Detection Of Scardovia wiggsiae In Thai Children With Early Childhood Caries
Prangkhae Suvansopee1,*, #, Anjalee Vacharaksa2, Waleerat Sukarawan3
1
DDS, Master of Science Program, Department of Pediatric Dentistry, Faculty of Dentistry,
Chulalongkorn University, Bangkok, Thailand
2
DDS, PhD, Lecturer, Department of Microbiology, Faculty of Dentistry, Chulalongkorn
University, Bangkok, Thailand
3
DDS, PhD, Lecturer, Department of Pediatric Dentistry, Faculty of Dentistry,
Chulalongkorn University, Bangkok, Thailand
*,#email: khae_dent@hotmail.co.th
Abstract
Early childhood caries (ECC) is a significant health concern of Thai population.
Cariogenic bacteria is the major factor in the disease progression. Streptococcus mutans
colonization was associated to ECC, however co-infection with other species could not be
negligible. Discovery of Scardovia wiggsiae associating to dentinal caries supported the
evidence of multispecies infection in carious lesions. The aim of this study was to used
polymerase chain reaction (PCR) to detect the prevalence of Scardovia wiggsiae and
Streptococcus mutans in a group of Thai children with ECC and caries-free. The plaque
samples were collected from 30 ECC and 30 caries-free children based on the patient
selection criteria. DNA isolation was performed. Endpoint PCR and SYBR Green-based
quantitative PCR (qPCR) were used to detect Scardovia wiggsiae and Streptococcus mutans.
Scardovia wiggsiae-specific products were sequenced and compared to GenBank database to
verify detection specificity. By endpoint PCR, the prevalence of Scardovia wiggsiae (14/30)
or Streptococcus mutans (30/30) was estimated in ECC children. Chi-square analysis showed
significantly differences in both bacteria between ECC and caries-free group (p<0.05). By
qPCR, the prevalence of Scardovia wiggsiae (19/30) or Streptococcus mutans (30/30) was
estimated in ECC children and significant differences were shown between the group of ECC
and caries-free (p<0.05).The qPCR was more sensitive than endpoint PCR in Scardovia
wiggsiae and Streptococcus mutans detection. In conclusion, Streptococcus mutans was
detected in all ECC children while Scardovia wiggsiae was detected in some ECC children.
The prevalence of Scardovia wiggsiae and Streptococcus mutans in ECC and caries-free
children was significantly different.
Keyword: Scardovia wiggsiae , Streptococcus mutans, Early childhood caries,
Endpoint PCR, quantitative PCR
Introduction
Early childhood caries (ECC) is a specific form of severe dental caries in infants and
young children. This type of caries initially presents with early lesion on smooth surface of
primary maxillary incisors and progress rapidly to other teeth in the oral cavity upon
sequence of tooth eruption, except the mandibular incisors that are usually protected in the
self-cleansing area by movement of tongue and salivary flow [1]. Historically, severe tooth
decay in young children was recognized as nursing caries, nursing bottle syndrome, night
bottle mouth, or baby bottle tooth decay [2]. These terms came from the cause of
inappropriate bottle-feeding. However, current evidence suggested that the bottle nursing was
not the only cause of disease, but several factors contributed to the etiology of ECC.
Therefore, the ‘early childhood caries’ is termed to better reflect to disease characteristics and
multifactorial etiology [3].
The American Academy of Pediatric Dentistry in 2011 defined ECC as the presence
of 1 or more carious, noncavitated or cavitated, lesions, 1 or more missing teeth due to caries,
or 1 or more filled surfaces in any primary tooth in a child under the age of 6 [3]. ECC is
considered a significant public health problem of Thailand as shown in the year 2012 national
oral health survey VII by Department of Health. The survey was conducted in aged 3 and 5
years to represent the caries status in primary teeth. In the group of 3 years old children, the
prevalence of dental caries was 51.7% with a decayed, missing, and filled teeth (dmft) score
equal to 2.7. Interestingly, 3.2% of children in this age group begin losing their teeth due to
caries. Moreover, the survey in children aged 5 years shown much higher incidence of dental
caries. Prevalence of dental caries was increased to 78.5% with dmft score of 4.4 and 8.2% of
5 years old children had loss their teeth [4]. The increasing number of dental caries in Thai
young children could reflect inadequate distribution of dental care and public oral health
information to some rural area.
Etiology of ECC involves contibuting factors including cariogenic substrates,
susceptible host, and cariogenic microorganisms. The tooth structure is affected when an
imbalance of the demineralization and remineralization occurs between tooth surface and the
adjacent plaque [5,6]. Heavy plaque accumulation may be an important sign for ECC [7].
Bacteria growth in dental biofilm tended to have high metabolic activity as well as natural
protection from saliva and environmental changes. As a result, the cariogenic bacteria
produced organic acid, which could demineralize the susceptible tooth structure [6].
Streptococcus mutans (Sm) of the mutans streptococci is the primary pathogen colonizing in
dental caries [8-10], and the key virulence factors of Sm in caries development has been
extensively studied. The water-insoluble extracellular polysaccharide, or the so-called
glucans, facilitated strong adherence of Sm to tooth structure. When lacking of exogenous
substrate, Sm produced intracellular polysaccharide for energy reserves. Additionally, Sm
metabolized a variety of sugars resulting in organic acids, particularly lactic acid, while Sm
showed extremely high acid tolerance. These characteristics supported Sm to grow under
acidified condition of tooth decay [6].
Previous studies using culture-dependent method have demonstrated some selected
bacteria, particularly Streptococcus species and Lactobacillus [9,1 1 -1 3 ], in association to
ECC. Currently, the use of molecular methods and sequencing technology has increased our
knowledge on non-culturable oral bacteria that might relate to oral health. In 2002, Becker et
al. discovered the novel bacteria species, including the gram-negative anaerobic species and
an unknown Bifidobacterium species, which were resided in deep carious lesions [14].
Recently, Tanner et al. reported Scardovia wiggsiae (Sw) as the potential cariogenic pathogen
in children with ECC in the presence or absence of Sm [15]. Sw is then identified as the
anaerobic gram-positive bacillus with acidogenic and aciduric properties that belongs to the
family Bifidobacteria [16].
Dental caries is bacterial infectious disease therefore transmission of the etiologic
bacteria may be spreading within a continent or globally. In Thailand, there is no previous
evidence relating Sw to dental caries, but Sm was known to be the primary cause of severe
ECC [17]. We postulated that Sw might be positive in some Thai children with ECC, but
varied from children in other continents based on differential environmental exposures, such
as food or drinks, or socio-economic background [18]. The purpose of this study was to use
endpoint polymerase chain reaction (endpoint PCR) and quantitative PCR (qPCR) to find the
prevalence of Sw and Sm in a group of Thai children with ECC, and then compared to cariesfree children.
Methodology
Study population
Children aged 2-6 years from the Pediatric Dentistry Department, Faculty of
Dentistry, Chulalongkorn University were recruited in this study. Children were healthy and
had not used antibiotic within the last 3 months. Study protocol was explained to the child’s
parent or guardian. Informed consent was signed before participation. Children with dmfs
score = 0 were assigned to caries-free group (n=30) and children with dmfs score ≥ 1 were
assigned to ECC group (n=30). The diagnosed as ECC was defined by the American
Academy of Pediatric Dentistry [3]. Children in ECC group had no history of operative
dental treatment. The study protocol was approved by the Human Ethics Committee of the
Faculty of Dentistry, Chulalongkorn University (HREC-DCU 2013-013).
Clinical examination and Plaque collection
Demographic data of children were collected. Posterior bitewing radiographs were
taken to evaluate proximal caries when proximal contact could not be clinically detected.
Dental caries were recorded using the decayed, missing, and filled surface (dmf-s) index.
Supragingival plaque samples were collected with sterile explorer from the buccal,
lingual, and accessible proximal surfaces of children with ECC or caries-free. In the ECC
children, the plaque samples were taken from non-carious tooth surface. Samples collected
from the same patient were pooled in 1.5 ml tube containing 1 ml sterile phosphate buffer
saline (PBS). Samples were stored at -20 C until DNA extraction could be performed.
DNA extraction
Plaque samples were pellet by centrifugation and storage buffer was removed. DNA
extraction was performed using Power BiofilmTM(MO BIO,CA,USA) DNA Isolation Kit,
according to the manufacturer’s instructions. DNA products were measured by
NanoDrop2000 Spectrophotometer (Thermo SCIENTIFIC, Wilmington DE, USA).
Bacterial-specific PCR analysis
Species-specific primers for endpoint PCR and quantitative PCR (qPCR) are shown in
Table 1. For testing the specificity of Sw-specific primers, DNA from one dental plaque
sample was amplified using the specific primers and the PCR product was shown to have
specific size as described in the previous study [15]. Two of the Sw-specific PCR products
were purified from DNA gel and submitted for sequencing. The BLAST search using the
nucleotide sequences of the positive band showed 100% match to Sw strain F0424 (Figure 1).
The plaque samples, which were positive for Sw and sequenced, were then used for positive
control in the following PCR.
For the endpoint PCR, 2µl of DNA (50 ng) was mixed with 12.5µl of TopTaq Master
Mix (Qiagen, Valencia, CA, USA), 0.4 µM of forward and reverse primer and the PCR water
was added to total volume of 25 µl. The PCR reaction was peformed by using DNA Engine®
Peltier Thermal Cycler (Bio-Rad, Hercules, CA, USA). The samples were amplified by preheating at 94°C for 3 minutes, amplification was performed for 35 cycles, with denaturation
at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and elongation at 72°C for 1
minintes, followed by a final extension at 72°C for 10 minutes. Amplicons were verified on
1.5% agarose gels with ethidium bromide and imaged by a digital imaging system.
(Molecular Imager Gel DocTMSystems, Biorad Laboratories Inc., CA, USA). A 100 base pair
DNA Ladder (Invitrogen™, Carlsbad, CA, USA) was used as a marker. The presence of a
band of the expected molecular size was scored as positive for the species. To confirmed the
specificities of the primers, some amplicons from plaque samples which Sw primers
amplified were sequenced and compared with those in the public databases.
For qPCR, each reaction tube contained 1 μl of DNA template(25 ng), 10 μl of Kapa
®
SYBR FAST qPCR Kit Master Mix (Kapa Biosystems™, Foster city, CA, USA), 0.2 μM of
forward and reverse primer, and the PCR water was added to total volume of 20 μl. The
quantitative PCR mixtures were analyzed using the Rotor-Gene Q™ Realtime PCR system
(QIAGEN, Valencia, CA, USA). The program consisted of an initial step at 95°C for 3
minutes, Then amplifications was performed for 40 cycles, at 95°C for 3 seconds and 60°C
for 25 seconds. The melting curve analysis was performed after qPCR amplification.
Statistical analysis
The differences in age and dmfs scores between the group of ECC and caries-free
children were evaluated by Student’s t-test. The differences in gender between two groups
were compared by Chi-squared test. The differences in a prevalence of Sw or Sm of ECC and
caries-free children were compared by Chi-squared test or Fisher’s exact test. All analyses
were performed using SPSS software version 20.0 (SPSS,Inc.,Chicago,USA).
Results
Demographic and caries status of study population
The groups of ECC and caries-free children recruited in this study showed no
differences in the mean age or gender by Student’s t-test or Chi-square test, respectively
(Table 2). In contrast, the mean (± SD) of dmfs scores of ECC children was 28.37 (±22.34),
while the score of caries-free children was zero.
The detection of Sw and Sm
Using endpoint PCR for detection, Sw- or Sm-specific PCR products were amplified.
PCR bands and product size were visualized and some selected samples were shown in
Figure 2. Positive and negative controls were added to ensure the specificity of PCR reaction.
When PCR band could not be seen on a photograph, the sample was considered to be
negative. The SYBR green signal was detected in qPCR reactions and species-specific
melting curves, as compared with positive controls, were shown in Figure 3. When the SYBR
green signal became a flat line similar to negative control, the sample was considered to be
negative.
The results from endpoint and qPCR detection were summarized in Table 3. The
prevalence of both Sw and Sm were significantly increased in the group of ECC of Thai
children regardless of the method of detection. Some ECC children were positive for Sw, but
Sm was detected in all ECC children. The detection methods used in this study clearly
affected the results. When endpoint PCR was used, none of the caries-free children was
positive for Sw, while 14 of ECC children were positive for Sw. Using qPCR, Sw was
detected in 2 and 19 of the caries-free and ECC children, respectively. Similarly, Sm was
detected in 13 or 22 of the caries-free children using endpoint or qPCR. This result suggested
that qPCR may be more sensitive compared to endpoint PCR. The Melting curve for Sw and
Sm of some plaque samples were shown in Figure 3.
Discussion and Conclusion
In this study, the group of ECC children was compared to caries-free children for the
prevalence of Sw and Sm. Both groups were demographically similar suggesting that the
colonization of Sw or Sm was unaffected by age or gender, but related to caries status. Sw was
associated with initial enamel lesions [19], and white-spot lesion under orthodontic
appliances [20]. Sw was also detected in deep dentinal caries in adults [21], suggesting a role
in caries progression. Consistently to previous reports, our result showed that the prevalence
of Sw in ECC children was significantly higher than caries-free children. This result
suggested that Sw might be related to ECC in Thai children. However, the role of Sw in
dental caries initiation and development remains to be studied.
Since Sm was detected in all ECC children while Sw could be detected in some ECC,
this could reflect to the limitation of DNA isolation and PCR bias. We used the mechanical
bead-beating method to extract DNA from dental plaque samples, which might be inadequate
to properly extract DNA from Sw and led to PCR bias. When the DNA isolation is optimized,
the prevalence of Sw can be studied to understand the role of Sw in dental caries. The
previous study suggested that Sw colonized on infected dentine of soft, active carious lesions
in both deciduous and permanent teeth [22]. Therefore, the positive detection in some
samples, but not all, might reflect the low abundance of Sw in dental plaque, while the
prevalence of Sw could be greater when sampling from carious dentine.
The non-culturable bacteria could be detected and studied by the use of molecular
technique. However, there are some limitations of using PCR methods. The accuracy of
detection depends largely on the specificity of the primers. Sequences of Sw-specific PCR
product not only matched to Sw, but also matched to some other Bifidobacteria. Primers for
Sw should be redesigned to improve its specificity. The sensitivity of the methods to detect
PCR products was also important. The sensitivity of qPCR was substantially higher than
endpoint PCR, since the SYBR green signal was detected during each cycle of the
amplification process as compared to the visualization of PCR products on electrophoresis
gel. Moreover, the study population was limited to the patients at the Faculty of Dentistry,
Chulalongkorn University. Thus generalization of these results to children in the rural area of
Thailand may be inapplicable.
In conclusion, Sw was detected in the plaque samples from Thai children by PCR in
this study. There were significant differences in the prevalence of Sw and Sm in ECC
compared to caries-free children suggesting their important roles in ECC. Sm can be detected
in all ECC children while the prevalence of Sw was lower. However, the efficacy in detection
method should be optimized.
Figure 1. Sequence analysis of the Sw-specific PCR product amplified from ECC dental plaque sample
showed 100% identities to partial sequence of Sw strain F0424 16S ribosomal RNA gene.
(Sequence ID:gbHM596282.1)
Figure 2. Detection of Sw (A) and Sm (B) in dental plaque samples by endpoint PCR. Lane M; 100 bp DNA
marker; P is positive control and N is negative control (distilled water); lanes 1-5 show PCR products from
plaque samples from different children in ECC group.
Figure 3. Melting curve for Sw demonstrated a peak at the melting temperature (Tm); 85.2-85.5 °C (A),
melting curve for Sm demonstrated a peak at the melting temperature (Tm); 81.5-81.8 °C (B)
Table 1. Specific primer of Streptococcus mutans and Scardovia wiggsiae
Primer (5’-3’)
Streptococcus mutans
Forward: TCG CGA AAA AGA TAA ACA AAC A
Reverse: GCC CCT TCA CAG TTG GTT AG
Scardovia wiggsiae
Forward: GTG GAC TTT ATG AAT AAG C
Reverse: CTA CCG TTA AGC AGT AAG
Size (bp)
References
479
Chen Z et al.[23]
172
Tanner AC et al.[24]
Table 2. Demographic and caries status of study population
Outcome variable
Mean age (years) ± SD a
Gender
Number of male (%)
Caries status
Mean dmfs ± SD
a
ECC (n=30)
Caries-free (n=30)
p value
3.72± 0.83
3.69±1.21
0.912 b
16(53.3%)
13(43.3%)
0.438 c
28.37±22.34
0
0.000b
SD = Standard deviation. bStudent’s t-test. cChi-square test.
Table 3. The detection of Sw and Sm
PCR
End-point PCR
qPCR
c
Bacteria
Sw
Sm
Sw
Sm
ECC children (%)
14 (46.7%)
30 (100%)
19 (63.3%)
30 (100%)
Caries free children
(%)
0 (0%)
13 (43.3%)
2 (6.7%)
22 (73.3%)
p value
<0.001c
<0.001c
<0.001c
0.005d
Chi-square test. dFisher’s Exact test.
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