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| Year : 2013 | Volume
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| Issue : 3 | Page : 342-346 |
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| Detection of putative periodontopathic bacteria in type 1 diabetic and healthy children: A comparative study |
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Ponnudurai Arangannal1, Santoshkumari2, Padma Krishnan3, Mamta Harilal Nichani1, Mahalakshmi Krishnan4, Vishnurekha Chamarthi1
1 Department of Paedodontics and Preventive Dentistry, Sree Balaji Dental College and Hospital, Bharath University, Pallikaranai, Chennai, Tamilnadu, India
2 Department of Paedodontics and Preventive Dentistry, Sree Balaji Dental College and Hospital, Bharath University, Pallikaranai; Department of Paedodontics and Preventive Dentistry, Sri Venkateswara Dental College and Hospital, Chennai, Tamilnadu, India
3 Department of Microbiology, Dr. A.L.M. PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, Tamilnadu, India
4 Department of Microbiology, Sree Balaji Dental College and Hospital, Bharath University, Pallikaranai, Chennai, Tamilnadu, India
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| Date of Submission | 28-Dec-2011 |
| Date of Decision | 08-Sep-2012 |
| Date of Acceptance | 01-Feb-2013 |
| Date of Web Publication | 12-Sep-2013 |
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 Abstract | | |
Aim: The aim of this study was to compare and assess the risk of periodontitis due to the presence of four putative periodontopathic bacteria (Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans) in type 1 diabetic and healthy children.
Materials and Methods: Fifty type 1 diabetic and 50 healthy children in the age group of 7-14 years were recruited for the study. Subgingival plaque samples collected from permanent first molars were subjected to polymerase chain reaction assay to detect 16S rRNA gene of P. gingivalis, T. forsythia, T. denticola and A. actinomycetemcomitans. The data were analyzed using Fisher exact test. The P < 0.05 was considered statistically significant.
Results: The prevalence of subgingival periodontal pathogens in diabetic and healthy children was 2% and 4% for P. gingivalis, 34% and 34% for T. denticola, 20% and 18% for A. actinomycetemcomitans and for T. forsythia, 4% and 34%, respectively. Significant statistical difference was not observed with regard to the prevalence of P. gingivalis, T. denticola, and A. actinomycetemcomitans among type 1 diabetic and healthy children (P = 1.00). Conversely, T. forsythia was less prevalent in diabetic children compared to healthy children.
Conclusion: Statistical significance was not observed for the prevalence of periodontopathic bacteria in type 1 diabetic subjects. The results of the present study thus reveal the absence of risk of periodontitis by these bacterial species in type 1 diabetic subjects. Keywords: A. actinomycetemcomitans, P. gingivalis, T. denticola, T. forsythia, type I diabetes
How to cite this article:
Arangannal P, Santoshkumari, Krishnan P, Nichani MH, Krishnan M, Chamarthi V. Detection of putative periodontopathic bacteria in type 1 diabetic and healthy children: A comparative study. Indian J Dent Res 2013;24:342-6 |
How to cite this URL:
Arangannal P, Santoshkumari, Krishnan P, Nichani MH, Krishnan M, Chamarthi V. Detection of putative periodontopathic bacteria in type 1 diabetic and healthy children: A comparative study. Indian J Dent Res [serial online] 2013 [cited 2023 Sep 29];24:342-6. Available from: https://www.ijdr.in/text.asp?2013/24/3/342/118000 |
Diabetes mellitus is a group of metabolic disorder characterized by hyperglycemia resulting from defect in Insulin secretion or absorption or both. There are four different types of Diabetes mellitus but vast majority of cases of diabetes fall in two categories. They are Type 1 Diabetes Mellitus (T1DM) and Type 2 Diabetes Mellitus (T2DM). [1] T1DM is due to autoimmune destruction of the insulin-producing pancreatic beta cells resulting in absolute deficiency of insulin. The diagnosis of Type 1 diabetes can be made at any age, but it usually manifests in childhood, adolescence or early adulthood, before the age of 20. [2] According to the American Diabetes Association, Type 1 diabetes is present in 5-10% of patients with diabetes. [2]
Among the long-term systemic complications of uncontrolled diabetes, periodontal diseases is the 6 th most common complication. [3] Periodontal disease is caused by gram-negative anaerobic periodontopathic subgingival microflora. [4],[5] In recent years, much interest has focused on this subgingival microflora and its role in destructive periodontal disease. The gram-negative putative periodontopathic bacteria (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans) are known causative agents for periodontitis [4] and identification of these putative pathogens can act as marker for onset of the periodontal diseases. [5],[6] These gram-negative periodontal pathogens especially P. gingivalis has been detected in high numbers in adult long-duration insulin-dependent diabetics. [7]
Detection of anaerobic bacteria by culture methods are challenging because of their specific growth requirements like anaerobic environment and certain technical barriers. [8],[9] Advances in molecular biology such as polymerase chain reaction (PCR) have enabled the identification of specific bacteria in large number of periodontitis cases. The PCR is a relatively simple, sensitive and rapid test for successful detection of oral anaerobic bacterial pathogens. [9],[10]
The presence of gram-negative periodontopathic bacteria and its relation with periodontal disease is widely studied in adult (Type 2) diabetic population [11],[12],[13],[14],[15],[16] but there are hardly any reports in children. Hence, the purpose of the present study was to compare the prevalence and assess the risk of the four periodontopathic bacteria (P. gingivalis, T. denticola, T. forsythia, and A. actinomycetemcomitans) among the type 1 diabetic children and healthy children/adolescents between the age group of 7 and 14 years, using PCR.
| Materials and Methods | |  |
The present study was cleared by the institutional ethical committee of Sree Balaji Dental College and Hospital, Chennai and the Department of Endocrinology and Diabetology, Institute of Child Health and Hospital for Children (ICHS), Chennai. Diabetic children and adolescents were screened by a single examiner in the Dental Department ICHS and selected based on inclusion and exclusion criteria.
Fifty children with type I diabetic were recruited for the study. Confirmed diagnosis of T1DM, age between 7 and 14 years, presence of all permanent first molars, were the inclusion criteria. History of diabetic related systemic complications, professional dental cleaning and periodontal therapy for past 6 months, antibiotic therapy for past 3 months and history of orthodontic treatment were the exclusion criteria. Fifty healthy children and adolescents attending the Department of Paedodontics and Preventive Dentistry, Sree Balaji Dental College and Hospital, Chennai were included as control group. Absence of systemic diseases, age between 7 and 14 years and presence of all permanent first molars were the inclusion criteria for the control group. Informed consent was obtained from the parents/guardian of all children enrolled for the study.
Intra-oral examination was carried out for all the 100 selected subjects and clinical parameters such as plaque index (Silness and loe 1964) and gingival index (Silness and Loe 1967) were recorded. The level of bleeding on probing was more in T1DM patient. The probing depths ranged 3-5 mm. Clinical attachment levels were not measured. Subgingival plaque was collected using sterile Gracey curette from the buccal surface of all first permanent molars after careful removal of supragingival plaque with sterile cotton roll. The plaque was immediately transferred to sterile tubes containing 500 μl of sterile phosphate buffered saline (pH 7.8). The samples were then transferred with necessary precautions to the Department of Microbiology, Institute of Basic Medical Sciences Chennai and stored at −20° till assay.
PCR analysis
The Subgingival plaque was micro-centrifuged at 8,000 rpm for 5 min, the supernatant was discarded and the pellet was re-suspended with 250 μl of lysis buffer. DNA extraction was performed by boiling-lysis method and the supernatant was used as template for PCR assay. Species specific primers for the target organisms have been used as suggested by previous studies. [8],[9] PCR reaction mixture (50 μl) for the detection of 16S rRNA of the four bacterial species contained 5 μl of ×10 PCR buffer (pH 8.4), 1 U Taq DNA polymerase (Bangalore genei, India.), 0.25 mMol/L of each deoxyribonucleotides (dNTP) (Medox Biotech India Pvt., Ltd.), 1.5 mM MgCl 2 (Sigma-Aldrich Pvt., Ltd.) , 0.5 μM of each primer (Sigma-Aldrich Pvt., Ltd.) and 5 μl of the template. Bacterial sequences submitted to Gen Bank under accession no's HQ112349, HQ112350, HQ202265 and HQ188689 were used as positive controls and sterile millipore water served as blank control. PCR was carried out for 35 cycle with each cycle consisting of denaturation (94°C for 5 min), annealing (50°C for 1 min) and primer extension (72°C for 1.5 min). Amplified PCR products were detected by 1.5% gel electrophoresis (Bio-rad, USA).
Statistical analysis
Fisher exact test (two-tailed) was used to calculate the P value. SPSS software 15 was used for all statistical analysis. In the present study, P < 0.05 was considered statistically significant. The odds ratio at 95% confidence interval was calculated to assess the association of the bacterial species to type I diabetes.
| Results | | |
PCR carried out with the subgingival plaque samples of type I diabetic children and healthy children amplified the 16S rRNA gene of P. gingivalis, T. forsythia, T. denticola and A. actinomycetemcomitans in the product size of 404 bp, 641b p, 316 bp and 443 bp, respectively. None of the samples showed non-specific amplification. [Table 1] shows the prevalence of four bacterial species in type I diabetic group and healthy subjects. The sample that was positive for P. gingivalis in type I diabetic and healthy subject was also positive for T. denticola. Two of the type I diabetic children showed co-occurrence of T. forsythia, A. actinomycetemcomitans and T. denticola. Co-occurrence of A. actinomycetemcomitans, and T. denticola was observed among six type I diabetic children. One healthy subject showed positive for all the four periodontopathic bacteria screened. T. forsythia and T. denticola co-occurred in eight healthy samples. A. actinomycetemcomitans and T. forsythia co-occurred in four healthy samples. The association between type I diabetes and healthy group with regard to P. gingivalis, T. denticola and A. actinomycetemcomitans is considered statistically insignificant as the two-tailed P = 1.000 by Fisher exact test. Conversely, good statistical difference was observed between type I diabetes and healthy group with regard to T. forsythia (two-tailed P = 0.0002 by Fisher exact test). [Table 2] shows the clinical parameters measured. Significant difference was not noticed in the PD between the T1DM and control group. The Plaque index was 0.35 and 0.37 for diabetic and healthy group, respectively. The gingival index was 0.88 and 0.44 for diabetic and healthy group respectively. The level of gingival bleeding (gingival index-0.88) on probing was more in T1DM children. Statistically significant difference was not observed for gingival index. The HbA1c Level of children withT1DM ranged 5.66-11.50%. About 45% of the T1DM children HbA1c readings were above 8.0%. The duration of diabetes among T1DM subjects ranged 3-7 years. The odds ratio (at 95% confidence interval) calculated for P. gingivalis, T. forsythia, T. denticola, and A. actinomycetemcomitans were 0.48 (0.043-5.58), 0.08 (0.017-0.37), 1 (0.44-2.29) and 1.13 (0.42-3.09) respectively. | Table 1: Percentage prevalence of periodontopathic bacteria among type I diabetes children and healthy subjects
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| Discussion | | |
The etiology of periodontal disease is a result of interaction between the plaque biofilm, microbial by products and the host response. [5] Bacteria are the primary etiological factor of periodontal disease. It is a proven fact that diabetes mellitus is a risk-factor for development of periodontitis in adults. [3] The association between periodontal disease and diabetes mellitus with respect to gram-negative anaerobes in adult population has been extensively studied. [14],[15],[16] while, data regarding prevalence of periodontopathic bacteria among type I diabetic children are lacking.
The PCR method of detection is advantageous over routinely used culture techniques, owing to their greater specificity and sensitivity, and less labor intensive. [10] The 16S rRNA detection by PCR method is accurate, reproducible, highly sensitive test for identification of highly fastidious gram negative pathogens. [9]
In the present study, 2% and 4% of P. gingivalis, 34% and 34% of T. denticola, 20% and 18% of A. actinomycetemcomitans, 4% and 34% of T. forsythia was detected in diabetic and healthy group respectively. P. gingivalis, T. denticola, and T. forsythia are principal red complex periodontopathogenic bacteria which exist as a consortium in subgingival biofilms. [4],[5] Despite red complex A. actinomycetemcomitans was also screened due to its association with aggressive periodontitis, which is mostly reported in adolescence. [17]
The results of the present study are well in concurrence with Matto et al. [18] who has reported P. gingivalis in 5% of the healthy children between 5 and 10 years of age. In contrast to the present study, Kulekci et al. [19] (12%) and Gafan et al. [20] (49%) found higher prevalence of P. gingivalis in healthy children. The present study found a very low prevalence of P. gingivalis (2%) among T1DM children compared to higher prevalence in few earlier reports among adult T1DM diabetes. [7],[15]
The prevalence rate (34%) observed in both the groups for T. denticola is well in concurrence with few previous studies. [19],[21]
Among the healthy children, prevalence rate of A. actinomycetemcomitans found in this study is in accordance with Kulekci et al. [19] who had reported a prevalence of 24%. In Type 1 diabetic children 20% of prevalence was detected for A. actinomycetemcomitans, which is high when compared to Trevonen et al. [15] study (6.5%). The higher prevalence in the present study may be due to the PCR method of detection in contrast to immunoassay method by Trevonen et al. [15]
The prevalence of T. forsythia in the present study among healthy children is well in agreement with Gafan et al., [20] Okada et al. [22] and Campus et al.[13] Gafan et al. [20] concluded that there is 2.3 times greater chance of developing gingivitis in absence of T. forsythia as this micro-organism is mostly associated with dental plaque of healthy individuals.
There was no significant statistical difference between type 1 diabetic and healthy children/adolescent in the prevalence of P. gingivalis, T. denticola, and A. actinomycetemcomitans. Thus, the results of this study are in agreement with various other previous reports. [7],[13],[15],[21] On the contrary, similar plaque level was observed in both the groups. Although, periodontal pathogens are necessary for progression of periodontal disease, their presence alone is not responsible for development of the disease. The failure to establish any statistically significant difference between the bacterial profile of type 1 diabetic and healthy children in the present study and other studies, [7],[13],[15],[21] suggest that factors other than micro-organism are needed to initiate the disease. Van winkelhoff et al. [23] had suggested that mere presence of periodontal pathogens in subgingival plaque were not enough to initiate the destructive periodontal disease and apparently susceptible host is needed to initiate the disease.
Cianciola et al. [24] have proposed that altered host immune response has a vital role in the development and progression of periodontal disease. He found 9.8% prevalence of periodontal disease in type 1 diabetic children when compared to 1.7% in control group even though there was no difference in the periodontal pathogens levels. In spite of similar prevalence of periodontopathic bacteria in the 2 groups, there was increased bleeding on probing in the gingiva of type 1 diabetic children, which was similar to the findings of two previous reports on periodontal changes among type I diabetic children. [25],[26] The similar prevalence among type I diabetic and healthy children in the present study with respect to bacterial profiles is well in agreement with Lalla et al. [27] Alteration in the function of immune inflammatory cells and hyper-responsiveness of macrophage and monocyte results in increased production of tumor necrosis factor (TNFα), IL-1 in response to the periodontal pathogens leading to prolonged inflammatory response and host tissue destruction. [23],[24],[28],[29] The results of the present study is well in agreement with the study of Salvi et al. [29] who found that there was no difference in the bacterial composition in both type 1 diabetic and non-diabetic individual. Only a few studies on the T1DM children with reference to periodontal pathogen have been reported, while, plenty of studies in adult T1DM and periodontitis have been reported. Hence, the results of the present study have been compared with adult T1DM. [27],[29]
In conclusion, the odds ratio of P. gingivalis, T. forsythia, T. denticola, and A. actinomycetemcomitans revealed a negative association to type I diabetes and hence the risk of periodontitis in this group is absent due to the presence of these bacterial species. A large cohort study may help in identifying the risk of periodontitis due to these periodontopathic bacteria in type I diabetes.
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Correspondence Address:
Mahalakshmi Krishnan
Department of Microbiology, Sree Balaji Dental College and Hospital, Bharath University, Pallikaranai, Chennai, Tamilnadu
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0970-9290.118000
[Table 1], [Table 2] |
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