龋病和牙周病患者唾液菌群和代谢产物的比较研究

Saliva microbiota and metabolite in individuals with caries or periodontitis

目的明确龋病和牙周炎来源的唾液菌群在物种组成、功能及代谢产物上的特点,为寻找可用于临床判断龋病和牙周炎发生的生物标志物提供依据。方法收集2022年3至6月就诊于第四军医大学口腔医学院牙体牙髓病科的10例高龋[龋失补牙数(decayed-missing-filled teeth,DMFT)≥6]患者[高龋(high caries,HC)组],牙周科的10例Ⅱ期A级~Ⅲ期C级牙周炎患者[牙周炎(periodontitis group,PG)组]和10名口腔健康个体[健康对照(healthy individuals,HH)组]的唾液样本。采集受试者的人口学基线、口腔内龋、牙周健康情况等信息,利用宏基因组测序和液相色谱质谱联用检测样本内微生物及其代谢产物。对测序数据进行分析获得各组样本的微生物物种分类学组成、功能基因及代谢产物的信息。各组受试人群的口腔基本情况及唾液样本采集均由同一名牙体牙髓病科主治医师完成。结果各组受试人群在年龄、性别等基线特征上差异均无统计学意义(P>0.05)。HC组DMFT(9.0±1.7)显著高于HH组和PG组(均为0)(F=243.00,P<0.001)。菌群分析显示,各组唾液菌群物种分类学组成在门水平均以变形菌门、厚壁菌门、拟杆菌门、放线菌门和梭杆菌门为主。在属水平,则主要由链球菌属、奈瑟菌属、罗氏菌属、普雷沃菌属等成员组成。差异分析显示,与HH组相比,HC组和PG组在属、种水平的物种分类学组成差异均有统计学意义(P<0.05)。在属水平,HC组和PG组相对丰度改变最显著的均为普雷沃菌属。而在种水平,HC组改变最显著的为苍白普雷沃菌;PG组改变最显著的为牙龈卟啉单胞菌。代谢产物分析显示,HC组共检出133种与HH组差异表达的代谢产物,其中差异最显著的代谢产物为3-羟基-1,5-二苯基戊烷-1-酮(P=0.001)。在PG组中,共检测出102种与HH组差异表达的代谢产物,其中差异最显著的代谢产物为N1-乙酰精胺(P=0.002)。HC组与PG组相比,代谢产物上差异最显著的代谢产物为D-氨基葡萄糖6-磷酸(P=0.006)。菌群代谢功能分析显示,HC组菌群糖代谢相关的功能基因丰度最高,其次为HH组,PG组的糖代谢相关功能基因最低。此外,与HH组相比,PG组内ABC转运体和磷酸转移酶系统等涉及糖转运相关的功能基因的丰度显著降低(P<0.05),在HC组中则显著升高(P<0.05)。结论唾液菌群在龋病、牙周炎及健康人群中存在显著异质性,且菌群的糖代谢能力改变与龋病和牙周炎的发生存在一定关联。苍白普雷沃菌及3-羟基-1,5-二苯基戊烷-1-酮可能作为龋病的潜在生物标志物;而牙龈卟啉单胞菌及N1-乙酰精胺可能作为牙周炎的潜在生物标志物。

Objective To detect and analyze the characteristics of oral microbiota in species composition,function and metabolism among caries,periodontitis and oral healthy individuals,hunting for the microbiome-derived biomarkers with specificity and sensitivity to estimate the occurrence of these two diseases.Methods Saliva samples were collected from 10 patients with high caries risk[decayed-missing-filled teeth(DMFT)≥6,HC group]in Department of Endodontics,10 patients with periodontitis of gradeⅡA-ⅢC(PG group)in Department of Periodontology and 10 oral healthy individuals(HH group)from School of Stomatology,The Fourth Military Medical University during from March 2022 to June 2022.A baseline examination was conducted on all participants,including their oral conditions of caries and periodontal health.Metagenomic sequencing(Illumina PE150 platform)and liquid chromatography-mass spectrometry were used to detect microorganisms and their metabolites in the samples respectively.The sequencing data were analyzed to obtain the information of microbial taxonomic composition,functional genes and metabolites in each group of samples.The basic oral conditions and saliva samples of subjects in each group were evaluated and collected by the same professional endodontist.Results There were no significant difference in baseline characteristics such as age and sex among the subjects in each group(P>0.05).DMFT in HC group(9.0±1.7)was significantly higher than that in HH group(0)and PG group(0)(F=243.00,P<0.001).Sequencing data analysis showed that the taxonomic compositions of salivary microbiota in each group were mainly Firmicutes,Bacteroidetes,Proteobacteria,Actinobacteria and Fusobacteria at the phylum level,and Streptococcus,Neisseria,Rothia,Prevotella at the genus level.Differential analysis showed that,compared with the HH group,HC group and PG group had significant differences in taxonomic composition(P<0.05),and the most significant among them was Prevotella.At the species level,Prevotella pallens was the most significant change in HC group,and Porphyromonas gingivalis in PG group.Metabolite analysis showed that there were significant differences in metabolites between HC group and PG group.The results showed that,compared with the HH group,the most significant metabolite change was 3-hydroxy-1,5-diphenylpentan-1-one in HC group(P=0.001)and N1 acetylspermine in PG group(P=0.002)respectively.Compared with the PG group,the metabolite of HC group with the most significant difference is D-glucosamine 6-phosphate(P=0.006).The metabolism gene function analysis showed that,the enrichment of carbohydrate metabolism related genes was highest in HC group,followed with HH group,and it was lowest in PG group.In addition,compared with the HH group,the abundance of functional genes related to glucose metabolism,such as ABC transporter and phosphotransferase system,were significantly decreased in PG group(P<0.05),but significantly increased in HC group(P<0.05).Conclusions There is a significant correlation between the alternation of carbohydrate metabolism of salivary microbiota with the occurrence of caries and periodontitis.In the future,Prevotella pallens and 3-hydroxy-1,5-diphenylpentan-1-one may be the potential biomarkers of caries;while Porphyromonas gingivalis and N1 acetylspermine work in the predictions of periodontitis.