Analysis of the Microbiota of Black Stain in the Primary Dentition

PLoS One. 2015 Sep 4;10(9):e0137030. doi: 10.1371/journal.pone.0137030. eCollection 2015.


Black tooth stain is a characteristic extrinsic discoloration commonly seen on the cervical enamel following the contour of the gingiva. To investigate the relationship between black tooth stain and the oral microbiota, we used 16S rRNA gene sequencing to compare the microbial composition of dental plaque and saliva among caries-free children with and without black stain. Dental plaque and saliva, as well as black stain, were sampled from 10 children with and 15 children without black stain. Data were analyzed using the pipeline tool MOTHUR. Student's t-test was used to compare alpha diversities and the Mann-Whitney U test to compare the relative abundances of the microbial taxa. A total of 10 phyla, 19 classes, 32 orders, 61 families and 102 genera were detected in these samples. Shannon and Simpson diversity were found to be significantly lower in saliva samples of children with black stain. Microbial diversity was reduced in the black stain compared to the plaque samples. Actinomyces, Cardiobacterium, Haemophilus, Corynebacterium, Tannerella and Treponema were more abundant and Campylobacter less abundant in plaque samples of children with black stain. Principal component analysis demonstrated clustering among the dental plaque samples from the control group, while the plaque samples from the black stain group were not and appeared to cluster into two subgroups. Alterations in oral microbiota may be associated with the formation of black stain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinomyces / classification
  • Actinomyces / genetics
  • Actinomyces / isolation & purification
  • Bacteroidetes / classification
  • Bacteroidetes / genetics
  • Bacteroidetes / isolation & purification
  • Cardiobacterium / classification
  • Cardiobacterium / genetics
  • Cardiobacterium / isolation & purification
  • Case-Control Studies
  • Child, Preschool
  • Corynebacterium / classification
  • Corynebacterium / genetics
  • Corynebacterium / isolation & purification
  • Dental Plaque / microbiology*
  • Female
  • Genes, Bacterial*
  • Haemophilus / classification
  • Haemophilus / genetics
  • Haemophilus / isolation & purification
  • Humans
  • Male
  • Microbiota / genetics*
  • Multigene Family
  • Phylogeny
  • RNA, Ribosomal, 16S / genetics*
  • Saliva / microbiology
  • Tooth Discoloration / microbiology*
  • Tooth, Deciduous / microbiology*
  • Treponema / classification
  • Treponema / genetics
  • Treponema / isolation & purification


  • RNA, Ribosomal, 16S

Grant support

This work was funded by the National Natural Science Foundation of China (81200762 to FC) and Peking University School of Stomatology (PKUSS20110301 to FC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.