The gut microbiome seems capable of influencing almost every system in the body. This occurs through direct microbe-immune interactions and microbial metabolites. The collection of all metabolites in the gut is known as the gut metabolome, which acts as a bridge between the gut microbiome and health/disease.
In ME/CFS there is some initial evidence for gut dysfunction - several studies indicate gut dysbiosis, infections and inflammation. These are all things which will affect gut metabolism, although there is no direct research here yet. Anecdotally however, altered ‘gut fermentation’ is often considered important. Here is a mini review of some recent research in this area - relating to evolution, diet and disease factors; some of which may be relevant in ME/CFS.
Animals have coevolved as superorganisms due to mutually beneficial (symbiotic) relationships with gut microbes. One of the primary evolutionary forces selecting for gut microbiota composition is diet. Like our own cells, microbes require and utilise a diverse array of substrates for metabolism and growth (diagram), which come from diet and host. Different microbes fulfil different metabolic niches and exist in mutualistic or competitive relationships. Microbial metabolism benefits the host by extending digestive capability, producing beneficial metabolites and inhibiting pathogens.
|Proposed metabolic pathways in F. prausnitzii, a major|
gut bacterium (>5% of gut microbiota)
In mammals, gut microbiota composition closely relates to dietary patterns (e.g. carnivore, omnivore, herbivore, etc.) 1,2. From an evolutionary perspective, ancestral mammals were carnivores, whereas nowadays most mammals are herbivores. This transition required a major shift in gut anatomy and microbiota composition, since plant foods contain many components which can only be degraded by microbes 2. For instance foregut fermenters such as ruminants (e.g. cattle) depend almost entirely on their gut microbiota to digest and ferment plant fibres in a special multi-compartment stomach. In contrast, other mammals and primates have a simple stomach, but concentrate microbes toward the end of the GI tract where they perform hindgut fermentation.
Humans (homo sapiens) are members of the hominid family (great apes) which descend from primates (order) and mammals (class). Gut microbes are present at relatively low levels in the stomach and throughout much of the small intestine, but greatly increase in number toward the terminal ileum and large intestine (colon). This allows for a microbe-based ‘second digestion’ of otherwise indigestible plant components (fibres and polyphenols) and the production of beneficial metabolites.
The adult human gut microbiome is dominated by bacteria from 2 major phyla (Firmicutes and Bacteroidetes) and contains 1000s of species. Relative to other mammals, the modern human gut microbiota most closely clusters with that of other omnivorous and frugivorous primates 1,2. Collectively the human gut microbiome contains around 150x more genes than the human genome, which encode diverse metabolic pathways. Combined, the human genome and microbiome form a metagenome which underlies a meta-organismal metabolism 3.
The body controls the distal and radial distribution and metabolism of gut microbes. The relatively sterile environment of the small intestine is maintained by the stomach acid barrier, intestinal mucus (entraps microbes) and antimicrobial secretions (antimicrobial peptides and IgA), as well as the migrating motor complex (MMC), which flushes mucus (and microbes) toward the colon 4. Microbial distribution and metabolism is also broadly determined by oxygen (O2) gradients 5. A small amount of O2 leaks through the epithelium, which can be used by some facultative/aerobic microbes, while the normally anoxic conditions of the colonic lumen, enable survival of obligate anaerobes and necessitate anaerobic metabolism.
The human gut microbiome is full of diverse genes for metabolising carbohydrates, which normally provide the major substrates for microbial fermentation 6–8. Dietary carbohydrates come in the form of simple sugars (e.g. glucose, fructose and lactose) and complex polysaccharides (starch and fibres). Most simple sugars and starches can be digested and absorbed in the small intestine, while indigestible carbohydrates (resistant starches and fibres) pass to the colon. Gut microbes hydrolyze these complex plant carbohydrates into their respective sugars, which greatly extends the digestive capability of the gut. Some commensal microbes (e.g. mucolytic bacteria) also break down host secreted carbohydrates (e.g. mucus and other glycans).
Microbial hydrolysis of complex carbohydrates typically releases 6 carbon (C6) hexose sugars (e.g. glucose, fructose and galactose) which are metabolised via various pathways. For instance, glucose is oxidised to pyruvate and reducing power (NADH), via the highly conserved glycolysis pathway 9. Several primary fermenters subsequently use lactic acid fermentation, which reduces pyruvate to lactate (C3). Other bacteria, and yeast (gut mycobiota) 10,11, can perform ethanol fermentation, generating ethanol (C2) and CO2. Some bacteria utilise metabolic pathways which generate specific short-chain fatty acids (SCFAs) – e.g. formate (C1), acetate (C2), propionate (C3) and butyrate (C4). For example, conversion of pyruvate (from glycolysis) to acetyl-CoA, can fuel the formation of acetate, formate and CO2. Alternately, acetogens can use CO2 and H (or formate, CHO2) to produce acetate. Other specific pathways are used to convert various substrates (e.g. glucose, lactate, acetate, etc.) to propionate and butyrate 12,13.
|Gut microbe cross-feeding and production of SCFAs|
Since most microbes lack the ability to fully oxidise carbohydrates, SCFAs represent terminal products of fermentation. Accumulation of SCFAs mildly acidifies the colon and inhibits pathogens. Up to 95% of SCFAs are absorbed by colon cells, where they provide fuel, strengthen barrier functions and prevent carcinogenesis. Butyrate in particular increases epithelial metabolism and O2 consumption 14 thereby limiting availability to gut microbiota and pathogens 15. SCFAs further pass into the lamina propria and blood where they favourably modulate systemic health (e.g. immunity, metabolism and brain function) 16.
The exact composition of the gut microbiome and metabolome varies between healthy individuals. However, in many intestinal and systemic disorders, gut microbiome and fermentation patterns can be far from normal, resulting in excessive gas, increased lactate, low SCFAs/butyrate, etc 16. This might be due to several factors.
Diet (nutrient balance)
Dietary carbohydrates fuel fermentation, the nature of which depends upon the chemical form and food context. Whole plant foods (nuts/seeds, grains, legumes, fruit and veg) contain high levels of indigestible carbs in the form of resistant starches and fibres - substrates for colonic SCFA production. Generally, non-starch polysaccharide (NSP) fibres favour production of acetate and propionate, while resistant starch favours butyrate 16–18. Intact (whole) grains/legumes and fibre may also stimulate small intestinal transit, thereby further pushing fermentation distally toward the colon 19,20. Plant foods contain other unique phytochemicals which may modulate fermentation patterns. In particular, plant polyphenols (blue-black pigments) mostly resist digestion and pass to the colon, where they inhibit pathogens/biofilms, have prebiotic effects (e.g. Bifidobacteria) and modulate SCFA output 21. Plant phytates may also have beneficial effects 22.
On the other hand, consumption of refined plant foods/carbs may promote dysfunctional fermentation patterns. Processed foods can be high in sugar and low in fibre (and other phytochemicals), thereby increasing sugar availability to the small intestine, while depriving the colon. Moreover, they often contain unnaturally high levels of fructose (i.e. high-fructose syrup), and a high fructose/glucose ratio, which promotes fructose malabsorption 23. High-dose fructose is osmotic and can actually distend the small intestine, in contrast to inulin (fructose polymer) which is fermented in the colon 24. In obesity, consumption of sugar is positively, and fibre negatively, associated with small intestinal bacterial overgrowth (SIBO) 25 (typically diagnosed via sugar challenge). Accordingly, several trials also suggest specific fibres can be beneficial in SIBO 20 or associated conditions (e.g. IBS 26,27 and IBD 17,28,29).
Dietary protein content greatly influences gut microbiota, since amino acids are used for energy metabolism and growth. Under normal conditions, a significant amount of dietary protein escapes host digestion and passes into the colon; which is greatly increased by maldigestion or high protein diets 30. Microbial metabolism of amino acids generates some beneficial compounds 31. However protein fermentation (aka. putrefaction) is associated with the growth of pathogens, formation of harmful metabolites and bad smells 30,32 (think rotting meat/eggs). Fermentation of branched amino acids yields branched-chain fatty acids (BCFAs); aromatic amino acids yield phenols; nitrogen waste yields ammonia (NH3); and sulfur is reduced to hydrogen sulfide (H2S) 16,30,32. Notably, H2S can inhibit butyrate utilisation (oxidation) by colon cells. Protein fermentation is normally suppressed by the presence of indigestible carbohydrates. However, low carb/high protein diets (e.g. weight-loss diets) promote a shift from a saccharolytic to putrefactive microbial metabolism and harmful metabolite profile in humans 30.
Dietary fats are not themselves fermented, but can still modulate fermentation patterns. High fat diets (30-50% total calories) impair carbohydrate fermentation by lowering SCFAs/butyrate 33, while increasing succinate and inflammation 34, and exacerbating autoimmunity 35. High fat diets also increase bile release 36, which promotes the growth of sulfur-reducing bacteria 37 and can inhibit colon butyrate uptake (via SMCT1) 38. These high fat diets used in research are mostly high in saturated fat, whereas unsaturated omega-3s appear to have more favourable effects 39,40. In a preliminary human case study, an omega-3-rich diet even increased some butyrate-producing bacteria 41. Another recent study on humans consuming an equilibrated diet (normal fat level) tested the consequences of acute fat malabsorption, which had no significant effect on microbiota or SCFAs, although did increase calprotectin (inflammation) and decrease antioxidant capacity 42.
Overall dietary patterns shape gut microbiota and metabolism. Generally, consumption of whole plant foods correlates with levels of SCFAs 7,8,13,16,43. Whereas extreme ketogenic (no carb) 44 or low carb/high protein diets 30 markedly shift gut microbiota and metabolism toward an unhealthy profile (↓ SCFAs, ↑ BCFAs, etc.). Similarly, long-term diet also determines the in vitro fermentation response to the prebiotic fibre inulin - consumption of plant foods (e.g. whole grains, beans and veg) correlated with SCFAs, while animal foods (i.e. dairy and processed meats) with BCFAs and ammonia 45. Finally, while pure plant-based vegan diets may have several favourable effects (e.g. low pathogens and TMA) 46, there were actually no differences between western omnivore and vegan diet patterns on SCFAs, suggesting westerners may possess a more restrictive microbiome than traditional agrarian cultures 47.
Many other complex factors can mess up gut fermentation patterns and undermine the effects of diet, as listed below. Some of these may be relevant in ME/CFS, perhaps especially in relation to gut infections and autonomic dysfunction.
- Antibiotics – Antibiotics seriously disrupt the normal gut microbiota, while promoting pathogen/yeast overgrowth and inflammation. Changes to the metabolome included suppressed SCFAs/butyrate and increased succinate 15,48,49.
- Sugar malabsorption – Malabsorption of sugars (e.g. lactose, fructose and sorbitol) may be quite common, especially with IBS-type symptoms 50,51, and promote abnormal fermentation patterns.
- Abnormal pH - Removal of the stomach acid barrier by acid-suppressing medications (e.g. PPIs) can promote gut dysbiosis and overgrowth of oral/upper GI bacteria (e.g. Streptococci) 52,53. Insufficient acidity can also impair protein digestion and increase protein fermentation 30. In active IBD there can be excessive acidity in the colon, which may inhibit lactate utilisation and SCFA production 54.
- Slow motility - Slow small intestinal transit may promote SIBO, which often involves an overgrowth of colon-type bacteria 55,56. Constipation can promote overgrowth of methanogenic archaea 57, the abundance of which may inversely correlate butyrate production 58.
- Immunodeficiency - Immunodeficiency disorders (e.g. CVID 59, HIV 60 and Crohn’s 61) are often associated with gut dysbiosis, overgrowth, infections and inflammation.
- Inflammation - Inflammation induces many changes to the gut environment (substrates, redox and antimicrobials) which result in a dysbiotic “inflammabiome” in IBD 62. In particular the release of oxidised electron acceptors (N/S-oxides) allows some pathogens (e.g. Enterobacteriaceae) to use anaerobic respiration and outcompete fermenting microbes 63.
- Oxidative stress – Tissue oxidative stress inhibits butyrate uptake 38 and gut motility 64, so may promote SIBO 65.
1. Ley, R. E., Lozupone, C. A., Hamady, M., Knight, R. & Gordon, J. I. Worlds within worlds: evolution of the vertebrate gut microbiota. Nat. Rev. Microbiol. 6, 776–88 (2008).
2. Ley, R. E. et al. Evolution of mammals and their gut microbes. Science 320, 1647–51 (2008).
3. Quercia, S. et al. From lifetime to evolution: timescales of human gut microbiota adaptation. Evol. Genomic Microbiol. 5, 1–9 (2014).
4. Johansson, M. E. V, Sjövall, H. & Hansson, G. C. The gastrointestinal mucus system in health and disease. Nat. Rev. Gastroenterol. Hepatol. 10, 352–61 (2013).
5. Circu, M. L. & Aw, T. Y. Redox biology of the intestine. Free Radic. Res. 45, 1245–66 (2011).
6. Kasubuchi, M., Hasegawa, S., Hiramatsu, T., Ichimura, A. & Kimura, I. Dietary Gut Microbial Metabolites, Short-chain Fatty Acids, and Host Metabolic Regulation. Nutrients 7, 2839–2849 (2015).
7. Simpson, H. L. & Campbell, B. J. Review article: dietary fibre-microbiota interactions. Aliment. Pharmacol. Ther. 42, 158–179 (2015).
8. Bach Knudsen, K. E. Microbial degradation of whole-grain complex carbohydrates and impact on short-chain Fatty acids and health. Adv. Nutr. An Int. Rev. J. 6, 206–13 (2015).
9. Court, S. J., Waclaw, B. & Allen, R. J. Lower glycolysis carries a higher flux than any biochemically possible alternative. Nat. Commun. 6, 8427 (2015).
10. Mukherjee, P. K. et al. Mycobiota in gastrointestinal diseases. Nat. Rev. Gastroenterol. Hepatol. 12, 77–87 (2014).
11. Schulze, J. & Sonnenborn, U. Yeasts in the gut: from commensals to infectious agents. Dtsch. Arztebl. Int. 106, 837–42 (2009).
12. Kettle, H., Louis, P., Holtrop, G., Duncan, S. H. & Flint, H. J. Modelling the emergent dynamics and major metabolites of the human colonic microbiota. Environ. Microbiol. 17, 1615–30 (2015).
13. Ríos-Covián, D. et al. Intestinal Short Chain Fatty Acids and their Link with Diet and Human Health. Front. Microbiol. 7, 185 (2016).
14. Kelly, C. J. et al. Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function. Cell Host Microbe 17, 662–71 (2015).
15. Rivera-Chávez, F. et al. Depletion of Butyrate-Producing Clostridia from the Gut Microbiota Drives an Aerobic Luminal Expansion of Salmonella. Cell Host Microbe 19, 443–454 (2016).
16. Verbeke, K. A. et al. Towards microbial fermentation metabolites as markers for health benefits of prebiotics. Nutr Res Rev 28, 42–66 (2015).
17. Pituch-Zdanowska, A., Banaszkiewicz, A. & Albrecht, P. The role of dietary fibre in inflammatory bowel disease. Prz. Gastroenterol. 10, 135–141 (2015).
18. Scott, K. P., Martin, J. C., Duncan, S. H. & Flint, H. J. Prebiotic stimulation of human colonic butyrate-producing bacteria and bifidobacteria, in vitro. FEMS Microbiol. Ecol. 87, 30–40 (2014).
19. Hovey, A. L., Jones, G. P., Devereux, H. M. & Walker, K. Z. Whole cereal and legume seeds increase faecal short chain fatty acids compared to ground seeds. Asia Pac. J. Clin. Nutr. 12, 477–82 (2003).
20. Furnari, M. et al. Clinical trial: the combination of rifaximin with partially hydrolysed guar gum is more effective than rifaximin alone in eradicating small intestinal bacterial overgrowth. Aliment. Pharmacol. Ther. 32, 1000–6 (2010).
21. Mosele, J. I., Macià, A. & Motilva, M.-J. Metabolic and Microbial Modulation of the Large Intestine Ecosystem by Non-Absorbed Diet Phenolic Compounds: A Review. Molecules 20, 17429–68 (2015).
22. Okazaki, Y. & Katayama, T. Dietary phytic acid modulates characteristics of the colonic luminal environment and reduces serum levels of proinflammatory cytokines in rats fed a high-fat diet. Nutr. Res. 34, 1085–91 (2014).
23. Latulippe, M. E. & Skoog, S. M. Fructose malabsorption and intolerance: effects of fructose with and without simultaneous glucose ingestion. Crit. Rev. Food Sci. Nutr. 51, 583–92 (2011).
24. Murray, K. et al. Differential effects of FODMAPs (fermentable oligo-, di-, mono-saccharides and polyols) on small and large intestinal contents in healthy subjects shown by MRI. Am. J. Gastroenterol. 109, 110–9 (2014).
25. Ierardi, E. et al. Macronutrient intakes in obese subjects with or without small intestinal bacterial overgrowth: an alimentary survey. Scand. J. Gastroenterol. 51, 277–80 (2016).
26. Rao, S. S. C., Yu, S. & Fedewa, A. Systematic review: dietary fibre and FODMAP-restricted diet in the management of constipation and irritable bowel syndrome. Aliment. Pharmacol. Ther. 41, 1256–70 (2015).
27. Moayyedi, P. et al. The effect of fiber supplementation on irritable bowel syndrome: a systematic review and meta-analysis. Am. J. Gastroenterol. 109, 1367–74 (2014).
28. Canani, R. B. et al. Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World J. Gastroenterol. 17, 1519–28 (2011).
29. M, C., T, T., K, N. & M, K. High Amount of Dietary Fiber Not Harmful But Favorable for Crohn Disease. Perm. J. 19, 58–61 (2015).
30. Yao, C. K., Muir, J. G. & Gibson, P. R. Review article: insights into colonic protein fermentation, its modulation and potential health implications. Aliment. Pharmacol. Ther. 43, 181–96 (2016).
31. Rothhammer, V. et al. Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and central nervous system inflammation via the aryl hydrocarbon receptor. Nat. Med. (2016). doi:10.1038/nm.4106
32. Windey, K., De Preter, V. & Verbeke, K. Relevance of protein fermentation to gut health. Mol. Nutr. Food Res. 56, 184–96 (2012).
33. Keenan, M. J. et al. Role of resistant starch in improving gut health, adiposity, and insulin resistance. Adv. Nutr. 6, 198–205 (2015).
34. Jakobsdottir, G., Xu, J., Molin, G., Ahrné, S. & Nyman, M. High-fat diet reduces the formation of butyrate, but increases succinate, inflammation, liver fat and cholesterol in rats, while dietary fibre counteracts these effects. PLoS One 8, e80476 (2013).
35. Haghikia, A. et al. Dietary Fatty Acids Directly Impact Central Nervous System Autoimmunity via the Small Intestine. Immunity 43, 817–829 (2015).
36. Murakami, Y., Tanabe, S. & Suzuki, T. High-fat Diet-induced Intestinal Hyperpermeability is Associated with Increased Bile Acids in the Large Intestine of Mice. J. Food Sci. 81, H216–22 (2016).
37. Shen, W., Gaskins, H. R. & McIntosh, M. K. Influence of dietary fat on intestinal microbes, inflammation, barrier function and metabolic outcomes. J. Nutr. Biochem. 25, 270–280 (2014).
38. Gonçalves, P. & Martel, F. Butyrate and colorectal cancer: the role of butyrate transport. Curr. Drug Metab. 14, 994–1008 (2013).
39. Ghosh, S. et al. Fish oil attenuates omega-6 polyunsaturated fatty acid-induced dysbiosis and infectious colitis but impairs LPS dephosphorylation activity causing sepsis. PLoS One 8, e55468 (2013).
40. Lam, Y. Y. et al. Effects of dietary fat profile on gut permeability and microbiota and their relationships with metabolic changes in mice. Obesity (Silver Spring). 23, 1429–39 (2015).
41. Noriega, B. S., Sanchez-Gonzalez, M. A., Salyakina, D. & Coffman, J. Understanding the Impact of Omega-3 Rich Diet on the Gut Microbiota. Case Rep. Med. 2016, 3089303 (2016).
42. Morales, P. et al. Impact of Dietary Lipids on Colonic Function and Microbiota: An Experimental Approach Involving Orlistat-Induced Fat Malabsorption in Human Volunteers. Clin. Transl. Gastroenterol. 7, e161 (2016).
43. De Filippis, F. et al. High-level adherence to a Mediterranean diet beneficially impacts the gut microbiota and associated metabolome. Gut gutjnl–2015–309957 (2015). doi:10.1136/gutjnl-2015-309957
44. David, L. a et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559–63 (2014).
45. Yang, J. & Rose, D. J. The impact of long-term dietary pattern of fecal donor on in vitro fecal fermentation properties of inulin. Food Funct. (2015). doi:10.1039/c5fo00987a
46. M, G.-B. & MC, Y. The Health Advantage of a Vegan Diet: Exploring the Gut Microbiota Connection. Nutrients 6, 4822–4838 (2014).
47. Wu, G. D. et al. Comparative metabolomics in vegans and omnivores reveal constraints on diet-dependent gut microbiota metabolite production. Gut 65, 63–72 (2016).
48. Ladirat, S. E. et al. Exploring the effects of galacto-oligosaccharides on the gut microbiota of healthy adults receiving amoxicillin treatment. Br. J. Nutr. 112, 536–46 (2014).
49. Lopez, C. A., Kingsbury, D. D., Velazquez, E. M. & B??umler, A. J. Collateral damage: Microbiota-derived metabolites and immune function in the antibiotic era. Cell Host Microbe 16, 156–163 (2014).
50. Goebel-Stengel, M. et al. Unclear abdominal discomfort: pivotal role of carbohydrate malabsorption. J. Neurogastroenterol. Motil. 20, 228–35 (2014).
51. Goldstein, R., Braverman, D. & Stankiewicz, H. Carbohydrate malabsorption and the effect of dietary restriction on symptoms of irritable bowel syndrome and functional bowel complaints. Isr. Med. Assoc. J. 2, 583–7 (2000).
52. Wallace, J. L. et al. Proton pump inhibitors exacerbate NSAID-induced small intestinal injury by inducing dysbiosis. Gastroenterology 141, 1314–22, 1322.e1–5 (2011).
53. Jackson, M. A. et al. Proton pump inhibitors alter the composition of the gut microbiota. Gut 65, 749–56 (2016).
54. Belenguer, A. et al. Rates of production and utilization of lactate by microbial communities from the human colon. FEMS Microbiol. Ecol. 77, 107–19 (2011).
55. Riordan, S. M. et al. Small intestinal mucosal immunity and morphometry in luminal overgrowth of indigenous gut flora. Am. J. Gastroenterol. 96, 494–500 (2001).
56. Bouhnik, Y. et al. Bacterial populations contaminating the upper gut in patients with small intestinal bacterial overgrowth syndrome. Am. J. Gastroenterol. 94, 1327–31 (1999).
57. Furnari, M. et al. Reassessment of the role of methane production between irritable bowel syndrome and functional constipation. J. Gastrointestin. Liver Dis. 21, 157–63 (2012).
58. Birt, D. F. et al. Resistant Starch : Promise for Improving Human Health. Am. Soc. Nutr. 4, 587–601 (2013).
59. Jørgensen, S. F. et al. Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation. Mucosal Immunol. (2016). doi:10.1038/mi.2016.18
60. Vyboh, K., Jenabian, M.-A., Mehraj, V. & Routy, J.-P. HIV and the Gut Microbiota, Partners in Crime: Breaking the Vicious Cycle to Unearth New Therapeutic Targets. J. Immunol. Res. 2015, 1–9 (2015).
61. Glocker, E. & Grimbacher, B. Inflammatory bowel disease: is it a primary immunodeficiency? Cell. Mol. Life Sci. 69, 41–8 (2012).
62. Stecher, B. The Roles of Inflammation, Nutrient Availability and the Commensal Microbiota in Enteric Pathogen Infection. Microbiol. Spectr. 3, (2015).
63. Winter, S. E. & Bäumler, A. J. Dysbiosis in the inflamed intestine: chance favors the prepared microbe. Gut Microbes 5, 71–3 (2014).
64. Pasqualetti, V. et al. Antioxidant activity of inulin and its role in the prevention of human colonic muscle cell impairment induced by lipopolysaccharide mucosal exposure. PLoS One 9, e98031 (2014).
65. Rana, S. V. et al. Relationship of cytokines, oxidative stress and GI motility with bacterial overgrowth in ulcerative colitis patients. J. Crohns. Colitis (2014). doi:10.1016/j.crohns.2014.01.007