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Available online at www.sciencedirect.com

Pediatric Dental Journal

journal homepage: www.elsevier.com/locate/pdj

Case Report

Black pigmentation in primary dentition: Case

report and literature review

Yukiko Takashima, Yuki Matsumi, Yoshie Yamasaki, Keiko Hirano,

Kanako Yanagida, Kazuyo Fujita, Michiyo Matsumoto-Nakano*
Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, Okayama, Japan

article info abstract

Article history: Black tooth staining is an extrinsic discoloration found in both primary and permanent
Received 27 March 2014 dentition, and seen as dark pigmented lines extending to the gingival margin or an
Received in revised form incomplete coalescence of dark dots that rarely extend beyond the cervical third of the
4 August 2014 crown. An association between black tooth staining and Actinomyces bacterial strains has
Accepted 18 September 2014 been reported, while black-pigmented bacteria associated with such staining are known to
Available online 22 November 2014 be harbored in the oral cavity. Prevotella intermedia and Prevotella nigrescens are black-
pigmented bacteria known to be dependent on the heme portion of hemoglobin as an
Keywords: iron source required for their growth. Recently, developments in molecular biological
Pigmentation techniques have enabled rapid and easy detection of periodontopathic bacterial species
Primary dentition using bacterial DNA extracted from oral specimens, such as plaque and saliva. Here, we
Prevotella intermedia report a case of black pigmentary deposition identified on all teeth of a 2-year-old girl, as
well as the results of analysis of the distribution of oral bacteria in saliva and plaque
specimens obtained from the patient using a molecular biological technique. In addition, a
literature search found a case of disease related to the oral bacteria detected in our patient.
We concluded that the bacteria detected in this case may have a strong relationship with
black pigmentation, although the route of bacterial infection and cause of staining remain
to be elucidated.
Copyright © 2014 The Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All
rights reserved.

deposition is caused by various deposits on teeth, such as

1. Introduction coloring by exogenous food, drink, or metal; endogenous
systemic disease and associated pulp necrosis; or adminis-
Tooth discoloration is a common dental finding that is asso- tered medicine [2]. Black tooth staining is an extrinsic discol-
ciated with clinical and esthetic problems [1]. Pigmentary oration seen in both primary and permanent dentition, and

* Corresponding author. Department of Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharma-
ceutical Science, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan. Tel.: þ81 86 235 6715; fax: þ81 86 235 6719.
E-mail address: mnakano@cc.okayama-u.ac.jp (M. Matsumoto-Nakano).
http://dx.doi.org/10.1016/j.pdj.2014.09.003
0917-2394/Copyright © 2014 The Japanese Society of Pediatric Dentistry. Published by Elsevier Ltd. All rights reserved.
 p e d i a t r i c d e n t a l j o u r n a l 2 4 ( 2 0 1 4 ) 1 8 4 e1 8 8 185

shown as pigmented dark lines extending to the gingival

margin or incomplete coalescence of dark dots that rarely
extend beyond the cervical third of the crown [3,4]. Such
staining has been speculated to be a form of dental plaque
different from other types based on contents that include
insoluble iron salt and high levels of calcium and phosphate
[4,5]. The black material is a ferric compound, most likely
ferric sulfide, that arises from the interaction of hydrogen
sulfide with iron in saliva or gingival fluid [4].
An association between black tooth staining and Actino-
myces bacterial strains has been reported [6,7]. In addition,
black-pigmented bacteria normally harbored in the oral cavity
are associated with such black stains [8]. Prevotella intermedia
and Prevotella nigrescens, black-pigmented bacteria, are
dependent on the heme portion of hemoglobin as an iron
source required for bacterial growth [9], and black pigmenta-
tion results from associated chemical reactions.
Developments in molecular biological techniques have
enabled rapid and easy detection of periodontopathic bacte-
rial species using bacterial DNA extracted from oral speci-
mens, such as plaque and saliva [10]. Based on analyses of
specimens collected from children and adolescents, the Fig. 1 e Oral photographs. (A) First visit. (B) After scaling
following 10 periodontal species are considered to be associ- and polishing.
ated with periodontal diseases: Porphyromonas gingivalis, Tan-
nerella forsythia, Campylobacter rectus, Eikenella corrodens,
Aggregatibacter actinomycetemcomitans, Capnocytophaga ochra-
cea, Capnocytophaga sputigena, and Treponema denticola, as well 3. Identification of oral bacteria in saliva
as the black-pigmented bacteria P. intermedia and P. nigrescens. specimens and review of literature
Other studies have found that polymerase chain reaction
(PCR) methods are quite sensitive and rapid for determining Whole saliva specimens were collected from the patient at 2
the prevalence of periodontal bacteria [11,12], and it has been years 2 months, 2 years 11 months, and 3 years 2 months of
speculated that a PCR assay system would be a useful tool for age. Subgingival dental plaque was also collected from the
analyzing the role of the black-pigmented Prevotella species in buccal side of both maxillary second primary molar teeth at 3
the mouth [8]. years 2 months of age and mixed with 1 ml of sterile saline.
Herein, we report a case of black pigmentary deposition Microbiological analyses were performed as previously
identified on all teeth of a 2-year-old girl as well as our anal- described [13]. Briefly, the specimens in sterile saline were
ysis of the distribution of related oral bacteria in saliva spec- centrifuged at 15,000 rpm for 5 min to pellet the bacterial cells,
imens using a molecular biological technique. and then bacterial genomic DNA was extracted from each
pellet using a DNA isolation kit (QIAGEN Sciences, German-
town, MD, USA). The samples were frozen at 20  C and stored
until use. Amplification of 16S ribosomal DNA (rDNA)
2. Case report occurred under standardized conditions using previously
published universal eubacterial primers. The primers used
The protocol of the present study was approved by the Ethics were PA 50 -AGA GTT TGA TCC TGG CTC AG-30 and PD 50 -GTA
Committee of Okayama University (No. 702). A girl of age 2 TTA CCG CGG CTG CTG-30 . Using a hot-start protocol, the
years 2 months visited our clinic for detailed examination of samples were denatured at 95  C for 15 min, followed by
pigmentation on the teeth, initially noticed by her guardian amplification consisting of a denaturation step at 95  C for
in January 2012 when the patient was approximately 1 year 5 40 s, annealing at 58  C for 1 min, and elongation at 72  C for
months of age. An intraoral examination showed that the 1 min. A total of 30 cycles was performed, followed by a final
first molar tooth had not yet emerged, with no caries lesions elongation step at 72  C for 10 min. The amplified fragments
or periodontal problems. Wide-ranging black pigmentation were cloned into a pGEM-T Easy vector (Promega, Madison,
was recognized on the surfaces of all teeth (Fig. 1). The WI, USA) and clones from each sample were randomly cho-
medical history and dietary habits of the patient were unre- sen. Sequencing was performed by Fasmac Co., Ltd. (Atsugi,
markable, there was no family history of dental diseases, and Kanagawa, Japan). For each of the 16S rDNA fragments, the
she had not taken any medicine up to that time. Based on sequences of all isolates were compared using a Japanese DNA
these findings, we diagnosed the black pigmentation as database (http://www.ddbj.nig.ac.jp/index-j.html) and their
foreign precipitation and decided to perform ultrasonic identities determined.
scaling and tooth flank polishing every 3 months to remove The numbers of mutans streptococci and lactobacilli or-
the pigmentation. Fig. 1B shows the appearance after ganisms in the specimens were measured using previously
treatment. described methods [14,15]. Briefly, the saliva and plaque
186 p e d i a t r i c d e n t a l j o u r n a l 2 4 ( 2 0 1 4 ) 1 8 4 e1 8 8
specimens were diluted with saline, inoculated onto Mitis
Salivarius agar plates containing bacitracin and lactobacillus-
selective (LBS) agar, and incubated anaerobically for 48 h at
37  C. The number of colonies on each plate was counted
using a microscope.
In samples obtained at the age of 2 years 2 months (n ¼ 24),
the frequency of detection of Neisseria meningitides (Nme),
Neisseria subflava (Ns), and other Neisseria sp. was 38% for each,
while Prevotella sp. was detected at a frequency of 28.6%,
Streptococcus mitis at 14.3%, Veillonella parvula (Vp) at 9.5%, and
Selenomonas artemidis (Sa) and Porphyromonas sp. each at 4.8%
(Fig. 2). In specimens obtained at 2 years 11 months of age
(n ¼ 30), the detection rate of Abiotrophia defectiva (Ad) was
21.7%, while that of Propionibacterium propionicum (Pp) and
Streptococcus sanguinis was 13% for each. In addition, Kingella
denitrificans (Kd), Neisseria mucosa (Nmu), Neisseria sp., and
Sphingomonas rhizogenes (Sr) were detected at a rate of 8.7%,
and Actinomyces odontolyticus (Ao), Lautropia mirabilis (Lm),
Leptotrichia sp., and Streptococcus sp. at a rate of 4.3%. On the
other hand, Prevotella sp. was not detected at this time. In
specimens obtained at 3 years 2 months of age (n ¼ 25), N.
mucosa was detected at a rate of 26.1%; Rothia aeria (Ra) at a
rate of 21.7%; Rothia mucilaginosa (Rm) and Neisseria sp. at a rate
of 8.7%; and Ad, Campylobacter curvus (Cc), Kd, Lm, Leptotrichia
sp., Prevotella sp., Pseudomonas sp., and S. mitis at a rate of 4.3%
(Fig. 2). The detection rate of Prevotella sp. was lower than that
seen in the first examination.
The detection rate in dental plaque samples at 3 years 2
months of age (n ¼ 25) was 29.4% for Leptotrichia sp., 17.6% for
S. sanguinis, 11.8% for Aggregatibacter sp., Nmu, and Pp; and
5.9% for Ad, Kd, and Neisseria sp. (Fig. 3). There were significant
differences between the saliva and plaque samples with re-
gard to the detection of oral bacteria.
Bacteria collected from the patient at 3 years 2 months of
age were investigated for their relationship to diseases using a
database (Table 1). Most in both the saliva and plaque speci-
mens were found to be gram-negative bacteria with detection
rates of 80.9% and 64.7%, respectively. In addition, several of
the bacterial strains were found to be related to genetic dis-
eases such as bacteremia and endocarditis.
On the other hand, mutans streptococci and lactobacilli
were not shown in either the saliva or plaque specimens,
Fig. 2 e Detection rates of oral bacteria in saliva specimens at 2 years 2 months, 2 years 11 months, and 3 years 2 months.
although oral streptococci strains were detected (data not
shown).
4. Discussion
The presence of black extrinsic tooth staining has been re-
ported to be associated with low caries occurrence in affected
patients as compared to individuals with healthy tooth sur-
faces [1,6]. Streptococcus mutans was not detected in the pre-
sent patient and no caries lesions were found, although
periodontal bacteria were present. Our findings support the
notion that there is no relationship between dental caries and
black staining. Therefore, we performed detailed examina-
tions of the oral microflora in our patient including the pres-
ence of the black-pigmented bacteria P. intermedia and P.
nigrescens, as they are known to have a strong relationship
with black staining on teeth [7,8]. In addition, these strains
have been proposed as possible periodontopathic bacteria in
some types of periodontitis [33,34].
Although inflammatory periodontal diseases are common
in children, they rarely result in loss of periodontal attach-
ment or resorption of alveolar bone [35]. Therefore, coloniza-
tion of periodontal bacteria has rarely been reported in
healthy children, although periodontitis seen in young ages is
classified as periodontitis as a manifestation of systemic dis-
ease, aggressive periodontitis, or chronic periodontitis [36]. In
the present patient, P. intermedia and P. nigrescens were
detected in saliva specimens, whereas neither of these strains
was detected in healthy children under 3 years of age in a
previous study [10]. In addition, P. intermedia and P. nigrescens
are considered to be dependent on the heme portion of he-
moglobin as a required iron source for their growth [9], and
black pigmentation is related to that function. The medical
history of our patient was unremarkable and no additional
iron supplementation had been taken. Other possible etio-
logical agents include dietary components, beverages, to-
bacco, mouth rinses, and other medicaments [37]. However,
no special substrates were ingested by our patient. On the
other hand, black tooth staining may be associated with high
salivary calcium and buffer capacity [38]. Furthermore, sali-
vary proteins, especially proline-rich protein, can mediate
 p e d i a t r i c d e n t a l j o u r n a l 2 4 ( 2 0 1 4 ) 1 8 4 e1 8 8 187

Fig. 3 e Detection rates of oral bacteria in plaque specimens at 3 years 2 months. Ad, Abiotrophia defectiva; Kd, Kingella
denitrificans; Nmu, Neisseria mucosa; Pp, Propionibacterium propionicum; Ss, Streptococcus sanguinis.

increased staining to interact with dietary constituents [39].
Additional analysis of saliva specimens will be needed to
clarify this mechanism.
After scaling at the age of 2 years 2 months, the rates of
detection of black-pigmented bacteria were decreased and
black staining disappeared in our patient. Therefore, we
concluded that these pigmented bacteria may have a rela-
tionship with black pigmentation.
S. sanguinis and S. mitis, which were also detected among
the oral streptococci harbored by our patient, have been
shown to be responsible for a variety of infectious complica-
tions including bacteremia and infective endocarditis [20].
Furthermore, both produce hydrogen peroxide (H2O2), which
is considered to play important roles in bacterial competition
in microbial communities such as oral biofilm [40]. On the
other hand, S. mutans was not detected; thus, it remains to be
elucidated if the phenomenon is caused by production of H2O2
by these organisms.
Taken together, black staining such as that seen in the
present case may have a strong relationship with the presence
of black-pigmented bacteria. Furthermore, a molecular bio-
logic technique, such as the one utilized in our study, may be
useful for diagnosis of the phenomenon as well as treatment.
However, the source of bacterial infection in our patient re-
mains to be found. Additional research about the infection
process and pathogenesis of black pigmentation is needed.

Table 1 e Summary of bacteria in saliva and plaque specimens at 3 years 2 months of age.
Detection rate (%) Related diseases References
Saliva Plaque
(n ¼ 25) (n ¼ 25)
Gram-positive bacteria
Abiotrophia defectiva e 5.9 Endocarditis [16,17]
Propionibacterium propionicum e 11.8 Acne [18]
Selenomonas artemidis 4.8 e Bacteremia [19]
Streptococcus mitis 14.3 e Endocarditis [20]
Streptococcus sanguinis e 17.6 Endocarditis [21]
Gram-negative bacteria
Aggregatibacter sp. e 11.8 Periodontitis, endocarditis, [22e24]
brain abscess
Kingella denitrificans 19 5.9 Endocarditis [25]
Leptotrichia sp. e 29.4 Bacteremia, endocarditis [26e28]
Neisseria sp. 19 5.9 e e
Neisseria meningitidis 9.5 e Nasopharynx [29,30]
Neisseria mucosa e 11.8 Bacteremia [31]
Neisseria subflava 9.5 e Bacteremia, endocarditis [32]
Porphyromonas sp. 4.8 e Periodontitis [12]
Prevotella sp. 28.6 e Periodontitis [8,9]
Veillonella parvula 9.5 e e e
188 p e d i a t r i c d e n t a l j o u r n a l 2 4 ( 2 0 1 4 ) 1 8 4 e1 8 8

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