Curr Hypertens Rep (2017) 19:33
DOI 10.1007/s11906-017-0725-2
GUT MICROBIOME, SYMPATHETIC NERVOUS SYSTEM, AND HYPERTENSION (M RAIZADA AND E M. SUMNERS, SECTION EDITORS)
Oral Microbiome and Nitric Oxide: the Missing Link
in the Management of Blood Pressure
Nathan S. Bryan 1 & Gena Tribble 2 & Nikola Angelov 2
# Springer Science+Business Media New York 2017
Abstract Having high blood pressure puts you at risk for
heart disease and stroke, which are leading causes of death
in the USA and worldwide. One out of every three Americans
has hypertension, and it is estimated that despite aggressive
treatment with medications, only about half of those medicat-
ed have managed blood pressure. Recent discoveries of the
oral microbiome that reduces inorganic nitrate to nitrite and
nitric oxide provide a new therapeutic target for the manage-
ment of hypertension. The presence or absence of select and
specific bacteria may determine steady-state blood pressure
levels. Eradication of oral bacteria through antiseptic mouth-
wash or overuse of antibiotics causes blood pressure to in-
crease. Allowing recolonization of nitrate- and nitrite-
reducing bacteria can normalize blood pressure. This review
will provide evidence of the link between oral microbiota and
the production of nitric oxide and regulation of systemic blood
pressure. Management of systemic hypertension through
maintenance of the oral microbiome is a completely new par-
adigm in cardiovascular medicine.
Keywords Gut microbiome . Blood pressure . Hypertension .
Nitric oxide . Diet . Nitrate . Nitrite
This article is part of the Topical Collection on Gut Microbiome,
Sympathetic Nervous System, and Hypertension
* Nathan S. Bryan
Nathan.bryan@bcm.edu
1
Department of Molecular and Human Genetics, Baylor College of
Medicine, The University of Texas Health Science Center at
Houston, Houston, TX, USA
2
Department of Periodontics, The University of Texas Health Science
Center at Houston, 7000 Fannin, Houston, TX 77030, USA
Hypertension
Human blood pressure regulation is complex, and despite de-
cades of research into the variables affecting resting blood
pressure, there is still a significant knowledge gap.
Hypertension is highly prevalent in the adult population in
the USA, especially among persons older than 60 years of
age and affects approximately one billion adults worldwide
[1, 2]. In the USA, about 77.9 million (one out of every three)
adults have high blood pressure. Among persons 50 years of
age or older, isolated systolic hypertension is the most com-
mon form of hypertension [4, 5], and systolic blood pressure
becomes more important than diastolic blood pressure as an
independent risk predictor for most all cardiovascular-related
disease including end-stage renal disease (ESRD) [69]. In
some age groups, the risk of cardiovascular disease doubles
for each increment of 20/10 mmHg of blood pressure, starting
as low as 115/75 mmHg. In addition to coronary heart diseases
and stroke, complications of raised blood pressure include
heart failure, peripheral vascular disease, renal impairment,
retinal hemorrhage, and visual impairment.
Worldwide, raised blood pressure is estimated to cause 7.5
million deaths, about 12.8% of the total of all deaths. This
accounts for 57 million disability adjusted life years
(DALYS) or 3.7% of total DALYS. Globally, the overall prev-
alence of raised blood pressure in adults aged 25 and over was
around 40% in 2008. The proportion of the worlds population
with high blood pressure, or uncontrolled hypertension, fell
modestly between 1980 and 2008. However, because of pop-
ulation growth and aging, the number of people with uncon-
trolled hypertension rose from 600 million in 1980 to nearly
one billion in 2008 [10].
Despite major advances in understanding the pathophysi-
ology of hypertension and availability of antihypertensive
drugs, suboptimal blood pressure control is still the most
33 Page 2 of 8
important risk factor for cardiovascular mortality. According
to the AHA 2013 Statistics Fact Sheet, although 75% of peo-
ple that know they have hypertension and are under current
treatment, only about 52% of those have it controlled. The
Systolic Blood Pressure Intervention Trial (SPRINT) showed
that among adults with hypertension but without diabetes,
lowering systolic blood pressure to a target goal of less than
120 mmHg, as compared with the standard goal of less than
140 mmHg, resulted in significantly lower rates of fatal and
nonfatal cardiovascular events and death from any cause [11].
Because blood pressure remains elevated in 50% of all treat-
ed hypertensive patients [12, 13], and more effective blood
pressure management can save lives, novel, efficacious, and
cost-effective therapeutic strategies are urgently required for
the treatment of hypertension.
Nitric Oxide
The discovery of endothelium derived relaxing factor (EDRF)
in 1980 [14] that was later identified as nitric oxide (NO)
[15, 16] revolutionized vascular biology and identified new
strategies for controlling blood pressure. NO is produced en-
dogenously from the five-electron oxidation of the guanidino
nitrogen of L-arginine by the enzyme isoform endothelial ni-
tric oxide synthase (eNOS) [17]. NO produced or generated in
the vasculature then diffuses into the underlying smooth mus-
cle causing these muscles to relax. This results in vasodilation,
causing a reduction in systemic blood pressure and an increase
in blood flow and oxygen delivery to specific vascular beds.
In healthy subjects, activation of eNOS causes vasodilation in
both muscular conduit vessels and resistance arterioles. In
contrast, in subjects with atherosclerosis or endothelial dys-
function, similar stimulation yields attenuated vasodilation in
peripheral vessels and causes paradoxical vasoconstriction in
coronary arteries, thus indicating a decrease in the production
and/or bioavailability of NO [18, 19]. Emerging evidence
shows that endothelial dysfunction and subsequent NO defi-
ciency are critically associated with the development of hy-
pertension and other forms of cardiovascular disease [20].
Hypertension accelerates atherosclerosis. Interestingly, endo-
thelial dysfunction can be demonstrated in patients with risk
factors for atherosclerosis in the absence of atherosclerosis
itself [21, 22]. Experimental and clinical studies provide evi-
dence that detective endothelial NO function is not only asso-
ciated with all major cardiovascular risk factors, such as hy-
perlipidemia, diabetes, hypertension, smoking, and the sever-
ity of established atherosclerosis but also has a profound pre-
dictive value for future atherosclerotic disease progression
[23]. These results indicate that essential hypertension is char-
acterized by an age-related reduction of nitric oxide produc-
tion and bioavailability.
Curr Hypertens Rep (2017) 19:33
Human Oral MicrobiomeBacterial Nitrate
Reduction
The human microbiome is composed of many different bac-
terial species, which outnumber our human cells ten to one
and provide functions that are essential for our survival. The
microbiota of the lower intestinal tract is widely recognized as
playing a symbiotic role in maintaining a healthy host physi-
ology [24] by participating in nutrient acquisition and bile acid
recycling, among other activities. In contrast, although the role
of oral microbiota in disease is well studied, specific contribu-
tions to host health are not well defined. A human nitrogen
cycle has been identified. This pathway, termed the
enterosalivary nitrate-nitrite-nitric oxide pathway, can posi-
tively affect nitric oxide homeostasis and represents a poten-
tial symbiotic relationship between oral bacteria and their hu-
man hosts [25, 26]. It is now recognized that the oral com-
mensal bacteria provide an important metabolic function in
human physiology by contributing a nitric oxide synthase
(NOS)-independent source of NO. This process is analogous
to the environmental nitrogen cycle, whereby soil bacteria
convert atmospheric nitrogen oxides to usable forms for plant
growth. Human nitrate reduction requires the presence of
nitrate-reducing bacteria as mammalian cells cannot effective-
ly reduce this anion.
Inorganic nitrite (NO2) and nitrate (NO3) are known as
food additives to cured meats and naturally occurring in green
leafy vegetables [27, 28] but also as oxidative end products of
endogenous NO production [29]. However, from research per-
formed over the past decade, it is now apparent that nitrate and
nitrite are physiologically recycled in blood and tissue to form
NO and other bioactive nitrogen oxides [30, 3133] and now
considered essential nutrients [34, 35]. As a result, they should
now be viewed as storage pools for NO-like bioactivity to be
acted upon when enzymatic NO production from NOS is insuf-
ficient, such as during anaerobic conditions or uncoupling of
NOS. The bioactivation of nitrate from dietary (mainly green
leafy vegetables) or endogenous sources (oxidation of NO) re-
quires its initial reduction to nitrite, and because mammals lack
specific and effective nitrate reductase enzymes, this conversion
is mainly carried out by commensal bacteria [36]. Dietary
nitrate is rapidly absorbed in the upper gastrointestinal tract. In
the blood, it mixes with the nitrate formed from the oxidation of
endogenous NO produced from the NOS enzymes. After a meal
rich in nitrate, the nitrate levels in plasma increase greatly and
remain high for a prolonged period of time (the plasma half-life
of nitrate is 56 h). The nitrite levels in plasma also increase after
nitrate ingestion after approximately 90 min [37] provided it can
be reduce by oral nitrate-reducing bacteria. Although much of
the nitrate is eventually excreted in the urine, up to 25% is
actively taken up by the salivary glands and is concentrated up
to 20-fold in saliva [37, 38]. In the mouth, commensal faculta-
tive anaerobic bacteria reduce salivary nitrate to nitrite during
Curr Hypertens Rep (2017) 19:33 Page 3 of 8 33

anaerobic respiration by the action of nitrate reductases [26, 36].

After a dietary nitrate load, the salivary nitrate and nitrite levels
can approach 10 mM and 12 mM, respectively [37]. When
saliva enters the acidic stomach (11.5 L per day), much of
the nitrite is rapidly protonated to form nitrous acid (HNO2;
pKa 3.3), which decomposes further to form NO and other
nitrogen oxides [32, 33]. A simplified human nitrogen cycle is
illustrated in Fig. 1. More recent studies indicate that nitrite does
not have to be protonated to be absorbed and is about 98%
bioavailable when swallowed in an aqueous solution [39].
Salivary nitrate is metabolized to nitrite via a two-electron
reduction, a reaction that mammalian cells are unable to per-
form, during anaerobic respiration by nitrate reductases pro-
duced by facultative and obligate anaerobic commensal oral
bacteria [40, 41]. As illustrated in Fig. 2, identification of
specific partial denitrifying bacteria in the oral cavity can pro-
vide optimal conditions for nitrate reduction with nitrite accu-
mulation in the saliva. Although a few nitrate-reducing bacte-
ria have been identified in the oral cavity [42], we have ana-
lyzed nitrate reduction by bacterial communities present in
tongue scrapings from healthy human volunteers during 4 days
of in vitro growth and performed a parallel metagenomic anal-
ysis of these samples to identify specific bacteria associated Fig. 2 The biological nitrogen cycle. Nitrate is reduced all the way down
to elemental nitrogen by a series of enzymatic steps from nitrate reductase
with nitrate reduction. Through 16S rRNA gene pyrosequenc- (NR), nitrite reductase [3], NO reductase (NOR), and nitrous oxide
ing and whole genome shotgun (WGS) sequencing and anal- reductase (N2OR). Identifying bacteria or communities that only reduce
ysis, we identified specific taxa that likely contribute to nitrate nitrate and/or nitrite will allow for nitrite accumulation and more efficient
reduction. Biochemical characterization of nitrate and nitrite NO
reduction by four candidate species indicates that complex
community interactions contribute to nitrate reduction [43]. worst reducing sample that belonged to the genera of interest
The bacterial communities had varying potential for nitrate and were identified through 16S rRNA gene pyrosequencing
reduction, and our study identified 14 candidate species that and analysis were Granulicatella adiacens, Haemophilus
were present in communities with the best nitrate reduction parainfluenzae, Actinomyces odontolyticus, Actinomyces
activity. The 14 species present at an abundance of at least viscosus, Actinomyces oris, Neisseria flavescens, Neisseria
0.1% in the best nitrate-reducing sample and at the highest mucosa, Neisseria sicca, Neisseria subflava, Prevotella
abundance in this sample compared to the intermediate and melaninogenica, Prevotella salivae, Veillonella dispar,
Veillonella parvula, and Veillonella atypica. Additionally,
Fusobacterium nucleatum and Brevibacillus brevis were des-
DIET ignated as species of interest even though they were not at a
NO3- relative abundance of at least 0.1% in the WGS best nitrate-
reducing sample [43]. Previously, the Doel et al., 2005, study
isolated and identified five genera of oral nitrate-reducing
BACTERIA bacterial taxa on the tongues of healthy individuals:
2 e- reduction
Veillonella, Actinomyces, Rothia, Staphylococcus, and
Oxyheme -
proteins NO2 Propionibacterium [42]. In our investigation, Veillonella spe-
cies were the most abundant group of nitrate reducers isolated
Oxygen, BACTERIA from the tongue, followed by Actinomyces spp. Veillonella
ceruloplasmin 1 e- reduction
Acid was the most abundant nitrate-reducing genus detected in
Deoxy-hemoproteins the original tongue scrapings, although Prevotella, Neisseria,
NO and Haemophilus were all found at a higher abundance than
NOS Actinomyces, highlighting the higher resolution of our study.
oxidation L-arginine
reduction This difference in resolution is likely due to our use of a
Fig. 1 The human nitrogen cycle whereby nitrate is serially reduced to sequencing-based approach, which allowed us to survey the
nitrite and NO providing the host with a source of bioactive NO native bacterial environment on the dorsal surface of the
33 Page 4 of 8
tongue without depending on the growth requirement neces-
sary for classic culture-based techniques. Metagenomic data
are important and informative in that one can determine what a
bacterial community is capable of doing, yet it is limited in the
sense that it cannot inform what the community is actually
doing, which can vary under different circumstances. Thus,
while each community had the same capacity for nitrate re-
duction, the true activity clearly differed between these com-
munities. Furthermore, as not all healthy donors had nitrate-
reducing bacteria in their oral cavity, there may be a significant
number of individuals who may not have a functioning
nitrate-nitrite-NO enterosalivary pathway to support systemic
health. The presence or absence of these select bacteria may
be a new determinant of nitrite and NO bioavailability in
humans and, thus, a new consideration for cardiovascular dis-
ease risk. Further studies on multi-species biofilms integrating
biochemical, metagenomic, and metatranscriptomic data will
answer these important questions and provide more informa-
tion regarding the community dynamics that contribute to oral
nitrate reduction that results in nitrite accumulation.
Nitrite-Based Signaling
The production of nitrite from nitrate-reducing bacteria may
have profound implications on the health of the human host.
Numerous studies have shown that nitrite produced from bac-
terial nitrate reduction is an important storage pool for NO in
blood and tissues when NOS-mediated NO production is in-
sufficient [44, 4548]. Nitrite is also an oxidative breakdown
product of NO that has been shown to serve as an acute marker
of NO flux/formation [49]. Nitrite is now recognized as a cell
signaling molecule that can act as a storage from of NO as well
as a NO independent signal [30, 50]. Nitrite is in steady-state
equilibrium with S-nitrosothiols [30, 51] and has been shown
to activate soluble guanylyl cyclase (sGC) and increase cGMP
levels in tissues [30] and lead to vasodilation [52, 53].
Therefore, it is an ideal candidate for restoring both cGMP-
dependent and cGMP-independent NO signaling. In addition
to the oxidation of NO, nitrite is also derived from reduction of
salivary nitrate by commensal bacteria in the mouth and gas-
trointestinal tract [54, 55], as well as from dietary sources,
such as meat, vegetables, and drinking water.
Since nitrate reduction by microbial communities generates
nitrite, it is of great importance to be able to recognize specific
bacteria in and on the body that are capable of generating
nitrite from nitrate. Once nitrite is formed, it can be utilized
as a substrate for NO production. Although largely inefficient,
there exists a number of nitrite-reducing system in mammals.
Nitrite reductase activity in mammalian tissues has been
linked to the mitochondrial electron transport system [56,
57], protonation [31], deoxyhemoglobin [58], and xanthine
oxidase [59, 60]. Nitrite can also transiently form S-
Curr Hypertens Rep (2017) 19:33
nitrosothiols (RSNOs) under both normoxic and hypoxic con-
ditions [61], and a recent study by Bryan et al. demonstrates
that steady-state concentrations of tissue nitrite and S-nitroso
species are affected by changes in dietary NOx (nitrite and
nitrate) intake [30]. Furthermore, enriching dietary intake of
nitrite and nitrate translates into significantly less injury from
myocardial infarction [44]. Previous studies demonstrated
that nitrite therapy given intravenously prior to reperfusion
protects against hepatic and myocardial ischemia/reperfusion
(I/R) injury [62]. Additionally, experiments in primates re-
vealed a beneficial effect of long-term application of nitrite
on cerebral vasospasm [63]. Moreover, inhalation of nitrite
selectively dilates the pulmonary circulation under hypoxic
conditions in vivo in sheep [64]. Topical application of nitrite
improves skin infections and ulcerations [65]. Furthermore, in
the stomach, nitrite-derived NO seems to play an important
role in host defense [36] and in regulation of gastric mucosal
integrity [66]. All of these studies, along with the observation
that nitrite can act as a marker of NOS activity [49], have
opened new avenues for the diagnostic and therapeutic appli-
cations of nitrite, especially in cardiovascular diseases, using
nitrite as a marker as well as an active agent. Oral nitrite has
also been shown to reverse L-NAME (NOS inhibitor)-in-
duced hypertension and serve as an alternate source of NO
in vivo [67]. In fact, a report by Kleinbongard et al. [68]
demonstrates that plasma nitrite levels progressively decrease
with increasing cardiovascular risk. Since a substantial portion
of steady-state nitrite concentrations in blood and tissue are
derived from dietary sources [30], modulation of nitrate in-
take along with optimal nitrate-reducing microbial communi-
ties may provide a first line of defense for conditions associ-
ated with NO insufficiency [50].
Eradication of Nitrate-Reducing Bacteria Eliminates
Health Benefits of Nitrate-Based Diets
and Interventions
In various animal models and in humans, dietary nitrate sup-
plementation has shown numerous beneficial effects, includ-
ing a reduction in blood pressure, protection against ischemia-
reperfusion damage, restoration of NO homeostasis with as-
sociated cardioprotection, increased vascular regeneration af-
ter chronic ischemia, and a reversal of vascular dysfunction in
the elderly [34, 35, 69, 70]. Some of these benefits were re-
duced or completely prevented when the oral microbiota were
abolished with an antiseptic mouthwash [69, 71]. Plasma and
salivary nitrite levels are abolished after a dietary nitrate load
in healthy subjects taking an antiseptic mouthwash [72]. It has
been reported that dietary nitrate reduces blood pressure in
healthy volunteers [46, 73], and that the effects are abolished
after rinsing with oral antiseptic mouthwash [74]. Both strong
and weak antibacterial agents suppress the rise in plasma
Curr Hypertens Rep (2017) 19:33
nitrite observed following the consumption of a high nitrate
diet and stronger antiseptics can influence the blood pressure
response during low-intensity exercise [75]. Additionally, it
was recently shown that in the absence of any dietary modifi-
cations, a 7-day period of antiseptic mouthwash treatment to
disrupt the oral microbiota reduced both oral and plasma ni-
trite levels in healthy human volunteers and was associated
with a sustained increase in both systolic and diastolic blood
pressure [74]. Oral nitrite exerts antihypertensive effects in
the presence of antiseptic mouthwash that disrupts the
enterosalivary circulation and reduction of nitrate [76].
Altogether, these studies firmly establish the role for oral
nitrate-reducing bacteria in making a physiologically relevant
contribution to host nitrite and thus NO levels, with
measureable physiological effects. It appears that providing
nitrite can overcome the absence of microbial nitrate
reduction.
Conclusion
Clearly, the potential for the enterosalivary nitrate-nitrite-NO
pathway to serve as a NO bioavailability maintenance system
by harnessing the nitrate reductase activity of specific commensal
bacteria calls for studies that may be profound and truly transfor-
mative. These studies will have the potential to (1) redefine the
meaning of healthy oral microbiome to include microbes asso-
ciated with NO production, (2) provide a new target for NO-based
therapies and open a new direction in cardiovascular research, and
(3) allow development of new diagnostics targeted at specific oral
microbial communities or select bacteria, the absence of which
may reflect a state of NO insufficiency and change the treatment
strategies for NO restoration in a number of different diseases.
With the loss of NO signaling and homeostasis being one of the
earliest events in the onset and progression of cardiovascular dis-
ease, targeting microbial communities early in the process may
lead to better preventative interventions in cardiovascular medi-
cine. This may also affect the way oral health professionals rec-
ommend oral hygienic practices. Manipulation of the human
microbiome as a therapeutic target for disease management is
on the near horizon. The oral cavity is an attractive target for
probiotic and/or prebiotic therapy because of the ease of access.
A full understanding of the enterosalivary nitrate-nitrite-NO path-
way will require the generation and integration of a complete set
of data from metagenomic, metatranscriptomic, metaproteomic,
and metametabolomic studies coupled to biochemical functional
assays. The potential to restore the oral flora as a means to provide
NO production is a completely new paradigm for NO biochem-
istry and physiology as well as for cardiovascular medicine and
dentistry.
There is a known correlation between oral health and sys-
temic disease [77]. Importantly, because oral NO production is
dependent on oral nitrate-reducing bacteria, these observations
Page 5 of 8 33
suggest that the link between oral health issues such as chronic
periodontitis and cardiovascular disease may be due in part to
decreased abundance of nitrate reducers and concurrent increase
of pathogenic bacterial species in the oral cavity. Disruption of
nitrite and NO production in the oral cavity may contribute to
the oral-systemic link between oral hygiene and cardiovascular
risk and disease. The identification of new biomarkers for NO
insufficiency and the exploitation of the oral microbiota to in-
crease cardiovascular health will be enabled by further charac-
terization of the enzymatic activities of native oral bacterial
communities from larger healthy cohorts and specific patient
populations. These cohorts should consist not only of specific
US population but also of other around-the-world (European,
Asian) populations. It is likely that the oral microbiomes of
different ethnic groups, even those within different regions of
the US, vary widely. It will be important to determine whether
different nitrate-reducing communities are more prevalent in
geographically dispersed healthy populations; likewise, it will
also be important to determine whether different nitrate-
reducing communities are lacking in specific patient populations
from around the world. If certain patient populations lack spe-
cific nitrate-reducing bacteria, personalized treatments to enrich
for nitrate reducers may be warranted. It may be time to discour-
age the use of antiseptic mouthwash. Additionally, while antibi-
otics are sometimes used to target specific bacterial species, it is
possible that potential deleterious effects of antibiotic usage on
nitrate-reducing communities may preclude the use of antibi-
otics in specific patient populations.
For the past 30 years, scientists have focused on NO
production/regulation at the level of nitric oxide synthase
(NOS), through the five-electron oxidation of L-arginine.
However, this pathway becomes dysfunctional with age and
disease [78]. The notion that this deficiency can be overcome
by targeting oral bacteria is profound and revolutionary.
Therapeutically, then, perhaps an effective strategy to promote
NO production and overcome conditions of NO insufficiency
may not be targeted at eNOS, but rather to target specific oral
nitrate-reducing bacterial communities and increasing the con-
sumption of nitrite and nitrate enriched foods and vegetables.
From a public health perspective, we may be able to make
better recommendations on diet and relevant host-microbe
communities to affect dramatically the incidence and severity
of a number of symptoms and diseases characterized by NO
insufficiency. Development of novel therapeutics or strategies
to restore NO homeostasis can have a profound impact on
disease prevention [79]. These new discoveries in the oral
bacterial microbiome suggest that an effective strategy to pro-
mote therapeutically NO production and overcome conditions
of NO insufficiency may not solely be to target NOS, but,
rather, to focus on understanding specific oral bacterial com-
munities and the optimal conditions for efficient oral nitrate
reduction. It appears from early studies that many individuals
may not have the optimal microbial communities for
33 Page 6 of 8
maximum nitrate reduction, causing a disruption in a critical
NO production pathway. Understanding and harnessing this
alternative pathway may prove to be a viable and cost-
effective strategy for maintaining NO homeostasis in humans.
Because NO signaling affects all organ systems and almost all
disease processes described to date, this novel approach to NO
regulation has the potential to affect the study and treatment of
many diseases across all organ systems.
Compliance with Ethical Standards
Conflict of Interest Dr. Bryan reports personal fees and other from
HumanN, Inc. In addition, he has a patent 8,298,589 with royalties paid to
University of Texas, a patent 8,303,995 with royalties paid to University of
Texas, a patent 8,435,570 with royalties paid to University of Texas, a patent
8,962,038 with royalties paid to University of Texas, a patent 9,119,823 with
royalties paid to University of Texas, and a patent 9,241,999 with royalties
paid to University of Texas. XXX declare no conflicts of interest relevant to
this manuscript. Drs. Tribble and Angelov declare no conflicts of interest
relevant to this manuscript.
Human and Animal Rights and Informed Consent This article does
not contain any studies with human or animal subjects performed by any
of the authors.
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