Year : 2010 | Volume
: 21 | Issue : 2 | Page : 248--252
16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque
Jaideep Mahendra1, Little Mahendra1, VM Kurian2, K Jaishankar3, R Mythilli1,
1 Department of Periodontics, Annamalai University, Chennai, India
2 Department of Cardiovascular Thoracic Surgery, Madras Medical Mission, Chennai, India
3 Department of Cardiology, Madras Medical Mission, Chennai, India
Department of Periodontics, Annamalai University, Chennai
Background: Atherosclerosis develops as a response of the vessel wall to injury. Chronic bacterial infections have been associated with an increased risk for atherosclerosis and coronary artery disease. The ability of oral pathogens to colonize in coronary atheromatous plaque is well known.
Aim: The aim of this study was to detect the presence of Treponema denticola, Porphyromonas gingivalis and Campylobacter rectus in the subgingival and atherosclerotic plaques of patients with coronary artery disease.
Materials and Methods: Fifty-one patients in the age group of 40-80 years with coronary artery disease were selected for the study. DNA was extracted from the plaque samples. The specific primers for T. denticola, C. rectus and P. gingivalis were used to amplify a part of the 16S rRNA gene by polymerase chain reaction.
Statistical Analysis Used: Chi-square analysis, correlation coefficient and prevalence percentage of the microorganisms were carried out for the analysis.
Results: Of the 51 patients, T. denticola, C. rectus and P. gingivalis were detected in 49.01%, 21.51% and 45.10% of the atherosclerotic plaque samples.
Conclusions: Our study revealed the presence of bacterial DNA of the oral pathogenic microorganisms in coronary atherosclerotic plaques. The presence of the bacterial DNA in the coronary atherosclerotic plaques in significant proportion may suggest the possible relationship between periodontal bacterial infection and genesis of coronary atherosclerosis.
|How to cite this article:|
Mahendra J, Mahendra L, Kurian V M, Jaishankar K, Mythilli R. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque.Indian J Dent Res 2010;21:248-252
|How to cite this URL:|
Mahendra J, Mahendra L, Kurian V M, Jaishankar K, Mythilli R. 16S rRNA-based detection of oral pathogens in coronary atherosclerotic plaque. Indian J Dent Res [serial online] 2010 [cited 2020 Feb 24 ];21:248-252
Available from: http://www.ijdr.in/text.asp?2010/21/2/248/66649
Coronary artery disease and periodontal diseases are common inflammatory conditions in the human population.  Chronic infections have been implicated as an increased risk factor for atherosclerosis and coronary artery disease.  However, it was not until the late 1970s, when Fabricant et al. showed that chickens experimentally infected with an avian herpes virus developed florid vascular lesions similar to those of human atherosclerosis. Subsequently, many investigators have reported observations implicating Chlamydia pneumoniae, Helicobacter pylori, Herpes Simplex Virus and cytomegalovirus as possible primary etiologic factors or cofactors in the pathogenesis of atherosclerosis, including ischemic heart disease and cerebrovascular disease. ,,,,
Atherosclerosis is believed to develop in response to vessel wall injury. Inflammation constitutes a major factor in the development of atherosclerosis, and plaque disruption followed by local thrombosis is responsible for the clinical presentation of acute coronary syndromes. ,, Damage to the endothelium is the main cause for development of the atherosclerotic plaque, which is an inflammatory and fibroproliferative response to a variety of insults sustained by it.
Although the classical risk factors like hypercholesterolemia, smoking, diabetes mellitus and hypertension account for the majority of the etiology, pathogenesis and clinical manifestations of atherosclerosis, but not all the cases of atherosclerosis, can be explained by commonly accepted risk factors.  The inflammation plays a major role in the development and progression of this disease.
Three basic lines of evidence suggesting a role of inflammation in atherosclerosis have been presented: (1) detection of the microorganisms in coronary atherosclerotic lesions by immunocytochemistry or molecular biology, (2) epidemiological evidence based on serological data implicating an association between coronary atherosclerotic disease and positive serology  and (3) studies of animal models suggesting that exposure to bacterial infection promotes atherosclerosis. 
Oral gram-positive and gram-negative bacteria have frequently been identified in bacteremia and may play a role in vascular diseases. The possible mechanism could be endothelial injury by oral microbial toxins and systemic inflammation triggered by oral infections. In addition, phagocytes in periodontal lesions may engulf various bacterial cells and their antigens. The bacterial cells and phagocytes may then penetrate the gingival tissues and get transported through circulation to the heart and adhere to the coronary artery endothelium. These deposited bacteria can then stimulate the release of inflammatory cytokines and initiate the formation of the characteristic foam cells associated with atherosclerosis. 
Recent studies have evaluated the presence of periodontal pathogens in diseased vessels and reported conflicting results.  Therefore, further evidence is required to clarify this relationship. Thus, we hypothesize that chronic periodontal infection may be involved in the development and progression of atherosclerosis. The present investigation was carried out to detect the presence of 16S rRNA of the pathogens, namely Treponema denticola, Porphyromonas gingivalis and Campylobacter rectus, in subgingival plaque and atherosclerotic plaques of the same patients undergoing coronary artery bypass grafting (CABG) using polymerase chain reaction (PCR).
Materials and Methods
Fifty-one patients (11 females and 40 males) in the age group of 40-80 years with chronic periodontitis were recruited consecutively from the Institute of Cardiovascular Disease, Madras Medical Mission, Chennai. These patients were suffering from CAD and were scheduled to undergo CABG. Exclusion criteria included other major systemic illness such as advanced malignancy, antibiotic intake and periodontal treatment in the previous 6 months.
The medical and dental history of each subject was obtained by an interview. Patients fulfilling the inclusion criteria were informed of the study and an informed consent was obtained from them. The ethics committee of the Madras Medical Mission approved the protocol of this study.
Collection of subgingival plaque samples
The samples were taken 1 day before the patients underwent the CABG. A periodontal examination was performed by a periodontist. Clinical evaluation included plaque index, probing depth index, periodontal index, total number of teeth present and clinical attachment loss. The deepest periodontal sites with periodontal depth ≥5 mm were selected for the microbial sampling. The teeth were gently dried with a sterile cotton swab. After removal of the supragingival plaque, the subgingival plaque samples were obtained with the help of a currette from the two deepest periodontitis sites and were pooled for analysis.
Collection of coronary atherosclerotic plaque sample
A biopsy was obtained from the coronary atherosclerotic plaque during the CABG. The surgeon excised one or two small bits of plaque (0.5-1 mm) from the edge of the coronary arteriotomy performed for anatomising the graft. To eliminate the blood contamination, the samples were placed in sterilized phosphate-buffered saline and mixed gently and tissue samples were transferred to fresh vials containing the transporting media. The samples were then homogenized by the tissue homogenizer as described by Saiki et al.
Both the subgingival plaque and the coronary atherosclerotic plaque samples were centrifuged for 10 min at 10,000 rpm. The supernatant was discarded and the resulting pellet was resuspended in 200 μl of lysis solution (100 mm Tris, 1.0 mm ethylenediammine tetraacetic acid, 1.0% Triton X-100, pH 7.8). The samples were kept in a boiling water bath for 10 min, allowed to cool and again centrifuged for 5 min at 10,000 rpm. The supernatant was collected as DNA template and stored at -70°C.
16S rRNA PCR amplification was carried out to detect the presence of the microorganism. PCR primers used in the study were designed as per the protocol of Larsen et al. PCR primers of the microorganisms in the study are as listed in [Table 1].
The upstream and downstream sequence primers were then verified for their species specificity by comparing the sequences with all the available 16S rRNA sequences in the RDP database. Ubiquitous primer was used as the positive control for PCR amplification. PCR was performed as described by Saiki et al. Ten microliters of DNA template of the sample was added to 40 μl of working stock reaction mixture containing 5 μl of 10x PCR buffer, 1.25 units of Taq DNA polymerase (0.4 μl), 0.2 mM (1 μl) of each deoxyribonucleotides (dNTP's), primers (1 μl) forward and (1 μl) reverse of the specific microorganisms and 31.6 μl of milli Q water. The PCR reaction was carried out using a PCR thermocycler (Applied Bio Systems , USA). The PCR temperature profile for all the four microorganismsincluded an initial denaturation of 95°C for 2 min, followed by 36 cycles of a denaturation step at 95°C for 30 s, annealing step at 60°C for 1 min, extension at 72°C for 1 min and the final step at 72°C for 2 min.
After amplification, 10 μl aliquot of the amplified PCR product was subjected to electrophoresis in a 0.75% agarose gel containing 0.5 μg/ml ethidium bromide in 1x TAE buffer. The gel was photographed under a 300-nm ultraviolet light trans-illuminator. A 100 bp DNA ladder (Bangalore Genei Pvt. Limited ) served as a molecular weight marker (BIORAD). The PCR-amplified products were sequenced in an automated sequencer (Genetic analyzer 3130; Applied Bio Systems). The sequencer data was blasted with available data in Gen Bank and compared for possible homologies.
The incidence of periodontal pathogens in subgingival, atherosclerotic plaques and both the sites are shown in the bar diagram [Graph 1]. [SUPPORTING:1]
The oral pathogens were detected in both the subgingival plaque and the coronary atherosclerotic plaque samples. Treponema denticola, Campylobacter rectus and Porphyromonas gingivalis were detected in 66.66%, 29.41% and 64.71% of subgingival plaque samples and in 49.01%, 21.51% and 45.10% of atherosclerotic plaque samples, rsepectively. In both subgingival plaque and coronary atherosclerotic plaque samples, Treponema denticola was detected in 39.21%, Campylobacter rectus in 11.76% and Porphyromonas gingivalis in 39.22%.
To explore the association between the presence of Treponema denticola, Campylobacter rectus and Porphyromonas gingivalis in both the plaque samples with the periodontal parameters, correlation coefficient was calculated and is shown in [Table 2].
Results revealed that there was a significant correlation between the plaque index and the presence of Treponema denticola in the atherosclerotic plaque (P≤0.05). There was also a significant correlation between Treponema denticola in the atherosclerotic plaque and the clinical attachment level and probing depth index (P≤0.05).
There was a statistically significant association between Campylobacter rectus in the atherosclerotic plaque and subgingival plaque with the total number of teeth and plaque index, respectively.
There was a highly significant association between plaque index and Russel's periodontal index for the presence of Porphyromonas gingivalis in subgingival and atherosclerotic plaque (P≤0.01). There was a statistically significant association between probing depth index and Porphyromonas gingivalis in both the plaque samples (P0≤0.05).
The above observations reveal that the amount of periodontal destruction directly correlates with the presence of the three microorganisms in both the plaque samples.
Chi-square analysis was carried out to find out the probability of the presence of the microorganisms in the subgingival plaque and coronary atherosclerotic plaque samples. The chi-square value was significant, inferring that Treponmema denticola, Campylobacter rectus and Porphyromonas gingivalis were more prevalent in the subgingival plaque, with a high probability of their presence in coronary atherosclerotic plaque as well [Table 2].
Periodontitis and atherosclerosis have many potential pathogenic mechanisms in common. Both the diseases have a complex causation, genetic and gender predisposition and might share common risk factors, such as age, education, smoking, social status and stress. 
A number of epidemiological studies have shown a statistical association between periodontitis and CAD. 
The present study was performed with the aim to investigate and compare the presence of three bacterial pathogens, namely Treponema denticola, Campylobacter rectus and Porphyromonas gingivalis in coronary atheromas recovered from patients undergoing CABG. Coronary plaque and subgingival plaque samples were collected in 51 patients and analyzed for universal bacterial primers followed by the specific primers for these microorganisms. The 16S rRNA-specific sequence of the above microorganisms was selected. Hence, the presence of both the bacterial DNA and the DNA for the specific microorganisms were ascertained. The result revealed the presence of bacterial DNA in the subgingival plaque and coronary atherosclerotic plaque samples. However, the prevalence of Treponema denticola, Campylobacter rectus and Porphyromonas gingivalis was detected in 66.66%, 29.41% and 64.71% of subgingival plaque samples and in 49.01%, 21.51% and 45.10% of coronary atherosclerotic plaque samples, respectively. In both the subgingival plaque and coronary atherosclerotic plaque samples, Treponema denticola was detected in 39.21%, Campylobacter rectus in 11.76% and Porphyromonas gingivalis in 39.22%, respectively.
Recently, studies have shown variations in the detection of putative periodontal pathogens in subgingival and atheromatous plaques using PCR assays. Cairo et al. and Ishihara et al. reported the presence of at least one target bacterial species in 75% of the samples. The results of this study favor the hypothesis that oral pathogens detected in the periodontal sites are also detected with a higher prevalence in the diseased coronary artery. This is in accordance with the study by Aimettiet al., in which the prevalence of Treponema denticola was 54.5% in subgingival plaques and atherosclerotic lesions. With the set of ubiquitous primers for the general detection of bacterial DNA, all the subgingival and atherosclerotic plaque samples were positive for the bacterial DNA. This proportion is greater than that reported by Aimetti et al. and Haraszthy et al.  and is similar to that presented by Fiehn et al., i.e. 100%.
We detected bacterial DNA of the above three microorganisms in the diseased coronary atherosclerotic arteries by 36 amplified cycles. According to the severity of the disease, these microorganisms may enter the circulation more easily and can subsequently get colonised in the coronary atherosclerotic plaque.
The chronic cyclic nature of periodontal disease provides multiple opportunities for repeated dissemination of pathogens in the blood. Oral pathogens would increase the incidence and severity of transient bacteremia through gingival ulceration and vascular changes in the periodontal tissues. The bacteremia could affect the endothelial integrity, metabolism of plasma lipoproteins, blood coagulations and platelet function. 
This study revealed the detection of oral bacterial DNA in coronary atherosclerotic plaques. The presence of common oral pathogens in the coronary atherosclerotic plaques in significant proportion may suggest the possible relationship between periodontal bacterial infection and genesis of coronary atherosclerosis. However, if this association is casual, a deeper evaluation of periodontal diseases is necessary. This work opens new insights regarding the potential risks of periodontal pathogens for atherosclerosis. Further molecular studies are required for better understanding of this association. Identifying the inflammatory bacteria associated with vascular pathogenesis will be beneficial to understanding the epidemiological link between periodontal disease and cardiovascular disease as well as in developing novel therapies for CVD .
We gratefully acknowledge Dr. Jebarani for her statistical analysis of the compiled data. We are thankful to Dr. Kumanan for his contribution in sequencing of the PCR products.
|1||Libby P. Inflammation in atherosclerosis. Nature 2002;420:868-74.|
|2||Hansson GK. Inflammation, Atherosclerosis, and Coronary Artery Disease. N Engl J Med 2005;352:1685-95. |
|3||Pesonen E, Siitonen O. Acute myocardial infarction precipitated by infectious disease. Am Heart J 1981;101:512-3. |
|4||Spodick DH, Flessas AP, Johnson MM. Association of acute respiratory symptoms with onset of acute myocardial infarction: Prospective investigation of 150 consecutive patients and matched control patients. Am J Cardiol 1984;53:481-2. |
|5||Saikku P, Leinonen M, Mattila K, Ekman MR, Nieminen MS, Mδkelδ PH, et al. Serological evidence of an association of a novel chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infarction. Lancet 1988;29:983-6. |
|6||Mattila KJ. Viral and bacterial infections in patients with acute myocardial infarction. J Intern Med 1989;225:293-6.|
|7||Mattila KJ, Valle MS, Nieminen MS, Valtonen VV, Hietaniemi KL. Dental infections and coronary atherosclerosis. Atherosclerosis 1993;103:205-11.|
|8||Woodhouse PR, Khaw KT, Plummer M, Foley A, Meade TW. Seasonal variations of plasma fibrinogen and factor VII activity in the elderly: Winter infections and death from cardiovascular disease. Lancet 1994;343:435-9.|
|9||Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115-26.|
|10||Vorchheimer DA, Fuster V. Inflammatory markers in coronary artery disease: Let prevention douse the flames. JAMA 2001;286:2154-6.|
|11||Rosenzweig A. Endothelial progenitor cells. N Eng J Med 2003;348:581-2. |
|12||Solberg LA, Enger SC, Hjermann I, Helgeland A, Holme I, Leren P, et al. Risk factors for coronary and cerebral atherosclerosis in the Oslo study. Atherosclerosis V. In: Gotto AM, Smith LC, Allen B, editors. New York, NY: Springer-Verlag; 1980. p. 57-62.|
|13||Buja LM. Does atherosclerosis have an infectious etiology? Circulation 1996;94:872-3.|
|14||Li L, Messas E, Batista EL Jr, Levine RA, Amar S. Porphyromonas gingivalis Infection accelerates the progression of atherosclerosis in a heterogenous Apolipoprotein E deficient murine model. Circulation 2002;105:861-7.|
|15||Mattila KJ, Valtonen VV, Nieminen M, Huttunen JK. Dental infection and the risk of new coronary events, Prospective study of patients with documented coronary artery disease. Clin Infect Dis 1995;20:588-92.|
|16||Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, et al. Primer mediated enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988;239:487-91.|
|17||Larsen N, Olsen GJ, Maidak BL, McCaughey MJ, Overbeek R, Macke TJ, et al. The ribosomal database project. Nucleic Acids Res 1993;21:3021-3.|
|18||Genco R, Offenbacher S, Beck J. Periodontal disease and cardiovascular disease: Epidemiology and possible mechanisms. J Am Dent Assoc 2002;133:14-22S.|
|19||Haraszthy VI, Zambon JJ, Trevisan M, Zeid M, Genco RJ. Identification of periodontal pathogens in atheromatous plaques. J Periodontol 2000;71:1554-60.|
|20||Cairo F, Gaeta C, Dorigo W, Oggioni MR, Pratesi C, Pini Prato GP, et al. Periodontal pathogens in atheromatous plaques: A controlled clinical and laboratory trial. J Periodontal Res 2004;39:442-6.|
|21||Ishihara K, Nabuchi A, Ito R, Miyachi K, Kuramitsu HK, Okuda K. Correlation between detection rates of periodontopathic bacterial DNA in carotid coronary stenotic artery plaque and in dental plaque samples. J Clin Microbiol 2004;42:1313-5.|
|22||Aimetti M, Romano F, Nessi F. Microbiological analysis of periodontal pockets and carotid atheromatous plaques in advanced chronic periodontitis patients. J Periodontol 2007;78:1718-23. |
|23||Fiehn NE, Larsen T, Christiansen N, Holmstrup P, Schroeder TV. Identification of periodontal pathogens in atherosclerotic vessels. J Periodontol 2005;76:731-6.|
|24||Fong IW. Infections and their role in atherosclerotic vascular disease. J Am Dent Assoc 2002;133:75-135.|