INTRODUCTION
Irritable bowel syndrome (IBS) is characterized by chronic gastrointestinal discomfort and abdominal pain with changes in bowel habits or stool consistency. IBS affects approximately 11.5% of the population, depending on the country or region (
1). Because of the high prevalence of IBS, symptoms contribute to changes in quality of life and increases in health care and economic burden (
2–5). There are four symptomic subtypes, IBS-C (constipation), IBS-D (diarrhea), IBS-A (alternating), and unspecified (IBS-U) (
6). Individuals with IBS-A experience alternating symptoms of chronic diarrhea and constipation. The criterion for diagnosis is symptom based and codified in the Rome IV criteria; there is not yet consensus on the underlying etiology of IBS (
7,
8). In addition, there are different factors that contribute to the various symptoms of IBS, including diet, immune response, host genetics, environmental stress, gut microbiome composition, and dysbiosis (
9,
10).
Currently, the role of the gut microbiome in IBS symptoms and recovery remains poorly understood. A “healthy” gut microbiome may be undefined, but there are microorganisms associated with an unhealthy microbiome, including microorganisms that induce inflammation or dysbiosis that contribute to the symptoms associated with IBS. Changes in microbiome composition also impact the microbial functional potential and metabolism, which may in turn affect host physiology. For example, studies indicate individuals experiencing IBS-C show microbiome signatures such as increased
Pseudomonas and
Bacteroides thetaiotaomicron with depletion of
Paraprevotella and significant associations with
Fusobacterium nucleatum and
Megamonas hypermegale (
11). In addition, research has characterized the microbiome of subjects with IBS-C with the biosynthetic pathways for sugar and amino acid metabolism; subjects with IBS-D had microbes that predominated in the pathways for nucleotides and fatty acid acid synthesis (
11). 16S rRNA amplicon sequencing studies have also described an enrichment of
Clostridiales,
Prevotella, and
Enterobacteriaceae; reduced microbial richness; and the presence of methanogens in IBS (
12,
13). However, amplicon studies can be subject to amplification bias, yielding variable results, and do not resolve species-level taxonomic classification. Alternatively, several studies limited by sample size and methodology have not shown a difference between a healthy cohort and individuals with IBS (
14).
Because of the differences in IBS symptoms that people experience and the individual nature of the syndrome, there is no standardized treatment or dietary recommendations to alleviate IBS symptoms (
15). The antibiotic rifaximin has been shown to be an effective treatment for IBS-D (
16,
17). However, rifaximin is ineffective for all IBS subtypes and antibiotic usage may be associated with an increased risk for IBS (
18–21). There are additional options for treatment, including pharmaceutical options and fecal transplants, but these options are not always feasible and can be invasive. The administration of live microbial organisms, in the form of probiotics, has gained popularity with patients to alleviate their symptoms. Probiotics can alter the microbiome of patients with and without IBS (
22,
23), depending on their endogenous microbiome (
24). Microbes not present in the current gut microbiome can also be reestablished through probiotic supplementation (
24). In individuals with IBS, there is correlative depletion of
Bifidobacterium and
Lactobacillus (
8). Therefore, reintroducing these microbes as probiotics into the gut of individuals with IBS may lead to phenotypic changes and reduction of IBS symptoms, as demonstrated by clinical trials (
25,
26). Treatment of subjects with IBS-D with
Bifidobacterium longum,
Bifidobacterium bifidum,
Bifidobacterium lactis,
Bifidobacterium infantis, and
Lactobacillus acidophilus resulted in a change in inflammation-related metabolites (
27). Individuals with IBS on a gluten-free diet with probiotic supplementation of
Lactobacillus and
Bifidobacterium spp. saw an overall improvement in symptoms (
28).
Here, we present a large-scale metagenomic study to characterize and compare the microbiome compositions and functional potentials of controls and individuals with IBS at baseline, as well as the microbiome changes associated with 4 months of daily synbiotic administration to subjects with IBS. Our primary goals were to (i) identify microbiome features associated with IBS and (ii) investigate whether synbiotics alter these IBS-associated microbiome features. We hypothesized that metagenomic features distinguish healthy from IBS microbiome subtypes and that daily synbiotic supplementation modulates the microbiomes of the individuals with IBS.
DISCUSSION
Although IBS is prevalent across the population, the underlying factors contributing to the syndrome make diagnosis and treatment challenging to define and standardize. Previous amplicon-based studies have identified changes in microbiome composition and diversity in individuals with IBS compared to a healthy control population (
29,
30). Concomitant with previous findings, our study corroborates the significant microbial community composition differences and diversity between healthy individuals and people with IBS. Unlike other studies, whole-metagenome shotgun sequencing enabled us to identify species and metabolic pathways associated with the dominant subtypes of IBS. In addition, our precision probiotics for individuals with IBS showed an increased relative abundance of probiotics detected in the gut microbiome across time. Of subjects with three time points, 91% had all three of the common probiotic species we included in formulations. Clinical studies that administer probiotics to individuals with IBS have shown reduced symptom severity and gut discomfort (
25,
27,
28). Although we did not find a significant change in alpha diversity or reduction of
Shigella spp. in the longitudinal IBS profiles with probiotic supplementation, there was a significant change in microbial species and pathways across time in IBS subtypes. Further research is needed to assess longitudinal changes in microbiome function in response to probiotics in IBS.
Individuals with IBS demonstrated a significant reduction in alpha diversity and predicted anti-inflammatory bacteria and a concomitant increased proportion of predicted proinflammatory bacteria, such as
Shigella. The reduction in alpha diversity may be due to medications such as antibiotics or overgrowth of specific bacteria (e.g., reference
31). However, there was no significant difference in the beta diversity in the microbiomes of subjects who have or have not taken antibiotics within the last 3 months of their stool sample collection. Consistent with IBS-A, IBS-C, and Crohn’s disease studies, we found lower relative abundances of the anti-inflammatory microbe
F. prausnitzii in individuals with IBS than in the healthy cohort (
29,
32–35). In contrast to previous amplicon-based studies that did not find a reduced abundance of
F. prausnitzii in IBS-D (
35–37), we detected
F. prausnitzii significantly reduced in IBS-D compared to controls.
F. prausnitzii enhances gut barrier protection and produces butyrate, a short-chain fatty acid (SCFA) essential for gut health (
29,
32,
38,
39).
Roseburia intestinalis has an anti-inflammatory role in the gut and is reduced in individuals with Crohn’s disease (
40,
41).
R. intestinalis was significantly reduced in IBS-C and IBS-D subtype (
Fig. 2).
Shigella spp., major contributors to diarrheal disease (
42) and associated with postinfectious IBS (
43), were found to be increased in the IBS subtypes (
Fig. 2).
The other differentially abundant microbes have an unclear role in IBS.
Ruminococcus lactaris is negatively correlated with interleukin-8 (IL-8) (
44) and is more abundant in a non-chronic kidney disease cohort (
45) but has also been shown to be associated with a high-fat diet in a murine diabetes model (
46).
Eubacterium rectale is a butyrate producer associated with infant gut microbiome development (
47) but is also associated with obesity and dysbiosis (
48). In a recent metagenomic assembly study of
E. rectale, there were different subspecies due to genetic and geographic dispersal in human populations, revealing differences in subspecies physiologies and metabolisms (
49).
Prevotella spp. are common in non-Western plant-rich diets (
50) and decreased in individuals with constipation (
51) but have also been associated with chronic inflammatory conditions (
52,
53). These studies indicate that the role of some microbes detected in this study is context and environment dependent.
Functional analysis identified pathways associated with each of the phenotypic classifications of IBS. The methanogenesis from an acetate pathway was associated with IBS-C (
Fig. 2). Methanogenesis contributes to methane production, which is correlated with the severity of constipation (
54) and may be useful as a diagnostic indicator of constipation-predominant IBS (
55,
56). Surprisingly, methanogenesis was also associated with IBS-D. Previous studies have demonstrated the reduction of methanogens in IBS-D (
57). The
Bifidobacterium shunt was also associated with IBS-C. The
Bifidobacterium shunt, also called the fructose-6-phosphate shunt, produces short-chain fatty acids (SCFAs) and other organic compounds (
58,
59). An overabundance of short-chain fatty acids, substrates for methanogenesis, may lead to gut symptoms in IBS. Depending on the chemical and microbial microenvironment, SCFAs can regulate the growth and virulence of enteric pathogens (
60). In addition, SCFA stimulates water absorption in the colon (
61). If too much water is absorbed, the stool becomes more solid, resulting in constipation. Thus, factors affecting host physiology in IBS may depend upon the microenvironments and microbes present in the gut. These findings suggest that future work should focus on formulating synbiotics that may reduce methanogenesis or regulate the production of SCFAs to improve IBS symptoms.
The enterobacterial common antigen (ECA) biosynthesis pathway was associated with IBS-A. The ECA is one of the components of the outer membrane of Gram-negative bacteria, and its association with IBS-A may indicate the increased presence of
Enterobacterales in the gut microbiome. Interestingly, the ECA may contribute to virulence and protect enteric pathogens from bile salts and antibiotics (
62–64). Bile acids protect the host from infection, contributing to overall gut intestinal health (
65). ECA protection against bile acids and antibiotics may make IBS-A challenging to treat with antibiotics and may contribute to dysbiosis. These results suggest that common antibiotic treatments for IBS may not be ideal for alleviating symptoms or treating the possible underlying microbiome triggers associated with IBS-A.
IBS is heterogeneous; a universal cocktail of probiotics may not comprehensively target all symptoms experienced by individuals with IBS. Therefore, individually formulated prebiotics and probiotics may be able to address the more common symptoms experienced by individuals with IBS. There were common strains included in formulas to specifically target constipation and diarrhea.
Bifidobacterium longum was included in formulations for constipation because studies have demonstrated treatment efficacy in stool frequency and consistency (
66,
67).
B. breve was included in formulations for diarrhea because it has been demonstrated to reduce severity and incidence of diarrhea (
68,
69). Although
B. longum did not increase in relative abundance across time, the presence of
B. longum may still promote gut health through cross-feeding mechanisms that lead to the production of short-chain fatty acids (
70,
71). Further investigation is needed to identify potential functional changes in microbiome metabolism with daily probiotic supplementation in IBS and whether symptoms associated with IBS can be improved.
There are several limitations to this current study. First, the self-reporting nature of IBS is a limitation to this study. For official diagnosis of IBS, the Rome IV criteria assess symptoms related to stool consistency and appearance, recurrent abdominal pain, and changes in bowel habits (
72,
73). Although the health and diet questionnaire included questions regarding gut symptoms and chronic conditions, a formal diagnosis was not verified. For potential lifestyle modifications in addition to probiotic supplementation, diet changes may also be an important factor in alleviating symptoms or changing the microbiome (
74–76). Low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) diet (LFD) and low-lactose diet (LLD) have been shown to reduce the IBS symptom severity score (IBS-SSS), and subjects on LFD had significantly less abdominal pain, bloating, and gas production (
75). These diet interventions were not accessed in this study. Second, this study was not designed to investigate longitudinal assessments of comprehensive gut issues experienced by the individuals with IBS. This hindered us in identifying whether gut symptoms were alleviated by daily probiotic supplementation or whether there were associations with certain probiotic formulations in improving certain symptoms in IBS. However, because the relative abundances of the common probiotics formulated for constipation and diarrhea were increased across time, these results may inform future studies. Additional research is also needed to determine the roles of specific pathways in the etiology of IBS.
In summary, we reported differentially abundant microbes and functional pathways associated with IBS and each IBS subtype relative to healthy controls. Of the microbes and pathways associated with each subtype, a subset was significantly changed in relative abundance across time in the IBS subtype populations. We also identified an increased relative abundance of probiotics in the gut microbiomes of people with IBS across time. These data may help inform future studies and therapeutic strategies by identifying important microbes and pathways associated with each IBS subtype. Probiotic strains or prebiotic ingredients can be formulated to target specific pathways or microbes that may be contributing to symptoms. Without these analyses, a blanket treatment may not resolve issues experienced by individuals with IBS. As IBS is a multifactorial syndrome, there is no one-size-fits-all approach to target all symptoms experienced by individuals with IBS. A combination of diet and probiotics may be needed to alleviate symptoms of IBS. Longitudinal monitoring of the gut microbiome is also important to understand changes associated with symptom progression. Further research is needed to identify the pathway benefits and interactions of prebiotic and probiotic supplementation with gut health and influence on IBS symptoms.