Human oral, gut, and plaque microbiota in patients with atherosclerosis Omry Korena,1, Aymé Spora,1, Jenny Felinb,c,1, Frida Fåkb,c, Jesse Stombaughd, Valentina Tremarolib,c, Carl Johan Behreb,c, Rob Knightd,e, Björn Fagerbergb,c, Ruth E. Leya,2, and Fredrik Bäckhedb,c,2 aDepartment of Microbiology, Cornell University, Ithaca, NY 14853; bSahlgrenska Center for Cardiovascular and Metabolic Research, University of Gothenburg, S-413 45 Gothenburg, Sweden; cThe Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, S-413 45 Gothenburg, Sweden; dDepartment of Biochemistry and Chemistry, University of Colorado, Boulder, CO 80309; and eHoward Hughes Medical Institute, University of Colorado, Boulder, CO 80309 Edited by Todd R. Klaenhammer, North Carolina State University, Raleigh, NC, and approved September 17, 2010 (received for review August 2, 2010) Periodontal disease has been associated with atherosclerosis, sug- Epidemiological studies support an association between cardio- gesting that bacteria from the oral cavity may contribute to the vascular disease and infections, such as periodontal disease and development of atherosclerosis and cardiovascular disease. Further- Chlamydia infections (14, 15). Dental disease has been associated more, the gut microbiota may affect obesity, which is associated with with elevated risk of myocardial infarction (14), and metabolic atherosclerosis. Using qPCR, we show that bacterial DNA was present activity of the gut microbiota was recently shown to relate to blood in the atherosclerotic plaque and that the amount of DNA correlated pressure (16). Furthermore, in a study where bacterial DNA was with the amount of leukocytes in the atherosclerotic plaque. To identified in atherosclerotic plaques, 51.5% of the patients tested investigate the microbial composition of atherosclerotic plaques and positive for Chlamydia in their atheromas (17). Several studies test the hypothesis that the oral or gut microbiota may contribute to suggest an oral source for atherosclerotic plaque-associated bac- atherosclerosis in humans, we used 454 pyrosequencing of 16S rRNA teria (18–21). However, to date, no single study has directly com- genes to survey the bacterial diversity of atherosclerotic plaque, oral, pared the microbial diversity of oral, gut, and atherosclerotic and gut samples of 15 patients with atherosclerosis, and oral and gut plaque microbiotas within individuals. This type of cross-site com- samples of healthy controls. We identified Chryseomonas in all ath- parison is essential given the high level of variability observed in the erosclerotic plaque samples, and Veillonella and Streptococcus in the microbiota between different subjects (1–6). majority. Interestingly, the combined abundances of Veillonella and Here, we characterized the atherosclerotic plaque, oral, and gut Streptococcus in atherosclerotic plaques correlated with their abun- microbiotas obtained from patients with atherosclerosis and dance in the oral cavity. Moreover, several additional bacterial phy- healthy controls by pyrosequencing their 16S rRNA genes. Our lotypes were common to the atherosclerotic plaque and oral or gut study addressed the following questions: Is there a core athero- samples within the same individual. Interestingly, several bacterial sclerotic plaque microbiota? Are bacteria present in the plaque taxa in the oral cavity and the gut correlated with plasma cholesterol also detectable in the oral cavities or guts of the same individuals? levels. Taken together, our findings suggest that bacteria from the Do the microbiotas of the oral cavity, gut, and atherosclerotic oral cavity, and perhaps even the gut, may correlate with disease plaque relate to disease markers such as plasma levels of apoli- markers of atherosclerosis. poproteins and cholesterol? Is an altered oral or fecal microbiota associated with atherosclerosis? Our findings revealed a number he human body is home to microbial ecosystems (microbiotas) of phylotypes common to the atherosclerotic plaque and oral and whose structure and function differ between different sites in gut samples within individuals, and that the abundances of spe- T fi the body (1–6). These microorganisms outnumber the number of ci c members of the oral and gut microbiota correlated with eukaryotic cells in the human body by at least an order of magni- disease biomarkers. tude (7). The gut microbiota is the best-studied human-associated Results ecosystem and has a major impact on our physiology, immune system, and metabolism. For instance, obese individuals generally Overall Comparison of the Human Oral, Gut, and Atherosclerotic have a less diverse gut microbiota, and some studies have observed Plaque Microbiotas. We surveyed the atherosclerotic plaque, oral reduced levels of the bacterial phyla Bacteroidetes (3), although cavity (swab from periodontium area), and gut (feces) bacterial communities of 15 patients with clinical atherosclerosis and 15 age- others have not (8, 9). Furthermore, germ-free mice have reduced and sex-matched healthy controls (Table 1). The 5′ variable regions adiposity and are resistant to diet-induced obesity (10, 11). Thus the (V1–V2) of the bacterial 16S ribosomal RNA (rRNA) gene were gut microbiota can be considered an environmental factor that PCR amplified using barcoded primers 27F and 338R (22). We affects obesity. However, the role of the human microbiome in generated a dataset of 380,501 high-quality 16S rRNA sequences obesity-related metabolic diseases such as atherosclerosis remains to be explored. Atherosclerotic disease, with manifestations such as myocar- This paper results from the Arthur M. Sackler Colloquium of the National Academy of dial infarction and stroke, is the major cause of severe disease Sciences, "Microbes and Health," held November 2–3, 2009, at the Arnold and Mabel and death among subjects with obesity. The disease is charac- Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. terized by accumulation of cholesterol and recruitment of mac- The complete program and audio files of most presentations are available on the NAS rophages to the arterial wall. It can thus be considered both Web site at http://www.nasonline.org/SACKLER_Microbes_and_Health. fl fi Author contributions: B.F. and F.B. designed research; O.K., A.S., J.F., and F.F. performed a metabolic and an in ammatory disease (12). Since the rst half research; J.S. and R.K. contributed new reagents/analytic tools; O.K., A.S., F.F., J.S., V.T., C.J.B., of the 19th century, infections have been thought to cause or B.F., R.E.L., and F.B. analyzed data; and O.K., A.S., R.K., R.E.L., and F.B. wrote the paper. promote atherosclerosis by augmenting proatherosclerotic The authors declare no conflict of interest. changes in vascular cells (13). These changes include increased This article is a PNAS Direct Submission. scavenger receptor expression and activity, enhanced uptake of 1O.K., A.S., and J.F. contributed equally to this work. cholesterol and modified LDL, increased expression of adhesion 2To whom correspondence may be addressed. E-mail: [email protected] or fredrik. molecules and inflammatory cytokines, and other effects, such as [email protected]. stimulating macrophages to express cytokines, leading to ath- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. erosclerotic plaque vulnerability (13). 1073/pnas.1011383107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1011383107 PNAS Early Edition | 1of7 Downloaded by guest on September 24, 2021 Table 1. Characteristics of study participants Controls (n = 15) Patients (n = 15) 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 ± 0.01 2.60 ± 1.36 0.019 Triglycerides, mmol/L (median [interquartile range]) 1.21 (0.75) 1.47 (0.80) 0.046 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 Antiplatelet treatment, n (%) 0 15 (100) NA NA, not applicable due to selection criteria. (n = 73: 15 patient and control fecal samples, 14 patient oral Pseudomonas luteola), was detected at high levels in the athero- samples, 15 control oral samples, and 14 atherosclerotic plaque sclerotic plaque samples and not at all in the gut or oral samples; samples). Sequences were assigned to species-level operational furthermore, nearest shrunken centroids (NSC) analysis revealed taxonomic units (OTUs) using a 97% pairwise-identity cutoff, and that this OTU was the most discriminant genus between sites (i.e., chimera checking revealed that 3.1% of total sequences were pu- driving the differences between the microbiotas of the different tative chimeras. One atherosclerotic plaque sample was excluded body sites; Fig. 3 A and B). In addition, three OTUs belonging to from the downstream analysis due to low sequence counts (<1,700 the genus Staphylococcus, three OTUs classified as Propioni- sequences). The final dataset included representatives of 13 bac- bacterineae, and one OTU belonging to the genus Burkholderia terial phyla; the majority of the sequences were classified as Fir- (Fig. 3 A and B)werespecific for atherosclerotic plaques and micutes (63.8%), Bacteroidetes (11.7%), Proteobacteria (15.4%), present in all samples.