|Year : 2012 | Volume
| Issue : 4 | Page : 462-466
16S rDNA-based metagenomic analysis of human oral plaque microbiota in patients with atherosclerosis and healthy controls
F Ismail1, C Baetzner1, W Heuer1, N Stumpp1, J Eberhard1, A Winkel1, I Ismail2, A Haverich2, M Stiesch1
1 Department of Prosthetic Dentistry and Biomedical Material Sciences, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
2 Department of Cardiac-, Thoracic-, Transplantation- and Vascular Surgery, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
|Date of Submission||16-Apr-2012|
|Date of Acceptance||21-Jul-2012|
|Date of Web Publication||24-Nov-2012|
Department of Prosthetic Dentistry and Biomedical Material Sciences, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover
Source of Support: None, Conflict of Interest: None
To address the question if an altered oral microbiota is associated with atherosclerosis. Twenty patients suffering from atherosclerosis and 10 controls were recruited. Clinical oral, medical and laboratory investigations were performed. Oral bacteria were collected and 16S rDNA was sequenced following Single strand conformation polymorphism.(SSCP) Probing pocket depths in patients were significantly elevated. The oral microbiota of patients and controls were dominated by Fusobacterium (16%/17%), Streptococcus (21%/14%), Prevotella (10%/12%), Enterococcus (12%/12%), Porphyromonas (8%/7%), TM7 (0%/7%) and Veillonella (6%/7%). Differences in diversity were not significant between groups.
The pathology of atherosclerosis may not be related to significant qualitative changes of the oral microbiota.
Keywords: Atherosclerosis, oral microbiota
|How to cite this article:|
Ismail F, Baetzner C, Heuer W, Stumpp N, Eberhard J, Winkel A, Ismail I, Haverich A, Stiesch M. 16S rDNA-based metagenomic analysis of human oral plaque microbiota in patients with atherosclerosis and healthy controls. Indian J Med Microbiol 2012;30:462-6
|How to cite this URL:|
Ismail F, Baetzner C, Heuer W, Stumpp N, Eberhard J, Winkel A, Ismail I, Haverich A, Stiesch M. 16S rDNA-based metagenomic analysis of human oral plaque microbiota in patients with atherosclerosis and healthy controls. Indian J Med Microbiol [serial online] 2012 [cited 2020 Oct 29];30:462-6. Available from: https://www.ijmm.org/text.asp?2012/30/4/462/103771
| ~ Introduction|| |
Atherosclerosis is the main pathological event of coronary heart and peripheral vascular diseases. Local inflammatory processes of the oral cavity and periodontal diseases in particular are well known as risk factors for these systemic diseases like coronary heart and peripheral vascular diseases.  Periodontal diseases are characterised by inflammatory processes of the tissues surrounding the teeth in response to bacterial accumulation that is, if untreated, followed by tooth loss.  Approximately 30% of adults in the United States are affected by moderate forms of periodontitis and 10% of the population is affected by severe forms of this disease.  Especially in patients suffering from periodontitis, the transmission of bacteria into blood is a prevalent event, because of the frequent bleeding events of the gingival tissues loaded with bacteria.  Recently, the human oral and gut microbiota was analysed by pyrosequencing of 16S rRNA (ribosomal ribonucleic acid) in patients suffering from atherosclerosis, showing that the oral cavity and even the gut may be a reservoir for bacteria found in atherosclerotic plaques.  These studies are essential given the microbiology of atherosclerotic plaques and the important effects of periodontitis for the initiation of atherosclerosis. However, these studies lack any information about the differences of the oral microbiota in patients suffering from atherosclerosis compared to healthy subjects addressing the question if qualitative changes of the oral microbiota are a predisposing factor for atherosclerosis.
Here, we characterised the oral microbiota obtained from patients with atherosclerosis and healthy controls by 16S rRNA-based metagenomics. Our study addressed the question if an altered oral microbiota is associated with atherosclerosis compared to healthy subjects with no atherosclerosis, including the simultaneous recording of selected periodontal health parameters.
| ~ Materials and Methods|| |
A total of 30 patients with no history of diabetes mellitus were recruited at a University-based hospital. Twenty patients with proven cardiovascular disease were treated at the Department of Cardiothoracic-, Transplantation- and Vascular Surgery of the Hannover Medical School between 2007 and June 2009. In addition, a group of 10 healthy age-matched control subjects were recruited from the Department of Prosthetic Dentistry and Biomedical Materials Science. Informed consent was obtained from all patients and the local Ethics Committee approved the study (no. 5253).
Patients were classified into the atherosclerotic lesion group with an angiographically documented multivessel coronary disease (stenosis ≥ 70%) suffering from severe angina pectoris symptoms (Canadian Cardiovascular Society class II) or evidence of ischemia requiring revascularisation by coronary-artery bypass grafting or with atherosclerotic disease involving the carotid or peripheral vessels documented by Doppler or Angio-CT (computer tomography) with ≥ to 70% stenosis. The severe disease status of all patients had required the respective surgical intervention procedure. The sampling of the oral bacterial deposits was performed approximately 2 to 5 days before the respective surgery was scheduled; the patients have used their medications for at least 4 months. Healthy controls showed no history of systemic diseases according to their medical history. In the patients and control groups, no participant had received antibiotics or any periodontal therapy during the last 6 months.
Clinical oral examination and sampling of oral bacteria
All subjects underwent a clinical oral examination conducted by one examiner (F.I.). Probing depth (PD) was recorded at four sites at the reference teeth 16, 11, 21, 26, 36, 31, 41 and 46. Plaque samples were collected using sterile filter papers from four sites with the deepest probing pocket depth. Prior to sampling, the tooth surfaces were gently dried by an air syringe and the teeth were isolated by cotton rolls. Paper points were carefully placed in the gingival sulcus to collect plaque and were placed into a 1.5-ml Eppendorf tube and kept at-80°C until analysis. Paper points that were contaminated with blood were discarded.
Clinical medical examination and laboratory assay
Baseline recordings consisted of an extensive medical history including parameters to evaluate the occurrence of coronary or peripheral artery vascular diseases, systemic embolic events and smoking status.
During routine medical investigations, venous blood was sampled from the cubital vein of each subject. The whole blood samples were centrifuged (2 000 rpm for 2 minutes) and serum was stored at-70°C in Eppendorf tubes until future processing. The quantitative measurement of C-reactive protein (hsCRP) in serum was performed using a turbidimetric assay that was used according to the manufacturer's instructions (CRP-Dynamic HIT, invicon).
DNA isolation, amplification and single strand conformation polymorphism analysis
Total genomic DNA (deoxyribonucleic acid) was isolated using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol for Gram-positive bacteria. A mechanical disruption step with a Precellys 24 bead mill (Bertin Technologies) was performed prior to spin-column DNA extraction. An approximately 500 bp fragment of the 16S rDNA was amplified using the universal primers 27f and the 5′-prime phosphorylated 521revP. PCR (polymerase chain reaction) was conducted on a TProfessional thermocycler (Biometra) following a 32-cycle standard PCR protocol. Amplicons were purified over silica spin-columns (QIAquick PCR Purification kit; Qiagen) and dried overnight at 40°C.
Single strand conformation polymorphism (SSCP) analyses were performed on a DCode Universal Mutation Detection System (Bio-Rad). The DNA fragments were electrophoretically separated on a 10% polyacrylamide gel at 350V (20°C) for 24 hours in 1x TBE buffer (Bio-Rad). DNA bands were visualised by silver-staining (Silver-Stain kit; Bio-Rad). Bands were excised and DNA was extracted and reamplified. PCR products were purified and subsequently sequenced (Seqlab). Obtained sequences were processed using the BioEdit software package (v7.0.9, Ibis Biosciences) and compared to GenBank database sequences from the National Center for Biotechnology Information (NCBI). For identification of the closest match, the NCBI Basic Local Alignment Search Tool as well as the SEQMATCH and CLASSIFIER tools from the Ribosomal Database Project were used. The 16S rDNA banding pattern of each sample was analysed using the Quantity One 1D-Analysis Software package (v4.6.5, Bio-Rad).
Mean and standard deviations were calculated for all clinical and laboratory parameters with the patient as the statistical unit. Only bacterial genera were included in the analyses that were found at least 2 times in all groups. The diversity-index represents the number of individual bands detected in polyacrylamide gels per patient. Statistical evaluation according to the number of bands per sample (diversity-index) was performed as described using the analysis software SPSS (v17.0, SPSS Inc.). For the comparison of intergroup differences, the Wilcoxon signed-rank test was used. Differences were reported significant for a P value ≤ 0.05.
| ~ Results|| |
Clinical and laboratory data
A total of 20 patients (age: 61.0 ± 8.4 years, 17 males, 10 current smokers) suffering from atherosclerosis of the coronary or peripheral vessels and 10 healthy controls (age: 55.4 ± 11.3 years, 6 males, 7 current smokers) were enrolled. The clinical and laboratory data are presented in [Table 1]. Compared to the healthy controls, the Body mass index (BMI) was significantly elevated in the atherosclerotic patient group (P = 0.038) and hsCRP concentrations of 9.2 ± 11.2 ng/ml were recorded in the atherosclerotic group. Evaluation of the oral parameters showed significant elevated PDs in the atherosclerotic group (3.2 ± 1.2 mm) compared to the control group (2.1 ± 0.8 mm; P = 0.007). Five patients of the atherosclerotic group showed PD ≥ 4 mm at least at four sites that did not belong to the same tooth. [Table 1] also presents the most frequently documented medications within the patient group that were ACE-inhibitors (angiotensin-converting-enzyme), acetylsalicylic acid and beta-blockers.
Composition of the oral microbiota in patients and healthy controls
The composition of the oral bacterial microbiota of patients [Figure 1]a detected by SSCP analysis was dominated by the genera Streptococcus (21%), Fusobacterium (16%), Enterococcus (12%), Prevotella (10%), Porphyromonas (8%) and Veillonella (6%). The oral microbiota of the healthy controls [Figure 1]c was dominated by the genera Fusobacterium (17%), Streptococcus (14%), Prevotella (12%), Enterococcus (12%), Porphyromonas (7%) TM7 (7%) and Veillonella (7%). The oral plaques of patients with atherosclerosis and increased PD [Figure 1]b were dominated by Fusobacterium (24%), Streptococcus (14%), Prevotella (10%), Enterococcus (14%), Porphyromonas (14%), Leptotrichia (10%) and Veillonella (10%). The genera Gemella and Haemophilus were found in three patients with atherosclerosis and no periodontal diseases and not once in the healthy controls. The genus TM7 was detected solely in the healthy control group. The distribution of bacterial genera within the patient and control groups did not reveal any genera that could discriminate between the atherosclerotic patient and the healthy control.
|Figure 1: The frequencies of the genera for (a) patients suffering from atherosclerosis (b) patients with probing pocket depth ≥ 4 mm and (c) healthy controls are depicted|
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Comparison of diversity of oral microbiota
A total of 35 different genera were found within the investigated groups. The highest number of different genera was found in the atherosclerosis group (n = 21) and the lowest number in the atherosclerosis group with increased PDs (n = 8). Twenty different genera were found in the control group. The diversity indices for patient (4.2 ± 0.8) and control groups (4.0 ± 0.6) are depicted in [Figure 2] and the statistical analysis revealed no significant differences between the two groups.
|Figure 2: The diversity-index for the patient and control group showed no significant differences. Means and standard deviations were calculated and tested for significant differences by the Wilcoxon signed-rank test (P ≤ 0.05)|
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| ~ Discussion|| |
Here, we used 16S rDNA-based metagenomics to evaluate the bacterial composition of dominant bacteria of the oral cavity in patients with atherosclerosis and healthy control subjects. This approach allowed a relatively comprehensive description of microbial communities associated with atherosclerosis and health. We found that oral plaque microbiota and the intergroup diversity are similar in patients suffering from atherosclerosis and in healthy controls represented by a core of genera highly diverse and variable between individuals. A subgroup analysis revealed significant lower diversity in patients with atherosclerosis in addition to increased probing pocket depths, which is an indicator for moderate periodontal diseases. The distribution of genera in patients and controls indicates that substantial qualitative changes to the oral microbiota may not be relevant for the development of atherosclerosis.
The study population included three traditional risk factors for cardiovascular diseases, increased BMI, high hsCRP levels and chronic infection.  For the present study, no patients with diabetes mellitus were included, because compared with non-diabetic patients, diabetic patients suffer from an increased incidence of infections and showed significant differences of the bacterial composition in chronic wounds.  HsCRP is a sensitive marker for inflammation and plays a significant role in endothelial dysfunction and elevated serum levels of CRP suggest an increased risk for myocardial infarction. 
The present study describes for the first time the oral microbiota of a patient group suffering from atherosclerosis and a healthy control group using the SSCP technique. In contrast to methods using specific 16S rDNA primers, the SSCP method is aimed to detect diverse dominant bacterial genera or species in a biofilm consortium with a detection limit of approximately 1 000 CFU/ml (colony-forming units/ml) as determined in a series of preliminary experiments (data not presented). To identify bacterial consortia in diverse habitats, a metagenomic approach is helpful, because of the potential identification of bacteria, irrespective of their taxonomical classification, which is not possible by searching for known genera or species. In contrast, this method is limited by the enormous complexity and costs for the necessary experimental procedures. In contrast to 454 pyrosequencing techniques, which are capable to potentially detect even single copies of bacterial DNA, but are also prone to contaminations during the sampling or laboratory procedures, SSCP techniques are capable to detect the dominant genera within a microbial community.
Enterococcus was present in a frequent number of oral plaque samples and may represent a previously unconsidered core member of oral plaque communities. Several genera were detected in few numbers solely in patients (Gemella and Haemophilus) or in healthy controls (TM7); however, their detection could not discriminate between the atherosclerotic patient and the healthy control. Interestingly, the periopathogen Porphyromonas gingivalis was found in nearly equal frequencies in diseased and healthy individuals; however, in patients with increased probing pocket depth, this bacteria was 2-fold increased compared to patients with inconspicuous PD values. These observations are in accordance with the current knowledge of oral biofilm composition in health and disease.  The genera Streptococcus was most frequently found in all patient groups, which is in agreement to several studies demonstrating a correlation between the detection of Streptococcus mutans in the oral cavity and in atheromatous plaques. These observations indicated that certain bacteria, potentially not periopathogens, are prominent pathological factors for the development of atherosclerosis. The presumption that oral bacteria others than periopathogens had impact on systemic diseases is facilitated by recent studies demonstrating a limited number of periopathogens in a comprehensive search for bacteria in atherosclerotic plaques. 
One intriguing observation was the low level of diversity in patients with atherosclerosis and increased probing pocket depths ≥ 4 mm. Thus, the pathobiology of the oral plaque in patients and controls as well as in patients with moderate periodontitis is not similar. A reduction of diversity may be associated with a polymicrobial infection that is characterised by a few key members required for the initiation of disease. This shift of the bacterial diversity is relevant with respect to data showing that a decrease of commensal bacteria is associated with an increased detection of opportunistic bacteria in the oral cavity.  In addition to this subgroup of patients with probing pocket depths ≥ 4 mm, patients suffering from atherosclerosis also showed increased levels of mean probing pocket depths, although no data giving the clinical attachment levels were available. This attribute is indicative for cumulative effects of repeated inflammatory processes associated with swelling of the gums and destructive processes of the tooth supporting structures in response to the accumulation of bacterial deposits on the tooth surfaces. In combination with other risk factors of this population like smoking, high blood pressure and increased BMI, periodontitis may at least in part contribute to the development of atherosclerosis in this population. Poor oral health conditions in patients suffering from coronary heart diseases compared to healthy subjects were shown by Meurman et al. 
In summary, we detected similar core bacterial members of the oral microbiota in patients and healthy controls. The oral cavity is a potential source for atherosclerotic plaque-associated bacteria; however, any correlation between specific bacteria in the oral cavity and atherosclerosis were not found and the pathology of atherosclerosis may not be related to significant qualitative changes in oral microbiota. Instead, an increased number of bacteria, altered virulence or host response may be more relevant mechanisms.
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[Figure 1], [Figure 2]
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