WO2012050513A1

WO2012050513A1 - Method for identifying a risk of cardiovascular disease by analysing oral microbiota

Info

Publication number: WO2012050513A1
Application number: PCT/SE2011/051214
Authority: WO
Inventors: Fredrik BÄCKHED
Original assignee: Baeckhed Fredrik
Priority date: 2010-10-11
Filing date: 2011-10-11
Publication date: 2012-04-19

Abstract

A method is provided for identifying an individual at risk of developing cardiovascular disease such as atherosclerosis and associated diseases, by determining the presence of specific bacterial groups or species in the individual's oral microbiota.

Description

METHOD FOR IDENTIFYING A RISK OF CARDIOVASCULAR DISEASE BY

ANALYSING ORAL MICROBIOTA

FIELD OF THE INVENTION

The present invention relates to a method for identifying an individual at risk of developing cardiovascular diseases, including atherosclerosis and associated diseases, by determining the presence of specific bacterial groups or species in the individual's oral microbiota.

BACKGROUND OF THE INVENTION

Within the body of a healthy adult, microbial cells are estimated to outnumber human cells by a factor of ten to one. These communities, however, remain largely unstudied, leaving almost entirely unknown their influence upon human development, physiology, immunity, nutrition and health.

Traditional microbiology has focused on the study of individual species as isolated units. However many, if not most, have never been successfully isolated as viable specimens for analysis, presumably because their growth is dependent upon a specific rnicroenvironment that has not been, or cannot be, reproduced experimentally. Among those species that have been isolated, analyses of genetic makeup, gene expression patterns, and metabolic physiologies have rarely extended to inter-species interactions or microbe-host interactions. Advances in DNA sequencing technologies have created a new field of research, called metagenomies, allowing comprehensive examination of microbial communities, even those comprised of uncultlvable organisms. Instead of examining the genome of an individual bacterial strain that has been grown in a laboratory, the metagenomic approach allows analysis of genetic material derived from complete microbial communities harvested from natural environments. For example, the gut microbiota complements our own genome with metabolic functions that affects human metabolism and may thus play an important role in health and disease (1-2).

The oral cavity forms an important part of the human microbiome, for its unique and diverse microbiota. More than 700 bacterial species or phylotypes, of which over 50% have not been cultivated, have been detected in the oral cavity.

Atherosclerotic disease, with manifestations such as myocardial infarction and stroke, is the major cause of severe disease and death among subjects with the metabolic syndrome. The disease is caused by accumulation of cholesterol and recruitment of macrophages to the arterial wall and can thus be considered both as a metabolic and inflammatory disease (3). Since the first half of the 19^th century infections have been suggested to cause or promote atherosclerosis by augmenting pro-atheroslerotic changes in vascular cells (4). These include increased scavenger receptor expression and activity, enhanced uptake of cholesterol and modified LDL, increased expression of adhesion molecules and inflammatory cytokines, and other effects such as stimulating macrophages to express cytokines, leading to atherosclerotic plaque vulnerability (4).

Epidemiological studies support an association between cardiovascular disease and infections, e.g. periodontal disease and Chlamydia infections (5-6). Dental health has been associated with elevated risk of myocardial infarction (5) and metabolic activity of the gut microbiota was recently shown to relate to blood pressure (7). Furthermore, in a recent study bacterial DNA was identified in all plaques investigated in patients with coronary heart disease, and 51.5% of the patients tested positive for Chlamydia in their plaque (8). Antibiotic treatment of patients with coronary heart disease has so far been unsuccessful (9-12).

However, these studies do not exclude a role of bacteria in the onset of disease. Several studies suggest an oral source for plaque-associated bacteria (13-15), however, to date no single study has directly compared the microbial diversity of oral, fecal and plaque

microbiotas.

There is a need for better ways to early identify a person having risk for cardiovascular diseases including atherosclerosis and associated diseases.

Several investigators have analyzed bacterial presence, for example in the oral cavity of humans, in relationship to different diseases and conditions. However, it has not previously been demonstrated that the bacterial amounts of specific groups or species in the person's oral microbiota correlates with atherosclerosis and related cardiovascular diseases.

In a clinical study, Karlsson and Ahrne et al administered a drink with a single strain probiotic to patients with atherosclerotic plaques (Atherosclerosis 208(1), 2010). The bacterial diversity in the gut was increased, something that was beneficial for the host. However, this study did not show a link between atherosclerosis and the presence of specific groups or species of bacteria in the oral microbiota.

Previous attempts focused on finding connections between bacteria and

atherosclerosis have mainly investigated single bacterial strains or the aggregate number of pathogens with which an individual is infected, so called "pathogen burden" (Epstein, Zhu et al, Arterioscler Thromb Vase Biol 2000;20:1417-1420). DEFINITIONS

All terms used in the present specification are intended to have the meaning usually given to them in the art. For the sake of clarity, some terms are also defined below.

The term "bacterial group" shall be construed as meaning a group of bacteria belonging to the same genus, family, order, class, or phylum of bacteria. A bacterial group thus includes at least one bacterial species; often several different bacterial species.

The term "abundance" of a bacterial group or species is defined as being an amount of at least 5%, preferably at least 10%, of the total microbial DNA in a body sample from an individual. Such a body sample is taken from the mouth, the gut or atherosclerotic plaques.

Throughout the text, the term "cardiovascular disease" is used to refer to vascular diseases (for a definition, see the United States National Library of Medicine, Medical Subject Headings (MeSH) C14.907) such as atherosclerosis, myocardial ischemia (including myocardial infarction) and stroke.

The term "computer-readable medium" as used herein refers to any medium that participates in providing instructions to a processor for execution. Such a medium may take many forms, including but not limited to non- volatile media, volatile media, and transmission media. Non-volatile media may include, for example, optical or magnetic disks. Volatile media may include dynamic memory. Transmission media may include coaxial cables, copper wire and fiber optics. Transmission media may also take the form of acoustic, optical, or electromagnetic waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media include, for example, a diskette, hard disk, magnetic tape, or other magnetic medium, a CD-ROM, CDRW, DVD, or other optical medium, a RAM, a PROM, and EPROM, a FLASH-EPROM, or other memory chip or cartridge, a carrier wave, or other medium from which a computer can read.

The term "metagenomics" refers to the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species.

SUMMARY OF THE INVENTION

For the first time, barcoded multiplexed pyrosequencing has been performed in patients with atherosclerosis and healthy controls to address the following questions: Is an altered oral or fecal microbiota associated with cardiovascular diseases, including

atherosclerosis? Are bacteria present in the plaque also detectable in the oral cavity or gastrointestinal tract of the same individual? The present invention discloses that several aspects of the plaque and oral microbiota are related to disease and disease biomarkers.

Herein, we show that atherosclerotic plaques in a human contain numerous bacteria from different phyla and that the abundance of specific ones correlates with the abundance of the same ones in oral microbiota of this person.

Surprisingly, it has been found that an abundance of at least one of the bacterial genera Veillonella, Streptococcus and the species Pseudomonas luteola in atherosclerotic plaques correlates with its abundance in the oral cavity and that its presence in the oral microbiota therefore can be used as a marker of disease for cardiovascular diseases, including

atherosclerosis and related disorders.

A primary object of the present invention is to analyze the presence of specific bacterial groups or species in the oral flora of a person to be used alone, or in combination with other measurements such as blood cholesterol and blood pressure, to better predict whether an individual is at risk of developing cardiovascular disease.

Thus, a method is disclosed for identifying an individual at risk of developing cardiovascular disease, comprising obtaining an oral sample from said individual, and determining the presence of specific bacterial groups or species in the oral sample of said individual, wherein the presence in said oral sample of at least one specific bacterial group or species indicates a risk of developing cardiovascular disease.

According to an embodiment of said method, the presence in said oral sample of at least one specific bacterial group or species in a total amount of at least 5% of the total microbial DNA in said oral sample indicates a risk of developing cardiovascular disease.

According to another embodiment of said method, the at least one specific bacterial group or species comprises Veillonella, Streptococcus or P. luteola.

In another embodiment of said method, the at least one specific bacterial group or species comprises Veillonella and Streptococcus. Further, P. luteola or Chlamydia may be comprised in combination with Veillonella and Streptococcus.

Another object of the present invention is to analyze the abundance of Veillonella or Streptococcus species in the oral flora of a person to be used alone, or in combination with each other, or with other measurements such as blood cholesterol and blood pressure, to better predict a person's risk of having cardiovascular disease such as atherosclerosis and associated diseases.

Another object of the present invention is to analyze the combined abundance of Veillonella, Streptococcus and P. luteola species in the oral flora of a person to be used alone, or in combination with other measurements such as blood cholesterol and blood pressure, to better predict a person's risk of having cardiovascular disease such as atherosclerosis and atherosclerotic associated disease.

Another object of the present invention is to analyze the combined abundance of Veillonella, Streptococcus and Chlamydia species in the oral flora of a person to be used alone, or in combination with other measurements such as blood cholesterol and blood pressure, to better predict a person's risk of having cardiovascular disease such as

atherosclerosis and atherosclerotic associated disease.

Another object of the present invention is to analyze the abundance of species Pseudomonas luteola in the oral flora of a person to be used alone, or in combination with other measurements such as blood cholesterol and blood pressure, to better predict a person's risk of having cardiovascular disease such as atherosclerosis and atherosclerotic associated disease.

Another object is to prevent from the risk of different cardiovascular disease in a person having high amounts of specific bacterial groups or species in the oral microbiota, by using selected anti-microbial treatment.

Thus, a method is disclosed for treatment or prevention of cardiovascular disease in an individual, comprising administering an antimicrobial agent to said individual to inhibit the presence of at least one of Veillonella, Streptococcus and P. luteola in said individual.

In an embodiment, said method may comprise inhibiting the presence of Veillonella and Streptococcus.

Another object is to prevent from the risk of cardiovascular disease in a person having high amounts of specific bacterial groups or species in the oral microbiota, by using vaccination to decrease said specific bacterial groups or species in the oral microbiota.

Thus, a method is disclosed for treatment or prevention of cardiovascular disease in an individual, comprising administering a vaccine against at least one of Veillonella,

Streptococcus and P. luteola in said individual.

As clarified above, the cardiovascular disease to be diagnosed, treated or prevented may for example be atherosclerosis.

It is a further object of the invention to provide products for said identification, vaccination, eradication or immune intervention. Thus, the invention further relates to an antimicrobial agent capable of inhibiting the presence of at least one of Veillonella, Streptococcus and P. luteola in an individual for use in the treatment or prevention of cardiovascular disease in said individual.

In an embodiment, said antimicrobial agent is capable of inhibiting the presence of Veillonella and Streptococcus.

Other objects and advantages of the present invention will become obvious to the reader and it is intended that these objects and advantages are within the scope of the present invention.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 : Bacterial diversity clustering by body habitat (mouth, plaque, gut) determined by sampling (oral, plaque, feces). The first two principal coordinates (PCI and PC2) from the principal coordinate analysis of unweighted UniFrac are plotted for each sample. The variance explained by the PCs is indicated in parentheses on the axes.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

A primary object of the present invention is to predict a person's risk of having cardiovascular disease such as atherosclerosis and associated diseases by analyzing the abundance of specific bacterial groups or species in a person's oral microbiota. This invention can be practiced for example by using sequencing such as barcoded multiplexed-454 sequencing to analyze the bacterial composition of the oral microbiota, alone or in combination with other analysis such as blood cholesterol, and blood pressure levels etc. in persons at risk for cardiovascular disease. The deep sequencing allows for a comprehensive description of microbial communities associated with cardiovascular disease such as atherosclerotic plaques.

The invention can also be practised using other methods for quantification of specific bacterial species or groups known in the art. These methods include, but are not limited to, quantitative PCR, ELISA, microarrays etc.

We have searched for OTUs (Operational Taxonomic Units) shared between oral and atherosclerotic plaque samples, and gut and atherosclerotic plaque samples, within the same individuals. Table 2 summarizes the OTUs that could be found in atherosclerotic plaques and at least one other body habitat in at least 2 patients. We detected Veillonella OTUs in all 13 patients (Table 2). In 11 of the 13 patients that provided oral samples, these OTUs could be detected in both the atherosclerotic plaques and the oral cavity samples of the same patients, and in two patients these OTUs were also detected in the gut. Streptococcus OTUs had a similar pattern of distribution: In 6 of the 10 patients Streptococcus OTUs were detected in the oral cavity and atherosclerotic plaques, and in 4 patients they could also be detected in the gut. The abundances of Veillonella and Streptococcus are correlated in oral cavity and atherosclerotic plaque.

Our findings show that bacteria predominantly affect atherosclerosis by activating the innate immune system. Indeed, atherosclerosis prone mice deficient in Toll-like receptor (Tlr) 2, Tlr4, or the adapter molecule MyD88 are resistant to the development of atherosclerosis (16-18). Furthermore, polymorphism in Tlr4 is associated with lower levels of proinflammatory cytokines, acute-phase reactants, carotid atherosclerosis, and a smaller intima- media thickness in the common carotid artery in humans (19). Taken together our results suggest that the presence of specific bacteria in the oral cavity may enter the blood stream and promote cardiovascular disease such as atherosclerosis by activation of Tlrs.

It has been disclosed, as demonstrated in the Examples, that there is a relationship between the abundance of specific bacterial groups or species in the oral flora and

cardiovascular disease such as atherosclerosis. These specific bacterial groups or species in the oral flora include, but are not limited to, Veillonella, Streptococcus, Chlamydia,

Pseudomonas luteola, Staphylococcus, Propionibacterineae and Burkholderia. Taking advantage of these discoveries, the present invention provides tools utilizing the oral microbiome as a diagnostic or prognostic biomarker for cardiovascular disease risk, a biomarker for drug discovery and a biomarker for the discovery of therapeutic targets involved in the regulation of bacterial amounts of specific groups or species in the oral flora. Such analysis of the abundance of specific bacterial groups or species, combined or separate, in the oral flora can be used alone or in combination with known risk factors for

cardiovascular disease such as blood cholesterol, blood pressure etc.

Decreased bacterial amounts of specific groups or species in the oral flora to beneficial levels may be accomplished by several suitable means generally known in the art. In one embodiment, an antimicrobial agent (an antibiotic) having efficacy against these bacteria in the flora may be administered. The susceptibility of the targeted species to the selected antibiotics may be determined based on culture methods or genome screening. Examples of antimicrobial agents that inhibit growth of Veillonella, Streptococcus, Chlamydia,

Pseudomonas luteola, Staphylococcus, Propionibacterineae and Burkholderia, are Metronidazoles, Fluoroquinolones, Penicillins, Cephalosporins, and Tetracyclins.

The actual effective amounts of compounds comprising a specific reduction of bacteria of the oral microbiota of the invention can and will vary according to the specific compounds being utilized, the mode of administration, and the age, weight and condition of the subject. Dosages for a particular individual subject can be determined by one of ordinary skill in the art using conventional considerations.

The present invention also encompasses use of the microbiome as a biomarker to construct microbiome profiles. Generally speaking, a microbiome profile is comprised of a plurality of values with each value representing the abundance of a microbiome biomolecule. The abundance of a microbiome biomolecule may be determined, for instance, by sequencing the nucleic acids of the microbiome as detailed in the examples. This sequencing data may then be analyzed by known software, as shown below.

A profile may be digitally-encoded on a computer-readable medium. A particular profile may be coupled with additional data about that profile on a computer readable medium. For instance, a profile may be coupled with data to analyze if the person is within a risk group, or for intervention; what therapeutics, compounds, or drugs may be efficacious for that profile. Conversely, a profile may be coupled with data about what therapeutics, compounds, or drugs may not be efficacious for that profile.

The microbiome profile from the host may be determined using DNA sequencing according to the invention. The reference profiles may be stored on a computer-readable medium such that software known in the art and detailed in the examples may be used to compare the microbiome profile and the reference profiles.

EXAMPLE 1

Patient and control groups

The patients were included from the Goteborg Atheroma Study Group biobank, containing carotid endarterectomies from patients who were operated for minor ischemic stroke, transient ischemic attack or amaurosis fugax as previously described in detail (20). The patients were consecutively included, and completed questionnaires covering previous and current diseases, life style factors and medication. Before surgery blood samples were drawn, plasma and serum aliquots were prepared and immediately frozen in -70°C. The excised endarterectomy specimens were immediately frozen in liquid nitrogen under sterile conditions. The subjects in the control group were obtained from two currently running population-based studies of men and women born 1937-40 (21-23). These studies were based on screening examinations of randomly selected population-based cohorts. The control subjects were matched to the patient group for sex, and had to fulfill criteria of feeling well and not suffering from past or current cerebrovascular disease. Each control subject came to the laboratory for information, and examinations identical to those performed in the patient group. Mouth swabs were obtained from both groups by a nurse and the patients were given material and instructions for providing fecal samples. The study was approved by the ethics committee. All patients gave written informed consent to participate after oral and written information.

EXAMPLE 2

DNA extraction

Genomic DNA was isolated from 100 mg of feces and pulverized plaque tissue using Viogene DNA extraction kit (Viogene, Sunnyvale, CA 94086, USA). Mouth swabs were soaked in 900 ml lysis buffer for 2h before DNA was isolated using the same kit. All samples were extensively homogenized using a bead beater at maximum speed for 3minutes. The remaining steps were performed as directed by the manufacturer. Extracted DNA was stored at -20°C.

EXAMPLE 3

PCR amplification of the VI- V2 region of bacterial 16S rRNA genes

Pyrosequencing was performed by amplifying the 16S rRNA genes with a forward primer containing the 454 Life Sciences primer B sequence (Roche) and the broadly conserved bacterial primer 27F and a reverse primer containing the 454 Life Sciences primer A sequence, a unique 12-nucleotide error-correcting barcode used to tag each PCR product, and the broad-range bacterial primer 338R (24-25). PCR reactions were carried out in quadruplicate 20 μΐ reactions with 0.3 μΜ forward and reverse primers, approximately 50 ng template DNA, and IX of HotStar Taq Plus Master Mix kit (Qiagen, Valencia, CA 91355, USA). Due to a low concentration of DNA extracted from plaque tissue a double PCR was performed where 5 μΐ plaque DNA was amplified in a 15-cycle reaction from which subsequently 2 μΐ served as template in a second otherwise identical 30-cycle PCR. Thermal cycling consisted of initial denaturation at 95°C for 2 minutes followed by 30 cycles of denaturation at 95°C for 20 seconds, annealing at 52°C for 20 seconds, and extension at 65°C for 60 seconds. Replicate amplicons were pooled, purified with Agencourt AMPure kit (Beckman Coulter, Danvers, MA, USA), and visualized on 1.0% agarose gels. EXAMPLE 4

Amplicon quantitation, pooling, and pyrosequencing

Am licon DNA concentrations were determined using a nanodrop. Following quantitation, cleaned amplicons were combined in equimolar ratios into a single tube with a final concentration of 16 ng/ml. Pyrosequencing was carried out using primer A on a 454 Life Sciences Genome Sequencer FLX instrument at Center for Metagenomic Sequence Analysis at KTH, School of Biotechnology in Stockholm, Sweden. EXAMPLE 5

Sequences were processed and analyzed using the QIIME pipeline using default parameters for each step except where specified (http://qiime.sourceforge.net/). Sequences were removed if lengths were <200, contained ambiguous bases, primer mismatches, homopolymer runs in excess of 6 bases, uncorrectable barcodes, or lacked the primer.

Remaining sequences were assigned to samples according to their barcodes. Similar sequences were binned into OTUs using cd-hit (26), with a minimum identity of 97%. A sequence was chosen to represent each OTU. Sequences belonging to OTUs detected in extraction control samples were removed from the entire dataset. Representative sequences from each OTU were aligned using PyNAST (a python-based implementation of NAST in QIIME) (27) and the Greengenes (28) database (aligned coreset 11/08/07) using a minimum percent identity of 75%. The LanemaskPH was used to screen out the hypervariable regions (29). A phylogenetic tree was constructed using FastTree (30). Taxonomy was assigned using the RDP classifier (31) with a minimum support threshold of 60% and the RDP classifier nomenclature.

To estimate amounts of certain communities, we employed rarefaction plots and phylogenetic diversity measures described in (32). To compare diversity between samples, we used the weighted and unweighted UniFrac distance metrics (33-34) using a random sample of 1,000 sequences per sample. To relate the OTU abundances to patient health data, we considered only OTUs containing at least two sequences, and that were classified at least to the phylum level.

EXAMPLE 6

Results from human study above We surveyed the atherosclerotic plaque, oral cavity (swab from periodontium area), and gut (feces) bacterial communities of 15 patients with clinical atherosclerosis and 15 age and sex matched healthy controls (Table 1). The 5' variable regions (V1-V2) of the bacterial 165^* ribosomal RNA (rRNA) gene were PCR amplified using barcoded primers 27F and 338R (25). We generated a dataset of 380,501 high-quality 165^* rRNA sequences (n = 73: 15 each patient and control fecal samples, 14 patient oral samples, 15 control oral samples, 14 atherosclerotic plaque samples). Sequences were assigned to "species-level" operational taxonomic units (OTUs) using a 97% pairwise-identity cutoff, and chimera checking revealed that 3.1%) of total sequences were putative chimeras. One atherosclerotic plaque sample was excluded from the downstream analysis due to low sequence counts (< 1,700 sequences). The final dataset included representatives of 13 bacterial phyla; the majority of the sequences were classified as Firmicutes (63.8%>), Bacteroidetes (11.7%), Proteobacteria (15.4%), and

Actinobacteria (6.4%).

We compared the overall bacterial community composition using the unweighted UniFrac distance metric, a phylogenetic tree-based metric ranging from 0 (distance between identical communities) to 1 (distance between totally different communities with no shared ancestry). This analysis revealed strong clustering of samples by body site, with the atherosclerotic plaque samples forming a distinct cluster apart from oral and fecal samples, indicating all three sites have distinct microbial communities (Fig. 1). We used the unweighted (qualitative) rather than the weighted (quantitative) version of the metric because it generally performs better for resolving human body sites (29). The average phylogenetic diversity (35) of the microbiotas was similar for atherosclerotic plaque (AP) and oral cavity (OC, ratio AP/OC= 1.09), but highest for the gut (G; AP/G=0.7, OC/G=0.64). Characterization of the atherosclerotic plaque microbiota

The atherosclerotic plaque contained low but detectable amounts of bacterial DNA: qPCR analysis revealed a positive correlation (p = 0.68, P = 0.009) between the amount of bacterial 165^* rDNA and the number of leukocytes within the atherosclerotic plaques, suggesting that the amount of bacteria contributes to the inflammatory status of the atherosclerotic plaque. Compared with the oral and gut samples, the atherosclerotic plaque contained significantly higher levels of Proteobacteria and fewer Firmicutes (Table 3). We detected several OTUs present in all atherosclerotic plaque samples, and which differentiated these samples from oral and fecal samples. 3 OTUs belonging to the genus Staphylococcus, 3 OTUs classified to the genus Propionibacterineae, and one OTU belonging to the genus Burkholderia (data not shown) were specific for atherosclerotic plaques and present in all samples. Chryseomonas (recently reclassified as Pseudomonas luteola), was detected at high levels in the atherosclerotic plaque samples. Together these observations support the notion that a 'core' microbiota composed of the same genus-level lineages exists in atherosclerotic plaque.

Composition of the oral microbiota and its relation to atherosclerosis and disease markers

The oral microbiota of patients and healthy controls was dominated by Firmicutes (69 and 76%, respectively, of OTUs classifiable to the phylum level), followed by Bacteroidetes (10 and 6%), Actinobacteria (9 and 10%), Fusobacteria (6 and 3%), Proteobacteria (5 and

4%>), and <1%, Spirochaeates, TM7, SRI and Tenericutes (data not shown). The NSC analysis did not reveal any species-level OTUs that could discriminate between the healthy and patient oral samples.

To search for correlations between the abundances of OTUs in the oral cavity and markers for cardiovascular disease, we required a minimum sequence count of 100 sequences per genus (across all samples, using 1,700 sequences randomly selected per sample) for inclusion in the analysis.

Composition of the gut microbiota and its relation to atherosclerosis and disease markers Because the fecal microbiota is representative of the gut microbiota (36), we characterized the bacterial diversity of fecal samples to determine whether gut microbiotas differed between patients and controls. Overall, the relative abundances of the phyla were similar between patients and controls, and NSC analysis did not reveal any OTUs whose abundances could differentiate the patients from the controls (Table 3). Compared to the oral and atherosclerotic plaque samples, gut samples contained significantly greater abundances of OTUs classified as members of the Lachnospiraceae family, and as the genera Ruminococcus and Faecalibacterium.

Inter- and intra-individual comparisons of atherosclerotic plaque, oral and gut microbiotas One of the main purposes of the study was to search for OTUs shared between oral and atherosclerotic plaque samples, and gut and atherosclerotic plaque samples, within the same individuals. Table 2 summarizes the OTUs that could be found in atherosclerotic plaques and at least one other body habitat in at least 2 patients. We detected Veillonella OTUs in all 13 patients (Table 2). In 11 of the 13 patients that provided oral samples, these OTUs could be detected in both the atherosclerotic plaques and the oral cavity samples of the same patients, and in two patients these OTUs were also detected in the gut. Streptococcus OTUs had a similar pattern of distribution: In 6 of the 10 patients Streptococcus OTUs were detected in the oral cavity and atherosclerotic plaques, and in 4 patients they could also be detected in the gut. The abundances of Veillonella and Streptococcus are correlated in oral cavity and atherosclerotic plaque.

Within individual patients, the atherosclerotic plaque samples contained additional OTUs that were also detected in oral and gut samples. For instance, additional OTUs detected in the atherosclerotic plaque and oral samples of the same individual for at least two patients include: Propionibacterium, Rothia, Burkholderia, Corynebacterium, Granulicatella, Staphylococcus and an unclassified OTU belonging to the Betaproteobacteria. In contrast, OTUs detected in the atherosclerotic plaque and gut samples of the same individual for at least two patients include: Bacteroides, an unclassified member of the Lachnospiraceae, Bryantella, Enterobacter, an unclassified Enterobacteriaceae, Ruminococcus and OTUs classified as Subdoligranulum (Table 2).

EXAMPLE 7

The method of the invention is used in a clinical setting to aid in the assessment if a person is in a risk group for developing cardiovascular disease, including arthrosclerosis and associated conditions. Oral swabs and blood samples are taken and other normal assessments such as blood pressure, BMI, waist size are made. The oral swabs are processed as described above and a value is determined for the presence of species Veillonella, Streptococcus and/or P. luteola in the oral flora. If bacteria of one or several of those species are found in a total amount of more than 5%, such as 10%, of the total microflora as described herein, this alone or in combination with clinically used risk values for the other variables, such as blood pressure, blood cholesterol etc, the person is considered at risk for developing a

cardiovascular disease, including atherosclerosis and should be further investigated and monitored.

EXAMPLE 8

Method for determining amount of Veillonella och Streptococcus using qPCR Mouth swabs were soaked in 900 ml lysis buffer for 2h before DNA was isolated using the same kit. The genomic DNA from the atherosclerotic plaque samples was extracted using the MOBIO PowerSoil DNA isolation kit. All samples were extensively homogenized using a bead beater at maximum speed for 3 minutes. The remaining steps were performed as directed by the manufacturer. All samples were diluted to 10 ng/ul and amplified using primers directed towards Streptococcus (Forward: 5 -GT AC AGTTGCTTC AGGACGTATC-3 and reverse 5-ACGTTCGATTTCATCACGTTG-3) and Veilonella (Forward: 5'- GTAACAAAGGTGTCGTTTCTCG-3^*) and reverse: 5'-

GCACCRTCAAATACAGGTGTAGC-3'). Data is expressed as ng/ng total DNA.

The present invention is not limited to the above-described preferred embodiments. Various alternatives, modifications and equivalents may be used. Therefore, the above embodiments should not be taken as limiting the scope of the invention, which is defined by the appending claims.

Table 1. Characteristics of study participants in example 1.

Table 1.

Controls Patients p-value

Males, n (%) 12 (80) 12 (80) NA Age, years 70.5±0.5 65.7±0.5 0.67

Current smoker, n (%) 0 6 (40) 0.017 Known diabetes, n (%) 0 4 (27) 0.10 Previous myocardial infarction, n (%) 0 4 (27) 0.10 Previous or current cerebrovascular disease NA No 15 (100) 0

Amaurosis fugax, n (%) 4 (27) Transitory ischemic attack, n (%) 6 (40) Stroke, n (%) 5 (33)

Known hypertension, n (%) 2 (13) 11 (73) 0.003

Systolic blood pressure, mm Hg 141±23 146±18 0.35

Diastolic blood pressure, mm Hg 80±12 77±12 0.60

Total cholesterol, mmol/L 5.56±1.12 4.67±1.53 0.026

HDL cholesterol, mmol/L 1.64±0.44 1.27±0.28 0.015

LDL cholesterol, mmol/L 3.35±.01 2.60±1.36 0.019

Triglycerides, mmol/L (median [interquartile 1.21 (0.75) 1.47 (0.80) 0.046 range])

Apolipoprotein AI, g L 1.43±0.19 1.32±0.21 0.19

Apolipoprotein B, g/L 1.10±0.30 0.98±0.35 0.25 hsCRP, mg/L (median [interquartile range]) 0.68 (3.01) 1.12 (3.73) 0.20

Statin treatment, n (%) 0 11 (73) NA

Antiplatlet treatment, n (%) 0 15 (100) NA

NA-not applicable due to selection criteria

Table 2.

OTUs shared among at leastotlsody sites ithin the same patient.

Columns at right indicate the number of patients for which the OTUs were found in both plaque + feces, plaque + mouth, or all three sites.

Number of patients (n=13):

Consensus Lineage (No. of different OTUs with the same consensus lineage) Plaque +

Plaque 4 Plaque +

Feces + Feces Mouth

Mouth

Firmicutes;Clostridia;Clostridiales;Veillonellaceae;Veillonella (7) 0 1 1 2 Firmicutes;Bacilli;Lactobacillales;Streptococcaceae;Streptococcus (17) 0 6 4

Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Propionibacterineae;Propionibacteriaceae (4) 0 8 0 Bacteroidetes;Bacteroidetes;Bacteroidales;Bacteroidaceae;Bacteroides (5) 6 0 0 Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;unclassified Lachnospiraceae(9) 6 0 0 Firmicutes;Clostridia;Clostridiales;Lachnospiraceae;Lachnospiraceae Incertae Sedis (4) 5 0 0 Firmicutes ; Clo stridia; Clo stridiales ; Lachno spiraceae ; Bry antella ( 1 ) 5 0 0

Actinobacteria;Actinobacteria;Actinobacteridae;Actinomycetales;Micrococcineae;Micrococcaceae;Rothia (2) 0 4 0 Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;Enterobacter (1) 4 0 0 Proteobacteria;Betaproteobacteria;Burldiolderiales;Burkholderiaceae;Burkholderia (1) 0 3 0

Proteobacteria;Gammaproteobacteria;Enterobacteriales;Enterobacteriaceae;unclassified Enterobacteriaceae (2) 3 0 0 Actmobacteria;Actinobacteria;Actmobacteridae;Actmomycetales;Corynebac 2) 0 2 0 Firmicutes;Bacilli;Lactobacillales;Carnobacteriaceae;Carnobacteriaceae 2;Granulicatella (1) 0 2 0 Firmicutes;Bacilli;Bacillales;Staphylococcaceae;Staphylococcus (3) 0 2 0 Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Ruminococcus (1) 2 0 0 Firmicutes;Clostridia;Clostridiales;Ruminococcaceae;Subdoligranulum (7) 2 0 0

Proteobacteria;Betaproteobacteria;unclassified Betaproteobacteria (1) 0 2 0

Table 3: Mean phylum abundances (%) for samples grouped by body habitat and health status. Plotted values are mean sequence abundances in each phylum for 1,700 randomly selected sequences per sample.

Oral Oral Feces Feces

Plaque (patient) (control) (patient) (control)

Firmicutes 14,693 69,325 76,310 80,375 78,395

Bacteroidetes 5,956 9,753 6,307 16,641 19,756

Actinobacteria 9,343 8,896 9,830 2,361 1,357

Fusobacteria 0,014 6,254 3,088 0,000 0,000

Proteobacteria 67,502 4,633 3,936 0,612 0,468

TM7 0,000 0,587 0,408 0,006 0,012

Spirochaetes 0,000 0,370 0,071 0,000 0,000

SRI 0,000 0,117 0,043 0,000 0,000

Tenericutes 0,000 0,059 0,005 0,000 0,000

Deinococcus-

Thermus 0,055 0,006 0,000 0,000 0,000

Acidobacteria 1,976 0,000 0,000 0,000 0,000

Gemmatimonadetes 0,330 0,000 0,000 0,000 0,000

Chloroflexi 0,131 0,000 0,000 0,000 0,000

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Claims

1. A method for identifying an individual at risk of developing cardiovascular disease, comprising

- obtaining an oral sample from said individual, and

- determining the presence of specific bacterial groups or species in the oral sample of said individual,

wherein the presence in said oral sample of at least one specific bacterial group or species indicates a risk of developing cardiovascular disease.

2. The method of claim 1, wherein the presence in said oral sample of at least one specific bacterial group or species in a total amount of at least 5% of the total microbial DNA in said oral sample indicates a risk of developing cardiovascular disease.

3. The method of claim 1 or 2, wherein the at least one specific bacterial group or species comprises Veillonella, Streptococcus or P. luteola.

4. The method of claim 3, wherein the at least one specific bacterial group or species comprises Veillonella and Streptococcus.

5. The method of claim 4, wherein the at least one specific bacterial group or species further comprises P. luteola.

6. The method of claim 4, wherein the at least one specific bacterial group or species further comprises Chlamydia.

7. A method for treatment or prevention of cardiovascular disease in an individual, comprising administering an antimicrobial agent to said individual to inhibit the presence of at least one of Veillonella, Streptococcus and P. luteola in said individual.

8. The method of claim 7, wherein the presence of Veillonella and Streptococcus is inhibited in said individual.

9. A method for treatment or prevention of cardiovascular disease in an individual, comprising administering a vaccine against at least one of Veillonella, Streptococcus and P. luteola in said individual.

10. The method of claim 9, wherein the vaccine acts against Veillonella and Streptococcus in said individual.

11. The method of any preceding claim, wherein the cardiovascular disease is

atherosclerosis.

12. An antimicrobial agent capable of inhibiting the presence of at least one of Veillonella, Streptococcus and P. luteola in an individual for use in the treatment or prevention of cardiovascular disease in said individual.