Methane-producing archaea have recently been associated with disorders of the gastrointestinal tract and dysbiosis of the resident microbiota. Some of these conditions include inflammatory bowel disease (Crohn’s disease (CD) and ulcerative colitis (UC)), chronic constipation, small intestinal bacterial overgrowth, gastrointestinal cancer, anorexia, and obesity. The causal relationship and the putative mechanism by which archaea may be associated with human disease are poorly understood, as are the strategies to alter methanogen populations in humans. It is estimated that 30–62% of humans produce methane detectable in exhaled breath and in the gastrointestinal tract. However, it is not yet known what portion of the human population have detectable methanogenic archaea. Hydrogen and methane are often measured in the breath as clinical indicators of intolerance to lactose and other carbohydrates. Breath gas analysis is also employed to diagnose suspected small intestinal bacterial overgrowth and irritable bowel syndrome, although standards are lacking. The diagnostic value for breath gas measurement in human disease is evolving; therefore, standardized breath gas measurements combined with ever-improving molecular methodologies could provide novel strategies to prevent, diagnose, or manage numerous colonic disorders. In cases where methanogens are potentially pathogenic, more data are required to develop therapeutic antimicrobials or other mitigation strategies.
- colorectal cancer
- irritable bowel syndrome
1.1. Methanogen diversity in the gastrointestinal tract
Archaea represent the third domain of life, in addition to Prokaryota, which they more or less physically resemble, and Eukaryota, with which they have more genetic similarities. Many archaea are classified as extremophiles, but those which live in the digestive tract of animals are known as methanogens. Archaeal diversity in the gastrointestinal tract (GIT) is far less than that of bacteria, and more specifically monogastrics have a much lower diversity as compared to herbivorous ruminant animals. In both host types, species belonging to the genus
Methanogens use hydrogen, in the form of free protons, H2 gas, NADH and NADPH cofactors, acetate, or formate, to reduce carbon dioxide and produce methane gas. Thus, methanogens rely on the by-products of bacterial fermentation of carbohydrates (i.e., carbon, hydrogen, acetate, formate, or methanol) as precursor materials required for methanogenesis and their own energy production. Dietary carbohydrates which are not broken down or absorbed by the host are available to bacteria for fermentation , and a large amount of unused carbohydrates may consequently increase bacterial fermentation and archaeal methanogenesis. A diet high in fiber and structural carbohydrates, which are largely indigestible to animal and human enzymes (i.e., cellulose, hemicellulose, and lignin), is associated with populations of
Methanogens also have a slower growth rate than bacteria, which is sensitive to concentrations of hydrogen required as an electron donor during methanogenesis, as well as other nutrients. Few methanogenic taxa are motile, and these are limited to the order Methanococcales, and the genera
1.2. Intestinal methane and the effect on the host
Colonic gases are among the most tangible features of digestion, yet physicians are typically unable to offer long-term relief from clinical complaints related to excessive gas and associated discomfort. Studies characterizing colonic gases have linked changes in volume or composition to individuals with gastrointestinal disorders (see below). These studies have suggested that hydrogen gas, methane, hydrogen sulfide, and carbon dioxide are by-products related to the interplay between hydrogen-producing fermentative bacteria and hydrogen consumers (reductive acetogenic bacteria, sulfate-reducing bacteria, and methanogenic archaea). The primary benefit of methanogenesis in the GIT is to decrease hydrogen (hydrogen gas, NADH, NADPH) resulting from carbohydrate fermentation by bacteria, protozoa, and fungi . Hydrogen gas in the intestines can shorten intestinal transit times of feces by 10–47% . Moreover, hydrogen has been shown to have antioxidant properties as an oxygen scavenger [21, 22]. It is possible that in the healthy colon, physiological hydrogen concentrations might protect the mucosa from oxidative insults, whereas an impaired hydrogen economy might facilitate inflammation or carcinogenesis.
However, excessive hydrogen in the GIT can be detrimental to commensal microorganisms. The decrease in hydrogen through the generation of inert methane gas helps to prevent hydrogen damage to host or symbiotic microbial cells . In ruminant animals, which have a four-chambered stomach, methanogens associated with ciliate protozoa act as a hydrogen sink , especially in the first two stomach chambers, the rumen and reticulum. There are a few commensal protozoan species that can be found in the human intestinal tract , but it is not yet known if they symbiotically interact with methanogens. Generally, this interaction only occurs with protozoa that have a hydrogenosome organelle, which metabolizes pyruvate and uses hydrogen ions as electron acceptors. In humans, the only protozoa that have a hydrogenosome are trichomonads, such as
Alternative hydrogen sinks in humans include sulfate-reducing bacteria (SRB), which produce hydrogen sulfide gas that is absorbed and detoxified by the liver, or acetogenic bacteria, which produce the short-chain fatty acid acetate that can be metabolized by the host or other microorganisms. Some of these pathways are mutually exclusive in humans, and either SRB or methanogens will be present in large numbers . Although higher hydrogen sulfide and SRB levels have been detected in patients with irritable bowel disease (IBD), and to a lesser extent in colorectal cancer (CRC), this colonic gas might have beneficial effects as a gaso-transmitter . Acetogens, on the other hand, have up to a 100 times higher hydrogen concentration threshold, and thus cannot out-compete methanogens for precursors [29, 30]. Consequently, acetogenesis is rare in the human GIT, and if present is usually restricted to the right colon .
Unlike hydrogen, there are as yet no known biological sinks for methane in the intestines , although methanotrophic bacteria exist in a variety of water and soil environments. Instead, some methane is excreted from the colon, and most is absorbed into the blood stream and expelled from the lungs via exhalation. This allows methane production to be indirectly and noninvasively measured, since breath methane concentration is correlated with methanogen cell density in the intestines . An undetectable concentration of breath methane does not equate to the absence of archaea, and therefore false-negative interpretations of breath gas analysis may result when breath methane is at undetectably low levels [33, 34]. Reported estimations suggest that between 30 and 62% of healthy humans produce detectable methane [31, 35]. The presence of methane gas in the intestines may influence or reduce intestinal transit time, and the correlation between breath methane production and transit time has been observed even in healthy individuals . This was further examined using animal models, in which the overabundance of methane gas caused a reduction in transit time while increasing intestinal contractions [20, 36], thus increasing pressure inside the intestine by an average of 137% . Alteration of intestinal motility may benefit slow-growing methanogen populations, which are limited by their ability to attach to host mucosal epithelia and maintain themselves in the intestines.
This increased gas production and resulting pressure cause bloating, discomfort, flatulence, or belching. In addition to detrimental physical effects, it has been speculated that methane potentially causes chemical and biological effects as a “gaso-transmitter” , in the same way that hydrogen sulfide affects smooth muscle activity  or nitrous oxide (N2O) is used in biological systems to control vascular tone . Studies using isolated gastrointestinal tissue suggest that this interaction is between methane and enteric nervous tissue, rather than the central nervous system . Clinically, hydrogen and methane measured in breath can indicate lactose and glucose intolerance, small-intestine bacterial overgrowth (SIBO), irritable bowel syndrome (IBS), or other gastrointestinal diseases [35, 36, 39–42]. Therefore, standardized breath gas measurements combined with ever-improving molecular methodologies could provide novel strategies to prevent, diagnose, or manage numerous colonic disorders as defined by the Rome III diagnostic criteria .
2. The role of archaea in metabolic disorders
Obesity in adults is most commonly defined using body mass index (BMI) (kg body weight/height in meters squared), and for Caucasian adults, is defined as a BMI of ≥30 kg/m2. For over a decade, shifts in intestinal bacteria diversity have been associated with weight gain or obesity in humans, generally following an increase in the proportion of Firmicutes , a decrease in Bacteroidetes, which has shown some anti-obesity influences [44–46], and with a shift in more minor phyla. Generally, this shift in intestinal bacteria leads to an increase in host energy harvest by improving polysaccharide digestion and host epithelial absorption which, in turn, causes weight gain [47–49]. Alternatively, a change in host genetics or immune system function can also cause a shift in bacterial diversity. The lack of host immune-modulating factors, such as Toll-like receptor 5 (TLR5) and fasting-induced adipocyte factor (Fiaf), produced insulin resistance, increased adiposity (especially visceral), and shifted GIT bacterial diversity and functionality in mice [49, 50]. Additionally, endotoxinemia, or the presence of microbial endotoxins (e.g., lipopolysaccharide-A (LPS)) in intestines or blood, has been shown to induce obesity, glucose intolerance, weight gain, and adiposity in response to a high-fat diet [51–53].
It would seem that bacterial diversity and density may have a specific role in metabolic dysbiosis, as treatment with oral antibiotics has been shown effective at improving fasting and oral glucose tolerance test (OGTT) levels in obese or insulin-resistant mice , or mitigating endotoxinemia and reducing cecal LPS concentrations in mice on a high-fat diet [51, 55]. Both obesity and diabetes are also correlated with low-grade chronic intestinal inflammation, likely caused by bacterial LPS. The presence of LPS, among other systemic immune responses, causes host macrophages to express pro-inflammatory cytokines, and in adipose-associated macrophages this only increases local insulin resistance and lipid storage [51, 53].
More recent studies have focused on the shifts in archaea associated with high-fat/high-calorie diets or weight gain, especially as
Obesity is strongly associated with an increased risk for diabetes mellitus, or type-2 diabetes, which is an inducible metabolic disease characterized by a lack of pancreatic production of insulin, or a resistance to insulin at the cellular level. Type-1 diabetes is an autoimmune disease characterized by the destruction of pancreatic beta cells which normally produce insulin. Diabetes can lead to a host of other health problems, most especially cardiovascular disease, renal failure, increased glaucoma and potential blindness, and reduced circulation, which increases the risk for ulcers and infection in the peripheral limbs. Few studies investigate the potential link between methanogens and diabetes. Type-1 diabetic patients with no complications showed a significant increase in intestinal transit time, although it was not associated with other gastric symptoms . Type-1 diabetes with an autonomic diabetic neuropathy complication affects heart rate, blood pressure, perspiration, or digestion. Some patients with this neuropathy have also been positive for SIBO [59, 60], which was associated with an increased daily insulin requirement , or detectable methane production, which was associated with a worse glycemic index . Breath methane producers, which had comparable BMI and baseline insulin resistance to non-methane producers, had higher serum glucose levels and a longer return to normal resting glucose after OGTT . The mechanistic relationship between methanogens, methane, and diabetes has yet to be explained.
3. The role of archaea in colon cancer
Colorectal cancer is the most commonly diagnosed malignancy in the Western World, being the fourth most common cancer diagnosis in the United States but the second leading cause of cancer-related deaths . In nonsmokers, it is the leading cause of cancer-related death in men and the second leading cause of cancer-related death in women (after breast cancer). The 5-year survival rate varies by stage and type, ranging from 53 to 92% . All colorectal cancers originate from adenomas or flat dysplasia, and are often asymptomatic, though occult bleeding may result and ultimately may be associated with an unexplained iron deficiency anemia. Large tumors in the distal or left colon may result in a compromised bowel lumen and potentially lead to symptoms including constipation, diarrhea, or bowel obstruction. The histopathology of CRC is complicated and involves a number of differently defined molecular pathways. There is evidence of microbial dysbiosis in CRC patients, as well as higher levels of breath methane in patients with CRC and premalignant polyps, as presented below.
Viral causative agents have been identified in a variety of cancers, but it is only recently that prokaryotic- or eukaryotic-causative or protective agents have been investigated. Cancer has been associated with a reduced bacterial diversity in the digestive tract , as well as in the mammary glands . Specific agents have been identified, which cause localized cancers through their molecular interactions with host cells , such as
There is some discussion about the change in the density of methanogens in individuals with colorectal cancer [33, 68, 69]. Methanogen density was shown to be inversely related to the fecal concentration of butyrate, a short-chain fatty acid produced by bacterial fermentation . Butyrate has been shown to provide energy for digestive tract epithelia cells, upregulate host immune system and mucin production, alter toxic or mutagenic compounds, and reduce the size and number of crypt foci, which are abnormal glands in intestinal epithelia that lead to colorectal polyps [71–73]. An altered gut microbiome in colorectal patients could shift bacterial fermentation away from butyrate production to something more favorable to methanogenesis.
Methane production was increased in patients with precancerous symptoms and colorectal cancer [39, 74], and was directly proportional to constipation but inversely proportional to diarrhea in chemotherapy patients . In the same study, pH was also directly proportional to constipation but inversely proportional to diarrhea in chemotherapy patients . Methane itself has not been shown to be carcinogenic. However, the oxidation of methane forms formaldehyde, which is carcinogenic . On the other hand, hydrogen sulfide gas produced by SRB has shown to promote angiogenesis (which tumors rely on), and has been shown to be genotoxic when DNA repair is inhibited . Colon cancer biopsies have shown an increase in the enzyme cystathionine-β-synthase (CBS), which allows host cancer cells to produce their own hydrogen sulfide, and a silencing of this gene was able to reduce tumor cell growth, proliferation, and migration .
4. The role of archaea in irritable bowel syndrome
The symptoms of IBS vary between patients, and may include diarrhea, constipation, excess flatus secondary to hydrogen or methane production, bloating, abdominal pain, and visceral hypersensitivity . Hydrogen sulfide gas from SRB was shown to increase luminal hypersensitivity . In addition, IBS is associated with changes in the diversity and density of intestinal bacteria [42, 81–83], as well as with an increase in hydrogen production . In some patients with IBS, the change in bacterial populations is amplified, leading to SIBO. SIBO is also seen in non-IBS patients, but it is much more prevalent in IBS patients, especially those with constipation as opposed to diarrhea [85, 86]. A common technique for the management of symptoms includes switching patients to a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) . Two-thirds of patients report symptoms linked to diet , especially gas production and bloating following ingestion of lactose , other carbohydrates, or fats [40, 88].
While the specific cause of IBS still remains unclear, the altered bacterial diversity causes a shift in carbohydrate fermentation and altered gas production. If this shift favors methanogenesis, the result is a decrease in transit time and an increase in constipation. The presence of methanogens in the digestive tract, and the production of methane, has been associated with patients with IBS, and especially with chronic constipation and reduced passage rate in the intestines (slow transit) [42, 85, 90]. Methanogen density was found to be lower in IBS patients as compared to controls [69, 91], although density and methane production were increased in IBS patients with constipation as compared to IBS patients without constipation .
5. The role of archaea in inflammatory bowel disease
Contrary to recent findings in patients with IBS, low methane production [35, 42] and lower methanogen density  were seen in patients with IBD, which includes the specific entities Crohn’s and ulcerative colitis. In contrast to IBS, IBD patients demonstrate chronic inflammatory changes in the colon (UC) or in the small bowel, or a combination of small bowel and colon involvement (CD).
Recently, it was demonstrated that two archaeal species normally found in the digestive system,
6. The role of archaea in other intestinal dysbiosis
There are many rare gastrointestinal diseases and general conditions of dysbiosis which are not well understood, but which may have a link to methane production in the intestines. Pneumatosis cystoides intestinalis (PCI) is a condition in which gas-filled cysts occur in the smooth muscle wall of the intestines, where it cannot be relieved by flatulence. It is believed to be caused by bacteria in the intestinal wall. Interestingly, patients with PCI have lower prevalence of breath methane production than patients with IBS, CD, UC, and even healthy control subjects .
Non-IBS constipated patients with slow transit were more likely to have detectable levels of breath methane (75 vs 44%) than constipated patients with normal transit, and both were more likely to have detectable breath methane than nonconstipated controls (28%) . This trend was also reported in other studies [56, 85].
Diverticulitis, a condition involving the herniation of the intestinal mucosal and submucosal layers back through the intestinal smooth muscle and creates pockets that harbor infections, has only been noted since the early 1800s . Interestingly, it is most common in the left colon in subjects from Western countries and the right colon in subjects from Asian countries , which is likely a function of the “Western diet.” Diverticulitis was associated with a high prevalence of methanogens in stool and high methane output , as well as fiber intake, age-associated changes in the colon wall, low colonic motility, and high intraluminal pressure; however, methane output was not associated with right colon diverticulitis . As methanogen density is higher in the left colon , an increase in methane production that reduced transit time and increased intraluminal pressure would seem to be a contributing factor to the development of left colon diverticulitis.
7. Mitigation strategies
IBS is the most common functional gastrointestinal disorder and affects up to 12–15% of adults in the United States. Roughly 1.6 million Americans currently suffer with CD or UC, collectively known as IBD. IBS adversely impacts quality of life and medical expenditures, with significant costs arising from health-care visits and reduced workplace productivity, while IBD is a chronic, relapsing, debilitating disease associated with both environmental and genetic factors. IBD affects one in 200 Americans (80,000 children) at an estimated direct cost of $1.84 billion dollars. Conventional therapy attempts to modulate the immune response in the gut as it relates to IBD, yet many individuals continue to require surgery to control their disease or address its complications. There is a longstanding belief that dysbiosis (altered microbial environment) in the GIT plays an important etiologic role in the pathogenesis of IBS and IBD. There is significant scientific and public interest in compositional understanding of the intestinal microbiome (the specific constellation of microorganisms populating the gut) to better understand the role of the microbiome in health and disease. The contribution of individual organisms, including archaea, in the pathogenesis of GI disease is complex because of the rudimentary understanding of the compositional components of the microbiome.
The control of methanogen populations has long been a strategy in livestock to improve animal dietary efficiency, as methane production is an energy sink, as well as to reduce greenhouse gas emissions. In ruminant livestock, as discussed in a review by Hook et al. , this is largely done by manipulating the diet to improve the digestibility of feed and increase passage rate through the digestive tract to both deprive methanogens of potential precursors and to manually flush them out of the system. A change in diet is a potential avenue for reducing methanogen populations in humans, as methanogenesis is associated with sugar-/starch-based diets in monogastrics . Environmental effects may also play a role, as children living near landfills, which had higher atmospheric methane than areas away from landfills, had a higher breath methane output and higher
Antibiotics have commonly been used to treat gastrointestinal disease or symptoms such as fasting and OGTT (glucose) levels , endotoxinemia and cecal LPS concentrations [51, 55], or global IBS symptoms . Archaea are largely resistant to antimicrobial agents, which target bacteria, as they have different cell wall components and structure, and the few antimicrobials which they are susceptible to have been summarized in a recent review . Notably,
Our increasing knowledge of the potential long-term effects on gut microbial diversity has led to a trend of alternative treatments or mitigating methods over antibiotics. A recent review of probiotics showed them to be effective in relieving digestive dysbiosis symptoms or treating gastrointestinal conditions [79, 81, 102, 103]. The use of prebiotics directly infused into the colon, such as short-chain fatty acids, however, did not increase colonic motility . While probiotics and other dietary additives have been used to reduce methanogenesis in ruminant livestock , the effect of probiotics on methanogen populations in humans has not yet been investigated. While current research suggests that methanogens and methane production may exacerbate symptoms, causative relations have only been shown in bacteria, and thus it is bacteria which should be the ultimate target for mitigation strategies in unhealthy populations.
Direct microbial remediation and mitigation have only been recently considered in human medicine with the advent of fecal transfer treatments from healthy donors. While this has mainly been aimed at remediating pathogenic bacterial populations, the implications for this technology to reduce methanogenesis and improve gastrointestinal conditions are clear. It may be possible to use fecal transfer treatments to increase the diversity of GIT archaea and thus promote competition to reduce methane production, to colonize with less-efficient methanogens, or to potentially increase competition by increasing SRB populations, which may have its own health implications for detoxifying hydrogen sulfate gas. Most interestingly, the transfer of fecal microbiota or cultures of specific methanogens has shown to also induce metabolic states in the recipients; fecal transfers, or colonization from parent to child, from overweight or pregnant individuals has been shown to increase weight gain in recipients [10, 16, 48, 105, 106]. While the possibility of this transfer to improve weight gain in severely malnourished individuals remains possible but not yet clinically applied, the more commercially appealing treatment of obesity using fecal transfers from lean individuals has yet to be explored.
Methane has been implicated in a number of gastrointestinal diseases, but methanogens have not yet been identified as causative agents. More work is needed in order to understand the interactions between archaea and host epithelia, as well as whether the root dysbiosis is caused by bacteria, archaea, or host epithelia. In addition, more sensitive, quick, and minimally invasive assessment techniques are needed to assess methane production, methanogen diversity, and methanogen density. In cases where methanogens are potentially pathogenic, more data are required to develop therapeutic antimicrobials or other mitigation strategies.
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