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ORIGINAL CONTRIBUTIONS
nature publishing group
see related editorial on page x
Methanogens in Human Health and Disease
Mark Pimentel, MD1, Robert P. Gunsalus, PhD2, Satish S.C. Rao, MD, PhD3 and Husen Zhang, PhD4
There is growing evidence that host/microbial interactions within the gut can have a profound impact on human
health and disease; in fact, the intestinal microflora have been shown to influence the innate physiology, biochemistry,
immunology, maturation of the vasculature, and gene expression in a host. Although most research has focused on
gut bacteria, current evidence suggests that the Archaea—an ancient domain of single-celled organisms—are resident
within the gut in high numbers, and have direct and indirect effects on the host. In particular, the methanogens are an
essential component of luminal intestinal microbial ecosystems. Methanogens oxidize hydrogen to produce methane
and ensure more complete fermentation of carbohydrate substrates, leading to higher production and adsorption of
short-chain fatty acids, which may lead to obesity. Methane, the key product of carbohydrate fermentation by the
methanogens, has long been thought to produce no ill effects in humans aside from gaseous distention. However,
recent evidence has linked methane production to the pathogenesis of constipation and irritable bowel syndrome
(IBS), as well as obesity. In particular, a significant percentage of patients with IBS and constipation excrete methane,
suggesting an overabundance of methanogenic archaea in their gut. Methane by itself may influence intestinal
transit and pH and facilitate development of constipation. If methane has a direct or indirect effect on intestinal
transit, attempting to manipulate methanogenic flora may serve as a novel therapeutic option. Thus, understanding
methanogens and their role in gut function/dysfunction is vital to our understanding of human health and disease.
Am J Gastroenterol Suppl 2012; 1:28–33; doi:10.1038/ajgsup.2012.6
INTRODUCTION
The total quantity of bacterial microbiota in the human host outnumber host cells by at least 100-fold. In the gut alone, the bacterial population is ~100 trillion and is composed of between 500
and 1,000 different species (1). Thus, it should be expected that
the host/microbial interactions within the gut exert effects on
human health and disease. In fact, the microbiota have direct and
indirect effects on host physiology, biochemistry, and immunology and influence host gene expression and development; additional studies in germ-free and gnotobiotic animals have found
that the microbiota have a direct effect on the enteric nervous
system, including alterations in the rate of gastric emptying and
intestinal transit, reductions in the expression of γ-aminobutyric
acid, and increases in cecal size (1). The profound effect of the
intestinal microflora has led to the proposal that they should be
considered a virtual organ (2,3).
The majority of recent interest in the effects and composition of human and animal intestinal microbiota have focused
on eubacteria and, to a lesser extent, on single-celled eukaryotes. Although once considered bacteria, the Archaea—an ancient
phylum of single-celled organisms lacking a nucleus or other
membrane-bound organelles—are resident within the gut in high
numbers and have direct and indirect effects on other resident
microbes and the human host. In particular, the methanogens—
anaerobes that respire hydrogen to produce methane—have
attracted considerable attention, both as contributors to host disease and health and as key mediators of the composition of gut
microbiota through a complex web of symbiotic, syntrophic, and
competitive relationships.
ARCHAEA
The archaea form an ancient microbial lineage that is as different from prokaryotic eubacteria as they are from eukaryotes.
Although some archaea, such as the halophiles and thermophiles,
are restricted to extreme environments, the methanogens are
widespread and are found in nearly every habitat in which
anaerobic biodegradation of organic compounds occurs, including the human and animal intestinal tracts (4,5), freshwater and
marine sediments, and anaerobic waste digesters (4). Acidophilic
methanogens are ubiquitous in peatlands, which are among the
largest natural sources of atmospheric methane (6). The methanogens are an essential component of luminal intestinal microbe
1
GI Motility Program, Cedars-Sinai Medical Center, Los Angeles, California, USA; 2Department of Microbiology, Immunology, and Molecular Genetics and the
UCLA Institute of Genomics and Proteomics, University of California, Los Angeles, Los Angeles, California, USA; 3Section of Gastroenterology/Hepatology, Medical
College of Georgia, Georgia Health Sciences University, Augusta, Georgia, USA; 4Department of Civil, Environmental, and Construction Engineering, University
of Central Florida, Orlando, Florida, USA. Correspondence: Mark Pimentel, MD, FRCP(C), GI Motility Program, Cedars-Sinai Medical Center, 8730 Alden Drive,
Suite 225E, Los Angeles, California 90048, USA. E-mail: [email protected]
This article was published as part of a supplement sponsored by the Gi Health Foundation, a nonprofit 501(c)(3) educational organization dedicated to increasing
awareness of the effect of gastrointestinal disorders in the United States. The foundation’s goal is to provide health professionals with the most current education
and information on gastrointestinal health.
The American Journal of GASTROENTEROLOGY Supplements
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Postinfectious Gut Dysfunction
ecosystems where they respire hydrogen to produce methane
and ensure more complete fermentation of substrates (5). Formate (7), methanol, (8), and acetate are also metabolized by some
methanogenic species.
A number of methanogens have been studied intensively. Methanobrevibacter ruminatum is found in ruminant species (e.g., goats,
sheep, cattle) in the rumen, where it is involved in fermentation of
feed. This pathway results in the production of between 250 and
500 l of methane in each animal per day. In humans, Methanobrevibacter smithii accounts for 94% of the methanogen population;
Methanosphaera stadtmaniae (9), Methanobrevibacter oralis, and
members of the order Methanosarcinales are also resident in the
gut (10). Methanogens are also found in the oral cavity (primarily M. oralis) and are particularly common at subgingival sites in
patients with periodontal disease (11).
METHANE AND METHANOGENESIS
Fermentation of dietary fiber by the intestinal bacterial community—primarily the Bacteroides and Firmicutes—results in the
generation of short-chain fatty acids (SCFAs) such as acetate,
propionate, and butyrate, which account for up to 10% of daily
caloric intake (see also the article by DiBaise et al. in this supplement) (12). The fermentation of fiber also produces formate, carbon dioxide, and hydrogen gas (13). The hydrogen gas produced
during fermentation is eliminated through three pathways: (i) via
flatus; (ii) absorption into the systemic circulation and respiratory
excretion; and (iii) metabolism by the colonic microbiota, which
is considered to be the major source of hydrogen disposal (14).
Hydrogen is metabolized by a range of bacterial and archaeal
flora in the gut, including the sulfate-reducing bacteria, which use
hydrogen to reduce sulfate to sulfide; the acetogenic bacteria, which
reduce carbon dioxide to acetate via molecular hydrogen; and the
methanogenic archaea, which convert hydrogen gas to methane
(Figure 1). In the colon of methanogenic humans, the methanogens are the primary hydrogen-consuming flora, reducing flatulence through 4:1 conversion of hydrogen gas to methane gas (15).
In humans, methanogenesis and sulfate reduction are the two
primary pathways of hydrogen metabolism in the colon. These
pathways are mutually exclusive in humans, such that only individuals with methane excretion demonstrate methanogenesis in
feces, whereas nonmethanogenic individuals show high levels of
sulfate reduction in the feces (16). Methanogenesis is clearly linked
to colonization of the gut by bacteria. Methane is not detected in
children until ~3 years of age, and its production peaks at age 10
when the adult distribution is reached. Based on the results of
breath testing, approximately one-third of healthy adults produce
methane; however, fecal incubation studies suggest a larger proportion of the population— ~72%—are methane producers (14).
In the gut, the methanogens exist in a syntrophic relationship
with other residents of the gut. For example, it has been shown
that accumulation of hydrogen gas inhibits eubacterial energy
production through reduction of the activity of nicotinamide
adenine dinucleotide dehydrogenases. Removal of this gas by
methanogens thus improves fermentation efficiency. In fact, cer© 2012 by the American College of Gastroenterology
H2-producing
bacteria
Sulfate-reducing
bacteria
Methanogens
ΔGo, :–152.2 (kJ/mol)
H2S
Acetogenic
bacteria
ΔGo, :–130 (kJ/mol)
CH4
80%
Liver
detoxification
Flatus
ΔGo, :–95 (kJ/mol)
Acetate
20%
Lung
Fecal
loss
Absorb
by colon and
metabolized
Figure 1. Pathways for hydrogen utilization in the gut. Adapted from
Sahakian et al. (14).
tain methanogens may act as the “power brokers” in the distal
gut community, regulating energy harvest from dietary glycans
and host energy storage (13). In a study conducted by Samuel
and Gordon (13), germ-free mice were colonized with Bacteroides thetaiotaomicron, which metabolizes polysaccharides with
or without the methanogen M. smithii or the sulfate-reducing bacterium Desulfovibrio piger. Notably, cocolonization with
M. smithii, but not D. piger, directed B. thetaiotaomicron to focus
on fermentation of dietary fructans to acetate and resulted in a significant increase in host adiposity. Syntrophic H2 production and
removal in obese humans have been linked to Prevotellaceae and
Methanobacteriales, respectively, which lead to a more complete
fermentation of indigestible dietary substances and more SCFA
production and absorption by the human gut (17).
METHANE, METHANOGENS, AND HUMAN DISEASE
Is there a role for methane in constipation-predominant
irritable bowel syndrome or chronic constipation?
Methane is a colorless, odorless, volatile, inert gas that has long
been thought to produce no ill effects in humans aside from discomfort from gaseous distention. However, increasing evidence
has linked methane production to various disease states. It is
unclear whether this is because of the methane itself or because of
the efficient removal of hydrogen from the bowel through methanogenesis.
In particular, the enteric microbiota may play a key role in the
pathogenesis of irritable bowel syndrome (IBS). Small intestinal
bacterial overgrowth (SIBO)—defined as a pathological increase
in the number of bacteria in the small bowel—may play a role in
the pathophysiology of IBS. In case–control studies, the prevalence
of positive tests for SIBO has ranged from < 20% (18) (using glucose breath testing) to > 80% (using lactulose breath testing) (19);
in systematic review and meta-analyses, positive tests for SIBO
were between 3.45- and 4.7-fold more common in patients with
IBS than in controls (20,21). Moreover, evidence suggests that an
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29
Pimentel et al.
episode of bacterial gastroenteritis increases the risk for IBS by
approximately sevenfold (22). Together, these data point toward a
role for gastrointestinal microbiota in the pathogenesis of IBS in at
least a subset of these patients.
Recently, the prevalence of methanogenic flora in patients with
SIBO was assessed by Attaluri et al. (23). In this study, SIBO was
diagnosed if hydrogen or methane increased by 20 p.p.m. over
baseline during the glucose breath test. The presence of methanogenic flora was defined as a baseline methane level of ≥3 p.p.m.
Breath testing identified methanogenic flora in 45% of patients
with SIBO, 30% of patients with fructose malabsorption, and 34%
of patients with lactose malabsorption, suggesting a higher yield
of methanogenic flora in patients with SIBO compared with those
with carbohydrate intolerance. Furthermore, the amount of methane produced (as measured by baseline, peak, and area under the
curve of methane production) was significantly higher in patients
with SIBO compared with patients with fructose or lactose malabsorption.
In another study, the presence of methanogenic flora was
significantly associated with chronic constipation (23). Furthermore, the amount of methane produced after a carbohydrate
challenge and its prevalence were both higher in patients with
slow-transit constipation compared with normal-transit constipation and nonconstipated controls. In addition, the amount of
methane produced was significantly correlated with colonic transit
time; slower colonic transit was associated with greater methane
production. Interestingly, stool frequency and stool consistency
were not associated with methane production. These observations were further corroborated by studies of colonic motility
that showed that methane producers had significantly decreased
colonic pressure wave activity (P < 0.05) than nonmethane producers, whereas the small bowel motility was similar between the
two groups (24).
In another study that examined the association of age and gender with methane production, women had a higher prevalence
of methanogenic flora than men in patients with chronic constipation, whereas men had a higher prevalence in patients with
IBS-D. Also, the prevalence was not influenced by age, suggesting
that methane production is independent of age (25).
The reproducibility of detecting methane in breath samples was
also assessed in a study of constipated patients. Baseline, peak,
and area under the curve of methane gas produced following a
lactulose challenge showed an excellent intrasubject reproducibility (intraclass correlation), suggesting that methane production is stable over time (26). In another intriguing study, small
bowel and colonic pH profiles were assessed in patients with
methanogenic flora and SIBO using a wireless pH and motility capsule. The pH profiles in the small bowel and colon were
similar between patients with SIBO, patients without SIBO, and
healthy controls (27). However, in constipated patients, the mean
colonic pH as well as the proximal colonic pH were significantly
lower in patients with methanogenic flora when compared with
the nonmethane producers (25). Finally, anorectal sensorimotor function was assessed in constipated patients with or without methanogenic flora. Patients with methanogenic flora had
The American Journal of GASTROENTEROLOGY Supplements
3.5
Constipation
3.0
Diarrhea
2.5
Severity score
30
2.0
1.5
1.0
0.5
0.0
H2
CH4 & H2
CH4
Gas pattern
Figure 2. Mean diarrhea and constipation severity scores of subjects with
small intestinal bacterial overgrowth (SIBO; N = 551) as a function of the
type of gas pattern produced on lactulose breath testing. P < 0.00001 for
trend in reduction of diarrhea with the presence of methane; P < 0.05 for
the trend toward increasing constipation with the presence of methane.
Reproduced from ref. 29 with kind permission from Springer Science +
Business Media.
weaker anal sphincter pressure and rectal hypersensitivity compared with nonmethanogenic patients (28).
These observations indicate that methane production is more
common in women, is not influenced by age, and is relatively stable in constipated subjects. Also, it significantly influences colonic
transit time, colonic pH, colonic motility, and anorectal sensorimotor function. All of these findings suggest a strong association
between methanogenesis and chronic constipation.
Even before the work by Attaluri et al. (25), Pimentel et al. (29)
studied consecutive patients undergoing lactulose breath testing
and were given a questionnaire to evaluate their gastrointestinal
symptoms. Breath test results were categorized according to pattern:
normal, hydrogen only, hydrogen and methane, and methane
only. When the entire group of subjects with SIBO was assessed,
diarrhea severity scores differed significantly depending on breath
test pattern, such that patients who excreted methane reported
significantly lower diarrhea scores than those who produced
hydrogen only (Figure 2). Conversely, higher constipation severity
scores were reported by subjects who produced methane. In fact,
if a breath test was methane positive, it was 100% associated with
constipation-predominant IBS. In contrast to IBS, the prevalence
of methane positivity was very low among patients with Crohn’s
disease or ulcerative colitis. These results were recently confirmed
and extended by Chatterjee et al. (30), who found that IBS subjects
with methane had a significantly higher mean constipation severity (P < 0.001). Notably, in this study the quantity of methane seen
on breath testing was directly proportional to the degree of constipation reported and a lower stool frequency (P < 0.01 for both).
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It is important to consider the above findings using an evidencebased medicine approach. A recent meta-analysis evaluated the
existing literature comparing the presence of methane and constipation (31). Although not all studies have confirmed these results,
most do to support a significant association. Moreover, this metaanalysis further described studies with objective measures of transit and again most studies supported an associated even here.
These studies also suggest that there is a strong relationship
between methane production and IBS pattern; however, they do
not permit determination of whether the relationship is causal or
incidental. For example, it is possible that methanogens may favor
the slow-transit environment, a hypothesis supported by studies
indicating that treatment with laxatives and bowel cleansing can
reduce or eliminate methane production in some patients (14).
As noted earlier, methanogens exist within a complex web of
syntrophic and competitive relationships; thus, it is possible that
methanogens could alter transit indirectly as a reflection of competition for hydrogen and the relative numbers of methanogens vs.
sulfate-reducing bacteria (14).
The possibility also exist that methane, in and of itself, is a bioactive molecule that can affect intestinal transit. In an animal model,
direct infusion of methane into the small intestine produced a
slowing of transit by an average of 59% compared with room air
(32). In part, the direct effect of methane on intestinal transit may
be mediated through serotonin, a neurotransmitter that, among
other functions, elicits peristaltic contractions in the gut. In one
study, lactulose breath testing was performed on subjects with
IBS, followed by a determination of serotonin before and after a
75-g oral glucose meal (33). Compared with hydrogen producers,
methane producers had significantly (P < 0.05) lower postprandial
serotonin levels. These data—although derived from a very small
(N = 18) study—suggest that methane-producing IBS patients have
reduced postprandial serotonin.
Although not directly studied, it is also possible that methanogen overgrowth relative to sulfide-reducing bacteria could enhance
abdominal pain by attenuating production of hydrogen sulfide, a
known gaseous neuromodulator with significant antinociceptive
effects (34).
MANIPULATION OF METHANE FOR THERPEUTIC
EFFECT
To date, there are limited data on manipulating the methanogenic
flora, either in clinical studies or in clinical practice. However, as
noted above, laxatives and bowel cleansing have both been shown
to reduce methane production (14).
Specific antibiotic therapy has been shown to attenuate the symptoms of nonconstipated-IBS through modulating effects on gut
flora. In two trials of rifaximin (35), patients received treatment with
rifaximin or placebo for 2 weeks and were followed for 10 weeks.
The primary end point—the proportion of patients who reported
adequate relief of global IBS symptoms—was achieved in a greater
percentage of patients in the rifaximin group than in the placebo
group in both trials; for the combined trials, the rate of achieving the
primary end point was 40.7% and 31.7%, respectively (P < 0.001).
© 2012 by the American College of Gastroenterology
Table 1. Methanogenic antibiotics and inhibitors
Antibiotic/inhibitor
Organisms
Reference
Neomycin
Methanococcus
mariplaudis
Argyle et al. (37)
Pseudomonic acid
Methanosarcina spp.
Methanobacterium
spp.
Boccazzi et al. (38),
Jenal et al. (39)
Puromycin
Methanosarcina spp
Methanococcus
mariplaudis
Gernhardt et al. (40)
8-Aza-2,6-diaminopurine All of the above species Pritchett et al. (41)
8-Aza-hypoxanthine
All of the above species Moore and Leigh (42)
2-Bromoethane sulfonate Most species
Gunsalus and Sligar (43)
2-Chloroethane sulfonate Most species
Gunsalus and Sligar (43)
Chloro/fluoromethanes
Most species
Gunsalus and Sligar (43)
Mevastatin, lovastatin
Methanobrevibacter
species
Miller and Wolin (9)
On a basis similar to the TARGET trials, the efficacy of antibiotic
therapy has also been evaluated in IBS patients with methane on lactulose breath test (36). A retrospective chart review was conducted
on patients with ≥3 p.p.m. of methane in their breath test who had
received 10 days of treatment with either neomycin alone, rifaximin alone, or a combination of the two drugs. Of the patients who
received combination therapy, 85% had a clinical response compared with 63% of patients who received neomycin alone (P = 0.15)
and 56% of patients who received rifaximin alone (P = 0.01). Notably, 87% of patients taking the combination eradicated methane on
their breath test compared with only 33% and 28% of patients in
the neomycin and rifaximin alone groups, respectively.
A number of other methanogenic antibiotics and inhibitors have
been described (Table 1).
Most methanogenic archaea are resistant to the majority of
antibiotics commonly used against the Gram-positive and Gramnegative bacteria. Three notable exceptions are puromycin, pseudomonic acid, and neomycin. The former is a peptidyl transfer
inhibitor of ribosome function in bacteria and eukaryotic cells and
likely acts via a similar mechanism in the archaea, whereas pseudomonic acid acts to block transfer RNA synthetase function. Neomycin, a well-described aminoglycoside protein synthesis inhibitor
in bacteria, is widely used as a selectable marker in the genetic
manipulation of methanogens. In pure cultures, it is 100% effective against M. smithii in the 50 μg/ml range. In contrast, rifaximin
did not demonstrate noticeable inhibition when tested at similar
concentrations (Gunsalus R. et al., unpublished data). It remains
unknown why single antibiotic treatment with neomycin or rifaximin alone is not adequate compared with their combined use in
the case of methanogens. However, there are many examples of this
problem in enteric bacterial colonization such as in the treatment of
Helicobacter pylori.
Several other methanogenic inhibitors have been described
(Table 1), including the chloro/fluoromethanes as well as 2-bromoand 2-chloro-ethane sulfonates that block key reactions leading to
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Pimentel et al.
methane biosynthesis. The statins mevastatin and lovastatin were
shown to inhibit growth of several rumen Methanobrevibacter isolates (strains Z4, Z8, ZA10) in the ~10 nmol/ml range. These two
eukaryotic 3-hydroxy-3-methylglutaryl-coenzyme A reductase
inhibitors presumably act by interfering with mevolonate synthesis
needed for isoprenoid lipid synthesis in methanogens.
CONCLUSIONS
An abundance of data point toward a critical role for the gut
microflora in regulating intestinal health. Methanogens play a
critical role in the intestinal ecosystem by metabolizing the hydrogen generated during the fermentation of carbohydrates. Thus,
the methanogens serve to reduce the volume of gas in the colon
considerably. Accumulating evidence suggests that methanogens,
through the production of methane, can directly influence intestinal transit; in fact, there appears to be a direct correlation between
methane production and constipation.
If methane has a direct or indirect impact on intestinal transit,
attempting to manipulate methanogenic flora may be a viable therapeutic option. Although methanogenic flora have not yet been a
specific target of therapy in either clinical studies or clinical practice, this area will require further research and development. However, the future may include inhibitors of methanogenesis, targeted
antimicrobial therapy, and even probiotics.
ACKNOWLEDGMENTS
We thank John Ferguson for editorial assistance in preparing the
manuscript for publication.
CONFLICT OF INTEREST
Guarantor of the article: Mark Pimentel, MD, FRCP(C).
Specific author contributions: Dr Pimentel initiated the concept
of the manuscript, and contributed to writing the manuscript and
critical revision of the manuscript. Dr Gunsalus aided in planning
this study, collecting and interpreting data, and in drafting the
manuscript. Dr Rao contributed to the idea, wrote manuscript, and
provided critical revision. Dr Zhang aided in interpreting data and
in drafting the manuscript. All authors have seen and approved the
final report.
Financial support: Mark Pimentel has received grant support from
Salix Pharmaceuticals and the Seaver Foundation. Robert P. Gunsalus
has received grant support from the Department of Energy (DOE)
and the National Institute of Health (NIH). Satish S.C. Rao has
received grant support from SmartPill and Forest Laboratories. An
independent medical educational grant from Salix Pharmaceuticals
was provided to support the development of this supplement. The
grantors did not review the manuscripts before publication, nor did
they provide input into the content of the supplement.
Potential competing interests: Mark Pimentel has received consulting fees from Salix Pharmaceuticals. Robert P. Gunsalus has also
received royalties from “Microbial Life” 2nd edn., an undergraduate
microbiology text book. Satish S.C. Rao has received consulting fees
from SmartPill and The Dannon Company. The remaining authors
declared no competing interests.
The American Journal of GASTROENTEROLOGY Supplements
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