| Abstract|| |
Background: The study was aimed to assess the prevalence of periodontal pathogens namely Tannerella forsythia (T.f), Campylobacter rectus (C.r), Eikenella corrodens (E.c), Porphyromonas gingivalis (P.g), Treponema denticola (T.d), Prevotella nigrescens (P.n) ,Aggregatibacter actinomycetemcomitans (A.a), P.g (fi mA gene) and Prevotella intermedia (P.i), in the subgingival and the atheromatous plaque of patients with coronary artery disease (CAD), and comparing them with the noncardiac subjects thereby indicating the role of periodontal pathogenic bacteria in the progression of atherosclerosis in south Indian population.
Materials and Methods: 51 cardiac and non cardiac subjects within the age group of 40-80 years,who met the eligibility criteria, were selected and categorized as the experimental and control group respectively. Total number of teeth was recorded, and oral hygiene was evaluated using Plaque Index and Oral Hygiene Index (OHI). Periodontal pocket depth and clinical attachment level were also assessed as a part of periodontal examination. Subgingival plaque samples were collected with the help of with Gracey's curette in both the groups. In experimental group, biopsy was obtained from the atherosclerotic plaque during Coronary artery bypass grafting CABG procedure. Both, subgingival and the coronary atherosclerotic plaque samples were subjected to polymerase chain reaction (PCR) analysis for identification of the periodontal bacteria.
Statistical Analysis: Mean, standard deviation and test of significance of quantitative variables such as periodontal parameters were compared between experimental group and control group. Kappa measures of agreement was done to analyze the relationship between the presence/absence of microorganisms in the subgingival and atherosclerotic plaque samples in the experimental group.
Results: The mean Plaque Index, Gingival Index, Russel's Periodontal Index, OHI, clinical attachment level, Pocket Depth Index was statistically significant in both the groups. Association of T.f, E.c, C.r, P.g, P.g (fi mA), T.d, P.i, P.n was significantly associated. A.a was absent in the control group, whereas only one patient in the experimental group was positive for the bacteria. Kappa analysis showed the significant association of periodontal bacteria T.f, C.r, P.g, P.g (fi mA), P.i and P.n in subgingival plaque and atherosclerotic plaque of the same patients of the experimental group.
Conclusion: It is concluded that CAD subjects had higher prevalence of periodontal pathogens in subgingival biofilms as compared to the non cardiac subjects. Further, the number of bacteria was significantly associated between the subgingival and atherosclerotic plaques of the cardiac patients in south Indian population.
Keywords: Atherosclerosis, chronic periodontitis, inflammation, periodontal bacteria
|How to cite this article:|
Mahendra J, Mahendra L, Nagarajan A, Mathew K. Prevalence of eight putative periodontal pathogens in atherosclerotic plaque of coronary artery disease patients and comparing them with noncardiac subjects: A case-control study. Indian J Dent Res 2015;26:189-95
Pathogenic periodontal microorganisms provide a significant and persistent Gram-negative bacterial challenge to the host.  The systemic exposures of orally derived bacteria or bacterial endotoxins exert a noxious effect that acts directly or indirectly on the vessel wall. In addition, lipopolysaccharides and the other products from Gram-negative bacteria may stimulate cytokine production, hyper-coagulability, and monocyte activation.  Recently, many researchers have focused their attention on the ability of periodontal pathogens to colonize in the atheromatous plaque.  Nevertheless, the clean correlation between the detection rates of periodontal bacteria DNA in atheromas and in subgingival plaque samples has not been established in the cardiac patients and has not been further compared with the systemically healthy subjects in South Asian population. Hence, an attempt has been made to assess the prevalence of periodontal pathogens namely Tannerella forsythia (T.f), Campylobacter rectus nbsp;Eikenella corrodens E.c), Porphyromonas gingivalis ), Treponema denticola T.d), Prevotella nigrescens P.n) and Aggregatibacter actinomycetemcomitans (A.a), P.g (fimA gene) and Prevotella intermedia P.i), in the subgingival and the atheromatous plaque of patients with coronary artery disease (CAD), and comparing them with the noncardiac subjects thereby indicating the role of periodontal pathogenic bacteria in the progression of atherosclerosis.
|How to cite this URL:|
Mahendra J, Mahendra L, Nagarajan A, Mathew K. Prevalence of eight putative periodontal pathogens in atherosclerotic plaque of coronary artery disease patients and comparing them with noncardiac subjects: A case-control study. Indian J Dent Res [serial online] 2015 [cited 2019 Sep 20];26:189-95. Available from: http://www.ijdr.in/text.asp?2015/26/2/189/159164
| Materials and Methods|| |
This study was conducted in "Department of Periodontia, Rajah Muthiah Dental College, Annamalai University, Chidambaram," "The Madras Medical Mission" (Institute of Cardiovascular Diseases), Mogappair, Chennai, "The Department of Animal Biotechnology," Tamil Nadu Veterinary and Animal Sciences University and "Post Graduate Institute of Basic Medical Sciences," University of Madras, Taramani, Chennai, India. The Institutional Human Ethics Committee of "The Madras Medical Mission" (Institute of Cardiovascular Diseases) had approved the protocol of this research work. Two hundred and fifty patients with CAD were screened for the experimental group, 50 patients were excluded due to the history of other systemic diseases, 100 patients refused to participate and in 49 patients, coronary plaque was not detected during the coronary bypass surgery. Finally, 51 patients met the eligibility for the study and were categorized as the experimental group [Figure 1]. Their consent was obtained for this study. Experimental group included patients between 40 and 80 years of age, generalized chronic periodontitis involving ≥30% sites with ≥5 mm of clinical attachment, presence of bleeding on probing and diagnosed of CAD, and recruited for coronary artery bypass grafting (CABG). Patients with history of smoking, abuse of tobacco, any other form of cardiac disease other than CAD, major systemic illness such as malignancy and respiratory diseases and long-term, recent or current regimen of medication (antibiotic or corticosteroids), which may interfere with the conduct of the study were excluded from the experimental group. On the other hand for the control group 51 noncardiac subjects within the age group of 40-80 years, both male and female with no history of cardiac ailments, and no history of other systemic diseases, which could interfere with the study were included in the present investigation. Patients with use of tobacco, any major systemic diseases such as malignancy, respiratory diseases or current regimen of medication which may interfere with the conduct of the study were excluded from the control group. Patient's consent was obtained before the participation of this study.
For the experimental group, periodontal examination was performed 1 day before the CABG. Total number of teeth was recorded, and oral hygiene was evaluated using Plaque Index and Oral Hygiene Index (OHI). Periodontal pocket depth and clinical attachment level were also assessed. Similar periodontal examination was also carried out for the control group.
Collection of the plaque samples
Teeth were gently dried. After the removal of supragingival plaque with the cotton swab, subgingival plaque samples were collected with Gracey's curette and transferred to the sterile vial containing transport media (500 μl of phosphate-buffered saline solution, pH-7.4). The samples, obtained from the two periodontitis sites (pocket depth >5 mm), which represented the deepest periodontal pockets, were pooled for analysis. In the case of the experimental group, the plaque samples were collected between 8 a.m. and 10 a.m. 1 day prior to the surgery.
Collection of the atherosclerotic plaque in coronary artery bypass grafting patients (experimental group)
Biopsy was obtained from the atherosclerotic plaque during CABG procedure. The surgeon excised a small bit of plaque (0.5-1 cm) from the cut edge of the coronary arteriotomy. To eliminate the blood contamination, the vascular endothelial 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 polymerase chain reaction (PCR) tissue homogenizer as described by Saiki et al. 
Polymerase chain reaction
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 ×10 PCR buffer, 1.25 units of Taq DNA polymerase (0.4 μl), 0.2 mM (1 μl) of each deoxyribonucleotides, 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 microorganisms included 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 ×1 TAE buffer. The gel was photographed under a 300-nm ultraviolet light trans-illuminator. A 100 bp DNA ladder (Bangalore Genei Pvt. Ltd.) served as a molecular weight marker (Bio-Rad). The PCR-amplified products were sequenced in an automated sequencer (Genetic analyzer 3130; Applied Bio Systems). The sequencer data were blasted with available data in Gen Bank and compared for possible homologies.
Mean, standard deviation and test of significance of quantitative variables such as periodontal parameters were compared between experimental group and control group. Student's independent t-test was used to calculate the P Value. Analysis of the relationship between the presence/absence of microorganisms in the subgingival plaque among the experimental and control groups was examined by carrying out Chi-square test. P ≤ 0.05 was considered as a level of significance. Kappa measures of agreement to analyze the relationship between the presence/absence of microorganisms in the subgingival and atherosclerotic plaque samples in the experimental group. Percentage of microorganisms in atherosclerotic plaque with subgingival plaque was calculated. P ≤ 0.05 was considered as a level of significance.
| Results|| |
The mean and standard deviation of the quantitative variables such as periodontal parameters were analyzed in the experimental and control groups [Table 2]. The mean Plaque Index, Gingival Index, Russel's Periodontal Index, OHI, clinical attachment level, Pocket Depth Index was statistically significant (P < 0.001).
The relationship of the periodontal pathogens in the subgingival plaque of experimental and control group was analyzed using Chi-square test. Results showed that the association of T.f, E.c, C.r, P.g, P.g (fimA), T.d, P.i, P.n was significantly associated [Table 3]. A.a was absent in the control group, whereas only one patient in the experimental group was positive for the bacteria. Hence, the analysis between the two groups was noncomparable.
|Table 3: Prevalence of periodontal microorganisms in subgingival plaque-group wise |
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Kappa analysis showed the significant association of periodontal bacteria T.f, C.r, P.g, P.g (fimA), P.i and P.n in subgingival plaque and atherosclerotic plaque of the same patients of the experimental group [Table 4]. The significant probability value indicated E.c and T.d did not show good agreement between the subgingival plaque and atherosclerotic plaque of the experimental group. Of 51 CAD patients, none of the atherosclerotic plaque samples were found positive for A.a. However, one subgingival plaque sample was found positive for the microorganism. Hence, there was no need for carrying out the analysis.
|Table 4: Presence of periodontal microorganisms in subgingival plaque and atherosclerotic plaque in the experimental group |
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In both the plaque samples, P.g had the highest prevalence than rest of the microorganisms [Table 5] and [Figure 2].
|Figure 2: Prevalence of periodontal microorganisms in subgingival plaque and atherosclerotic plaque - Experimental group|
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|Table 5: Prevalence of periodontal microorganisms in subgingival plaque and atherosclerotic plaque in the experimental group |
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| Discussion|| |
Atheroclerosis develops in response to vessel wall injury.  Apart from traditional risk factors such as hypertension, hypercholesterolemia, diabetes mellitus, marked obesity, smoking and physical inactivity, chronic infections have also been associated with an increased risk of atherosclerosis and CAD.  Bacteremia following dental procedures has been well-documented in the progression of atherosclerosis. Though various studies have shown association between atherosclerosis and periodontitis, the results seem to be conflicting. Hence, the present study was undertaken to examine the presence of the bacterial DNA of periodontal pathogens namely T.f, C.r, E.c, P.g, P.g (fimA gene), T.d, A.a, P.i and P.n in the subgingival plaque and atherosclerotic plaque of the patients undergoing CABG and compare them with the controls with no history of cardiac ailments. In our study we assessed all the periodontal parameters among the cardiac patients and compared them with the noncardiac patients and found that the mean value of the parameters, i.e. Plaque Index, Gingival Index, Russel's Periodontal Index, OHI, clinical attachment level and Probing Pocket Depth Index was higher in CAD patients than the control group [Figure 3]. The results of this study were in concurrence with the findings of Beck et al.,  Danesh et al.  and Morrison et al.  According to Padilla et al.,  all the patients with CAD had pocket depth of 3.92-5.5 mm and the probing depth index was much higher in these patients as compared to controls. The patients with CAD had higher plaque scores and gingival index.
We detected eight periodontal pathogens in the subgingival plaque of the experimental and control groups. These periodontal pathogens were found with a higher prevalence in the experimental group when compared to the control group [Table 3] and [Figure 4]. Thus this suggested the contribution of these bacteria in the progression of atherosclerosis. However, since the detection rate of T.f was more or less the same in both the groups, no association could be found pertaining to this pathogen. It may be attributed to the smaller sample size and the method chosen to detect the microorganism. This is one of the few studies to examine the prevalence of the pathogenic periodontal bacteria in the subgingival plaque of the patients with CAD and comparing them with the detection rate of the same periodontal bacteria in the subgingival plaque of the subjects without any cardiac ailments.
|Figure 4: Prevalence of periodontal microorganisms in subgingival plaque-group wise|
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To analyze the association of periodontal pathogens in subgingival and atherosclerotic plaque of the patients with CAD, the percentage prevalence and the measures of agreement (kappa) were calculated and results were found to be favorable. The percentage of the periodontal bacteria in the subgingival plaque was significantly correlated with the periodontal pathogens T.f, C.r, P.g, P.g (fimA gene), A.a, P.i and P.n in the atherosclerotic plaque samples [Table 4]. Our study concurs with the studies done by Zaremba et al.,  Nakano et al.,  Ford et al.,  Ishihara et al.  which showed a significant association between the presence of the microorganism in the subgingival and atherosclerotic plaque samples. This is one of the very few studies to detect P.g and P.n in the coronary plaque samples of the cardiac patients. However, E.c did not show a significant association between the subgingival and atherosclerotic plaque samples though its prevalence was higher in the subgingival plaque of the same patients. We also detected the periodontal bacteria only in the atherosclerotic plaque without the presence in the subgingival plaque. The presence of the above periodontal pathogens only in the atherosclerotic plaque was probably due to the fact that the periodontal microbiota in the subgingival plaque is always constantly changing and is never consistent. In our study, P.g (fimA) was to found to have a significant association between subgingival and atherosclerotic plaque samples in the CAD patient thereby suggesting its major role in the progression of atherogenesis. Our finding supported the animal study conducted by Chou et al.  who demonstrated that P.g fimbria-mediated invasion up regulates inflammatory gene expression in Human Aortic Endothelial Cells and in aortic tissues indicating that invasive P.g infection accelerates inflammatory response directly in the aorta.
Of 51 patients with CAD, only one patient was positive for A.a in the subgingival plaque whereas no bacterial DNA for A.a could be detected in the atherosclerotic plaque. This finding was in concurrence with the study carried out by Zhong et al.,  Zhang et al.  and Okuda et al.  who could not detect the presence of A.a in the atherosclerotic plaque. However, our study was in contrast with the study done by Aimetti et al.,  Padilla et al.  who detected A.a in the atherosclerotic plaque. A.a has been suggested to contribute to the pathogenesis of coronary heart disease and antibody levels against this pathogen in serum have been viewed as indicators of the risk for this disease. A.a probably invades endothelial cells via a mechanism dependent on the engagement of the platelet-activating factor receptor by bacterial phosphorylcholine. However, direct invasion of vascular endothelium by A.a in the development and progression of atherosclerosis still remains questionable.
Further, the present study indicated that a good agreement existed between the percentage prevalence of the periodontal pathogens, in particular P.g, P.g fimA, P.i, T.f, P.n and C.r in the subgingival and atherosclerotic plaque samples suggesting that the presence of these micoorganisms in the subgingival plaque will have significant chances of their presence in atherosclerotic plaque as well, thereby contributing to the progression of atherosclerosis and vice-versa. Thus it is suggested that the higher the abundance of periodontal pathogens in the subgingival microbiota, the greater the number of bacteria that are likely to enter the circulation, with possible involvement of some bacteria in the pathogenesis of atheromatous plaques.
One of the limitations of the present study was that, the methodology applied was in accordance with the technology available at hand. The analysis of the study can be further undertaken by adopting varied technologies available in this regard. In addition, the study was a reterospective analysis performed on 51 patients undergoing CABG. The subjects selected for this study were predominantly from the urban and semi-urban areas in and around the city of Chennai, India. In the future, longitudinal studies can be undertaken targeting a larger population from the rural areas since the rural Indian population, having poor oral hygiene, in general, can provide further substantiating results towards this study.
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Dr. Jaideep Mahendra
Department of Periodontology, Meenakshi Ammal Dental College, Maduravoyal, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]