| BackgroundAs an irreversible chronic progressive disease,periodontitis is one of the main causes leading to tooth loss in adults.During the transition from periodontal health status to periodontitis,oral microbial community structure also changed significantly.Recent studies have shown that oral microorganisms not only affect the local oral environment and systemic system,but also the systemic system affects the community structure of oral microorganisms.However,many cohort studies of periodontitis and oral microbes did not paid much attention to the interference of systemic factors.ObjectiveThis study examined the changes in the composition of the oral microbial community and the functional analysis in various periodontal conditions to the exclusion of systemic factors and local factors.MethodsFrom July 2019 to November 2019,566 saliva samples were collected from the Health Management Center of the First Affiliated Hospital of Zhengzhou University,and the subjects were given a general oral examination and basic information gathering.Following rigorous diagnosis and exclusion procedures,112 subjects were enrolled,including 61 periodontal healthy cohort,30 patients with Ⅰ&Ⅱ stage periodontitis and 21 patients with III&IV stage periodontitis.1.Using 16S rRNA technology to detect changes in the oral microbiota of different populations.Independent T-test was used for analyzing difference of continuous variables in subjects’ basic information,and chi-square test for difference of categorical variables.According to the OTUs analysis result of the rank sum test,the sample sequence is randomly selected.The Brary-Curtis distance based on OTUs was used to perform permutational multivariate analysis of variance(PERMANOVA)to analyze the factors causing differences between cohorts.The Shannon index and Observed Species(Obs)index showed the alpha diversity of bacteria by using R program package Vegan,and based on the Bray-Curtis distance matrix,Principal Coordinates Analysis(PCoA)for differences in the beta diversity of microbial communities between cohorts.Linear discriminant analysis Effect Size(LEfSe)was used to find biomarkers with statistical differences between cohorts.Spearman correlation analysis was used to analyze the correlation between clinical index data of subjects(such as age,BMI,smoking,etc.)and relative abundance of different microflora.2.Using PICRUSt(Phylogenetic Investigation of Communities by Reconstruction of Unobserved States)to predicate functional differences.In combination with the Kyoto Encyclopedia of Genes and Genomes(KEGG),PICRUSt was used to obtain annotation results of microbiome and gene functions,and rank-sum test for difference analysis to compare the differences in microbiome gene functions between the different cohorts.Results1.Statistical analysis showed that gender,age,BMI,dietary habit,smoking status,alcohol consumption,daily brushing frequency,daily flossing and malocclusion had no statistical significance among different groups(P>0.05).2.Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria and Fusobacteria ranked the top five in the abundance of salivary bacteria in chronic periodontitis cohort and periodontal healthy cohort.3.The results of multivariate analysis using PERMANOVA showed that in the basic information of chronic periodontitis and periodontal healthy cohort.,grouping had a significant effect on the results of bacterial community(P<0.05),while other basic information had no significant effect on the structure of bacterial community(P>0.05).In addition,all the basic information of the Ⅰ&Ⅱ stage periodontitis group and Ⅲ&Ⅳstage periodontitis group had no significant effect on bacterial community structure(P>0.05).4.There was no significant difference in alpha diversity of salivary flora between periodontal healthy cohort.and chronic periodontitis cohort(P>0.05),but there was significant difference in beta diversity between periodontal healthy cohort and chronic periodontitis cohort(P<0.05).There were no significant differences in alpha and beta diversity in salivary bacteria between cohorts with different degrees of chronic periodontitis(P>0.05).5.LEfSe analysis showed that there were significant differences in 16 genera between the chronic periodontitis cohort and the periodontal healthy cohort.15 genera were enriched in the chronic periodontitis cohort,including Bifidobaclerium,Streptobacillus,Tannerella,Treponema,Bacteroides Lactobacillus,Lactococcus,Oscillibacter,Selenomonas,Barnesiella,Coprobacter,Parabacteroides,Flavonifractor,Parasutterella and Senegalimassilia.Haemophilus was enriched in the periodontal healthy cohort(LDA>2).Spearman correlation analysis showed that three genera enriched in the chronic periodontitis cohort,including Lactobacillus,Bifidobacteria and Flavonifractor,were positively correlated with the characteristics of the subjects(e.g.,malocclusion and daily flossing).In the chronic periodontitis cohort,Selenomonas and Treponema were negatively correlated with gender and daily flossing,respectively.Haemophilus enriched in the periodontal healthy group was significantly negatively correlated with daily flossing.LEfSe analysis showed that there were significant differences in the four genera between the mild or moderate periodontitis cohort and the severe periodontitis cohort.The bacteria enriched in Ⅰ&Ⅱ stage periodontitis group included Corynebacterium,Streptobacillus and Victivallis.Eggerthella was enriched in severe periodontitis cohort(LDA>2).Spearman correlation analysis showed that Streptococcus enriched in Ⅰ&Ⅱ stage periodontitis group was positively correlated with daily brushing frequency,and Victivallis was negatively correlated with age.6.KEGG Orthology(KO)analysis showed that 171 functional genes were different between chronic periodontitis cohort and periodontal healthy cohort,among which 50 genes were significantly different(P<0.025),and 34 genes were concentrated in chronic periodontitis group.At the same time,25 functional enzymes were found to be different between Ⅰ&Ⅱ stage periodontitis group and Ⅲ&Ⅳ stage periodontitis group.There were six functional enzymes with significant differences(P<0.025),five functional enzymes were enriched in Ⅰ&Ⅱ stage periodontitis group and one functional enzymes were enriched in Ⅲ&Ⅳ stage periodontitis group.ConclusionIn this study,we found that there were significant differences at microbial community and functional genes in saliva samples between the periodontal health cohort and the chronic periodontitis cohort under excluding systemic and local factors,but no significant differences between the Ⅰ&Ⅱ stage periodontitis group and theⅢ&Ⅳ stage periodontitis group. |
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