Bile acid dysmetabolism and microbial dysbiosis coincide with Clostridioides difficile infection in pediatric inflammatory bowel disease

Abstract

Pediatric inflammatory bowel disease (IBD) patients are at increased risk for Clostridioides difficile infection. This review highlights bile acid dysmetabolism, driven by gut microbiota dysbiosis and reduced microbial production of secondary bile acids, as a key factor linking pediatric IBD to increased CDI susceptibility. We examine interactions among IBD, dysbiosis, bile acid metabolism, and C. difficile, and discuss emerging microbiome-based interventions aimed at restoring bile acid homeostasis.

Background

Inflammatory bowel diseases (IBD), which include Crohn’s disease (CD) and ulcerative colitis (UC), are debilitating chronic inflammatory conditions of the gastrointestinal tract with a multifactorial pathogenesis that includes interactions between host inflammation and commensal gut microbiota¹. Recent reports demonstrate that the prevalence of pediatric IBD more than doubled from affecting 33/100,000 patients in 2007 to 77/100,000 in 2016 in the United States². This mirrors reported worldwide prevalence increases in pediatric IBD³. A well-documented association in IBD is periods of disruption in the gut microbial community, termed “dysbiosis”, which occur in tandem with altered microbial metabolite production and host responses⁴. Throughout this review, the term “dysbiosis” refers to a microbial community that differs from the usual homeostatic equilibrium of that community⁵. Importantly, periods of dysbiosis are recognized as both drivers of ongoing inflammation in patients with IBD as well as the consequence of exacerbated inflammatory disease states⁶. Microbial metabolism of host-derived primary bile acids is a key metabolic function with direct impacts on both the gut microbial community and host regulation of inflammation⁷.

Bile acids play several key roles in defense of the host from the enteric pathogen Clostridioides difficile. This Gram-positive, anaerobic, spore-forming, and toxin-producing bacterium is the causative agent of Clostridioides difficile infection (CDI). Patients with CDI are estimated to cost the United States’ healthcare system $4.8 billion annually while causing approximately half a million infections and 29,000 deaths⁸. Classically, altered bile acid metabolism coincides with CDI⁹ and is associated with developing infection in immune-competent individuals following use of antimicrobials^8,10,11. The landscape for treatment in immune-competent individuals has radically shifted with the advent of microbiome-directed therapeutics, including the use of fecal microbiota transplantation for recurrent CDI (rCDI)¹² and the recently FDA-approved products Rebyota¹³ and Vowst¹⁴. Though not strictly antibiotic-induced as observed in immune-competent individuals, bile acid dysmetabolism during states of dysbiosis still occurs in IBD patients⁴, including pediatric IBD patients^4,15–29. Moreover, this relationship between bile acid dysmetabolism and CDI has been increasingly studied in the last decade within the pediatric IBD population as a biological axis that can be leveraged for CDI prevention and treatment in this unique patient demographic.

This review aims to highlight and provide mechanistic insight into the role bile acids play in CDI within the pediatric IBD population. That is accomplished by: (1) outlining mechanisms of microbial diversification of the bile acid pool; (2) illustrating that dysbiosis in pediatric IBD patients includes loss of microbes capable of bile acid pool diversification; (3) demonstrating the multiple mechanisms bile acids utilize to directly impact C. difficile pathogenesis; and (4) highlighting the paucity of literature tying these concepts together to explain the overarching implications for susceptibility to CDI in pediatric IBD patients. Throughout this discussion, a mixture of translational evidence spanning in vitro laboratory science, animal model research, and clinical evidence from patients will all be summarized to tie these themes together. Ultimately, this niche of discovery can lead to high-yield interventions correcting bile acid dysmetabolism in pediatric IBD patients aimed at mitigating CDI morbidity and mortality.

Microbial-derived secondary bile acids create a diverse bile acid pool with host signaling potential

The composition of the intestinal bile acid pool is dictated by gut microbial metabolism^30,31. Host primary bile acids (PBAs) are first synthesized from cholesterol in the liver and then secreted into the gastrointestinal tract at the level of the duodenum via biliary excretion³². In humans, this PBA pool consists of cholic acid (CA) and chenodeoxycholic acid (CDCA) (Fig. 1a), which can be unconjugated or conjugated to the amino acids taurine or glycine. It has also been reported that mice produce additional PBAs, including muricholic acids, that are distinct from those found in humans, although these bile acids can be detected in the feces of other species³³. The classic functions of bile acids are to aid in the emulsification and absorption of fat from the diet through contributing to micelle formation³⁴ and to act as natural detergents within the gut, including the disruption of microbial cell membranes³⁵. The vast majority (95%) of bile acids are reabsorbed through enterohepatic recirculation, largely through receptor-mediated absorption in the ileum³⁶. Though a modest degree of microbial modification can occur prior to ileal reabsorption³⁷, the largest microbiota biomass is present in the large intestine, where the bulk of microbial bile acid modification occurs along with passive reabsorption of bile acids^30,31,38,39. The dynamic biogeography of both the gut microbiota and host physiology along the intestinal tract is a critical consideration when evaluating this process, especially in patients with IBD^39,40. Ultimately, in health, PBAs are metabolized by gut microbes into microbial-derived secondary bile acids (SBAs), such that human fecal bile acid composition generally contains >95% SBAs and <5% PBAs³⁸. Though lacking a distinct, consistent definition in the existing literature, the authors of this review consider deviations from these expected proportions to represent the presence of bile acid dysmetabolism. However, clear proportional cutoffs or the presence or absence of certain bile acids entirely from the bile acid pool should be considered when assessing for bile acid dysmetabolism.

Fig. 1. Microbial generation of secondary bile acids.

a Molecular structure of the host-derived primary bile acid cholic acid (CA). b Molecular reaction of microbial bile salt hydrolase (BSH) deconjugating taurocholic acid (TCA) to cholic acid (CA). c Molecular conversion of cholic acid (CA) to deoxycholic acid (DCA; left) and chenodeoxycholic acid (CDCA) to lithocholic acid (LCA; right) via the microbial bai operon. d Two-step molecular conversion of chenodeoxycholic acid (CDCA) to ursodeoxycholic acid (UDCA) using 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH).

Classically, for a PBA to be available for microbial modification, it must first be deconjugated⁴¹. PBAs are exclusively deconjugated by gut microbes through a “gatekeeper” reaction via bile salt hydrolase (BSH) enzyme activity (Fig. 1b)^{32,38,41–43}. Many bacterial genera contain BSH genes, including Clostridium, Lactobacillus, Bifidobacterium, Bacteroides, Enterococcus, and Stenotrophomonas^30,32,43, though a total of 119 genera containing BSH have been identified from a metagenomic study of 11 publicly available datasets⁴⁴. BSH conservation across multiple bacterial taxa affords a large degree of functional redundancy, ensuring the gatekeeping step is preserved across a wide range of gut microbiota community structures⁴⁵. BSH enzyme affinity and functional capacity can vary depending on the microbe harboring the gene, with metagenomic sequencing revealing unique clusters of BSH genes in people with IBD⁴⁶. Measurement of BSH activity using a novel bioluminescent technique was recently described and showed potential to discriminate between IBD patients with active versus quiescent disease⁴⁷. Moreover, it was recently demonstrated that microbial BSHs can orchestrate the SBA pool at large and ultimately impact colonization resistance in a mouse model of CDI⁴¹. Microbes harboring BSHs also often participate in other metabolic processes, such as short-chain fatty acid (SCFA) production, where the SCFA butyrate was recently described to also promote colonization resistance for CDI through modulation of the bile acid pool⁴⁸. Recently, BSH has also been reported to generate novel bacterial amino acid bile acid amidates that are directly sensed by bile-acid-activated host receptors⁴⁹. So, while BSH activity opens the door to further SBA generation and diversification, the process should not be viewed homogenously but rather as having the potential to steer overall bile acid pool composition. The resulting bile acid pool can then impact CDI dynamics through both direct interactions with C. difficile as well as altering host signaling through bile acid-activated receptors.

Microbial generation of SBAs occurs through multiple metabolic mechanisms (dehydroxylation, oxidation, epimerization, esterification) with an end result of a diversified bile acid pool³⁰. The largest driver of these processes, 7α-dehydroxylation, is a multistep process encoded by the bile acid inducible (bai) operon, where the SBA deoxycholic acid (DCA) is produced from CA or the SBA lithocholic acid (LCA) is produced from CDCA (Fig. 1c)⁵⁰. Only four culturable members of the human gut microbiome have been described to contain the bai operon: Clostridium scindens, Clostridium hylemonae, Clostridium sordelli, and Peptacetobacter (formerly Clostridium) hiranonis^30,32. An additional microbe in mice, Extibacter muris, was recently described to produce DCA and LCA⁵¹. Further, recent metagenomic studies leveraging Hidden Markov Models have identified the bai operon from currently unculturable gut microbes within the order Oscillospirales, including from the family Oscillospiraceae⁵² as well as Ruminococcaceae and Lachnospiraceae⁵³. From 16S rRNA gene amplicon sequencing data, Oscillospiraceae from IBD patient fecal samples have been shown to positively correlate with the SBAs DCA and LCA, demonstrating there may be 7α-dehydroxylation performed by these microbes in IBD patients⁵⁴. Furthermore, recent metagenomic work has highlighted that there are likely additional contributing microbes from taxonomic clades that are not yet recognized, and that these microbes specifically differ between IBD patients and healthy individuals⁵⁵. Ultimately, the ability of the gut microbiota to perform 7α-dehydroxylation is a key determinant in generating SBAs.

Additional microbial diversification of SBAs occurs through epimerization and oxidation reactions utilizing hydroxysteroid dehydrogenase enzymes⁵⁶, which have recently been reviewed⁵⁷. An important example of this is the conversion of CDCA to ursodeoxycholic acid (UDCA) through 7α- and 7β-hydroxysteroid dehydrogenases (Fig. 1d)⁵⁸. To a lesser extent, microbial esterification of bile acids contributes to the diversification of SBAs, though the microbial mechanisms of esterification remain unknown⁵⁹. An additional barrier to consider for microbial interaction with bile acids is the sulfonation of bile acids that can occur during enterohepatic recirculation, where host hepatocytes sulfonate bile acids prior to biliary excretion⁶⁰. To overcome this barrier, gut microbes can possess the ability to desulfonate bile acids, making them available for further microbial modification⁶¹. Lastly, the recent discovery that gut microbiota can create novel amino acid conjugated bile acids, known as microbial conjugated bile acids (MCBAs), adds a new layer to microbial diversification potential of the bile acid pool^62,63. Since initial discovery in 2020, as many as 118 novel MCBAs have been reported in humans^64,65. Moreover, their occurrence has been demonstrated in pediatric IBD patients, including reduced presence in pediatric IBD patients who receive antibiotics⁶⁶. Together, these microbial mechanisms yield a diverse bile acid pool with the potential to directly modulate host cell responses through host bile acid receptors^33,67.

Though reviewed elsewhere⁶⁷, host bile acid-activated receptors include nuclear receptors, such as the farnesoid X receptor (FXR), and membrane-bound G protein-coupled receptors, such as the Takeda G protein-coupled receptor 5 (TGR5). Additional bile acid activated nuclear receptors include the vitamin D receptor (VDR)⁶⁸, pregnane x receptor (PXR)⁶⁹, and constitutive androstane receptor (CAR)⁶⁷ while additional membrane bound receptors include α5β1 integrin⁷⁰ and sphingosine-1-phosphate receptor 2 (S1PR2)⁷¹. Host physiology can be regulated through these receptors, including de novo hepatic bile acid synthesis⁷², cellular metabolism⁷³, intestinal permeability in IBD⁷⁴, and intestinal innate inflammation mediated through NF-κB signaling^75–77. Receptor activity is dictated by varying ligand affinity depending on the bile acids encountered, with conjugation status playing a role to determine receptor agonism or antagonism in some instances^67,78,79. In addition, MCBAs have also been shown to act as agonists of host bile acid-activated receptors⁶². Thus, the composition of the bile acid pool, which is dependent upon the gut microbiota, regulates the degree of agonism and antagonism for these host receptors. Importantly, this provides a method of bidirectional communication between the host and gut microbiota, resulting in complex physiologic changes³¹. Leveraging these communication pathways has been demonstrated to reduce the severity of CDI with the FXR agonist obeticholic acid, regulating the bile acid pool of obese mice⁸⁰ and UDCA, mitigating host NF-κB inflammation⁸¹. In this context, FXR represents a particularly compelling host bile acid receptor due to its potential role in regulating inflammation and maintaining intestinal barrier function in pediatric IBD^75,82,83. This is especially true given prior findings that adults with IBD have reduced FXR expression^84,85 and haplotypes in the FXR gene are associated with IBD⁸⁶.

Therefore, both the microbial generation of SBAs and the host mechanisms for sensing and responding to the resulting bile acid pool play key roles in a collaborative physiology between the host and gut microbiota. Dysregulation of this physiology in pediatric IBD patients will subsequently be discussed (Section “Bile acid dysmetabolism occurs during dysbiosis in pediatric IBD patients”) as well as the direct impacts of this physiology on C. difficile pathogenesis (Section “Bile acids directly impact Clostridioides difficile pathogenesis”) and the current characterization and clinical application of this dynamic in pediatric IBD patients with CDI (Section “Bile acid dysmetabolism promotes CDI in pediatric IBD patients”).

Bile acid dysmetabolism occurs during dysbiosis in pediatric IBD patients

Lack of microbial-derived SBAs has been recognized as a hallmark of fecal bile acid dysmetabolism in pediatric IBD patients for at least the past 30 years¹⁵. Across multiple IBD patient cohorts that are either exclusively pediatric^{15–29,87–89} or include a mixture of pediatric and adult IBD patients⁴, the lack of microbial-derived SBAs recovered in feces has been repeatable (Table 1). This finding was further supported by a recent systematic review of seven pediatric IBD fecal metabolomics studies⁹⁰. Notably, these studies used targeted fecal metabolomics as a surrogate for intraluminal ecology of the gastrointestinal tract, relying on a range of analytical approaches that each carry their own advantages and inherent biases. Simultaneously with bile acid dysmetabolism discoveries, much attention has also been devoted to identifying alterations in the gut microbiota of pediatric IBD patients^{4,28,29,87,88,91–98}. Although not directly related to CDI, dysbiosis-associated bile acid dysmetabolism in pediatric IBD has been correlated with the presence of psychological disorders, suggesting an emerging role for the gut-brain axis and underscoring the importance of bile acid homeostasis in pediatric health²³.

Table 1.

Literature reporting fecal bile acid data in pediatric IBD patients

Author year	Pediatric study population	Bile acids	Bile acid method	Microbiota	Microbiota method	CDI	Interventions assessed
Edjerhamn et al.15	HC = 5, CD = 2, UC = 16,	+	GLC-MS	−	−	−	−
Jacobs et al.16	HC = 54, CD = 26, UC = 10	+	LC-MS	+	16S	−	−
Kolho et al.17	C = 29, CD = 36, UC = 20, IBDU = 13	+	UPLC-MS	−	−	−	−
aLloyd-Price et al.4	C = 14, CD = 33, UC = 15	+	LC-MS	+	MetaG, 16S	−	−
Bushman et al.18	HC = 38, IBD = 27, IBD + CDI = 23, M + CDI = 9	+	UPLC-MS	+	MetaG	+	−
Connors et al.19	CD = 17	+	LC-MS/MS	+	MetaG, 16S	−	EEN
Diederen et al.20	CD = 43	+	HPLC	+	16S	−	EEN
Rotondo-Trivette et al.21	HC = 29, UC = 23, UC + CDI = 16, CDI = 10	+	LC-MS	+	MetaG, 16S	+	−
Wang et al.22	HC = 20, CD = 29	+	UPLC-MS/MS	+	16S	−	IFX
Feng et al.23	HC = 14, CD = 39	+	LC-MS/MS	+	16S	−	−
Verburgt et al.24	HC = 26, CD = 54	+	LC-MS	+	MetaG	−	EEN
Xiao et al.25	CD = 12	+	UPLC-TQMS	+	MetaG	−	EEN
Chen et al.26	CD + CDI = 4, UC + CDI = 1, CDI = 3	+	LC-MS	+	16S	+	FMT
Lv et al.27	HC = 27, CD = 27	+	LC-MS/MS	+	MetaG, qPCR	−	EEN
Chen et al.28	HC = 11, aIBD = 37, rIBD = 33	+	LC-MS/MS	+	16S	−	−
Lo et al.29	HC = 28, UC 48	+	LC-MS	+	MetaG	−	FMT

16S 16S rRNA gene amplicon sequencing, aIBD active inflammatory bowel disease, C control, CD Crohn’s Disease, CDI C. difficile infection, GLC-MS gas liquid chromatography, HC healthy control, HPLC high-performance liquid chromatography, IBD inflammatory bowel disease, IBDU unclassified inflammatory bowel disease, LC-MS liquid chromatography and mass spectrometry, LC-MS/MS liquid chromatography and tandem mass spectrometry, M malignancy, MetaG metagenomic sequencing, qPCR quantitative polymerase chain reaction, rIBD remitting inflammatory bowel disease, UC ulcerative colitis, UPLC-MS ultra-performance liquid chromatography and mass spectrometry, UPLC-MS/MS ultra-performance liquid chromatography and tandem mass spectrometry, UPLC-TQMS ultra-performance liquid chromatography with triple quadrupole mass spectrometry.

^aIncludes pediatric and adult patients analyzed together.

Conceptually, while the gut microbiota of pediatric IBD patients differs from healthy patients at baseline, periods of dysbiosis occur sporadically with a variety of triggers that can include exacerbation of intestinal inflammation, antibiotic use, and dietary alterations^{4,87,88,91,93,95}. It is important to consider that pediatric patients with IBD have a documented baseline IBD-associated dysbiosis when compared to healthy individuals^88,93,95 or individuals with non-IBD gastrointestinal illness^4,91,92. Nevertheless, these altered microbial communities appear to reach a stable equilibrium during periods of quiescent disease in pediatric IBD patients. The authors describe this state as a relative eubiosis, characterized by a microbial community structure that remains distinct from that of healthy individuals but does not actively incite disease activity. In contrast to quiescent disease states, following diagnosis, pediatric IBD patients with either UC^94,97 or CD^88,95,97 may experience periods in which further microbial dysbiosis coincides with disease exacerbation. It remains difficult to discern if these dysbiotic changes are the cause or the consequence of worsening disease activity, though there is evidence that greater baseline microbiota instability increases the risk for future disease activity flares in adult CD patients⁹⁹. Thus, future longitudinal studies with greater temporal resolution will be important for clarifying the causal nature of these changes in pediatric IBD patients. Disease-associated dysbiosis also occurs in non-clinical first-degree relatives of pediatric IBD patients, leading to the suggestion that a genetic component of disease-associated dysbiosis also exists¹⁶. Moreover, alterations in the gut microbiota are noted in pre-clinical states of IBD prior to the emergence of clinical IBD¹⁰⁰. Indeed, it is also possible that the gut microbial community structure promotes or further triggers inflammation as well, such as reported expansion of Proteobacteria associated with increased DUOX2 activity, an enzyme that generates hydrogen peroxide⁹². In IBD patients, longitudinal studies that employ multi-omic study designs have subsequently captured that periods of microbial dysbiosis coincide with bile acid dysmetabolism^4,88,101. In the analysis by Heinken et al., known bai harboring microbes, C. scindens and C. hylemonae, were positively associated with SBA concentration⁸⁸. Similarly, Lloyd-Price et al. identified unclassified Subdoligranulum species and members of the Roseburia genus as being metagenomically and metatranscriptomically associated with bile acid dysmetabolism⁴. Interestingly, Subdoligranulum (within the family Ruminococcaceae) and Roseburia (within the family Lachnospiraceae) belong to families that include currently unculturable microbes that have been identified by metagenomics to harbor the bai operon⁵³. Both families have also been positively associated with SBA concentrations in a mouse model of CDI, supporting their role in promoting colonization resistance¹¹. These findings suggest that the unculturable organisms identified by Lloyd-Price et al. may either directly mediate SBA production through bai operon activity or indirectly promote SBA generation by helping to establish and maintain a microbial community favorable to bile acid transformation.

In pediatric IBD, Enterococcus is a component of the gut microbiota consistently shown to thrive. A systematic review of 41 studies characterizing pediatric IBD-associated dysbiosis found a consistent enrichment of Enterococcus, whereas Anaerostipes, Blautia, Coprococcus, Faecalibacterium, Roseburia, Ruminococcus, and Lachnospira were significantly depleted in most studies⁹⁶. Importantly, the analyzed studies captured pediatric patients with IBD (n = 2188) with an array of disease activity (n = 19 studies of active disease, n = 11 studies with a combination of active and remissive disease, n = 10 studies with unreported disease activity, and n = 1 study with remissive disease only)⁹⁶. Given the heterogeneity of the IBD patient cohorts included in these studies, it is difficult to distinguish dysbiotic features that characterize baseline disease from those associated with periods of exacerbation, especially within pediatric IBD patients. Consequently, the authors of the systematic review were unable to identify consistent gut microbial signatures linked to increased disease activity⁹⁶. Separately, Enterococcus relative abundance via 16S rRNA gene amplicon sequencing was successfully applied to a machine learning approach to discriminate pediatric IBD patients from those with irritable bowel syndrome (IBS)¹⁰². Although all members of the gut microbiota must tolerate the membrane-disruptive effects of bile, Enterococcus faecium possesses several specialized mechanisms that enhance resistance to the detergent activity of bile acids, potentially conferring a competitive advantage during states of bile acid dysmetabolism^103,104. Moreover, it has been demonstrated that the SBAs DCA and LCA both inhibit the growth of Enterococcus in vitro, while the PBAs CA and CDCA do not¹⁰⁵. Thus, in periods of dysbiosis-associated bile acid dysmetabolism (characterized by reduced SBAs), Enterococcus likely maintains an additional growth advantage. Furthermore, E. faecium recovered from adult UC patients induces inflammation in a mouse model of IBD, while wild-type E. faecium isolates do not, suggesting strain-specific variation in Enterococcus may be a driver of inflammation in the IBD disease process¹⁰⁶. Enterococcus also enhances C. difficile pathogenicity via metabolic cross-talk that uses the arginine deiminase pathway to provide ornithine for C. difficile metabolism¹⁰⁷, creating a competitive advantage for C. difficile¹⁰⁸. Separately, host-derived heme from CDI damage promotes Enterococcus faecalis fitness during infection¹⁰⁹. It is unsurprising, then, that Enterococcus is a gut microbial feature associated with CDI in adult IBD patients^110,111 and is increased in relative abundance in pediatric UC patients with a history of CDI compared to those without a history of CDI²¹. In summary, the evidence suggests that Enterococcus is a hallmark of pediatric IBD-associated dysbiosis, possesses mechanisms that likely enhance its fitness during periods of bile acid dysmetabolism, and may contribute to the development of CDI.

The metabolic consequences of dysbiosis for pediatric IBD patients have led to several studies reporting clinical outcomes associated with the resolution of dysbiosis and subsequent bile acid dysmetabolism. From one study (n = 70 CD, n = 23 UC), patients reported abdominal pain was associated with a lack of longitudinal microbial community stability, demonstrating an adverse clinical outcome of dysbiosis in the absence of differing calprotectin, a biomarker of intestinal inflammation⁹⁷. Separately, it was explored whether gut microbiome features could be predictive of treatment response (n = 20 CD) using both 16S rRNA gene amplicon sequencing and shotgun metagenomics, with both approaches identifying key microbial taxa significantly correlated with treatment responders¹¹². Notably, the interventions in this study were heterogeneous and uncontrolled. While this reflects the realities of clinical practice and the individualized nature of the patient microbiomes, it also limits the ability to draw broad predictive conclusions. Infliximab, an anti-Tumor Necrosis Factor α (TNFα) monoclonal antibody used in the treatment of IBD¹¹³, was associated with increased relative abundances of the BSH-containing taxa Blautia and Collinsella in a cohort of newly diagnosed pediatric Crohn’s disease patients (n = 29)²². Simultaneously, infliximab treatment also coincided with a significant increase in the proportion of unconjugated fecal bile acids as well as the ratio of SBAs to PBAs²², suggesting restoration of normal microbial-mediated bile acid metabolism.

One intervention that has garnered significant attention for pediatric CD patients is the application of exclusive enteral nutrition (EEN). This intervention involves exclusive consumption of a liquid formulation as the only source of nutrition, with protein supplied in either polymeric or elemental form¹¹⁴. Several meta-analyses comparing EEN with corticosteroid therapy have reported either comparable efficacy in inducing remission in pediatric CD patients^115,116 or limited evidence suggesting that EEN may be superior for remission induction¹¹⁷ and improving short-term mucosal inflammation¹¹⁸. Although not discussed in detail here, dietary interventions other than EEN are also used in pediatric Crohn’s disease. One such approach is the Crohn’s Disease Exclusion Diet (CDED), which combines a highly restrictive whole-food diet with a partial enteral nutrition¹¹⁹. Recent evidence supports its efficacy in inducing remission in pediatric CD patients while offering greater practicality and feasibility than EEN¹¹⁹.

From a bile acid perspective, a longitudinal study of treatment-naïve pediatric Crohn’s disease patients (n = 17) undergoing EEN found that all treatment responders exhibited SBA-predominate fecal bile acid profiles at baseline, whereas a subset of non-sustained responders and non-responders displayed PBA-predominant profiles both before and during treatment¹⁹. Non-sustained responders and non-responders had differing gut microbial communities associated with the fecal bile acid profile type, and those with SBA-predominant profiles, based on metagenomic sequencing, had significantly greater bai operon gene recovery¹⁹. These findings suggest that evaluating bile acid dysmetabolism simultaneously with the gut microbial community structure may hold predictive utility for response to EEN in pediatric IBD patients. In a separate study of treatment-naïve pediatric Crohn’s disease patients (n = 43), patients who ultimately responded or did not respond to EEN had significantly different baseline fecal microbiota composition²⁰. Bile acids reported as percentage SBA ranged in healthy pediatric patients from 85.3–96.3% in feces, while CD pediatric patients ranged widely from 46.9–94.7%²⁰. Interestingly, the lower bound of the SBA percentage range increased to 88.3% by completion of EEN therapy, but declined to 36.4% at follow-up after treatment cessation²⁰. These findings suggest that EEN may transiently promote conditions favorable for SBA production in certain individuals. Two additional pediatric investigations (total n = 39 CD) corroborate improvement of SBA proportion with 2 months of EEN therapy^25,27 that coincided with improved Pediatric Crohn’s Disease Activity Index (PCDAI)²⁷. Specifically, correction of bile acid dysmetabolism in one instance was characterized by increases of the SBAs hyocholic acid and α-muricholic acid²⁵. Interestingly, further work in this study found that hyocholic acid both attenuated inflammatory responses of peripheral CD4+ T cells from CD patients and also reduced inflammation in a mouse model of IBD, likely through action of multiple host bile acid-activated receptors²⁵. Separately, a study (n = 54 CD) evaluating 6 weeks of treatment with both EEN and a combination of Crohn’s Disease Exclusion Diet and Partial Enteral Nutrition demonstrated that EEN treatment significantly decreased the percentage of PBAs²⁴. Lastly, among a cohort of pediatric Crohn’s disease patients (n = 27), 12 patients with moderate-to-severe disease were treated with EEN for 8 weeks, leading to remission (PCDAI < 10) in 91.7% of patients (11/12)¹²⁰. Within this population, 16S rRNA gene amplicon sequencing demonstrated that Ruminococcus torques relative abundance was associated with remission, prompting further investigation in a murine IBD model¹²⁰. Addition of R. torques to the murine model alleviated colitis, improved intestinal barrier function, and rescued fecal SBA concentrations as well as the expression of the host bile acid-activated receptor FXR¹²⁰. Collectively, these studies demonstrate both the clinical efficacy of EEN and provide mechanistic insights into its capacity to improve dysbiosis and bile acid dysmetabolism in pediatric IBD patients. However, clinical responses are not universal, highlighting the need for further investigation of non-responders. Identifying the microbial and host factors that predict poor response to EEN may facilitate more personalized therapeutic strategies and ultimately improve pediatric IBD patient outcomes.

In summary, there is clear evidence that a baseline dysbiotic state is present in pediatric IBD patients that can be distinguished from both healthy individuals^88,93,95 as well as non-IBD gastrointestinal illness patients^4,91,92. However, it is possible for this microbial community to exist with quiescent disease activity as a relative eubiotic population^{4,88,94,95,97}. Current evidence supports the notion that episodes of increased disease activity in pediatric IBD are associated with more dramatic perturbations of the gut microbial ecosystem^{4,88,94,95,97}. Furthermore, microbial community changes are associated with a bile acid dysmetabolism characterized by reduced microbial-derived SBAs, whether or not there is a change in disease activity^{4,15–29,87–89}. A common feature of dysbiosis that occurs in pediatric IBD patients is a disproportionate overabundance of Enterococcus, though loss of many anaerobic microbes also occurs⁹⁶. Within the pediatric IBD microbial ecosystem, only a limited number of taxa capable of generating SBAs through the bai operon have been identified, including species that positively correlate with fecal SBA concentrations⁸⁸. Additional taxa, such as Subdilogranulum and Roseburia, have also been associated with SBA levels despite not being known to harbor the bai operon⁴. Though several therapeutic interventions have shown promise to mitigate the consequences of IBD-associated dysbiosis, particularly EEN^{19,20,24,25,27,120}, there remains a population of pediatric IBD patients who do not respond or who intermittently experience a dysbiotic state that coincides with bile acid dysmetabolism. A significant consequence for this pediatric patient population with dysbiosis and lack of microbial-derived SBAs is the predisposition to CDI^121,122. The next section describes how SBAs can directly impact C. difficile pathogenesis.

Bile acids directly impact Clostridioides difficile pathogenesis

Multiple stages of C. difficile’s life cycle within a human host are influenced by the intestinal environment (Fig. 2), which has recently been extensively reviewed¹²³. In this section, we emphasize the known interplay between C. difficile and bile acids within the gastrointestinal tract, which shapes the current conceptual framework of C. difficile pathogenesis, with relevance to bile acid dysmetabolism in pediatric IBD.

Fig. 2. Direct effects of bile acids on Clostridioides difficile pathogenesis.

The three primary life stages leading to CDI are depicted left to right as “germination”, “outgrowth”, and “toxin production and activity.” Bile acids that promote a particular life stage are listed above with a curved straight arrowhead and specific bile acids that inhibit a particular life stage are listed below with an inhibitory arrowhead. Primary bile acids are contained in red ovals, and secondary bile acids are contained in blue ovals. Primary bile acids: CDCA chenodeoxycholic acid, CA cholic acid, TCA taurocholic acid; Secondary bile acids: DCA deoxycholic acid, GDCA glycodeoxycholic acid, GLCA glycolithocholic acid, iDCA isodeoxycholic acid, iLCA isolihocholic acid, LCA lithocholic acid, UDCA ursodeoxycholic acid.

Pathogenesis begins with C. difficile spore germination, a process which has long been known to be governed by interaction with intestinal host-derived bile acids^124–126. C. difficile contains pseudoproteases CspC and CspA which recognize bile acid germinants as well as other molecules that act as co-germinants^127–129. Primarily, the conjugated host-derived PBA taurocholic acid (TCA) promotes C. difficile spore germination through recognition by CspC^127,130. During early stages of CDI, host biliary secretion of TCA is induced, creating conditions that favor continued C. difficile germination and outgrowth¹³¹. This feed-forward effect has been shown to be mitigated by the bile acid sequestrant cholestyramine, which leads to reduced spore germination and delayed C. difficile colonization¹³¹. Due to its speculated higher binding affinity relative to TCA, CDCA can inhibit C. difficile spore germination by competitively preventing TCA from binding¹³². Additionally, bile salt analogs inhibit C. difficile spore germination both in vitro and in rodent models, including a recent study substituting phenylamine onto CA^133,134. Indeed, other important co-germinant and environmental factors mediate C. difficile spore germination in concert with bile acids, including the amino acid glycine, calcium, pH, and temperature^{128,135–138}.

Following spore germination, the subsequent outgrowth of vegetative C. difficile cells leads to toxin production, which ultimately produces CDI clinical signs¹²³. Experimental in vitro studies have demonstrated that C. difficile spore germination, vegetative outgrowth, and toxin production are inhibited by several bile acids, including DCA, LCA, UDCA, isodeoxycholic acid (iDCA), isolithocholic acid (iLCA), ω-muricholic acid (ωMCA), and hyodeoxycholic acid (HDCA), underscoring the importance of bile acid composition in impacting susceptibility to CDI¹³⁹. Importantly, inhibition can vary depending on the ribotype of C. difficile¹³⁹. At lower concentrations, DCA induces biofilm formation by C. difficile, promoting survival as opposed to inhibiting growth¹⁴⁰. Further, UDCA inhibits C. difficile spore germination and outgrowth in a dose-dependent manner in vitro⁸¹. Two of the main bacteria capable of PBA conversion to SBAs via 7α-dehydroxylation, C. scindens and C. sordellii, have also been shown to produce tryptophan-derived antimicrobial compounds with activity against C. difficile in vitro¹⁴¹. Notably, this antimicrobial activity was enhanced by the SBAs DCA and LCA, both of which can be produced by these microbes, whereas the PBA CA did not have a similar enhancing effect¹⁴¹.

Aside from direct pathogen interactions, bile acids also directly impact the toxins secreted by C. difficile, which is the primary mechanism mediating clinical CDI via cytotoxicity to intestinal epithelial cells⁸. The C. difficile toxin TcdB has been shown to interact with the SBAs LCA and DCA, along with their glycine- and taurine-conjugated forms, resulting in inhibition of the conformational changes required for toxin function¹⁴². PBAs likewise demonstrate inhibitory activity against TcdB, but with considerably lower efficacy, exhibiting effects that are approximately one order of magnitude weaker than those mediated by SBAs¹⁴². Total bile acid concentration contributes to the suppression of TcdB activity, although reductions in total bile acid concentrations can permit the emergence of breakthrough toxin activity¹⁴³. In a dose-dependent manner, the SBA UDCA significantly decreases C. difficile toxin activity and outgrowth in vitro, linking decreased pathogen growth with the observed decreased toxin activity⁸¹. In multiple C. difficile strains in vitro, the PBAs TCA and CA were shown to increase toxin activity, while the PBA CDCA reduced toxin activity¹⁴⁴. Though not a direct effect on toxin activity, the conjugated SBA tauroursodeoxycholic acid (TUDCA) was recently shown to decrease toxin-induced apoptosis in vitro with Caco-2 cells exposed to TcdA and TcdB¹⁴⁵. Taken together, disruptions in the bile acid pool may influence not only C. difficile colonization and growth but also the activity of its major toxins, reinforcing the multifaceted role of bile acids in CDI susceptibility.

In summary, PBAs, particularly TCA and CA, promote C. difficile spore germination and toxin production, whereas a diverse array of microbial-derived SBAs suppress C. difficile pathogenesis across multiple stages of its life cycle, including spore germination, outgrowth, and toxin production. These observations support a conceptual framework in which the composition of the intestinal bile acid pool serves as a critical regulator of colonization resistance against C. difficile. In pediatric IBD, dysbiosis-associated bile acid dysmetabolism is characterized by depletion of SBA-producing microbes and a subsequent accumulation of PBAs, shifting the bile acid pools from one that restrains C. difficile to one that promotes its survival and virulence. Collectively, these findings suggest that bile acid dysmetabolism may represent a key mechanistic link between the dysbiotic microbiome of pediatric IBD patients and their increased susceptibility to CDI.

Bile acid dysmetabolism promotes CDI in pediatric IBD patients

Similar to adults with IBD, pediatric IBD patients experience a higher incidence of CDI than the general pediatric population^{121,122,146,147}. Pediatric CD and UC patients experience comparable rates of CDI^146,148,149, in contrast to adult IBD patients, where some studies have reported a higher incidence among UC patients^150,151. Although the incidence of CDI among hospitalized pediatric patients in the United States declined between 2013 and 2019, concurrent chronic gastrointestinal disease accounted for more than one-third of all pediatric CDI cases¹⁵². Compared to age- and sex-matched pediatric controls without IBD, hospitalized pediatric IBD patients exhibit an almost 70-fold greater incidence of CDI¹⁵³. Despite this substantial increase in risk, some reports support that hospitalized pediatric patients with malignancies may experience an even higher incidence of CDI¹⁵⁴. Additionally, the incidence of community-acquired CDI among children nearly doubled between 2012 and 2015¹⁵⁵. This trend may be particularly relevant to pediatric IBD populations, as a recent retrospective Romanian case series of 55 pediatric IBD patients identified six cases of CDI, all of which were community-acquired¹⁵⁶. Similarly, a single-center study from Taiwan reported significant increases in both community-acquired and healthcare-associated pediatric CDI cases¹⁵⁷.

Recent meta-analysis identified 5-aminosalicyclic acid (5-ASA) as the only medication risk factor for CDI in pediatric IBD patients, while proton pump inhibitors, antibiotics, biologics, immunomodulatory medications, and systemic steroids were not identified as risk factors¹⁵⁸. However, the authors cautioned against overinterpreting this finding, noting that approximately 70% of the observed effect size was driven by a single study¹⁵⁹, while the remaining contribution came from two studies^154,160 that did not support 5-ASA as a risk factor. This clinical risk factor also contrasts with findings from the dextran sodium sulfate (DSS) murine IBD model¹⁶¹, where 5-ASA has been shown to improve microbial dysbiosis and bile acid dysmetabolism¹⁶². Outside of medication, the other risk factor identified for CDI in pediatric IBD patients was active underlying exacerbation of intestinal inflammation¹⁵⁸. A major challenge in IBD patients is that clinical manifestations of active disease often overlap with those of CDI, making clinical differentiation difficult. Consequently, a recent review highlighted the need for improved tools capable of distinguishing pediatric IBD flares from CDI in pediatric IBD patients¹⁶³. To further complicate matters, pediatric IBD patients may be asymptomatically colonized with C. difficile and also may proceed to develop overt CDI later¹⁶⁴. This makes it particularly challenging to distinguish true cases of active CDI from IBD flares in which C. difficile is present as a colonizing organism rather than the underlying cause of disease¹⁶³. Overall, this creates a substantial diagnostic challenge, and distinguishing between these states is further complicated by the fact that many of the same host and microbial factors that contribute to IBD pathogenesis also promote susceptibility to CDI. Fecal bile acid dysmetabolism is a recognized feature of pediatric IBD (see Section “Bile acid dysmetabolism occurs during dysbiosis in pediatric IBD patients”) and was recently highlighted in a systematic review⁹⁰. Ultimately, the interplay between chronic intestinal inflammation, microbial dysbiosis, and bile acid dysmetabolism leads to a loss of colonization resistance against C. difficile, rendering pediatric IBD patients particularly vulnerable to CDI (Fig. 3).

Fig. 3. Conceptual framework of bile acid dysmetabolism and gut dysbiosis in pediatric IBD CDI.

Periods of relative eubiosis are depicted in blue and characterized by a microbial community of increased diversity and a predominance of secondary bile acids without CDI occurring, although asymptomatic C. difficile colonization is possible (left). Periods of dysbiosis are depicted in red and characterized by a microbial community of decreased diversity and overrepresentation of Enterococcus and a predominance of primary bile acids occurring leading to loss of colonization resistance against C. difficile and ultimately CDI. Triggering factors to cause dysbiosis are listed with the top arrow, while interventions associated with the resolution of bile acid dysmetabolism and dysbiosis are listed with the bottom arrow.

Fecal bile acid dysmetabolism is associated with pediatric IBD patients who develop CDI^18,21. Importantly, fecal bile acid composition serves as a surrogate marker of the intestinal bile acid pool and may not fully represent the localized bile acid environment encountered by C. difficile at the sites of spore germination, vegetative growth, and toxin production. Among pediatric UC patients, those with a prior history of CDI (n = 16) demonstrated significant reductions in microbial-derived SBAs, specifically LCA and UDCA, relative to healthy controls (n = 29) and UC patients without previous CDI (n = 23)²¹. In this study, the history of CDI was defined as a positive fecal PCR test for C. difficile, with most patients also having a positive toxin assay²¹. The observed bile acid dysmetabolism coincided with reduced abundance of BSH, HSDH, and bai operon genes detected via metagenomic sequencing²¹. While the microbes contributing to these functional pathways were not taxonomically resolved or discussed²¹, 16S rRNA gene amplicon data revealed reduced abundance of several genera known to contain BSH and HSDH genes, namely Blautia, Dorea, Roseburia, and Ruminococcus²¹. Interestingly, the inclusion of a non-IBD pediatric CDI cohort (n = 10) revealed no detectable difference in BSH, HSDH, and bai operon gene abundances relative to healthy controls, highlighting a potential distinct relationship between bile acid metabolism and CDI in the setting of pediatric IBD²¹. Importantly, no patients in the study had active CDI, and those with a history of CDI (with or without UC) had experienced clinical resolution for at least 1 month duration²¹. Taken together, this suggests that correction of dysbiosis and functional microbiota-mediated recovery of bile acid metabolism is impaired in pediatric UC patients compared to non-IBD pediatric patients. Thus, these data demonstrate that even in the absence of clinical disease from CDI, there are likely lasting components of dysbiosis and bile acid dysmetabolism following CDI in pediatric IBD patients that may perpetuate susceptibility to CDI.

In a separate multi-omic study of pediatric patients, including UC and CD patients with CDI (n = 23), UC and CD patients without CDI (n = 27), and healthy controls (n = 38), fecal bile acid dysmetabolism was a defining feature of the CDI cohort¹⁸. Cases of CDI were defined by the presence of at least three watery stools per day for at least 24 h, or an increase in stool frequency relative to baseline, together with either a positive glutamate dehydrogenase assay or PCR detection of the toxin A/B gene¹⁸. Compared with the non-CDI groups, patients with CDI exhibited significantly lower concentrations of the SBAs LCA and DCA and elevated concentrations of PBAs CA and CDCA¹⁸. Notably, these alterations persisted throughout the 8-week follow-up period, suggesting ongoing dysbiosis and bile acid dysmetabolism that may reflect the combined effects of underlying IBD and antimicrobial treatment for CDI¹⁸. Metabolomics additionally identified increased concentrations of the novel metabolite isocaproyltaurine in IBD patients with CDI relative to those without CDI¹⁸. The authors proposed that isocaproyltaurine may serve as a potential biomarker for distinguishing pediatric IBD patients with CDI from those experiencing an IBD flare in the absence of C. difficile infection¹⁸, addressing a well-recognized diagnostic challenge in clinical practice¹⁶³. Isocaproyltaurine likely represents a combination of a unique Stickland-derived amino acid metabolite produced by C. difficile¹⁶⁵ and taurine, with the taurine component thought to originate from host cell damage¹⁸. Alternatively, taurine-conjugated bile acids could provide an additional taurine source, and C. difficile has been shown to increase host secretion of TCA early in CDI¹³¹. While not evaluated in this study, the mechanisms responsible for restoring microbial eubiosis and bile acid homeostasis after CDI in pediatric IBD patients remain unclear. Such recovery may be counteracted, at least in part, by the microbiome perturbations induced by antimicrobial therapies used to treat CDI.

A separate contributing factor to bile acid homeostasis within the pediatric IBD population is the contribution of bile acid malabsorption (BAM), which has been reviewed in the context of adults with IBD^166,167. Briefly, inflammation from IBD reduces the expression of receptors within the ileum that allow for enterohepatic recycling of bile acids, primarily the apical sodium-dependent bile acid transporter (ASBT)¹⁶⁸. Consequently, the intraduodenal bile acid pool becomes significantly reduced and impairs lipid digestion and can lead to hypermotility of the gut¹⁶⁶. This clinical entity can be diagnosed via gold standard radiolabeled nucleotide administration^167,169, though the serum biomarker C4, a precursor to bile acid synthesis, can also be utilized¹⁷⁰. Importantly, targeted therapeutics are available for BAM, including bile acid sequestrants such as cholestyramine¹⁷¹. More recently, alternative therapeutic approaches with tropifexor, colesevelam, and liraglutide have also demonstrated efficacy^172–174. Although data from pediatric IBD patients remains limited, one study of pediatric patients with CD (n = 44) and UC (n = 14) identified BAM in 23% of CD patients (n = 10) and one UC patient based on serum C4 concentrations¹⁷⁵. Therefore, BAM represents an additional contributor to disrupted bile acid homeostasis in pediatric IBD patients and should be further evaluated in the context of CDI in pediatric IBD patients.

Interventions targeting microbiome modification instead of pathogen-directed antimicrobial treatment have shown promise to treat CDI and promote C. difficile colonization resistance by resolving lasting dysbiosis and subsequent bile acid dysmetabolism¹⁷⁶. In the general population of adult CDI cases, practice guidelines from the American Gastroenterological Association (AGA) suggest use of fecal microbiota transplantation (FMT) after a second recurrence of CDI treated with appropriate antimicrobials¹⁷⁷. However, these guidelines do not recommend FMT for adult patients with IBD except within the context of a clinical trial¹⁷⁷. Separate guidelines issued by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA) recommend FMT not be offered until three instances (two recurrences) of CDI in adult patients without IBD, and do not make separate recommendations for adults with IBD¹⁷⁸. It should also be noted that there is evidence for the use of FMT for fulminant or severe CDI in adults without IBD¹⁷⁹. Regarding recurrent CDI in pediatric IBD patients, FMT has demonstrated efficacy similar to that reported in pediatric populations without IBD, with roughly 80% of cases resolving after a single FMT¹⁸⁰. Recent data from a cohort of eight pediatric cases with recurrent CDI ($\ge$3 recurrences), including individuals with CD (n = 1), UC (n = 4), and no underlying IBD (n = 3), demonstrated successful resolution of infection following a single administration of fresh FMT via colonoscopy²⁶. C. difficile toxin B PCR remained negative at both 10 and 20 weeks and 6 months after FMT treatment²⁶. Despite only a transient increase in gut microbial diversity following FMT, resolution of bile acid dysmetabolism was sustained in all cases²⁶. Longitudinal microbiota dynamics in this cohort were characterized by 16S rRNA gene amplicon sequencing, a method that does not provide information on the abundance of bai operons within these microbial populations¹⁸¹. Thus, it is possible that only a small number of key bai operon containing microbes successfully engrafted following FMT, whereas other donor-derived microbes failed to establish long-term colonization or produce a sustained increase in microbial diversity. In a more recent longitudinal study from a cohort of pediatric UC patients without CDI (n = 48), FMT was associated with sustained restoration of fecal bile acid metabolism, with metagenomic analysis at 48 weeks post-treatment revealing increased microbial genes involved in bile acid transformation²⁹. Further elucidation of the mechanistic drivers of FMT success, including microbial engraftment of donor-derived microbes and correction of bile acid dysmetabolism, will aid in the successful integration and optimization of FMT as a treatment modality for CDI in pediatric IBD patients.

Overall conclusions and future directions

The relationship between bile acid metabolism and CDI in pediatric IBD patients represents a compelling example of translational microbiome medicine. Our mechanistic understanding of the microbial-mediated bile acid metabolism in health provides a foundation for interpreting the bile acid dysmetabolism that emerges in the dysbiotic microbiota of pediatric IBD patients. Further refinement of this framework will require a more comprehensive characterization of bile acid profiles across disease states, including the degree of bile acid dysmetabolism present at baseline and the dynamic changes that accompany flares of disease activity. Addressing these knowledge gaps remains an important area for future investigation.

The extensive effects of bile acids on multiple life stages of C. difficile, including spore germination, vegetative outgrowth, and toxin activity, further strengthen the biological rationale linking bile acid dysmetabolism to increased CDI susceptibility in pediatric IBD. Although only a limited number of studies have directly examined the intersection of pediatric IBD, bile acid metabolism, and CDI, a substantial body of basic science and clinical evidence supports this conceptual framework and highlights its translational potential.

Collectively, evidence in the current literature supports a model in which dysbiosis-driven alteration in bile acid metabolism serves as a mechanistic link connecting pediatric IBD to increased vulnerability to CDI. This framework creates opportunities to move beyond conventional antimicrobial approaches and toward targeted microbiome-based interventions designed to restore bile acid homeostasis and restore colonization resistance against C. difficile. Continued development and evaluation of translational strategies will be essential for reducing the burden of CDI and improving outcomes in this high-risk pediatric population.

Building upon this framework, we put forward the notion that a dynamic and bidirectional relationship exists between the gut microbiota and inflammatory disease activity in pediatric IBD. Although preliminary, current evidence suggests that pediatric IBD patients harbor a characteristic baseline microbiota that is distinct from that of healthy individuals^88,93,95 and patients with non-IBD gastrointestinal illness^4,91,92. During periods of quiescent disease, this microbial community appears capable of reaching a relatively stable equilibrium state that, despite differing from healthy controls, may retain sufficient functional capacity to support bile acid homeostasis. However, disease exacerbations are frequently accompanied by further disruption of the microbial ecosystem^88,94,95,97. These dysbiotic shifts are associated with the loss of microbial function required for SBA production, including depletion of microbial genes involved in SBA generation^4,88,101. The resulting bile acid dysmetabolism, characterized by reduced SBA abundance and accumulation of PBAs, shifts the intestinal ecosystem from one that supports colonization resistance to one that promotes C. difficile spore germination, outgrowth, and toxin production. We therefore suggest a conceptual model in which bile acid dysmetabolism represents a key pathogenic pathway linking the IBD-associated dysbiotic microbiome and the increased risk of CDI observed in pediatric IBD patients (Fig. 3). Given this framework, there are a limited number of pediatric IBD cases with CDI in the literature (Table 1), making it difficult to reach generalized conclusions of how the pediatric population differs from adult cohorts in the context of IBD-microbiome-bile acids-CDI axis. This emphasizes the need for additional dedicated studies in pediatric populations to further elucidate pediatric-specific features to guide future intervention strategies.

Future work in this area should also continue to utilize and integrate multi-omics approaches that simultaneously evaluate the gut microbiota, metabolome, and host factors to capture this dynamic disease interaction. This will facilitate studies aimed at generating predictive insights, particularly through the identification of biomarkers that can improve the diagnosis of CDI in pediatric IBD patients and inform therapeutic decision-making, including interventions such as FMT. Characterization of beneficial mechanisms of FMT and determinants of microbial engraftment in pediatric IBD patients are additional open questions that benefit from this translational approach. Similarly, future mechanistic studies should investigate the biological basis underlying the potential clinical benefits of dietary interventions, probiotics, and bile acid sequestrants. Separately, the identification of metabolomic biomarkers capable of distinguishing active CDI from exacerbations of underlying IBD accompanied by C. difficile colonization remains a critical unmet clinical need. Identification of isocaproyltaurine¹⁸ is an example toward this end, but remains to be translated into widespread clinical practice. Future multi-omic studies should also examine host responses mediated through bile acid-activated receptors such as FXR and TGR5, with the goal of elucidating how these signaling pathways contribute to microbial dysbiosis and bile acid dysmetabolism in pediatric IBD. Finally, characterization and application of the novel MCBAs⁶² bioactivity for both host inflammation regulation and impacts on C. difficile pathogenesis are likely other areas for continued investigation with translational relevance in the context of IBD. Ultimately, the development of targeted interventions that leverage the bile acid pool to reduce CDI risk in pediatric IBD patients is an attainable translational medicine objective with the potential for significant and rapid patient benefit.

Author contributions

Both authors (J. Rowe and J. Winston) equally contributed to conceptualization, literature review, figure generation, synthesis of material, and drafting of this manuscript.

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Competing interests

The authors declare no competing interests.