REVIEWS

Role of gut microbiota in atherosclerosis

Annika Lindskog Jonsson1 and Fredrik Bäckhed1,2
Abstract | Infections have been linked to the development of cardiovascular disease and
atherosclerosis. Findings from the past decade have identified microbial ecosystems residing
in different habitats of the human body that contribute to metabolic and cardiovascular-related
disorders. In this Review, we describe three pathways by which microbiota might affect
atherogenesis. First, local or distant infections might cause a harmful inflammatory response that
aggravates plaque development or triggers plaque rupture. Second, metabolism of cholesterol
and lipids by gut microbiota can affect the development of atherosclerotic plaques. Third,
diet and specific components that are metabolized by gut microbiota can have various effects
on atherosclerosis; for example, dietary fibre is beneficial, whereas the bacterial metabolite
trimethylamine-N‑oxide is considered harmful. Although specific bacterial taxa have been
associated with atherosclerosis, which is supported by increasing mechanistic evidence, several
questions remain to be answered to understand fully how the microbiota contributes to
atherosclerosis and cardiovascular disease. Such knowledge might pave the way for novel
diagnostics and therapeutics based on microbiota.

1
Wallenberg Laboratory
for Cardiovascular and
Metabolic Research,
Department of Molecular
and Clinical Medicine,
Institute of Medicine,
University of Gothenburg,
Bruna Stråket 16,
41345 Gothenburg, Sweden.
2
Novo Nordisk Foundation
Center for Basic Metabolic
Research, Section for
Metabolic Receptology and
Enteroendocrinology, Faculty
of Health Sciences, University
of Copenhagen, Copenhagen,
DK‑2200, Denmark.
Correspondence to F.B.
fredrik@wlab.gu.se
doi:10.1038/nrcardio.2016.183
Published online 1 Dec 2016
The human body hosts a vast number of micro­organisms
including bacteria, viruses, protozoa, archaea, and fungi,
which constitute the commensal microbiota that mainly
resides in the gut. Although the number of bacterial
species varies between individuals, at higher taxo­nomic
levels (such as phyla) the microbiota is similar between
individuals. In contrast to other environments, the
gut microbiota is dominated by two bacterial phyla,
Bacteroidetes or Firmicutes, which constitute >90% of
the taxa present in the human gut, with a lower abun-
dance of Actinobacteria, Cyanobacteria, Fusobacteria,
Proteobacteria, and Verrumicrobia1,2 (FIG. 1).
Humans have co-evolved with these microorganisms
in a mutualistic fashion. Whereas microorganisms benefit
from a constant supply of human substrates, the human
hosts benefit from microbial activities such as production
of essential vitamins (such as vitamin B and K), diges-
tion of carbohydrates, ‘education’ of the immune sys-
tem, and production of signalling molecules including
short-chain fatty acids and secondary bile acids. Bacterial
genes (microbiome) in the gut outnumber human genes
by at least two orders of magnitude3. The microbiota can
influence host physiology, but external factors such as
genetics, diet, pharmacological compounds, lifestyle,
and hygiene also affect the microbial composition4.
Despite these beneficial effects described above, stud-
ies in the past 10 years have demonstrated that the micro-
biota is associated with several diseases including obesity
and type 2 diabetes mellitus as well as atherosclerosis and
cardiovascular disease (CVD) in humans. However, when
studying the microbiota and its role in disease, it is impor-
tant to keep in mind Koch’s postulates, which describe
the criteria that should be fulfilled in order to deter-
mine a causative relationship between a microorganism
and a disease (BOX 1). Given that these postulates were
established in the late nineteenth century, they should
be adapted to incorporate the increased knowledge of
host–microorganism interaction, and should include
the altered microbiome rather than only one pathogenic
species. Different methods exist to study microbiota and
its effect on host metabolism, such as animal models and
modern culture-independent techniques (including bac-
terial 16S rRNA gene sequencing), which provide a huge
amount of data that need interpretation (BOX 2).
Periodontitis and gut microbial metabolites have
been shown to modulate CVD, although the under­
lying mechanisms still remain unclear. Atherosclerosis
is a complex disease involving both inflammatory and
metabolic pathways and might be modulated by the gut
microbiota. In this Review, we discuss the microbial-­
related processes that could affect the development of
atherosclerosis, including the infection-induced inflam-
matory response, altered host lipid metabolism, and the
interaction between specific dietary components and
microbial metabolites.
Role of oral and gut microbiota in CVD
The development of atherosclerotic plaques can be
affected by a distant infection or by a direct infection
of vessel wall cells. This mechanism is supported by

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Key points with this condition, the number of Lactobacillales and

• Microorganisms that reside in the human body, the majority of which colonize the gut,
might affect host physiology in various ways
• Bacteria from the gut or the oral cavity might translocate to atherosclerotic plaques
and could affect the development of atherosclerosis and cardiovascular disease
• Microbial transplantations in mice influence diet-enhanced susceptibility to
atherosclerosis and thrombosis
• Dietary components can either alter the composition of gut microbiota or be processed
into metabolites that can delay or accelerate the development of atherosclerosis
• Trimethylamine N‑oxide is a potentially harmful bacterial metabolite that influences
cholesterol metabolism and thrombosis activity
• To determine the role of the (gut) microbiota in cardiovascular disease and
atherosclerosis, the underlying mechanisms need to be further elucidated
the finding of bacterial DNA in the plaques5,6. Bacteria
observed in the atherosclerotic plaques are also found
at other body sites, predominantly the oral cavity
and the gut, which might thus serve as reservoirs of
these potentially pathogenic microorganisms (FIG. 1).
An established association exists between perio­dontal
disease and CVD7 as well as between poor dental health
and acute myocardial infarction 8. In a large study
that included almost 12,000 participants, poor oral
hygiene was associated with an increased risk of fatal
and nonfatal CVD events (such as myocardial infarc-
tion, coronary artery bypass graft surgery, coronary
angioplasty, stroke, and heart failure) and low-grade
inflammation, as demonstrated by increased concentra-
tions of C‑reactive protein and fibrinogen9. A study of
92 individ­uals demonstrated that patients with sympto-
matic atherosclerosis (myocardial infarction or stroke)
had a higher abundance of Anaeroglobus in the oral
cavity (around 50% of patients) compared with healthy
controls (around 25% of individ­uals)10. Several oral
bacteria detected in atherosclero­tic plaques, including
Porphyromonas gingivalis and Aggregatibacter actino­
mycetemcomitans, led to increased lesions in animal
models following oral or intravenous infection, which
could be explained by an increase in proatherogenic
mediators11–13. Although the mechanism is unclear,
these findings indicate that bacteria from the oral cavity
are associated with CVD and might trans­locate to the
vessel, where they might affect growth and stability of
the atherosclerotic plaque.
In addition to the oral cavity, the gut is a likely
source of microorganisms that could influence CVD.
Using shotgun sequencing, faecal samples from
13  healthy individ­u als and 12 patients with symp-
tomatic atherosclero­s is (myocardial infarction or
cerebro­vascular events) were found to differ in several
aspects. Indeed, the genus Collinsella was enriched in
patients with symptomatic atherosclerosis, whereas
Eubacterium and Roseburia were enriched in healthy
controls14 . In the patients, the altered microbial com-
position was also associated with increased abundance
of genes involved in inflammatory processes14. The
abundance of gut microorganisms such as Bacteroides,
Clostridium, and Lactobacillales has been shown to pre-
dict coronary artery disease15. Furthermore, in patients
the ratio of Firmicutes to Bacteroidetes increased com-
pared with control, whereas the number of Bacteroidetes
decreased16. These findings need to be corroborated
in independent cohorts, and mechanistic studies are
required to determine whether the altered microbiota
causes, or merely reflects, coronary artery disease.
Infection and inflammation
Infections can affect the development of atherosclero-
sis via two predominant mechanisms: direct infection
of vessel wall cells, which creates a site prone to lesion
development, or an indirect infection at a site distant
from the atherosclerotic plaque, which activates the
immune system that in turn increases the systemic
inflammatory status.
Plaque microbiota. The presence of bacterial DNA in
human atherosclerotic plaques has been demonstrated
with various techniques and in different patients. One
of the most common bacteria found in atherosclerotic
plaques is Chlamydia pneumoniae17,18, which causes
respir­atory tract infection. Furthermore, C. pneumoniae
has been detected in postmortem coronary arteries and
endarterectomy specimens of living patients, but this
bacterium was not associated with histological assess-
ment of plaque stability (or lesion severity) or clinical
parameters19–21. By contrast, C. pneumoniae correlates
with cytotoxic CD8+ T cells in symptomatic plaques and
is associated with increased C‑reactive protein levels in
serum22,23. In addition to C. pneumoniae, >50 bacterial
species were detected in atherosclerotic plaques, whereas
no bacteria were found in control tissues5.
Improved sequencing technology and bioinformatics
analyses of bacterial 16S rRNA genes have provided a
detailed view of the bacterial content of atherosclero­
tic plaques. Whereas the majority of bacterial DNA
belongs to Firmicutes and Proteobacteria phyla 6,24
(FIG. 1), bac­terial DNA from taxa belonging to poten-
tially pathogenic families such as Helicobacteraceae and
Neisseriaceae was shown to be more abundant in athero­
sclerotic plaques of symptomatic patients than in those
of asymptomatic ones25. However, this finding needs
to be corroborated in larger studies. Given that athero-
sclerotic plaques from asymptomatic patients could be
vulnerable and at risk of rupture, it would be interesting
to correlate plaque morphology with bacterial content.
Although the presence of bacterial DNA in athero­
sclerotic plaques is established, whether an infection
initiates or augments their development is uncertain.
Taking into account Koch’s postulates, only a few infec-
tious agents have been shown to be potentially associated
with atherosclerosis. Microorganisms such as A. actino­
mycetemcomitans, C. pneumoniae, Helicobacter pylori,
and P. gingivalis might contribute to atherosclerosis by
increasing lesion areas in animal models26; however,
these data need to be validated in humans.
Infectious agents. Since the first half of the nineteenth
century, infections of either viral or bacterial origin
have been considered as a cause of atherosclerosis27.

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These infectious agents include bacteria such as C. pneu­ To test whether a causal relationship exists between
moniae and H. pylori, periodontal microorganisms, and bacterial infection and CVD, several randomized, placebo-­
viruses including cytomegalovirus, hepatitis viruses, controlled trials have investigated whether treatment
influenza viruses, and human i­ mmunodeficiency virus26. with antibiotics has beneficial effects (TABLE 1). Although
The potential association between acute infection these trials showed no benefit in patients with coronary
and cardiovascular events was investigated in a study artery disease31–36, negative outcomes in such studies are
that included >40,000 patients28. The risk of myo­cardial to some extent inconclusive as the treatment duration
infarction or stroke was found to be increased after might have been too short or, if pathogens initiated the
systemic infection of the respiratory tract and infec- disease process, the treatment might have been too late.
tion of the urinary tract. This effect was most prom- Furthermore, the antibiotics used in these studies do not
inent within the first days of infection, and the risk target all bacteria residing in the human body. Further
gradually decreased afterwards28. In a meta-analysis studies are thus required to investigate the involvement
including >10,000 patients, seroprevalence of C. pneu­ of bacteria in the initiation of the athero­sclerotic process;
moniae correlated with an increase in the levels of sev- however, obtaining such evidence might not be feasible
eral inflammatory markers, such as C‑reactive protein owing to the long experimental duration and the risk of
(measured by high-affinity assay), IL‑6, and fibrinogen, spreading antibiotic resistance.
compared with seronegative patients with atherosclero­ Long-term treatment with antibiotics might cause
sis29. By contrast, in a study involving 1,100 patients, adverse effects, given that their administration early
Escherichia coli-related gastroenteritis did not increase in life to mice alters commensal gut microbiota and
the risk of cardiovascular events (myocardial infarc- induces obesity and insulin resistance 37, which in
tion, stroke, or heart failure) compared with 11,000 humans can cause cardiovascular disease. Interestingly,
controls30. Thus, specific bacterial infections, followed antibiotic treatment also affects lipid and cholesterol
by activation of the immune system and increased lev- metabolism 37, but whether atherosclerosis is also
els of pro­atherogenic inflammatory molecules, might increased is unknown. Other studies have suggested that
contribute to plaque vulnerability and thrombus patients exposed to a large number of pathogens have
formation (FIG. 2). an increased risk of CVD27,38. This evidence could also
help to explain the lack of effect of antibiotics, given that
some bacteria are resistant, and viruses are not targeted
by antibiotic treatment39. Although no direct evidence
from large clinical studies indicates that bacteria cause
CVD, this h ­ ypothesis cannot be ruled out.
Firmicutes
• Gram-positive bacteria
• Most abundant phylum in the Toll-like receptors and atherosclerosis. Regardless of
gut and oral cavity, and also the infection site (local or systemic), the immune system
Proteobacteria present in atherosclerotic plaques
• Include the genera Anaeroglobus,
responds to microbial components, and several inflam-
• Gram-negative bacteria matory pathways are activated. One of these pathways
Clostridium, Eubacterium,
• Most abundant phylum Lactobacillales, and Roseburia
in atherosclerotic involves Toll-like receptors (TLRs). TLRs are a family
plaques • The ratio of Firmicutes to of highly conserved membrane pattern-recognition
Bacteroidetes in patients with
• Include the genera CAD is higher than in controls receptors, which have an important role in the immune
Chryseomonas and
Helicobacter host defence system that protects against invading
pathogens40,41. For example, stimulation with lipopoly-
Bacteroidetes saccharides (LPS) — potent endotoxins that are part of
• Gram-negative bacteria mainly the outer membrane of Gram-negative bacteria — leads
present in the gut, and to a lesser to the recruitment of adaptor proteins such as myeloid
extent, in the oral cavity and
Actinobacteria atherosclerotic plaques differentiation primary response protein MYD88 to the
• Gram-positive bacteria • Include the genus Bacteroides cytoplasmic domain of TLRs. The recruitment of adap-
• Found in atherosclerotic and the species Porphyromonas
plaques, the oral cavity, tor proteins triggers downstream signalling cascades that
gingivalis
and the gut • The number of Bacteroidetes lead to production of proinflammatory cytokines and
• Include the genus decreases in patients with CAD chemokines40. LPS induce a low-grade inflammatory
Collinsella compared with healthy individuals state and aggravate the progression of atherosclerosis42.
Moreover, saturated fatty acids also act as non-­microbial
ligands for TLR4. These lipids are associated with an
Figure 1 | Body sites of microbiota that influences atherosclerosis. Bacterial DNA altered microbiota, elevated plasma LPS levels, and
detected in atherosclerotic plaques might derive from bacteriaNature Reviews
residing | Cardiology
in the oral cavity metabolic disease42–44. Furthermore, genetic deletion
or the gut. The description of the dominant phylum in each body habitat is based on a of Tlr4 and/or Myd88 in Apoe−/− mice was associated
previous publication6. Firmicutes is the most abundant phyla in the gut and oral cavity,
with reduced macrophage inflammatory response and
whereas Proteobacteria is most abundant in plaques. The methods for microbiota
characterization and taxonomic profiling are described in BOX 2. Specific bacterial taxa aortic lesions45,46.
in atherosclerotic plaques are also present in the oral cavity or gut6, and altered microbial In agreement with the aforementioned animal stud-
composition in the gut is associated with symptomatic atherosclerosis14. Bacteria in ies, TLR4 expression is increased in human atherosclero­
plaques, oral cavity, or gut might initiate or augment atherosclerosis, but causal evidence tic lesions, primarily in macrophages47,48. However,
is lacking. CAD, coronary artery disease. a 2012 meta-analysis found no association between

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Asp299Gly, a known TLR4 polymorphism that dimin-
ishes inflammatory responses49, and risk of athero-
sclerosis50. Although convincing animal data linking
inflammatory signalling to atherosclerosis have been
obtained, evidence in humans remains scarce.
Microbial effects on lipid metabolism
In addition to infections and bacteria in atherosclerotic
plaques or other body sites, the gut microbiota also
influences CVD through the regulation of host metabo-
lism, including cholesterol and lipid metabolism (FIG. 2).
Indeed, gut microbiota is now thought to have a piv-
otal role in low-grade inflammatory metabolic diseases
such as obesity and diabetes51–53, which are associated
with CVD54.
Microbiota-regulated plasma lipids in CVD. A few
studies have investigated the gut microbiota and plasma
lipids in relation to CVD. Patients with atherosclerosis
have altered lipid metabolism, and bacterial taxa in the
oral cavity and gut were found to correlate with plasma
cholesterol levels6, potentially serving as disease bio-
markers. In addition, bacterial richness (that is, n­ umber
of bacterial taxa identified) and specific taxa were associ­
ated with BMI and levels of triglycerides and HDL,
whereas no such clear association was found with levels
of total cholesterol or LDL55. Compared with germ-free
mice, the levels of cholesterol and triglycerides in plasma
of conventionally raised mice are reduced, whereas they
are increased in adipose tissue and liver56. These find-
ings support a relationship between the composition
of the gut microbiota and plasma levels of cholesterol
and lipids.
Bile acid signalling. The gut microbiota can affect lipid
and cholesterol metabolism through bile acids and the
bile acid receptor (also known as farnesoid X receptor,
FXR). Bile acids are important for absorption of dietary
lipids and fat-soluble vitamins. Bile acids are synthesized
from cholesterol in the liver, stored in the gallbladder,
and released into the intestine after ingestion of a meal57.
Box 1 | Koch’s postulates
Koch’s postulates, developed in the late nineteenth century, describe four criteria
that should be fulfilled in order to determine a causative relationship between a
microorganism and a disease:
• The microorganism should be present in all cases of disease
• The microorganism should not be found in healthy individuals
• After isolation and propagation in culture, the microorganism should cause the
disease in an infected organism
• The microorganism, reisolated from the infected organism, should be identified
as the original disease-causing agent
Technical advances in microbiology have led to the understanding that a single
pathogen is not the only cause of a disease, as resistor microorganisms (that is, those
mediating resistance to disease) and microbial consortia are also important122. Thus,
Koch’s postulates should be adapted to include a holistic view that takes into account
whole microbial communities and their interaction with pathogenic microorganisms.
Furthermore, metabolic diseases are associated with altered microbial communities
and with the interaction between microorganisms, so the contribution of individual
pathogens might be of secondary importance in these cases.
Primary bile acids formed in the liver are conjugated to
either taurine or glycine, whereas intestinal deconjuga-
tion by bacterial bile salt hydrolase and further metab-
olism in the colon lead to the formation of secondary
bile acids. Germ-free mice have larger gallbladders and
higher levels of bile acids than those of conventionally
raised mice, including a differential bile acid profile
characterized by increased prevalence of conjugated
primary bile acids and absence of secondary bile acids58.
These data indicate the importance of gut microbial
­regulation of bile acid composition.
Bile acids can act as signalling molecules that bind
to FXR, a nuclear receptor that regulates several physio­
logical processes such as glucose and lipid metabo-
lism57,59. In the liver, bile acid-mediated activation of
FXR regulates triglyceride metabolism, especially the
production of VLDL and de novo lipogenesis. However,
FXR signalling in the small intestine has also been
demonstrated to regulate steatosis and obesity60–63. The
relative effect of microbial-induced FXR signalling on
liver and small intestine needs to be clarified59.
Conflicting data exist on the effect of FXR on athero­
sclerosis development. Nevertheless, diet is an impor-
tant factor that needs to be taken into consideration.
FXR-deficient mice fed a chow diet have impaired HDL
metabolism and reverse cholesterol transport, and, as a
consequence, develop hypercholesterolaemia64. Other
studies that used FXR agonists in atherosclerosis-­
prone mice have also suggested a protective role of this
receptor in atherosclerosis65–67, and this effect could be
weight-­dependent65. Accordingly, Apoe and Fxr double-­
knockout mice had a worse plasma lipid profile and
larger athero­sclerotic lesions than Apoe−/− mice68. By con-
trast, deletion of Fxr in mice lacking apolipo­protein E
or the LDL receptor led to reduced aortic lesions and
decreased plasma LDL cholesterol, suggesting a non-
protective role for FXR69,70. In addition, bacteria not
only influence bile acid signalling through FXR in the
intestine and liver, but possibly also directly in the vas-
culature, given that FXR has been shown to be expressed
in aortic vascular smooth muscle cells and endothelial
cells71–73. To date, the role of microbial regulation of bile
acids and FXR in athero­genesis is not fully understood,
and several Fxr-expressing tissues might be involved.
Most current data are based on animal studies, and
human data are scarce.
Diet and microbial metabolites
The gut microbiota interacts with dietary components74,
and data obtained in the past 5 years suggest that micro-
bial metabolites can promote or prevent CVD (FIG. 2).
Atherosclerosis-promoting diet. A large proportion of
humans have switched from a healthy diet to a Western
diet rich in cholesterols, fats, and sugars. Several compo-
nents of the Western diet can promote inflammation75,
and changes in dietary pattern have been linked to meta­
bolic diseases and atherogenesis54. Moreover, saturated
fats in the Western diet increase endotoxin activity
in plasma compared with a healthy diet, which could
be attributed to a change in microbiota composition

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Box 2 | Methods to study the microbiota
The effect of bacteria on host metabolism can be studied using germ-free animals,
usually mice, which are completely free of any germs in the gut and other body sites.
In contrast to conventionally raised animals, germ-free animals enable the investigation
of the microbiota’s effect on specific conditions. Germ-free animals can also be
colonized with specific bacteria to investigate microbial effects on host physiology,
and faecal transplantations are used to determine whether a trait is transmissible
through an intact microbiota. An important consideration is that germ-free animals
have an immature immune system owing to the lack of microorganisms. An alternative
model is the depletion of the gut microbiota by antibiotic treatment. This procedure
is methodologically easier than using germ-free animals, and it does not require
germ-free facilities. However, antibiotics might cause adverse effects.
Given that many bacteria in the gut have not yet been isolated and cultured,
culture‑independent technologies are used to characterize the microbiota. These
methodologies, which have revolutionized studies on microbial ecology, are generally
based on microbial 16S rRNA gene sequencing to determine taxonomic profiles
including diversity and abundance. Shotgun metagenomic sequencing is used to
investigate the microbiome and provides information on functional capacity in addition
to taxonomy. RNA, proteins, and metabolites can be investigated using
metatranscriptomics, metaproteomics, and metabolomics, respectively, providing a
holistic view of host–microorganism interaction. However, it is essential to bear in mind
that many studies to date have not demonstrated a causal relationship, but rather
associations, between bacteria and diseases.
and/or increased translocation of microorganisms across
the intestinal b­ arrier52,76,77. In Ldlr−/− mice, Western diet
induces changes in circulating metabolites and inflam-
matory markers, indicating an altered cholesterol
homeo­stasis and oxidative stress, which could be partly
reversed by switching the mice to a fibre-rich chow diet78.
Circulating LPS levels and aortic lesion sizes are also partly
reduced through a microbial-­dependent mech­anism78,79,
in agreement with the reduced TLR4‑dependent pro­
inflammatory signalling discussed above. By contrast,
germ-free Apoe−/− mice fed a low-cholesterol diet had
increased aortic lesion sizes compared with convention-
ally raised mice, whereas no difference was found in mice
fed a high-cholesterol diet80. Accordingly, atherosclero-
sis development in germ-free Apoe−/− mice fed a Western
diet was similar to that of conventionally raised mice81.
Thus, a diet-­dependent microbial mechanism might be
involved in the development of atherosclerosis but, on the
basis of the data discussed above, the microbiota does not
have a causal role in atherosclerosis. However, it cannot
be excluded that microbiota induces atherosclerosis and
CVD ­indirectly by causing chronic metabolic dysfunc-
tion or, as discussed below, by producing proatherogenic
molecules from d ­ ietary precursors82,83.
TMAO. Dietary components are also involved in the
atherogenic process. One such example is the proathero­
genic metabolite trimethylamine N‑oxide (TMAO)
generated from microbial metabolism of phosphatidyl-
choline, which is common in red meat, shellfish, and eggs
(FIG. 2). Phosphatidylcholine is metabolized by the gut
microbiota to generate trimethylamine (TMA), which is
further processed in the liver by flavin mono­oxygenases,
leading to the production of TMAO. Plasma levels of
phosphatidylcholine-related metabolites and other
TMAO-producing molecules, including l­ -carnitine and
γ‑butyrobetaine, have been associated with an increased
risk of CVD84–87. Accordingly, increased levels of TMAO
have been correlated with an increased risk of cardio-
vascular events88, prevalent CVD85,89,90, poor prognosis91,
and increased risk of death92. Moreover, TMAO levels
are higher in patients with chronic heart failure than in
healthy controls93.
A study in mice published in 2015 revealed that a­ ortic
lesion size positively correlated with plasma TMAO but
inversely correlated with choline, and that the com-
bined variations of plasma metabolites accounted for
up to 40% of the variation in lesion size94. Apoe−/− mice
treated with phosphatidylcholine metabolites resulted
in increased aortic lesions, which was dependent on the
gut microbiota84. Microbial capacity to produce TMA
seems to be important for atherosclerosis development;
indeed, the aortic lesions of Apoe−/− mice transplanted
with microbiota from high-TMAO-producing mouse
strains (C57BL/6J) are increased compared with those
of Apoe−/− mice transplanted with microbiota from
low-TMAO-producing strains (NZW/LacJ)95. Human
intestinal isolates have been screened to identify ­bacterial
strains that can produce large amounts of TMAO96.
Nine bacterial strains with TMA-producing properties
in vitro led to accumulated plasma TMAO levels when
transplanted into germ-free mice96. Taken together,
these experimental studies in mice support the potential
­harmful effect of TMAO on the cardiovascular system.
To date, the mechanisms by which TMAO contrib-
utes to atherosclerosis are only partially understood
and might involve multiple pathways. In choline-fed
Apoe−/− mice, the number of macrophages in aortic
lesions as well as foam cell formation are reduced when
TMA prod­uction is suppressed by antibiotic treatment84.
By contrast, different concentrations of TMAO did
not affect foam cell formation in mouse macrophages
in vitro97. Therefore, the role of TMAO in foam cell
­formation needs to be clarified.
TMAO can also contribute to atherosclerosis by
inhibiting reverse cholesterol transport. However, the
mechanisms underlying such an effect are unclear, and
might not involve changes in the expression of choles-
terol transporters in macrophages85. TMAO was also
shown to induce several atherosclerosis-­promoting
inflammatory proteins in vascular cells, including
prostaglandin G/H synthase 2 (also known as cyclo­
oxygenase 2), IL‑6, E‑selectin, and intra­cellular adhesion
molecule 1 through activation of nuclear f­ actor‑­κB98.
In addition to atherosclerosis, TMAO might also
affect thrombosis as increased levels of TMAO are
associated with higher rates of thrombotic events and
platelet activation99.
Sex might be an important factor to consider when
studying TMAO synthesis, because expression of
dimethylaniline monooxygenase [N‑oxide-forming] 3
(also known as flavin monooxygenase 3; FMO3) is
increased in human and mouse females compared
with males100. In agreement with these data, germ-free
female mice colonized with TMA-producing bacterial
strains had host-related and sex-related higher levels of
plasma TMAO and increased activity of hepatic FMO3
compared with male mice96. Knock down of Fmo3 in

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different mouse strains results in decreased TMAO Pharmacological elimination or reduction of plasma
plasma levels, altered lipid and cholesterol metabolism, TMAO is a potential therapeutic approach to reduce
and reduced atherosclerotic lesion areas101–103. FMO3 athero­s clerotic cardiovascular events. However, the
is regulated by FXR, and injection of FXR ligands in resulting accumulation of TMA might induce trimethyl­
mice induces the expression of FMO3 and TMAO100, aminuria, also known as fish odour syndrome104. Thus,
thus potentially explaining the reduced atherosclerosis it would be preferable to target the microbiota and TMA
in Fxr-deficient Apoe−/− mice65–67. Furthermore, TMAO synthesis directly. Inhibition of choline trimethylamine-­
decreases the total pool of bile acid in chow-fed mice85. lyase, the microbial enzyme that converts choline to
Taken together, these data indicate that FMO3 is dif- TMA, was demonstrated to reduce TMAO plasma
ferentially regulated by sex, is a target of FXR, and is levels and aortic lesions in mice105, but data in humans
­important for lipid and cholesterol metabolism. are lacking. Similarly, the antiatherosclerotic molecule
resveratrol increases the abundance of bifidobacteria
and lactobacilli in Apoe−/− mice. This increase is associ-
ated with reduced TMAO plasma levels through inhib­
Microbiota ition of microbial TMA production, but the mechanisms
are unclear106.
Infection Metabolism Diet and microbial metabolites Although a number of studies have pointed to an
Invading Choline/carnitine Dietary fibre association between plasma levels of choline, TMAO,
bacteria Lipids
and CVD88–93, available data are still inconsistent. After
bariatric surgery, increased plasma levels of TMAO are
LPS observed despite an improved metabolic profile, which
could be explained by increased intestinal abundance
Choline
Cholesterol TMA-lyase Short-chain of Pseudomonas107. Furthermore, Apoe−/− mice express-
TMA
Macrophage fatty acids: ing human cholesteryl ester transfer protein, which is
• Acetate essential for reverse cholesterol transport, were used
• Butyrate
• Propionate to investigate the role of l-carnitine, which can also be
Proatherogenic FXR
↑ FMO3 TMAO metabolized to TMAO and promote atherosclerosis85.
cytokines
Cyp7a1 ↓ RCT Mice fed a Western diet supplemented with l-carnitine
Cyp27a1 ↑ Foam cells had increased TMAO plasma levels and decreased aortic
Platelet ↓ Systemic
Foam cell ↓ Bile inflammation lesion sizes, with no changes in plasma lipid profile97,
activation
acids ? thus suggesting a protective role for l-carnitine and
TMAO in atherosclerosis. Why l­ -carnitine feeding
gave rise to opposite phenotypes in two different lab-
oratories is currently unclear, although these studies
Plaque rupture used different animal models (mice fed a chow diet85
Atherosclerosis or a Western diet97).
Figure 2 | Microbiota-related pathways in atherosclerosis. Microbiota can affect
Nature Reviews
atherosclerosis by three different pathways. First, bacterial infection activates| Cardiology
the Dietary prevention of atherosclerosis. Diet modula-
immune system causing a harmful inflammatory response. Both local infections by tion with specific dietary components might also be
bacteria invading the atherosclerotic plaque and distant infection can lead to a used to prevent atherosclerosis. However, most studies
proatherogenic response. Infections lead to an increase in proinflammatory cytokines have prov­ided only association data, and mechanistic
and chemokines, which could be mediated by Toll-like receptor 4 expressed in evidence is lacking. Dietary fibre is thought to provide
macrophages. The proinflammatory response, regardless of bacterial infection site health benefits as fibre is considered to lower the risk
(vessel wall or distant site), might affect the progression of atherosclerosis and of CVD. In humans, this effect could be attributed to a
potentially also vulnerability of the plaque. Second, microbiota can influence decreased level of plasma LDL108. Moreover, in healthy
atherosclerosis through altered cholesterol metabolism. The microbiota alters the levels
people, an inverse correlation exists between dietary
of serum triglycerides and cholesterol, and bacterial taxa in the oral cavity and gut
correlate with serum cholesterol6. Gut bacteria are also important in modifying bile acids, fibre and CVD mortality109. However, a limited number
which function as signalling molecules through the farnesoid X receptor (FXR). of human studies have been performed in this field, and
Activation of FXR induces flavin monooxygenase 3 (FMO3) in the liver; this enzyme the effects of diet­ary fibre might be indirectly related to
converts trimethylamine (TMA) to trimethylamine N‑oxide (TMAO). Third, specific CVD through the regulation of glucose metabolism.
dietary  components and microbial metabolites can lead to the production of both Prebiotics are plant-derived fibres that improve the
beneficial and harmful molecules. These pathways might contribute to the development composition or function of the gut microbiota. Indeed,
of atherosclerotic plaques and might also augment the disease, causing plaque rupture Apoe−/− mice fed inulin, a prebiotic polysaccharide, have
and thrombosis. Increased serum levels of TMAO, a bacterial-derived metabolite from reduced cholesterol levels and atherosclerotic lesions
choline and carnitine, are associated with CVD. Some effects of TMAO are directly compared with control mice110. Probiotics are live bac-
related to atherogenesis, such as the inhibition of reverse cholesterol transport (RCT)
teria with health benefits. Lactobacilli are common pro­
and the increase in foam cell formation, and indirect metabolic effects such as the
inhibition of enzymes involved in bile acid synthesis (cholesterol 7α‑monooxygenase biotics that have been evaluated in CVD animal models
[Cyp7a1] and sterol 26‑hydroxylase, mitochondrial [Cyp27a1])85 might also affect plaque with various outcomes, probably owing to strain-specific
development. The effects of dietary fibre and the bacteria-induced formation of effects. For example, consumption of a specific strain
short-chain fatty acids such as acetate, butyrate, and propionate are not clear, but might of Lactobacillus plantarum had beneficial effects in a
involve an overall reduction in systemic inflammation. LPS, lipopolysaccharide. small number of patients, including increased intestinal

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Table 1 | Clinical trials on antibiotic treatment for cardiovascular disease

Antibiotic Number Groups compared End points Conclusion
(treatment of
duration) patients
Azithromycin 7,747 Patients with CAD, serum-positive to Death, nonfatal No significant risk
(12 weeks)33 Chlamydia pneumoniae, treated with reinfarction, coronary reduction
antibiotic or placebo revascularization, angina
Azithromycin 4,012 Patients with CAD, serum-positive Death from CAD, No benefit
(1 year)34 and serum-negative to C. pneumoniae, nonfatal MI, coronary
treated with antibiotic or placebo revascularization, angina
Gatifloxacin 4,162 Patients with CAD, serum-positive Death, MI, angina, No benefit
(~2 years)35 and serum-negative to C. pneumoniae, revascularization, stroke
treated with antibiotic or placebo
Clarithromycin 4,373 Patients with CAD, serum-positive Mortality, MI, angina All-cause mortality
(2 weeks)36 and serum-negative to was higher in the
C. pneumoniae, treated with antibiotic antibiotic group than
or placebo in the placebo group
as a result of higher
CAD mortality
CAD, coronary artery disease; MI, myocardial infarction.

bacterial diversity111, and decreased inflammatory risk
factors for atherosclerosis112. Furthermore, although
some studies showed that lactobacilli reduce blood cho-
lesterol levels in mice and humans113–115, three different
strains of Lactobacillus reuteri were shown to have no
effect on cholesterol levels or atherosclerotic lesions in
Apoe−/− mice116,117.
An example of a bacterium with beneficial effects
on atherosclerosis is Akkermansia muciniphila, which
is part of the normal microbiota and is important in
protecting the intestinal barrier in diet-induced obe-
sity118–120. Apoe−/− mice fed a Western diet supplemented
with A. muciniphila have reduced atherosclerotic lesions,
an effect that could be attributed to an improved intes-
tinal barrier and inflammatory profile121. To summar­
ize, data from mouse studies are conflicting, possibly
because of different bacterial strains used. Furthermore,
different dietary sources might explain the discrepancies
between various studies. Large studies in patients with
atherosclerosis are needed to determine any potential
beneficial effect of probiotics. The modification of bac-
terial composition through specific dietary components,
or regulation of microbial metabolite formation, might
have long-term effects on the development of athero-
sclerosis, whereas short-term treatments might not be
clinically effective.
Conclusions
Atherosclerosis is a complex disease, and character-
izing the underlying mechanisms is, therefore, diffi-
cult. However, increasing evidence indicates that the
micro­biota has a role in this disease. The microbiota
might influence atherosclerosis by promoting plaque
development via activation of the immune system,
alteration of cholesterol metabolism, and production of
bacterial metabolites (such as TMAO; FIG. 2). Together,
these processes might initiate, but more likely sustain
and aggrav­ate, an ongoing process of plaque growth
and rupture. Specific bacterial infections seem to be of
minor importance in atherosclerosis progression and the
presence of bacterial DNA in the athero­sclerotic plaque
might reflect instead a systemic infection and/or bacterial
uptake by macrophages. However, ­bacterial ­infections
might contribute to plaque rupture.
Whether CVD and atherosclerosis can be success-
fully treated by targeting the microbiota is still not clear.
Long-term treatment with antibiotics is unfeasible and
could cause severe adverse effects. Although adherence
to strict diets is generally low, dietary interventions,
for example with fibre, might be more practical in
atherosclerosis prevention compared with antibiotics.
Modulation of microbiota composition or the regulation
of pathways that produce proatherogenic metabolites,
such as TMAO and other molecules, might also be novel
therapeutic strategies.
New insights into how atherosclerosis can be affected
by commensal microbiota have been provided over the
past decade. However, the interaction between host and
microorganisms throughout the human lifetime is a
crucial factor in the slow progression of atherosclerosis.
The challenge is to determine whether specific bacterial
taxa and/or metabolites are the cause or consequence
of the disease. To answer this question and to deter-
mine the disease mechanisms, linking the microbiota
(or microbial communities) to specific functions is of
critical importance.

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Acknowledgements
The authors thank Anna Hallén, Walleneberg Laboratory at
University of Gothenburg, Sweden, for assistance with figures
and artwork. The authors’ laboratory is supported by grants
from AFA-insurances, the Ragnar Söderberg’s Foundation,
the Swedish Foundation for Strategic Research, the Swedish
Heart Lung Foundation, the Swedish Research Council,
Torsten Söderberg’s, and LUA-ALF grants from Västra
Götalandsregionen. F.B. is a recipient of ERC consolidator
Grant 2013 (European Research Council, Consolidator grant
615362-METABASE).
Author contributions
Both authors researched data for the article, discussed its
content, and wrote, reviewed, and edited the manuscript
before submission.
Competing interests statement
F.B. is founder and shareholder of Metabogen AB. A.L.J.
declares no competing interests.

NATURE REVIEWS | CARDIOLOGY ADVANCE ONLINE PUBLICATION | 9

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