*Article* **The Oral Microbiome in Children with Black Stained Tooth**

**Ji Young Hwang 1, Hyo-Seol Lee 2, Jaehyuk Choi 3, Ok Hyung Nam 2, Mi Sun Kim 2,4 and Sung Chul Choi 2,\***


Received: 25 September 2020; Accepted: 9 November 2020; Published: 13 November 2020

**Abstract:** Black stain (BS) is a characteristic extrinsic discoloration, which occurs along the third cervical line of the buccal and/or lingual surfaces of teeth, particularly in the primary dentition of humans. BS is produced by oral bacteria and byproducts of saliva, but there is a controversy about related bacteria. The aim of this study was to identify the oral microbiome in tooth BS using pyrosequencing. It was hypothesized that the oral microbiome of BS in children might be related to black-pigment producing bacteria. Supragingival dental plaque was obtained from six children (mean 8.1 years) with BS and four children (mean 8.3 years) without BS. The bacterial metagenome was obtained by pyrosequencing. The BS group contained 348 operative taxonomic units (OTUs), whereas the control group had 293 OTUs. Microbial abundance and diversity were significantly higher in the BS group (*p* < 0.05). In the heatmap, the correlation between samples was the same as the BS scale. At the genus level, six genera—*Abiotrophia, Eikenella, Granulicatella, Neisseria, Porphyromonas* and *Streptococcus*—were significantly different between the two groups (*p* < 0.05). We suggested that compositional changes in the oral microbiome are essential, and several species in the genus *Neisseria, Porphyromonas* and *Streptococcus* may be major contributors for BS formation. Although the number of subjects was relatively limited, our study is the first species-level analysis of pyrosequencing data in BS formation.

**Keywords:** black stain; oral microbiota; pyrosequencing; *Neisseria*; *Porphyromonas*; *Streptococcus*
