|Year : 2015 | Volume
| Issue : 2 | Page : 134-137
The significance of gtf genes in caries expression: A rapid identification of Streptococcus mutans from dental plaque of child patients
Apurva Mishra1, Ramesh K Pandey1, Natesan Manickam2
1 Department of Pediatric and Preventive Dentistry, Faculty of Dental Sciences, King George Medical University, Lucknow, Uttar Pradesh, India
2 Principal Scientist, Enviornmental Bio technology Division, Indian Institute of Toxicology Research, Lucknow, Uttar Pradesh, India
|Date of Web Publication||15-Apr-2015|
Dr. Ramesh K Pandey
Department of Pediatric and Preventive Dentistry, Faculty of Dental Sciences, King George Medical University, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Rapid phylogenetic and functional gene (gtfB) identification of S. mutans from the dental plaque derived from children. Material and Methods: Dental plaque collected from fifteen patients of age group 7-12 underwent centrifugation followed by genomic DNA extraction for S. mutans. Genomic DNA was processed with S. mutans specific primers in suitable PCR condtions for phylogenetic and functional gene (gtfB) identification. The yield and results were confirmed by agarose gel electrophoresis. Results: 1% agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1 kbp indicating the presence of S. mutans in the test sample. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bp when compared to 100 bp standards. Conclusion: With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques.
Keywords: 16S rRNA, bacterial primers, dental plaque, PCR, S. mutans
|How to cite this article:|
Mishra A, Pandey RK, Manickam N. The significance of gtf genes in caries expression: A rapid identification of Streptococcus mutans from dental plaque of child patients. J Indian Soc Pedod Prev Dent 2015;33:134-7
|How to cite this URL:|
Mishra A, Pandey RK, Manickam N. The significance of gtf genes in caries expression: A rapid identification of Streptococcus mutans from dental plaque of child patients. J Indian Soc Pedod Prev Dent [serial online] 2015 [cited 2020 Jul 3];33:134-7. Available from: http://www.jisppd.com/text.asp?2015/33/2/134/155126
| Introduction|| |
Dental plaque is tooth-associated biofilm consisting of microbial community and a matrix of polymer of bacterial and host origin. The bacterium, such as Streptococcus mutans, found in plaque plays an important role in induction of dental caries. Prevention of dental caries has been considered as an important task for the health professionals. National Health Survey 2004 in India has shown dental caries in 51.9% in 5-year-old children, 53.8% in 12-year-old children, and 63.1% in 15-year-old children.  Scientific research have been focused on application of molecular biology techniques for rapid detection of cariogenic bacteria, and thus making progress in identifying the best techniques for preventing and diagnosing dental caries. Streptococcus mutans has been identified as the principal causative agent for dental caries, producing extracellular glucosyltransferases (gtf) leading to formation of glucan and plaque.  Bacterial identification using 16S ribosomal RNA (rRNA) is a universal bacterial identification method.  The objective of present study is rapid phylogenetic detection of S. mutans in dental plaque using 16S rRNA and polymerase chain reaction (PCR) techniques and functional gene (gtf) identification. The approach in the present study can be used in future research for detecting the effect of dental restorations, or materials on plaque microbial community.
| Materials and Methods|| |
The dental plaque samples were collected from 15patients of age group 7-12 years attending the Outpatient Department of Paediatric and Preventive Dentistry, King George's Medical University, Lucknow. The consent for the participation in the present study was obtained from the parents/guardians of the patients. Patients with a systemic diseases or having a history of antibiotic treatment within previous 6months were excluded from the present study.
Dental plaque was collected by brushing maxillary molars with a sterile toothbrush for a minute. Plaque adhered to the toothbrush was collected in a sterile tube by washing the toothbrush with sterile distilled water several times. The tubes were immediately stored at −20°C, prior to extraction of genomic deoxyribonucleic acid (DNA).
The dental plaque was harvested in Luria Broth (LB), centrifugation was done at 1,600g for 20 min. The supernatant was discarded and the cell pellet was used for DNA extraction using Nucleopore gDNA fungal bacterial mini kit (Genetrix Biotech).
- Take 50-100 mg (wet weight) of cells suspended in phosphate buffer and add 750 μl of lysis buffer.
- Vortex the thrashing bead tube for 5 min.
- Centrifuge the tube at 10,000 for 1 min.
- Transfer upto 400 μl supernatant to a shredder column placed ina collection tube and centrifuge at 7,000 rpm fora minute.
- Add 1,200 μl of DNA binding buffer to the filtrate.
- Transfer 800 μl of the mixture to spin column and centrifuge at 10,000 for a minute.
- Discard the supernatant and repeat the centrifuge.
- Add 200 μl of prewash buffer and centrifuge at 10,000 for a minute.
- Add 500 μl of wash buffer and centrifuge at 10,000 for a minute.
- Put spin column to a fresh centrifuge tube and add 100 μl of elution buffer, allow it to stand for a minute and centrifuge at 10,000 for a minute to elute pure DNA.
PCR amplification was performed in a reaction mixture (25 μl) consisting of PCR beads that contained the required reagents, 25 pmol of each primer and 20-40 ng of DNA solution in a thermal cycler (Eppendroff, India). Positive and negative controls were added for each set of PCR.
Detection of the presence of Streptococcus using PCR, using primers pair specific to 16S rDNA [Table 1] specific to Streptococcus,  was carried out to detect Streptococcus. The target sequence of 16S rDNA was amplified by using PCR mixture (total volume 25 μl) containing 3mM MgCl 2 , 0.4 mMdNTPs, 5U of Taq DNA polymerase, 1μl of each primer, 2.5 μl of × 10 PCR buffer, and 1 μl of template DNA. The PCR program consisted of initial denaturation at 95°C for 15 min, 35 cycles involving denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min followed by a final extension at 72°C for 10 min. All reaction mixtures were placed and stored at 4°C.
|Table 1: Sets of primers used for phylogenetic and functional gene amplification using S. mutans genomic DNA|
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Functional gene identification
Total volume of 25μl of PCR mixture was made identical to the previous mixture. GtfB primers  were used and PCR amplication was carried using following cycling protocol: One cycle at 94°C for 5 min followed by 38 cycles at 94°C for 30 s, 48°C for 30 s, and 72°C for 1 min. In the negative control reaction to check for DNA contamination, reverse transcriptase was not added into the mix. (Reverse transcriptase is an important enzyme for the reaction. Absence of enzyme does not carry out reaction, hence acted as a negative control against our test sample.)
| Results|| |
Genomic DNA (gDNA) extracted from S. mutans was analyzed on 1% agarose gel electrophoresis and found to be good quality and quantity off or further use [Figure 1]. Total gDNA isolated from dental plaque underwent PCR amplification using primer pair specific to 16S rDNA of the genus Streptococci. One percent agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1kbp [Figure 2]. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bpwhen compared to 100 bp standards, depicting the presence of functional genes (gtf) in S. mutans [Figure 3].
|Figure 1: Qualitative assessment of genomic deoxyribonucleic acid (DNA) of Streptococcus mutans on agarose gel electrophoresis|
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|Figure 2: Amplification of 16S gene using S. mutans genomic DNA as template (lane 1, 2, and 3) against 1,000 base pair ladder (lane 4)|
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| Discussion|| |
Microbial assay of the clinically derived dental plaque is mainly done by Gram staining, biochemical tests, and colony morphology identification; but these features of bacteria may change under stress or with evolution.  Under stress a common bacteria may show an unusual phenotype, thus identifying the bacterial phenotype based on these characteristics or inexperience in unusual colonial morphology may lead to false identification of the bacteria. Overview of literature did not reveal significant literature regarding frequency and misinterpretation of laboratory errors in bacterial identification and their impact on patient's healthcare.
Ono et al., 1994 reported PCR methods to be more sensitive for detection than conventional culture techniques. Igarashi et al., (2000) in their study observed that PCR was able to detect low numbers of bacterial species with a detection limit of as few as 25-100 cells as compared to conventional culture technique. The present study thus describes the successful identification of both phenotype and presence of functional gene (gtf) in S. mutans directly derived from the dental plaque. S. mutans harbors three distinct gtfgenes expressing for glucosyltransferase enzyme. The gtfB and gtfC genes encode enzymes that produce mostly water-insoluble linked glucans, whereas the gtfD gene, encodes an enzyme that synthesizes water-soluble glucans.  In particular, insoluble glucan production has been shown to greatly increase the ability of these organisms to colonize tooth surfaces, initiating dental caries. Therefore in present study, gtfB was targeted as a functional gene and after phenotypic identification of Streptococcus from the genomic DNA obtained from dental plaque, a PCR amplification was done both with positive and negative controls with species-specific gtf primers. The amplification observed in 1.2% agarose gel electrophoresis depicts the presence of gtfB genes, hence confirming the presence of S. mutans in the dental plaque samples. Moreover, the amount of bacterial load can also be determined using real time PCR.
| Conclusion|| |
Mutans streptococci are the prime etiological factors in initiation of dental caries in humans. With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques. Inspite of availability of resources, gene-based identification is still expensive; being the biggest restrain for the present technique.
The present study provides a platform for future research to develop an effective chair side tool in rapid identification of bacteria of dental plaque matrix.
| References|| |
National Oral Health Survey and Fluoride Mapping. An Epidemiological Study of Oral Health Problems and Estimation of Fluoride Levels in Drinking Water. Dental Council of India, New Delhi, 2004;32:67-78.
Yamashita Y, Bowen WH, Burne RA, Kuramitsu HK. Role of the Streptococcus mutans
gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun 1993;61:3811-7.
Patel JB. 16S rRNA gene sequencing for bacterial pathogen identification in clinical laboratory. Mol Diagn 2001;6:313-21.
Sato T, Matsuyama J, Kumagai T, Mayanagi G, Yamaura M, Washio J, et al
. Nested PCR for detection of mutans streptococci in dental plaque. Lett Appl Microbiol 2003;37:66-9.
Yoshida A, Kuramitsu HK. Streptococcus mutans
biofilm formation: Utilization of agtfB promoter - green fluorescent protein (PgtfB: gfp) construct to monitor development. Microbiology 2002;148:3385-94.
Ochman H, Lerat E, Daubin V. Examining bacterial species under the specter of gene transfer and exchange. Proc Natl Acad Sci U S A 2005;102:6595-9.
Hanada N, Kuramitsu HK. Isolation and characterization of the Streptococcus mutans
gtfD gene, coding for primer-dependent soluble glucan synthesis. Infect Immun 1989;57:2079-85.
[Figure 1], [Figure 2], [Figure 3]