Role of the microbiome in essential metabolism in the human gut and its implications for Clostridioides difficile infection

Functions of the gut microbiota and colonization resistance e term gut microbiome describes the diverse group of microorganisms (microbiota) including bacteria, archaea, fungi, algae, and small protists that share the human intestinal space, as well as their genomes and environment [1]. e gut bacterial microbiota in most healthy individuals primarily comprise a diversity of Bacteroidetes (Bacteroides and Prevotella spp.) and Firmicutes (Lactobacillus, Bacillus, Clostridium, and Ruminococcus spp.) phyla, with a lower abundance of Proteobacteria and Actinobacteria [2].


Functions of the gut microbiota and colonization resistance
e term gut microbiome describes the diverse group of microorganisms (microbiota) including bacteria, archaea, fungi, algae, and small protists that share the human intestinal space, as well as their genomes and environment [1].
e gut bacterial microbiota in most healthy individuals primarily comprise a diversity of Bacteroidetes (Bacteroides and Prevotella spp.) and Firmicutes (Lactobacillus, Bacillus, Clostridium, and Ruminococcus spp.) phyla, with a lower abundance of Proteobacteria and Actinobacteria [2].
A rich and diverse gut microbiome provides resistance to colonization of the gut by exogenous organisms and prevents expansion of potential pathogenic organisms within the gut through a variety of mechanisms [2, 3]: Bile acids were initially thought to be only involved in lipid digestion in the intestinal lumen and cholesterol solubilization in the bile but have now seem to have a range of other e ects: contributing to the homeostasis of lipids, glucose and other metabolic substrate, in addition to a ecting immune system functions as well as gut microbiome composition [15].Modi cation of bile acids is a Dysbiosis is a perturbation, usually due to antibiotic treatment, in the microbiota composition, abundance, and/or diversity, which can have signi cant impacts on the functionality of the gut microbiome.One consequence of dysbiosis is the loss of colonization resistance to pathogens [2], such as Clostridioides di cile, a spore-forming,-Gram-positive, obligate anaerobic bacterium.Patients with C. di cile infection (CDI) typically have reduced richness and diversity in the gut microbiome [4][5][6]] and perturbed microbiota composition, in particular decreased members from the Clostridia and Bacteroidia classes and increased members from the Gammaproteobacteria and Bacilli classes, most commonly including Enterobacterales and Enterococcacea families [7][8][9][10][11].ese perturbations are frequently worsened by treatment with standard-of-care prescription CDI antibiotics.Consequences of this dysbiosis include metabolomic changes that are more favorable for mucosa-adhered C. di cile spores to germinate to their active vegetative form, which can produce toxins in the colon that disrupt the intestinal epithelial barrier and result in a host in ammatory response [12][13][14].Among the metabolites implicated in this cascade of CDI infection are bile acids and short-chain fatty acids (SCFA).

Impact of REBYOTA TM (RBL)
e rst FDA-approved microbiota-based therapeutic for preventing CDI recurrence, REBYOTA TM (fecal microbiota, live-jslm; 'RBL') (Rebiotix Inc., Roseville, MN) is a commercially prepared, FDA-approved microbiota-based live biotherapeutic.In a pivotal Phase 3 trial (PUNCH CD3, NCT03931941), RBL was evaluated for the prevention of CDI recurrence as a randomized, double-blinded, placebo controlled (2:1) study, in which treatment response was de ned as absence of CDI recurrence at 8 weeks a er blinded investigational treatment.In that study, RBL met the prespeci ed epithelial integrity and barrier function while reducing in ammation and oxidative stress [32][33][34].
e type and quantity of intestinal bacteria are the key factors determining the type and concentration of SCFAs.erefore, one manifestation of intestinal dysbiosis in CDI patients is the decreased abundance of SCFA-producing bacteria, as the Firmicutes phyla produces butyrate (Eubacterium rectale, Roseburia inulinivorans, Faecalibacterium prausnitzii) whereas the Bacteroidetes phyla produce acetate (Blautia obeum) and propionate (Bacteroides uniformis, Bacteroides vulgatus) [30].Depletion of these taxa are strongly associated with respective SCFA derangement.[35,36].
T. Chopra et al.
addition to a ecting immune system functions as well as gut microbiome composition [15].Bile acids are excreted to the upper digestive tract as glycine or taurine conjugates (bile salts), most of which are reabsorbed in the distal ileum.Most of the primary bile salts that reach the colon are metabolized to secondary bile acids by commensal gut microbiota, which helps resist C. di cile colonization because primary bile acids, especially taurocholic acid, can stimulate spore germination via CspC pseudoprotease binding.It has been reported that some secondary bile acids inhibit spore germination.Further, several secondary bile acids may have a role in inhibiting C. di cile growth and/or toxin activity [16][17][18][19][20][21][22].e primary-to-secondary bile acid conversion has been demonstrated to be important in CDI, as patients with CDI have lower secondary bile acids compared with healthy controls and higher primary bile acids in patients with recurrence versus those experiencing a rst episode [20,23].Additionally, recovery from CDI seems to be correlated with recovery of secondary bile acids with multiple secondary bile acids in the large intestine shown to inhibit C. di cile spore germination and growth at physiological concentrations [24,25].

RBL responders have higher secondary bile acids and lower primary bile acids
at trial included exploratory analyses of the microbiome composition of administered RBL doses and patient fecal samples from before and a er assigned study treatment, as well as liquid chromatography tandem mass spectrometry (LC-MS) analysis of 36 bile acid in patient fecal samples [43].
ose analyses indicated repopulation and restoration of the gut microbiome with  among patients that responded to RBL.Concurrently, the ratio of secondary to primary (S:P) bile acids was calculated, and it was found that patients responding to RBL had a higher S:P ratio, when compared with pretreatment or placebo, with changes occurring as early as one week post treatment [48]. is shi in bile acid compositions may help suppress spore germination and C. di cile toxin production, underpinning the clinical e cacy to prevent CDI recurrence.
e bile acid shi s are also consistent with the microbiome changes, in that many of the taxa increased a er RBL are known to carry bile acid metabolism genes [48].SCFA changes with RBL were not

Conclusion
• e importance of a broad consortium of Bacteroidetes and Firmicutes is manifest in their critical role in colonization resistance and immune modulation [49] • e essential ability of these taxa to metabolize bile acids and SCFAs confers colonization resistance to various pathogens including C. di cile • Additionally, SCFAs may regulate several leukocyte functions including production of cytokines (TNF-α, IL-2, IL-6 and IL-10), eicosanoids and chemokines • Restoration of the normal gut microbiome is viewed as very bene cial • RBL has been shown to restore the essential metabolic processes for a healthy microbiome and avoid recurrent CDI

•
Competition for key nutrients • Production of inhibitory bile acids • Production of short-chain fatty acids • Lowering the luminal pH • Production of bacteriocins • Maintenance and enhancement of intestinal barrier function • Host immune modulation © The Author(s) 2023.Published by Oxford University Press for the Infectious Diseases Society of America.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/),which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.which gut microbiota provide colonization resistance to C. di cile.Liver-produced primary bile acids were initially thought to be only involved in lipid digestion in the intesti-nal lumen and cholesterol solubilization in the bile, but have now been shown to have a range of other e ects: contributing to the homeostasis of lipids, glucose and other metabolic substrate, in • The S:P ratio in responders is significantly different than in non-responders • The S:P ratio with RBL is significantly different than with placebo treatment• Statistical significance among the various groups was driven by changes in the RBL-responder groupFootnote: S = secondary, P = primary, BL = baseline, 1W = 1 week, 4W = 4 week, 8W = permeability, and inhibits intestinal in ammation and bacterial translocation[26,[28][29][30].Acetate, enhances host immune function, stimulates immune cells to secrete anti-in ammatory cytokines, and mitigates the toxicity of C. di cile toxin A to colonocytes to ameliorate CDI[30, 31].Finally, propionate promotes intestinal Antibiotic therapy is the standard course of treatment for rCDI[37], however, this represents a therapeutic dilemma as many antibiotics disrupt the gut microbiome, including rst-line options for rCDI[38].Although guideline recommended antibiotics can provide some sustained clinical response, they are unable to restore the dysbiosis.emicrobiomedisruptionscausedby antibiotics can then lead to the decreased production of secondary bile acids and SCFAs[20,[39][40][41], which favors the germination and growth of C. di cile[2, 12].erefore, treatment methods that can e ciently restore the diversity of the gut microbiome a er antibiotics and thereby in uence colonization resistance and immune modulation are likely to prevent CDI recurrence.
SCFAs are produced through bacterial fermentation of indigestible carbohydrates or dietary ber.SCFAs are an important energy source for intestinal epithelial cells and play a vital role in maintaining epithelial function and colonization resistance.reemicrobial-derivedSCFAs in particular (butyrate, acetate, and propionate) have been associated with CDI resistance[17, 26, 27].Butyrate, the main energy source for the colonic cells to proliferate and maintain the gut barrier, is metabolized through Β-oxidation, and helps maintain an anaerobic milieu inside the colon[16].It enhances the host innate antimicrobial immunity and cytokine-mediated immune response, maintains intestinal epithelial integrity, increases the expression of tight junction proteins, prevents epithelial damage by C. di cile toxins, reduces intestinal

Role of the microbiome in essential metabolism in the human gut and its implications for Clostridioides difficile infection
Dr. Teena Chopra Detroit Medical Center and Vibra Hospital, Detroit MI -tchopra@med.wayne.edu