**1. Introduction**

Black stain (BS) is a characteristic, extrinsic dental discoloration that occurs on the cervical third of the tooth and follows the contour of the gingival margin, particularly in primary dentition [1]. BS is thought to be a special form of dental plaque related to calcification because it contains an insoluble ferric salt, which is likely ferric sulfide, and has high calcium and phosphate contents [2]. The ferric sulfide may be formed through a reaction between hydrogen sulfide produced by bacterial action and iron in saliva or gingival exudates.

BS occurs at any age, but there is a higher prevalence in childhood that decreases in adolescence and adulthood [3]. Studies have shown no difference in occurrence between the sexes [1]. Researchers have been interested in BS because of its correlation with the low caries experience [2,4]. However, a recent study of more than 3000 people found no association between dental caries and BS [5].

Many studies have examined BS-related bacteria, but the causative bacteria is still not clear [1,3]. Early ultrastructural examinations of BS demonstrated that the black material is a ferric salt,

probably ferric sulfide, formed by the reaction between sulfide produced by bacterial action and iron in the saliva or gingival exudate [2,3]. The most commonly cultivated bacteria from BS teeth were *Actinomycetes* with the detection of rarely pigmented Gram-negative rods [6]. Black-pigmented bacteria were found primarily in extrinsic tooth stains [6,7]. Among black-pigmented bacteria, *Prevotella intermedia* and *Prevotella nigrescens* are the ones most frequently found in the oral cavity and associated with BS [3]. Soukos et al. [6] reported that *Porphyromonas gingivalis*, *P. intermedia* and *P. nigrescens* were related to BS using DNA–DNA hybridization for 40 taxa, whereas Saba et al. [7] suggested that *P. gingivalis* and *Prevotella melaninogenica* were absent and *Actinomycetes* sp. and *Aggregatibacter actinomycetemcomitans* were present in BS by means of polymerase chain reaction (PCR) and electrophoresis gel on the agarose medium for 200 people. This conflict was partly caused by limitations in the analysis methods. As a result, more advanced analysis methods are needed.

Recent advances in sequencing technologies, such as 454 pyrosequencing, have provided a deep understanding of the metagenomic diversity of the human oral microbiome [8]. Pyrosequencing can identify bacterial sequences and their amounts to give both qualitative and quantitative information of the oral microbiome, including uncultivable microorganisms [8]. For example, high-throughput pyrosequencing has revealed that dental plaques pooled from adults contain approximately 10,000 microbial phylotypes, with the ultimate diversity of oral microbiomes being estimated to contain approximately 25,000 phylotypes [9].

The aim of this study was to identify the oral microbiome in BS using pyrosequencing and to test our hypothesis that the oral microbiome of BS in children might be related to black-pigmented bacteria.
