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Gut Microbes
Gut Microbes
Gut Microbes
1949-0976
1949-0984
Taylor & Francis

39224076
10.1080/19490976.2024.2393766
2393766
Version of Record
Review Article
Review
Bile acids impact the microbiota, host, and C. difficile dynamics providing insight into mechanisms of efficacy of FMTs and microbiota-focused therapeutics
A. S. MCMILLAN AND C. M. THERIOT
GUT MICROBES
https://orcid.org/0000-0002-0115-0225
McMillan Arthur S. a b
https://orcid.org/0000-0002-1895-8941
Theriot Casey M. b
a Genetics Program, Department of Biological Sciences, College of Science, North Carolina State University , Raleigh, NC, USA
b Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University , Raleigh, NC, USA
CONTACT Casey M. Theriot cmtherio@ncsu.edu Department of Population Health and Pathobiology, College of Veterinary Medicine, Research Building 406, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607
3 9 2024
2024
3 9 2024
16 1 2393766Integra29 8 2024
Integra29 8 2024
27 3 2024
12 8 2024
13 8 2024
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
2024
The Author(s)
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

ABSTRACT

Clostridioides difficile is a major nosocomial pathogen, causing significant morbidity and mortality worldwide. Antibiotic usage, a major risk factor for Clostridioides difficile infection (CDI), disrupts the gut microbiota, allowing C. difficile to proliferate and cause infection, and can often lead to recurrent CDI (rCDI). Fecal microbiota transplantation (FMT) and live biotherapeutic products (LBPs) have emerged as effective treatments for rCDI and aim to restore colonization resistance provided by a healthy gut microbiota. However, much is still unknown about the mechanisms mediating their success. Bile acids, extensively modified by gut microbes, affect C. difficile’s germination, growth, and toxin production while also shaping the gut microbiota and influencing host immune responses. Additionally, microbial interactions, such as nutrient competition and cross-feeding, contribute to colonization resistance against C. difficile and may contribute to the success of microbiota-focused therapeutics. Bile acids as well as other microbial mediated interactions could have implications for other diseases being treated with microbiota-focused therapeutics. This review focuses on the intricate interplay between bile acid modifications, microbial ecology, and host responses with a focus on C. difficile, hoping to shed light on how to move forward with the development of new microbiota mediated therapeutic strategies to combat rCDI and other intestinal diseases.

KEYWORDS

Clostridioides difficile
recurrent CDI
microbiota
bile acids
fecal microbiota transplantation
nuclear receptors
Molecular Biotechnology Training Program at NCSU the National Institute of General Medical Sciences of the National Institutes of Health R35GM149222 A.S.M was funded by the Molecular Biotechnology Training Program at NCSU (NIH NCSU MBTP T32 GM133366). C.M.T. was funded by the National Institute of General Medical Sciences of the National Institutes of Health under award number [R35GM149222].
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pmcIntroduction

Clostridioides difficile is a Gram-positive, spore-forming, anaerobic pathogen that causes C. difficile infection (CDI), a major healthcare-associated infection with significant morbidity and mortality worldwide. It is classified as an urgent threat by the Centers for Disease Control and Prevention (CDC), based on gaps in research and treatment despite aggressive actions being taken.1 In 2017, there were approximately 462,100 cases and 20,500 deaths related to CDI.2 Approximately, half of these cases were classified as community-associated as opposed to healthcare-associated, although community-associated CDI represents only about a fifth of in-hospital deaths.2

A major risk factor for CDI is antibiotic usage, which kills protective gut microbes that are able to provide colonization resistance.3,4 The standard of care for patients with CDI is treatment with vancomycin or fidaxomicin, with a preference for fidaxomicin due to its preservation of the gut microbiota and reduced incidence of recurrence.5 Treatment with vancomycin and to a much lesser degree fidaxomicin continues to alter the gut microbiota allowing C. difficile to reestablish infection, leading to recurrent CDI (rCDI) in approximately 25% of patients after initial treatment.3,6 The definition for rCDI is recurrence of diarrhea and a positive C. difficile test within 8 weeks of completing CDI directed therapy.7 The risk of recurrence can increase by 20–25% for subsequent recurrences.8,9 Fecal microbiota transplantation (FMT), a treatment derived from healthy donor stool, has emerged as an effective therapy for rCDI, with cure rates exceeding 90%.6 The goal of this therapy is to restore colonization resistance that is lost after antibiotic treatment.

Recently, the FDA approved the first microbiota-derived therapeutics for the treatment of rCDI. These microbiota-focused therapeutics source healthy stool in such a way as to reduce the risk of introducing harmful bacteria and aim to standardize the processing of stool for FMT.10,11 Products such as Rebyota and VOWST are derived from healthy donor stool, with VOWST having an additional ethanol purification to select for spore forming bacteria.10,11 Live biotherapeutic products (LBPs) composed of bacterial consortia, such as MET-2 and VE303, are also being investigated.12,13 As of February 2024, MET-2 is in phase 1 clinical trials for rCDI and ulcerative colitis (UC), while VE303 is in phase 2 clinical trials for rCDI. However, the specific mechanisms underlying the efficacy of FMTs and LBPs as well as the mechanisms through which gut microbes provide colonization resistance are still an area of active investigation.

A proposed mechanism that we hypothesize contributes to the efficacy of microbial-based therapies is microbiota mediated alterations in the gut bile acid pool (Figure 1(a)), which impacts C. difficile, the microbiota, and the host. Bile acids exert antimicrobial effects against C. difficile and other members of the gut microbiota through different mechanisms such as disruption of the bacterial cell membrane.4,22,23 Microbial-derived secondary bile acids are able to inhibit different stages of the C. difficile life cycle, such as spore germination, vegetative outgrowth, toxin expression, production, and activity4,17,19,22,24,25 (Figure 1b). Bile acids also impact the host immune response through interactions with nuclear receptors like farnesoid X receptor (FXR), Takeda G-protein receptor 5 (TGR5), pregnane X receptor (PXR), and retinoic acid-related orphan receptor γT (RORγT).26 Bile acid-induced activation of these receptors plays a role in immunity, specifically the differentiation of Th17 and Treg cells.27 Figure 1. The gut microbiota modifies bile acids which are able to inhibit different stages of the C. difficile life cycle. (a) Basic overview of the main bile acid altering enzymes discussed in this review. BSHs deconjugate and reconjugate bile acids with an amino acid or directly exchange the conjugated amino acid for another. Amino acids can be host or microbial conjugated. The bai operon removes a 7α-hydroxyl group producing secondary bile acids. HSDHs dehydroxylate bile acids resulting in an oxo-bile acid, in this case a oxo-secondary bile acid. (b) Effect of bile acids on different stages of the C. difficile life cycle in vitro. TCA mediated spore germination of C. difficile spores (grey arrow)14–16 is inhibited by various bile acids (red box).17,18 outgrowth of vegetative C. difficile is impacted by a variety of bile acids (red box).17,18 the production of toxin is inhibited by bile acids (red box) through reducing expression of toxin or toxin activity.15,17,19 bile acids also bind directly to C. difficile toxin, reducing its toxicity in the host (red box).21 abbreviations: AA, amino acid; Ala, alanine; BSH, bile salt hydrolase; bai, bile acid inducible; CA, cholate; CDCA, chenodeoxycholate; DCA, deoxycholate; glu, glutamate; gly, glycine; HDCA, hyodeoxycholate; his, histidine; HSDH, hydroxysteroid dehydrogenase; iDCA, isodeoxycholate; iaLCA, isoallolithocholate; iLCA, isolithocholate; LCA, lithocholate; phe, phenylalanine; ser, serine; tau, taurine; trp, tryptophan; tyr, Tyrosine; UDCA, ursodeoxycholate; αMCA, α-muricholate; βMCA, β-muricholate; ωMCA, ω-muricholate; 3-oxo LCA, 3-oxolithocholate.

There are also non-bile acid mediated microbial interactions through which the gut microbiota may protect against C. difficile colonization, including the production of metabolites and exchange of nutrients between C. difficile and members of the gut microbiota. Other metabolites, such as short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate return after FMT.28 Butyrate inhibits C. difficile growth and promotes epithelial barrier integrity and modulates the host immune responses.29–31 Butyrate was also found to increase C. difficile toxin production and sporulation.31 Additionally, many members of the gut microbiota are able to compete for nutrients, such as amino acids important for Stickland fermentation with C. difficile.32 Whereas others like Enterococcus are able to provide C. difficile with nutrients it requires for growth, including ornithine.33

In this review, we will focus on how bile acid modifications carried out by members of the gut microbiota impact the pathogen, microbiome, and host response with a lens on C. difficile. We will also consider the role of competition of nutrients and metabolite production by the microbiota. We will highlight more recent work that provides evidence for how members of the gut microbiota maybe contributing to the efficacy of FMTs and LBPs for the treatment of rCDI and other intestinal diseases.

Bile acids and C. difficile

Bile acids are synthesized from cholesterol in the liver, conjugated with an amino acid, stored in the gallbladder, and secreted when the host eats a meal.34 Their role during digestion is to act as surfactants, allowing for the absorption of dietary fats and vitamins. Once they reach the terminal ileum, ~95% of bile acids are reabsorbed through enterohepatic circulation.35 Gut bacteria modify bile acids during intestinal transit, thereby changing the chemistry and diversity of the bile acid pool. Modifications made by the microbiota include deconjugation of the conjugated amino acid, reconjugation or a swap of the conjugated amino acid, dehydrogenation, dehydroxylation, and epimerization. Changes made to the sterol core, rather than the conjugated amino acid, mark the conversion of a primary bile acid to a secondary bile acid. In-depth reviews that explore bile acid modifications in humans and animals can be seen here.36,37

Various bile acids directly impact multiple stages of the C. difficile life cycle in vitro (Figure 1(b)). Bile acids such as taurocholate (TCA) are germinants of C. difficile spores14−34], while other bile acids such as chenodeoxycholate (CDCA) inhibit spore germination14,15,17,22,38 (Figure 1b). Microbially derived secondary bile acids are able to inhibit C. difficile spore germination, vegetative cell growth, and toxin activity17,19,20 (Figure 1b). The mechanisms through which bile acids inhibit growth are attributed to their antimicrobial and detergent-like properties, which change based on how they are modified.34 C. difficile is exquisitely sensitive to many of these modifications. Additionally, conjugated and unconjugated forms of the primary bile acids, cholate (CA) and CDCA, as well as secondary bile acids, deoxycholate (DCA) and lithocholate (LCA), can bind to C. difficile toxin TcdB mitigating its cell toxicity21 (Figure 1(b)). The combined repetitive oligopeptide (CROP) region of TcdB is essential for bile acid binding, which causes major conformational changes when bound.21 Several reviews have previously detailed the relationship between C. difficile and the bile acid pool and its relevance in colonization resistance here.3,25,39–43

Bile acids that are modified by the gut microbiota are associated with successful FMTs, potentially indicating their importance in rCDI (Table 1).18,28,54,55 The gut microbiota encodes a variety of bile acid altering enzymes that act on either the sterol core or the amino acid conjugated to it, including, but not limited to, bile salt hydrolases (BSHs), hydroxysteroid dehydrogenases (HSDHs), and those in the bile acid inducible (bai) operon (Figure 1(a)). These modifications and specifically how they help or hinder C. difficile are discussed in more detail below.Table 1. Efficacy of FMT, microbiota-focused therapeutics, and LBPs on rCDI and their impact on the bile acid pool and the microbiome.

Intervention	Impact on rCDI	Impact on bile acid pool	Impact on microbiome	References	
FMT	81–93% prevention of C. difficile recurrence in human studies.	Reduction in primary bile acids including TCA, GCA, CA, TCDCA, GCDCA, CA.

Increase in secondary bile acids including DCA, LCA, and HDCA.

Reduction in primary MCBAs, loss of histidine, serine, and valine conjugates.

Increase in secondary MCBAs, increase in glutamate, tryptophan, and tyrosine conjugates.

	Increase in alpha diversity.

Decreases in Enterobacteriaceae, Lactobacillaceae, Enterococcaceae, Veillonellaceae, and Megaspharaceae.

Increases in Lachnospiraceae, Acutalibacteraceae, Ruminococcaceae, Oscillospiraceae, Anaeroboracaceae, Butyricicoccaceae, CAG-74, Bacteroidaceae, Rikenellaceae, Bifidobacteriaceae, Coriobacteriaceae, and Eggerthellaceae.

	6,18,28,44–47	
Rebyota	88% prevention of C. difficile recurrence in phase 3 clinical trial.	Significant reduction in TCA, GCA, and CA.

Significant increase in DCA and LCA.48

Reduction in conjugated and deconjugated primary bile acids.

Increase in secondary bile acids, particularly deconjugated.

	Increase in alpha diversity.

Decreases in Gammaproteobacteria and Bacilli.

Increases in Bacteroidia and Clostridia.

	48–50	
VOWST	87.1% prevention of C. difficile recurrence in phase 3 clinical trial	Increase in secondary bile acids compared to placebo.

Increase in LCA and DCA correlates with engraftment of VOWST-based microbes.

	Engraftment of enriched taxa, Clostridia and Erysipelotrichia, observed after treatment with VOWST.

Significant increase in abundance of other organisms alongside VOWST-based microbes, namely Bacteroidetes.

	51,52	
VE303	86.2% prevention of C. difficile recurrence in phase 2 clinical trial	Bile acid abundances return to pre-vancomycin treatment levels in healthy volunteers.

Increase in deconjugated secondary bile acids coupled with decrease in conjugated primary bile acids.

	VE303 strains displaced native Clostridia including Blautia spp. and Erysipelotrichaceae bacterium 21_3.

Expedited return of native gut microbiota after cessation of antibiotics in healthy volunteers.

	13,53	
MET-2	79% prevention of C. difficile recurrence in phase 1 clinical trial	Not evaluated.

	Increase in alpha diversity after treatment with MET-2.

Increase in Lachnospiraceae, Bifidobacteriaceae, Ruminococcaceae, Erysipelotrichaceae, and Coriobacteriaceae.

Decrease in Enterobacteriaceae, Lactobacillaceae, Streptococcaceae, and Fusobacteriaceae.

	12	

Bile salt hydrolases (BSHs)

BSHs are encoded by a variety of microbial Phyla including Bacillota, Bacteroidota, Actinomycetota, and Euryarchaeota.56 BSHs are traditionally known to cleave host-conjugated amino acids, either glycine or taurine, from conjugated bile acids.57 This first step is often a prerequisite for other bile acid modifications.57 The simple conversion of conjugated bile acids to deconjugated bile acids increases the inhibition of C. difficile vegetative growth, and the bile acid is more inhibitory as the conversions get more complex.15,58

Historically, it was proposed that the purpose of microbial BSHs was to detoxify conjugated bile acids, thereby providing a fitness benefit for colonization in the harsh gut environment by the microbes that encode them.59 Additionally, it was suggested that BSHs provide nutrients in the form of liberated amino acids (either taurine or glycine) for the microbes that encode them or the surrounding gut microbiota.60 However, recent studies leveraging both Gram-positive Lactobacillus and Gram-negative Bacteroides found that bile acids become more toxic to bacteria after deconjugation.61,62 Lactobacillus gasseri and Lactobacillus acidophilus each have a taurine-specific BSH and a glycine-specific BSH.61 Knockouts of these BSHs resulted in increased growth of these strains in vitro in the presence of certain bile acids including conjugated forms of CA, particularly in L. gasseri.61 Utilizing these knockout strains, the production of deconjugated bile acids by these BSHs decreased the membrane integrity of both microbes.61 BSH knockouts of L. gasseri and L. acidophilus also outcompeted their wild-type counterparts in gnotobiotic mouse models.61 BSH knockout strains of Bacteroides thetaiotaomicron behave similarly. In a recent paper, different combinations of B. thetaiotaomicron’s bile acid altering enzymes, including two BSHs and one HSDH, were knocked out, and the resulting phenotypes were evaluated.62 BSH knockouts of B. thetaiotaomicron showed an increase in growth in the presence of different bile acids, particularly in conjugated forms of DCA, when compared to wildtype.62 BSH knockout strains of B. thetaiotaomicron also showed broad changes in their transcriptome including carbohydrate, amino acid, lipid, and energy metabolism when compared to wildtype.62 Counter to the dogma that BSHs liberate amino acids to use as a nutrient, B. thetaiotaomicron altered the expression of its polysaccharide utilization loci (PULs) in the presence of bile acids rather than amino acid metabolism.62 The specific PUL that changes in expression was dependent on the bile acid B. thetaiotaomicron encountered.62 Most bile acids increased expression of a starch utilization PUL in B. thetaiotaomicron, while the secondary bile acid DCA had the broadest impact by activating PULs involved in the degradation of α-mannans, host and dietary glycans, mucins, rhamnogalacturonan I and II, and starch.62 These findings indicate that BSH mediated changes to bile acids modify the toxicity of bile acids as well as signaling bacteria to utilize different nutrients in the gut.

Recent work has indicated that BSHs may play an additional role in the production of bile acids conjugated with a variety of non-canonical amino acids, outside of glycine and taurine, collectively referred to as microbially conjugated bile acids (MCBAs) or bacterial bile acid amidates (BBAAs).15,63,64 A screen of 70 bacterial species identified 27 different species across multiple Phyla capable of conjugating amino acids to -CA, -CDCA, or -DCA.65 A recent survey of 654 unique BSHs from the microbiota in the Integrated Gene Catalog identified a selectivity loop that dictates substrate preference of BSHs in multiple taxa.15 The ‘G-X-G’ motif within the selectivity loop prefers taurine conjugated bile acids while the ‘S-R-X’ motif prefers glycine conjugated bile acids.15 Further characterization of electrostatic interactions of the selectivity loop implicates the ‘Y-S-R-G’ motif as a preference for aromatic MCBAs.66 A taurine-specific lactobacilli BSH cocktail was given to mice in a CDI model and was able to inhibit spore germination and growth of C. difficile in the small and large intestines. This was associated with the deconjugation of primary bile acids as well as the production of primary MCBAs (i.e., MCBAs with a primary bile acid sterol core).15 These primary MCBAs, specifically alanine, phenylalanine, serine, tryptophan, and tyrosine conjugated CA (Ala/Phe/Ser/Trp/Tyr-CA), glutamate, histidine, tryptophan conjugated CDCA (Glu/His/Trp-CDCA), and phenylalanine-βMCA (Phe-βMCA) inhibit TCA mediated spore germination15 (Figure 1b). C. difficile vegetative growth was inhibited by Tyr-βMCA and Phe-βMCA.15 MCBAs also inhibit the expression of TcdA, specifically phenylalanine, and tyrosine conjugated CA (Phe/Tyr-CA) as well as phenylalanine and tyrosine conjugated βMCA (Phe/Tyr-βMCA).15 Host conjugated primary and secondary bile acids also impact different stages of the C. difficile lifecycle as seen in Figure 1(b).

Bacteria encoding BSHs are significantly associated with MCBA production, and most bacterial isolates that produce at least one MCBA have a bsh homologue.63 Unconjugated bile acids also upregulate transcription of bsh genes in a strain known to produce MCBAs, Bifidobacterium longum NCTC 11,818.63 BSH inhibitors were able to significantly decrease the production of MCBAs by B. longum in vitro and biochemical assays with the purified BSH encoded by this organism showed both acyltransferase (i.e., TCA to AA-CA) and reconjugation activity (i.e., CA to AA-CA).63 This novel activity of BSHs was also confirmed in a strain of Bacteroides fragilis using genetic knockouts and complementation, confirming this activity occurs in BSHs encoded across Actinomycetota and Bacteroidota.63 In newborn infants, the production of MCBAs increased as their microbiota developed from birth to 1 year and positively correlated with colonization of bsh encoding bacteria.63

In a different study, Clostridium perfringens BSH also demonstrated acyltransferase activity and reconjugation activity.64 This BSH produced MCBAs conjugated with most amino acids except for aspartate and proline.64 Further, in vitro analysis determined 19 of 29 strains of bacteria across Actinomycetia, Verrucomicrobia, Gammaproteobacteria, Bacilli, and Clostridia produced at least one MCBA.64 This activity was especially prevalent in the Lachnospiraceae Family.44,64 Hierarchical clustering of bacterial conjugation profiles indicated little association between phylogeny of these organisms and BSH activity.64 Sequence analysis indicated amino acid substitution at position 82 in the active site of BSHs, separate from the selectivity loop, may dictate the diversity of amino acids that BSHs can conjugate to bile acids to produce MCBAs.64 There is evidence that MCBAs enter enterohepatic circulation and therefore should have an opportunity to interact with a variety of host receptors.64 Though MCBA concentrations are lower than liver conjugated bile acids in healthy individuals, MCBAs have been observed in concentrations equal to or higher than primary bile acids in the stool of human bariatric surgery patients.64,67 In human bariatric surgery, a patient’s total MCBA pool concentration was approximately 77.7 μM compared to the total primary conjugated and secondary bile acid pool concentration of approximately 34.2 μM.64

Primary MCBAs, are also enriched in patients with inflammatory bowel disease (IBD), and cystic fibrosis.68,69 In IBD, there is no change in the level of secondary MCBAs (i.e., MCBAs with a secondary bile acid sterol core). This is in contrast to rCDI patients before and after FMT, where there is a shift from high primary MCBAs pre-FMT to high secondary MCBAs post-FMT.44 The BSHs in highest abundance post-FMT were encoded by many members of the Lachnospiraceae Family.44

Differences have been observed in how C. difficile, members of the gut microbiota, and the host respond to bile acids conjugated with taurine, as opposed to glycine, and now to non-canonical amino acids.15,19,61,62 In light of recent discoveries that challenge conventional wisdom, the precise role of BSHs encoded by the gut microbiota is an area of new investigation and could have major implications for the design of microbiota-focused therapeutics.

Hydroxysteroid dehydrogenases (HSDHs)

HSDHs that act on bile acids are encoded by a variety of organisms including Bacillota, Pseudomonadota, Bacteroidota, Actinomycetota, and Archaea.44,70,71 HSDHs convert hydroxyl groups to ketones at positions 3, 7, or 12 in either the α or β orientation, and 7α-HSDHs are the most studied.72,73 Although they can be components of a larger pathway, such as the bai operon, these enzymes are also found independently in some organisms.74 In some cases, HSDHs have activity on conjugated bile acids.62 It is hypothesized that the keto bile acids produced by HSDHs are further modified to produce epimers or remove the hydroxyl group, and thus are often referred to as intermediates. Recently, advances in mass spectrometry have allowed for the detection of these keto bile acids and epimers in urine and serum, indicating these bile acids enter enterohepatic circulation and may be interacting with host receptors.75

Epimers of LCA arising from modifications of HSDHs, including 3-oxo LCA, isoLCA (iLCA), alloLCA (aLCA), 3-oxo aLCA, and isoalloLCA (iaLCA) have been found to be enriched in the stool of centenarians, implicating their role in sustained health.20 These bile acids are bactericidal and cause damage similar to β-lactam antibiotics.20 These bile acids are also able to decrease carriage of C. difficile and other Gram-positive pathogens in vivo.20 Colonization of a prolific producer of these epimers, Odoribacteraceae St21, significantly increased fecal iaLCA levels while reducing C. difficile shedding to non-detectable levels in a mouse model genetically modified to have similar bile acid profiles to humans.20

In vitro studies have shown that 3-oxo LCA, iLCA, and iaLCA effectively inhibit the growth of C. difficile19,20 (Figure 1(b)). As each modification is made, there is a decrease in minimum inhibitory concentration (MIC) of C. difficile. While CDCA exhibits an MIC of 1.25 mM, this value drops to 0.1 mM with the conversion to 3-oxo LCA, and even further to 0.03 mM and 0.02 mM with the subsequent conversions to iLCA and iaLCA, respectively.19 Even at subinhibitory concentrations, iLCA and iaLCA significantly reduce toxin expression by C. difficile.19 Moreover, these bile acids, along with their precursor LCA, diminish the toxin activity of C. difficile in vitro.19 iLCA and iaLCA also exhibit a more pronounced impact on the growth of C. difficile compared to other commensal gut microbes, particularly Gram-negatives, while causing minimal effects on host cell viability.19

HSDHs can also function on cholesterol-derived steroids. Gut microbe-encoded 3β-HSDH can degrade estradiol and testosterone, leading to depression in patients.76,77 Encoding HSDH provides a range of fitness benefits to microbes, including detoxifying bile acids and being able to use bile acids as electron donors for the electron transport chain.72,78,79 In patients undergoing FMT for rCDI, significantly higher levels of HSDH-modified bile acids such as 3-oxo LCA, iLCA, and 12-oxo LCA were observed post-FMT compared to pre-FMT.44 Random Forest Analysis showed that these bile acids were among the most significantly different and important metabolites.44 As our understanding of HSDHs evolves, these enzymes continue to be crucial for altering cholesterol-derived biomolecules in the gut.

The bile acid inducible (bai) operon

The bai operon is encoded by <1% of metagenome-assembled genomes (MAGs) identified in the gut of healthy humans.80 Bacillota, specifically Ruminococcaceae and Lachnospiraceae, are the most abundant bai operon-encoding bacteria.80,81 The bai operon comprises a series of genes involved in the removal of the 7α-hydroxyl group from bile acids. This operon is comprised of six core genes that carry out 7α-dehydroxylation: baiB, baiA, baiCD, baiE, baiF, and baiH, as well as baiG which encodes a transporter to bring bile acids into the cell.82,83 Other less characterized genes associated with this operon have also been described in some organisms including baiI, baiN, baiJ, baiK, baiL, baiM, and baiP.82,84–86 The removal of the 7α-hydroxyl group results in the conversion of unconjugated primary bile acids, such as CA and CDCA, into the secondary bile acids DCA and LCA, respectively. Recent findings have identified some of the accessory genes associated with the bai operon, namely baiJ and baiP, as having an important role in the 7α-dehydroxylation of CDCA.87 It is known that organisms encoding the bai operon carry out 7α-dehydroxylation, however there are still many unknowns regarding the regulation and the substrate specificity of the different enzymes in this operon.88

Antibiotic-induced changes to the microbiota can modulate colonization resistance against C. difficile in mice and in humans. Often, these changes are due to the loss of 7⍺-dehydroxylating bacteria such as Clostridium scindens ATCC 35704, which has been associated with resistance to C. difficile in a secondary bile acid-dependent manner.89 Additionally, C. scindens ATCC 35704 has been shown to inhibit CDI in patients undergoing hematopoietic stem cell therapy, the radiation from which alters the gut microbiota in a way similar to antibiotics.89 While antibiotics alter the structure of the gut microbiota, they also alter the gut metabolome. Alongside antibiotic-induced decreases in secondary bile acids, glucose, free fatty acids, and dipeptides, there are increases in primary bile acids, amino acids, and sugar alcohols.4 The metabolites that increased post antibiotics in mice were able to support C. difficile TCA-mediated spore germination and vegetative growth with amino acids C. difficile is auxotrophic for, and sugars including mannitol, fructose, sorbitol, raffinose, and stachyose in vitro.4 These findings were further confirmed in a mouse model where broad-spectrum antibiotics resulted in a significant loss of secondary bile acids and members of the Lachnospiraceae and Ruminococcaceae, and an increase in C. difficile germination and outgrowth ex vivo.22

The inhibition of multiple stages of the C. difficile life cycle by secondary bile acids has been reviewed previously.25,40 Briefly, secondary bile acids produced by the bai operon including DCA, iDCA, LCA, iLCA, and hyodeoxycholate (HDCA) were found to inhibit TCA-mediated spore germination, growth, and toxin activity in several clinically relevant strains of C. difficile in vitro17,22 (Figure 1(b)). Work has also been done to determine whether bai-encoding bacteria can produce sufficient quantities of secondary bile acids to inhibit C. difficile in vitro. Four strains of Lachnospiraceae, C. scindens VPI 12708, C. scindens ATCC 35704, Clostridium hiranonis TO-931, and Clostridium hylemonae TN-271 were characterized for their ability to inhibit C. difficile in a series of in vitro assays.90 While C. difficile outcompeted these strains in co-culture, supernatants of both C. scindens strains grown in CA were able to produce enough DCA to inhibit C. difficile growth.90 This was associated with CA induced increased expression of the bai genes in strains of C. scindens, and this was not observed with supernatants of C. hiranonis or C. hylemonae grown in CA.90

In human studies, bai operon genes are found in significantly higher abundance in patients without CDI than those with CDI.91 Secondary bile acid profiles are also distinct in CDI patients, which have low secondary bile acid abundance compared to non-colonized or asymptomatically colonized C. difficile patients, which had higher levels of secondary bile acids.92 Post-FMT samples in patients being treated for rCDI had a significant increase in baiA genes, primarily those encoded by the Lachnospiraceae Family.44 Post-FMT samples also had a significant increase in secondary bile acids, specifically DCA, LCA, HDCA, epideoxycholate (EDCA), 3-oxo LCA, iLCA, and 12-oxo LCA.44 Of these secondary bile acids that returned post-FMT, targeted metabolomics identified the return of many MCBAs with DCA as the sterol core (Table 1).44 Evaluating the abundance of other secondary bile acid amidates (i.e., MCBAs with a secondary bile acid core), such as those with an LCA sterol core, is still difficult due to the lack of synthesized standards.

Microbiota-focused therapeutics have also been observed to restore the secondary bile acid pool. rCDI patients treated with Rebyota showed a shift from dominance of primary bile acids to secondary bile acids, specifically driven by an increase in DCA and LCA (Table 1).93 Many spore-forming bacteria encode the bai operon, highlighting its role in products that select for spores such as VOWST or communities of spore formers such as VE303. Secondary bile acids and the bacteria that produce them play an important role in restoring colonization resistance against CDI, and it is important that these organisms and the enzymes they encode are further biochemically characterized.

Bile acids and the gut microbiota

Inhibition of growth by bile acids is not unique to C. difficile. Primary and secondary bile acids also exert varying effects on microbial growth across different taxa. As stated previously, bile acids inhibit bacteria by disrupting their membrane integrity.4,22,23 However, the concentration of bile acid required to disrupt membrane integrity can vary greatly by species. For example, glycocholate (GCA), which is generated by the host, has a MIC of 20 mM for B. thetaiotaomicron, Lactobacillus acidophilus, Lactobacillus gasseri, and Staphylococcus aureus.61,62,94 However, once GCA is converted to CA by BSHs, the MIC changes to 10 mM, 5 mM, 2.5 mM, and 20 mM, respectively.61,62,94 When CA is converted to DCA, via 7α-dehydroxylating bacteria, the MIC changes again to 0.625 mM, 10 mM, 10 mM, and 1 mM, respectively.61,62,94 The largest difference in MIC among the strains listed here was observed in the 7α-dehydroxylation of CA to DCA. This variation in response to secondary bile acids highlights how small changes in the bile acid pool can impact the fitness of members of the microbiome.

A recent study using gnotobiotic mice used a consortium of over 100 strains, denoted hCom1a, to demonstrate nuances of niche establishment with and without C. scindens ATCC 35704, and C. hylemonae DSM 15053, the only microbes in this consortium that perform 7α-dehydroxylation. By simply removing these two organisms, >100-fold changes in the relative abundance of eight strains in the consortia were observed.95 The removal of the 7α-dehydroxylating strains not only resulted in the elimination of 7α-dehydroxylated bile acids, but also an increase in other modifications to the 7-hydroxyl groups such as dehydrogenation and epimerization.95 The taxonomic shift associated with this was very specific, with decreases in Ruminococcus, Roseburia, and Eubacteria and increases in Veillonella, Clostridium, and Dorea. These changes appear to be species specific, as many other organisms at the same genus level in this consortium were not affected to such a degree.

Investigating genes involved in 7α-dehydroxylation, namely the bai operon, has been difficult due to the lack of genetic tools for the microbes that encode it. However, recent work has been able to clone a bai operon into C. sporogenes and determined a minimal gene set required to perform 7α-dehydroxylation.82 This allowed for characterization of each enzyme in the reductive arm of 7α-dehydroxylation.82 This opens up the possibility for future studies to include strains with the appropriate background as a control, rather than removing 7α-dehydroxylating bacteria or using closely related species.

In fact, most bile acids directly modify the structure of the gut microbial community. Mice exogenously fed host-derived primary bile acids, TCA and GCA, had large changes to the microbial composition of their gut, marked by a significant decrease in the abundance of Verrucomicrobia.96 In addition, mice fed the microbial-derived secondary bile acid UDCA had a significant increase in Lachnospiraceae as well as increases in Lactobacillus, Clostridiales, and unclassified Bacillota alongside decreases in Ruminococcaceae.97 The administration of bile acids to mice significantly influenced both the bile acid pool and the synthesis of host bile acids.96,98 Bile acids given therapeutically to humans are also associated with changes to the microbiota. Patients with colorectal adenomatous polyps treated with UDCA had significant shifts in their microbial community composition, marked by an increase in Faecalibacterium prausnitzii coupled with a decrease in Ruminococcus gnavus.99

MCBAs can also differentially impact the microbiota. A prolific MCBA producer Enterocloster bolteae grows better in most MCBAs with CA core than unconjugated CA,64 although this was not the case for all MCBA producers. For example, Phe-CA decreased the growth of the MCBA producer Lacrimispora indolis while increasing the growth of another MCBA producer of Lactiplantibacillus plantarum, but the opposite was true of TCA.64 MCBA dependent inhibition was extended to a total of 18 strains and the inhibition of each species was dependent on the amino acid conjugated to CA in vitro.64 Of the MCBAs, Phe-CA and Leu-CA had the strongest levels of inhibition for the most species, stronger than TCA and GCA, indicating that MCBA generation increases the toxicity of bile acids.64 Generally, the greater the hydrophobicity of the conjugated amino acid in the MCBA the greater the antimicrobial properties.64 Species dependent inhibition by MCBAs was also confirmed in vivo. Mice fed the MCBAs Phe-CA and Ser-CA had significant shifts in the beta diversity of their fecal microbiota, though this effect was subtle and lost when comparing between groups at individual timepoints.64 Changes to the chemical structure of bile acids by gut microbes play an important role in shaping gut ecology at the species level and are an important factor when considering new therapies for CDI and other intestinal diseases.

Bile acids and the host response

Bile acids not only interact with the microbiota but also with the host. These interactions are reviewed in Collins et al. 2023.100 During enterohepatic circulation, bile acids can directly interact with the host through nuclear receptors, such as FXR and PXR, which regulate the production of bile acids in the host. This can also influence other receptors such as RORγT that alters host physiology, such as immunity, by modulating Treg and Th17 cell differentiation.27,45,101–104 TGR5 also interacts with bile acids.103,105 Among these, FXR and TGR5 are the only receptors whose known agonists are solely bile acids, and they both react differently depending on which bile acid is bound.101,103 The differential-binding ability of bile acids to host receptors has been reviewed by Fiorruci et al. 2021.104

FXR has differential activation depending on the bile acid it encounters in the order of CDCA>CA>LCA>DCA.104,106 Both murine and human FXR are activated by MCBAs, again with varying responses to each bile acid, the highest activation is with CDCA conjugates.63 Increased FXR signaling is associated with successful FMT, though FXR signaling does not appear to be significantly altered in patients with primary CDI.32,45

FXR activation regulates the production of bile acids by the host through controlling the expression of the gene that encodes the rate limiting enzyme of host bile acid synthesis: Cyp7a1.101 FXR may also modulate the nutritional environment in the gut, specifically the amount of taurine present through increase of host taurine synthesis and bile acid amidation.107 This plays an important role in modulating blood cholesterol levels and the amount of bile acid present in the gut. Diet can also modulate bile acid levels, for example dietary polyphenols modulate bile acid metabolism and signaling pathways.108 Not only is FXR important in regulating bile acid synthesis, but it has been shown to be important in modifying the immune response. FXR can activate the NLRP3 inflammasome in inflammatory macrophages, thereby modulating aspects of the innate immune system.109 FXR is also able to stabilize the transcriptional regulator, NCoR, on the NF-kB binding site of IL-1B, preventing its transcription and reducing the inflammatory response.110 Certain diseases, such as IBD, also have higher expression of FXR increasing sensitivity to fiber-induced changes to microbiota-derived bile acids such as increased CA and CDCA.111 Treatments targeted at modifying the microbiota to restore bile acid metabolism will also directly impact the host through receptors such as FXR.

Bile acids, while toxic to certain microbes such as C. difficile, can also be toxic to the host. PXR can be activated by various xenobiotics including pharmaceuticals, nutraceuticals, dietary factors, environmental chemicals, as well as secondary bile acids.112 PXR regulates the gut liver axis through bidirectional interactions with the gut microbiome under different conditions, to reduce the amount of toxic bile acids. MCBAs, particularly those conjugated to glutamate, have been observed to interact with PXR in mouse organoids and human cell culture reporter models.63

RORγT is particularly interesting with regard to CDI as this bile acid receptor modulates T cells and is activated differently depending on which bile acid is present.113 The microbial-derived bile acid isoDCA promotes the generation of colonic FOXP3 regulatory T cells expressing RORγT through interaction with dendritic cells.113 RORγT also plays a role in the differentiation of Th17 and Treg cells in response to microbially derived bile acids.27 Diet also plays a role in RORγT signaling.102 In mice, a change in diet due to weaning alters RORγT signaling and was associated with an increase in Clostridia, a class of organisms rich in 7α-dehydroxylation of bile acids.114 Diet is also important as malnutrition has also been observed to increase the number of cells expressing RORγT.115 Increases in RORγT cells have been observed in mouse models of FMT for IBD.116

Leveraging the microbiota to alter bile acids to modulate nuclear receptors may be able to impact inflammation and immunity in patients with CDI. However, it is essential to acknowledge that bile acids are not always beneficial to the host. Certain bile acids or an excess of bile acid such as CDCA, DCA, and LCA have been observed to increase inflammation in the gut, so it will be important to be able to control the concentration delivered.117–119 Inflammation, including that resulting from CDI, has wide reaching impacts including increasing host-derived microbial nutrients in the gut, impacting the colonization of microbes, and affecting the metabolome.119,120

Non-bile acid dependent microbial interactions and C. difficile

Cross feeding, or the production of essential nutrients for C. difficile by gut microbes, is important to consider when studying how C. difficile establishes colonization. Amino acids are some of the most important nutrients to consider in cross-feeding, as C. difficile is auxotrophic for six amino acids: cysteine, isoleucine, leucine, proline, tryptophan, and valine.121 Most cases of cross feeding involve C. difficile using nutrients produced by other gut microbes while generating minimal nutrients in return.122 In silico models predict that C. difficile consumes amino acids produced by commensal organisms such as Bifidobacterium longum, including proline, glutamate, leucine, tyrosine, alanine, serine, glutamine, and methionine while only providing aspartate in return.122 Enterococci cross feeds C. difficile with amino acids including leucine and ornithine, resulting in a high C. difficile load and worse disease in a mouse model of CDI.33 These findings were mirrored in CDI patients colonized with vancomycin-resistant Enterococcus (VRE).33 While Enterococci is involved in cross feeding, it can also limit the amino acids available to C. difficile. Competition for arginine between Enterococcus faecalis OG1RF and C. difficile causes stress in C. difficile, which in turn increases virulence factor expression and promotes disease.33 C. sardiniense also cross feeds C. difficile with amino acids including ornithine, leading to an increase in pathogen biomass and disease in a mouse model.123

Host factors also play an important role in nutrient-mediated support of C. difficile. The gut environment of pre-FMT rCDI patients contains higher levels of host-associated acylcarnitines, which other members of the gut microbiota can use for growth.124 This suggests that C. difficile may be engaging in cross-feeding by liberating host molecules via toxin production.44,124 Hydroxyproline is the main ingredient of collagen and is an amino acid-rich nutrient that C. difficile likely liberates from the host via toxin mediated inflammation. Toxin mediated inflammation disrupted collagen networks, which supported C. difficile growth.120 Hydroxyproline is converted to proline, a key nutrient for C. difficile, by hypD and proC, genes widely found in many commensals and in C. difficile.125 The presence of hydroxyproline affects the metabolic gene expression of both C. difficile and commensal Clostridia strains including C. scindens VPI 12708, C. hylemonae TN 271, and C. hiranonis TO 931, suggesting that it influences nutrient competition and adaptation within the gut environment.125 Specifically, proline reductase genes are upregulated in C. difficile, while hydroxyproline is present in vitro.125 This increase in proline reductase genes was not consistent across the commensal species tested indicating hydroxyproline may be preferred by specific species.125 A mouse model comparing wild type C. difficile to hypD knockout C. difficile noted changes in the microbiota, mainly as an increase in Lachnospiraceae.125

Nutrient competition between members of the gut microbiota and C. difficile is another mechanism by which colonization resistance can be maintained. Gut communities with members that compete with pathogens for key nutrients have been shown to restore colonization resistance against Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium in a mouse model.126 Since C. difficile and many other Bacillota use Stickland fermentation to generate energy, substrates such as glycine, isoleucine, leucine, proline, and hydroxyproline are often the cause of competition between microbes.121,127–129 In a mouse model of CDI, competition for Stickland metabolites between C. difficile and commensals like C. hiranonis 10542, Clostridium leptum ATCC 29065, or C. scindens VPI 12708 is enough to prevent CDI-related weight loss.32 As these organisms encode a bai operon, which may be producing inhibitory bile acids, the ability for these organisms to compete with C. difficile and prevent CDI-related weight loss was validated in a knockout mouse that does not produce CA.32 There was also an increase in Stickland metabolites, namely 5-aminovalerate which is a byproduct of proline fermentation, in germfree mice monoassociated with the commensal strains.32 In another study, monocolonization of germ-free mice with the C. scindens ATCC 35704 prior to C. difficile challenge delayed clinical signs of disease and colonic damage for a few weeks, and did not prevent CDI.130 In gnotobiotic mice with a defined consortium, C. scindens ATCC 35704 also did not prevent colonization of C. difficile.130,131

While these strains contain the bai operon and, therefore, may produce inhibitory secondary bile acids, other strains such as Paraclostridium bifermentans that do not encode the bai operon also compete with C. difficile, and increases survival in a CDI mouse model, indicating that competition for nutrients between C. difficile and its close relatives may prevent or reduce disease.32,123

Non-toxigenic strains of C. difficile are also able to colonize the gut of rCDI patients and reduce CDI recurrence, likely by competing for the same nutrients.132 Considering that many of these Bacillota are lost during antibiotic treatment, they are a population which therapies often seek to reestablish with the hope of increasing colonization resistance. For example, a strain of C. scindens ATCC 35704 produces an antimicrobial alkaloid derived from L-tryptophan and oxaloacetaldehyde, 1-acetyl-β-carboline, that inhibits C. difficile.133 Another microbial metabolite, Urolithin A, has been observed to reduce the expression of C. difficile toxin and repair epithelial damage.134 These metabolites and the bacteria that produce them are important to consider when designing novel therapeutics against C. difficile.

Rational design and standardization of microbiota-focused therapeutics to combat rCDI

While FMT serves as a last resort in treating patients with rCDI, the lack of knowledge of specific mechanisms that mediate its success impedes the development of microbiota-focused therapeutics. Defining which bacteria are needed for a successful FMT is a challenge. We are still not sure if the ability of donor strains to inhabit the recipient after an FMT, a process called engraftment, is required for successful treatment. A recent meta-analysis found that the clinical success of FMTs for rCDI was associated with donor strain engraftment and convergence of microbial species abundance.135 Although this process is taxonomically biased, some taxa have more difficulty engrafting than others.136 However, engraftment is not always required for successful treatment with FMTs or LBPs. Some microbiota-focused therapeutics and LBPs used to treat rCDI do not always show complete engraftment observe increases in beneficial taxa even if they are not included in the formulation.51,53 Additionally, benefits from probiotic strains, such as lactobacilli, can be observed even in the absence of engraftment, as they are passing through, but still providing a function.33 The complexities of FMT, including the intricacies of engraftment and variable roles of individual bacterial taxa, underscore the need for further research to decipher the mechanisms behind its success.

Attempts to systematically reestablish the functions that are lost after antibiotic treatment with regard to CDI are ongoing. Rebyota (RBX2660) is a product produced by Ferring Pharmaceuticals that achieved FDA approval in 2022 and became the first microbiota-based therapeutic to enter the market. Rebyota is pathogen screened feces that has been filtered after suspension in a solution of saline and polyethylene glycol 3350 mix.11,137,138 Additional screens ensuring a minimal dose of Bacteroides and a maximum dose of polyethylene glycol 3350 were also performed.11,137,138 This product is similar to FMT as it utilizes minimal processing thereby keeping the microbiome of the donor mostly intact. Patients treated with Rebyota have similar changes to the bile acid pool as those observed in FMT studies, a reduction of primary bile acids with an increase in secondary bile acids (Table 1).48,49 While this is a critical first step in standardizing the process of introducing beneficial bacteria to treat rCDI, there are still potentially unnecessary bacteria in this preparation. VOWST, another microbiota-focused therapeutic previously known as SER-109, produced by SERES Therapeutics also attained FDA approval in 2023. VOWST is produced using an ethanol-based purification method to enrich spore forming cells, namely Bacillota.10,51 This method has a similar success rate of 88% for the treatment of rCDI compared to Rebyota 87.1% in their respective clinical trials.50,52 This is similar to the success rates reported for FMT which range from 81% to 93%.6,46 VOWST was able to restore the presence of secondary bile acids after treatment, similar to Rebyota and FMTs (Table 1).51 Patients treated with VOWST also had an increase in the amount of Bacteroides, which was absent from the treatment administered.51 This highlights that a successful microbiota-focused therapeutic does not necessarily have to contain all of the bacteria necessary to treat rCDI. The bacteria administered might play an important role in changing the existing gut ecology, which provides resistance against C. difficile.

While the trajectory of development for products like Rebyota or VOWST is a top-down approach starting with healthy donor feces, LBPs composed of defined consortia aim to build an effective set of microbes from the bottom up. The Nubiyota product, Microbial Ecosystem Therapeutic-2 (MET-2), is comprised of 40 strains of bacteria and has shown promise in phase 1 clinical trials to treat rCDI as well as phase 2 clinical trials for depression and general anxiety disorder.12,139 Similar to FMT and other LBPs, MET-2 is associated with an increase in alpha diversity post-treatment (Table 1).12 VE303, developed by Vedanta Biosciences, is a formulation of eight Clostridia strains. In a recent phase 2 trial, this consortia achieved a rCDI remission rate of 86.2% in the high-dose group, an outcome consistent with FMT and other microbiota-focused therapeutics.13 After treatment with VE303 in patients who recently received antibiotics there was an increase in deconjugated bile acids and secondary bile acids, as well as a decrease in conjugated primary bile acids, indicating a return of microbes encoding bile acid altering enzymes (Table 1).53 In a first of its kind dose–response study on vancomycin-treated patients receiving VE303, the microbiota recovered significantly quicker in high-dose cohorts (8.0 × 109 CFU/day) vs low-dose cohorts (≤4.0 × 109 CFU/day).53 A common theme among LBPs is smaller communities being biased toward Bacillota in which most 7α-dehydroxylation occurs.90,140

Microbiota-focused therapies beyond rCDI

FMTs and LBPs have shown promise in other diseases outside of rCDI. Perhaps, the most direct translational diseases include other bowel and liver diseases such as IBD, including Ulcerative Colitis (UC) and Crohn’s disease (CD), and NAFLD that are all associated with shifts in the bile acid pool.141,142 The guidelines set by the American Gastroenterological Association (AGA) state FMTs are recommended for the treatment of rCDI in immunocompetent and mildly immunocompromised adults, while microbiota-based therapeutics such as Rebyota and VOWST are recommended for use only in immunocompetent adults, however, no microbiota-based therapeutic is recommended for IBD at this time.7 Understanding the overlap between rCDI and other intestinal diseases will aid in our understanding of how to best leverage microbiota-based therapeutics. IBD is marked by increases in primary bile acids such as CA and CDCA, alongside decreases in abundance of secondary bile acids such as DCA and LCA, similar to changes observed in rCDI.44,141 Changes to the serum bile acid pool in NAFLD also include an increase in primary bile acids and secondary bile acids as well as a decrease in conjugated bile acids overall in serum.142

Increased levels of secondary bile acids and SCFAs were found in UC patients who responded to FMT in a randomized trial.143 An open label clinical trial comparing gastroscopy and colonoscopy for delivery of FMTs to treat CD achieved 66.7% remission rates and increased the taxonomic diversity in the gut.144 In cases where CDI co-occurs with IBD, FMTs have been observed to have a positive effect on both diseases at the same time.145 Liver-related diseases such as NAFLD, including nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) have shown promise with microbiota-focused therapies. A clinical trial using a consortium of eight probiotic strains, called VSL#3, including Streptococcus thermophilus, Bifidobacterium breve, two strains of Bifidobacteria animalis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus helveticus improved NAFLD.146 FMTs have also been evaluated for other diseases in which gut bacterial bile acid metabolism is also thought to be important, such as diabetes mellitus.47

The gut-brain axis is another active area of research in the context of bile acid metabolism. Bile acids share structural similarities with other cholesterol-derived hormones, and as such research in bile acid modifications may be applicable to these structurally similar molecules. One such instance is polycystic ovarian syndrome (PCOS), which is characterized by an excess of androgens.147 In studies using mice colonized with microbiomes from women with PCOS, it was observed that there was a disruption of ovarian function coupled with altered bile acid metabolism compared to mice colonized with a control microbiome.147 As mentioned previously, there are also links to depression, with gut encoded HSDHs being implicated, as well as MET-2 the defined consortia in clinical trials used to treat depression.76,77,139 A reduction in symptoms associated with autism spectrum disorder (ASD) has also been observed alongside changes in blood neurotransmitter levels in open label clinical trials.148 Validation of the open-label trials, ideally through double blinded randomized controlled trials will be critical in further evaluating the efficacy of FMT for these diseases. Other neurological disorders such as Alzheimer’s, stroke, epilepsy, Tourette Syndrome, diabetic neuropathy, and Guillain–Barre are also being investigated, although limited evidence for efficacy is currently available.149

Conclusions and future directions

C. difficile is a major healthcare-associated pathogen with significant morbidity and mortality. CDI is associated with antibiotic usage, which disrupts the native gut microbiota, diminishing colonization resistance against C. difficile. A deeper understanding of the underlying mechanisms facilitating FMTs and LBPs, which aim to reinstate colonization resistance, will allow for targeted approaches to their development. We have reviewed C. difficile’s high sensitivity to bile acids, and the microbial modifications that enhance this susceptibility. These modifications, mediated by the gut microbiota through enzymes like BSHs, HSDHs, and those in the bai operon play a pivotal role in altering bile acid profiles. Moreover, these modifications not only influence C. difficile, but the composition of the microbiota and host responses as well. Beyond bile acid modifications, other mechanisms such as cross-feeding, nutrient blocking, and the production of antimicrobial metabolites warrant consideration for optimizing FMT and LBP therapies. Ongoing efforts in this optimization process include the FDA approved Rebyota, a product comprised pathogen screened feces, and VOWST, a product comprised feces enriched for spore-forming bacteria, as well as ongoing clinical trials using defined bacterial consortia such as VE303 and MET-2. These products have been shown to alter the gut microbial community as well as the bile acid pool reflecting changes after an FMT. FMTs and LBPs have demonstrated potential efficacy in treating various other diseases such as IBD including UC and CD, NAFLD, PCOS, ASD, Alzheimer’s, stroke, epilepsy, Tourette syndrome, diabetic neuropathy, and Guillain–Barre syndrome.

The diversity of modifications to the bile acid pool has exploded recently to include MCBAs and newer modifications are continuing to be discovered.150 Exploring the impact of these novel bile acids in the context of rCDI prevention and treatment is one of the newest uncharted territories, warranting the need for comprehensive studies. Host produced and microbial-derived bile acids play a critical role in inhibiting C. difficile spore germination, growth, and toxin production, and continued evaluation of each bile acid’s role in these processes will be critical in developing therapies for rCDI. By leveraging the different antimicrobial effects bile acids have on different members of the microbiota, new therapies have the potential to shape not only pathogen dynamics, but commensal dynamics. This highlights the need to not only evaluate how bile acids impact pathogens, but also commensal organisms in the gut. Alongside these changes to the microbiome, bile acids also interact with host receptors and can mediate immune responses, highlighting their importance in developing therapies for a variety of diseases. More research is needed to understand how MCBAs and other non-canonical modified bile acids interact with host receptors and the consequences this has for host immunity and the gut microbiota. Other than bile acid mediated interactions with C. difficile, the microbiome, and the host, competition for nutrients and cross-feeding in the gut is becoming an important mechanism to evaluate while developing microbiota-focused therapeutics. Investigating the intricate connections between these elements will be important for developing successful microbial-focused therapeutics.

Disclosure statement

C.M.T. consults for Vedanta Bioscicens, Inc., Summit Therapeutics, and Ferring Pharmaceuticals, Inc. and is on the Scientic Advisory Board for Ancilia Biosciences.
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References

1. CDC. Antibiotic resistance threats in the United States, 2019. Atlanta (GA): U.S. Department of Health and Human Services, CDC; 2019.
2. Guh AY, Mu Y, Winston LG, Johnston H, Olson D, Farley MM, Wilson LE, Holzbauer SM, Phipps EC, Dumyati GK, et al. Trends in U.S. Burden of Clostridioides difficile infection and outcomes. N Engl J Med. 2020;382 (14 ):1320–22. doi:10.1056/NEJMoa1910215.32242357
3. Pike CM, Theriot CM. Mechanisms of colonization resistance against Clostridioides difficile. J Infect Dis. 2021;223 (12 Suppl 2 ):S194–S200. doi:10.1093/infdis/jiaa408.33326565
4. Theriot CM, Koenigsknecht MJ, Carlson PE, Hatton GE, Nelson AM, Li B, Huffnagle GB, Z. Li, J., Young VB. Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to clostridium difficile infection. Nat Commun. 2014;5 (1 ):3114. doi:10.1038/ncomms4114.24445449
5. Johnson S, Lavergne V, Skinner AM, Gonzales-Luna AJ, Garey KW, Kelly CP, Wilcox MH. Clinical practice guideline by the infectious diseases society of America (IDSA) and society for healthcare epidemiology of America (SHEA): 2021 focused update guidelines on management of Clostridioides difficile infection in adults. Clin Infect Dis. 2021;73 (5 ):e1029–e1044. doi:10.1093/cid/ciab549.34164674
6. van Nood E, Vrieze A, Nieuwdorp M, Fuentes S, Zoetendal EG, de Vos WM, Visser CE, Kuijper EJ, Bartelsman JFWM, Tijssen JGP, et al. Duodenal infusion of donor feces for recurrent clostridium difficile. N Engl J Med. 2013;368 (5 ):407–415. doi:10.1056/NEJMoa1205037.23323867
7. Peery AF, Kelly CR, Kao D, Vaughn BP, Lebwohl B, Singh S, Imdad A, Altayar O. AGA clinical practice guideline on fecal microbiota–based therapies for select gastrointestinal diseases. Gastroenterology. 2024;166 (3 ):409–434. doi:10.1053/j.gastro.2024.01.008.38395525
8. Kelly CP. Can we identify patients at high risk of recurrent clostridium difficile infection? Clin Microbiol infect. 2012;18 Suppl 6 : p. 21–27. doi:10.1111/1469-0691.12046.
9. Nelson WW, Scott TA, Boules M, Teigland C, Parente A, Unni S, Feuerstadt P. Health care resource utilization and costs of recurrent Clostridioides difficile infection in the elderly: a real-world claims analysis. J Managed Care & Specialty Pharm. 2021;27 (7 ):828–838. doi:10.18553/jmcp.2021.20395.
10. Khanna S, Sims M, Louie TJ, Fischer M, LaPlante K, Allegretti J, Hasson BR, Fonte AT, McChalicher C, Ege DS, et al. SER-109: an oral investigational microbiome therapeutic for patients with recurrent Clostridioides difficile infection (rCDI). Antibiotics (Basel. Antibiotics. 2022;11 (9 ):1234. doi:10.3390/antibiotics11091234.36140013
11. Lee C, Louie T, Bancke L, Guthmueller B, Harvey A, Feuerstadt P, Khanna S, Orenstein R, Dubberke ER. Safety of fecal microbiota, live-jslm (REBYOTA ™) in individuals with recurrent Clostridioides difficile infection: data from five prospective clinical trials. Therap Adv Gastroenterol. 2023;16 :17562848231174277. doi:10.1177/17562848231174277.
12. Kao D, Wong K, Franz R, Cochrane K, Sherriff K, Chui L, Lloyd C, Roach B, Bai AD, Petrof EO, et al. The effect of a microbial ecosystem therapeutic (MET-2) on recurrent Clostridioides difficile infection: a phase 1, open-label, single-group trial. Lancet Gastroenterol Hepatol. 2021;6 (4 ):282–291. doi:10.1016/S2468-1253(21)00007-8.33631102
13. Louie T, Golan Y, Khanna S, Bobilev D, Erpelding N, Fratazzi C, Carini M, Menon R, Ruisi M, Norman JM, et al. VE303, a defined bacterial consortium, for prevention of recurrent Clostridioides difficile infection: a randomized clinical trial. JAMA. 2023;329 (16 ):1356–1366. doi:10.1001/jama.2023.4314.37060545
14. Bhattacharjee D, Francis MB, Ding X, McAllister KN, Shrestha R, Sorg JA. Reexamining the germination phenotypes of several Clostridium difficile strains suggests another role for the CspC germinant receptor. J Bacteriol. 2015;198 (5 ):777–786. doi:10.1128/JB.00908-15.26668265
15. Foley MH, Walker ME, Stewart AK, O’Flaherty S, Gentry EC, Patel S, Beaty VV, Allen G, Pan M, Simpson JB, et al. Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut. Nat Microbiol. 2023;8 (4 ):611–628. doi:10.1038/s41564-023-01337-7.36914755
16. Sorg JA, SoneR%nshein AL. Bile salts and glycine as cogerminants for Clostridium difficile spores. Journal of Bacteriology. 2008;190 (7 ):2505–2512.18245298
17. Thanissery R, Winston JA, Theriot CM. Inhibition of spore germination, growth, and toxin activity of clinically relevant C. difficile strains by gut microbiota derived secondary bile acids. Anaerobe. 2017;45 :86–100. doi:10.1016/j.anaerobe.2017.03.004.28279860
18. Weingarden AR, Dosa PI, DeWinter E, Steer CJ, Shaughnessy MK, Johnson JR, Khoruts A, Sadowsky MJ. Changes in colonic bile acid composition following fecal microbiota transplantation are sufficient to control Clostridium difficile germination and growth. PLoS One. 2016;11 (1 ):e0147210. doi:10.1371/journal.pone.0147210.26789728
19. Kisthardt SC, Thanissery R, Pike CM, Foley MH, Theriot CM. The microbial-derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota. J Bacteriol. 2023;205 (9 ):e0018023. doi:10.1128/jb.00180-23.37695856
20. Sato Y, Atarashi K, Plichta DR, Arai Y, Sasajima S, Kearney SM, Suda W, Takeshita K, Sasaki T, Okamoto S, et al. Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians. Nature. 2021;599 (7885 ):458–464. doi:10.1038/s41586-021-03832-5.34325466
21. Tam J, Icho S, Utama E, Orrell KE, Gómez-Biagi RF, Theriot CM, Kroh HK, Rutherford SA, Lacy DB, Melnyk RA, et al. Intestinal bile acids directly modulate the structure and function of C. difficile TcdB toxin. Proc Natl Acad Sci U S A. 2020;117 (12 ):6792–6800. doi:10.1073/pnas.1916965117.32152097
22. Theriot CM, Bowman AA, Young VB, Ellermeier CD. Antibiotic-induced alterations of the gut microbiota alter secondary bile acid production and allow for Clostridium difficile spore germination and outgrowth in the large intestine. mSphere. 2016;1 (1 ):1(1. doi:10.1128/mSphere.00045-15.
23. Oberkampf M, Hamiot A, Altamirano-Silva P, Bellés-Sancho P, Tremblay YDN, DiBenedetto N, Seifert R, Soutourina O, Bry L, Dupuy B, et al. C-di-amp signaling is required for bile salt resistance, osmotolerance, and long-term host colonization by Clostridioides difficile. Sci Signal. 2022;15 (750 ): p. eabn8171. doi:10.1126/scisignal.abn8171.36067333
24. Aias M, Azrad M, Saad G, Leshem T, Hamo Z, Rahmoun LA, Peretz A. Different bile acids have versatile effects on sporulation, toxin levels and biofilm formation of different Clostridioides difficile strains. J Microbiol Methods. 2023;206 :106692. doi:10.1016/j.mimet.2023.106692.36809809
25. Winston JA, Theriot CM. Impact of microbial derived secondary bile acids on colonization resistance against Clostridium difficile in the gastrointestinal tract. Anaerobe. 2016;41 :44–50. doi:10.1016/j.anaerobe.2016.05.003.27163871
26. Cheung KCP, Ma J, Loiola RA, Chen X, Jia W. Bile acid-activated receptors in innate and adaptive immunity: targeted drugs and biological agents. Eur J Immunol. 2023;53 (8 ):2250299. doi:10.1002/eji.202250299.
27. Hang S, Paik D, Yao L, Kim E, Trinath J, Lu J, Ha S, Nelson BN, Kelly SP, Wu L, et al. Bile acid metabolites control T(H)17 and T(reg) cell differentiation. Nature. 2019;576 (7785 ):143–148. doi:10.1038/s41586-019-1785-z.31776512
28. Seekatz AM, Theriot CM, Rao K, Chang Y-M, Freeman AE, Kao JY, Young VB. Restoration of short chain fatty acid and bile acid metabolism following fecal microbiota transplantation in patients with recurrent Clostridium difficile infection. Anaerobe. 2018;53 :64–73. doi:10.1016/j.anaerobe.2018.04.001.29654837
29. Wang S, Xiang L, Li F, Deng W, Lv P, Chen Y. Butyrate protects against Clostridium difficile infection by regulating bile acid metabolism. Microbiol Spectr. 2023;11 (4 ):e0447922. doi:10.1128/spectrum.04479-22.37350595
30. Pensinger Daniel A, Fisher AT, Dobrila HA, Van Treuren W, Gardner JO, Higginbottom SK, Carter MM, Schumann B, Bertozzi CR, Anikst V, et al. Butyrate differentiates permissiveness to Clostridioides difficile infection and influences growth of diverse C. difficile isolates. Infect Immun. 2023;91 (2 ):e00570–22. doi:10.1128/iai.00570-22.36692308
31. Baldassare MA, Bhattacharjee D, Coles JD, Nelson S, McCollum CA, Seekatz AM. Butyrate enhances Clostridioides difficile sporulation in vitro. J Bacteriol. 2023;205 (9 ):e0013823. doi:10.1128/jb.00138-23.37655912
32. Aguirre AM, Yalcinkaya N, Wu Q, Swennes A, Tessier ME, Roberts P, Miyajima F, Savidge T, Sorg JA. Bile acid-independent protection against Clostridioides difficile infection. PloS Pathog. 2021;17 (10 ):e1010015. doi:10.1371/journal.ppat.1010015.34665847
33. Smith AB, Jenior ML, Keenan O, Hart JL, Specker J, Abbas A, Rangel PC, Di C, Green J, Bustin KA, et al. Enterococci enhance Clostridioides difficile pathogenesis. Nature. 2022;611 (7937 ):780–786. doi:10.1038/s41586-022-05438-x.36385534
34. Begley M, Gahan CG, Hill C. The interaction between bacteria and bile. FEMS Microbiol Rev. 2005;29 (4 ):625–651. doi:10.1016/j.femsre.2004.09.003.16102595
35. Dawson PA, Karpen SJ. Intestinal transport and metabolism of bile acids. J Lipid Res. 2015;56 (6 ):1085–1099. doi:10.1194/jlr.R054114.25210150
36. Ridlon JM, Gaskins HR. Another renaissance for bile acid gastrointestinal microbiology. Nat Rev Gastroenterol Hepatol. 2024;21 (5 ):348–364. doi:10.1038/s41575-024-00896-2.38383804
37. Rowe JC, Winston JA. Collaborative metabolism: gut microbes play a key role in canine and feline bile acid metabolism. Vet Sci. 2024;11 (2 ):94. doi:10.3390/vetsci11020094.38393112
38. Sorg JA, Sonenshein AL. Inhibiting the initiation of clostridium difficile spore germination using analogs of chenodeoxycholic acid, a bile acid. J Bacteriol. 2010;192 (19 ):4983–4990. doi:10.1128/JB.00610-10.20675492
39. Theriot CM, Young VB. Interactions between the gastrointestinal microbiome and Clostridium difficile. Annu Rev Microbiol. 2015;69 (1 ):445–461. doi:10.1146/annurev-micro-091014-104115.26488281
40. Reed AD, Theriot CM. Contribution of inhibitory metabolites and competition for nutrients to colonization resistance against Clostridioides difficile by commensal clostridium. Microorganisms. 2021;9 (2 ):371. doi:10.3390/microorganisms9020371.33673352
41. Theriot CM, Young VB. Microbial and metabolic interactions between the gastrointestinal tract and Clostridium difficile infection. Gut Microbes. 2014;5 (1 ):86–95. doi:10.4161/gmic.27131.24335555
42. Yadegar A, Pakpour S, Ibrahim FF, Nabavi-Rad A, Cook L, Walter J, Seekatz AM, Wong K, Monaghan TM, Kao D. Beneficial effects of fecal microbiota transplantation in recurrent Clostridioides difficile infection. Cell Host Microbe. 2023;31 (5 ):695–711. doi:10.1016/j.chom.2023.03.019.37167952
43. Sorbara MT, Pamer EG. Interbacterial mechanisms of colonization resistance and the strategies pathogens use to overcome them. Mucosal Immunol. 2019;12 (1 ):1–9. doi:10.1038/s41385-018-0053-0.29988120
44. McMillan AS, Zhang G, Dougherty MK, McGill SK, Gulati AS, Baker ES, Theriot CM. Metagenomic, metabolomic, and lipidomic shifts associated with fecal microbiota transplantation for recurrent Clostridioides difficile infection. bioRxiv. 2024. doi:10.1101/2024.02.07.579219.
45. Monaghan T, Mullish BH, Patterson J, Wong GK, Marchesi JR, Xu H, Jilani T, Kao D. Effective fecal microbiota transplantation for recurrent Clostridioides difficile infection in humans is associated with increased signalling in the bile acid-farnesoid X receptor-fibroblast growth factor pathway. Gut Microbes. 2019;10 (2 ):142–148. doi:10.1080/19490976.2018.1506667.30183484
46. Cammarota G, Ianiro G, Gasbarrini A. Fecal microbiota transplantation for the treatment of Clostridium difficile infection: a systematic review. J Clin Gastroenterol. 2014;48 (8 ):693–702. doi:10.1097/MCG.0000000000000046.24440934
47. Bustamante JM, Dawson T, Loeffler C, Marfori Z, Marchesi JR, Mullish BH, Thompson CC, Crandall KA, Rahnavard A, Allegretti JR, et al. Impact of Fecal Microbiota Transplantation on Gut Bacterial Bile Acid Metabolism in Humans. Nutrients. 2022;14 (24 ):5200. doi:10.3390/nu14245200.36558359
48. Papazyan R, Ferdyan N, Srinivasan K, Gonzalez C, Shannon WD, Blount K, Fuchs BC. Human fecal bile acid analysis after investigational microbiota-based live biotherapeutic delivery for recurrent Clostridioides difficile infection. Microorganisms. 2023;11 (1 ):135. doi:10.3390/microorganisms11010135.36677428
49. Blount KF, Shannon WD, Deych E, Jones C. Restoration of bacterial microbiome composition and diversity among treatment responders in a phase 2 trial of RBX2660: an investigational microbiome restoration therapeutic. Open Forum Infect Dis. 2019;6 (4 ):ofz095. doi:10.1093/ofid/ofz095.31024971
50. Khanna S, Assi M, Lee C, Yoho D, Louie T, Knapple W, Aguilar H, Garcia-Diaz J, Wang GP, Berry SM, et al. Efficacy and safety of RBX2660 in PUNCH CD3, a phase III, randomized, double-blind, placebo-controlled trial with a bayesian primary analysis for the prevention of recurrent Clostridioides difficile infection. Drugs. 2022;82 (15 ):1527–1538. doi:10.1007/s40265-022-01797-x.36287379
51. McGovern BH, Ford CB, Henn MR, Pardi DS, Khanna S, Hohmann EL, O’Brien EJ, Desjardins CA, Bernardo P, Wortman JR, et al. SER-109, an investigational microbiome drug to reduce recurrence after Clostridioides difficile infection: lessons learned from a phase 2 trial. Clin Infect Dis. 2021;72 (12 ):2132–2140. doi:10.1093/cid/ciaa387.32255488
52. Feuerstadt P, Louie TJ, Lashner B, Wang EEL, Diao L, Bryant JA, Sims M, Kraft CS, Cohen SH, Berenson CS, et al. SER-109, an oral microbiome therapy for recurrent Clostridioides difficile infection. N Engl J Med. 2022;386 (3 ):220–229. doi:10.1056/NEJMoa2106516.35045228
53. Dsouza M, Menon R, Crossette E, Bhattarai SK, Schneider J, Kim Y-G, Reddy S, Caballero S, Felix C, Cornacchione L, et al. Colonization of the live biotherapeutic product VE303 and modulation of the microbiota and metabolites in healthy volunteers. Cell Host Microbe. 2022;30 (4 ):583–598 e8. doi:10.1016/j.chom.2022.03.016.35421353
54. Chen LA, Oliva-Hemker M, Radin A, Weidner M, O’Laughlin BD, Sears CL, Javitt NB, Hourigan SK. Longitudinal bile acid composition changes following faecal microbiota transplantation for clostridioides difficile infection in children with and without underlying inflammatory bowel disease. J Crohns Colitis. 2023;17 (8 ):1364–1368. doi:10.1093/ecco-jcc/jjad057.36988432
55. Mullish BH, McDonald JAK, Pechlivanis A, Allegretti JR, Kao D, Barker GF, Kapila D, Petrof EO, Joyce SA, Gahan CGM, et al. Microbial bile salt hydrolases mediate the efficacy of faecal microbiota transplant in the treatment of recurrent Clostridioides difficile infection. Gut. 2019;68 (10 ):1791–1800. doi:10.1136/gutjnl-2018-317842.30816855
56. Jones BV, Begley M, Hill C, Gahan CGM, Marchesi JR. Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome. Proc Natl Acad Sci USA. 2008;105 (36 ):13580–13585. doi:10.1073/pnas.0804437105.18757757
57. Foley MH, O’Flaherty S, Barrangou R, Theriot CM. Bile salt hydrolases: gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract. PloS Pathog. 2019;15 (3 ):e1007581. doi:10.1371/journal.ppat.1007581.30845232
58. Aguirre AM, Adegbite AO, Sorg JA. Clostridioides difficile bile salt hydrolase activity has substrate specificity and affects biofilm formation. NPJ Biofilms Microbiomes. 2022;8 (1 ):94. doi:10.1038/s41522-022-00358-0.36450806
59. De Smet I, Van Hoorde L, Vande Woestyne M, Christiaens H, Verstraete W. Significance of bile salt hydrolytic activities of lactobacilli. J Appl Bacteriol. 1995;79 (3 ):292–301. doi:10.1111/j.1365-2672.1995.tb03140.x.7592123
60. Begley M, Hill C, Gahan CG. Bile salt hydrolase activity in probiotics. Appl Environ Microbiol. 2006;72 (3 ):1729–1738. doi:10.1128/AEM.72.3.1729-1738.2006.16517616
61. Foley MH, O’Flaherty S, Allen G, Rivera AJ, Stewart AK, Barrangou R, Theriot CM. Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization. Proc Natl Acad Sci U S A. 2021;118 (6 ):e2017709118. doi:10.1073/pnas.2017709118.33526676
62. McMillan AS, Foley MH, Perkins CE, Theriot CM. Loss of bacteroides thetaiotaomicron bile acid-altering enzymes impacts bacterial fitness and the global metabolic transcriptome. Microbiol Spectr. 2024;12 (1 ):e0357623. doi:10.1128/spectrum.03576-23.38018975
63. Rimal B, Collins SL, Tanes CE, Rocha ER, Granda MA, Solanki S, Hoque NJ, Gentry EC, Koo I, Reilly ER, et al. Bile salt hydrolase catalyses formation of amine-conjugated bile acids. Nature. 2024;626 (8000 ):859–863. doi:10.1038/s41586-023-06990-w.38326609
64. Guzior DV, Okros M, Shivel M, Armwald B, Bridges C, Fu Y, Martin C, Schilmiller AL, Miller WM, Ziegler KM, et al. Bile salt hydrolase acyltransferase activity expands bile acid diversity. Nature. 2024;626 (8000 ):852–858. doi:10.1038/s41586-024-07017-8.38326608
65. Lucas LN, Barrett K, Kerby RL, Zhang Q, Cattaneo LE, Stevenson D, Rey FE, Amador-Noguez D. Dominant bacterial phyla from the human gut show widespread ability to transform and conjugate bile acids. mSystems. 2021;6 (4 ):e0080521. doi:10.1128/msystems.00805-21.
66. Malarney KP, Chang PV. Electrostatic interactions dictate bile salt hydrolase substrate preference. Biochemistry. 2023;62 (21 ):3076–3084. doi:10.1021/acs.biochem.3c00210.37883888
67. Shalon D, Culver RN, Grembi JA, Folz J, Treit PV, Shi H, Rosenberger FA, Dethlefsen L, Meng X, Yaffe E, et al. Profiling the human intestinal environment under physiological conditions. Nature. 2023;617 (7961 ):581–591. doi:10.1038/s41586-023-05989-7.37165188
68. Gentry EC, Collins SL, Panitchpakdi M, Belda-Ferre P, Stewart AK, Carrillo Terrazas M, Lu H-H, Zuffa S, Yan T, Avila-Pacheco J, et al. Reverse metabolomics for the discovery of chemical structures from humans. Nature. 2024;626 (7998 ):419–426. doi:10.1038/s41586-023-06906-8.38052229
69. Quinn RA, Melnik AV, Vrbanac A, Fu T, Patras KA, Christy MP, Bodai Z, Belda-Ferre P, Tripathi A, Chung LK, et al. Global chemical effects of the microbiome include new bile-acid conjugations. Nature. 2020;579 (7797 ):123–129. doi:10.1038/s41586-020-2047-9.32103176
70. Doden H, Sallam LA, Devendran S, Ly L, Doden G, Daniel SL, Alves JMP, Ridlon JM. Metabolism of oxo-bile acids and characterization of recombinant 12α-hydroxysteroid dehydrogenases from bile acid 7α-dehydroxylating human gut bacteria. Appl Environ Microbiol. 2018;84 (10 ):84(10. doi:10.1128/AEM.00235-18.
71. Tang S, Pan Y, Lou D, Ji S, Zhu L, Tan J, Qi N, Yang Q, Zhang Z, Yang B, et al. Structural and functional characterization of a novel acidophilic 7α-hydroxysteroid dehydrogenase. Protein Sci. 2019;28 (5 ):910–919. doi:10.1002/pro.3599.30839141
72. Hylemon PB, Sherrod JA. Multiple forms of 7-alpha-hydroxysteroid dehydrogenase in selected strains of Bacteroides fragilis. J Bacteriol. 1975;122 (2 ):418–424. doi:10.1128/jb.122.2.418-424.1975.236279
73. Ridlon JM, Harris SC, Bhowmik S, Kang D-J, Hylemon PB. Consequences of bile salt biotransformations by intestinal bacteria. Gut Microbes. 2016;7 (1 ):22–39. doi:10.1080/19490976.2015.1127483.26939849
74. Bortolini O, Medici A, Poli S. Biotransformations on steroid nucleus of bile acids. Steroids. 1997;62 (8–9 ):564–577. doi:10.1016/S0039-128X(97)00043-3.9292932
75. Zhu P, Zhang J, Chen Y, Yin S, Su M, Xie G, Brouwer KLR, Liu C, Lan K, Jia W, et al. Analysis of human C24 bile acids metabolome in serum and urine based on enzyme digestion of conjugated bile acids and LC-MS determination of unconjugated bile acids. Anal Bioanal Chem. 2018;410 (21 ):5287–5300. doi:10.1007/s00216-018-1183-7.29907951
76. Li D, Sun T, Tong Y, Le J, Yao Q, Tao J, Liu H, Jiao W, Mei Y, Chen J, et al. Gut-microbiome-expressed 3β-hydroxysteroid dehydrogenase degrades estradiol and is linked to depression in premenopausal females. Cell Metab. 2023;35 (4 ):685–694 e5. doi:10.1016/j.cmet.2023.02.017.36933555
77. Li D, Liu R, Wang M, Peng R, Fu S, Fu A, Le J, Yao Q, Yuan T, Chi H, et al. 3β-hydroxysteroid dehydrogenase expressed by gut microbes degrades testosterone and is linked to depression in males. Cell Host & Microbe. 2022;30 (3 ):329–339.e5. doi:10.1016/j.chom.2022.01.001.35108497
78. Watanabe M, Fukiya S, Yokota A. Comprehensive evaluation of the bactericidal activities of free bile acids in the large intestine of humans and rodents. J Lipid Res. 2017;58 (6 ):1143–1152. doi:10.1194/jlr.M075143.28404640
79. Hofmann AF. Chemistry and enterohepatic circulation of bile acids. Hepatology. 1984;4 (5 Suppl ):4S–14S. doi:10.1002/hep.1840040803.6384004
80. Vital M, Rud T, Rath S, Pieper DH, Schlüter D. Diversity of bacteria exhibiting bile acid-inducible 7α-dehydroxylation genes in the human gut. Comput And Struct Biotechnol J. 2019;17 :1016–1019. doi:10.1016/j.csbj.2019.07.012.31428294
81. Takamine F, Imamura T. Isolation and characterization of bile acid 7-dehydroxylating bacteria from human feces. Microbiol And Immunol. 1995;39 (1 ):11–18. doi:10.1111/j.1348-0421.1995.tb02162.x.7783673
82. Funabashi M, Grove TL, Wang M, Varma Y, McFadden ME, Brown LC, Guo C, Higginbottom S, Almo SC, Fischbach MA, et al. A metabolic pathway for bile acid dehydroxylation by the gut microbiome. Nature. 2020;582 (7813 ):566–570. doi:10.1038/s41586-020-2396-4.32555455
83. Cai J, Rimal B, Jiang C, Chiang JYL, Patterson AD. Bile acid metabolism and signaling, the microbiota, and metabolic disease. Pharmacol Ther. 2022;237 :108238. doi:10.1016/j.pharmthera.2022.108238.35792223
84. Lee JW, Cowley ES, Wolf PG, Doden HL, Murai T, Caicedo KYO, Ly LK, Sun F, Takei H, Nittono H, et al. Formation of secondary allo-bile acids by novel enzymes from gut firmicutes. Gut Microbes. 2022;14 (1 ):2132903. doi:10.1080/19490976.2022.2132903.36343662
85. Harris SC, Devendran S, Alves JMP, Mythen SM, Hylemon PB, Ridlon JM. Identification of a gene encoding a flavoprotein involved in bile acid metabolism by the human gut bacterium clostridium scindens ATCC 35704. Biochim Biophys Acta Mol Cell Biol Lipids. 2018;1863 (3 ):276–283. doi:10.1016/j.bbalip.2017.12.001.29217478
86. Ridlon JM, Hylemon PB. Identification and characterization of two bile acid coenzyme a transferases from clostridium scindens, a bile acid 7alpha-dehydroxylating intestinal bacterium. J Lipid Res. 2012;53 (1 ):66–76. doi:10.1194/jlr.M020313.22021638
87. Meibom KL, Marion S, Volet C, Nass T, Vico-Oton E, Menin L, Bernier-Latmani R. BaiJ and BaiB are key enzymes in the chenodeoxycholic acid 7α-dehydroxylation pathway in the gut microbe Clostridium scindens ATCC 35704. Gut Microbes. 2024;16 (1 ):2323233. doi:10.1080/19490976.2024.2323233.38465624
88. Ridlon JM, Devendran S, Alves JM, Doden H, Wolf PG, Pereira GV, Ly L, Volland A, Takei H, Nittono H, et al. The ‘ in vivo lifestyle’ of bile acid 7α-dehydroxylating bacteria: comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice. Gut Microbes. 2020;11 (3 ):381–404. doi:10.1080/19490976.2019.1618173.31177942
89. Buffie CG, Bucci V, Stein RR, McKenney PT, Ling L, Gobourne A, No D, Liu H, Kinnebrew M, Viale A, et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature. 2015;517 (7533 ):205–208. doi:10.1038/nature13828.25337874
90. Reed AD, Nethery MA, Stewart A, Barrangou R, Theriot CM. Strain-dependent inhibition of Clostridioides difficile by commensal clostridia carrying the bile acid-inducible (bai) operon. J Bacteriol. 2020;202 (11 ):. doi:10.1128/JB.00039-20.
91. Solbach P, Chhatwal P, Woltemate S, Tacconelli E, Buhl M, Gerhard M, Thoeringer CK, Vehreschild MJGT, Jazmati N, Rupp J, et al. BaiCD gene cluster abundance is negatively correlated with Clostridium difficile infection. PLoS One. 2018;13 (5 ):e0196977. doi:10.1371/journal.pone.0196977.29738579
92. Robinson JI, Weir WH, Crowley JR, Hink T, Reske KA, Kwon JH, Burnham CAD, Dubberke ER, Mucha PJ, Henderson JP, et al. Metabolomic networks connect host-microbiome processes to human Clostridioides difficile infections. J Clin Invest. 2019;129 (9 ):3792–3806. doi:10.1172/JCI126905.31403473
93. Papazyan R, Fuchs B, Blount K, Gonzalez C, Shannon B. Rapid restoration of bile acid compositions after treatment with RBX2660 for recurrent Clostridioides difficile infection—results from the PUNCH CD3 phase 3 trial. (Electronic)). 1039. p. 2328–8957.
94. Sannasiddappa TH, Lund PA, Clarke SR. In vitro antibacterial activity of unconjugated and conjugated bile salts on staphylococcus aureus. Front Microbiol. 2017;8 :1581. doi:10.3389/fmicb.2017.01581.28878747
95. Wang M, Osborn LJ, Jain S, Meng X, Weakley A, Yan J, Massey WJ, Varadharajan V, Horak A, Banerjee R, et al. Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome. Cell. 2023;186 (13 ):2839–2852 e21. doi:10.1016/j.cell.2023.05.037.37352836
96. Zheng X, Huang F, Zhao A, Lei S, Zhang Y, Xie G, Chen T, Qu C, Rajani C, Dong B, et al. Bile acid is a significant host factor shaping the gut microbiome of diet-induced obese mice. BMC Biol. 2017;15 (1 ):120. doi:10.1186/s12915-017-0462-7.29241453
97. Winston JA, Rivera A, Cai J, Patterson AD, Theriot CM. Secondary bile acid ursodeoxycholic acid alters weight, the gut microbiota, and the bile acid pool in conventional mice. PLoS One. 2021;16 (2 ):e0246161. doi:10.1371/journal.pone.0246161.33600468
98. Winston JA, Rivera AJ, Cai J, Thanissery R, Montgomery SA, Patterson AD, Theriot CM. Ursodeoxycholic acid (UDCA) mitigates the Host inflammatory response during clostridioides difficile infection by altering gut bile acids. Infect Immun. 2020;88 (6 ):. doi:10.1128/IAI.00045-20.
99. Pearson T, Caporaso JG, Yellowhair M, Bokulich NA, Padi M, Roe DJ, Wertheim BC, Linhart M, Martinez JA, Bilagody C, et al. Effects of ursodeoxycholic acid on the gut microbiome and colorectal adenoma development. Cancer Med. 2019;8 (2 ):617–628. doi:10.1002/cam4.1965.30652422
100. Collins SL, Stine JG, Bisanz JE, Okafor CD, Patterson AD. Bile acids and the gut microbiota: metabolic interactions and impacts on disease. Nat Rev Microbiol. 2023;21 (4 ):236–247. doi:10.1038/s41579-022-00805-x.36253479
101. Wang H, Chen J, Hollister K, Sowers LC, Forman BM. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol Cell. 1999;3 (5 ):543–553. doi:10.1016/S1097-2765(00)80348-2.10360171
102. Song X, Sun X, Oh SF, Wu M, Zhang Y, Zheng W, Geva-Zatorsky N, Jupp R, Mathis D, Benoist C, et al. Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis. Nature. 2020;577 (7790 ):410–415. doi:10.1038/s41586-019-1865-0.31875848
103. Pols TW, Noriega LG, Nomura M, Auwerx J, Schoonjans K. The bile acid membrane receptor TGR5: a valuable metabolic target. Dig Dis. 2011;29 (1 ):37–44. doi:10.1159/000324126.21691102
104. Fiorucci S, Distrutti E, Carino A, Zampella A, Biagioli M. Bile acids and their receptors in metabolic disorders. Prog Lipid Res. 2021;82 :101094. doi:10.1016/j.plipres.2021.101094.33636214
105. Ibrahim E, Diakonov I, Arunthavarajah D, Swift T, Goodwin M, McIlvride S, Nikolova V, Williamson C, Gorelik J. Bile acids and their respective conjugates elicit different responses in neonatal cardiomyocytes: role of Gi protein, muscarinic receptors and TGR5. Sci Rep. 2018;8 (1 ):7110. doi:10.1038/s41598-018-25569-4.29740092
106. Lew JL, Zhao A, Yu J, Huang L, de Pedro N, Peláez F, Wright SD, Cui J. The farnesoid X receptor controls gene expression in a ligand- and promoter-selective fashion. J Biol Chem. 2004;279 (10 ):8856–8861. doi:10.1074/jbc.M306422200.14684751
107. Miyazaki T, Ueda H, Ikegami T, Honda A. Upregulation of taurine biosynthesis and bile acid conjugation with taurine through FXR in a mouse model with human-like bile acid composition. Metabolites. 2023;13 (7 ):824. doi:10.3390/metabo13070824.37512531
108. Tveter KM, Villa-Rodriguez JA, Cabales AJ, Zhang L, Bawagan FG, Duran RM, Roopchand DE. Polyphenol-induced improvements in glucose metabolism are associated with bile acid signaling to intestinal farnesoid X receptor. BMJ Open Diabetes Res Care. 2020;8 (1 ):e001386. doi:10.1136/bmjdrc-2020-001386.
109. Hao H, Cao L, Jiang C, Che Y, Zhang S, Takahashi S, Wang G, Gonzalez FJ. Farnesoid X receptor regulation of the NLRP3 Inflammasome underlies cholestasis-associated sepsis. Cell Metab. 2017;25 (4 ):856–867 e5. doi:10.1016/j.cmet.2017.03.007.28380377
110. Vavassori P, Mencarelli A, Renga B, Distrutti E, Fiorucci S. The bile acid receptor FXR is a modulator of intestinal innate immunity. J Immunol. 2009;183 (10 ):6251–6261. doi:10.4049/jimmunol.0803978.19864602
111. Arifuzzaman M, Won TH, Yano H, Uddin J, Emanuel ER, Hu E, Zhang W, Li T-T, Jin W-B, Grier A, et al. Dietary fiber is a critical determinant of pathologic ILC2 responses and intestinal inflammation. J Exp Med. 2024;221 (5 ):221. doi:10.1084/jem.20232148.
112. Lv Y, Luo Y-Y, Ren H-W, Li C-J, Xiang Z-X, Luan Z-L. The role of pregnane X receptor (PXR) in substance metabolism. Front Endocrinol (Lausanne). 2022;13 :959902. doi:10.3389/fendo.2022.959902.36111293
113. Campbell C, McKenney PT, Konstantinovsky D, Isaeva OI, Schizas M, Verter J, Mai C, Jin W-B, Guo C-J, Violante S, et al. Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells. Nature. 2020;581 (7809 ):475–479. doi:10.1038/s41586-020-2193-0.32461639
114. Al Nabhani Z, Dulauroy S, Marques R, Cousu C, Al Bounny S, Déjardin F, Sparwasser T, Bérard M, Cerf-Bensussan N, Eberl G, et al. A weaning reaction to microbiota is required for resistance to immunopathologies in the adult. Immunity. 2019;50 (5 ):1276–1288 e5. doi:10.1016/j.immuni.2019.02.014.30902637
115. Bhattacharjee A, Burr AHP, Overacre-Delgoffe AE, Tometich JT, Yang D, Huckestein BR, Linehan JL, Spencer SP, Hall JA, Harrison OJ, et al. Environmental enteric dysfunction induces regulatory T cells that inhibit local CD4+ T cell responses and impair oral vaccine efficacy. Immunity. 2021;54 (8 ):1745–1757 e7. doi:10.1016/j.immuni.2021.07.005.34348118
116. Britton GJ, Contijoch EJ, Spindler MP, Aggarwala V, Dogan B, Bongers G, San Mateo L, Baltus A, Das A, Gevers D, et al. Defined microbiota transplant restores Th17/RORγt + regulatory T cell balance in mice colonized with inflammatory bowel disease microbiotas. Proc Natl Acad Sci U S A. 2020;117 (35 ):21536–21545. doi:10.1073/pnas.1922189117.32817490
117. Chen W, Ding M, Ji L, Yao J, Guo Y, Yan W, Yu S, Shen Q, Huang M, Zheng Y, et al. Bile acids promote the development of HCC by activating inflammasome. Hepatol Commun. 2023;7 (9 ):7(9. doi:10.1097/HC9.0000000000000217.
118. Sun L, Zhang Y, Cai J, Rimal B, Rocha ER, Coleman JP, Zhang C, Nichols RG, Luo Y, Kim B, et al. Bile salt hydrolase in non-enterotoxigenic bacteroides potentiates colorectal cancer. Nat Commun. 2023;14 (1 ):755. doi:10.1038/s41467-023-36089-9.36765047
119. Vich Vila A, Hu S, Andreu-Sánchez S, Collij V, Jansen BH, Augustijn HE, Bolte LA, Ruigrok RAAA, Abu-Ali G, Giallourakis C, et al. Faecal metabolome and its determinants in inflammatory bowel disease. Gut. 2023;72 (8 ):1472–1485. doi:10.1136/gutjnl-2022-328048.36958817
120. Fletcher JR, Pike CM, Parsons RJ, Rivera AJ, Foley MH, McLaren MR, Montgomery SA, Theriot CM. Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota. Nat Commun. 2021;12 (1 ):462. doi:10.1038/s41467-020-20746-4.33469019
121. Karasawa T, Ikoma S, Yamakawa K, Nakamura S. A defined growth medium for Clostridium difficile. Microbiol (Read). 1995;141 (Pt 2 ):371–375. doi:10.1099/13500872-141-2-371.
122. Jenior ML Leslie JL, Kolling GL, Archbald-Pannone L, Powers DA, Petri Jr WA, Papin JA. Systems-ecology designed bacterial consortium protects from severe Clostridioides difficile infection. bioRxiv. 2023. doi:10.1101/2023.08.08.552483.
123. Girinathan BP, DiBenedetto N, Worley JN, Peltier J, Arrieta-Ortiz ML, Immanuel SRC, Lavin R, Delaney ML, Cummins CK, Hoffman M, et al. In vivo commensal control of Clostridioides difficile virulence. Cell Host & Microbe. 2021;29 (11 ):1693–1708.e7. doi:10.1016/j.chom.2021.09.007.34637781
124. Lemons JMS, Conrad M, Tanes C, Chen J, Friedman ES, Roggiani M, Curry D, Chau L, Hecht AL, Harling L, et al. Enterobacteriaceae growth promotion by intestinal acylcarnitines, a biomarker of dysbiosis in inflammatory bowel disease. Cell Mol Gastroenterol Hepatol. 2024;17 (1 ):131–148. doi:10.1016/j.jcmgh.2023.09.005.37739064
125. Reed AD, Fletcher JR, Huang YY, Thanissery R, Rivera AJ, Parsons RJ, Stewart AK, Kountz DJ, Shen A, Balskus EP, et al. The stickland reaction precursor trans-4-hydroxy-l-proline differentially impacts the metabolism of Clostridioides difficile and commensal clostridia. mSphere. 2022;7 (2 ):e00926–21. doi:10.1128/msphere.00926-21.35350846
126. Spragge F, Bakkeren E, Jahn MT, Araujo EBN, Pearson CF, Wang X, Pankhurst L, Cunrath O, Foster KR. Microbiome diversity protects against pathogens by nutrient blocking. Science. 2023;382 (6676 ):eadj3502. doi:10.1126/science.adj3502.38096285
127. Jenior ML, Leslie JL, Young VB, Schloss PD. Clostridium difficile Colonizes Alternative Nutrient Niches during Infection across Distinct Murine Gut Microbiomes. mSystems. 2017;2 (4 ):. doi:10.1128/mSystems.00063-17.
128. Fletcher JR, Erwin S, Lanzas C, Theriot CM. Shifts in the gut metabolome and Clostridium difficile transcriptome throughout colonization and infection in a mouse model. mSphere. 2018;3 (2 ):3(2. doi:10.1128/mSphere.00089-18.
129. de Vladar HP. Amino acid fermentation at the origin of the genetic code. Biol Direct. 2012;7 (1 ):6. doi:10.1186/1745-6150-7-6.22325238
130. Graham M, DiBenedetto N, Delaney ML, Lavin R, Pavao A, Yeliseyev V, Bry L. Clostridium scindens colonization of gnotobiotic mice promotes a chronic unresolving infection with Clostridioides difficile. bioRxiv. 2022. doi:10.1101/2022.06.12.495821.
131. Studer N, Desharnais L, Beutler M, Brugiroux S, Terrazos MA, Menin L, Schürch CM, McCoy KD, Kuehne SA, Minton NP, et al. Functional intestinal bile acid 7α-dehydroxylation by Clostridium scindens associated with protection from Clostridium difficile infection in a gnotobiotic mouse model. Front Cell Infect Microbiol. 2016;6 :191. doi:10.3389/fcimb.2016.00191.28066726
132. Gerding DN, Meyer T, Lee C, Cohen SH, Murthy UK, Poirier A, Van Schooneveld TC, Pardi DS, Ramos A, Barron MA, et al. Administration of spores of nontoxigenic clostridium difficile strain M3 for prevention of recurrent C difficile infection: a randomized clinical trial. JAMA. 2015;313 (17 ):1719–1727. doi:10.1001/jama.2015.3725.25942722
133. Kang JD, Myers CJ, Harris SC, Kakiyama G, Lee I-K, Yun B-S, Matsuzaki K, Furukawa M, Min H-K, Bajaj JS, et al. Bile acid 7α-dehydroxylating gut bacteria secrete antibiotics that inhibit clostridium difficile: role of secondary bile acids. Cell Chem Biol. 2019;26 (1 ):27–34 e4. doi:10.1016/j.chembiol.2018.10.003.30482679
134. Ghosh S, Erickson D, Chua MJ, Collins J, Jala VR. The microbial metabolite urolithin a reduces Clostridioides difficile toxin expression and toxin-induced epithelial damage. mSystems, 2024;9 (2 ):e0125523. doi:10.1128/msystems.01255-23.
135. Porcari S, Benech N, Valles-Colomer M, Segata N, Gasbarrini A, Cammarota G, Sokol H, Ianiro G. Key determinants of success in fecal microbiota transplantation: from microbiome to clinic. Cell Host Microbe. 2023;31 (5 ):712–733. doi:10.1016/j.chom.2023.03.020.37167953
136. Ianiro G, Punčochář M, Karcher N, Porcari S, Armanini F, Asnicar F, Beghini F, Blanco-Míguez A, Cumbo F, Manghi P, et al. Variability of strain engraftment and predictability of microbiome composition after fecal microbiota transplantation across different diseases. Nat Med. 2022;28 (9 ):1913–1923. doi:10.1038/s41591-022-01964-3.36109637
137. Dubberke ER, Lee CH, Orenstein R, Khanna S, Hecht G, Gerding DN. Results from a randomized, placebo-controlled clinical trial of a RBX2660—A microbiota-based drug for the prevention of recurrent Clostridium difficile infection. Clin Infect Dis. 2018;67 (8 ):1198–1204. doi:10.1093/cid/ciy259.29617739
138. Feuerstadt P, Harvey A, Yoho DS, Garcia-Diaz JB, Knapple WL, Bancke L. Retrospective analysis of the safety and efficacy of fecal microbiota, live-jslm (REBYOTATM) administered under enforcement discretion to patients with Clostridioides difficile infection. Open Forum Infect Dis. 2023;10 (5 ): p. ofad 171. doi:10.1093/ofid/ofad171.
139. Chinna Meyyappan A, Forth E, Milev R. Microbial ecosystem therapeutic-2 intervention in people with major depressive disorder and generalized anxiety disorder: phase 1, open-label study. Interact J Med Res. 2022;11 (1 ):e32234. doi:10.2196/32234.35060914
140. Kim KH, Park D, Jia B, Baek JH, Hahn Y, Jeon CO. Identification and characterization of major bile acid 7α-dehydroxylating bacteria in the human gut. mSystems. 2022;7 (4 ):e0045522. doi:10.1128/msystems.00455-22.35736002
141. Franzosa EA, Sirota-Madi A, Avila-Pacheco J, Fornelos N, Haiser HJ, Reinker S, Vatanen T, Hall AB, Mallick H, McIver LJ, et al. Gut microbiome structure and metabolic activity in inflammatory bowel disease. Nat Microbiol. 2019;4 (2 ):293–305. doi:10.1038/s41564-018-0306-4.30531976
142. Caussy C, Hsu C, Singh S, Bassirian S, Kolar J, Faulkner C, Sinha N, Bettencourt R, Gara N, Valasek MA, et al. Serum bile acid patterns are associated with the presence of NAFLD in twins, and dose-dependent changes with increase in fibrosis stage in patients with biopsy-proven NAFLD. Aliment Pharmacol Ther. 2019;49 (2 ):183–193. doi:10.1111/apt.15035.30506692
143. Paramsothy S, Nielsen S, Kamm MA, Deshpande NP, Faith JJ, Clemente JC, Paramsothy R, Walsh AJ, van den Bogaerde J, Samuel D, et al. Specific bacteria and metabolites associated with response to fecal microbiota transplantation in patients with ulcerative colitis. Gastroenterology. 2019;156 (5 ):1440–1454 e2. doi:10.1053/j.gastro.2018.12.001.30529583
144. Yang Z, Bu C, Yuan W, Shen Z, Quan Y, Wu S, Zhu C, Wang X. Fecal microbiota transplant via endoscopic delivering through small intestine and colon: No difference for crohn’s disease. Dig Dis Sci. 2020;65 (1 ):150–157. doi:10.1007/s10620-019-05751-y.31367877
145. Allegretti JR, Kelly CR, Grinspan A, Mullish BH, Hurtado J, Carrellas M, Marcus J, Marchesi JR, McDonald JA, Gerardin Y, Silverstein M. Inflammatory bowel disease outcomes following fecal microbiota transplantation for recurrent C. difficile infection. Inflamm Bowel Dis. 2021;27 (9 ):1371–1378. doi:10.1093/ibd/izaa283.33155639
146. Derosa G, Guasti L, D’Angelo A, Martinotti C, Valentino MC, Di Matteo S, Bruno GM, Maresca AM, Gaudio GV, Maffioli P, et al. Probiotic therapy with VSL#3® in patients with NAFLD: a randomized clinical trial. Front Nutr. 2022;9 :846873. doi:10.3389/fnut.2022.846873.35685888
147. Qi X, Yun C, Sun L, Xia J, Wu Q, Wang Y, Wang L, Zhang Y, Liang X, Wang L, et al. Gut microbiota–bile acid–interleukin-22 axis orchestrates polycystic ovary syndrome. Nat Med. 2019;25 (8 ):1225–1233. doi:10.1038/s41591-019-0509-0.31332392
148. Li N, Chen H, Cheng Y, Xu F, Ruan G, Ying S, Tang W, Chen L, Chen M, Lv L, et al. Fecal microbiota transplantation relieves gastrointestinal and autism symptoms by improving the gut microbiota in an open-label study. Vol. 11 . Front Cell Infect Microbiol; 2021. p. 759435. doi:10.3389/fcimb.2021.759435.
149. Vendrik KEW, Ooijevaar RE, de Jong PRC, Laman JD, Van Oosten BW, Van Hilten JJ, Ducarmon QR, Keller JJ, Kuijper EJ, Contarino MF. Fecal microbiota transplantation in neurological disorders. Front Cell Infect Microbiol. Vol. 10 ; 2020. p. 98. doi:10.3389/fcimb.2020.00098.32266160
150. Mohanty I, Mannochio-Russo H, Schweer JV, El Abiead Y, Bittremieux W, Xing S, Schmid R, Zuffa S, Vasquez F, Muti VB, Zemlin J. The underappreciated diversity of bile acid modifications. Cell. 2024;187 (7 ):1801–1818.e20. doi:10.1016/j.cell.2024.02.019.38471500
