猖獗龋患者的病因和口腔微生物种类分析:1例报告

Analysis of causes and whole microbial structure in a case of rampant caries

目的分析患者口腔全微生物种类,对猖獗龋患者提出细菌学预防与治疗手段。方法抽血查免疫全套和血常规,获取全菌斑(A)、龋损(Q)、唾液(T)、黏膜(N)样本经原代涂片镜检,并传代分离培养后采用形态学、梅里埃鉴定仪、16S r DNA序列进化分析鉴定微生物种类;进一步采用Q-PCR分析变异链球菌的含量,DGGE分析微生物组成和结构。结果血液免疫全套基本正常,但Ig E、中性分叶核细胞增高。原代样本涂片发现唾液中有大量极长链的细菌,经培养发现唾液中总菌含量最高,龋损样本中溶血菌含量最高,变异链球菌在全菌斑中最多。梅里埃和测序鉴定出33株细菌,全菌斑中分离出多种链球菌和韦德纤毛菌、龋损菌斑中多次分离到变异链球菌、具核梭杆菌和苏黎世链球菌,唾液样本中分离出多种乳杆菌。q PCR发现变异链球菌含量明显增高;变性梯度凝胶电泳(DGGE)显示该患者细菌多样性明显降低,但丰度明显增强;条带切胶测序检出多种纤毛菌。结论患者免疫功能基本正常,血液检测呈现感染血象,病因疑与细菌感染有关。可能由于唾液减少导致变异链球菌、多种纤毛菌等致病微生物过度繁殖、细菌多样性减少而引起生态失衡。

Objective To analyze the whole microbial structure in a case of rampant caries to provide evidence for its prevention and treatment. Methods Clinical samples including blood, supragingival plaque, plaque in the caries cavity, saliva,and mucosal swabs were collected with the patient’s consent. The blood sample was sent for routine immune test, and the others samples were stained using Gram method and cultured for identifying colonies and 16 S r RNA sequencing. DNA was extracted from the samples and tested for the main cariogenic bacterium(Streptococcus mutans) with q PCR, and the whole microbial structure was analyzed using DGGE. Results The patient had a high levels of Ig E and segmented neutrophils in his blood. Streptococci with extremely long chains were found in the saliva samples under microscope. Culture of the samples revealed the highest bacterial concentration in the saliva. The relative content of hemolytic bacterium was detected in the samples, the highest in the caries cavity; C. albicans was the highest in the dental plaque. In addition, 33 bacterial colonies were identified by VITEK system and 16 S r DNA sequence phylogenetic analysis, and among them streptococci and Leptotrichia wade were enriched in the dental plaque sample, Streptococcus mutans, Fusobacterium nucleatum, and Streptococcus tigurinus in the caries cavity, and Lactobacillus in the saliva. S. mutans was significantly abundant in the mucosal swabs, saliva and plaque samples of the caries cavity as shown by q PCR. Compared to samples collected from a healthy individual and another two patients with rampant caries, the samples from this case showed a decreased bacterial diversity and increased bacterial abundance shown by PCR- DGGE profiling, and multiple Leptotrichia sp. were detected by gel sequencing. Conclusion The outgrowth of such pathogenic microorganisms as S. mutans and Leptotrichia sp., and dysbiosis of oral microbial community might contribute to the pathogenesis of rampant caries in this case.