Analysis Of Subgingival Plaque Microorganism In Chronic Moderate And Severe Periodontitis With Alzheimer’s Disease Patients

Objective: To analyze the differences of the cognitive scores and periodontal clinical indicators in patients who have moderate and severe periodontitis with Alzheimer’s disease(AD),which is using Illumina High-throughput sequencing(HTS)technology to analyze and compare the structural characteristics,diversity and metabolic function of subgingival plaque for patients.Based on the community level of microorganism,this paper wants to discuss the characteristics of subgingival plaque under different diseases and briefly discuss the relation between chronic periodontitis and Alzheimer’s disease.Methods: According to the diagnostic and inclusion criteria.From December 2017 to December 2018,collecting 44 cases of moderate and severe periodontitis with or without Alzheimer’s disease and healthy people who were over 45 years old.Those samples were enrolled in the Department of Neurology,the Medical Examination Center of Affiliated Hospital of Zunyi Medical University and the Department of Periodontology Affiliated Dental Hospital of Zunyi Medical University.The subjects were divided into three groups,moderate and severe periodontitis with Alzheimer’s disease group(n=14),moderate and severe periodontitis group(n=15),healthy control group(n=15).The three groups were labeled as AP group,P group,and N group respectively.Recording the basic information,the number of remaining teeth,periodontal clinical indicators and cognitive scores of the subjects.Subgingival plaque samples were collected and frozen at-80℃ environment,then would be sent to Wuhan Huada Gene Company to extract DNA from subgingival plaque samples,amplify 16 S r DNA V4 region and use Illumina Hi Seq2500 PE250 platform to test the sequence.The structure,diversity and metabolic function of subgingival plaque were analyzed by some software,like QIIME,Mothur,SPSS and so on.Results:1.The comparison of basic information of subjects in the three groups showed that there was no significant difference between the three groups in terms of gender,education level,blood pressure(systolic/diastolic blood pressure)and fasting blood-glucose(P>0.05);on the age,the AP group were significantly greater than the N and P group(P<0.05).In the MMSE scale score,the AP group was significantly larger than the N group and the P group(P<0.05).2.The comparison of the remaining teeth number,bleeding on probing(BOP%),probing the depth(PD)and clinical attachment loss(CAL)of the three groups,the results showed that AP group had a significantly less than remaining teeth number N,P(P<0.05);BOP(%),PD and CAL in AP group were significantly greater than those in N group and P group(P<0.05).BOP(%),PD and CAL in group P were significantly greater than those in group N(P<0.05).3.A total of 2711,831 Clean Tags were obtained through Illumina Hi Seq2500 platform for high-throughput sequencing,with an average of 61,632 Clean Tags per sample and an average length of 252 bp Tags.44 subgingival plaque samples were clustered to produce 1130 OTUs at 97% similarity,with an average of 259 OTUs per sample.A total of 24 phylums,41 classes,58 orders,134 families,289 genus and 379 species were obtained by comparison with RDP database.4.The results of Beta diversity comparison among the three groups showed that the AP group was more similar to the P group.The comparison results of Alpha diversity index showed that the species richness and evenness of subgingival plaque community in AP group were significantly higher than those in P group and N group(P<0.05);The species richness and evenness of the flora in group P were lower than those in group N,although the differences were not significant(P>0.05).5.The results of PICRUST analysis showed that there are significant differences among the groups of AP-P,AP-N and P-N.The metabolic activity types of bacteria in patients were significant,including 15/9,17/14 and 31/29,respectively.It is indicating that the metabolic activity of subgingival plaque flora in AP group may be more active than that in patients with moderate and severe periodontitis.6.Through Venn and core-pan analysis,there were 23,36 and 42 core microorganism in each group: AP<P<N,respectively;Among the core microorganism uniquely belonging to the AP group,only Prevotella oulorum,Prevotella saccharolytica,Lachnoanaerobaculum saburreum belonged to it.And the increase of the Prevotella oulorum might be related to AD.In addition,six core set of microbes in the Fusobacterium nucleatum、Porphyromonas_gingivalis 、 Tannerella forsythia 、 Treponema denticola 、 Prevotella intermedia 、Campylobacter rectus(classic periodontal pathogens)average relative abundance sort found size,are all P>AP>N,and compare differences between the three groups,found the Porphyromonas gingivalis and Treponema denticola of AP group and N group significantly smaller than that of P group(P<0.05).7.At the phylum level,the Actinobacteria and Firmicutes in the AP group and the N group were significantly greater than those in the P group(P<0.05).In group P,Spirochaetes and Synergistetes were significantly larger than those in group AP and group N(P<0.05).Bacteroidetes in AP group and P group were significantly larger than those in N group(P<0.01).Proteobacteria in N group was significantly larger than that in P group and AP group(P<0.01).Fusobacteria showed no significant difference in each group.Redundancy analysis was used to analyze the correlation between each phylum,MMSE scores and periodontal clinical indicators.The results showed that Spirochetes,Synergistetes and Proteobacteria were positively correlated with MMSE score.While Fusobacteria,Bacteroidetes,Firmicutes and Actinobacteria were negatively correlated.On the other hand,Spirochetes,Synergistetes,Fusobacteria,Bacteroidetes are positively correlated with BOP(%),PD and CAL,while Actinobacteria and Proteobacteria are negatively correlated.8.(1)At the level of genus,the increase of 1 dominant genus(Leptotrichia)and 3 nondominant genus(Delftia,Eubacterium and Saccharibacteria_genera_incertae_sedis)may be related to AD;The increase of 1 dominant genus(Tannerella),2 non-dominant genus(Desulfobulbus and Schwartzia)and decrease of 5 dominant genus(Corynebacterium,Rothia,Neisseria,Aggregatibacter,Lautropia),10 non-dominant genus(Acinetobacter,Brevundimonas,Enhydrobacter and so on)might be related to the moderate and severe periodontitis.(2)At the species level,the increase of 3 dominant species(Campylobacter_gracilis,Prevotella nigrescens,Prevotella maculosa)might be related to AD.The increase of 4 dominant species(Fusobacterium nucleatum,Treponema medium,Treponema socranskii,Prevotella intermedia)and the decrease of 10 dominant species,including Corynebacterium_martensii,Capnocytophaga gingivalis,Neisseria subflava,Lautropia mirabilis and so on,may be related to the moderate and severe periodontitis.Conclusion:1.Periodontitis patients with AD have worse periodontal health;2.Subgingival plaque flora in moderate and severe periodontitis with AD patients is composed of 23 core microorganism,withing 6 classical periodontal pathogens’ lower relative abundance;3.Preliminary results indicate that the increase of 4 bacterial genus(Leptotrichia,Delftia,Eubacterium,Saccharibacteria_genera_incertae_sedis)and 4 bacterial species(Prevotella_oulorum,Campylobacter gracilis,Prevotella nigrescens,Prevotella maculosa)in subgingival plaque community of patients with moderate and severe periodontitis may be associated with AD.