基于全长16s测序技术鉴定成年人龋齿牙菌斑微生物

Identification of Initial Colonizing Bacteria in Dental Plaques from Young Adults Using Full-Length 16S rRNA Gene Sequencing6.519

mSystems . 2019 Sep 3;4(5):e00360-19. doi: 10.1128/mSystems.00360-19.

Abstract

Development of dental plaque begins with the adhesion of salivary bacteria to the acquired pellicle covering the tooth surface. In this study, we collected in vivo dental plaque formed on hydroxyapatite disks for 6 h from 74 young adults and identified initial colonizing taxa based on full-length 16S rRNA gene sequences. A long-read, single-molecule sequencer, PacBio Sequel, provided 100,109 high-quality full-length 16S rRNA gene sequence reads from the early plaque microbiota, which were assigned to 90 oral bacterial taxa. The microbiota obtained from every individual mostly comprised the 21 predominant taxa with the maximum relative abundance of over 10% (95.8 ± 6.2%, mean ± SD), which included Streptococcus species as well as nonstreptococcal species. A hierarchical cluster analysis of their relative abundance distribution suggested three major patterns of microbiota compositions: a Streptococcus mitis/Streptococcus sp. HMT-423-dominant profile, a Neisseria sicca/Neisseria flava/Neisseria mucosa-dominant profile, and a complex profile with high diversity. No notable variations in the community structures were associated with the dental caries status, although the total bacterial amounts were larger in the subjects with a high number of caries-experienced teeth (≥8) than in those with no or a low number of caries-experienced teeth. Our results revealed the bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults.IMPORTANCE Selective attachment of salivary bacteria to the tooth surface is an initial and repetitive phase in dental plaque development. We employed full-length 16S rRNA gene sequence analysis with a high taxonomic resolution using a third-generation sequencer, PacBio Sequel, to determine the bacterial composition during early plaque formation in 74 young adults accurately and in detail. The results revealed 21 bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults, which include several streptococcal species as well as nonstreptococcal species, such as Neisseria sicca/N flava/N mucosa and Rothia dentocariosa Given that no notable variations in the microbiota composition were associated with the dental caries status, the maturation process, rather than the specific bacterial species that are the initial colonizers, is likely to play an important role in the development of dysbiotic microbiota associated with dental caries.

Keywords: 16S rRNA; PacBio Sequel; dental plaque; initial colonizer; saliva.

牙菌斑是在牙齿表面形成的一种微生物生物膜，是龋齿和牙周炎的直接病因，也是造成牙齿脱落的主要原因。牙菌斑微生物群的发展是微生物有序附着在牙齿表面的动态过程，这种长期积累导致生物量的增加和组成的变化，同样会导致口腔疾病的风险增加。虽然已有针对口腔微生物群差异的研究，但是没有关于亚洲人群早期牙菌斑细菌组成的报道。

二代测序技术读长短，但吞吐率比Sanger测序高的多，使得我们能够同时获得数十种复杂菌群样品的全面信息。此外，基于Pacbio平台的三代测序技术能够获得平均长度~10kb的长测序读数，目前已有部分研究利用这一平台对复杂微生物群落展开全长16s rRNA基因分析。

Identification of Initial Colonizing Bacteria in Dental Plaques from Young Adults Using Full-Length 16S rRNA Gene Sequencing

1. 样本采集

3. 定量PCR分析

使用引物806F-926R进行早期牙菌斑样品中细菌总数的定量PCR分析

为确定所获得的的牙菌斑样本不是简单的唾液细菌群落，通过扩增16s rRNA的V1-V2区，将早期牙菌斑样品的微生物组成与唾液样本微生物群进行比较，基于UniFranc的PCoA图显示（图1），早期牙菌斑微生物群表现出与唾液细菌种群不同的群落结构，在种水平上，牙菌斑样本中的OTU数量显著低于唾液样本，但早期唾液样本中OTU相对丰度显著更高，这些差异表明，早期牙菌斑是通过选择性附着在羟基磷灰石上生长形成的独特细菌群落。

2. 牙菌斑微生物群组成

研究通过Pacbio测序，对全长16s rRNA序列进行分析，获得了141个已识别的分类单元，但所有受试者的早期牙菌斑微生物并没有出现交叉。同时，从每个个体获得的微生物群构成了21个主要类群，最大相对丰度超过10%，意味着这些特定类群在早期牙菌斑微生物类群中占大多数。S. mitis/Streptococcus sp. HMT-423和N. sicca/flava/mucosa是在早期噬菌斑样品中观察到的两个主要分类模式，受试者的细菌组成可以大致分为三种模式：S.mitis/ Streptococcus sp.HMT-423占主导地位、N.sicca/ flava/mucosa占主导地位和具有较高多样性的复杂组成，这一结果与16s rRNA V1-V2区测序结果一致，说明这些物种参与了早期牙菌斑的定殖。

图2. 受试者早期牙菌斑中21个主要类群的相对丰度

3. 易患龋齿相关的微生物群个体差异

图3. 不同龋齿数量受试者的细菌组成差异