Simón-Soro Aurea, Guillen-Navarro Miriam, Mira Alex
FISABIO Foundation, Center for Advanced Research in Public Health, Valencia, Spain.
FISABIO Foundation, Center for Advanced Research in Public Health, Valencia, Spain;
J Oral Microbiol. 2014 Oct 24;6:25443. doi: 10.3402/jom.v6.25443. eCollection 2014.
Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms.
To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay.
Non-cavitated enamel caries lesions (n=15) and dentin caries lesions samples (n=12) were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition.
A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.02-0.73% of the total bacterial community.
The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci detected confirms that they are a minority and questions its importance as the main etiological agent of tooth decay. Future experimental work should be performed to confirm the cariogenicity of the identified bacteria.
确定龋损中的微生物种类有助于明确龋齿的病因。然而，龋损中相当一部分细菌尚未得到培养，分子方法的应用也仅限于基于DNA的方法，该方法能检测活跃的和不活跃或已死亡的微生物。
确定疾病进展不同阶段龋损中基于RNA的、具有代谢活性的细菌组成，以便提供一份潜在的龋齿病原体清单。
从13名个体中收集非龋洞性釉质龋损样本（n = 15）和牙本质龋损样本（n = 12）。提取RNA并构建cDNA，用于扩增16S rRNA基因。使用Titanium-plus化学方法对所得的780 bp聚合酶链反应产物进行焦磷酸测序，并利用获得的序列确定细菌组成。
每个样本平均获得4900条16S rRNA基因序列，平均读长为661 bp，从而全面了解龋损中活跃的细菌群落。细菌多样性估计表明，龋洞中的微生物群高度复杂，每个样本包含70至400种具有代谢活性的细菌。这些细菌群落的组成在个体之间以及同一个体的不同龋损之间存在差异。此外，釉质和牙本质龋损的细菌构成不同。乳酸杆菌几乎仅在牙本质龋洞中发现。链球菌在釉质龋的总活跃菌群中占40%，在牙本质龋中占20%。然而，变形链球菌仅占细菌群落总数的0.02 - 0.73%。
数据表明龋齿的病因与组织有关，且该疾病具有明确的多微生物起源。检测到的变形链球菌比例较低，证实它们是少数，这对其作为龋齿主要病原体的重要性提出了质疑。未来应开展实验工作以证实所鉴定细菌的致龋性。
