Prevelance of periodontopathogenic bacteria in subgingival biofilm and atherosclerotic plaques of patients undergoing coronary revascularization surgery.

AIM: The objective of the present study was to detect the presence of specific periodontopathogenic bacteria in the coronary plaque of patients with coronary artery disease and to find out the significant association between the periodontal status and the presence of pathogenic bacteria in the coronary plaque.
MATERIALS AND METHODS: The study population consisted of 51 patients with chronic generalized periodontitis undergoing coronary artery bypass grafting. Periodontal parameters were recorded and deoxyribonucleic acid was extracted from the subgingival plaque and coronary atherosclerotic plaque samples of the same patients. Polymerase chain reaction was used to amplify the part of 16S ribosomal ribonucleic acid (rRNA) gene to detect the presence of Aggregatibacter actinomycetemcomitans (Aa), Tannerella forsythia (Tf), Porphyromonas gingivali (Pg), Porphyromonas gingivalis (fimA) gene and Treponema denticola (Td).
RESULTS: Aa, Tf, Pg, Pg (fimA) gene and Td were detected in 0%, 31.4%, 45.1% 39.2% and 51% of atherosclerotic plaque samples, respectively. Tf was detected in 19.6%, Pg in 39.2%, Pg (fimA) gene in 33.3% and Td in 35.3% of both, subgingival plaque and atherosclerotic plaque samples. Periodontal parameters correlated with the presence of bacteria in coronary plaque. Aa could not be detected in coronary plaque samples.
CONCLUSIONS: The study confirmed the detection of Red complex bacteria in coronary plaque samples and these bacteria correlated with the severity of periodontal destruction.

INTRODUCTION

Periodontal disease is one of the most common chronic bacterial infections of the tooth supporting structures, which is predominantly associated with gram negative bacteria that exist in the subgingival biofilm.[1] Atherosclerosis, on the other hand, is a highly prevalent disease in humans with significant morbidity and mortality.[2] Previous data suggests that subjects with the chronic periodontitis are at a significantly increased risk for developing coronary artery disease (CAD), some of the studies could not substantiate the correlation in this regard. Apart from the known risk factors, such as smoking, diabetes, high blood pressure and socio-economic factors, the chronic periodontal inflammation is said to be an important contributing factor for cardiovascular diseases.[3]

The important risk markers for cardiovascular disease, such as Plasma fibrinogen level, C-reactive protein, total white blood corpuscles count have been reported to be elevated in patients with chronic periodontitis.[145] Animal and human studies have found an association between the periodontal inflammation and the incidence of coronary events. Though the studies performed in the past have shown the presence of periodontal bacteria in coronary plaque, but most of these have shown conflicting results.[3] Moreover, very few studies have shown its association with the amount of periodontal destruction.

Hence to explore further, the present study was undertaken to evaluate the presence of the 16S rRNA gene of Red complex bacteria (Tf, Pg, Td), Pg (fimA) and Aa in the subgingival and the atherosclerotic plaques of periodontitis patients with CAD undergoing coronary artery bypass grafting (CABG). The periodontal status also compared with the presence of pathogenic bacteria in coronary plaque.

MATERIALS AND METHODS

Patients selection

One hundred and fifty patients with CAD, scheduled to undergo CABG in the Institute of Cardiovascular Diseases, Madras Medical Mission, Chennai, India were recruited consecutively for the study, out of which fifty one patients (40 males and 11 females) in the age group of 40-80 years (age: 61 ± 8.75 years) with chronic generalized periodontitis were selected based on following specific criteria. They were diagnosed with chronic generalized periodontitis with atleast 30% of sites with clinical attachment loss (CAL) and alveolar bone exceeding 1/3 of the root in atleast 30% of the entire dentition.[6] These patients were suffering from CAD and were scheduled to undergo CABG. They were cooperative and readily accepted for the study.

Exclusion criteria included smokers, hypertensive patients, those with respiratory diseases, diabetes or any other major systemic diseases that could affect the periodontium. Subjects with antibiotic intake or a history of periodontal treatment carried out in the previous 6 months were also excluded from the study. This was done to rule out the confounding factors in atherogenesis other than periodontal diseases. The informed consent was obtained from the subjects. The Ethics Committee of the Madras Medical Mission approved the protocol of this study. Medical and dental history of each subject was obtained by an interview. The periodontal examination of all the subjects included Plaque Index,[7] Gingival Index,[8] Clinical Attachment Level and Pocket Depth Index[9] and Oral Hygiene Index.[10]

Subgingival plaque collection

The subgingival plaque samples were taken a day before patients underwent the CABG. The two deepest periodontal sites with periodontal depth ≥ 5 mm were selected for 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 curette and were pooled for analysis.

Collection of atherosclerotic plaque

A biopsy was obtained from the diseased coronary artery during the CABG procedure. The surgeon excised one or two small bits of atherosclerotic plaque (0.5-1 mm) from the edge of the coronary arteriotomy performed for anatomizing the graft. To eliminate the blood contamination, the plaque samples were placed in sterilized phosphate-buffered saline and mixed gently and was transferred to fresh vials containing the transporting media. The samples were then homogenized by the tissue homogenizer as described by Saiki et al.[11]

Deoxyribonucleic acid (DNA) extraction

Both the subgingival plaque and the coronary atherosclerotic plaque samples were centrifuged for 10 min at 10,000 rpm (Remi Centrifuge, Mumbai, India). The supernatant was discarded and the resulting pellet was resuspended in 200 μl of lysis solution (100 mM Tris, 1.0 mM ethylenediamine tetra-acetic 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.

Polymerase chain reaction (PCR) amplification

16S rRNA PCR amplification was carried out to detect the presence of the bacteria. PCR primers used in the study were designed as per the protocol of Larsen et al.[12] PCR primers of microorganisms in the study are as listed in Table 1.

Table 1

PCR primers

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.[11] Briefly, 10 μl 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 (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 Biosystems, CA, USA). The temperature profile of the bacteria Tf, Pg and Td was 95°C (2 min), 36 cycles of 95°C (30 s), 60°C (1 min), 72°C (1 min) and 72°C (2 min). The temperature profile for Aa was 95° C (2 min), 36 cycles of 94°C (30 s), 30 cycles, annealing at 55°C (1 min), 72°C (2 min) and final extension at 72°C (10 min). Pg (fimA gene) had a temperature profile of 94°C (5 min), 35 cycles of denaturation 94°C (1 min), 50°C (1 min), 72°C (1.5 min) and final 72°C (7 min).

After amplification, 10 μl aliquot of the amplified PCR product was subjected to electrophoresis is 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 (Bio-Rad Laboratories, CA, USA). The gel images in the figures represented the presence of bacterial DNA of Tf, Pg, Pg (fimA) gene and Td in the subgingival plaque and atherosclerotic plaque of the same patient [Figure 1].

Figure 1

16S ribosomal ribonucleic acid-based polymerase chain reaction detection of Tannerella forsythia (a), Porphyromonas gingivali (b), P. gingivalis (fimA) gene (1c) and Treponema denticola (d). (a) Lane 1 – Deoxyribonucleic acid (DNA) ladder – 100 bp, Lane 2 and 4 – Subgingival plaque samples, Lane 3 and 5 – Coronary artery plaque samples, Lane 6 – Negative control. (b) Lane 1 – DNA ladder – 100 bp, Lane 2 and 4 – Subgingival plaque samples, Lane 3 and 5 – Coronary artery plaque samples, Lane 6 – Negative control. (c) Lane 1 – DNA ladder – 100 bp, Lane 2, 4 and 6 – Subgingival plaque samples, Lane 3, 5 and 7 – Coronary artery plaque samples, Lane 8 – Negative control. (d) Lane 1 – DNA ladder – 100 bp, Lane 2, 4 and 6 – Subgingival plaque samples, Lane 3, 5 and 7 – Coronary artery plaque samples, Lane 8 – Negative control

Sequencing of the PCR products

The PCR-amplified products were sequenced in an automated sequencer (Genetic analyzer; Applied Biosystems, CA, U.S.A.). The sequencer data was blasted with available data in GenBank and compared for possible homologies.

Statistical analysis

The association between the periodontal parameters and presence of Pg (fimA), Aa and the red complex (Td, Tf and Pg) in the atherosclerotic plaque samples, was analyzed by the Student's independent t-test. Percentage of microorganisms in the atherosclerotic plaque and the subgingival plaque was calculated. P ≤ 0.05 was considered as a level of significance. Kappa analysis was applied to access the association between the presence of the bacterial species in the subgingival biofilm and atherosclerotic plaques.

RESULTS

The study showed the presence of bacteria, in all the subgingival and atherosclerotic plaque samples. All the samples were positive for bacterial DNA (100%). In the PCR analysis, Aa sequence of the 16SrRNA gene was detected only in one subgingival plaque sample and could not be detected in any of the atherosclerotic plaque samples. Pg (fimA), Td, Tf and Pg were detected in 62.7%, 66.7%, 43.1% and 64.71% of subgingival plaque samples and in 39.2%, 51%, 31.4% and 45.10% of atherosclerotic plaque samples, respectively. In both subgingival plaque and atherosclerotic plaque samples, Pg (fimA) was detected in 33.3%, Td was detected in 35.3%, Tf in 19.6% and Pg in 39.22% [Figure 2, Bar Diagram]. We found a significant association of Pg (P - 0.003) and Pg (fimA) (P - 0.008) in subgingival plaque and coronary plaque samples [Table 2].

Figure 2

The incidence of periodontal pathogens in subgingival plaque, atherosclerotic plaque and both sites

Table 2

Kappa analysis for the presence of the microorganisms in subgingival and atherosclerotic plaque samples

The present study further showed a statistically significant relationship between the presence of Tf in the atherosclerotic plaque with gingival index (P - 0.03) and probing pocket depth (P - 0.01) [Table 3]. However, all the periodontal parameters analyzed were non-significant [Table 3] with the presence of Td, Pg and Pg (fimA), in the atherosclerotic plaque samples.

Table 3

Students test of the selected periodontal parameters for the presence of microorganisms in atherosclerotic plaques

DISCUSSION

Periodontal diseases are one of the most common bacterial infections characterized by inflammation and the subsequent destruction of the tooth supporting tissues. The disease is associated with a widely diverse and complex subgingival microorganisms encompassing Gram-positive and Gram-negative bacteria, facultative and anaerobic organisms.

Dental plaque has been considered as the reservoir of the most pathogenic bacteria. The past evidence states that dental plaque is the primarily etiological factor for the progression of periodontal diseases. These microorganisms have also been associated with the various systemic diseases as well. Several microorganisms including Chlamydia pneumoniae and Cytomegalovirus have been implicated in the infectious etiology of atherosclerosis. Likewise, the chronic periodontal infections have also been reported to increase the risk for CAD.[13] It has been postulated that chronic infections can contribute to the development of atheromas by direct (platelet aggregation, invasion of endothelial cells and endothelial injury) and indirect (induction of intracellular adhesion molecules, production of antibodies to lipopolysaccharide as well as cytokines and dysfunction of the immune system) mechanisms.[14]

In recent years, many researchers have focused their attention on the ability of periodontal pathogens to colonize in the atheromatous plaque in order to substantiate the potential role of the periodontal bacteria in the progression of atherosclerosis. Nevertheless, a clear correlation between the detection rates of periodontopathic bacterial DNA in atheromas and periodontal pockets has not been established.

In view of the above, the present study was undertaken to assess the presence of the bacterial DNA of putative periodontal pathogens in the subgingival and coronary plaque of patients with chronic periodontitis and to compare the periodontal status with the presence of periodontal bacteria in the coronary plaque.

The study revealed the presence of the red complex bacteria (Td, Tf and Pg) in 51%, 31.4% and 45.10% of atherosclerotic plaque samples, respectively. Pg was detected in 24.4% of subgingival plaque and 45.10% of atherosclerotic plaque, showing a significant correlation of the microorganism between the two plaque samples thus indicating its potential role toward the progression of coronary events. The studies by Nakano et al.,[15] Ford et al.[16] Zhong et al.,[17] Gotsman et al.[18] and Zhang et al.[19] also identified Pg in 7.4%, 52%, 38.7%, 86%, 33% from atherosclerotic plaque samples, respectively. Haraszthy et al.[20] examined 50 carotid endarterectomy samples using PCR and found the presence of Pg in 26% of the samples.

We detected Pg (fimA) in 33.3% of both the subgingival and coronary plaque samples. The presence of Pg (fimA) was found to be significantly associated among both the plaque samples [Table 2]. Our finding supports the animal study conducted by Chou et al.,[21] who demonstrated that Pg fimbria-mediated invasion upregulates inflammatory gene expression in (Human Aortic Endothelial Cells) and in aortic tissues. Pg is one of the major causative factor in periodontal disease and its fimbriae (fimA), a filamentous component located on the cell surface, plays a vital role in the colonization and invasion of the periodontal pathogen in the aortic tissues.[22]

We also detected Td in 51% of the atherosclerotic plaque samples; however, it remained non-significant [Table 2]. Probably, a larger sample size is further needed to elucidate this relationship. Zaremba et al.[23] identified 55% of Td only in subgingival plaque samples. Nakano et al.[15] and Gotsman et al.[18] detected T. denticola in 44.4% and 85% of the atherosclerotic plaque samples respectively. It is suggested that Td can degrade the intracellular matrix.[19] In addition to the motility of Td, it is possible that protease activity may also play a role in their penetration into the periodontal tissues by infiltrating between the gingival cells and the blood stream. Padilla et al.[24] showed that oral Treponema penetrated endothelial cells. The results of our study were in contrast to the findings of Padilla et al.[24] who could not detect Td in the coronary plaque samples. In the present study showed the detection of Tf in 43.1% of the subgingival plaques and 31.4% of atherosclerotic plaques. This was in contrast with Padilla et al.[24] and Okuda et al.,[25] who could not identify Tf in the coronary plaque samples.

In our study, only one patient was positive for Aa in the subgingival plaque sample whereas 16S rRNA gene for the microorganism could not be detected in the atherosclerotic plaque. Our study was in contrast with the studies performed by Aimetti et al.,[26] Padilla et al.,[24] Ishihara et al.,[27] Marques da Silva et al.,[28] and Cairo et al.,[29] who detected Aa in 33.3%, 58%, 29%, 7.1% and 5% of the subgingival plaque samples, respectively. However, the present study was in concurrence with the study done by Zhong et al.,[17] Zhang et al.,[19] and Okuda et al.,[25] who could not detect the presence of Aa in atherosclerotic plaque samples.

Aa 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. Aa probably invades endothelial cells through a mechanism dependent upon the engagement of the platelet-activating factor receptor by bacterial phosphorylcholine. The bacterium possesses a number of putative virulence factors including a leucotoxin (LtxA) that target and destroy the host immune cells, such as monocytes and neutrophils.[28] We were not able to detect Aa, in our plaque samples. The reason could be that Aa being more prevalent in the younger age group, was absent in our samples as the age of the selected subjects was between 40 and 80 years in this study.

In the present study, the mean of the selected periodontal parameters namely gingival index, oral hygiene index, mean probing pocket depth, clinical attachment level, number of teeth present and the presence of the periodontal pathogens in the atherosclerotic plaque of the cardiac patients was analyzed [Table 3]. Tf, showed a significant association with the gingival index suggesting that with an increase in the gingival inflammation, the above microorganism is more liable to be prevalent in the coronary plaque. Tf was also associated with an increase in the probing pocket depth. Tf has been reported to express a number of enzymatic and proteolytic activities that could contribute to its ability to compete effectively in the complex biofilm of the subgingival pocket.[15]

The previous studies have also shown the significant association between the periodontal parameters and the presence of the microorganisms. Aimetti et al. detected higher prevalence of periodontal pathogens in the coronary plaque with increased pocket depth.[26] Emingil et al.[30] investigated whether poor periodontal condition was associated with an acute episode of myocardial infarction (AMI) among chronic coronary heart disease patients. The proportion of patients with bleeding on probing and probing pocket depth ≥4 mm was found to be significantly associated with AMI. Similarly, Gotsman et al.[18] determined the relationship between periodontal disease measures and CAD. Patients with severe CAD had significantly more periodontal destruction than those with mild CAD. Logistic regression analysis showed that percentage of teeth with CAL more than 5mm was significantly associated with CAD severity. Patients with CAD had higher plaque scores and gingival index. In our study, the correlation of Td, Pg and fimA gene with periodontal parameters in the present study was not found to be significant. The reason could be a small sample size. In addition, Aa, could not show any significant association with the periodontal parameters.

Overall, the present study indicated that a good agreement existed between the percentage prevalence of the periodontal pathogens, in particular Pg and Pg fimA in the subgingival and atherosclerotic plaque samples suggesting that the presence of these microorganisms in the subgingival plaque will have significant chances of their presence in atherosclerotic plaque as well contributing to the progression of atherosclerosis and vice-versa.

CONCLUSION

It is suggested that the bacteria growing in the biofilm become resistant to the phagocytosis and host immune system. The ulcerated pocket epithelium becomes the portal of entry of the periodontal pathogens and their products to the general circulation in small and major blood vessels.[31] On the other hand, the significant virulence factors of these bacteria are able to evade the host clearance mechanisms, participate in the tissue destruction and enter the general circulation in small and major blood vessels thus contributing to the coronary events. However, at this point of time, it is difficult to pin point whether it is the stand-alone role of individual periodontal pathogen or the total periodontal burden as a whole, which contribute toward atherosclerosis. Further molecular studies are required to elucidate the role of each periodontal pathogen in a complex form for a better understanding of this association. In future, large cross –sectional and longitudinal studies are required to elicit the role of Aa, the microorganisms of red complex and their virulence factors in the pathogenesis of atherosclerosis.