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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.
* 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
specimens were diluted with saline, inoculated onto Mitis although oral streptococci strains were detected (data not
Salivarius agar plates containing bacitracin and lactobacillus- shown).
selective (LBS) agar, and incubated anaerobically for 48 h at
37 C. The number of colonies on each plate was counted
using a microscope. 4. Discussion
In samples obtained at the age of 2 years 2 months (n ¼ 24),
the frequency of detection of Neisseria meningitides (Nme), The presence of black extrinsic tooth staining has been re-
Neisseria subflava (Ns), and other Neisseria sp. was 38% for each, ported to be associated with low caries occurrence in affected
while Prevotella sp. was detected at a frequency of 28.6%, patients as compared to individuals with healthy tooth sur-
Streptococcus mitis at 14.3%, Veillonella parvula (Vp) at 9.5%, and faces [1,6]. Streptococcus mutans was not detected in the pre-
Selenomonas artemidis (Sa) and Porphyromonas sp. each at 4.8% sent patient and no caries lesions were found, although
(Fig. 2). In specimens obtained at 2 years 11 months of age periodontal bacteria were present. Our findings support the
(n ¼ 30), the detection rate of Abiotrophia defectiva (Ad) was notion that there is no relationship between dental caries and
21.7%, while that of Propionibacterium propionicum (Pp) and black staining. Therefore, we performed detailed examina-
Streptococcus sanguinis was 13% for each. In addition, Kingella tions of the oral microflora in our patient including the pres-
denitrificans (Kd), Neisseria mucosa (Nmu), Neisseria sp., and ence of the black-pigmented bacteria P. intermedia and P.
Sphingomonas rhizogenes (Sr) were detected at a rate of 8.7%, nigrescens, as they are known to have a strong relationship
and Actinomyces odontolyticus (Ao), Lautropia mirabilis (Lm), with black staining on teeth [7,8]. In addition, these strains
Leptotrichia sp., and Streptococcus sp. at a rate of 4.3%. On the have been proposed as possible periodontopathic bacteria in
other hand, Prevotella sp. was not detected at this time. In some types of periodontitis [33,34].
specimens obtained at 3 years 2 months of age (n ¼ 25), N. Although inflammatory periodontal diseases are common
mucosa was detected at a rate of 26.1%; Rothia aeria (Ra) at a in children, they rarely result in loss of periodontal attach-
rate of 21.7%; Rothia mucilaginosa (Rm) and Neisseria sp. at a rate ment or resorption of alveolar bone [35]. Therefore, coloniza-
of 8.7%; and Ad, Campylobacter curvus (Cc), Kd, Lm, Leptotrichia tion of periodontal bacteria has rarely been reported in
sp., Prevotella sp., Pseudomonas sp., and S. mitis at a rate of 4.3% healthy children, although periodontitis seen in young ages is
(Fig. 2). The detection rate of Prevotella sp. was lower than that classified as periodontitis as a manifestation of systemic dis-
seen in the first examination. ease, aggressive periodontitis, or chronic periodontitis [36]. In
The detection rate in dental plaque samples at 3 years 2 the present patient, P. intermedia and P. nigrescens were
months of age (n ¼ 25) was 29.4% for Leptotrichia sp., 17.6% for detected in saliva specimens, whereas neither of these strains
S. sanguinis, 11.8% for Aggregatibacter sp., Nmu, and Pp; and was detected in healthy children under 3 years of age in a
5.9% for Ad, Kd, and Neisseria sp. (Fig. 3). There were significant previous study [10]. In addition, P. intermedia and P. nigrescens
differences between the saliva and plaque samples with re- are considered to be dependent on the heme portion of he-
gard to the detection of oral bacteria. moglobin as a required iron source for their growth [9], and
Bacteria collected from the patient at 3 years 2 months of black pigmentation is related to that function. The medical
age were investigated for their relationship to diseases using a history of our patient was unremarkable and no additional
database (Table 1). Most in both the saliva and plaque speci- iron supplementation had been taken. Other possible etio-
mens were found to be gram-negative bacteria with detection logical agents include dietary components, beverages, to-
rates of 80.9% and 64.7%, respectively. In addition, several of bacco, mouth rinses, and other medicaments [37]. However,
the bacterial strains were found to be related to genetic dis- no special substrates were ingested by our patient. On the
eases such as bacteremia and endocarditis. other hand, black tooth staining may be associated with high
On the other hand, mutans streptococci and lactobacilli salivary calcium and buffer capacity [38]. Furthermore, sali-
were not shown in either the saliva or plaque specimens, vary proteins, especially proline-rich protein, can mediate
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.
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]. is considered to play important roles in bacterial competition
Additional analysis of saliva specimens will be needed to in microbial communities such as oral biofilm [40]. On the
clarify this mechanism. other hand, S. mutans was not detected; thus, it remains to be
After scaling at the age of 2 years 2 months, the rates of elucidated if the phenomenon is caused by production of H2O2
detection of black-pigmented bacteria were decreased and by these organisms.
black staining disappeared in our patient. Therefore, we Taken together, black staining such as that seen in the
concluded that these pigmented bacteria may have a rela- present case may have a strong relationship with the presence
tionship with black pigmentation. of black-pigmented bacteria. Furthermore, a molecular bio-
S. sanguinis and S. mitis, which were also detected among logic technique, such as the one utilized in our study, may be
the oral streptococci harbored by our patient, have been useful for diagnosis of the phenomenon as well as treatment.
shown to be responsible for a variety of infectious complica- However, the source of bacterial infection in our patient re-
tions including bacteremia and infective endocarditis [20]. mains to be found. Additional research about the infection
Furthermore, both produce hydrogen peroxide (H2O2), which 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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