Presence of Tannerella forsythia in patients with chronic periodontal disease and atherosclerosis arterosklerozom

Background/Aim. Periodontal disease is an inflammatory disease that occur in the tissues surrounding the teeth in response to bacterial biofilm accumulation (dental plaque). Among others, Tannerela forsythia ( Tf ) was recognized as one of the most significant and specific bacterial species in periodontal pocket („red it should be considered as potential risk factor for atherosclerosis. Accordingly, it would be necessary to control periodontal disease in order to reduce mortality and morbidity associated with atherosclerosis. with coton rolls, scalled carefully su-pragingivaly, and air dried. A sterile paper inserted the apical each selected for 60 sec-onds, and transfered immediatelly to a sterile Eppendorf tube and the on until the analysis. Samples from the arteries were taken during the surgery (endarterectomy, PTCA) and transfered immediatelly to a sterile Eppendorf tube with Tris-EDTA, as transport medium, and kept in the refrigerator on -20° C until the analysis by PCR method.


Introduction
Periodontal disease is affecting up to 90% of the worldwide population 1 . It is an inflammatory disease that occurs in the tissues surrounding the teeth in response to bacterial biofilm accumulation (dental plaque). It is well known that more than 500 virulent microbial species from dental biofilm are mainly responsible for periodontal disease. Although several bacterial species are currently recognized as causally associated with periodontal disease, subgingival colonization is not sufficient for the disease to occur 2 . It is well known that abnormal host response to periodontal disease with specific genetic predisposition and detrimental environment exposures are likely important determinants of suspectibility, e.g. age, poor oral hygiene, cigarette smoking, pregnancy, obesity, stress and systemic conditions such as diabetes mellitus, osteoporosis and rheumatoid arthritis 3 .
Atherosclerosis is a progressive narrowing of arteries that may lead to occlusion as a consequence of lipid deposition 2 . It underlies coronary heart disease (80%), as well as myocardial and cerebral infarctions, therefore having a big socio-economic importance 4 . Onset of atherosclerosis is thought to be due to endothelium function disturbances, platelet activation, and oxidative changes in plasma lipoproteins. Several possible biological mechanisms, including common genetic variants may explain the link between cardiovascular diseases and periodontitis 5 . It is believed that there are multiple mechanisms underlying these links, with inflammatory, infectious, immune, and genetic components 4 . The link between periodontal disease and atherosclerosis was first established around 30 years ago when De Stefano et al. 6 reported an increased risk of atherosclerotic plaque formation in a group of patients with periodontitis (25 % higher) based on 14 years of research including 9,760 individuals aged between 25 to 74 years. Recent attention has been directed towards the potential contribution of chronic inflammatory processes that may amplify vascular inflammation in atherosclerosis, and periodontal disease is recognized as a chronic inflammatory disease 4,7,8 . Today, the American Hearth Association defines this contribution with level A evidence.
Tannerela forsythia (Tf) is one of the most significant and specific bacterial species in periodontal pocket ("red complex" bacteria). Increasing evidence, over the past decade, suggests a link between periodontal disease and atherosclerosis, where Tf, mainly present in diseased periodontal pockets, can enter the systemic circulation directly, and may be present in distant organs, such as aterosclerotic blood vessels. Lypopolysaccharides and other products from Tf cell breakdown may stimulate inflammatory cytokines, upregulate endothelial adhesion molecules and induce a prothrombotic enviroment, that can enhanced risk of an atherosclerossis 9 . The causal relationship between periodontal disease and atherosclerosis can be detected through bacterial presence at the diseased sites detected by real-time polymerase chain reaction (PCR) 10 .
Therefore, the aim of the present study was to detect the presence of Tf in subgingival plaques and atheromatous plaques obtained from different blood vessels.
Patients were recruited only if they were with at least four periodontal pockets. Periodontitis was diagnosed if a patient exhibited clinical attachment level (CAL) > 1 mm and periodontal pocket depth (PPD) > 3 mm, at least at three sites in two different quadrants. According to CAL, patients with diagnosed periodontitis were classified into three subgroups: patients with moderate chronic periodontitis (CP) (CAL = 3-4 mm) and severe CP (CAL ≥ 5 mm). Periodontitis was defined as localized or generalized depending on the number of affected sites 11 .
Exclusion criteria were: systemic diseases, antibiotic intake and periodontal treatment in the previous three months. The medical and dental history of each subject was obtained by interview. Smoking and alcohol use were detected by self-report. Patients fulfilling the inclusion criteria were informed of the study and signed informed consent form that was approved by the Ethics Committee of the Faculty of Medicine in Kosovska Mitrovica, Serbia.

Subgingival and atheromatous plaque sample
On the same day of the surgical intervention, a complete periodontal examination was performed by a single periodontist. Clinical examinations included plaque index (PI) (according to Silness Lӧu), gingival index (GI) (according to Lӧe Silnes), sulcus bleeding index (according to Mühleman-Son) and periodontal probing depth (PPD). Subgingival plaque samples were collected using the paper point technique (Periopaper, Amityville, Pro Flow, NY, USA) from the bottom of two out of four present periodontal pockets. Each sample site was isolated with coton rolls, scalled carefully supragingivaly, and air dried. A sterile paper point was inserted into the apical extent of each selected pocket, kept for 60 seconds, and transfered immediatelly to a sterile Eppendorf tube and kept in the refrigerator on -70°C until the analysis. Samples from the arteries were taken during the surgery (endarterectomy, PTCA) and transfered immediatelly to a sterile Eppendorf tube with Tris-EDTA, as transport medium, and kept in the refrigerator on -20° C until the analysis by PCR method.

PCR analysis
Presence of Tf in periodontal pockets and atherosclerotic vessels was detected using PCR with positive control -Tf American Type Culture Collection (ATCC) 43037. The negative control was sterile distilled water instead of template DNA. The positive control consisted of DNA from pure cultures of Tf ATCC 43037. Colonies obtained from cultures were suspended in sterile water, centrifuged and subjected to DNA extraction.

Statistical analysis
Continuous variables were presented as means ± standard deviations (SD) or median and range and categorical variables were expressed as absolute and relative frequencies. The normality of the data distribution was confirmed by the Shapiro-Wilk tests. The Mann-Whitney U-test and Kruskal-Wallis test were used to compare differences between two groups and for three or more groups, respectively. Categorical data between study groups were analyzed using the chi-squared (χ 2 ) test. Results were considered statistically significant when p-values were less than 0.05. Statistical analysis was performed with the Statistical Package for the Social Science Program (version 22, SPSS Inc., Chicago, IL, USA).

Results
In subgingival plaque samples, Tf was detected in 68 (75.3%) of the patients, while in atherosclerotic plaque samples, Tf was present in 48 (53.3%) of the patients (Table 1). In subgingival and atheromatous plaque samples of patients with atherosclerotic carotid arteries, Tf was detected in 79.3% and 58.6% of the patients, respectively. In case of patients with a. abdominalis aneurisms, Tf was present in sublingval and aterosclerotic plaque samples of 80% and 60% of the patients, respectively. In patients with atherosclerotic a. femoralis, Tf was present in subgingival plaques of 70% of the patients, while in atheromatous plaques of this blood vessel only in 10% of the patients. At the same time, Tf was present in subgingival plaques in 75% of patients with atherosclerosis of a. iliaca, while no Tf was present in the a. iliaca plaque samples. On the contrary, Tf was isolated in subgingival plaques of 79.3% of patients with atherosclerotic coronary arteries, and also in atherosclerotic plaque samples of 69% of these patients. In atheroslerotic and subgingival plaque samples of patients with atherosclerotic mammary arteries, Tf was found in both cases in 50% of the patients ( Table 2).
Among patients with atheromatous plaque positive on Tf, 8.3% were with moderate and 91.7% with severe gingival inflammation, which was statistically significant difference in relation to patients with no presence of the bacterium in atheromatous plaques. Further, among patients with positive atheromatous plaque on Tf, 22.9% and 77.1% had moderate and severe form of periodontal disease, respectively, which was also statistically significant difference in relation to patients with no presence of Tf in atheromatous plaques (Table 3).   Presence of Tf was also analyzed in relation to sociodemographic characteristics of the patients. According to our results, age, gender, level of education and presence of bad habits (smoking and alcohol use) were not statistically significant regarding presence of Tf (Table 4).

SD -standard deviation.
Tf demonstrated the following distribution in blood vessels of the patients with atherosclerosis: the highest prevalence was in coronary arteries (41.7%), followed by carotid arteries (35.4%) and aneurisms of a. abdominalis (12.5%), a. mammaria (8.3%) and a. femoralis (2.1%) while in a. iliaca Tf was not detected at all (Table 5). There were no statistically significant differences in values of PI, GI, sulcus bleeding index and PPD among patients with or with no presence of Tf in atheromatous plaques of different blood vessels (Table 6).

Discussion
Periodontal disease represents chronic inflammation in tooth supportive tissues (periodontal ligament, connective tissue and alveolar bone), that, if left untreated, leads to periodontal pocket formation and consequent bone loss. It is unclear which pathogens initiate the disease, but several species including anaerobic Gram negative bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia are most strongly associated with the destruction of periodontium, and are routinely found in subgingival plaques in patients with chronic periodontitis 12 . To date, many researches are pointing on correlation between periodontal disease and systemic health. It has been suggested that periodontitisassociated bacteraemias and systemic dissemination of inflammatory mediators produced in the periodontal tissues may cause a systemic inflammation. Therefore, it is consider that periodontal disease can contribute as a risk factor for cardiovascular diseases, endocrine disturbances (e.g. diabetes mellitus), premature birth and low weight on birth, etc. 13,14 . Atherosclerosis, a progressive disease of medium and large elastic and muscular arteries can lead to ischemic lesions of brain, heart or extremities and can result in thrombosis and infarction of affected vessels 4 . It is considered the primary cause of heart disease and stroke and is the underlying cause of around 50% of all deaths in western societies 15 .
Among 90 patients included in this study, 61 of them (67.8%) were males, which make a prevalence of periodontal disease and atherosclerosis higher in men. Patients' mean age was 59.2 years. That is in correlation with common understanding of periodontal disease progress. Even thought, the age itself is not predetermining risk factor for periodontal disease 16 due to lower number of elastic and collagen fibers, and mitotic activity of fibroblasts is usually seen in adults over 40 years. Maybe the reason for the presence of periodontal disease in older persons can be partially explain by association between poor oral hygiene habits and systemic diseases [17][18][19] .
Presence of periopathogen Tf in atherosclerotic blood vessels showed a significant correlation in regards to degree of periodontal inflammation. Consequently, prevalence of Tf was statistically significantly higher in patients with moderate and severe periodontal disease when compared to patients with average PPD. Presence of Tf strongly correlated with level of periodontal inflammation and BOP.
Results from this study are pointing on strong correlation between periopathogens and their presence in atherosclerotic plaques, which is in accordance with many published studies on this topic [20][21][22][23][24] . So far, studies confirmed the presence of Tf in 30˗61.9% patients with atherosclerotic a. carotis 24,25 , and in 86% of patients with a. abdominalis aneurism 26 . In our study, among 90 patients Tf was detected with highest prevalence in coronary arteries (41.7%) and carotid arteries (35.4%), and a. abdominalis aneurisms (12.5%), followed by a. mammaris (8.3%) and a. femoralis (2.1%), while in a. iliaca Tf was not detected at all. Therefore, our results are pointing that most probably the periopathogens after entering the systemic circulation are located in the circulation nearby heart blood vessels. Tf presence in far away blood vessels are less, but not unimportant.

Conclusion
The present study suggests strong relationship between periodontal inflammation and atherogenesis, therefore it should be considered as potential risk factor for arherosclerosis. It is, however necessary to control periodontal disease in order to reduce mortality and morbidity associated with atherosclerosis and myocardial infarction.