with those in H group and P group;Porphyromonas and Treponema in AP and P groups were higher than those in H group,with statistical significance（P<0.05）;Prevotella in AP group was significantly higher than that in H group（P<0.05）;At the species level of subgingival plaque,Haemophilus_parainfluenzae,Haemophilus_influenzae,Rothia_aeria,and Rothia_dentocariosa in the AP and P groups were significantly lower than those in the H group（P<0.05）,Among them,Haemophilus_parainfluenzae and Haemophilus_influenzae belonging to the Haemophilus and Rothia_aeria and Rothia_dentocariosa belonging to the Rothia;T.denticola and Treponemas_socranskii was significantly higher than that in H group（P<0.05）,Among them,T.denticola and Treponemas_socranskii belonging to the Treponema.（3）At the genus level of fecal microflora,the relative abundance of Bifidobacterium and Clostridium_sensu_stricto₁ in AP group was lower than that in H group and P group;The Escherichia-Shigella in the P group was significantly greater thanthe H group（P<0.05）;At species level,the number of Escherichia_coli in P group was also significantly higher than that in H group（P<0.05）,Escherichia_coli belonging to Escherichia-Shigella;Faecalibaculum_rodentium and Clostridiaceae_bacterium_DJF_VR76 in the H.f group were significantly greater than those in the AP.f group（P<0.05）,Faecalibaculum_rodentium belonging to Faecalibacterium,Clostridiaceae_bacterium_DJF_V-R76 belonging to Ruminococcus,but Faecalibacterium and Ruminococcus were not statistical significance between three groups（P>0.05）.Conclusion: 1.The composition of subgingival plaque and fecal microflora in H,P and AP groups were different,and the three groups had their own unique dominant flora,and the species composition was different,indicating that subgingival microbe and gut microbe were disturbed in periodontitis.2.Subgingival plaque and fecal microbes share a common microbe,oral microbes may colonize the gut ectopicly,and the number of intestinal probiotics decreases and the number of pro-inflammatory bacteria increases under periodontitis,suggesting the cause of periodontitis Oral pathogenic bacteria may change the structure and function of the intestinal flora by colonizing the intestinal tract,and then have a certain connection with AD.