irritablebowelsyndrome (IBS) is a chronic heterogeneous gastrointestinal (GI) disorder of multifactorial (genetic, physiological, psychosocial and environment) origin, which afflicts nearly 11% of the world population (20). Recently revised Rome criteria (Rome IV) define IBS as “recurrent abdominal pain on average at least one day a week in the last 3 mo associated with two or more of the following: 1) related to defecation, 2) associated with a change in frequency of stool, and 3) associated with a change in form (consistency) of stool; symptoms should have persisted for at least six months” (https://ibsimpact.wordpress.com/2016/06/09/new-rome-iv-diagnostic-criteria-for-irritable-bowel-syndrome-ibs-unveiled-may-2016/). IBS was thought to be more prevalent in Western countries than in developing non-Western countries (20, 25, 48, 114, 149), although a recent study refutes this claim (24). In North America, up to 20% of the middle-aged population are affected while the reported prevalence is ~7% in South Asia (20, 114). Interestingly, the prevalence of IBS is higher in affluent Asian cities than in the poorer regions, which raises suspicion on the role of Western influence in these regions (42).

The human GI tract is home to trillions of microbes referred to as the gut microbiota. The role of gut microbiota in modulating GI physiology has been described in early gnotobiotic studies, which highlight the mutualistic relationship between gut microbiota and the host. Gut microbiota are involved in vital processes such as development of the host immune system (83, 111), maintenance of normal GI physiology (124), and fermentation of undigested carbohydrates (81). Alterations in gut microbiota structure and composition have been implicated in various GI tract disorders including IBS (50, 107). In fact, studies estimate that nearly 10% of IBS cases begin after an episode of gastroenteritis and leads to postinfectious IBS (PI-IBS), whose microbial signature closely resembles to that of diarrhea-predominant IBS (IBS-D) patients (49). Futhermore, a recent study has shown that symptom severity in IBS is negatively associated with microbial richness and a distinct microbial signature (132). Although several studies that have compared gut microbiota composition in patients with IBS to healthy patients have failed to provide consistent results (summarized in Table 1), some key findings include an increase in Firmicutes to Bacteroidetes ratio (50, 65, 107), decrease in Lactobacilli and Bifidobacteria population (8, 60, 79), and increase in Streptococci and Ruminococcus species (45, 57, 107, 117). A more consistent finding has been decreased alpha diversity (within individual differences); however, this may be nonspecific as decreased alpha diversity has also been reported in other metabolic and GI diseases such as obesity, type 2 diabetes, inflammatory bowel disease (Crohn’s disease, ulcerative colitis), and necrotizing enterocolitis (31, 80, 85, 87, 137). The variability in findings among studies is possibly due to the heterogeneous nature of the disease, differences in study design, differing methods for sample collection and data analysis, small sample size, and lack of longitudinal data. In addition to predominantly correlational studies, recent studies have evaluated the association of individual members of gut microbiota with symptoms paving the way for future mechanistic studies (112, 117). In spite of the need for better characterization of the gut microbiota in larger cohorts with longitudinal sampling, the above findings highlight an important role for this forgotten organ in IBS.

Below we first present an overview of evidence, supporting the effect of risk factors such as host genetics, stress, diet, antibiotics, and early childhood experience known to be associated with IBS on gut microbiota composition and function.

Host Genetics Shape Gut Microbiota The role of host genetics in IBS is highlighted by studies that show familial clustering of IBS. Although the familial pattern could result from a multitude of factors including host genetics, and environmental factors such as social learning and childhood experiences (68, 69, 71, 98), studies show that the relative contribution of host genetics is at least as significant as the environment (61, 68, 91, 126). Interestingly, both host genetics and the environment can shape and influence the gut microbiota (14). Using 16S rRNA based sequencing of stool samples from monozygotic twins and marital partner and unrelated individuals that were living in the same environment and had comparable feeding habits, Zoetendal et. al. (148a) found greater similarity among gut microbiota profile in monozygotic twins than unrelated individuals. Furthermore, findings from a recent study suggest that the heritable fraction of gut microbiota is temporally stable, being shaped mostly by host physiology and less by environmental factors (41). In another five separate studies that evaluated concordance (probability that 2 people with shared gene will develop similar disease) of IBS between twin pairs from various geographical regions found that the heritability (proportion of phenotypic variance attributable to genetic variance) contribution ranged anywhere between 0 and 57% (67, 68, 89, 91, 113), supporting a genetic contribution to IBS. In summary, these studies show that host genetics plays a prominent role in predisposition to IBS. Further host genetics also shape the gut microbiota composition. While gut microbiota changes are heritable and dependent on host physiology, future studies are needed to determine if gut microbiota changes as a result of host gene variations play a role in IBS pathogenesis.

Stress Modulates Gut Microbiota Composition Chronic psychological stress influences the onset, duration, and severity of symptoms in IBS (26, 96, 132, 145). In a recent study authors found that anxiety and depression was correlated with the severity of IBS and this was reflected on the microbial signature (132). The authors had also previously reported a correlation between Actinomycetales and depression in patients with IBS. In rodents, chronic stress models such as maternal separation, prolonged restraint stress, and social disruption either early in life or during adulthood has been shown to significantly alter gut microbiota composition (6, 9, 96) via various mechanisms, which include 1) increasing circulating proinflammatory cytokines level (6), 2) disrupting the intestinal barrier (116), and 3) increasing the activity of the hypothalamus-pituitary-adrenal (HPA) axis (3, 28, 63). In humans, depression due to chronic stressful life events is associated with overrepresentation of bacteria from Enterobacteriaceae family (52), while psychological stress is associated with reduction in Lactobacilli spp. and increase in Escherichia coli and Pseudomonas spp. (73). These gut bacteria have been implicated in the development of IBS (59, 133, 134). A recent study shows that stress-induced changes in gut microbiota can also be transferred to offspring. In monkeys, for example prenatal stress is associated with decrease in beneficial Bifidobacterium and Lactobacillus in the GI tract of the offspring (7). Together, these findings suggest that both prenatal stress and postnatal stress modulate gut microbiota and support the potential role of gut microbiota as a mediator of stress in development of IBS.

Diet Alters Gut Microbiota Composition Dietary intolerance is common in IBS patients with symptoms being triggered by specific foods, such as dairy products, grains, and fat (93). In fact, population-based studies have estimated that up to 70% of IBS patients suffer from symptoms of perceived food intolerance (72, 90, 93). While the mechanisms by which dietary ingredients trigger symptoms remain unclear, both long-term and short-term dietary changes have a significant impact on the gut microbiota, raising the possibility that gut microbes mediate the effect of diet on IBS symptoms. Long-term dietary patterns have been shown to shape the gut microbial composition. For example, long-term protein and animal fat intake is associated with increase in Bacteroides, while a long-term carbohydrate intake is associated with increase in Prevotella spp. (147). Similar enrichment in Prevotella at the expense of Bacteroides is also noted in children from Burkina Faso in rural Africa consuming primarily an agrarian diet compared with European children that consume modern Western diet rich in animal fat and protein (30). Short-term dietary changes are also associated with alterations in gut microbiota, which can be seen as early as a day after diet change and are mostly reversible. Interestingly, patients with IBS experience intermittent short-lasting exacerbations and it is tempting to hypothesize that short-term diet-related microbial changes might in part be responsible for the waxing and waning of symptoms (29). Microbial metabolism of dietary ingredients can lead to generation of metabolites such as organic acids, ammonia, methane, and hydrogen sulfide (108, 131), which may contribute to IBS symptoms. These dietary metabolites also support a dysbiotic microbiota with expansion of members within Gammaproteobacteria and at the same time suppress the growth of healthy gut microbiome (108). Dietary modifications such as low FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) diet and other forms of dietary intervention including conventional dietary advice (16) are associated with improvement in symptoms and change in gut microbiota composition (43, 44), suggesting that while dietary ingredients and their metabolism by gut microbiota represent a mechanism via which diet influences IBS symptoms, the amount and timing of consumption of different dietary ingredients possibly play a vital role in symptom generation.

Antibiotics Administration Causes Short-Term and Long-Term Changes in Gut Microbiota Diversity Antibiotic consumption is associated with development of IBS (82, 86, 139, 143). Adults and infants who consume antibiotics for non-GI diseases are at a greater risk of reporting bowel symptoms such as recurrent abdominal pain (139, 142). In fact, a study shows that patients who consume antibiotics for non-GI disorder are three times as likely to report functional bowel symptoms 4 mo later than those who did not take antibiotics (82). Gut microbiota are an obvious target for antibiotics taken for non-GI illnesses. Antibiotics in addition to affecting target pathogens also cause short-term and long-term changes in gut microbial diversity (51, 103, 128). Microbiota alterations due to antibiotic administration depend on several factors, including the spectrum of the agent, dosage, and the duration of treatment. A recent study showed that intravenous injection of β-lactam antibiotics, ampicillin/sulbactam, and cefazolin in a single patient for 14 days not only led to large short-term oscillatory fluctuations in microbial diversity but also long-term change in microbial diversity and composition (103). Bacterial genera such as Enterococcus, Blautia, Faecalibacterium, and Akkermansia bloomed at different stages of antibiotics treatment while taxa such as Actinobacteria, Betaproteobacteria, Streptococcaceae, Lachnospiraceae, Porphyromonadaceae, and Clostridiales were completely lost and did not reappear even 40 days after cessation of the antibiotic treatment (40, 103). Together these studies highlight the role of antibiotics in shaping gut microbiota composition.

Early Childhood Experiences Alter Gut Microbiota Composition Later in Life Early life events such as privileged childhood living conditions, emotional and sexual abuse are associated with IBS (86, 115). Bradford et al. (17) in a recent study found that IBS patients reported early adverse life events more often than control patients. Interestingly, early childhood experiences also have a significant impact on gut microbiota. Since bacterial colonization during and shortly after birth has a significant impact on the composition of gut microbiota later in life, events such as mode of delivery (C-section or natural delivery), feeding (formula feeding or breast feeding), hygiene, and pet exposure have a significant influence on microbiota assembly. Infants that are delivered via C-section have microbiota composition predominated by Staphylococci, Coryneabacterium, and Propionibacterium spp, while vaginally born infants are colonized mostly by Lactobacillus, Bacteroides, Bifidobacterium, Prevotella, and Sneathia (5, 36, 97). Furthermore, breast-fed newborns possess gut microbiota dominated by Bifidobacterium (15), which is considered a beneficial microbe compared with formula fed, who have higher level of proinflammatory Gammaproteobacteria (15, 97). These studies raise the possibility that early childhood experiences impact gut microbiota diversity and composition. Further studies are however needed to determine if changes in gut microbiota resulting from early life adverse events contribute to development of IBS later in life. In summary, these findings suggest that the predominant risk factors underlying IBS can affect gut microbiota composition and function. In the next part of the review, we will highlight the effect of gut microbiota on pathophysiological mechanisms underlying IBS, including the gut brain axis (GBA), GI motility, visceral sensation, epithelial barrier function, and immune activation.

Effect of Gut Microbiota on the GBA The GBA is a bidirectional communication network that works through neural, endocrine, and immune pathways between the emotional and cognitive centers of the brain and the GI tract. GBA dysregulation is a common feature in the pathogenesis of IBS (64). Emerging evidence suggests that gut microbiota and their products can modulate the GBA (28). In a seminal study, Sudo et al. (127) found that postnatal gut microbial colonization programs the development of the HPA axis to regulate stress response. They found that germ-free (GF) mice exhibited significantly higher corticosterone and lower brain-derived neurotrophic factor (BDNF) levels in the cortex and the hippocampus compared with specific pathogen-free (SPF) mice, when subjected to restraint stress. Interestingly, monocolonization of GF mice with Bifidobacterium infantis reversed these effects highlighting the influence of individual gut microbial members on HPA axis regulation (127). Additionally, a recent study in healthy volunteers found that consumption of a fermented milk product containing Bifidobacterium, Lactobacilli, and Streptococcus thermophiles alters brain connectivity and function (135), supporting the effect of gut microbes on the GBA. One way by which microbiota alter brain function and behavior is via free fatty acid production (FAA) (140). As an example, propionic acid produced by gut bacteria readily crosses the blood-brain barrier and influences brain function and behavior in animals (120, 140). Besides FAA, gut microbes such as Lactobacilli and Bifidobacteria can also generate γ-amino butyric acid (GABA), an inhibitory neurotransmitter in the human brain (33). These studies highlight the important role of gut microbiota and their metabolites in modulating the GBA.

Effect of Gut Microbiota on Visceral Sensation Visceral hypersensitivity (VH), a prominent feature in IBS patients, is characterized by reduced pain threshold to a painful stimuli. In a recent study, Crouzet et al. (27) found that GF rats colonized with gut microbiota from IBS patients displayed reduced pain threshold to colonic distension, when compared with GF rats that were colonized with gut microbiota from healthy individuals. Although the mechanisms by which microbiota transfer leads to VH in rats remain unclear, there was a significant decrease in Bifidobacteria and an increase in Enterobacteriaceae and other sulfate-reducing bacteria (27). Concomitantly, there was higher hydrogen excretion and sulfide production, the two principal components of pronociceptive gasotransmitter hydrogen sulfide (H 2 S) (27, 96). Excitatory acid-sensitive nociceptors called transient receptor potential vanilloid type 1 (TRPV1) channel proteins that are found in the vagal and spinal afferents are important mediators of VH in IBS (23, 53, 88, 146). Interestingly probiotic Lactobacillus reuteri exerts an antinociceptive effect via TRPV1 highlighting a potential role for gut microbiota/microbial products in VH (102).

Effect of Gut Microbiome on GI Motility GI motility requires complex coordination among neurons, interstitial cells of Cajal, smooth muscle, and the immune cells to facilitate digestion of nutrients and movement of unwanted waste along the length of the GI tract. Alteration in GI motility is a hallmark of IBS. Gut microbiota and its metabolites can influence GI motility by affecting one of several pathways involving enteric neurons, glia, or enteric muscularis macrophages. For example, gut microbiota-derived lipopolysaccharide (LPS) and microbiota products such as the short-chain fatty acids (SCFAs) promote enteric neuronal survival (4, 125). In addition SCFAs also affect neurotransmitter release and influence the cross talk between enteric neurons, smooth muscles and muscularis macrophages to regulate GI motility (56, 92, 125). Microbiota and their products also affect the development, maturation, and generation of mucosal enteric glial cells, which might play a role in regulating GI motility (12, 54). Recently, gut microbiota bile acid metabolism has been implicated in GI motility (37) and their interaction with the enteric nervous system (32). The role of gut microbiota in regulating GI motility in IBS is further supported by interventional studies using probiotics. For example, a fermented dairy product containing Bifidobacterium lactis has been found to accelerate GI transit and improve symptoms in patients with constipation-predominant IBS (2), while a probiotic mixture containing various strains of Lactobacillus and Bifidobacterium has been shown to improve stool consistency and overall symptoms in IBS-D patients (62). Overall, these studies not only highlight the importance of gut microbiota in regulating GI motility but also underscore how probiotics can be therapeutically used to treat GI dysmotility in IBS.

Effect of Gut Microbiota on Intestinal Barrier Dysfunction Integrity of the intestinal epithelial barrier is essential for maintaining adequate nutrient transport and providing a barrier against pathogens residing in the gut lumen. While the gut microbiota and their metabolites play an important role in maintaining the integrity of the epithelial barrier, alterations in gut microbiota in turn can disrupt the epithelial barrier (58). Increased intestinal permeability is a feature of IBS-D and has been linked to symptom generation (19, 38, 148). Bacterial derived SCFAs help in maintenance of intestinal barrier structure and function (100, 101, 129). Butyrate, a bacterial derived SCFA, for example, prevents bacterial translocation by increasing expression of tight junction proteins such as claudin, occludin, and zonula occludens proteins (105). This is particularly interesting given the fact that reduction in butyrate producing gut bacteria has been observed in patients with IBS (106). Furthermore, Lactobacillus rhamnosus GG, a probiotic strain has been shown to induce claudin expression in neonatal mice suggesting that exposure to bacteria early in life promotes the maturation and development of the epithelial barrier (55, 99). In addition to affecting tight junction proteins along the epithelial barrier, gut microbiota and their metabolites also regulate mucus layer (35, 123). Mucus layer forms the barrier between the lumen and the epithelial cells and prevents pathogen access to the epithelial surface (136). Changes in the amount and/or the composition of mucus may lead to inflammatory responses (70). Mucin degraders such as Ruminococcus torques and Ruminococcus gnavus are associated with severity of bowel symptoms in IBS (75, 78, 130, 133). Furthermore, a multispecies probiotic formulation containing a combination of Lactobacillus rhamnosus GG, L. rhamnosus Lc705, Propionibacterium freudenreichii ssp.Shermanii JS, and Bifidobacterium breve Bb99 decreases mucolytic Ruminococcus torques in IBS possibly by upregulating cell-surface mucin secretion and preventing its adherence to the epithelial layer (74, 76, 77, 94).

Effect of Gut Microbiota on Immune Activation Immune activation underlying IBS has been an important area of investigation with several studies showing low-grade inflammation and infiltration of inflammatory cells notably the mast cells (MCs) and macrophages in the intestinal mucosa of IBS patients (10, 11, 22, 144). MCs regulate innate immunity through expression of pattern recognition receptors such as Toll-like receptors (TLRs). Notable examples of TLR expression in MCs associated with IBS include TLR2 and TLR4 (13, 18). Bacterial components in the gut such as flagellin and LPS can act as TLR ligands. Interestingly, an increased serum level of antibodies specific to flagellin and LPS concomitant with increased TLR (TLR4 and TLR5) expression has been observed in IBS patients (34). Activation of mast cells through TLR ligands causes release of inflammatory mediators such as histamine, tryptase, and prostaglandin E 2 (PGE 2 ). Release of these inflammatory mediators is also increased in IBS patients (11). These mediators acting through a G-coupled receptor called protease-activated receptors (PAR) can influence excitability of enteric neurons and potentially lead to VH in IBS (21). Together these studies suggest increased innate immune system activity can be driven by gut bacteria or their components. The resulting mast cell activation can potentially play a role in symptom generation in IBS. Besides increased innate immune system activity, immunoregulatory molecules including pro- and anti-inflammatory cytokines are also altered in patients with IBS. For example, an increase in proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and IL 1β, IL-6, and IL-8] level (46, 118, 121) and a decrease in anti-inflammatory cytokine (IL-10) have been reported in in both plasma and peripheral blood mononuclear cell (PBMC) of IBS patients (95, 118, 141; reviewed in Ref. 47). Probiotics have been investigated for their ability to restore cytokine balance. A placebo-controlled randomized clinical trial in patients with IBS showed that a 12-wk course of B. infantis resulted in an increase in the IL-10/IL-12 ratio similar to levels found in healthy patients (95). Furthermore, probiotic Bifidobacterium longum administration reduced proinflammatory TNF-α and probiotic combination of Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacterium bifidum, and Lactobacillus bulgaricus decreased TNF-α, interferon-γ (IFN-γ), and PGE 2 levels in mice (109, 138). Together, these studies suggest that probiotics modulate both innate and adaptive immunity to potentially impart a therapeutic benefit and treat immune dysregulation in IBS (18, 34, 84, 119). In addition to probiotics, microbial metabolites of dietary nutrients have been shown to be anti-inflammatory. Two recent studies describe the role of gut microbial metabolites of dietary tryptophan, which act as aryl hydrocarbon receptor (AHR) ligands and can improve inflammation in peripheral GI tissues as well as the central nervous system highlighting the importance of microbial metabolites as mediators of their effect on the immune system (66, 110).