|Year : 2016 | Volume
| Issue : 2 | Page : 106-110
Prevalence of Porphyromonas gingivalis and its relationship with herpesvirus in Indian subjects with chronic periodontitis: A cross-sectional study
Vinayak M Joshi, Kishore G Bhat, Manohar S Kugaji, Preeti S Ingalagi
Department of Molecular Biology and Immunology, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India
|Date of Web Publication||15-Jul-2016|
Dr. Vinayak M Joshi
Department of Molecular Biology and Immunlogy, R.S. No. 47A/2, Bauxite Road, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Porphyromonas gingivalis (P. gingivalis) is a periodontal pathogen that is commonly harbored in the dental plaque of humans. The aim of this study was to look into the prevalence of P. gingivalis and its association with herpesvirus in Indian subjects. This is probably the first study on the association of this bacterium with herpesvirus in Indians. Materials and Methods: This cross-sectional study consists of 200 subjects, with 100 subjects each in the healthy group and the chronic periodontitis (CP) group. Upon plaque collection, one portion of the samples was immediately plated, on culture media that is selective for P. gingivalis. Total colony-forming units (CFU)/mL from each plate was recorded. The remaining plaque sample was subjected to DNA extraction and polymerase chain reaction (PCR) was performed using specific primers for Cytomegalovirus (CMV) and Epstein–Barr virus (EBV). The data are analyzed using the chi-square test, Spearman's rho correlation coefficient, and Mann–Whitney U test. Results: P. gingivalis was detected in 66% of the subjects with CP and in 40% in the healthy group, and this difference was statistically significant (P = 0.00023). The correlation of clinical parameters with P. gingivalis showed a significant positive correlation, indicating that higher levels of clinical parameters were associated with higher CFUs of P. gingivalis in culture. The comparison of the presence of P. gingivalis between herpesvirus-negative and -positive cases showed that CMV-positive cases had significantly higher levels of this bacterium. Conclusions: The results of this study confirmed the earlier finding of P. gingivalis presence in significantly higher levels in CP subjects and in CMV-positive sites. In addition, there was a positive association of the bacterium with clinical parameters.
Keywords: Chronic periodontitis (CP), culture, Cytomegalovirus (CMV), Epstein–Barr virus (EBV), polymerase chain reaction (PCR), Porphyromonas gingivalis
|How to cite this article:|
Joshi VM, Bhat KG, Kugaji MS, Ingalagi PS. Prevalence of Porphyromonas gingivalis and its relationship with herpesvirus in Indian subjects with chronic periodontitis: A cross-sectional study. J Int Clin Dent Res Organ 2016;8:106-10
|How to cite this URL:|
Joshi VM, Bhat KG, Kugaji MS, Ingalagi PS. Prevalence of Porphyromonas gingivalis and its relationship with herpesvirus in Indian subjects with chronic periodontitis: A cross-sectional study. J Int Clin Dent Res Organ [serial online] 2016 [cited 2018 May 20];8:106-10. Available from: http://www.jicdro.org/text.asp?2016/8/2/106/173791
| Introduction|| |
Porphyromonas gingivalis (P. gingivalis) is a periodontal pathogen that is commonly harbored in the dental plaque of humans. Among the periopathogens causing chronic periodontitis (CP), P. gingivalis is considered as a “keystone” pathogen for its ability to disrupt host protective mechanisms.P. gingivalis is found in the dental plaque of both healthy individual and subjects with periodontitis. Conclusions from previous studies considered all associates of a particular species of bacteria to possess equal pathogenic potential. However, not all strains of P. gingivalis are equally pathogenic. Studies on the carriage rates of P. gingivalis have shown their presence to be higher in South Asian periodontitis patients as compared to those in the United States (USA).,,
The herpesviruses are known to be present in a greater number in most cases of periodontal disease as compared to those in healthy individuals. However, there has been a vast variation in the number of the viruses found, depending on factors such as the clinical status of the subjects, the techniques used to identify the virus, and the geographical location. The data on the prevalence of P. gingivalis in Indian subjects are scarce and there are no data on the association of these bacteria with herpesvirus in these subjects according to our knowledge. Therefore, the aim of this study was to look into the prevalence of P. gingivalis and its association with herpesvirus in Indian subjects.
| Materials and Methods|| |
The current study, consisting of 100 subjects each in the healthy group and in the CP group, is a cross-sectional study conducted to understand the relationship between P. gingivalis and herpesvirus [Cytomegalovirus (CMV) and Epstein–Barr virus (EBV)]. All the subjects included in the study were screened to meet the inclusion and exclusion criteria. Subjects with a history of tobacco use, those undergoing orthodontic therapy, or those with a history of antibiotic use or professional cleaning within the previous 3 months were excluded. Those subjects requiring antibiotic coverage or those with a history of immunosuppressant medications, bisphosphonates, steroids, or diabetes were also excluded. Subjects in the healthy group included individuals with periodontal health and with probing depth ≤3 mm.
In the CP group, individuals with CP were selected according to the classification laid down by the American Association of Periodontology. Selected subjects were informed of the details of the study and consent was obtained before the start of the study. The study was reviewed and approved by the Ethical Committee of the institute.
Clinical procedure and sample collection
After detailed examination, pocket depth (PD), clinical attachment loss (CAL), plaque, and gingival indices  were recorded using a UNC-15 probe (Paradise Dental Technologies, Missoula, MT, USA). To collect the subgingival plaque the selected area was isolated, air-dried, and supragingival plaque removed with cotton rolls. Universal curette was used to collect the plaque from the deepest area accessible during probing. In each subject, 6 teeth were selected from both anterior and posterior teeth, and the plaque was pooled into the same vial.
Culturing and PCR assay
Upon plaque collection, one portion of the samples was immediately plated following a 10-fold dilution, on blood agar (BA) and kanamycin blood agar (KBA), which is selective for P. gingivalis. The plates were stored in an anaerobic jar for a period of 5 days, following which the plates were examined for the presence of P. gingivalis. Colonies of P. gingivalis appear as tiny black colonies, which were confirmed with biochemical tests. Total colony-forming units (CFU)/mL from each plate was recorded [Figure 1].
|Figure 1: arrows showing isolated black pigment colonies of P. gingivalis with surrounding hemolysis|
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The other portion of the plaque sample was subjected to DNA extraction using a modified proteinase K method. This technique involves the use of lysis buffer containing detergents and proteinase K (10 mg/mL) (Chromous Biotech, Bangalore, Karnataka, India). The DNA isolate was transferred in a separate tube and stored at −20°C for future analysis.
Polymerase chain reaction
Specific primers for the structural proteins of CMV and EBV were used for polymerase chain reaction (PCR). The primers used were as follows:
CMV Primer sequences: 5'-ACG TGT TAC TGG CGG AGT CG −3' as forward and '-TTG AGT GTG GCC AGA CTG AG −3' as reverse
EBV Primer sequences: 5'-AGC ACT GGC CAG CTC ATA TC −3' as forward and 5'-TTG ACG TCA TGC CAA GGC AA −3' as reverse 
The PCR reaction mixture (Qiagen, Duesseldorf, Germany) included 10× PCR buffer, dNTP mix 10 mM each, 5 mM of MgCl2, and Taq DNA polymerase 1.5 units/reaction to which primer concentration of 2.5 pmol and DNA templates ≤1 µg/reaction were added. The thermal cycler conditions used were: Initial denaturation at 95°C for 5 min followed by 45 cycles of denaturation at 95°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 30 s, and a final step of 72°C for 5 min. PCR amplification was performed in a Veriti thermal cycler (Applied Biosystems, Grand Island, NY, USA).
Agarose gel electrophoresis of amplified samples on 2% agarose was used to detect the PCR products. A 100-base pairs (bp) DNA ladder was loaded onto the gel simultaneously with the samples. The gel was visualized after staining with 0.5 µg/mL ethidium bromide for 30 min and the results were recorded by gel doc system (Major Science, Saratoga, CA, USA). The molecular size of 368 bp for CMV and 326 bp for EBV was detected by comparing the band position with the 100-bp DNA ladder.
The results of the healthy group and the CP group were compared taking into consideration the presence and absence of P. gingivalis by chi-square test. The correlation of the clinical parameters with P. gingivalis was done using Spearman's rho correlation coefficient. For the comparison of P. gingivalis between CMV- and EBV-positive and negative cases, the Mann–Whitney test was applied. Any difference of P< 0.05 was considered as statistically significant. All calculation was done using SPSS software package (SPSS for Windows, Version 11.0, released 2001. SPSS Inc., Chicago, IL, USA).
| Results|| |
In this study, using culture, P. gingivalis was detected in 66% of the subjects with CP and in 40% in the healthy group, and this difference was statistically significant (P value = 0.00023) [Table 1].
|Table 1: Comparison of healthy and CP groups with culture outcome in P. gingivalis bacteria|
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Correlation of clinical parameters with P. gingivalis showed a significant positive correlation, indicating that higher levels of clinical parameters were associated with higher CFUs of P. gingivalis in culture. However, when the analysis was done separately for healthy and for CP groups, the relations were not statistically significant [Table 2].
|Table 2: Spearman's correlation test applied to know the relationship between clinical parameters and the CFUs of PG|
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The comparison of the presence of P. gingivalis between herpesvirus-negative and -positive cases showed that CMV-positive cases had significantly higher levels of this bacterium (P value = 0.023). Although EBV-positive cases showed a higher level of P. gingivalis by culture, the difference was not statistically significant [Table 3]. However, there was no significant difference in the comparison of P. gingivalis between the CMV- and EBV-positive and -negative cases when the data were analyzed separately for healthy and diseased individuals [Table 4].
|Table 3: Comparison of P. gingivalis between the CMV- and EBV-negative and -positive cases compare by Mann–Whitney test|
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|Table 4: Comparison of P. gingivalis between the CMV- and EBV-negative and -positive cases compare by Mann–Whitney test in periodontally healthy and diseased individuals|
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| Discussion|| |
The current study employed anaerobic culture for the identification of P. gingivalis and a single PCR method using specific primers with no evidence of cross-reactivity or nonspecific amplification. The viruses could be detected with a minimum of 102 copies of plasmid template and not lower. Anaerobic culturing was used for detection of P. gingivalis, which required levels of viable bacteria higher than 103 for culture to be positive. Another limitation of the study was the use of qualitative PCR for assessment of the virus rather than the quantitative method. The qualitative assessment of herpesvirus does not allow the determination of possible relation to levels of P. gingivalis.
This study confirmed the earlier reports of the presence of P. gingivalis in healthy and CP subjects. The prevalence of P. gingivalis varied between 16% and 56% in the USA,,,, whereas the prevalence in Sri Lanka, Thailand,, and China  was between 40% and 100%. In this study, although the healthy subjects had a notable amount of P. gingivalis present, the results were in line with Fagri et al. and Torrungruang et al., who had similar findings in subjects from South Asia. Other studies from India had similar results as this study, which found the prevalence of P. ginigivalis in both healthy and diseased samples, and the difference was significant.,
The clinical parameters showed signThe clinical parameters showed significantly higher values in P. gingivalis-positive sites as compared to negative sites, but no such correlation existed when the healthy and CP groups were analyzed separately. This only indicated that the P. gingivalis levels may be more closely related to overall “healthy and diseased” status than individual clinical parameters. The study by Torrungruang et al. in Thai subjects noted that a significantly higher percentage of CP subjects had P. gingivalis and found significant associations with the presence of CP. According to Tribble et al.,P. gingivalis is a common organism known to be found in healthy plaque and in deep pockets. P. gingivalis colonizing the plaque biofilms in healthy gingival sulcus is considered less pathogenic and is known to evade the host immune system. Deep pockets in periodontal disease provide an ideal environment for the survival and propagation of P. gingivalis. There are different strains of P. gingivalis, each exhibiting different amounts of virulence. Hence, not all the strains of P. gingivalis detected in the periodontal pockets are equally pathogenic. The variation in the results between studies may be because of the difference in the study subjects, the different phenotypes of the bacterium, and changes in bacterial detection methods.
Herpesvirus is known to be present in most adults even without causing an overt disease. A report by Kothari et al. from India showed that the seroprevalance of the some of the herpesviruses is higher than in other, developed countries., In the present study, the comparison of P. gingivalis between the CMV- and EBV-negative and -positive cases in periodontally healthy and CP individuals showed no significant difference. However, CMV-positive cases had significantly higher levels of P. gingivalis and EBV-positive cases had higher levels of the bacterium when an overall analysis of all study subjects was done. Previous reports have shown the existence of higher levels of periodontal pathogens in CMV- and EBV-positive sites. Saygun et al. found CMV and EBV to be positively associated with occurrence of P. gingivalis, Tannerella forsythia, and Campylobacter rectus in young adults with advanced periodontitis. Several other reports from USA, Brazil, Colombia, and Italy  have found increased periodontal pathogens in herpesvirus-positive periodontally diseased subjects. This study found a higher mean for EBV-positive cases who had P. gingivalis in the CP group as compared to the healthy subjects, which was similar to a recent report from Japan. This dissimilarity in the results of this study from other earlier reports can be because of various reasons such as the techniques used, the type of periodontal diseases, and ethnic or geographical factors. The sample size of the study can also influence the finding, particularly when only a few subjects are involved and the results from them are extrapolated onto a larger population. In our study, culture was used for identification of P. gingivalis, which could have been identified at higher levels, probably with the use of much more sensitive methods. Different PCR primers and the variation in the protocols may also lead to differences in the ability to detect. Another limitation of the study was the use of qualitative PCR for assessment of the virus rather than the quantitative method. The qualitative assessment of herpesvirus does not allow the determination of possible relation to levels of P. gingivalis. To our knowledge, this is probably the first study exploring the relationship of P. gingivalis and herpesvirus in Indian subjects.
| Conclusion|| |
The results of this study confirmed the earlier finding of P. gingivalis presence in significantly higher levels in CP subjects and in CMV-positive sites, and the association of the bacterium with clinical parameters. Further studies need to be planned to look into the association of herpesvirus with other periodontal pathogens, along with the use of more sensitive techniques for identification of the pathogens.
We would like to thank Dr. Ravi Shirahatti for the help with statistics and results during the writing of this manuscript.
(Dr. Ravi Shirahatti, Associate Professor, Department of Public Health Dentistry, Maratha Mandal's NGH Institute of Dental Sciences and Research Centre, Belgaum, Karnataka State).
Financial support and sponsorship
No financial or support received in any form for this study.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, et al
. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe 2011;10:497-506.
Tribble GD, Kerr JE, Wang BY. Genetic diversity in the oral pathogen Porphyromonas gingivalis: Molecular mechanisms and biological consequences. Future Microbiol 2013;8:607-20.
Mombelli A, Gmür R, Frey J, Meyer J, Zee KY, Tam JO, et al
. Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in young Chinese adults. Oral Microbiol Immunol 1998;13:231-7.
Papapanou PN, Baelum V, Luan WM, Madianos PN, Chen X, Fejerskov O, et al
. Subgingival microbiota in adult Chinese: Prevalence and relation to periodontal disease progression. J Periodontol 1997;68:651-66.
Papapanou PN, Teanpaisan R, Obiechina NS, Pithpornchaiyakul W, Pongpaisal S, Pisuithanakan S, et al
. Periodontal microbiota and clinical periodontal status in a rural sample in southern Thailand. Eur J Oral Sci 2002;110:345-52.
Slots J. Herpesviral-bacterial interactions in periodontal diseases. Periodontol 2000 2010;52:117-40.
Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999;4:1-6.
Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condtion. Acta Odontol Scand 1964;22:121-35.
Löe H. The Gingival Index, the plaque index and the retention index systems. J Periodontol 1967;38(Suppl):610-6.
Murray P, Baron E, Jorgensen J, Landry M, Pfaller M. Manual of Clinical Microbiology. 9th
ed. Washington, D.C.: ASM Press; 2007. p. 911-32.
Verkuil E, Belkum A, Hays J. Principles and Technical Aspects of PCR Amplification. New York: Springer; 2008. p. 34.
Shin CH, Park GS, Hong KM, Paik MK. Detection and typing of HSV-1, HSV-2, CMV and EBV by quadruplex PCR. Yonsei Med J 2003;44:1001-7.
Umeda M, Chen C, Bakker I, Contreras A, Morrison JL, Slots J. Risk indicators for harboring periodontal pathogens. J Periodontol 1998;69:1111-8.
Wolff LF, Aeppli DM, Pihlstrom B, Anderson L, Stoltenberg J, Osborn J, et al
. Natural distribution of 5 bacteria associated with periodontal disease. J Clin Periodontol 1993;20:699-706.
Beck JD, Koch GG, Zambon JJ, Genco RJ, Tudor GE. Evaluation of oral bacteria as risk indicators for periodontitis in older adults. J Periodontol 1992;63:93-9.
Grossi SG, Zambon JJ, Ho AW, Koch G, Dunford RG, Machtei EE, et al
. Assessment of risk for periodontal disease. I. Risk indicators for attachment loss. J Periodontol 1994;65:260-7.
Preus HR, Anerud A, Boysen H, Dunford RG, Zambon JJ, Löe H. The natural history of periodontal disease. The correlation of selected microbiological parameters with disease severity in Sri Lankan tea workers. J Clin Periodontol 1995;22:674-8.
Torrungruang K, Bandhaya P, Likittanasombat K, Grittayaphong C. Relationship between the presence of certain bacterial pathogens and periodontal status of urban Thai adults. J Periodontol 2009;80:122-9.
Faghri J, Moghim S, Abed AM, Rezaei F, Chalabi M. Prevalence of Porphyromonas gingivalis and Bacteroides forsythus in chronic periodontitis by multiplex PCR. Pak J Biol Sci 2007;10:4123-7.
Mahalakshmi K, Krishnan P, Chandrasekaran SC. Prevalence of periodontopathic bacteria in the subgingival plaque of a south indian population with periodontitis. JCDR 2012;6:747-52.
Joshi VM, Vandana KL. The detection of eight putative periodontal pathogens in adult and rapidly progressive periodontitis patients: An institutional study. Indian J Dent Res 2007;18:6-10.
Wilson M, Lopatin D, Osborne G, Kieser JB. Prevalence of Treponema denticola and Porphyromonas gingivalis in plaque from periodontally-healthy and periodontally-diseased sites. J Med Microbiol 1993;38:406-10.
Kothari A, Ramachandran VG, Gupta P, Singh B, Talwar V. Seroprevalence of cytomegalovirus among voluntary blood donors in Delhi, India. J Health Popul Nutr 2002;20:348-51.
Embil JA, Haldane EV, MacKenzie RA, van Rooyen CE. Prevalence of cytomegalovirus infection in a normal urban population in Nova Scotia. Can Med Assoc J 1969;101:78-81.
Enders G, Daiminger A, Lindemann L, Knotek F, Bäder U, Exler S, et al
. Cytomegalovirus (CMV) seroprevalence in pregnant women, bone marrow donors and adolescents in Germany, 1996-2010. Med Microbiol Immunol 2012;201:303-9.
Saygun I, Kubar A, Ozdemir A, Yapar M, Slots J. Herpesviral-bacterial interrelationships in aggressive periodontitis. J Periodontal Res 2004;39:207-12.
Contreras A, Umeda M, Chen C, Bakker I, Morrison JL, Slots J. Relationship between herpesviruses and adult periodontitis and periodontopathic bacteria. J Periodontol 1999;70:478-84.
Imbronito AV, Okuda OS, Maria de Freitas N, Moreira Lotufo RF, Nunes FD. Detection of herpesviruses and periodontal pathogens in subgingival plaque of patients with chronic periodontitis, generalized aggressive periodontitis, or gingivitis. J Periodontol 2008;79:2313-21.
Botero JE, Parra B, Jaramillo A, Contreras A. Subgingival human cytomegalovirus correlates with increased clinical periodontal parameters and bacterial coinfection in periodontitis. J Periodontol 2007;78:2303-10.
Santangelo R, D'Ercole S, Graffeo R, Marchetti S, Deli G, Nacci A, et al
. Bacterial and viral DNA in periodontal disease: A study using multiplex PCR. New Microbiol 2004;27:133-7.
Kato A, Imai K, Ochiai K, Ogata Y. Prevalence and quantitative analysis of Epstein-Barr virus DNA and Porphyromonas gingivalis associated with Japanese chronic periodontitis patients. Clin Oral Investig 2015;19:1605-10.
Combs DR, Reilly EA, Dawson DR 3rd
, Avdiushko SA, Danaher RJ, Miller CS. Detection of human cytomegalovirus in dental plaque from individual periodontal sites by real-time polymerase chain reaction. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:840-4.
[Table 1], [Table 2], [Table 3], [Table 4]