Abstract
Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum–periodonticum–simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60–100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0 %, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum–periodonticum–simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.
Introduction
Atherosclerosis has been defined as a progressive process that causes focal thickening of large- to medium-sized muscular and large elastic arteries (Pucar et al., 2007). In addition, several risk factors have been associated with its progression, such as hypertension, hypercholesterolaemia, diabetes, marked obesity, smoking, physical inactivity and chronic bacterial infection (Taylor-Robinson et al., 2002; Pucar et al., 2007). The development of atheromatous plaques seems to be relevant to cardiovascular disease as a result of endothelial cell damage and by maintenance of the inflammatory reactions in the walls of blood vessels (Libby et al., 2002).Studies have demonstrated a close association between cardiovascular disease and periodontitis, and oral hygiene and periodontal status are closely related to the occurrence of heart attacks (Kozarov et al., 2006; Aimetti et al., 2007; Johansson et al., 2008). A relationship between periodontal status and cardiovascular diseases has been suggested, where the periodontium represents the source of inflammatory mediators, in addition to micro-organisms systemically disseminated by the blood flow (Li et al., 2000).
Considerable evidence supports a plausible set of mechanisms by which periodontopathic bacteria may directly or indirectly contribute to cardiovascular disease, such as blood platelet aggregation, enhanced low-density cholesterol and lipoprotein deposition in the artery walls, invasion of cardiac and carotid endothelium, and the high level of inflammatory mediators in the circulation and tissues (Dorn et al., 1999; Sharma et al., 2000; Libby et al., 2002). However, evidence that periodontal infections contribute to or are decisive factors in the development of atherosclerotic plaques is circumstantial, and an epidemiological association is not proof of a causal link between pathogens and cardiovascular disease, although bacterial presence at the diseased site is one of the requirements to determine a causal relationship (Cairo et al., 2008).
As a result of the high sensitivity of PCR and other molecular methods, the presence of micro-organisms inadvertently introduced into the blood flow or as a result of accidental contamination during endarterectomy may be confused with the presence of these pathogens within atheromatous plaques. However, with the advent of real-time PCR, it is possible to quantify the microbial DNA in order to differentiate the transient presence of micro-organisms colonizing and infecting the vascular walls and atheromatous lesions.
In recent years, studies have implicated Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Prevotella nigrescens, Porphyromonas gingivalis and Tannerella forsythia in connective tissue attachment loss and periodontal inflammation (Ebersole et al., 2008; Herrera et al., 2008), whilst Fusobacterium nucleatum represents a bridge between the first and the late colonizers of oral subgingival biofilms (Kolenbrander, 2000). In this study, the presence of these micro-organisms in atheromatous plaques obtained from coronary arteries from patients with chronic periodontitis and from periodontally healthy subjects was determined.
Methods
Patients.. Forty-four adult patients (35 males and 9 females), from 36 to 82 years old (mean age 60±11.3 years), displaying cardiovascular disease and seen at the Evangelic Hospital of Londrina (Londrina, Paraná, Brazil) and Hospital de São José do Rio Preto (São José do Rio Preto, SP, Brazil), were evaluated. Clinical samples were collected from March 2005 to December 2007. Patients fulfilling the inclusion criteria were informed of the study and signed an informed consent form that was approved by the Ethics Committee in Research of the University of São Paulo (no. 270/02).Initially, before endarterectomy of coronary arteries, all patients underwent a clinical interview in order to obtain information about their identification, age, disease history, and medical and familial histories. Patients were submitted to a complete periodontal examination by a single periodontist, collecting data on tooth loss, plaque index (full-mouth plaque score), bleeding on probing (full-mouth bleeding score), probing depth, gingival recession and clinical attachment level at six sites (mesial, mid- and distal sites of oral and facial surfaces) per tooth, excluding third molars, using a manual periodontal probe (PCP UNC-15; Hu-Friedy).
The periodontal conditions of the patients are presented in Table 1: 39 patients displayed generalized chronic periodontitis with attachment loss exceeding 5 mm in ≥30 % of the periodontal sites, whilst five patients were periodontally healthy (Tonetti & Claffey, 2005). Exclusion criteria included self-medication history, diabetes, autoimmune disease or other systemic pathology, and any periodontal or antibiotic therapy during the last 3 months.
Table 1. Clinical parameters at baseline in patients with chronic periodontitis and periodontally healthy subjects submitted to endarterectomy of coronary arteries Values are means±SD.
All patients submitted to endarterectomy presented diffuse atherosclerotic disease and multiple stenoses with distal and diffuse involvement with reversible ischaemia, documented by scintigraphy and analysis of echocardiography, under conditions considered inoperable by conventional methods, or advanced atherosclerotic disease with viable myocardium.
Atheromatous plaque sample collection.. Atheromatous plaques from coronary arteries were removed surgically and placed in vials containing 10 ml sterile DNA-free saline solution and stored at –20 °C. Only atheromatous plaques were removed and processed in order to avoid damage to vascular walls. A sagittal section was made through the middle of the atherosclerotic plaque. Approximately 100 mg tissue was placed in a vial containing 5 ml RNAlater (Ambion; Applied Biosystems) for DNA extraction by using a Charge Switch gDNA Mini Tissue kit (Invitrogen) according to the manufacturer's instructions. DNA was stored at –20 °C until amplification by real-time PCR. Purified genomic DNAs from A. actinomycetemcomitans ATCC 29523, F. nucleatum ATCC 10953, Porphyromonas gingivalis ATCC 33277, Prevotella intermedia ATCC 25611 and T. forsythia ATCC 43037 were used as positive controls. DNA concentrations were determined spectrophotometrically by measuring the A260 (model DU-640; Beckman Instruments).
Quantitative analysis by real-time-PCR.. Real-time PCR assays were carried out using a Rotor Gene 6000 (Corbett Life Science). For amplification reactions, duplicate samples were routinely used and assays were performed in a total volume of 25 µl containing 12.5 µl 2x Taqman Universal Master Mix (Applied Biosystems), 0.2 µl each forward and reverse primer (final concentration 200 nM each), 0.1 µl Taqman probe (final concentration 100 nM), 2 µl template DNA solution and an appropriate volume of sterilized DNase- and RNase-free water. Amplification reactions were performed in a thermocycler programmed as follows. For detection of total bacterial DNA for A. actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and F. nucleatum, an initial denaturation at 95 °C for 10 min was followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. For T. forsythia, an initial denaturation at 95 °C for 10 min was followed by 45 cycles at 95 °C for 15 s and 55 °C for 1 min. The negative control was a PCR TaqMan Master Mix without DNA. The primer/probe sets used are shown in Table 2.
Table 2. Specific primers and probes used in the real-time PCR
The total number of bacterial cells was also determined using the TaqMan PCR procedure with a universal primer/probe set. The standard curve was analysed for each evaluated bacterium and using the universal primer/probe set against a serial dilution of each bacterial DNA corresponding to 102–107 cells, and showed an error value range of 0.0288–0.0695. A sample was considered positive for a target micro-organism when the fluorescence emitted by the sample was at least 50 % higher than the limit of detection previously established for the micro-organism and 50 % above the background fluorescence.
fimA genotyping for Porphyromonas gingivalis... Samples of atheromatous plaques were assayed to detect fimA genotypes for Porphyromonas gingivalis. The tests were performed basically as described previously by Amano et al. (1999) and Nakagawa et al. (2000, 2002b). Table 3 lists the PCR primers used. A ubiquitous primer set that matches almost all bacterial 16S rRNA genes was used as a positive control, and Porphyromonas gingivalis species-specific primers (16S rRNA gene-specific) were used for fimA typing. All primers were custom-made by Invitrogen.
Table 3. Primer sets used for Porphyromonas gingivalis fimA genotyping
PCR amplification was performed in a volume of 25 µl containing 1x PCR/Mg2+ buffer (Boehring Mannheim), 0.2 mM each dNTP (Pharmacia Biotech), 0.5 U Taq DNA polymerase (Invitrogen), 0.4 µM each primer pair (Invitrogen) and 10 ng template. Amplification was performed in a DNA thermal cycler (GeneAmp PCR System 9700; Perkin Elmer) at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min, with a final extension at 72 °C for 7 min. Amplification products were analysed by electrophoresis in 1 % agarose gels in 1x TBE buffer [1 M Tris/HCl (pH 8.4), 0.9 M boric acid, 0.01 M EDTA; Invitrogen], stained with 0.5 mg ethidium bromide ml–1.
Statistical analysis.. Mean values±SD were calculated for each bacterial species and for total eubacteria. Statistical analyses were performed using the software SPSS, version 13. Differences between clinical and microbiological qualitative parameters were evaluated using a Mann–Whitney, χ2 or Fisher's exact test. For evaluation of the quantitative microbial parameters, data obtained from patients harbouring these bacteria were also computed. A difference of P <0.05 was considered statistically significant.
Results And Discussion
By using the universal and specific primer/probe sets, total bacterial DNA and periodontopathic bacteria DNA were detected in 94.9 and 92.3 %, respectively, of atherosclerotic samples from patients with periodontitis. DNA of the targeted periodontal bacteria represented a mean of 47.3 % of the total bacterial DNA observed in atheromas, and Porphyromonas gingivalis DNA comprised 18.8 % of the microbial DNA in these samples (Table 4), whilst fusobacteria were not detected. Bacterial DNA was found in 80 % of atheromatous plaques recovered from periodontally healthy subjects, and Porphyromonas gingivalis was the only targeted micro-organism detected in these atheroma samples from healthy subjects (20 %).Table 4. Bacterial distribution in atheromatous plaques from 44 patients submitted to endarterectomy of coronary arteries
Prevotella intermedia (59.0 %), Porphyromonas gingivalis (53.8 %) and A. actinomycetemcomitans (46.2 %) were the most prevalent bacteria, followed by T. forsythia (25.6 %) and Prevotella nigrescens (17.9 %), in the atheromas from patients with periodontitis (Table 4). Moreover, in most of the samples, the presence of DNA from more than one periodontal pathogen was observed, particularly A. actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia (Table 5). The concomitant detection of two or more species was observed in 64.1 % of the clinical samples from periodontitis patients, whilst the presence of a single species was detected in 28.2 % of the samples.
Table 5. Bacterial associations in 44 atheromatous plaques obtained from coronary arteries
The presence of these periodontopathic bacteria in atherosclerotic plaques was not related to age (P=0.188), sex (P=0.633), number of teeth (P=0.352) or tobacco use (P=0.082) (all using Fisher's exact test). The presence of DNA of the targeted micro-organisms (mean±SD) is shown in Fig. 1. In our study, Porphyromonas gingivalis was detected in the highest numbers, followed by Prevotella intermedia and A. actinomycetemcomitans. The DNA levels of T. forsythia and Prevotella nigrescens were similar (P=0.171).
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The presence of periodontopathic bacteria in atheromatous plaques from coronary arteries is associated with the concept that periodontal bacteria such as A. actinomycetemcomitans and especially Porphyromonas gingivalis are able to invade endothelial cells and induce a chronic vascular inflammation (Yuan et al., 2008). Moreover, A. actinomycetemcomitans, F. nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and other oral anaerobes and microaerophiles can degrade immunoglobulin, preventing the action of the complement system, and can produce some toxic components such as endotoxins and exotoxins (Fives-Taylor et al., 1999; Brook, 2007). These factors may help micro-organisms to maintain their viability during bacteraemia, which occurs frequently as a result of surgical procedures, tooth brushing and other dental procedures (Dorn et al., 1999; Daly et al., 2001).
Studies have associated the presence of Porphyromonas gingivalis, A. actinomycetemcomitans, T. forsythia and Prevotella intermedia DNA in atheromatous plaques with the severity of periodontal disease, increasing with the age of the patients (Haraszthy et al., 2000; Pucar et al., 2007). In the present study, the prevalence of bacterial DNA in atheromas was higher than that reported by Ishihara et al. (2004) and Padilla et al. (2006), but similar results were reported by Lehtiniemi et al. (2005) and Kozarov et al. (2006) using a quantitative SYBR Green PCR assay. Bacterial DNA was observed in 94.9 % of atheromas from periodontitis patients and in 80.0 % of atheromas from periodontally healthy subjects. This result is in agreement with the results of Fiehn et al. (2005) and Aimetti et al. (2007), who reported the presence of bacterial DNA in 100 and 94 %, respectively, of the atheromas analysed. Moreover, the target periodontopathic bacterial DNA evaluated here represented 47.3 % of the total bacterial DNA found in atheromatous samples from periodontitis patients and 7.2 % of the DNA detected in atheromas from periodontally healthy subjects.
Due to the sensitivity of PCR for detecting microbial DNA, doubts have been raised about sample contamination as a result of bacteraemia or handling of samples (Sanz et al., 2004). However, because of the high level of microbial DNA observed in most of the atheromatous plaques, it is unlikely that these patients suffered accidental contamination; instead, it suggests a stable and lasting microbial colonization of atheromas. It is well known that Gram-negative anaerobes can evade the immune system and that they possess proteolytic activity associated with acute and suppurative infections, particularly abscesses and cellulitis (Brook, 2008), but it has not been observed in tissue removed during endarterectomy.
The high prevalence of Porphyromonas gingivalis, Prevotella intermedia and A. actinomycetemcomitans may reflect the periodontal condition of the patients, as most of the infected atheromas were obtained from chronic periodontitis patients and these micro-organisms are highly prevalent in patients with periodontal pockets, subgingival plaque, insertion loss, periodontal bone loss and inflammation (Ebersole et al., 2008; Herrera et al., 2008).
Genotyping of the gene fimA was performed for the 21 atheromatous samples that were positive for Porphyromonas gingivalis, and this gene was detected in the atheromas from these patients. The genotype fimA II was detected in 11 (52.4 %) of the atheromatous plaques from patients with periodontitis and in only one clinical sample obtained from a periodontally healthy subject, whereas fimA IV was detected in six (28.6 %) of the atheromas. Interestingly, genotype fimA V, which is considered to be relatively rare in patients with chronic periodontitis (Nakagawa et al., 2002b; Miura et al., 2005; Enersen et al., 2008), was observed in four (19.0 %) of the atheromatous plaques. In addition, a recent study suggested the involvement of genotypes fimA II and fimA IV in the initiation and progression of cardiovascular diseases (Nakano et al., 2008). Moreover, both fimA genotypes also represent those most commonly observed in periodontitis (Miura et al., 2005; Zhao et al., 2007; Enersen et al., 2008). Theoretically, the genotypes of the fimA gene show different responses to bacterial virulence and to periodontal treatment. As Porphyromonas gingivalis genotype fimA II shows an increased capacity to adhere to and invade human epithelial cells (Nakagawa et al., 2002a) and to colonize the gingival crevice (van der Ploeg et al., 2004), the significant occurrence of this genotype in atheromatous plaques may reflect these differences in virulence, although this hypothesis remains to be evaluated.
The predominance of these three micro-organisms in the clinical samples may be due to their ability to persist in vascular tissue in which latent intracellular bacteria are transformed to a viable state (Li et al., 2008). In addition, periodontal patients harbouring Porphyromonas gingivalis have shown higher levels of lipids, low-density lipoprotein and total cholesterol in the bloodstream, which seem to be associated with the development of atheromatous plaques (Cutler et al., 1999; Cairo et al., 2008).
The development of atheromas induces fibrosis, as well as cholesterol and lipoprotein deposition, producing a reduced redox potential suitable for the development of anaerobic bacteria such as Porphyromonas gingivalis, T. forsythia and Prevotella intermedia, which are considered to be the most prevalent oral pathogens in atherosclerotic plaques (Fiehn et al., 2005). On the other hand, the high prevalence of Gram-negative oral bacteria in atherosclerotic tissues may induce the secretion of several cytokines associated with the development of cardiovascular diseases by their LPS (Fiehn et al., 2005).
Studies in elderly patients with chronic periodontitis have shown high detection rates of periodontopathic bacteria in atheromatous plaques; in contrast, in young patients, these bacteria are rarely present (Kozarov et al., 2006). The difference observed in the literature on the detection of periodontopathic bacteria from atherosclerotic plaques may be explained by the methodologies employed, periodontal status, bacterial biofilm composition, socio-economic and educational levels, and the ethnic characteristics of the populations examined (Fiehn et al., 2005).
It is important to note that the presence of DNA from more than one periodontal species in atheromas may be relevant in the progression of atherosclerosis, as immunological data support the suggestion that association of three or more different micro-organisms is epidemiologically implicated in the pathogenesis of atherosclerosis and may lead to myocardial infarction (Haheim et al., 2008). Our results suggest that microbial infections in the coronary arteries are essentially mixed, as 64.1 % of the clinical samples from periodontitis patients showed the presence of at least two different periodontopathic bacteria in the atherosclerotic samples.
As most infections associated with oral micro-organisms are mixed, with different species establishing ecological relationships with each other and with the host, it is possible that the mixed contamination detected in 64.1 % of atheromatous plaques from patients with periodontitis may reflect the complex ecological interactions that these organisms maintain in the oral cavity (Kolenbrander, 2000; Brook, 2007).
On the other hand, asymptomatic bacteraemia produced by periodontopathic bacteria may accelerate the progression of atheromatous plaques (Ishihara et al., 2004). Thus, taken together with our results, this suggests that periodontal disease-associated or non-associated bacteria reach the bloodstream, playing a direct or indirect role in the pathogenesis of cardiovascular diseases. The results shown here reinforce the importance of periodontal bacteria as a possible contributing factor in the development of cardiovascular diseases. Knowledge of the micro-organisms present in atheromas from patients with chronic periodontitis is relevant in the prevention and treatment of cardiovascular infections that appear to be produced, in part, by these oral periodontopathic bacteria.
Acknowledgements
This study was supported by grants from the Fundação do Amparo à Pesquisa do Estado de São Paulo (FAPESP) Proc. No. 04/03199-3 and 07/51016-3.References
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