# The Oral Microbiome in Children with Black Stained Tooth
## 4 Discussion  
Conventional BS therapy has been focused on the mechanical removal of black pigments themselves because the exact mechanism of BS prevalence had yet to be identified [1]. In this study, we used pyrosequencing analysis of supragingival dental plaque of children with and without BS to understand the microbiological aspects of BS, which may lead to better prevention and treatment for BS. The genera _Porphyromonas_, _Streptococcus_ and _Neisseria_, were abundant in the microbiome of children with BS, while the genera _Corynebacterium_, _Prevotella_ and _Selenomonas_g1_ were abundant in children without BS in this study. This study suggests that dynamics in the oral microbiome may cause the formation of BS in children, and the microbial population in the above genera would play important roles in changing microbial dynamics. The genus _Porphyromonas_ was significantly abundant in the BS group (Figure 2). All the species belonging to this genus exhibited a similar pattern in abundance (Figure 3). Four _Porphyromonas_ sp. showed a trend to increase as the BS scales increased (Figure 4). _Porphyromonas_ is an obligate anaerobic bacterium that produces a black pigment called porphyrin [11]. This pigment can protect the bacterial cell from oxidative stress by reducing oxygen. _P. gingivalis_ is one of the most well-known species that produces black pigments [12] and is implicated in the progression of periodontal disease [4]. However, this species was not one of the pathogens observed in this study. _Porphyromonas catoniae_ is the most common species in this genus (Figure 3A). It is frequently found in the oral microbiota of healthy children [9] and is known to be a pioneer bacterium that is established in the mouths of babies [13]. In addition, it is a non-pigmented species [14]. However, it is possible that this species may play an opportunistic role in the formation of BS because this species exhibits activities of diverse cellular enzymes like trypsin, which is important in bacterial colonization [13]. The other _Porphyromonas_ spp. may contribute to the accumulation of black pigments. The genus _Streptococcus_ was also observed abundantly in the BS group (Figures 2 and 3B). _S. cristatus_, _S. sanguuins_, _S. oralis_, _S. mitis_ and _S. gordonii_ are known as oral biofilm bacteria that colonize the tooth surface [15; 16]. Since these species are normal tooth microbiome and do not produce black pigments, they may contribute to the formation of microbial beds on teeth and interact with other species producing black pigments. Although the genus _Streptococcus_ makes up 10% of the total microbiome, _S. mutans_ was detected in only one subject (BS2, five reads). This rare detection of _S. mutans_ is consistent with a recent report of BS patients where _S. mutans_ was not found. This may be due to the antagonistic effect of the predisposed _Streptococcus_ spp. _S. sanguinis_ and _S. gordonii_ that are known to inhibit the growth of _S. mutans_ by producing hydrogen peroxide [17]. A recent study using pyrosequencing suggests that the genus _Streptococcus_ was most abundant but showed no difference between the groups [18]. Another pyrosequencing study reported that this genus was observed frequently in subjects having both caries and pigments, but not in subjects having only pigments [19]. Interestingly, a recent microbiome study showed that the population of _P. catoniae_ was decreased in children with caries and increased in children with healthy oral status [9]. Since the _P. catoniae_ population was found to be increased in our BS group, it may have a negative influence on caries development (Figure 3A). _Veillonella dispar_ and _V. rodentium_ were identified as positive candidates for BS formation (Figure 4). _V. dispar_ produces hydrogen sulfide (H\({}_{2}\)S) from \({}_{\text{L}}\)-cysteine [20]. The genus _Actinomyces_ can produce H\({}_{2}\)S and has been known to be a strong candidate for BS formation.