The Gut Microbiome and Colonic Motility Disorders: A Practical Framework for the Gastroenterologist

Abstract

Purpose of Review

Colonic motility disorders may be influenced by the gut microbiota, which plays a role in modulating sensory and motor function. However, existing data are inconsistent, possibly due to complex disease pathophysiology, fluctuation in symptoms, and difficulty characterizing high-resolution taxonomic composition and function of the gut microbiome.

Recent Findings

Increasingly, human studies have reported associations between gut microbiome features and colonic motility disorders, such as irritable bowel syndrome and constipation. Several microbial metabolites have been identified as regulators of colonic motility in animal models. Modulation of the gut microbiota via dietary intervention, probiotics, and fecal microbiota transplant is a promising avenue for treatment for these diseases.

Summary

An integration of longitudinal multi-omics data will facilitate further understanding of the causal effects of dysbiosis on disease. Further understanding of the microbiome-driven mechanisms underlying colonic motility disorders may be leveraged to develop personalized, microbiota-based approaches for disease prevention and treatment.

Introduction

Colonic motility disorders, including functional diarrhea, constipation, irritable bowel syndrome (IBS), and fecal incontinence (FI), are among the most common gastrointestinal (GI) indications for clinic and emergency department visits in the U.S.[1] IBS, the archetypal GI motility disorder, has been estimated to affect 10–15% of the population with annual direct costs of over $1 billion dollars alone.[2, 3] Like other disorders of brain-gut interaction, colonic motility disorders are characterized by a complex pathophysiology involving both motor and sensory dysfunction. The colon represents one of the body’s primary interfaces with the microbial milieu and accumulating evidence suggests that the gut microbiota may play a crucial role in the development of colonic dysmotility syndromes.

Evidence from germ-free animal models (animals raised to be microbiologically sterile) suggests that the gut microbiota has a profound influence on the modulation of gut sensory and motor function.[4, 5] In addition, other factors that may affect the colonic motility such as diet[6] and host genetics[7] are associated with alterations in the gut microbiome and may exert their effects at least partially via modulation of the microbiome. The advances in novel tools for identification and quantification of the microbiome, primarily next-generation sequencing, has revolutionized studies of the gut microbiome by enabling investigations of microbial changes in a culture-independent manner. Our initial understanding of these microbial changes has been derived using 16 S ribosomal RNA (rRNA) sequencing. While such techniques have expanded our understanding of the altered microbiota seen in IBS and constipation compared to healthy controls, findings from these studies have been inconsistent[8, 9]—likely at least in part of the inability of 16 S rRNA analyses to provide more than genus-level comparisons. Indeed, individual species and strains within the same genus can have markedly different functions. The recent emergence of whole shotgun metagenomic sequencing (WGS) techniques provides not only a higher taxonomic resolution (i.e. ability to discern species- and strain-level data) but also the ability to perform functional characterization of the gut microbiome to assess how altered microflora may influence physiology.[10, 11].

In this review, we focus on the most recent evidence pertinent to the gut microbiome in colonic motility disorders and highlight future directions translating this knowledge to therapeutic prevention and treatment strategies.

The Gut Microbiome in Colonic Motility Disorders

Irritable Bowel Syndrome

Irritable bowel syndrome (IBS) is one of the most common lower GI disorders comprising abdominal pain associated with altered bowel habits (diarrhea, constipation, or a mix of the two). The pathophysiology is believed to be multifactorial but is not yet fully understood. Altered gut microbiota have been hypothesized as a potential contributor to IBS based on the observations that acute gastroenteritis, which leads to marked shifts in microbiota, is a strong risk factor for IBS[12]. Patients with post-infectious IBS are characterized by alterations in the microbiome including enriched Bacteroidetes and depleted Clostridia.[13].

Much of the data on the role of gut microbiota in IBS is derived from cross-sectional studies reporting differences in overall microbiome configuration and specific bacterial groups among IBS patients compared to healthy controls. In a comprehensive systematic review, Pittayanon et al. reviewed studies published through 2018 comparing the gut microbiome of adult or pediatric patients with IBS with that of healthy individuals.[8] A total of 24 studies that assessed fecal or mucosal microbiota using bacterial culture, 16 S rRNA, and other targeted next-generation sequencing methods were included. While most studies report some degree of dysbiosis associated with IBS, a major finding of this data synthesis was that there is significant heterogeneity between studies and a distinct lack of concordant results. Nevertheless, some IBS-related changes in the gut microbiome were reported consistently across multiple studies (≥ 3) at broad order and genus levels, including increased abundances of Bacteroides, Enterobacteriaceae, and Lactobacillaceae, as well as decreased abundances of Bifidobacterium, suggesting a potential signal for microbial markers associated with disease state.

More recent studies using WGS sequencing have facilitated a deeper understanding of changes in the gut microbiome associated with IBS by discerning high-resolution taxonomic composition at both species and strain levels and creating a functional profile of these biosamples (Table 1). To our knowledge, the first-of-its-kind study was conducted in a Dutch case-control cohort, in which stool samples from 412 patients with IBS were compared to those from 1,025 controls.[14] In line with previous studies, IBS patients had an increase in several Streptococcus species (belonging to the order Lactobacillales) and decrease in butyrate-producing bacteria, including Faecalibacterium prausnitzii. They also found IBS was associated with an increased strain diversity in pathogenic species and reduced strain diversity in beneficial species. Last, taxonomic changes were accompanied with altered microbial pathways including decreased fermentation of pyruvate to butanoate, a butyrate precursor, and increased fermentation and carbohydrate degradation. A subsequent study identified enrichment of amino acid biosynthesis/degradation pathways in IBS patients, accompanied by changes in fecal metabolites such as amino acids, dodecanedioic acid, and purine nucleosides.[10] Within IBS patients, IBS severity was found to be associated with altered gut microbiota hydrogen function that was correlated with microbiota enzymes involved in animal carbohydrate metabolism.[11].

Table 1.

Summary of recent studies of gut microbiota and colonic motility disorders^*

Study	Study population	Sample	Sequencing method	Findings
Irritable bowel syndrome
Vich Vila et al. 2018[14]	412 IBS and 1025 controls	Fecal	WGS	↓ Butyrate-producing bacteria (e.g., Faecalibacterium prausnitzii) and strain diversity in beneficial species ↑ Streptococcus and strain diversity in pathogenic species ↑ Fermentation and carbohydrate degradation pathways
Zhao et al. 2020[41]	277 IBS-D and 84 controls	Fecal	WGS	↑ Clostridia and genomes in bile acid synthesis/excretion among IBS-D patients with high bile acid excretion in feces ↑ Ruminococcus, Clostridium, Eubacterium, Dorea, Bifidobacterium, Escherichia, and Bilophila ↓ Alistipes and Bacteroides
Xu et al. 2020[70]	22 IBS-D and 15 controls	Fecal	WGS	↑ Functional genes in trehalose and maltose hydrolase and fucose permease ↑ Escherichia coli
Jeffery et al. 2020[10]	80 IBS and 65 controls	Fecal	WGS, metabolomics	Differences in fecal microbiome (e.g., enrichment of amino acid biosynthesis/degradation pathways) and fecal metabolome (e.g., amino acids, dodecanedioic acid, and purine nucleosides) from controls, independent of symptom-based subtypes of IBS. Fecal metabolome can distinguish patients with IBS with vs. those without bile acid malabsorption.
Mars et al. 2020[15]	51 IBS and 24 controls (longitudinal samples)	Fecal	WGS, metabolomics	↑ Streptococcus spp. in IBS-C and IBS-D compared to controls ↑ ≤ 20Lactobacillus spp. associated with IBS-D severity ↓ Short-chain fatty acid levels in IBS-C compared to controls ↑ Tryptophan and tryptamine levels in IBS-D compared to controls ↓ Hypoxanthine levels in IBS-D and IBS-C ↑ Functional capacity for hypoxanthine utilization and breakdown and an upregulation of the purine salvage pathway in colonic epithelium Some specific alterations in gut microbiome (e.g., diversity, Halobiforma nitratireducens) and microbial metabolites (primary bile acids) were linked to flares in the patients.
Tap et al. 2021[11]	149 IBS and 52 controls	Fecal	WGS	IBS symptom severity was associated with altered gut microbiota hydrogen function, which was correlated with animal carbohydrate metabolism.
Jabbar et al. 2021[16]	62 IBS and 31 controls	Mucus samples from sigmoid colon biopsies	Metaproteomics	Colonization of pathogenic Brachyspira species was significantly more common in IBS and detected in 40% of IBS-D patients.
Chronic constipation
Zhu et al. 2014[71]	8 constipation and 14 controls, obese children (mean age: 11 and 13 years)	Fecal	16 S rRNA gene pyrosequencing	Differences in alpha- and beta-diversity metrics ↑ Lachnospiraceae, Blautia, Coprococcus, Ruminococcus, Anaerotruncus, and Clostridium ↓ Prevotella
De Meij et al. 2016[72]	76 children with constipation and 61 controls (median age: 8.0 and 8.6 years)	Fecal	PCR-based IS-pro	↑ Bacteroides fragilis, Bacteroides ovatus, Parabacteroides spp., Bifidobacterium longum, Proteus mirabilis ↓ Alistipes finegoldii, Ruminococcus spp.
Parthasarathy et al. 2016[23]	25 constipation (13 FC, 6 IBS-C, 6 mixed IBS) and 25 controls	Mucosal, fecal	16 S rRNA	The overall composition of colonic mucosal microbiota was associated with constipation, independent of colonic transit. Patients with constipation had more Bacteroidetes in mucosal microbiota. The profile of fecal microbiota was associated with colonic transit and methane production, but not constipation.
Mancabelli et al. 2017[46]	68 constipation and 79 controls	Fecal	16 S rRNA and WGS	↑ Faecalibacterium, Ruminococcaceae, Paraprevotella, Clostridiales, and Ruminiclostridium ↓ Bacteroides, Roseburia, and Coprococcus 3 ↑ Pathways in hydrogen production, methanogenesis and glycerol degradation ↓ Pathways in methylglyoxal degradation and carbohydrate, fatty acid, and lipid metabolism
Tian et al. 2020[73]	18 constipation and 17 controls	Fecal	16 S rRNA	Differences in alpha- and beta-diversity metrics ↑ Ruminococcus torques, Parabacteroides gordonii, Bacteroides coprophilus, Bacteroides caccae, and Bacteroides fragilis ↓ Prevotella stercorea, Prevotella copri, and Roseburia intestinalis ↓ Levels of acetate
Guo et al. 2020[74]	61 constipation and 48 controls	Fecal	16 S rRNA	Differences in alpha- and beta-diversity metrics ↑ Bacteroides, Prevotella, Butyricimonas, Ruminococcus, and Lactococcus ↓ Akkermansia, Atopobiu, Thermus, Dehalobacterium, and Veillonella

^*We only included recent studies using WGS for IBS since earlier studies using 16 S rRNA have been reviewed previously.[8]

WGS, whole shotgun metagenomic sequencing; IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome with diarrhea; IBS-C, irritable bowel syndrome with constipation; FC, functional constipation

In another study, Mars et al. used an innovative approach by integrating longitudinal multi-omics data from the gut microbiome, metabolome (metabolites produced by the microbiome), host genetics, transcriptome (the full range of messenger RNA produced by gut bacteria), and epigenome (record of the chemical changes to the DNA and histone proteins of an organism).[15] They found IBS subtypes and symptom severity were associated with specific changes in the composition and function of the gut microbiome and metabolome. For example, significantly higher abundances of multiple Streptococcus spp. were shown in both IBS with constipation (IBS-C) and diarrhea (IBS-D) patients, and severity of IBS-D was associated with a higher abundance of ≥ 20Lactobacillus spp. Metabolomic analysis suggested that short-chain fatty acids (SCFAs), including propionate, butyrate, and acetate, were significantly lower in IBS-C patients compared to controls, whereas tryptophan and tryptamine were increased in IBS-D patients. Some specific alterations in gut microbiome (e.g., diversity, Halobiforma nitratireducens) and microbial metabolites (primary bile acids) were linked to flares in the patients. Finally, hypoxanthine levels were consistently decreased over time in IBS subtypes, along with increased functional capacity for hypoxanthine utilization and breakdown and an upregulation of the purine salvage pathway in colonic epithelium.

While the majority of gut microbiome studies rely on analysis of fecal samples that largely reflect the luminal microbial community, it has been suggested that microbial community of the colonic inter mucus layer may play a role in the pathogenesis of IBS as well. A metaproteomic analysis of colon mucus samples identified colonization of pathogenic Brachyspira species in 40% of IBS-D patients but not in any healthy individuals, and Brachyspira-associated IBS was associated with distinct clinical, histological, and molecular characteristics.[16] Recent evidence has also shed light on new hypotheses that alterations of other microorganisms, particularly viruses and fungi, may also contribute to IBS and its related symptoms.[17-19].

Constipation

Chronic constipation is another highly prevalent colonic motility disorder, simplistically defined as 2 or more of the following: fewer than three spontaneous bowel movements per week, straining, lumpy or hard stools, sensation of anorectal obstruction or blockage, sensation of incomplete defecation, and/or manual maneuvers required to defecate with symptoms lasting at least 3 months.[20] By definition, pain is not a prominent feature in chronic constipation, which has been used to differentiate chronic constipation from IBS-C.

There is some evidence from animal experiments that methane may delay intestinal motility,[21] and methane production measured by breath test is associated with delayed transit time in patients with chronic constipation.[22] Thus, some have argued that higher numbers of methanogenic bacteria and corresponding methane production may be underlying the pathogenesis of chronic constipation. However, compared to higher-quality evidence on the altered gut microbiome in IBS, relatively fewer studies using modern microbiome sequencing techniques have been conducted in chronic constipation.

Parthasarathy et al. examined the mucosal-associated vs. stool microbiota in relation to symptoms, colonic transit, and methane production in 25 women with constipation and 25 healthy women (controls).[23] They found that the overall composition of colonic mucosal microbiota was associated with constipation symptoms independent of colonic transit and discriminated between patients with constipation and controls with 94% accuracy. Patients with constipation had more abundant Bacteroidetes. In contrast, stool microbiota was associated with colonic transit and methane production, but not constipation symptoms. In another study, patients with constipation showed a depleted representation of Bacteroides, Roseburia, and Coprococcus 3, as well as an over-representation of multiple genera belonging to the Ruminococcaceae family, such as Faecalibacterium. The authors further performed WGS sequencing in a subset of participants, and found that patients with constipation had higher abundances of genes involved in production of methane and hydrogen, and lower abundances of genes involved in degradation of methylglyoxal (a potential toxic metabolite that has been implicated in many diseases including IBS[24]). Stool from patients with constipation was also enriched with genes associated with degradation of glycerol, which is known to relieve constipation by osmotic regulation.[25].

As in IBS, systematic reviews of existing studies regarding gut microbiota in chronic constipation have demonstrated a lack of clear microbial signature of chronic constipation.[9, 26] Future large-scale studies integrating multi-omics such as metabolomics and functional profiling of the microbial community will be warranted to further our understanding of the pathways underlying gut microbiota and chronic constipation.

Fecal Incontinence

The etiology of fecal incontinence (FI) among people whose symptoms could not be attributed to an underlying organic disorder is poorly understood. Epidemiological studies have shown that bowel disturbances such as diarrhea and IBS rather than obstetric injury are the main risk factors for FI.[27, 28] To the best of our knowledge, microbial changes in patients suffering from FI have not been investigated before. An ongoing study is being conducted to determine the associations of the gut microbiota composition and stool metabolites with FI within the Controlling Anal Incontinence in women by Performing Anal Exercises with Biofeedback or Loperamide (CAPABLe) trial.[29].

Bidirectional Interplay between Gut Microbiota and Motility and Microbial Metabolites

Perhaps the most fascinating aspect of our widening understanding of the role of the gut microbiota in colonic motility disorders is the relationship between gut microbiota composition and function and GI physiology. It is well recognized that there is a bidirectional relationship between gut microbiota and GI motility/transit.[5] On one hand, gut microbes can influence GI transit. Experiments using germ-free rat models revealed that microbes stimulated or suppressed small intestinal transit depending on the species.[30] On the other hand, GI motility represents one of the major control systems of gut microbiota through the sweeping of excessive bacteria from the lumen, via which it may shape the gut microbiota composition and function.[5] This is consistent with the r/K selection theory of microbial ecology.[31] For example, when GI transit slows, as with constipation, species better adapted to grow more rapidly in competitive environments (i.e., limited resources; K-selected) will dominate. Conversely, as GI transit accelerates, species better adapted to persist during reduced competition (i.e., excess resources; r-selected) will dominate.[32].

In an elegant study, Kashyap et al. used highly controlled mouse models to investigate the complex interrelationship between transit time, microbiota, and host dietary intake.[32] By accelerating or decelerating host GI transit with polyethylene glycol or loperamide, respectively, they demonstrated altered gut microbial communities with changes in gut transit time, which returned to baseline after discontinuing the treatments. Conversely, introducing a complex, fecal microbiota from a healthy human into germ-free mice significantly shortened GI transit time and increased colonic contractility; this effect was dependent on the type and amount of carbohydrate in the diet. Specifically, administration of dietary cellulose, as nonfermentable polysaccharides, shortened GI transit time similarly in germ-free and humanized mice compared to standard diet, suggesting that this effect was independent of the presence of gut microbes. In contrast, compared to standard diet, a diet enriched with fermentable fructooligosaccharides shortened GI transit time in germ-free mice, but increased transit time in humanized mice, the latter of which was accompanied by lower fecal SCFA levels. Since the quantity and complexity of carbohydrates was higher in the standard diet relative to the FOS-enriched diet, these results suggest that GI transit time is at least partly affected by microbe-mediated carbohydrate fermentation.

Several microbial products have been identified as regulators of GI motility (Fig. 1), and may play a role in the development of colonic motility disorders including SCFAs, bile acids, tryptamine, and several gaseous byproducts such as methane, hydrogen sulfide, and hydrogen gas.[33].

Fig. 1. Overview of the role of gut microbiota-derived metabolites in gastrointestinal motility and sensation.

Reprinted from “Metabolites and microbial composition of stool of women with fecal incontinence: Study design and methods”, by L. A. Arya, 2018, Neurourol Urodyn, 37(2), 634 – 41. Copyright 2018 by John Wiley and Sons. Reprinted with permission

SCFAs

SCFAs - in particular, acetate, propionate, and butyrate, are produced by fermentation of dietary carbohydrates by a group of colonic bacteria. SCFAs play a role in modulating the serotonergic pathway in the colon. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter with pleiotropic effects including modulation of GI motility.[34] In murine models, butyrate and acetate can stimulate 5-HT biosynthesis by increasing the rate-limiting enzyme tryptophan hydroxylase.[35] In rats, intraluminal administration of SCFAs into the proximal colon resulted in accelerated colonic transit mediated by 5-HT release, with this effect abolished by a 5-HT₃ receptor antagonist.[36] 5-HT released from the basal membrane of intestinal enterochromaffin cells then interact with receptors from neurons in the enteric nervous system and regulate motility.[37] These findings are consistent with observations of microbial changes in patients with IBS, including decreased abundance of butyrate-producing bacteria, altered carbohydrate metabolism microbial pathways, and decreased fecal and colonic SCFA levels.[11, 14, 15].

Beyond GI motility, 5-HT has also been recognized as a critical regulator of the gut-brain-microbiome axis signaling.[37] The vagus nerve can sense 5-HT and connects the GI tract to the nucleus of the solitary tract and the dorsal raphe nucleus, the latter of which houses the majority of the brain’s 5-HT neurons. These areas then interact with the emotion-regulating brain networks that influence mood. Additionally, SCFAs can also directly stimulate free fatty acid receptors on multiple cell types and modulate down-stream regulation of gut-brain signaling.

Bile Acids

The primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) are deconjugated from taurine or glycine and dehydroxylated by colonic bacteria, producing secondary bile acids including deoxycholic acid (DCA) and lithocholic acid (LCA). Presence of bile acids in the colonic lumen results in colonic secretion of water and electrolytes[38] and induction of high amplitude propagated contractions.[39] Indeed, bile acid malabsorption is an increasingly-recognized cause of diarrhea,[40] and patients with IBS-D have enriched bile-acid transforming genomes and bacteria as well as higher fecal levels of bile acids.[15, 41].

Tryptamine

Tryptamine is derived by microbial metabolism of dietary tryptophan. Prior studies have demonstrated that tryptamine activates the 5-HT receptor-4 (5-HT₄) resulting in increased fluid secretion in the proximal colon and accelerated GI transit in gnotobiotic mice.[42] Additionally, fecal levels of tryptophan and tryptamine are significantly higher in IBS-D patients, indicating that tryptamine could in part be responsible for the increased water content of stools in IBS-D.[15].

Gaseous Byproducts

Several microbial gaseous products may influence GI motility. Methane has been demonstrated to delay small intestinal transit in animal experiments, possibly by augmenting smooth muscle contractility.[21] Producers of methane in humans are thought to primarily include Archaea and a relatively small subset of methanogenic bacteria (e.g., Bacteroides thetaiotaomicron and Clostridium spp.).[43] Hydrogen sulfide is another compound with documented effects on GI motility. It is produced by sulfate-reducing bacteria and has been shown to inhibit the contractile activity of smooth muscle in experimental studies.[44] Finally, hydrogen has been reported to significantly shorten transit in the proximal and distal colon.[45] Thus far, only limited human data support the link between gut microbiome production of methane and hydrogen and colonic motility disorders such as constipation.[46].

Therapeutic Modulation of the Gut Microbiota in Colonic Motility Disorders

With the accumulating evidence of gut microbiome alterations in colonic motility disorders from cross-sectional studies, a number of randomized controlled trials (RCTs) have investigated the effects of therapeutic modulation of the gut microbiota, including dietary interventions, probiotics, and more recently fecal microbiota transplant (FMT).

Diet

Dietary fiber has been advocated for the management of chronic constipation and IBS, and a fermentable oligosaccharide, disaccharide, monosaccharide and polyol (FOD-MAP) restricted diet has been recommended for IBS.

The most recent American College of Gastroenterology Monograph reviewed the evidence in support for the use of fiber in the management of IBS, including for overall symptom improvement with modest quality of evidence.[47] In addition, the effects of fiber vary according to its solubility and fermentability. A seminal trial found that soluble, slowly-fermentable fiber (e.g., psyllium) was more effective than insoluble fiber (e.g., bran) for overall symptom improvement.[48] In a systematic review of seven RCTs, fiber supplementation was shown to be beneficial in mild to moderate chronic constipation in five studies, although RCTs with a larger sample size and longer duration of intervention are needed.[49].

A weak recommendation was made for the use of low FODMAP diet, as opposed to gluten-free or exclusion diet, in the management of IBS based on very low quality of evidence given the high risk of bias, relatively small number of patients included in the trials, and significant heterogeneity.[47] Despite the short-term symptomatic benefits, concerns have been raised regarding the potentially undesirable effects of the low FODMAP diet on the gut microbiota. This was then tested in several subsequent RCTs (Table 2).[50-52] In addition to an expected effect on reducing the abundance of carbohydrate-fermenting bacteria Bifidobacteria,[50, 52] which was considered not ideal, the low FODMAP diet also led to increases in other bacterial groups such as Clostridium, Eubacterium, and Faecalibacterium[52], increases in Actinobacteria richness and diversity,[52] changes in bacteria involved in gas consumption,[51] as well as altered metabolome such as reduced urine levels of histamine.[51] The low FODMAP diet was also found to decrease saccharolytic fermentation activity as derived using SCFA levels, and interestingly, higher saccharolytic fermentation activity is associated with a higher symptom burden and a favorable therapeutic response to the low FODMAP diet.[52] Such data provides support for a diet-microbiota interaction in IBS and highlights the potential for a personalized dietary therapeutic intervention based on an individual’s gut microbiome profile.

Table 2.

Summary of randomized controlled trials of effects of dietary interventions on the gut microbiome/metabolome and symptoms in colonic motility disorders

Study	Study design	Study population	Intervention and duration	Sample	Sequencing method	Primary outcome	Findings
Staudacher et al. 2017 [50]	2 × 2 factorial RCT	104 IBS	4 groups given dietary advice for 4 weeks: 27 Sham diet/placebo; 26 sham diet/probiotic; 24 low FODMAP diet/placebo; 27 low FODMAP diet/probiotic	Fecal	Quantitative PCR and 16 S rRNA	Relief of symptoms; Bifidobacterium abundances	1) Low FODMAP diet improved IBS symptoms compared to sham diet. 2) Abundance of Bifidobacteria was lower in low FODMAP diet than sham diet, but restored with coadministration of a probiotic.
Mclntosh et al. 2017[51]	Parallel-group RCT	40 IBS	2 groups instructed follow dietary advice for 3 weeks: 20 low FODMAP diet; 20 high FODMAP diet	Fecal and urine	16 S rRNA for stool microbiome; lactulose breath test and mass spectrometry for urine metabolome	IBS SSS; hydrogen and methane from lactulose breath test; urine metabolites; gut microbiome	1) Reduced IBS-SSS in low FODMAP diet group but not in the high FODMAP group 2) ↓ in hydrogen in low FODMAP 3) Separation of metabolome between two groups (histamine, p-hydroxybenzoic acid, azelaic acid) 4) Low FODMAP diet increased Actinobacteria richness and diversity; and had higher relative abundance of bacteria involved in gas consumption
Zhang et al. 2021[52]	Parallel-group RCT	108 IBS-D	2 groups instructed to follow diets for 3 weeks: 54 FODMAP diet; 54 traditional diet	Fecal	16 S rRNA	≥ 50-point reduction in IBS SSS	1) No difference in percentage of participants achieving ≥ 50-point reduction in IBS SSS 2) Earlier symptomatic improvement in stool frequency and excessive wind 3) ↓ Bifidobacterium and ↑ Clostridium, Eubacterium, and Faecalibacterium 4) Higher saccharolytic fermentation activity was associated with a higher symptom burden and a favorable response to FODMAP

RCT, randomized controlled trial; IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome with diarrhea; FODMAP, fermentable oligosaccharides, disaccharides, monosaccharides, and polyols; SSS, severity scoring system

Probiotics

The American Gastroenterological Association’s Technical Review as part of the recently-released clinical guidelines on probiotics reviewed studies assessing the efficacy of probiotics for both children and adults with IBS.[53] A total of 55 trials testing 44 different probiotic species/strains or combinations of species/strains were identified. For the majority of probiotics, evidence supporting their use was derived from a single trial with relatively small sample sizes, variable quality, and risk of bias. Some specific species or strains (mostly Bifidobacteria species) or particular combinations have shown benefits in the improvement of global IBS symptoms and abdominal pain scores. These results were consistent with an earlier meta-analysis of 19 trials reporting a significant benefit in improvement of IBS symptoms from multistrain probiotics among adults with IBS.[54] However, one of the better-conducted RCTs to date demonstrated significant improvement in IBS symptoms using a more rigorous endpoint than most studies with a single-species, inactivated strain.[55] In a meta-analysis of 15 RCTs evaluating the use of probiotics in constipation, multispecies probiotics, but not single-species probiotics, were found to significantly reduce the gut transit time, increase stool frequency, and improve stool consistency.[56].

Despite these positive results, higher quality studies on the use of probiotics in functional bowel diseases have generally found lower effect sizes than earlier, uncontrolled pilot studies. With the mechanism by which probiotics improve symptoms in colonic motility disorders remaining largely unknown, the optimal probiotic may depend more on an individual’s microbiome and clinical phenotype than any one strain showing benefit across all patients with a given disorder.

Fecal Microbiota Transplant

Seven RCTs investigating the efficacy of FMT in IBS have already been conducted, four of which showed an effect of FMT on symptom improvement in IBS while the other three showed no effect.[57] However, as with other interventions, there was substantial heterogeneity between studies—including variations in dose and duration of the treatment, route of administration, outcome measured, and study population—which precludes a definite conclusion of overall efficacy of FMT use. Certain techniques may be more beneficial than others. FMT from donor stool delivered using colonoscopy or nasojejunal were superior to autologous FMT, whereas FMT administration via capsules was not effective.[58, 59] The effect may be dose-dependent, with greater doses more likely achieving improvement of IBS symptoms.[57, 59] One RCT has been conducted to examine the effects of FMT for treatment of chronic constipation.[60] In patients with slow-transit constipation, compared to conventional treatment (education, behavioral strategies, and oral laxatives), FMT treatment over 6 days led to a significantly higher clinical improvement rate and cure rate.

Future Directions

Despite studies demonstrating alterations of the gut microbiome in IBS or chronic constipation, we are still far from fully understanding the role of gut microbiome in disease pathogenesis. Below we propose strategies for improved study designs and novel hypotheses that need to be tested for future studies.

Longitudinal Multi-omics Data Collection and Microbiome Sampling

One of the challenges in this field has been to disentangle the causal relationship between the microbiota and the clinical condition. The bidirectional relationship between gut microbiota and GI motility precludes making firm conclusions when the data is derived from cross-sectional studies. Ideally, a prospective study with longitudinal sample collections prior to disease onset as well as during the disease would more definitively address these questions. The recent work by Mars et al. provides a good example of using longitudinal samples to capture the fluctuation of symptoms in IBS.[15] Furthermore, colonic motility disorders are complex diseases that are heterogeneous clinical manifestations. An integration of multi-omics data including molecular profiles of the host (e.g., serum metabolomics and serological biomarkers, mucosal gene expression, etc.) and microbial activity (e.g., metagenomics, metatranscriptomics, and metabolomics of the gut microbiome) will facilitate a comprehensive view of functional dysbiosis in the gut microbiome in the disease and impacts on host biology.[61].

Of course, parallel advances in how we study the gut microbiome are also required for rigorous and robust findings. Differences in sampling approaches, including subtle technical variation introduced by the use of different fixatives or collection devices, or sequencing approaches (e.g., 16S rRNA vs. WGS) likely contributes to the inconsistencies observed across studies. Indeed, a major goal of the Microbiome Quality Control Project is to develop universal sampling approaches to overcome these limitations.[62] Nonetheless, while fecal sampling empowers several study design advantages including being non-invasive and allowing at-home sampling, especially before significant perturbation introduced by bowel preparation prior to clinical procedures, this measure provides only a proxy or ‘snapshot’ of the gut microbiome present in the lower GI tract, but may miss particularly important mucosal-associated microbiota. Further, microbial composition varies between different regions of the gastrointestinal tract and mucosal sampling may require multiple biopsy sites for a more holistic view of the inter-microbial associations central to disease processes.[63] Microbe-microbe interactions captured both within fecal communities and those living at the mucosal interface are important to advancing our understanding of the role of the gut microbiome in gut motility. Future studies should make a considered effort to sample both fecal and mucosal surfaces for multi-omic profiling, and we echo calls for innovation in these spaces for precise sampling microbial communities.[64].

While acknowledging that the addition of more ‘big data’ to the already vastly complex gut microbiome seems to further complicate existing -omics challenges, the collection of additional molecular endpoints will allow for deeper characterization, interrogation, and validation of potential causal microbiome-host relationships within the context of the same individual. Our opinion is that by pairing multiple microbiome sampling approaches and pursuing deeper molecular characterization of both the host and the gut microbiome within the same individuals that stronger protective or health-associated microbial signatures may emerge while clarifying the specific processes associated with disease states and dysbiosis.

Leveraging the Gut Microbiome for Personalized Treatment and Specialized Functions

Overcoming these challenges in disentangling the complexities of host-microbe interactions in health and disease will likely lead to advances in identification of microbiota-based biomarkers, therapeutic targets, and mechanisms for prevention. As discussed, the emerging links between microbiota and the gut-brain axis are providing promising avenues by which we can revolutionize the care of patients with motility disorders. Prior work has demonstrated that fecal metagenomes and metatranscriptomes are relatively stable over time while remaining individualized,[65] suggesting that there exist individual-level microbial features that may be leveraged for non-invasive biomarker development. These microbiome-based biomarkers may empower precision medicine approaches to identify individuals likely to respond to specific motility treatments or predict likelihood for detrimental side-effects or provide sentinel-like indicators for disease progression or remission. Similarly, altering or engineering gut microbiota directly, beyond broad-spectrum approaches like probiotics or FMT, may be one avenue to modify existing treatment approaches. Utilization of commensal bacteria (i.e. Escherichia coli, Lactobacillus, Lactococcus, or Bacteroides) as ‘chassis’ for delivery and engraftment of specialized synthetic biology processes within the microbiome[66] has been proposed for prevention and treatment of a spectrum of several gastrointestinal disorders, ranging from appetite modulation[67] to motility dysfunction.[68] One promising area of with recent developments is engineering the microbiome to increase the availability of diet-derived or other microbially-produced bioactive metabolites[42, 69] With specific respect to motility, promising proof-of-principle has been provided where germ free mice were colonized with a strain of Bacteroides thetaiotaomicron that was engineered to specifically produce tryptamine through gene transfer of tryptophan decarboxylase from Ruminococcus gnavus. By leveraging that B. thetaiotaomicron is a common, genetically tractable commensal and would readily colonize the gut, Bhattarai et al. were able to establish an effective increase in tryptamine levels that significantly accelerated GI transit. These transformative findings highlight the immense potential microbial engineering has in acting as a vehicle to supplement treatment approaches and deliver innovative strategies to improve GI health and motility.

Conclusions

Although our understanding of the role of the microbiome in colonic motility disorders is in some ways still in its infancy, there is enormous potential for transformative discoveries identifying not just which microbes are present in health and disease but more importantly their underlying function with use of longitudinal, multi-omics data. Only then can we truly understand how these microbiome-driven mechanisms can be translated to the clinic to improve both gut motility and sensation.