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ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 3  |  Page : 290-295
 

Isolation of Scardovia wiggsiae using real-time polymerase chain reaction from the saliva of children with early childhood caries


1 Department of Pedodontics and Preventive Dentistry, Subharti Dental College, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh, India
2 Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India

Date of Web Publication24-Sep-2018

Correspondence Address:
Dr. Preetika Chandna
Subharti Dental College, Swami Vivekanand Subharti University, Meerut, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISPPD.JISPPD_225_17

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   Abstract 


Aim: This study aimed to quantitatively assess the levels of Scardovia wiggsiae in caries-free and early childhood caries (ECC)- and severe ECC (SECC)-affected children using real-time polymerase chain reaction (RT-PCR). Methods: Forty-five children aged <71 months were randomly recruited from the Outpatient Clinic at the Department of Pedodontics and Preventive Dentistry at Subharti Dental College and Hospital, Meerut, India. Fifteen children suffering from ECC, 15 with SECC, and 15 children without ECC were enrolled in the study. About 1–2 mL of unstimulated saliva was collected and subjected to microbial analysis using RT-PCR. Results: The SECC group (n = 15) was found to have significantly higher mean relative 16s rRNA expression of S. wiggsiae (3.67) than both ECC (n = 15) and controls (n = 15) (1.69 and 0.85, respectively). S. wiggsiae was detected in 86.7% of the SECC and 60% ECC group and was detected negligibly in the control (caries free) group. The correlation of decayed, missing, or filled surface levels with 16s rRNA levels showed significant positive correlation with 16S rRNA in both ECC and SECC patients. Conclusion: Salivary levels of S. wiggsiae were significantly associated with ECC in children. S. wiggsiae represents a new frontier in the microbial etiology of ECC. This may lead to the development of new antimicrobial agents targeted to this organism and improve the treatment of ECC.


Keywords: Detection, early childhood caries, genes, real-time polymerase chain reaction, Scardovia wiggsiae


How to cite this article:
Chandna P, Srivastava N, Sharma A, Sharma V, Gupta N, Adlakha VK. Isolation of Scardovia wiggsiae using real-time polymerase chain reaction from the saliva of children with early childhood caries. J Indian Soc Pedod Prev Dent 2018;36:290-5

How to cite this URL:
Chandna P, Srivastava N, Sharma A, Sharma V, Gupta N, Adlakha VK. Isolation of Scardovia wiggsiae using real-time polymerase chain reaction from the saliva of children with early childhood caries. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2019 Nov 12];36:290-5. Available from: http://www.jisppd.com/text.asp?2018/36/3/290/241967





   Introduction Top


Dental caries is complicated and multifactorial and often begins to develop as early as infancy. Early childhood caries (ECC) is a virulent form of the disease that is a public health problem which continues to affect infants and preschool children worldwide.[1] ECC has a diverse etiology involving microbiologic, dietary, behavioral, and socioeconomic factors. The infectious component of ECC is based on its microbiologic component which has been studied extensively in an attempt to prevent and control its transmission.

Most of the microbiology in clinical studies of ECC focuses on mutans streptococci (MS) and lactobacilli (LB), which are routinely detected using selective-culture-based methods.[2] However, the microbiota of biofilms taken from ECC patients has been acknowledged to contain a broad diversity of bacteria. Some studies support the view that caries can develop in the absence of MS.[3],[4],[5] Newer molecular methods have shown that the traditional MS and LB species appear to be less important or missing, which suggests that additional species other than MS and LB may also be responsible for ECC. Thus, several bacterial species, either alone or as a group, other than MS may also play major roles in caries development.[3],[6],[7],[8]

Some of the discrepancies in detection of the etiologic microorganism responsible for ECC result from technical differences between methods, resulting in Actinomyces, Bifidobacterium, and Scardovia species being underestimated.[9],[10]

Molecular methods such as the polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis have been used to examine bacterial profiles in ECC and demonstrate differences in the microbial community between children with and without ECC.[11],[12],[13] The major species associated with severe ECC (SECC) identified by Tanner et al. include Streptococcus mutans, Scardovia wiggsiae, Parascardovia denticolens, Veillonella parvula, Streptococcus sobrinus, and Actinomyces gerencseriae. S. wiggsiae has been shown to be significantly associated with SECC in the presence and absence of S. mutans detection.[14] The aim of this study was to identify and assess the levels of S. wiggsiae isolated from the saliva of caries free, and ECC-affected children using real-time PCR (RT-PCR).


   Methods Top


Study population

Forty-five children aged <71 months were randomly recruited from the Outpatient Clinic at the Department of Pedodontics and Preventive Dentistry at Subharti Dental College and Hospital, Meerut, India. The inclusion criteria used stated that the children included must be medically healthy, have a full primary dentition, have not used antibiotics within the preceding 3 months, and written informed consent provided by the parent or caregiver be available for the child's participation in the study. The study design, protocol, and informed consent were approved by the Institutional Ethical Committee.

Fifteen children suffering from ECC, 15 with SECC and 15 children without ECC who fulfilled the inclusion criteria were enrolled and the study procedure was carefully explained to the children. The caries-free children were designated as the control group and the ECC and SECC groups as the study groups. The presence of decayed, missing, or filled surfaces (dmfs) in all enrolled children was scored according to the World Health Organization (WHO, 1997).[15] Teeth were considered decayed if there was an unmistakable cavity, undermined enamel, or a detectably softened floor or wall. White spots were considered as sound tooth surfaces. Radiographs were exposed only when there was any doubt if a child had proximal caries or not. ECC was diagnosed when there were >1 dmfs in any primary tooth. SECC was diagnosed as when a child younger than 3 years had any sign of smooth surface caries, when 3–5-year-old child had >1 dmfs in primary maxillary anterior teeth, or when there were >4, 5, and 6 dmfs in a 3-, 4-, and 5-year-old child, respectively.[16]

Sample collection

A total of 1–2 mL of unstimulated saliva was collected from all the children. Samples were collected in a sterile vial and kept frozen at −80°C until microbial analysis.

Microbial analysis using real-time-polymerase chain reaction

Saliva samples of all children enrolled in the study were analyzed by RT-PCR for the detection of S. wiggsiae. RNA extraction was done by Qiagen spin column kit (Qiagen, Chatsworth, CA, USA). cDNA was made from RNA using reverse transcriptase. PCR for S. wiggsiae was performed using forward 5′-GTGGACTTTATGAATAAGC-3′ and reverse primer 5′-CTACCGTTAAGCAGTAAG-3′ of 16sRNA of S. wiggsiae.

RNA isolation from saliva sample

Before PCR, the frozen saliva samples were thawed. Five volumes of QIAzol Lysis Reagent were added to the saliva sample and mixed by vortexing or pipetting up and down. A tube containing the lysate at room temperature (15°C–25°C) was placed for 5 min. Chloroform of an equal volume to the starting sample to the tube containing the lysate and capped securely and vortexed vigorously for 15 s. The tube containing the lysate was placed at room temperature (15°C–25°C) for 2–3 min and then centrifuge for 15 min at 12,000 × g at 4°C. After centrifugation, the sample separates into three phases: an upper-colorless, aqueous phase containing RNA; a white interphase; and a lower-red, organic phase. The upper aqueous phase was transferred to a new collection tube. Transfer of any interphase material was avoided. Next, 1.5 volumes of 100% ethanol are added and mixed thoroughly by pipetting up and down several times (without centrifugation). Up to 700 μl of the sample was pipetted, including any precipitate that may have formed, into an RNeasy MinElute spin column in a 2 ml collection tube. The lid was closed gently and centrifuged at 8000 × g (10,000 rpm) for 15 s at room temperature (15°C–25°C). The flow through was discarded. This was repeated for the remainder of the sample. About 700 μl buffer RWT was added to the RNeasy MinElute spin column. The lid was closed gently and centrifuged for 15 s at 8000 × g (10,000 rpm) to wash the column and the flow through was discarded. About 500 μl buffer RPE was pipetted onto the RNeasy MinElute spin column. The lid was closed gently and centrifuged for 15 s at 8000 × g (10,000 rpm) to wash the column. The flow through was discarded. About 500 μl of 80% ethanol was pipette onto the RNeasy MinElute spin column. The lid was closed gently and centrifuged for 2 min at 8000 × g (10,000 rpm) to wash the spin column membrane. The collection tube with the flow through was discarded. The RNeasy MinElute spin column was placed into a new 2 ml collection tube. The lid of the spin column was opened and centrifuged at full speed for 5 min to dry the membrane. The collection tube with the flow through was discarded. The RNeasy MinElute spin column was placed in a new 1.5 ml collection tube. About 14 μl of DNase-free water was added directly to the center of the spin column membrane. The lid was closed gently and centrifuged for 1 min at full speed to elute the RNA. The DNase treatment was given to RNA followed by quantification using nanodrop spectrophotometer.

DNase treatment of the isolated RNA

The treatment of RNA was done using RNase-free DNase I (Thermo Fisher Scientific, Waltman, MA, USA). About 1 μl of 10X DNase I buffer was added to 8 μl solution containing 1.5 μg of RNA and nuclease-free water. Next, 1 μl of DNase I was added to RNA and incubated at 37°C for 30 min. This was followed by addition of 1 μl of 25 mM EDTA to the RNA and incubated at 65°C for 10 min.

Preparation of cDNA (reverse transcription)

The DNase-treated RNA so obtained from the cells was used to synthesize complementary DNA (cDNA) using RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltman, MA, USA) that was further used as template to analze the amplification using primers specific to the different molecules. 20 μl of cDNA was prepared from the RNA using the following protocol:

  1. 1 μg of RNA was used to synthesize cDNA. Thus, volume of RNA solution corresponding to this amount of RNA was added to a separate tube, which was treated with diethyl pyrocarbonate
  2. Nuclease-free water was added to the tube making up the volume to 11.5 μl
  3. To this solution, 1 μl of Random Hexamer (Thermo Fisher Scientific, Waltman, MA USA) was added, following which the tube was incubated at 70°C for 5 min. This was done to denature the secondary structures of RNA, to allow priming of the Random Hexamer to the single strand of RNA. The tube was immediately chilled on ice after the incubation was over
  4. Next, 7.5 μl of reaction mixture was added to the tube. The reaction mixture contained following components – 5X RT Buffer – 4.0 μl, 20 μM Deoxynucleotides (dNTPs) – 2.0 μl, RNase Inhibitor (40 units/μl) – 0.5 μl, and Revertaid RT enzyme (200 units/μl) – 1.0 μl. The tube was incubated at following temperatures −25°C for 10 min, 42°C for 60 min, and 70°C for 10 min.


Primer designing and standardization

For PCR analysis [Table 1], primers were designed using the Primer3 software. To ensure the specificity of selected primers, BLAST was done to align the primers (Sigma-Aldrich Corporation, Bengaluru, India) with the genome sequence in the database and specificity of the sequences was checked.
Table 1: Primer sequences and their annealing temperatures used for polymerase chain reaction analysis

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Annealing temperature of the primers was standardized by putting up gradient PCR in Eppendorf thermocycler, at temperatures of 5°C higher and lower than the melting temperature (tm) of the primers. A 20 μl reaction was set up for gradient PCR utilizing TaQ buffer, forward primer, reverse primer, dNTPs, Taq, and nuclease-free water.

The reaction mix was added in the 0.2 ml PCR tubes followed by addition of 1.0 μl template cDNA. The final contents was mixed gently without forming bubbles and kept it in Eppendorf PCR machine under following thermal cycling conditions that included initial denaturation at 95°C for 5 min followed by denaturation at 95°C for 20 s, annealing at 54°C for 20 s, extension at 72°C for 20 s, and finally, final extension at 72°C for 10 min.

The temperatures on which annealing were done to standardize primers were 49.1°C, 50.3°C, 51.2°C, 52.2°C, 53.3°C, and 54.4°C. The optimum annealing temperature was found to be 540°C and further used for quantitative PCR (qPCR) analysis. This was followed by 2% agarose gel electrophoresis to check for the specificity and the intensity of the desired product size.

2% agarose gel electrophoresis

The PCR products were analyzed by running on 2% agarose gel in 1X TAE buffer. The procedure followed was as follows:

  1. An amount of agarose corresponding to 2% was weighed and added to conical flask containing 1X TAE
  2. The mixture was boiled in a microwave till clear solution was obtained
  3. This was allowed to cool, after which ethidium bromide was added and the gel was carefully poured on casting plate with an appropriate comb
  4. The gel was allowed to solidify and then immersed in 1X TAE
  5. The samples were loaded in respective wells and allowed to run till bromophenol blue reached the two-third of the gel
  6. The gel bands were analyzed in gel doc.


Quantitative polymerase chain reaction of the prepared cDNA

Maxima SYBR Green qPCR Master Mix (2X) (Thermo Fisher Scientific, Waltman, MA, USA) was used to perform the relative expression analysis of the different molecules using the Bio-Rad RT-PCR machine (Bio-Rad, Hercules, CA, USA). A 20-μl reaction protocol was set up for this purpose. The reaction mixture was added to the RT-PCR strip tubes followed by addition of 1-μl template cDNA. The final contents was mixed gently without forming bubbles and kept it in Bio-Rad RT-PCR machine under following thermal cycling conditions. The steps for this included initial denaturation at 95°C for 5 min followed by denaturation at 95°C for 20s, annealing at 54°C for 20 s, and finally, extension at 72°C for 20 s. Data acquisition was done at annealing step.

Statistical analysis

Comparison between two groups was done by applying Mann–Whitney Test. Correlation analysis was done using Spearman Correlation for nonparametric data. P < 0.05 was considered statistically significant.


   Results Top


16s rRNA primers were used for detection of S. wiggsiae. The primers for 16s rRNA of S. wiggsiae were standardized using gradient PCR and the optimum annealing temperature was found to be 54°C [Figure 1] which was further used for qPCR analysis.
Figure 1: Gradient polymerase chain reaction gel image for optimization of annealing temperature

Click here to view


16s rRNA of S. wiggsiae was represented as relative mRNA expression. The comparison between two groups was conducted using the Mann–Whitney test (P < 0.05 was considered statistically significant). The representative gel image showing relative mRNA expression of 16s rRNA of S. wiggsiae in ECC patients and controls in saliva samples are shown in [Figure 2]. S. wiggsiae was detected in 86.7% of the SECC (n = 15) and 60% ECC group (n = 15) and was detected negligibly in the control (caries free) group.
Figure 2: Representative gel image showing relative mRNA expression of 16s rRNA of Scardovia wiggsiae in severe early childhood caries and early childhood caries patients and controls in saliva samples

Click here to view


The mean dmfs scores were 0, 4, and 10 in the control, ECC, and SECC groups, respectively. The values of 16s rRNA for different groups are shown in [Table 2]. The mean relative mRNA expression of 16s rRNA of S. wiggsiae was found to be significantly higher in patients (2.68) as compared to controls (0.85). The SECC group (3.67) was found to have significantly higher relative mRNA expression than both ECC and controls (1.69 and 0.85). The ECC group was also observed to have statistically significant higher 16s rRNA expression in comparison to controls.
Table 2: Relative 16s rRNA expression among early childhood caries, severe early childhood caries, and control groups

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The correlation of dmfs levels with 16s rRNA levels [Table 3] showed significant positive correlation with 16S rRNA in both ECC and SECC patients. This represents that patients with high dmfs score have high relative expression of 16S rRNA and vice versa.
Table 3: Correlation among relative mRNA expression of 16S rRNA and dmfs score in early childhood caries and severe early childhood caries patients

Click here to view



   Discussion Top


In the present study, RT-PCR was used to detect S. wiggsiae in the saliva of caries-free and ECC-affected children. PCR is a useful tool in modern molecular epidemiology and requires less time than the microbiological identification protocol.[17] Molecular techniques such as PCR have revealed many novel, presumed unculturable, and taxa in oral infections.[9] Munson et al.[9] identified 44 taxa that were detected by the molecular method alone from dentinal carious lesions. Out of these, 31 taxa were previously undescribed. RT-PCR with species-specific primers may provide an accurate and sensitive method for detection and quantification of individual species and bacterial populations as well as total bacteria.[18],[19]

Bifidobacteriaceae are acidogenic and aciduric, Gram-positive, pleomorphically branched, nonmotile, nonspore-forming, and nonfilamentous rods that can be grouped on the basis of one of six different ecological niches that they occupy: the human intestine, oral cavity, food, the animal gastrointestinal tract, the insect intestine, and sewage.[20] Many diverse species of Bifidobacteria are members of the human gut flora, whereas the oral species appear to be limited to Bifidobacterium dentium, P. denticolens, Scardovia inopinata, and S.wiggsiae although others including Bifidobacterium subtile, Bifidobacterium longum, Bifidobacterium scardovii, Alloscardovia omnicolens, Bifidobacterium adolescentis, and Bifidobacterium urinalis have been isolated from oral samples but generally only at a low prevalence.[21],[22] Bifidobacteria and related genera, Scardovia and Parascardovia, have been sporadically isolated or detected in the oral cavity usually, but not always, in relation to active caries.[22] The microorganism S. wiggsiae (domain: Bacteria, phylum: Actinobacteria, class: Actinobacteridae, order: Bifidobacteriales, family: Bifidobacteriaceae, genus: Scardovia, Species: Wiggsiae) was named in honor of Lois Wiggs, American microbiologist, for her contributions to anaerobic microbiology. Strains corresponding to this group have subsequently been isolated from both root and occlusal caries.[23],[24] Phylotype Scardovia C1 was described in a study of the microbiota associated with dentinal caries on the basis of 16S rRNA gene sequence comparisons.[9] Downes et al. also used 16s rRNA sequence to isolate Scardovia species from root and dentinal caries lesions and stated that 16S rRNA gene sequence analysis gave the most significant relation with the novel species, S. wiggsiae.[25] 16s rRNA gene sequence analysis was used in this study to identify S. wiggsiae strains from saliva samples of caries-free and ECC-affected children.

In the present study, S. wiggsiae was isolated from saliva samples of children with ECC and SECC at significantly higher levels as compared to the control group. The levels of S. wiggasiae also showed positive correlation with DMF levels, with higher correlation seen for SECC patients than ECC patients. Beighton et al. similarly isolated S. wiggsiae from saliva samples, but from an older population, and showed that bifidobacteria such as S. wiggsiae play a role in the caries process.[26] Other investigators have similarly shown that salivary levels of bifidobacteria and Scardovia are significantly associated with caries experience in children.[22],[27] Further, the present study showed that levels of mRNA were statistically significantly different between ECC and SECC group which indicated that a greater proportion of S. wiggsiae was present in SECC-affected children as compared to ECC-affected children.


   Conclusion Top


Within the limits of this study, it may be concluded that S. wiggsiae is associated with ECC and SECC in children. However, further research utilizing a larger sample size and a more detailed microbiologic spectrum may show more definitive results in terms of proportion of S. wiggsiae present in the total microbiologic flora in children with ECC and SECC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Munson MA, Banerjee A, Watson TF, Wade WG. Molecular analysis of the microflora associated with dental caries. J Clin Microbiol 2004;42:3023-9.  Back to cited text no. 9
    
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Tanner AC. Anaerobic culture to detect periodontal and caries pathogens. J Oral Biosci 2015;57:18-26.  Back to cited text no. 10
    
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Ling Z, Kong J, Jia P, Wei C, Wang Y, Pan Z, et al. Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing. Microb Ecol 2010;60:677-90.  Back to cited text no. 11
    
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Li Y, Ge Y, Saxena D, Caufield PW. Genetic profiling of the oral microbiota associated with severe early-childhood caries. J Clin Microbiol 2007;45:81-7.  Back to cited text no. 12
    
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Tanner AC, Mathney JM, Kent RL, Chalmers NI, Hughes CV, Loo CY, et al. Cultivable anaerobic microbiota of severe early childhood caries. J Clin Microbiol 2011;49:1464-74.  Back to cited text no. 14
    
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World Health Organization. Oral Health Surveys: Basic Methods. 4th ed. Geneva, Switzerland: WHO; 1997.  Back to cited text no. 15
    
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Definition of Early Childhood Caries. Council on Clinical Affairs. 2016. Available from: http://www.aapd.org/media/policies_ guidelines/d_ecc.pdf. [Last cited on 2017 Jun 29].  Back to cited text no. 16
    
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Loyola-Rodriguez JP, Martinez-Martinez RE, Flores-Ferreyra BI, Patiño-Marin N, Alpuche-Solis AG, Reyes-Macias JF, et al. Distribution of Streptococcus mutans and Streptococcus sobrinus in saliva of Mexican preschool caries-free and caries-active children by microbial and molecular (PCR) assays. J Clin Pediatr Dent 2008;32:121-6.  Back to cited text no. 17
    
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Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ, et al. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 2000;38:1747-52.  Back to cited text no. 18
    
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Hata S, Hata H, Miyasawa-Hori H, Kudo A, Mayanagi H. Quantitative detection of Streptococcus mutans in the dental plaque of Japanese preschool children by real-time PCR. Lett Appl Microbiol 2006;42:127-31.  Back to cited text no. 19
    
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Modesto M, Biavati B, Mattarelli P. Occurrence of the family bifidobacteriaceae in human dental caries and plaque. Caries Res 2006;40:271-6.  Back to cited text no. 21
    
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Kaur R, Gilbert SC, Sheehy EC, Beighton D. Salivary levels of bifidobacteria in caries-free and caries-active children. Int J Paediatr Dent 2013;23:32-8.  Back to cited text no. 22
    
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Mantzourani M, Fenlon M, Beighton D. Association between bifidobacteriaceae and the clinical severity of root caries lesions. Oral Microbiol Immunol 2009;24:32-7.  Back to cited text no. 23
    
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Mantzourani M, Gilbert SC, Sulong HN, Sheehy EC, Tank S, Fenlon M, et al. The isolation of bifidobacteria from occlusal carious lesions in children and adults. Caries Res 2009;43:308-13.  Back to cited text no. 24
    
25.
Downes J, Mantzourani M, Beighton D, Hooper S, Wilson MJ, Nicholson A, et al. Scardovia wiggsiae sp. nov. isolated from the human oral cavity and clinical material, and emended descriptions of the genus Scardovia and Scardovia inopinata. Int J Syst Evol Microbiol 2011;61:25-9.  Back to cited text no. 25
    
26.
Beighton D, Al-Haboubi M, Mantzourani M, Gilbert SC, Clark D, Zoitopoulos L, et al. Oral bifidobacteria: Caries-associated bacteria in older adults. J Dent Res 2010;89:970-4.  Back to cited text no. 26
    
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Vacharaksa A, Suvansopee P, Opaswanich N, Sukarawan W. PCR detection of Scardovia wiggsiae in combination with Streptococcus mutans for early childhood caries-risk prediction. Eur J Oral Sci 2015;123:312-8.  Back to cited text no. 27
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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