涎腺导管菌群和口腔菌群多样性与涎石病的相关性研究

A correlation study of the diversity of oral and salivary gland ductal microbiota and sialolithiasis

目的探究涎腺导管结石患者是否存在导管内或口腔菌群失调,探讨细菌与涎石病发生的相关性。方法收集20例涎腺导管结石(1例腮腺结石,19例下颌下腺结石)患者的导管结石样本、导管内唾液及口腔内唾液样本作为实验组,收集20例健康志愿者导管内唾液及口腔内唾液样本作为对照组。提取样品的细菌DNA采用酶链聚合反应进行扩增。采用Roche高通量454焦磷酸测序技术对细菌16S rRNA V1~V3可变区的PCR扩增子进行测序,进一步利用生物信息学分析方法对测序数据进行挖掘,明确样本中细菌组分、群落结构,比较实验组与对照组间差异性。结果成功对唾液及结石样本进行了DNA测序分析,样本稀疏曲线显示测序深度充分,测序覆盖深度(Coverage指数)满足实验要求。实验组与对照组的导管内唾液及口腔内菌群的生物多样性无差异,结石样本与导管内菌群生物多样性无差异(P>0.05)。唾液腺导管内菌群的生物多样性指标香农指数、Chao指数、ACE指数均大于口腔内菌群(P<0.05)。导管内菌群结构比较显示:细菌门水平,梭杆菌门在实验组中显著高于对照组(P<0.05);细菌属水平,普氏菌属、卟啉单胞菌属及奈瑟氏菌属在实验组中均显著减少(P<0.05)。口腔内菌群结构比较显示:在细菌门水平,变形菌门在实验组中显著高于对照组(P<0.05);细菌属水平,普氏菌属、链球菌属及韦永氏球菌属在实验组中均显著低于对照组(P<0.05)。结论结石样本中的微生物菌群结构及多样性与唾液腺导管内相似,唾液腺导管内微生物多样性显著高于口腔内。涎石病患者与健康对照人群的口腔及唾液腺导管微生物群落结构存在显著差异。涎石病患者的口腔及唾液腺导管内存在菌群失调情况。

Objective To investigate whether there are intraductal or oral dysbacteriosis in patients with sialolithiasis, and to explore the relationship between bacteria and the occurrence of sialolithiasis. Methods A total of 20 patients with sialolithiasis(1 parotid and 19 submandibular gland) were enrolled in the study as the experimental group. Samples of stone, salivary in oral cavity or intraduct were collected. A total of 20 healthy volunteers were included in the study as the control group. Samples of salivary in oral cavity or intraduct were collected. The bacterial DNA of the extracted samples was amplified by enzyme chain polymerization. The Roche high-throughput 454 pyrosequencing technology was used to sequence the PCR amplicons of the 16 S rRNA V1-V3 variable region of bacteria. The sequencing data were further analyzed by using bioinformatics methods to identify the bacterial components and communities. The differences between the experimental and control groups were compared. Results DNA sequencing of saliva and stone samples was successfully performed. Sample sparse curves showed that the sequencing depth was sufficient, and the coverage coverage depth(Coverage index) met the experimental requirements. There was no difference in the biological diversity of intraductal saliva and intraoral microbiota between the experimental group and the control group. There was also no difference in the biological diversity between the stone sample and the intraductal saliva(P>0.05). The Shanno index, Chao index, and ACE index were higher in intraductal saliva than oral cavity(P<0.05). Intraductal microbiota structure comparison showed that Fusobacterium in the experimental group was significantly higher than the control group(P<0.05). At genus level, Prevotella, Porphyromonas and Neisseria were significantly reduced in the experimental group(P<0.05). The comparison of bacterial structures in the oral cavity showed that at the phylum level, Proteobacteria was significantly higher in the experimental group than in the control group(P<0.05). At genus level, Proteus, Streptococcus, and Veillonococcus in the experimental group were significantly lower than the control group(P<0.05). Conclusion The structure and diversity of microbiota in the stone sample are similar to salivary gland duct. The microbial diversity in the salivary gland duct is significantly higher than oral cavity. There is a significant difference in the microbial community between sialolithiasis patients and controlled subjects. There is a dysbacteriosis in the oral and salivary gland ducts of sialolithiasis patients.