The Differences Of Subgingival Microbiota And Blood Cell Indexes In Different Periodontal Inflamed Surface Areas And Comparison With The Effect Of Non-surgical Periodontal Therapy

Objectives:Depending on the individual,the role of microorganisms and autoimmune state in the etiology of periodontitis varies.Calculated using Periodontal Epithelial Surface Area(PESA)and Bleeding on Probing(BOP),Periodontal Inflamed Surface Area(PISA)can more precisely reflect the true inflammatory burden brought on by periodontitis.Microbial infection and host immune response are important links in the pathogenesis of periodontitis,and a comprehensive assessment of subgingival microbiota and peripheral blood cell indicators can help to provide a more complete picture of disease risk.The recovery status of patients after non-surgical periodontal therapy can be predicted by longitudinal studies.In this study,the PISA was used to estimate the burden of periodontal inflammation.In order to determine the relationships between periodontal inflammation,microbiota,and host systemic immunity,the peripheral blood cells and subgingival microbiota of patients with various PISA were examined.The markers of the impact of periodontal non-surgical therapy were discovered through the comparative analysis of the condition before and after treatment.Materials and Methods:According to the inclusive and exclusive criterions,90individuals were recruited from Hospital of Stomatology,China Medical University between 2021-2022.All the individuals were divided into no-or-mild,moderate and severe inflammation group according to PISA≤400mm~2,400mm~2<PISA≤1000mm~2and PISA>1000mm~2.Subgingival plaques were collected.After DNA extraction,DNA extraction and PCR amplification,DNA library construction and sequencing were carried out.The taxonomy of each operational taxonomy unite(OTU)representative sequence was analyzed at the individual level.Peripheral blood samples were collected for blood cell count and blood cell indexes were obtainted.For the 28 patients who completed the treatment according to the therapy plan and reexamined after 6 weeks,subgingival plaques were collected again for sequencing.The composition,alpha diversity and beta diversity of baseline microbiota were analyzed.Linear discriminant analysis of effect size(LEf Se)was used to screen different bacteria,and Spearman was used to analyze the correlation of indicators.PISA and subgingival microbiota before and after treatment were compared.Besides,patients were divided into good-recovery group and poor-recovery group according to the rate of change in PISA(ΔPISA%)>70%and≤70%,also the subgingival microbiota and peripheral blood cell indexes were compared and correlation analysis was conducted between the two groups.Results:1.The PISA of subjects with periodontal health or gingivitis was20.66～392.34mm~2,while the PISA of patients with periodontitis was377.76～4604.98mm~2.The PESA,PISA and BOP increased in order from no-or-mild to moderate to severe grouped by PISA and the differences were statistically significant(P<0.05).2.A total of 1321482 sequences were obtained by subgingival microbiota sequencing,which were clustered into 14 phyla,133 genera and 9093 OTUs.The total relative abundance of periodontal pathogenic genera accounted for 20.40%,24.54%and 25.60%of no or mild group,moderate group and severe group respectively.3.Alpha diversity analysis showed no significant differences among different PISA groups,while principal co-ordinate analysis(PCo A)of beta diversity revealed significant differences in microbiota structure and significantly different bacteria were found at the genus level through LEf Se:in no-or-mild group Streptococcus,Campylobacter,Cardiobacterium and Haemophilus were significantly enriched while in severe groups Porphyromonas,Filifactor,Rikenellaceae＿RC9＿gut＿group and Treponema were enriched(LDA≥2,P<0.05).4.There were 36 significant correlations between genera and PISA,including 22positive correlations and 14 negative correlations.Among the periodontal pathogens,Porphyromonas,Eubacterium＿nodatum＿group,Tannerella,Treponema and Parvimonas were positively correlated with PISA,while Campylobacter was negatively correlated with PISA.5.In blood cell indexes,significant differences were showed in white blood cell count(WBC),neutrophil count(NEU),neutrophil-lymphocyte ratio(NLR)and systemic immune-inflammation index(SII)among different PISA groups(P<0.05).6.In analysis of correlation between blood cell indexes and periodontal indexes,WBC,NEU,platelet distribution width(PDW)and NLR showed positive weak correlation with BOP(|r|>0.2,P<0.05).In the analysis of association with subgingival microbiota,WBC was weakly correlated with Pseudopropionibacterium,Porphyromonas and Tannerella,PDW was weakly correlated with Lentimicrobium,Peptostreptococcus,Fretibacterium,and NEU was weakly correlated with Porphyromonas(|r|>0.3,P<0.05).7.After non-surgical periodontal therapy,PISA of the patients decreased from 1174.86(515.26,1651.05)mm~2to 304.12(159.88,479.69)mm~2which proved periodontal conditions were significantly improved(P<0.05).The alpha diversity of subgingival microbiota and the total relative abundance of periodontal pathogenic genera decreased(P<0.05).No significant correlation was found between periodontal recovery status and baseline blood cell indexes(P>0.05).8.According toΔPISA%>70%and≤70%,the patients were divided into good-recovery and poor-recovery group.LEf Se analysis showed that Veillonella,Actinomyces,Butyrivibrio,unclassified＿p＿Firmicutes,unclassified＿o＿Veillonellales-Selenomonadales and Brachymonas were enriched in the poor-recovery group,while Pseudopropionibacterium,unclassified＿f＿Peptostreptococcaceae and Bergeyella were enriched in the good-recovery group(LDA≥2,P<0.05).9.The correlation analysis between periodontal recovery and subgingival microbiota indicated that Lautropia,Aggregatibacter,Rothia,Leptotrichia,Granulicatella and F0058 were correlated withΔPESA%weakly,while Lautropia,Aggregatibacter,Lachnoanaerobaculum and Cardiobacterium correlated withΔBOP%weakly in correlation analysis(|r|>0.3,P<0.05).Conclusion:1.Streptococcus,Campylobacter,Cardiobacterium and Haemophilus were significantly enriched in no-or-mild group,while in severe groups Porphyromonas,Filifactor,Rikenellaceae＿RC9＿gut＿group and Treponema were enriched.2.The relative abundance of multiple genera were correlated with PESA,PISA and BOP.And blood cells count indexs include NEU、WBC、PDW and NLR were correlated with BOP weakly.3.The poor recovery after non-surgical periodontal therapy may be related to the enrichment of Veillonella,Actinomyces and Butyrivibrio,while the good recovery may be related to Pseudopropionibacterium and Bergeyella.4.The increase ofΔPESA%after non-surgical periodontal therapy was positively correlated with the abundance of Aggregatibacter and Leptotrichia,and negatively correlated with the abundance of Lautropia,Rothia and Granulicatella at baseline.The increase ofΔBOP%was positively correlated with the abundance of Lautropia and Cardiobacterium,and negatively with Aggregatibacter and Lachnoanaerobaculum.