Compositions and methods for microbiome dysbiosis

Abstract

Aspects herein, inter alia, concern the ability of certain bacterial consortia to reconstitute a microbiome and re-introduce beneficial metabolites into the microbiome. Certain aspects relate to compositions comprising bacteria capable of the same.

Description

COMPOSITIONS AND METHODS FOR MICROBIOME DYSBIOSIS

[0001] This application claims priority of U.S. Provisional Application Nos. 63 / 472,758 filed June 13, 2023 and 63 / 543,437 filed October 10, 2023, both of which are hereby incorporated by reference in their entirety. BACKGROUND I. Field of the Disclosure

[0002] This disclosure relates to the field of microbiology, pathology, and medicine. II. Background

[0003] The intestinal microbiota is composed of hundreds of microbial species that impact the health of the mammalian host by optimizing immune functions and maximizing mucosal barrier defenses. A diverse microbiota reduces the risk of infection by depleting essential nutrients, producing or modifying metabolites and secreting bacteriocins that inhibit or kill potential pathogens. Loss of microbiota diversity has been associated with increased incidence of infection,1severe graft versus host disease,2and increased mortality following hematopoietic cell transplantation.3Bacterial species belonging to the Bacteroidota and Bacillota (formerly Bacteroidetes and Firmicutes) phyla represent the major populations inhabiting the lower intestinal tract of healthy, antibiotic naive individuals. Species belonging to the Pseudomonadota (formerly Proteobacteria) and Actinomycetota phyla also play key roles in the intestinal microbiota ecosystem.4Within the Bacillota phylum, bacterial species belonging to the Lachnospiraceae, Oscillobacteriaceae and Ruminococcaceae families are the most consequential for immune modulation and colonization resistance2.

[0004] The global burden of chronic liver disease (CLD) continues to rise due to the shifting epidemiology of disease causes.1–4CLD is often clinically silent until an initial decompensation event,5,6after which care is largely supportive without the ability to significantly modify the underlying disease with current therapies. Due to the high rates of infection (including culture negative infections) in patients with liver disease, empiric, broad spectrum antibiotics are commonly prescribed.7–9This practice is associated with high levels of antibiotic resistant organisms, a finding that correlates with poor clinical outcomes.9–11

[0005] The gut microbiome contributes to human health and disease, including liver disease. There is bidirectional anatomic communication between the liver and the gut via the portal vein and biliary tree. Preclinical studies have implicated the gut microbiome as a potential driver of non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease.12–14While robust clinical evidence linking the microbiome to progression of liver disease is201720021.2 - 1 -lacking, multiple observational studies have reported various gut microbiome “signatures” of advanced fibrosis and cirrhosis.15–19Observational studies have also associated the gut microbiome composition with complications of end stage liver disease, including death.11,13,16,20

[0006] To further understand the contribution of the gut microbiome to complications of liver disease, an observational study in the Duchossois Family Institute (DFI) performed shotgun metagenomic sequencing and targeted metabolomic analyses on 847 fecal samples from 262 unique patients with various liver diseases and 22 healthy donors (Adapted in FIG. 3A)11. This study confirmed other groups’ published findings that patients with liver disease have a wide range of gut microbiome compositions, including lower alpha-diversity and increased burden of potentially pathogenic taxa (e.g. Enterobacteriaceae and Enterococcus) compared to healthy controls. With detailed analysis of the timing of stool samples relative to medication administration, it was demonstrated that much of the decreased alpha-diversity and expansion of potentially pathogenic and antibiotic resistant taxa occurs after the use of broad- spectrum antibiotics.

[0007] Many of these samples also contained very low (and frequently undetectable) concentrations of beneficial metabolites, including short chain fatty acids (SCFA, e.g. butyrate) and secondary bile acids (e.g. deoxycholic acid) (FIGs. 3A and 3C). Importantly, loss of microbiome alpha-diversity and low levels of fecal metabolites both associate with poor outcomes, including death11. Therefore there is a need for microbiome intervention, specifically reconstitution of the gut microbiome with commensal organisms that produce beneficial metabolites including SCFA and secondary bile acids, to reduce the incidence of systemic infections and prolong survival in patients with advanced liver disease. SUMMARY OF THE INVENTION

[0008] Aspects of the present disclosure address needs in the art by providing methods and compositions for reconstituting microbiota and / or re-establishing microbiota-derived metabolites in order to treat or prevent disease, including diseases associated with microbiome dysbiosis.

[0009] Disclosed herein are compositions comprising a collection of bacteria. The collection of bacteria may be bacteria from one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or a combination thereof. In certain aspects, one or more bacteria of DFI Consortia A, one or more bacteria of201720021.2 - 2 -DFI Consortia B1, one or more bacteria of DFI Consortia B2, one or more bacteria of DFI Consortia C1, one or more bacteria of DFI Consortia C2, one or more bacteria of DFI Consortia D1, one or more bacteria of DFI Consortia D2, one or more bacteria of DFI Consortia E1, one or more bacteria of DFI Consortia E2, and / or one or more bacteria of DFI Commensal Consortia are excluded from the collection.

[0010] In certain aspects, the collection of bacteria comprises at least two bacteria that produce butyrate. In certain aspects, the collection of bacteria comprises at least one bacteria that can deconjugate conjugated primary bile acids. In certain aspects, the collection of bacteria comprises at least one bacteria that can convert primary to secondary bile acids. In certain aspects, the collection of bacteria comprises at least one bacteria that can modify secondary bile acids. The bacteria that produce butyrate may include Anaerostipes hadrus DFI.4.30, Anaerostipes caccae DFI.7.76, Clostridium symbiosum DFI.5.64, Coprococcus comes DFI.3.84, Anaerobutyricum hallii DFI.9.38, and / or Eubacterium rectale DFI.5.28. The bacteria that can deconjugate conjugated primary bile acids may include Bifidobacterium longum DFI.7.60, Bifidobacterium pseudocatenulatum DFI.7.61, and / or Bifidobacterium bifidum DFI.7.51. The bacteria that can convert primary to secondary bile acids may be Clostridium scindens SL.1.22. The bacteria that modify secondary bile acids may include Bacteroides thetaiotaomicron DFI.6.40, Bacteroides ovatus MSK.18.37, Bacteroides cellulosilyticus DFI.5.60, Phocaeicola vulgatus DFI.4.81, Parabacteroides distasonis DFI.5.23, Parabacteroides merdae DFI.4.73, and / or Eggerthella lenta DFI.5.72.

[0011] Disclosed are compositions comprising 2 bacteria of DFI Consortia A. Disclosed are compositions comprising 3 bacteria of DFI Consortia A. Disclosed are compositions comprising 4 bacteria of DFI Consortia A. Disclosed are compositions comprising 5 bacteria of DFI Consortia A. Disclosed are compositions comprising 6 bacteria of DFI Consortia A. Disclosed are compositions comprising 7 bacteria of DFI Consortia A. Disclosed are compositions comprising 8 bacteria of DFI Consortia A.

[0012] Disclosed are compositions comprising 2 bacteria of DFI Consortia B1. Disclosed are compositions comprising 3 bacteria of DFI Consortia B1. Disclosed are compositions comprising 4 bacteria of DFI Consortia B1. Disclosed are compositions comprising 5 bacteria of DFI Consortia B1. Disclosed are compositions comprising 6 bacteria of DFI Consortia B1. Disclosed are compositions comprising 7 bacteria of DFI Consortia B1. Disclosed are compositions comprising 8 bacteria of DFI Consortia B1.201720021.2 - 3 -

[0013] Disclosed are compositions comprising 2 bacteria of DFI Consortia B2. Disclosed are compositions comprising 3 bacteria of DFI Consortia B2. Disclosed are compositions comprising 4 bacteria of DFI Consortia B2. Disclosed are compositions comprising 5 bacteria of DFI Consortia B2. Disclosed are compositions comprising 6 bacteria of DFI Consortia B2. Disclosed are compositions comprising 7 bacteria of DFI Consortia B2. Disclosed are compositions comprising 8 bacteria of DFI Consortia B2.

[0014] Disclosed are compositions comprising 2 bacteria of DFI Consortia C1. Disclosed are compositions comprising 3 bacteria of DFI Consortia C1. Disclosed are compositions comprising 4 bacteria of DFI Consortia C1. Disclosed are compositions comprising 5 bacteria of DFI Consortia C1. Disclosed are compositions comprising 6 bacteria of DFI Consortia C1. Disclosed are compositions comprising 7 bacteria of DFI Consortia C1. Disclosed are compositions comprising 8 bacteria of DFI Consortia C1.

[0015] Disclosed are compositions comprising 2 bacteria of DFI Consortia C2. Disclosed are compositions comprising 3 bacteria of DFI Consortia C2. Disclosed are compositions comprising 4 bacteria of DFI Consortia C2. Disclosed are compositions comprising 5 bacteria of DFI Consortia C2. Disclosed are compositions comprising 6 bacteria of DFI Consortia C2. Disclosed are compositions comprising 7 bacteria of DFI Consortia C2. Disclosed are compositions comprising 8 bacteria of DFI Consortia C2.

[0016] Disclosed are compositions comprising 2 bacteria of DFI Consortia D1. Disclosed are compositions comprising 3 bacteria of DFI Consortia D1. Disclosed are compositions comprising 4 bacteria of DFI Consortia D1. Disclosed are compositions comprising 5 bacteria of DFI Consortia D1. Disclosed are compositions comprising 6 bacteria of DFI Consortia D1. Disclosed are compositions comprising 7 bacteria of DFI Consortia D1. Disclosed are compositions comprising 8 bacteria of DFI Consortia D1.

[0017] Disclosed are compositions comprising 2 bacteria of DFI Consortia D2. Disclosed are compositions comprising 3 bacteria of DFI Consortia D2. Disclosed are compositions comprising 4 bacteria of DFI Consortia D2. Disclosed are compositions comprising 5 bacteria of DFI Consortia D2. Disclosed are compositions comprising 6 bacteria of DFI Consortia D2. Disclosed are compositions comprising 7 bacteria of DFI Consortia D2. Disclosed are compositions comprising 8 bacteria of DFI Consortia D2.

[0018] Disclosed are compositions comprising 2 bacteria of DFI Consortia E1. Disclosed are compositions comprising 3 bacteria of DFI Consortia E1. Disclosed are compositions comprising 4 bacteria of DFI Consortia E1. Disclosed are compositions comprising 5 bacteria of DFI Consortia E1. Disclosed are compositions comprising 6 bacteria of DFI Consortia E1.201720021.2 - 4 -Disclosed are compositions comprising 7 bacteria of DFI Consortia E1. Disclosed are compositions comprising 8 bacteria of DFI Consortia E1.

[0019] Disclosed are compositions comprising 2 bacteria of DFI Consortia E2. Disclosed are compositions comprising 3 bacteria of DFI Consortia E2. Disclosed are compositions comprising 4 bacteria of DFI Consortia E2. Disclosed are compositions comprising 5 bacteria of DFI Consortia E2. Disclosed are compositions comprising 6 bacteria of DFI Consortia E2. Disclosed are compositions comprising 7 bacteria of DFI Consortia E2. Disclosed are compositions comprising 8 bacteria of DFI Consortia E2.

[0020] Disclosed are compositions comprising 2 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 3 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 4 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 5 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 6 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 7 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 8 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 9 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 10 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 11 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 12 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 13 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 14 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 15 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 16 bacteria of DFI Commensal Consortia. Disclosed are compositions comprising 17 bacteria of DFI Commensal Consortia.

[0021] Certain aspects also concern compositions, including compositions comprising bacteria disclosed herein, that comprise at least one metabolite (including any metabolite described herein), at least one antibiotic, and / or at least one immunosuppressant. In certain aspects, the metabolite comprises a fatty acid, an amino acid, a primary bile acid, a secondary bile acid, a sugar, or a combination thereof. In some aspects, the composition comprises 103, 104, 105, 106, 107,108, 109, 1010, 1011, 1012colony forming units (or any range derivable therein) of at least one bacteria.

[0022] In certain aspects, the composition also comprises 10% sucrose. In certain aspects, the composition also comprises 5% trehalose. In certain aspects, the composition also comprises 10% sucrose and 5% trehalose.201720021.2 - 5 -

[0023] In certain aspects, the composition also comprises at least, at most, or approximately 8%, 9%, 10%, 11%, or 12% sucrose, or any range derivable therein. In certain aspects, the composition also comprises at least, at most, or approximately 3%, 4%, 5%, 6%, or 7% trehalose, or any range derivable therein. In certain aspects, the composition also comprises 8%, 9%, 10%, 11%, or 12% sucrose, or any range derivable therein, and 3%, 4%, 5%, 6%, or 7% trehalose, or any range derivable therein.

[0024] Also disclosed are methods of reestablishing commensal organisms in a patient’s microbiome, methods of altering a patient’s microbiome, methods of increasing a metabolite in a microbiome of a patient, methods of reversing microbiome dysbiosis, and methods of treating a patient (including methods of treating liver disease in a patient). The methods disclosed herein can include 1, 2, 3, 4, 5 or more of any of the following steps administering an effective amount of a collection of bacteria (including any collection of bacteria disclosed herein) to a patient, administering an effective amount of a composition comprising a collection of bacteria (including any collection of bacteria disclosed herein) to a patient, introducing commensal bacteria (including any collection of bacteria disclosed herein) to a microbiome, measuring the levels of one or more bacteria (including any collection of bacteria disclosed herein) in a sample from a microbiome of a patient (such as a stool sample), and measuring the levels of one or more metabolites (including any metabolites disclosed herein) in a sample from a microbiome of a patient (such as a stool sample).

[0025] Disclosed are methods of treating a disease in a patient, the method comprising administering to the patient an effective amount of one or more of DFI Commensal Consortia bacteria. In some aspects, the disease comprises a liver disease and / or an infection. In some aspects, the patient has or will receive a transplant. In some aspects, the transplant comprises a liver transplant or cell transplant.

[0026] In certain aspects, the patient has or will receive a liver transplant. In certain aspects, the patient has, has been diagnosed with, or is suspected of having, chronic liver disease. In some aspects, the patient has received a cell transplant. In some aspects, the stem cell transplant comprises an allogeneic hematopoietic cell transplantation.

[0027] In some aspects, a stool sample from the patient has been measured to have low fecal taxonomic alpha diversity. In some aspects, a stool sample from the patient has or has been determined to have less than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 µM or mM, or any range derivable therein, of201720021.2 - 6 -butyrate. In some aspects, a stool sample from the patient has or has been determined to have more than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any range derivable therein, µM of taurocholic acid. In some aspects, a stool sample from the patient has or has been determined to have less than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or any range derivable therein, µM of deoxycholic acid. In some aspects, a stool sample from the patient has or has been determined to have less than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or any range derivable therein, µM of alloisolithocholic acid.

[0028] In some aspects, a stool sample from the patient has or has been determined to have less than 700 µM butyrate and less than 10 µM deoxycholate. In some aspects, a stool sample from the patient has or has been determined to have reduced butyrate and / or deoxycholic acid levels compared to a standard. In some aspects, a stool sample from the patient is or has been determined to be substantially free of butyrate and / or deoxycholic acid.

[0029] In certain aspects, the composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 times per day. In some aspects, the composition is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some aspects, the days are consecutive. In some aspects, the days are every other day.

[0030] Also disclosed are methods of detecting microbiome dysbiosis in a patient. In some aspects, the method comprises measuring a level of one or more DFI Commensal Consortia bacteria in a sample from the patient. In some aspects, one or more bacteria of the DFI Commensal Consortia are not measured. In some aspects, the patient has, has been diagnosed with, or is suspected of having, liver disease or COVID-19. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.201720021.2 - 7 -

[0032] FIG.1 shows DFI Symbiotic Bacterial Strain Bank (also referred to as General Cell Bank) contains approximately 1,700 strains that include the species belonging to diverse phylogenetic families. The number of isolates within each family is indicated in parentheses.

[0033] FIG.2 shows manufacture, encapsulation, bottling, storage, consortium assembly, and dosing for each drug product.

[0034] FIGs. 3A-3C show fecal metagenomic and metabolomic data from patients hospitalized with liver disease. Fecal samples from hospitalized patients with liver disease (n=262) and healthy donors (n=22) were analyzed by shotgun metagenomics and targeted metabolomics. (A) Relative taxa abundance by shotgun metagenomics is shown for each sample. Metagenomic alpha-diversity was quantified using the inverse Simpson metric, and samples were grouped by whether they had low alpha-diversity (defined as lowest 1 / 3 of invSimpson values) or had medium or high alpha-diversity (top 2 / 3 of invSimpson values). Within alpha-diversity groupings, samples are arranged from left to right in order of increasing butyrate concentrations. Quantitative targeted metabolite concentrations for each corresponding sample are shown below the metagenomic data. (B) Abundance of commensal organisms contained within the proposed consortia are shown below each corresponding sample. Compared to healthy donor samples and samples with higher alpha-diversity, fecal samples with low alpha-diversity and low metabolite levels have very low abundances (and often times absent) of the commensal bacteria that the inventors propose administering. (C) Select metabolite concentrations were plotted for each grouping of alpha-diversity within the liver disease cohort or healthy donor. Units for SCFA are mM, and units for BA derivatives are in g / mL. Each point represents a single value. Median and interquartile range are indicated by the line and box, respectively. Statistical comparisons between individual groups were analyzed using the Wilcoxin rank sum as indicated by brackets. P-values are adjusted for multiple comparisons and represented as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0035] FIG.4 shows Pre-MCB Manufacturing Process. Healthy donors were screened, and specimens (either fecal samples or material obtained during a screening colonoscopy) were collected, aliquoted and serially diluted prior to inoculating on rich media under anaerobic conditions. After growth, multiple individual colonies were picked and subjected to WGS. At this point, individual strains were selected and grown on rich agar plates prior to storage at - 80°C in 20% glycerol to form the General Cell Bank (or Symbiotic Strain Bank). After serial passages in GMP-grade media (described in detail below), the pre-master cell bank was formed.201720021.2 - 8 -

[0036] FIG.5 shows generation of pre-master cell bank. Validated stocks of isolates from General Cell Bank / SBSB were repeatedly cultured in GMP-grade media to make pre-master cell bank stocks. The media used in the SBSB stocks was diluted to 10-11.

[0037] FIG. 6 shows Master Cell Bank (MCB) generation. Passage from pre-MCB to generate MCB will take place in the cGMP facility

[0038] FIG.7 shows LBPs Manufacturing Process Flow Diagram. The MCB is inoculated into GMP-grade media for a seed culture. After growth, the seed culture is transferred to the bioreactor where it ferments, and is then concentrated by tangential flow filtration (TFF) to make the drug substance. The drug substance is lyophilized and encapsulated to make the drug product.

[0039] FIG.8 shows drug substance (DS) manufacturing process. The MCB is inoculated into GMP-grade media in an anaerobic chamber to make a seed culture in GMP-grade media. After growth, the seed culture is subjected to in process testing (IPT) prior to transfer to the bioreactor where it ferments. The fermentation product undergoes IPT prior to then being purified and concentrated by tangential flow filtration (TFF) in either 10% sucrose or 10% sucrose / 5% trehalose to make the drug substance. The drug substance undergoes Quality Control (QC) testing and can be stored at -80°C. Drug substance is lyophilized, encapsulated, bottled, sealed, and labeled to make the drug product.

[0040] FIG. 9 shows drug substance (DS) manufacturing process with low volume culturing. Strains that grow to high densities with low-volume culturing will undergo the same process as above (FIG. 8) but with a static culture instead of the bioreactor. The static culture product will be subjected to IPT prior to passing through to TFF and QC for final drug substance.

[0041] FIG. 10 shows drug product manufacturing process. Drug product manufacturing begins when the Drug Substance (i.e. fermentation or static culture growth product concentrated by TFF in a buffer) begins lyophilization. After lyophilization, the product is milled into a fine powder and encapsulated. These capsules are then bottled, sealed, and labeled prior to storage at -80°C. In process testing and quality control is performed where indicated prior to Drug Product release.

[0042] FIG. 11 shows a bottle, cap, and seal structure for certain processes and compositions disclosed herein.

[0043] FIG. 12 shows Stability of drug product at -80°C at the indicated timepoints was determined by measuring the CFU per capsule in each of the 8 strains within Consortium A.201720021.2 - 9 -This process is ongoing, and will be performed on drug product for up to 24 months post- manufacturing.

[0044] FIGs.13A-13D show strain and consortia capacity to perform metabolic functions. A) Experimental design schematic. Individual strains were grown in media containing a known concentration of a precursor bile acid molecule in order to test the ability to produce a specific product by catalyzing a specific step of bile acid metabolism. Strains were harvested in the late log phase of growth, and then quantitative, targeted mass-spectrometry was performed on culture supernatant. B and C) Bifidobacterium species (B. longum, B. pseudocatenulatum, and B. bifidum) were grown in media containing conjugated primary bile acids and supernatant was tested specifically for primary bile acids. C. scindens was grown in media containing primary bile acids and culture supernatant was tested for secondary BA. C) E. lenta was grown in the presence of LCA and supernatant was tested for 3-oxo-LCA. The concentration of the reaction product (i.e. produced bile acid) is graphed. Conversion efficiency was calculated by dividing the product concentration by the precursor concentration and is shown as a percent. D) Butyrate concentration was measured in culture supernatant of each of the six butyrate producing strains at the late log phase of growth.

[0045] FIGs.14A-14E show strain and consortia capacity to perform metabolic functions. A) Experimental design schematic. The members of each consortium were inoculated (in a 1:1 ratio) into media containing 10uM of conjugated primary bile acid, either taurocholic acid or taurochenodeoxycholic acid. Consortia were grown to the late log-phase phase of growth, and supernatant was subjected to targeted metabolomics to determine the ability of the consortia to generate butyrate and specific bile acid metabolites. B) Culture pellets were subjected to 16S sequencing, and the relative abundance of each species in the final culture is shown. C and D) The concentration of each specified bile acid metabolite was measured in the culture supernatant by mass spectrometry and is graphed. Conversion efficiency was calculated by dividing the specified bile acid metabolite by the starting concentration of the conjugated primary bile acid. Conversion efficiency for TCA and TCDCA was calculated by subtracting 100% - (end concentration / starting concentration) and is shown in red. All other conversion efficiencies are relative to the starting conjugated primary bile acid (black text). E) Graph of butyrate concentration in the supernatant of each consortium at the late log phase of growth.

[0046] FIGs.15A-15E show strain and consortia capacity to perform metabolic functions. A) Experimental design schematic. Germ free mice were gavaged with each consortium (in a 1:1 ratio for each species) for three consecutive days. After 14 days, fecal pellets were collected and subjected to 16S sequencing and targeted metabolomics. B) 16S sequencing results were201720021.2 - 10 -averaged over each group (n = 4) for each experiment and are shown as a relative abundance. C and D) Concentration of each indicated metabolite is graphed. Percentages indicate the relative amount of the indicated metabolite in fecal pellets compared to control mice (germ- free mice that were not inoculated, n=14). When values exceed ten times the concentrations found in germ-free control mice, labels indicate “>10x”. E) Butyrate concentration for mice that engrafted at least one butyrate producer.

[0047] FIG. 16 shows a mechanism of action for therapeutic compositions disclosed herein. Schematic of steps in butyrate and secondary bile acid metabolism and mechanism of action in the gut and liver. Dietary polysaccharides are broken down by polysaccharide metabolizing bacteria to monosaccharides which are fermented to butyrate by butyrate producing bacteria. Conjugated bile acids are formed by the liver and secreted into the gut via the biliary tree where they are deconjugated by bacteria into primary bile acids. Primary bile acids are dehydroxylated at the 7 position by C. scindens forming secondary bile acids, which are further modified by bacteria into modified secondary bile acids. Butyrate, secondary bile acids, and modified secondary bile acids restrict the growth of gut pathogens, augment mucosal barrier integrity, and modulate the immune system reducing gut inflammation.

[0048] FIG. 17 shows genotypic analysis for antibiotic resistance using CARD. Whole genome sequences of each organism were compared to resistance genes in the CARD database.63Perfect hits indicate 100% homology to an established resistance gene. Strict hits correspond to genes with partial homology to antimicrobial resistance genes, but have not been confirmed phenotypically.64One perfect hit for dfrF was identified in C. scindens. Thirteen genes with partial homology were identified that could cause resistance to fluoroquinolones, tetracyclines, vancomycin, mupirocin, clindamycin, erythromycin, and rifamycins.

[0049] FIG. 18 shows toxicology results from studies disclosed herein. Mice were sacrificed 15 days after colonization with each consortium, and a segment of both small intestine (ileum) and colon was fixed in formalin and embedded in paraffin prior to sectioning and staining with hematoxylin and eosin (H&E) for histology. Representative sections of H&E stained sections are shown. Images were acquired at 20x magnification. DETAILED DESCRIPTION I. Definitions

[0050] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.201720021.2 - 11 -

[0051] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0052] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment or aspect.

[0053] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0054] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of” excludes any element, step, or ingredient not specified. The phrase “consisting essentially of” limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of” or “consisting essentially of.”

[0055] The term “isolated” encompasses a bacterium or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and / or (2) produced, prepared, purified, and / or manufactured by the hand of man. Isolated bacteria may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or any range derivable therein, or more of the other components with which they were initially associated. In some aspects, isolated bacteria are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any range derivable therein, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components.

[0056] The terms “purify,” “purifying” and “purified” refer to a bacterium or other material that has been separated from at least some of the components with which it was associated201720021.2 - 12 -either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. In some aspects, “purified” is used interchangeably with “enriched”. A bacterium or a bacterial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the bacterium or bacterial population, and a purified bacterium or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or any range derivable therein, or above about 90% and still be considered “isolated.” In some aspects, purified bacteria and bacterial populations are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any range derivable therein, or more than about 99% pure. In the instance of bacterial compositions provided herein, the one or more bacterial types present in the composition can be independently purified from one or more other bacteria produced and / or present in the material or environment containing the bacterial type. Bacterial compositions and the bacterial components thereof are generally purified from residual habitat products.

[0057] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term “unit dose” or “unit dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0058] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure. II. Therapeutic Compositions

[0059] Certain aspects relate to compositions comprising a bacteria. The bacteria may be one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof. In some aspects, the therapeutic composition comprises, consists of, or consists essentially of a bacteria, including one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI201720021.2 - 13 -Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof, in a unit dosage.

[0060] The unit dosage may be any dosage sufficient for the desired effect of the therapeutic composition. The unit dosage may be the bacteria combined in an amount that is not found in nature. In some aspects, the bacteria, including one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof, are enriched, isolated, or purified to an amount and / or concentration that is not found in nature. In some aspects the unit dosage comprises between 1×103to 9×1016colony forming units (CFU) of the bacteria. In some aspects the unit dosage comprises at least, at most, or about 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×1013, 9×1013, 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, 2×1015, 3×1015, 4×1015, 5×1015, 6×1015, 7×1015, 8×1015, 9×1015, 1×1016, 2×1016, 3×1016, 4×1016, 5×1016, 6×1016, 7×1016, 8×1016, 9×1016, or any range derivable therein, CFU of the bacteria. In another aspect, the disclosure relates to compositions comprising an isolated or purified population of one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof. Therapeutic compositions and methods of administering one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof may involve such unit dosages. Moreover, a unit dosage may be given multiple times over a time period as discussed below.

[0061] In some aspects, the composition comprises a bacteria, including any bacteria disclosed herein such as one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof,201720021.2 - 14 -and a second composition, which can include a metabolite (including any metabolite described herein), antibiotic, and / or immunosuppressant.

[0062] The therapeutic compositions can be formulated for administration, including as pharmaceutical formulations, e.g., formulated for oral administration; suppository administration; or injection such as via the intravenous, intramuscular, subcutaneous, or intraperitoneal routes. Such compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0063] In certain aspects, the therapeutic composition, which may include a bacteria, including any bacteria disclosed herein, and a saponin, including any saponin disclosed herein, is formulated for oral administration. The formulation for oral administration may comprise a pill, capsule, suspension, drink, or the like. In some aspects, the saponin is administered through food.

[0064] In some aspects, the therapeutic composition comprises fecal transplant. The fecal transplant may comprise one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof. In some aspects, the fecal matter is administered in a dose of 50 g. In some embodiments, the fecal matter is administered in a dose of at least, at most, or exactly 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75.80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, or 400 g (or any derivable range therein). In certain aspects, the fecal transplant comprises fecal matter collected from a patient that has not received an antibiotic in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, weeks, months, and / or years (and any range derivable therein) prior to collecting the fecal matter. In certain aspects, the fecal transplant comprises fecal matter that comprises a measurable amount of one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof. In certain aspects, the fecal transplant comprises fecal matter that comprises a therapeutically effective amount of one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof.

[0065] The pharmaceutical formulations suitable for injectable use include sterile aqueous solutions or dispersions; formulations including, for example, aqueous propylene glycol; and201720021.2 - 15 -sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In certain aspects, the formulation is stable under the conditions of manufacture and storage and preserved against the contaminating action of non-therapeutic microorganisms.

[0066] A pharmaceutical composition or formulation can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of unintended microorganisms can be brought about by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In certain aspects, the formulation includes isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0067] Injectable solutions may be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required. In certain aspects encompassing powders for the preparation of injectable solutions, the therapeutic composition(s) are vacuum-dried and / or freeze-dried, which yield a powder of the active ingredient, plus any additional desired ingredient.

[0068] The present disclosure also provides a pharmaceutical composition comprising one or more microbial cultures as described above. The bacterial species therefore are present in the dose form as live bacteria, whether in dried, lyophilized, or sporulated form. This may be preferably adapted for suitable administration; for example, in tablet or powder form, potentially with an enteric coating, for oral treatment.

[0069] In particular aspects, the composition is formulated for oral administration. Oral administration may be achieved using a chewable formulation, a dissolving formulation, an encapsulated / coated formulation, a multi-layered lozenge (to separate active ingredients and / or active ingredients and excipients), a slow release / timed release formulation, or other suitable formulations known to persons skilled in the art. Although the word “tablet” is used herein, the formulation may take a variety of physical forms that may commonly be referred to by other terms, such as lozenge, pill, capsule, or the like.

[0070] While the compositions of the present disclosure are preferably formulated for oral administration, other routes of administration can be employed, however, including, but not201720021.2 - 16 -limited to, intracolonic, subcutaneous, intramuscular, intradermal, transdermal, intraocular, intraperitoneal, mucosal, vaginal, rectal, and intravenous.

[0071] In another aspect, the disclosed composition may be prepared as a suppository. The suppository may include but is not limited to the bacteria and one or more carriers, such as polyethylene glycol, acacia, acetylated monoglycerides, carnuba wax, cellulose acetate phthalate, corn starch, dibutyl phthalate, docusate sodium, gelatin, glycerin, iron oxides, kaolin, lactose, magnesium stearate, methyl paraben, pharmaceutical glaze, povidone, propyl paraben, sodium benzoate, sorbitan monoleate, sucrose talc, titanium dioxide, white wax and coloring agents.

[0072] In some aspects, the composition may be prepared as a tablet. The tablet may include the bacteria and one or more tableting agents (i.e., carriers), such as dibasic calcium phosphate, stearic acid, croscarmellose, silica, cellulose and cellulose coating. The tablets may be formed using a direct compression process, though those skilled in the art will appreciate that various techniques may be used to form the tablets.

[0073] In other aspects, the composition may be formed as food or drink or, alternatively, as an additive to food or drink, wherein an appropriate quantity of bacteria is added to the food or drink to render the food or drink the carrier.

[0074] The compositions of the present disclosure may further comprise one or more prebiotics known in the art, such as lactitol, inulin, or a combination thereof.

[0075] In some aspects, the composition may further comprise a food or a nutritional supplement effective to stimulate the growth of one or more bacteria, including any bacteria disclosed herein, present in the gastrointestinal tract of the subject. In some aspects, the nutritional supplement is produced by a bacterium associated with a healthy human gut microbiome. III. Commensal Bacteria

[0076] Certain aspects herein concern one or more commensal bacteria, including the DFI Commensal Consortia. The commensal bacteria described herein can be identified using whole genome sequencing, which may be found using an NCBI accession number. One skilled in the art can re-generate a bacteria using the whole genome sequence starting from publicly available biological samples using gene editing available in the art. The commensal bacteria include:201720021.2 - 17 -A. Bile acid deconjugators and acetate producers

[0077] Bile acid deconjugators and acetate producers include, but are not limited to Bifidobacterium longum DFI.7.60 (NCBI accession number CP143939.1), Bifidobacterium pseudocatenulatum DFI.7.61 (NCBI accession number CP143938), Bifidobacterium bifidum DFI.7.51 (NCBI accession number JAKNHM000000000).

[0078] Bacterial species belonging to the Bifidobacterium genus are available over the counter in pharmacies and health-food markets as orally ingested probiotics.13Experimental studies have demonstrated associations between the presence of Bifidobacterium species and responses to cancer immunotherapy,14reduction of liver injury following D-glactosamine administration,15enhanced fiber metabolism16and amelioration of bowel inflammation.17Although oral ingestion of Bifidobacterium strains is of low risk, rare cases of bacteremia have occurred in premature infants, highly immunocompromised individuals at the extremes of age and patients with gastrointestinal barrier defects.18–20Comparison of genomes of Bifidobacterium strains isolated from bloodstream infections with common probiotic strains did not identify genes associated with invasiveness, suggesting that systemic infections resulted principally from states of severe immunocompromise or secondary to intestinal barrier defects.18–20B. Secondary bile acid producer

[0079] Secondary bile acid producers include, but are not limited to, Clostridium scindens SL.1.22 (NCBI accession number CP143935)

[0080] C. scindens encodes the bai operon that converts primary to secondary bile acids,21and impacts the lower GI tract’s composition because secondary bile acids and their derivatives can inhibit the growth of some commensal bacterial species.22There are no published manuscripts describing infections caused by C. scindens. C. Polysaccharide metabolizers and secondary bile acid modifiers

[0081] Secondary bile acid modifiers (including those that metabolize polysaccharides) include, but are not limited to, Bacteroides thetaiotaomicron DFI.6.40 (NCBI accession number CP143941.1), Bacteroides ovatus MSK.18.37 (NCBI accession number201720021.2 - 18 -JAHOIB000000000), and Bacteroides cellulosilyticus DFI.5.60 (NCBI accession number JAJCMC000000000).

[0082] Species of the Bacteroides genus are prevalent colonizers of the lower GI tract and metabolize complex polysaccharides and produce a range of metabolites that positively impact the host and that influence microbiota composition by cooperating and competing with other microbial species.23Members of the Bacteroides fragilis group can cause clinical infections, though usually in settings of polymicrobial infection associated with more virulent bacteria such as Escherichia coli and occur almost exclusively as a result of intestinal barrier disruption.24B. thetaiotaomicron has been shown to reduce inflammation in mouse models of inflammatory bowel disease,25while enhancing gut graft-versus-host-disease in stem cell transplant patients receiving treatment with carbapenem antibiotics.26In the gut, B. ovatus produces acetate, propionate, succinate and GABA and metabolizes tryptophan.27In patients receiving metformin, B. ovatus can sequester and lead to deficiency of vitamin B12.28Some evidence suggests that B. ovatus can reduce LPS-induced inflammation.29Studies of bacterial associations with colorectal cancer demonstrated an association of B. ovatus with benign but not malignant tumors.30In a mouse study, B. ovatus administration increased the severity of experimental malaria but did not lead to hyperparasitemia.31

[0083] Secondary bile acid modifiers (including those that metabolize polysaccharides) also include, but are not limited to, Phocaeicola vulgatus DFI.4.81 (NCBI accession number CP143952.1)

[0084] P. vulgatus is a common member of the microbiota that can metabolize complex polysaccharides and produce acetate, propionate and succinate. Correlative studies have demonstrated a negative correlation with bone mineral density and P. vulgatus density in fecal samples.32In a study of patients with treated hepatitis C infection, while prevalence of P. vulgatus did not correlate with stage of liver fibrosis, transcription of P. vulgatus glycan degradation genes correlated with the presence of liver fibrosis.33However, the patients in this study were significantly different than the population in multiple ways. Clinically, all patients studied were in the outpatient setting, only a subset of the patients had cirrhosis, and those with cirrhosis were all well-compensated (Childs-Pugh Class A). Additionally, HCV-related cirrhosis is far less common than other etiologies in the inpatient hepatology population. From a microbiome perspective, these patients all had relatively diverse microbiomes compared to the severely dysbiotic patients the inventors will target in a hospitalized study population. In contrast to the patients here with low diversity, patients in the HCV study had P. vulgatus in201720021.2 - 19 -their gut microbiomes regardless of fibrosis stage. This is similar to the healthy donors, suggesting that P. vulgatus is an important component of the normal gut microbiome. Additional discussion of this study is below in the section on A. hadrus.

[0085] Secondary bile acid modifiers (including those that metabolize polysaccharides) also include, but are not limited to, Parabacteroides distasonis DFI.5.23 (NCBI accession number CP143949.1) and Parabacteroides merdae DFI.4.73 (JANGCV000000000).

[0086] P. distasonis has been associated with reduced development of rheumatoid arthritis,34reduced hepatic fibrosis,35reduced progression of non-alcoholic steatohepatitis36and reduced obesity and metabolic syndrome (in mice).37P. merdae prevalence has been associated with reduced cardiovascular disease.38D. Secondary bile acid modifiers

[0087] Secondary bile acid modifiers also include, but are not limited to, Eggerthella lenta DFI.5.72 (NCBI accession number JANGCM000000000)

[0088] E. lenta is an extensively studied member of the intestinal microbiota that encodes enzymes that mediate reduction of a wide range of molecules, including secondary bile acids.39E. lenta has been associated with inflammatory diseases such as colitis and arthritis. In the case of arthritis, using a mouse model, increased frequencies of E. lenta do not induce arthritis, however once arthritis has developed E. lenta can enhance auto-antibody production. Along similar lines, E. lenta can increase bowel inflammation by expressing the cgr2 gene, which reduces steroidal glycocides and enhances Th17 responses.40The inventors have searched the genome of E. lenta DFI.5.72 and do not detect the cgr2 gene. Multiple case reports and series have documented cases of bacteremia caused by E. lenta that are almost exclusively associated with bowel perforation or leakage and are responsive to antibiotic treatment.41–44E. Butyrate producers

[0089] Butyrate producers include, but are not limited to, Anaerobutyricum hallii DFI.9.38 (NCBI accession number CP143936).

[0090] Bacterial strains belonging to the Anaerobutyricum genus are butyrate producing obligate anaerobes that are common members of the colonic microbiota. A. soehngenii has been administered to patients with metabolic syndrome by duodenal infusion and demonstrated to be safe and improves glucose metabolism.45,46Prevalence of A. hallii in fecal samples has201720021.2 - 20 -been correlated with enhanced depletion of meat-derived dietary carcinogen.47There are no published reports of infections caused by Anaerobutyricum species.

[0091] Butyrate producers also include, but are not limited to, Anaerostipes hadrus DFI.4.30 and Anaerostipes caccae DFI.7.76 (NCBI accession number CP143937).

[0092] Dietary fiber supplementation in diabetic patients has been shown to reduce hemoglobin A1C and increased the frequency of A. hadrus in fecal samples.48Studies in patients receiving chemotherapy for esophageal cancer demonstrated that A. hadrus and Bifodobacterium pseudocatenulatum abundances were correlated with reduced incidence of febrile neutropenia.49On the other hand, administration of A. hadrus to mice with DSS- induced colitis worsened inflammation while administration to control mice did not lead to colonic pathology.50Investigation of portal vein blood and fecal samples from patients with treated hepatitis C infection has associated intestinal A. hadrus transcription with higher concentrations of free fatty acids in the portal vein, which is associated with liver fibrosis.33However, this observational study is unable to draw causative conclusions. It is also not conclusive if the possible product of A. hadrus transcriptional activity (free fatty acids) is implicated in disease progression as other studies have shown that long chain fatty acids in the intestinal lumen correlate with lower alcohol-induced liver injury in mice and lower intestinal FA synthesis genes correlate with alcohol abuse in humans.51The alcohol model may be more relevant to the patients who overwhelmingly have liver disease from alcohol use as compared to a small minority with liver disease from HCV. Bloodstream or other serious infections caused by Anaerostipes are rare, with only one systemic graft and bloodstream infection caused by A. caccae having been reported.52

[0093] Butyrate producers also include, but are not limited to, Clostridium symbiosum DFI.5.64 (NCBI accession number CP143946).

[0094] The abundance of C. symbiosum has been reported to increase as colorectal neoplasia advances from adenoma to early and advanced colorectal cancer.53There is no evidence, however, of causation. C. symbiosum infection is rare and only described following colon injury.54

[0095] Butyrate producers also include, but are not limited to, Coprococcus comes DFI.3.84 (NCBI accession number CP143955).

[0096] There are no documented infections caused by C. comes. One report indicates that C. comes can inactivate an Angiotensin-Converting Enzyme inhibitor and reduce its effectiveness in lowering blood pressure in hypertensive rats.55201720021.2 - 21 -

[0097] Butyrate producers also include, but are not limited to, Eubacterium rectale DFI.5.28 (NCBI accession number CP143947.1)

[0098] Increased prevalence of E. rectale has been associated with reduced incidence of gastrointestinal B cell lymphoma in patients and transfer of E. rectale to mice has been shown to reduce bowel inflammation.56E. rectale has been associated with reduced inflammation in mice infected with HSV-157and reduced mortality in patients with COVID-19,58and enhanced responses to cancer immunotherapy.59In contrast, E. rectale has been suggested to serve as a driver of colorectal cancer and enhancer of colitis in some mouse strains.60There are no reports of systemic infection with E. rectale. F. DFI Commensal Consortia

[0099] The DFI Commensal Consortia consists of Bifidobacterium longum DFI.7.60 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143939.1), Bifidobacterium pseudocatenulatum DFI.7.61 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143938), Bifidobacterium bifidum DFI.7.51 (https: / / www.ncbi.nlm.nih.gov / nuccore / JAKNHM000000000), Clostridium scindens SL.1.22 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143935), Bacteroides thetaiotaomicron DFI.6.40 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143941.1), Bacteroides ovatus MSK.18.37 (https: / / www.ncbi.nlm.nih.gov / nuccore / JAHOIB000000000), Bacteroides cellulosilyticus DFI.5.60 (https: / / www.ncbi.nlm.nih.gov / nuccore / JAJCMC000000000), Phocaeicola vulgatus DFI.4.81 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143952.1), Parabacteroides distasonis DFI.5.23 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143949.1), Parabacteroides merdae DFI.4.73 (https: / / www.ncbi.nlm.nih.gov / nuccore / JANGCV000000000), Eggerthella lenta DFI.5.72 (https: / / www.ncbi.nlm.nih.gov / nuccore / JANGCM000000000), Anaerostipes hadrus DFI.4.30 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143954), Anaerostipes caccae DFI.7.76 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143937), Clostridium symbiosum DFI.5.64, (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143946), Coprococcus comes DFI.3.84 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143955), Anaerobutyricum hallii DFI.9.38 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143936), and Eubacterium rectale DFI.5.28 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143947.1).201720021.2 - 22 -1. Consortia Strain Composition:

[0100] Consortia were designed to 1) produce butyrate and 2) facilitate bile acid metabolism from conjugated primary bile acids (which are secreted by the liver) to bioactive secondary and modified secondary bile acids. Consortia A, B1, B2, C1, C2, D1, D2, E1, and E2 each contain 8 different bacterial strains, including two different organisms that produce butyrate, at least one organism that can deconjugate conjugated primary bile acids, one that can convert primary to secondary bile acids, and one that can modify secondary bile acids. Strains that metabolize a wide range of polysaccharides are also included in each consortium. In certain aspects, strain engraftment is variable. In certain aspects, functional redundancy is incorporated into the consortia (e.g.2 butyrate producers per consortium) for a total of 8 bacterial strains per consortium. In some aspects, unique consortia were assembled to change the members based on the metabolic capacity of consortium A according to the adaptive trial design. The composition of each of the DFI Consorita (A through E2) are shown in the following table Consortia Compositions Consortia Function Organism A B1 B2 C1 C2 D1 D2 E1 E2 B lon um DFI760 x x x X x x x x x x x x201720021.2 - 23 -IV. Administration of Therapeutic Compositions

[0101] Certain aspects concern the administration of therapies and therapeutic compositions, including any therapeutic composition described herein that includes one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof. The therapies may be administered in any suitable manner known in the art. The therapy provided herein may comprise administration of a combination of therapeutic composition, such as a first composition and a second composition. In some aspects, the first composition comprises one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof. In some aspects, the second composition comprises a metabolite, antibiotic, and / or immunosuppressant. The first and second compositions may be administered sequentially (at different times) or simultaneously (at the same time). In some aspects, the first and second compositions are administered as separate compositions. In some aspects, the first and second compositions are administered as the same composition.

[0102] In some aspects, the first therapeutic composition and the second therapeutic composition are administered substantially simultaneously. In some aspects, the first therapeutic composition and the second therapeutic composition are administered sequentially. In some aspects, the first therapeutic composition, the second therapeutic composition, and a third therapy, which may be an antibiotic, are administered sequentially or simultaneously. In some aspects, the first and second therapeutic compositions are administered concurrently and the third therapy is administered sequentially, before and / or after, with the first and second therapeutic compositions. In some aspects, the first therapeutic composition is administered before administering the second therapeutic composition. In some aspects, the first therapeutic composition is administered after administering the second therapeutic composition.

[0103] Aspects of the disclosure relate to compositions and methods comprising therapeutic compositions. The different therapies may be administered in one composition or in more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of the agents may be employed.

[0104] In some aspects, the first composition, which may be a composition comprising one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and201720021.2 - 24 -DFI Commensal Consortia, or subsets of bacteria selected thereof, and optionally the second composition, which may be a composition comprising a metabolite, antibiotic, and / or immunosuppressant, are administered prophylactically. The first composition and optionally the second composition may be administered before the administration of a treatment that causes a disease or disorder. In some aspects, the first composition and optionally the second composition are administered before the administration of a an antibiotic (including any antibiotic described herein). The first composition and optionally the second composition may be administered prophylactically, as described herein, at any time before the administration of the antibiotic, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, hours, days, weeks, and / or months (or any range derivable therein) before the administration of the antibiotic.

[0105] In some aspects, the first composition, which may be a composition comprising one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof, and optionally the second composition are administered substantially simultaneously with another composition. The first composition and optionally the second composition may be administered substantially simultaneously with the administration of a treatment that causes a disease or disorder. In some aspects, the first composition and optionally the second composition are administered substantially simultaneously and / or before the administration of an antibiotic (including any antibiotic described herein). In certain aspects, the antibiotic is administered over a time period, which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, hours, or more days, weeks, months, and / or years (and any range derivable therein). In such aspects, the first composition and optionally the second composition may be administered over all or part of the time period.

[0106] In some aspects, the first composition, which may be a composition comprising one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof, and optionally the second composition, which may be a metabolite, antibiotic, and / or immunosuppressant composition, are administered after the administration of another composition. The first composition and optionally the second composition may be administered after the administration of a treatment that causes a disease or disorder. In some aspects, the first composition and optionally the second composition are administered after the administration of an antibiotic (including any antibiotic described herein). The first composition and optionally the second composition may be administered at any time after the administration of the antibiotic, including 1, 2, 3, 4, 5, 6,201720021.2 - 25 -7, 8, 9, 10, 11, 12, or more, hours, days, weeks, and / or months (and any range derivable therein) after the administration of the antibiotic.

[0107] In certain aspects, the first composition and optionally the second composition— administered before, simultaneously with, or after a treatment that causes a disease or disorder—is administered in an amount that prevents, reduces the severity of, or treats the disease or disorder. In certain aspects, the first composition and optionally the second composition—administered before, simultaneously with, or after an antibiotic—is administered in an amount that prevents, reduces the severity of, or treats the disease or disorder caused by the antibiotic. Such amount may be referred to herein as a therapeutically effective amount.

[0108] The therapeutic agents of the disclosure may be administered by the same route of administration or by different routes of administration. In some aspects, the therapeutic composition is administered intracolonically, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the patient, the patient's clinical history and response to the treatment, and the discretion of the attending physician.

[0109] The treatments may include various “unit doses.” Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, is within the skill of determination of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. In some aspects, a unit dose comprises a single administrable dose.

[0110] In some aspects, a single dose of the first therapeutic composition, which comprises one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof, is administered. In some aspects, multiple doses of the first therapeutic composition are administered. In some aspects, the first therapeutic composition is administered at a dose of between 1×105to 9×108CFUs, or any range derivable therein. In some aspects, the first therapeutic composition is administered201720021.2 - 26 -at a dose of at least, at most, or about 1×103, 2×103, 3×103, 4×103, 5×103, 6×103, 7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104, 8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105, 9×105, 1×106, 2×106, 3×106, 4×106, 5×106, 6×106, 7×106, 8×106, 9×106, 1×107, 2×107, 3×107, 4×107, 5×107, 6×107, 7×107, 8×107, 9×107, 1×108, 2×108, 3×108, 4×108, 5×108, 6×108, 7×108, 8×108, 9×108, 1×109, 2×109, 3×109, 4×109, 5×109, 6×109, 7×109, 8×109, 9×109, 1×1010, 2×1010, 3×1010, 4×1010, 5×1010, 6×1010, 7×1010, 8×1010, 9×1010, 1×1011, 2×1011, 3×1011, 4×1011, 5×1011, 6×1011, 7×1011, 8×1011, 9×1011, 1×1012, 2×1012, 3×1012, 4×1012, 5×1012, 6×1012, 7×1012, 8×1012, 9×1012, 1×1013, 2×1013, 3×1013, 4×1013, 5×1013, 6×1013, 7×1013, 8×1013, 9×1013, 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, 2×1015, 3×1015, 4×1015, 5×1015, 6×1015, 7×1015, 8×1015, 9×1015, 1×1016, 2×1016, 3×1016, 4×1016, 5×1016, 6×1016, 7×1016, 8×1016, 9×1016, or any range derivable therein, CFU of the bacteria.

[0111] In some aspects, a single dose of the second therapeutic composition, which may comprise a metabolite, antibiotic, and / or immunosuppressant. In some aspects, multiple doses of the second therapeutic composition are administered. In some aspects, the second therapeutic composition is administered at a dose of 1 µg / kg to 1 mg / kg, or any range derivable therein, or between 1 mg / kg and 100 mg / kg, or any range derivable therein. In some aspects, the second therapeutic composition is administered at a dose of at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or any range derivable therein, µg / kg or mg / kg.

[0112] The quantity to be administered, both according to number of treatments and unit dose, depends on the treatment effect desired. An effective dose is understood to refer to an amount necessary to achieve a particular effect.

[0113] Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each patient. Factors affecting dose include physical and clinical state of the patient, the route of administration, the intended goal of treatment (alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance or other therapies a subject may be undergoing.

[0114] It will be understood by those skilled in the art and made aware that dosage units of µg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of µg / ml or mM (blood levels). It is also understood that uptake is species and organ / tissue201720021.2 - 27 -dependent. The applicable conversion factors and physiological assumptions to be made concerning uptake and concentration measurement are well-known and would permit those of skill in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies and results described herein.

[0115] In certain instances, it will be desirable to have multiple administrations of the composition, e.g., 2, 3, 4, 5, 6 or more (and any range derivable therein) administrations, including administrations at any relevant amount such as all or a portion of a unit dosage of the composition. The administrations can be given every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, weeks, or months, including all ranges there between.

[0116] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-infective agents and vaccines, can also be incorporated into the compositions.

[0117] Administration of the compositions will typically be via any common route. This includes, but is not limited to oral, suppository, or intravenous administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intranasal administration. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.

[0118] The desired dose of the composition of the present disclosure may be presented in multiple (e.g., two, three, four, five, six, or more) sub-doses administered at appropriate intervals throughout the day.

[0119] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.201720021.2 - 28 -V. Methods of Treatment

[0120] Provided herein are methods for treating or delaying progression of certain diseases or disorders by administration of compositions, such as compositions comprising bacteria, including one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or subsets of bacteria selected thereof to a patient. In some aspects, the patient has been administered, is currently being administered, or will be administered an antibiotic. In certain aspects, the disease or disorder comprises an liver disorder. In certain aspects, the disease or disorder is associated with or caused by a change in a metabolite, including any metabolites described herein such as butyrate, taurocholic acid, deoxycholic acid and / or alloisolithocholic

[0121] The term “treatment” or “treating” means any treatment of a disease in a mammal, including: (i) preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease; (ii) suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease; (iii) inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; and / or (iv) relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance. In some aspects, the treatment may exclude prevention of the disease.

[0122] In certain aspects, the patient administered a therapeutic composition is identified as having, or is at risk of having, a disease, or disorder. In certain aspects, the patient administered a therapeutic composition is identified as having, or is at risk of having, a certain response to a treatment. The response to the treatment may be an infection, which may be an antibiotic-resistant infection.

[0123] In certain aspects, the administration of a therapeutic composition to a patient alters the microbiome, including altering the bacteria in the microbiome, and / or microbiome environment in the patient. In some aspects, the administration of the therapeutic composition decreases the amount of at least one bacteria and / or genus of bacteria. VI. Methods of Determining Microbiome Composition

[0124] In some aspects, the methods relate to obtaining a microbiome profile of a patient. In some aspects, obtaining a microbiome profile comprises the steps of or the ordered steps of:201720021.2 - 29 -i) obtaining a sample obtained from a subject (e.g., a human subject), ii) isolating one or more bacterial species from the sample, iii) isolating one or more nucleic acids from at least one bacterial species, iv) sequencing the isolated nucleic acids, and v) comparing the sequenced nucleic acids to a reference nucleic acid sequence. When performing the methods necessitating genotyping, any genotyping assay can be used. For example, this can be done by sequencing the 16S or the 23S ribosomal subunit or by metagenomics shotgun DNA sequencing associated with metatranscriptomics.

[0125] In some aspects, obtaining the microbiome profile of a patient is used to monitor the need of administering the therapeutic compositions described herein to the patient. In certain aspects, obtaining the microbiome profile of a patient is used to monitor the efficacy of the therapeutic compositions administered to the patient. In certain aspects, the patient is or is not administered a therapeutic composition based on the obtained microbiome profile of the patient. In certain aspects, the patient is administered a therapeutic composition because the obtained microbiome profile has an increased and / or decreased amount of one or more bacteria species and / or genus of bacteria when compared to a standard.

[0126] In certain aspects, the standard for comparison of the microbiome profile is a microbiome profile from a healthy individual. The healthy individual may be a patient that has not received an antibiotic in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1213, 14, or more days, weeks, months, and / or years (and any range derivable therein). In certain aspects, the healthy individual is a patient that does not have a diagnosed intestinal disorder.

[0127] Methods for determining microbiome composition may include one or more microbiology methods such as sequencing, next generation sequencing, wester blotting, comparative genomic hybridization, PCR, ELISA, etc.

[0128] Certain aspects relate to methods for screening for specific genes and metabolic features of interest for certain therapies. In some aspects, the method includes building bacterial consortia using mechanistic insights on how to inhibit certain bacterial gut colonization based on the methods and examples disclosed herein. VII. Kits

[0129] Certain aspects of the disclosure also encompass kits for performing the methods of the disclosure. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: enzymes, reaction tubes, buffers, detergent, primers, probes, antibodies.201720021.2 - 30 -

[0130] In a particular aspect, these kits may comprise a plurality of agents for assessing or identifying microorganisms, wherein the kit is housed in a container. The kits may further comprise instructions for using the kit for assessing sequences, means for converting and / or analyzing sequence data to generate prognosis.

[0131] Kits may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a composition which includes a probe that is useful for prognostic or non-prognostic applications, such as described above. The label on the container may indicate that the composition is used for a specific prognostic or non-prognostic application, and may also indicate directions for either in vivo or in vitro use, such as those described above. The kit may comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0132] Further kit aspects relate to kits comprising the therapeutic compositions of the disclosure. The kits may be useful in the treatment methods of the disclosure and comprise instructions for use. VIII. Bacteria Isolation and Culture

[0133] In some aspects, bacteria, including any bacteria described herein, are isolated and / or purified from a source. The bacteria may be isolated and / or purified from any suitable source. In some aspects, the source is a human sample. In some aspects, the source is a non- human sample, such as a rodent sample. The one or more bacteria genus and / or species may be isolated and / or purified using any method known in the art. The isolated and / or purified bacteria may then be formulated into therapeutic compositions, including the therapeutic compositions described herein. Before or after being isolated and / or purified, the bacteria may be characterized, including by sequencing to determine the composition and identity of the isolated and / or purified bacteria. The bacteria may be characterized by 16S rRNA or 23S rRNA sequencing.

[0134] In certain aspects, the methods described herein of treating a patient and methods comprising administering bacteria to a patient further comprise isolating and / or purifying the bacteria prior to the treatment and / or administration. The purified and / or isolated bacteria may be used in the methods described herein.

[0135] In some aspects, the isolated and / or purified bacteria may be cultured for at least between about 1 days and about 40 days, for at least between about 5 days and about 35 days,201720021.2 - 31 -for at least between about 5 days and 21 days. The bacteria may be cultured to generate sufficient quantity of bacteria to reach a unit dosage. The bacteria may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or any range derivable therein, or more days. The bacteria may be cultured in the presence of a liquid culture medium, such as an LB broth or other suitable medium for culturing bacteria. Typically, the medium may comprise a basal medium formulation as known in the art. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the bacteria cultured. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing the bacteria are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of bacteria. The bacteria may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, or any range derivable therein, and may be in a humidified incubator. IX. Examples

[0136] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. Example 1 – Design

[0137] To conduct pre-clinical studies of human-derived commensal bacterial species and clinical trials of microbiota reconstitution in patients with damaged microbiome compositions and functions, the DFI established the Symbiotic Bacterial Strain Bank (SBSB), as shown in FIG. 1. The DFI SBSB (https: / / dfi.cri.uchicago.edu / biobank / ) contains nearly 1,700 bacterial strains derived from healthy human donors at Memorial Sloan Kettering Cancer Center (MSKCC) and at the University of Chicago (UC). Donors were screened for absence of recent antibiotic use, history of colitis or inflammatory bowel diseases and autoimmune diseases. Bacterial strains were isolated from healthy donors by anaerobically culturing dilutions of201720021.2 - 32 -freshly collected fecal samples or during routine screening colonoscopy. All strains have undergone whole genome sequencing, and genomes have been assembled from Illumina sequencing reads. Whole genome sequences were used for taxonomic identification of each of the isolates in the DFI SBSB and have been submitted to NCBI. The inventors have optimized in vitro bacterial strain cultivation, metabolite production, and cooperativity upon co-culture with other commensal bacterial species using a variety of media compositions.

[0138] Clinical studies of microbiota reconstitution with commensal bacterial strains are limited. While experimental studies have implicated short chain fatty acid production, bile acid modification, B vitamin and tryptophan metabolite production with health benefits, little is known about which commensal strains or strain combinations are optimal. The inventors are at early stages of knowing which strains to choose for specific functions such as butyrate production (there are many to choose from) and the understanding of how recipient differences and residual microbiota compositions impact commensal strain engraftment and function remains a black box.

[0139] To enable studies of the safety and effectiveness of commensal bacterial strain manufacturing and administration to human recipients with compromised microbiota compositions, the inventors have constructed and equipped a two-suite cGMP facility to manufacture capsules containing individual symbiotic bacterial strains from the DFI Symbiotic Bacterial Strain Bank.

[0140] The proposed MARCO Trial will begin to identify bacterial strains and strain combinations that engraft and function in recipients with documented microbiota deficiencies. Name of the Drug and All Active Ingredients For Aspects Herein3.1

[0141] Drug Name: Commensal Bacterial Strains

[0142] Active Ingredients: Bile acid deconjugator and acetate producer: Bifidobacterium longum DFI.7.60 Bifidobacterium pseudocatenulatum DFI.7.61 Bifidobacterium bifidum DFI.7.51 Secondary bile acid producer: Clostridium scindens SL.1.22 Polysaccharide metabolizer and secondary bile acid modifier: Bacteroides thetaiotaomicron DFI.6.40 Bacteroides ovatus MSK.18.37 Bacteroides cellulosilyticus DFI.5.60201720021.2 - 33 -Phocaeicola vulgatus DFI.4.81 Parabacteroides distasonis DFI.5.23 Parabacteroides merdae DFI.4.73 Secondary bile acid modifier: Eggerthella lenta DFI.5.72 Butyrate producer: Anaerostipes hadrus DFI.4.30 Anaerostipes caccae DFI.7.76 Clostridium symbiosum DFI.5.64 Coprococcus comes DFI.3.84 Anaerobutyricum hallii DFI.9.38 Eubacterium rectale DFI.5.28

[0143] The drug is a lyophilized live commensal bacterial strains (Live Biotherapeutic Product). Example 2 - Formulation of the Dosage Forms to be Used

[0144] The manufacturing process for each bacterial strain has three main phases; Master cell bank formation and Drug Substance followed by Drug Product manufacturing. The manufacturing phases are fully described herein (CMC - Chemistry, Manufacturing and Control section). In short, each bacterial strain is cultured, concentrated, lyophilized and encapsulated using individual controlled processes that result in distinct Drug Substances and Drug Products that are evaluated for identity, stability, and purity prior to release. Each bacterial strain is batch manufactured independently over a 7- to 10-day time period. After each production cycle, the cleanroom suite is decontaminated via vapor hydrogen peroxide and prepared for the next batch manufacturing. Each batch yields between 120 to 300 capsules.

[0145] During encapsulation, Enteric capsules (Lonza, size 0) will be filled with one strain of lyophilized, live bacterial cells to a target strength of 1x105- 1x1010colony forming units (cfu) and then sealed. The inventors will seal 7 individual capsules containing a specific strain in a Child-Resistance bottle with Push-N-Turn Caps produced by the Drug PLASTICS company. Bottles will be induction foil sealed, and closed with a tamper proof cap. As an example, if a batch yields 140 capsules, 20 bottles of 7 capsules each are produced. This process is repeated for each of the 8 bacterial strains constituting a specific consortium.

[0146] The inventors have chosen both the dose strength and 7-day duration of treatment based on published data on another commensal consortia, VE3039. This study demonstrated that consortia engraftment was most efficient when given at a higher dose for a longer period of time9. The optimal dose in this published study was the maximum dose given (1x109cfu per201720021.2 - 34 -commensal strain per dose, total of 8x109cfu / dose) for the maximum number of days (14 days) for a total dose of 1.1x1011cfu per course of commensal consortia. This dose was safe, well- tolerated and was able to engraft in antibiotic-treated healthy volunteers. Lower doses (either a single day or fewer cfu / dose) were also safe and tolerated but did not achieve the same level of sustained engraftment. Based on this study, the inventors have chosen a maximal single dose of 1x1010cfu / strain (maximum total dose of 8x1010cfu) for a maximum total dose of 5.6x1011cfu / course of commensal consortia administered over 7 days. The lower range of the proposed dose (1 x 105cfu / dose) accounts for potential loss of viability during manufacturing, although all strains that are in Consortium A have been fermented, concentrated, lyophilized and encapsulated at higher counts (i.e. > 107cfu / strain / dose).

[0147] A single dose of a specific consortium consists of 8 distinct capsules representing each member of the consortium, placed into one single bottle. Prior to initiating the 7-day course of drug administration, capsules containing the 8 bacterial strains, will be placed into 7 bottles (one for each of the seven doses), sealed and delivered for administration to study subjects every 24 to 72 hours over the course of 10 days. The rationale for this dosing schedule is a practical consideration to allow for missed doses due to patient availability (e.g. for holidays or during medical appointments) and staff availability for drug delivery (e.g. on weekends or holidays). All seven doses will be given at least 1 day apart from one another and within 7-10 days of the first dose. Seven consecutive days of Commensal Consortia administration would be an acceptable dosing regimen under this protocol.

[0148] The DFI cGMP has two identical suites enabling the team to manufacture 2 strains simultaneously. Manufacturing and subsequent suite / equipment sterilization, on average, requires 10 days for each strain, thus the inventors anticipate that capsules for an 8-member consortium can be completed in 40 days. Given the stability of drug product upon storage at - 80°C, the inventors anticipate that the drug product from a prep of 8 strains will be available for use for at least 4-6 months.

[0149] Capsules will be ingested orally. Example 3 - Objectives and Duration of the Proposed Clinical Investigation(s)

[0150] The objective of this clinical trial is to test the safety of commensal bacterial strain combinations designed to reconstitute microbiota compositions and metabolite production in patients with liver disease and profound dysbiosis. The adaptive trial design will allow safety to be studied for three different combinations of bacteria, and their ability to engraft and function metabolically will be determined. The inventors will enroll 24 patients and follow201720021.2 - 35 -each patient for 12 months. The inventors anticipate completing enrollment within 12 months of trial initiation. Thus, the anticipated duration of the proposed trial, including time for data analysis, is 24 to 30 months. Summary of Previous Human Experience

[0151] This is a first in human study with bacterial strains that were isolated from healthy human donors. Thus, every strain included in this IND has colonized humans without causing evidence of toxicity. Nevertheless, there is no previous experience administering these specific bacterial strains to individuals with depleted microbiome compositions.

[0152] There is, however, extensive experience administering complex microbial populations to patients with dysbiosis. Fecal microbiota transplantation (FMT) facilitates clearance of recurrent Clostridioides difficile infection21. FMT has also shown promise in a number of other disease states including graft versus host disease in allogeneic hematopoietic cell transplantation,10recurrent urinary tract infection,11colonization with drug resistant organisms, and multiple liver-related complications including hepatic encephalopathy,12,13severe alcoholic hepatitis,14and alcohol cravings.15,16FMT is generally well-tolerated, with the majority of adverse events being mild and transient.17However, rare, unintentional transmission of antibiotic-resistant pathogens has occurred, including one report of death caused by transmission of drug-resistant E. coli to participants in a clinical trial.18Despite careful donor selection and stool screening, the inability to completely define the composition of feces remains a concern.

[0153] Two live biotherapeutics (REBYOTATMand VOWSTTM) were recently FDA- approved for treatment of recurrent C. difficile. While these two products have favorable safety profiles,19,20they are stool-derived products that rely on stool donors with variable microbial compositions. As such, the active “ingredient” is incompletely defined, and dosing, clinical efficacy, and side-effect profiles may vary between batches. Logistic issues of scalability and implementation may arise since these rely on stool donors.

[0154] An alternate approach, which the inventors propose in this application, is to use well-defined bacterial consortia that contain commensal organisms from healthy donors. This approach overcomes problems of heterogeneity, safety, and scalability, as every dose contains only the intended organisms at defined doses. Commensal consortia are currently being investigated by Vedanta (VE303) for the treatment of recurrent Clostridioides difficile infection (CDI) and Seres (SER-155) for prevention of bloodstream and antibiotic resistant pathogens.201720021.2 - 36 -Studies of SER-155 are still enrolling patients and data has not been reported yet (NCT04995653). VE303 has been shown to be safe in a Phase 1 trial with healthy volunteers and both safe and effective in a Phase 2 trial of patients with recurrent C. difficile colitis.21,22VE303 is also being investigated for treatment of hepatic encephalopathy (NCT04899115), although no results are available. VE303 engraftment in recipients is markedly variable and remains largely unexplained.21,22Engraftment of administered bacterial strains may depend on residual microbiota composition, gut metabolite concentrations, diet, and the recipient’s immune competence. The unpredictability of engraftment resulting from variation between recipients remains a gap in the understanding of how best to administer next-generation probiotics.

[0155] Studies that compare the engraftment and in vivo function of different, carefully defined consortia, therefore, will provide insights into the complex relationship between the human microbiota, microbial-derived / modified metabolites, and health. Example 4 - General Investigational Plan

[0156] The global burden of chronic liver disease (CLD) continues to rise due to the shifting epidemiology of disease causes.1–4CLD is often clinically silent until an initial decompensation event,5,6after which care is largely supportive without the ability to significantly modify the underlying disease with current therapies. Due to the high rates of infection (including culture negative infections) in patients with liver disease, empiric, broad spectrum antibiotics are commonly prescribed.7–9This practice is associated with high levels of antibiotic resistant organisms, a finding that correlates with poor clinical outcomes.9–11

[0157] The gut microbiome contributes to human health and disease, including liver disease. There is bidirectional anatomic communication between the liver and the gut via the portal vein and biliary tree. Preclinical studies have implicated the gut microbiome as a potential driver of non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease.12–14While robust clinical evidence linking the microbiome to progression of liver disease is lacking, multiple observational studies have reported various gut microbiome “signatures” of advanced fibrosis and cirrhosis.15–19Observational studies have also associated the gut microbiome composition with complications of end stage liver disease, including death.11,13,16,20

[0158] To further understand the contribution of the gut microbiome to complications of liver disease, the inventors completed an observational study in the Duchossois Family Institute (DFI) where the inventors performed shotgun metagenomic sequencing and targeted201720021.2 - 37 -metabolomic analyses on 847 fecal samples from 262 unique patients with various liver diseases and 22 healthy donors (Adapted in FIG. 3A)11. This study confirmed other groups’ published findings that patients with liver disease have a wide range of gut microbiome compositions, including lower alpha-diversity and increased burden of potentially pathogenic taxa (e.g. Enterobacteriaceae and Enterococcus) compared to healthy controls. With detailed analysis of the timing of stool samples relative to medication administration, the inventors demonstrated that much of the decreased alpha-diversity and expansion of potentially pathogenic and antibiotic resistant taxa occurs after the use of broad-spectrum antibiotics.

[0159] Many of these samples also contained very low (and frequently undetectable) concentrations of beneficial metabolites, including short chain fatty acids (SCFA, e.g. butyrate) and secondary bile acids (e.g. deoxycholic acid) (FIGs. 3A and 3C). Importantly, loss of microbiome alpha-diversity and low levels of fecal metabolites both associate with poor outcomes, including death11. The inventors therefore hypothesize that microbiome intervention, specifically reconstitution of the gut microbiome with commensal organisms that produce beneficial metabolites including SCFA and secondary bile acids, may reduce the incidence of systemic infections and prolong survival in patients with advanced liver disease.

[0160] As discussed herein, interventions that target the gut microbiome include antibiotic treatment, administration of prebiotics, probiotics or synbiotics, and fecal microbiota transplantation (FMT). Antibiotics are commonly prescribed to patients with decompensated cirrhosis, and the prebiotic lactulose is used to treat hepatic encephalopathy (HE)11,21,22. Probiotics, synbiotics, and FMT are not currently standard of care for treatment of liver disease or its complications but are the subject of ongoing studies23,24.

[0161] Given the defined composition, reproducibility, and favorable safety profiles of probiotics, in both healthy volunteers25and in patients with varying liver diseases,26–3228,2930,3132the inventors have constructed consortia containing commensal bacteria from healthy donors from the DFI SBSB outlined in FIG.1.

[0162] The inventors are positioned to identify and manufacture well-defined probiotic strains for microbiome augmentation. From the commensal bacteria strains in the DFI SBSB, the inventors have selected 17 strains for inclusion in 9 different 8-member commensal consortia. The strains the inventors have chosen have high abundances in healthy donors and in patients with liver disease who have high alpha-diversity, but they are significantly reduced (and commonly absent) in patients with low alpha-diversity and low levels of butyrate and deoxycholic acid (FIG. 3B). These 17 strains grow in combination and cooperate to generate SCFA and multiple bile acid metabolites that are present in the intestines of healthy individuals201720021.2 - 38 -and are susceptible to multiple commonly used clinical antibiotics. The DFI recently constructed a current Good Manufacturing Practice (cGMP) facility that is equipped to grow, lyophilize, encapsulate, and store each of these well-defined gut commensal bacteria to generate live biotherapeutic products (LBP) for human use.

[0163] The MARCO trial is a phase 1b, open label single center clinical trial in patients with liver disease and reduction / loss of butyrate and deoxycholate concentrations in fecal samples. The objectives of this study are to i) test the safety and tolerability of well-defined commensal consortia and ii) test the ability of consortia to engraft, persist, and produce beneficial metabolites in patients with liver disease and dysbiosis.

[0164] Indication to be Studied

[0165] Treatment of profound dysbiosis as marked by low butyrate and deoxycholic acid production in patients with liver disease.

[0166] General Approach for Evaluation of Treatment

[0167] Patients hospitalized for complications of liver disease at the University of Chicago Medical Center are being enrolled in an ongoing fecal collection study , enabling identification of patients with ≤ 700 µM butyrate and ≤ 10 µM deoxycholate in fecal samples. Patients who meet clinical inclusion and exclusion criteria who also have reduced butyrate and deoxycholate will be approached to participate in the Microbiota Augmentation to Reestablish Commensal Organisms (MARCO) Trial.

[0168] Patients who choose to participate will be given 7 doses of a well-defined commensal consortium containing 8 different bacterial strains that produce beneficial metabolites and that are often depleted in patients with liver disease. During the administration and observation phases of the study, patients will be monitored for adverse events and questioned about symptoms to determine patient tolerability of the administered commensal consortium. Fecal samples will be collected at multiple timepoints during the year after consortium administration, and these samples will be subjected to shotgun metagenomic sequencing and targeted metabolomics to determine strain engraftment and persistence and metabolite production. Details about the enrollment, consortium administration, and follow up including measures of safety, tolerability, strain engraftment and metabolite production are discussed below.

[0169] Number of Subjects to be Evaluated

[0170] The inventors will enroll a total of 24 patients in two stages. In the 1stStage, 8 patients will be enrolled, and in the 2ndStage 16 patients will be enrolled. The treatment for each stage is outlined in detail below.201720021.2 - 39 -

[0171] Based on i) enrollment data from the ongoing observational study, ii) the inclusion and exclusion criteria (Protocol), and iii) fecal metabolite thresholds (butyrate ≤ 700 µM and deoxycholic acid ≤ 10 µM), the inventors anticipate being able to approach 75 patients for enrollment within 1 year. This would allow for an approximately 1:3 approach-to-enrollment rate for the goal of 24 patients.

[0172] Drug Related Risks

[0173] First-in-human studies of any drug may come with unanticipated safety consequences. In the case of Commensal Consortia, these include the following:

[0174] Likely: Mild-to-moderate gastrointestinal symptoms (e.g. bloating and gaseous distension)

[0175] Less likely: Nausea and vomiting from Commensal Consortia Administration

[0176] Gene transfer of virulence factors and antibiotic resistance genes

[0177] Unlikely but potentially serious: Infection from administered organism(s)

[0178] Allergic reaction to the drug product

[0179] Patients with liver disease, and particularly those with dysbiosis, are at increased risk for adverse events11,13,16,18,19,33,34. However, studies that involved administration of probiotics to patients with cirrhosis have reported only self-limited gastrointestinal symptoms (e.g. bloating and gaseous discomfort), and there have been no serious treatment-related adverse events, including infectious complications. These studies have been conducted with patients with a wide range of liver disease severity, extending from outpatients with mild hepatic encephalopathy (HE) to patients admitted to the intensive care unit (ICU) with acute- on-chronic liver failure (ACLF) and MELD ≥3526–32. While unlikely, the possibility of an infection caused by an organism in the Commensal Consortia would be a serious adverse event that could be life threatening. Each subject will be followed by a hepatologist who is well- versed in the recognition and treatment of complications of liver-disease, including infections. In the unlikely event of a treatment-associated infection, bacterial strains constituting the proposed commensal consortia have been screened for the absence of transferable antibiotic- resistance genes and for culture sensitivity to a panel of clinically available antibiotics.

[0180] Transfer of virulence factor and antimicrobial resistance genes to pathogenic bacteria is a theoretical risk. That risk was mitigated by reviewing the genomes of species contained in the general cell bank for potentially problematic genes and selecting organisms with favorable genotypes.

[0181] Allergy to a drug ingredient is possible but improbable. Each of the selected strains is derived from healthy donor’s fecal sample and belong to species that are common members201720021.2 - 40 -of the human gut microbiome. These bacterial strains have not been associated with allergic responses. Bacterial lyophilization occurs in buffers containing sucrose and trehalose, which are also not associated with allergic responses. The lyophilized product will be encapsulated in a capsule (Lonza) that contains inert compounds, is commonly used in drug delivery, and has not been reported to cause allergy. To minimize this theoretical risk, the inventors will take a full allergy history of each patient prior to enrollment. Safety monitoring plan

[0182] Based on the existing literature and the data demonstrating favorable genotypic and phenotypic behavior of the chosen organisms, the risks from Commensal Consortia administration are likely low. Nevertheless, the liver disease patient population carries additional risk factors. The inventors have therefore limited enrollment to patients with MELD ≤ 30 who do not meet criteria for acute-on-chronic liver failure (ACLF) at the time of enrollment. Additionally, participants will be followed by a hepatologist well-versed in the treatment of liver disease and vigilant for any potential adverse events related to commensal consortia administration.

[0183] The inventors will collect solicited and unsolicited adverse events (AEs) following completion of commensal consortium administration. Patients will be given a symptom diary to solicit AEs during consortium administration and for 7 days following administration. They will also be contacted by phone every 24 hours during administration and for 7 days (1 week) after completing administration to solicit AEs through the symptom diary. They will then be contacted by phone at 2 weeks and monthly for 12 months after administration to solicit AEs. During these phone calls, the inventors will document all concomitant medication use. Telephone contact will also include a call from a member of the clinical team approximately 6 months after the commensal consortium administration to record any SAEs, new medical conditions / diagnoses or changes in medical conditions / medications since last study contact. Patients will be monitored during each contact with patients. Both anticipated and unanticipated adverse events and problems will be formally monitored and recorded. Unanticipated serious adverse events or problems will be reported to the Biological Sciences Division / University of Chicago Medicine Institutional Review Board (https: / / biologicalsciences.uchicago.edu / irb-home), as per local reporting requirements, and the FDA (within 15 days; or 7 days for unexpected fatal or life-threatening events or transmission of infectious agent). Anticipated and less serious adverse events will be submitted annually in reports to the FDA. The Sponsor will be responsible for monitoring the safety and efficacy of this treatment and complying with the reporting requirements.201720021.2 - 41 -

[0184] The sponsor will convene a safety monitoring board independent of this protocol (composed of infectious diseases and / or gastroenterology specialists) who will review the outcomes of patients treated under this protocol after the initial cohort of 8 patients completes therapy and every 6 months thereafter. This board will have the power to halt the treatment of patients under this protocol if it is determined that safety concerns exist. The FDA will be notified within 48 hours if the expanded access IND is halted for review. Example 5 - Chemistry, Manufacturing and Control Information Introduction

[0185] This drug is a consortium of commensal bacterial strains. Each bacterial strain was cultured from a fecal sample collected from a healthy human donor or obtained during a screening colonoscopy and belongs to species commonly detected in the fecal microbiota of healthy humans. Each strain is grown in monoculture in an anaerobic fermenter, concentrated into a sucrose or sucrose + trehalose buffer, lyophilized, and placed into an enteric coated capsule for release in the recipient’s intestine. Bacterial strains will be administered to recipients as a consortium of 8 distinct strains for 7 doses over 10 days. Mitigation of Potential Human Risk

[0186] The potential risks associated with administration of Commensal Consortia are discussed herein and include:

[0187] Likely: Mild-to-moderate gastrointestinal symptoms (e.g. bloating and gaseous distension)

[0188] Less likely: Nausea and vomiting from Commensal Consortia Administration

[0189] Gene transfer of virulence factors and antibiotic resistance genes

[0190] Unlikely but potentially serious: Infection from administered organism(s) and / or allergic reaction to the drug product

[0191] The inventors have mitigated potential human risks by the following:

[0192] Restricting inclusion of commensal bacterial strains to those cultured from feces of healthy human donors with no evidence of gastrointestinal or other systemic inflammatory diseases. All bacterial strains included are common colonizers of the human intestine.

[0193] Infections caused by intestinal commensal microbes occur almost exclusively when they are inoculated into a sterile space, such as the bloodstream or peritoneal cavity, following gut perforation or transmural malignancy. To minimize this risk patients with intestinal barrier defects including those with gastrointestinal bleeding, enteropathy, inflammatory bowel disease, graft versus host disease, or intestinal malignancy will be excluded.201720021.2 - 42 -

[0194] To ensure any possible infection caused by a member of the consortia are easily treatable, the inventors have performed phenotypic antimicrobial sensitivity testing of all the strains in accordance with Clinical Laboratory and Standards Institute (CLSI) standards and results are included herein.

[0195] To reduce the risk of transmission of genes encoding virulence factors or antimicrobial resistance, each member of the consortia has been whole genome sequenced (WGS) and queried against databases for virulence factors, toxins, and antimicrobial resistance genes. The results of those analyses and links to NCBI WGS results are listed herein.

[0196] Each commensal strain consortium has been tested in gnotobiotic mouse models for evidence of bowel inflammation or epithelial damage and results are included herein.

[0197] To mitigate risk for allergy, patients will be screened for history of allergy to all components of the drug product. Drug Substance Description of Drug Substance

[0198] The Drug Substance consists of 17 bacterial strains that were cultured from fecal samples obtained from healthy human donors. Manufacturer

[0199] All unit operations for Drug Substances (DS) manufacturing will occur at a cGMP facility. The Facility consists of two fully isolated and equipped cleanroom suites. Within each cleanroom suite, temperature, humidity, differential pressure, and airflow are monitored and controlled.

[0200] Equipment used for manufacturing processes are maintained, validated and calibrated at specified intervals for safe, effective, and consistent operation. A team of dedicated operators, engineers, and scientists who are trained for their respective and applicable functions in areas of safety, aseptic standards, and manufacturing processes will perform unit operations within the cGMP Facility. Control of Raw Materials

[0201] For production use, raw materials are GMP-grade and / or suitable for their intended use. If GMP-grade material is not commercially unavailable, the usage is defaulted to the next available highest standard recommended for manufacturing for phase 1B clinical trials and reviewed for suitability. All materials are quarantined and subjected to inspection upon arrival and prior to consumption. The Certificate of Analysis (CoA) is compared to the approved raw material specification before being released by Quality Assurance (QA).

[0202] Isolation of SBSB (General Cell Bank) strains:201720021.2 - 43 -

[0203] Bacterial strains were cultured in anaerobic chambers upon dilution of approximately 100 mg feces in 900µl of sterile phosphate buffered saline (PBS). Dilution tubes were prepared with 900µl of sterile PBS and 100µl of sample was transferred (i.e. a 10-fold dilution) serially into 10 tubes down to a final 10-10dilution. Following serial dilution, 50µl of each dilution was plated onto the agar plates and incubated anaerobically at 37°C for 2-5 days. Plates were evaluated for the number of colonies, and plates with 100-150 colonies were chosen for colony harvesting. Colonies were picked with a sterile probe and split between a single well of a PCR plate and onto a labeled replica plate. Colony PCR was performed on the samples in 96-well plate using 16S rRNA primers, and purified amplicons were Sanger sequenced for genus / species identification and determining purity. Replica plates were incubated at 37°C, and specific isolates were selected for purification based on 16S rRNA sequencing results. Replica plate colonies were re-streaked twice to ensure purity and whole genome sequenced for strain characterization.

[0204] Table 7.1: Isolates and primary stock media Isolates Media (Agar)

[0205] Isolates for the SBSB / General Cell Bank were obtained from healthy donor fecal samples or samples from screening colonoscopy exams. The samples were inoculated onto201720021.2 - 44 -multiple different media types, and the isolates shown were grown and isolated on the indicated agar or media type. This media was used in both the initial strain isolation and in the replicate stock generation. Replicate stock generation of SBSB strains:

[0206] Primary stocks of newly isolated bacterial strains were generated by plating a 100 µl strain culture on specified agar plates (Table 7.1) for 24-48 hours until bacterial lawns were formed. Lawns were transferred into 8 to 10 cryovials with pre-aliquoted PBS and glycerol stock to obtain a final concentration of 20% glycerol in each vial. Pre-master and Master Cell Bank generation:

[0207] The medium formulation components used for manufacturing of the pre-Master and Master Cell Bank (MCB) and Drug Substance are listed in Appendix Table 7.2. The media were used for strain passaging, seed inoculum, fermentation and lyophilization. Whenever possible, chemicals are free of animal-derived compounds. In cases where animal-free components are unavailable, the inventors have obtained transmissible spongiform encephalopathies / bovine spongiform encephalopathies (TSE / BSE) free statements to justify use in the phase 1B clinical trial. The GMP Optimized Media used for cultivation is strain dependent. Initially, base media is manufactured, and additional supplements are added from stock solutions to prepare media prior to use for various bacterial strains. Control of the Starting Material

[0208] The Master Cell Bank (MCB) used for production of drug substance was manufactured from a well-defined and controlled pre-MCB. The Pre-MCB was made from isolates in the General Cell Bank (GCB) in a controlled research environment, using GMP manufacturing processes and protocols, with defined raw materials and sterile supplies, as described herein. Fecal specimens were obtained from healthy human donors who were screened for lack of antibiotic exposure and intestinal, autoimmune, inflammatory or metabolic disorders. Feces were cultured under strict anaerobic conditions on a variety of rich media designed for cultivation of commensal bacterial species. FIG. 4 outlines the Pre-MCB manufacturing processes. Pre-Master Cell Bank:

[0209] The pre-master cell bank for the MARCO Trial commensal strains was generated by culturing each strain repeatedly in GMP-grade medium (Table 7.2). For each strain, 50 µl of the primary General Cell Bank stock was inoculated into 5ml GMP-grade medium, and cultured for 24 hours. Then 40µl of culture was transferred into 40ml GMP grade medium, achieving a 1:1000 dilution of inoculum. Repeat culturing for two additional cycles at 1:1000201720021.2 - 45 -dilutions resulted in a net 10-11dilution of primary stock media. Freezer stocks of the pre- master cell bank strains were generated by transferring 500 µl of the final dilution culture into freezer tubes containing 500 µl of glycerol stock to achieve a final concentration of 20% glycerol prior to freezing at -80°C. Three tubes of each strain were selected for whole genome sequencing to validate the pre-master cell bank stocks. The pre-master cell bank contains 30- 40 tubes of each MARCO Trial strain. This process is depicted in FIG.5. Table 7.2: Isolates and optimized growth media Isolate and strain ID Base Media Supplementg used for manufacturing. Information on the GMP Grade Optimized Media used for each bacterial strain for manufacture of the pre-master cell bank, master cell bank and Drug Substance is shown. Master Cell Bank:

[0211] The MCB was manufactured in the cGMP environment by passaging the pre-MCB once with GMP Grade Optimized Media (FIG. 6). The MCB includes samples used for manufacturing, samples used for QC release testing and samples collected for QC and QA retains. Prior to use, several quality control release tests (Refer to Table 7.3) will be performed on the MCB, thus making it suitable for Phase 1b clinical trial use.

[0212] To generate the master cell bank, the contents of one pre-master cell bank freezer tube (1.0 ml) will be inoculated into 100 ml of GMP grade medium and cultured to late log phase (6 to 18 hours of culture depending on strain). Upon reaching late log phase, the inventors will generate 50 MCB aliquots by mixing 2 ml of culture with 2 ml of glycerol stock prior to201720021.2 - 46 -freezing at -80°C in a total volume of 4 ml and a glycerol concentration of 20%. All steps of master cell bank generation will be conducted in a Vaporized Hydrogen Peroxide sterilized anaerobic chamber to enhance viability while reducing the risk of contamination. To determine the composition of the master cell bank, one freezer vial will be thawed, gram stained, cultured and subjected to whole genome sequencing and metabolomic analysis to ensure purity, genomic integrity (i.e. presence of critical genes for desired functions) and metabolic capacity (i.e ability to produce short-chain fatty acids and to modify bile acids) (Table 7.3).201720021.2 - 47 -

[0213] Table 7.3: Release Testing for Master Cell Bank Manufacturing Step Control Criteria Justification t d o k in s na uacu g o e ug u sa ce

[0214] The process flow diagram depicted in FIG.7, outlines the intended manufacturing processes for Live Biotherapeutic Products Manufacturing. The three main phases in the manufacturing processes are, Master Cell Bank, Drug Substance and Drug Product manufacturing. The intended final product is a non-sterile, bioburden reduced, orally ingested201720021.2 - 48 -drug product. The drug product is a lyophilized form of bacteria (single strain) in a single capsule, which is placed in a small, controlled bottle, with an aluminum seal, along with a tamper-proof cap and appropriate labeling.

[0215] The commensal bacteria strains used for production are sensitive to oxygen, thus Master Cell Bank and Drug Substance production activities are carried out within controlled anaerobic environments. Throughout the manufacturing processes, characterization, IPT (In Process Testing) and QC (Quality Control) Release Testing have been established to assess process performance and ensure product quality for use in Phase 1b clinical trials.

[0216] Where possible, manufacturing operations are conducted in closed-processing systems (bioreactor, tangential flow filtration (TFF), lyophilization (LYO), etc.). When this is not achievable, open processing is carried out within certified anaerobic chambers and laminar flow biosafety cabinets.

[0217] Each strain of bacteria is prepared, grown, fermented, concentrated, and lyophilized using individual controlled processes resulting in independent and unique Drug Substances and Drug Products that are released, characterized and evaluated for identity, stability, purity, and safety. Manufacturing Process and In-Process Testing

[0218] The Drug Substance manufacturing process is presented step by step in FIG. 8. Bacterial strains used to manufacture drug substances will be initially inoculated and grown in single-use shake flasks using GMP Grade Optimized Media. The mixture will then be further cultured using fermentation methods in an enclosed bioreactor. Once fermentation is completed the mixture will be purified and concentrated using the Tangential Flow Filtration (TFF) system.

[0219] For bacterial strains that can grow to high densities through lower-volume culturing, the fermentation process is eliminated as shown in FIG.9.

[0220] Drug Substance Process

[0221] Steps 1 & 2: Inoculum, Seed Optimization, Fermentation, & IPT

[0222] During the manufacturing process, individual bacterial strains are grown in a Sartorius STR 50L bioreactor using sterile single use bags filled with GMP Grade Optimized Media in their individual optimized conditions to achieve the desired Optical Density (OD600) and concentration of CFUs.201720021.2 - 49 -

[0223] The process starts with the inoculation of a shake flask filled with GMP Grade Optimized Media to create the seed inoculum from the Master Cell Bank. This seed inoculum is grown for 12-24 hours in a shaker incubator to optimal OD600and concentration of CFUs before pumping into the 50L bioreactor. The bioreactor is filled with GMP Grade Optimized Media and oxygen levels are lowered and controlled with the continuous sparging of nitrogen gas. When conditions in the bioreactor are optimal, the seed inoculum is introduced and, thus, begins the batch fermentation process. Depending on the bacterial strain, the process can take 24-72 hours to complete. During the in-process batch fermentation, dissolved oxygen, pH and temperature levels are continuously monitored and regulated. Characterization and In-Process Testing (IPT) is performed before, during, and after fermentation (Table 7.4).

[0224] Steps 1 & 2 (Low Volume Culturing): Inoculum, Seed Optimization, Low Volume Culturing (instead of fermentation), & IPT

[0225] During manufacturing, strains can also be grown under low volume conditions in the anaerobic chamber. Seed inoculum will be prepared from the master cell bank to a volume of 100ml to optimal OD600 and concentration of CFUs. Seed inoculum will be incubated in an incubator shaker at 37°C in the anaerobic chamber. This culture will be inoculated into two sterile bottles with 1L GMP Grade Optimized Media that have been pre-reduced in the anaerobic chamber. Cells will be harvested after 5-10 hours, depending on the isolate. This process will be used for isolates that are rapid growers that achieve high cell density within a short duration without continuous pH monitoring and control. This process will be performed in a controlled environment (anaerobic chamber) under anaerobic conditions with controlled temperature. Characterization and In-Process Testing (IPT) is performed before and after this process as described in Table 7.4.

[0226] Step 3: Purification, Concentration and Diafiltration within TFF System

[0227] Tangential Flow Filtration (TFF) is a quick and efficient method used to concentrate and buffer exchange bacterial cultures using ultrafiltration membranes. To begin the process, the bioreactor is connected via sterile connectors and tubing and the finished batch fermentation product is pumped into the anaerobic chamber where the Repligen TFF system is located. The Repligen TFF system allows for the monitoring, data collection, and automatically adjusts the pressures within the system. Transmembrane pressure (TMP) is monitored via sensors at the feed, retentate, and permeate ports and is automatically adjusted throughout the process. During the process, the bacterial culture is concentrated and, by diafiltration exchange, the culture media and its components is replaced by the buffer solution (either 10% sucrose or 10% sucrose and 5% trehalose) for subsequent lyophilization. The Drug Substance (DS) has been created201720021.2 - 50 -when the fermentation product from the bioreactor has been concentrated by a factor of 20X and 5 diafiltration volumes with the buffer solution (either 10% sucrose or 10% sucrose and 5% trehalose) have been achieved.

[0228] When 2L low volume cultures are ready for harvest, culture flasks will be connected to the TFF system (refer to table 7.4 for release criteria of the static culture) and contents transferred into the TFF feed carboy via a peristaltic pump. The TFF process will be conducted, concentrating the bacterial culture by diafiltration exchange, replacing the culture media and its components with the buffer solution (either 10% sucrose or 10% sucrose and 5% trehalose) to create the DS for subsequent lyophilization.

[0229] Table 7.4 outlines the quality control (QC) release testing that are performed to assess process performance and ensure the quality of the Drug Substance. After the release tests are completed the samples are stored at -80oC to preserve viability.

[0230] Table 7.4: Drug Substance Manufacturing Process and Release Testing Manufacturin Contro g Step l Criteria Justification of e of d . of201720021.2 - 51 -Manufacturin Contro g Step l Criteria Justification e in is d al og Description and Composition

[0231] The drug product consists of 8 lyophilized bacterial strains, each contained in an individual capsule. The final drug product, consisting of 8 capsules that each contain a distinct bacterial strain, will be administered orally to study participants every 24 to 72 hours over the course of 10 days.201720021.2 - 52 -Drug Product Process Step 1: Lyophilization

[0232] Lyophilization (freeze-drying) removes water from the concentrated drug substance, providing product stability and the ability to encapsulate it. The lyophilization buffer is either a 10% sucrose solution or a 10% sucrose & 5% trehalose solution (i.e. the TFF buffer solution), which acts as a lyoprotectant by maintaining the integrity of bacterial cell membranes and preserving viability of bacterial strains. The lyophilization process involves three main steps:

[0233] Freezing of the concentrated drug substance at -80° C following TFF.

[0234] Primary lyophilization at low temperatures (-40°C to -25°C) under vacuum.

[0235] Desorption stage where remaining water is slowly sublimated and actively monitored by sensors within the lyophilization chamber to develop a freeze dried ‘cake’ of concentrated drug product.

[0236] Within an ISO 5 cleanroom environment, the lyophilized drug product is then taken off the lyo trays with a sterile spatula and placed into the vessel of the IKA Multidrive Basic Mill. The vessel is sealed, placed onto the IKA Multidrive Basic Mill, and milled at 3,000 RPM for 3 minutes. The milled product is a fine, homogenized powder that is then placed onto the capsule filler to start the capsule filling process. Steps 2, 3 & 4: Encapsulation, IPT, Bottling, Sealing, Labeling, & Storage at -80oC

[0237] Encapsulation of lyophilized product is performed using a sterilized ProFiller Capsule filling apparatus. Lonza capsules are assembled in the pre-autoclaved ProFiller. In order to proceed with filling each capsule, following a defined protocol, capsule caps are decoupled. Lyophilized product is homogenized using IKA MultiDrive Basic Mill and placed on the capsule filler. Then, using a flat sterile spatula, the contents are carefully filled into each capsule body. Contents are gently pressed down using a Tamper to ensure consistent amounts of product are placed in each capsule. Capsules are reassembled and locked in place. The encapsulation process is performed in a biological safety cabinet (BSC) to prevent contamination. Capsules are bottled by induction sealing in a tamper-proof bottle with a hand- tight cap. Upon completion, bottles will be labeled and stored at -80oC to preserve viability. Stability tests are performed periodically by determining viability of the Drug Product over time (Table 7.8).

[0238] Table 7.5 outlines the quality control release testing that will be performed to assess process performance and ensure the quality of the Drug Product. The capsules the inventors intend to use for production are manufactured by Lonza. These capsules (Capsulgel Enprotect201720021.2 - 53 -Sz 0 Caddy) are well defined, and controlled. Manufacturer testing is for capsule dissolution and disintegration are shown in Table 7.9. The inventors will also perform USP<701> on Drug Product to determine capsule disintegration of the product.

[0239] Bioburden and enumeration of specified non-product organisms will be tested in the MCB, Drug Substance, and Drug Product using USP<61> and USP<62> tests at the times indicated in Tables 7.3, 7.4, and 7.5. Although USP<61> and USP<62> are not designed for testing with products containing live organisms such as ours, the inventors have performed these tests on the Drug Product of each strain in Consortium A, and results are shown in Tables 7.6, 7.7 and 7.8. These have demonstrated Passing results for all tests with the exception of the QC organism C. sporogenes, which is an obligate anaerobe similar to the product. Since all of the organisms in the MCB, drug substance, and drug product are anaerobes, distinguishing growth of the organisms from other obligate anaerobes such as C. sporogenes using USP<62> is not possible. Therefore, instead of performing USP<62> to detect the presence of contaminating obligate anaerobes, the inventors use an alternative approach to enumerate obligate anaerobe contamination. This entails extracting DNA from MCB, Drug Substance and Drug Product and spiking with defined amounts of DNA from another obligate anaerobe (such as C. sporogenes; e.g. 0%, 0.01%, 0.1%, and 1% C. sporogenes DNA) followed by shotgun short-read sequencing. This approach will demonstrate that obligate anaerobe contamination, if present, can be accurately deteremined even at low levels. For MCB and DS, a passing result will only be obtained with 0 non-MCB or non-DS organism growth in USP<61>. For DP, which is a non-sterile oral medication, a passing result requires non-product bioburden less than the recommended thresholds for USP<61> / USP<62> testing as shown in Table 7.5.

[0240] Table 7.5: Quality Control Release Testing for Drug Product Manufacturing Manufactu Control Criteria Justification te he & nd201720021.2 - 54 -Manufactu Control Criteria Justification ring Step ns r. at ne in an > ia te he ty st201720021.2 - 55 -Manufactu Control Criteria Justification ring Step he to a t. re In er ed nt al201720021.2 - 56 -Manufactu Control Criteria Justification ring Step of ng > ct ill ng m in ct ng201720021.2 - 57 -

[0241] Table 7.6: USP<61> microbial enumeration testing results from Drug Product for Consortium A USP 61: Microbial Enumeration f l n s, a

[0242] Table 7.7: USP<61> percent recovery for suitability from Drug Product for Consortium A USP61: % Recovery for Suitability201720021.2 - 58 -C. comes 85 61.5 96.7 133.3 A h dr 90 962 130 896 ngia: ult. USP62: % Recovery of Specified Microorganism s ng ia: % F)

[0244] Table 7.9: Capsule stability and protection against acid exposure Dissolution Performance Disintegration Performance Mechanical Robustness ed al201720021.2 - 59 -in acid stage (HCL 0.1N) for minutes in the buffer stage properties in all test up to 2 hours. (PH 6.8). conditions. er ty en ne. g Method Attribute Tested Criteria 0 or s. no he es .

[0247] The intended bottles for this application are PharmaSure 40 cc Artistic Series Wide Mouth Pharmaceutical Round Bottles. These bottles have a capacity of 40 ml / cc / 1.4 oz. The height, the width and the depth of each bottle are 59.54 mm, 38.1 mm and 38.1 mm, respectively. The bottles will be paired with PharmaSure 33mm SecuRX Ribbed Side Caps. The bottles and caps are manufactured by Drug PLASTICS Company.

[0248] Capsules are contained in a 7-day unit dose, 40 mL opaque white HDPE wide- mouth pharmaceutical bottle with a foil seal and threaded ribbed cap. Bottles are made from food grade HDPE resin and colorant. Caps are made from food grade polypropylene with U.S. FDA 21 CFR 177.1210 compliant foil liner. The inner liner consists of a laminate structure of201720021.2 - 60 -0.0015” Heat Seal (bottle-facing), 0.005” PET, 0.001” Foil, 0.035” backing and 0.035” Pulp. Please reference FIG.11 for bottle and cap structure. Stability Testing

[0249] FIG. 12 outlines the results obtained on capsules stored at -80oC to determine stability of drug product and assess if the quality (i.e. cell viability) is compromised by long- term storage under the above conditions. Viability will be evaluated at time intervals post- lyophilization by enumerating colony-forming units on appropriate agar plates. This test will help determine the shelf-life of the product. Drug Product stability data have been determined for ≥ 1 month for the 8 strains constituting Consortium A, which will be administered in first stage of the MARCO Trial. Stability data for those 8 strains, and the additional 9 strains that will be considered in the second stage of the trial will be collected on an on-going basis as part of the stability protocol (months 1, 2, 3, 4, 6, 9, 12, 15, 18, 21, and 24). Prior to any strain being administered in the clinical trial, there will be a minimum of 1 month passing stability data, with stability studies on-going in parallel. If there is a loss of >2 log10 CFU or if there is < 105CFU per capsule, this will lead to rejection. In addition, USP <61> and USP <62> testing will be integrated into the stability protocol for bioburden and enumeration of non-product organisms every 12 months for the Drug Product, which is stored at -80°C. USP<701> will also be integrated into the stability protocol for disintegration testing after every 4 months of storage at -80°C. If capsules do not fully disintegrate in ≤ 30 minutes using USP<701>, the Drug Product will be rejected. Results through 7 months are shown in FIG.12.

[0250] The inventors will also perform a stability protocol to test the stability of drug product at room temperature after removing from storage at -80°C. Briefly, after removal from storage at -80°C, drug product will be plated for CFU hourly for 5 hours post-removal. The results for one strain, B. ovatus MSK.18.37, are outlined in Table 7.12. This test will be performed on each drug product. Table 7.12: -80oC to Room Temperature Stability Target s 0201720021.2 - 61 -

[0251] Table 7.12: Stability of drug product after removing from -80°C to room temperature at the time of removal and hourly for 5 hours after removal.

[0252] Table 7.13 lists the major equipment used for DS and DP manufacturing processes. Table 7.13: Manufacturing Equipment Equipment Vendor Model Purpose n to d

[0253] Appendix Table 7.1: Definitions, Abbreviations and Terms Terms Definitions201720021.2 - 62 -Terms Definitions LYO Lyophilization e,201720021.2 - 63 -

[0254] Appendix Table 7.2: Raw Material for preparation of MCB, Drug Substance and Media Formulation Item Description / Manufacturer Cat# Grade Acceptance Criteria Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O201720021.2 - 64 -Item Description / Manufacturer Cat# Grade Acceptance Criteria Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O Q O201720021.2 - 65 -

[0255] Appendix Table 7.3: Liquid YT Media Components Liquid Stocks for 1L of YT LSolids for 1L of YT201720021.2 - 66 -

[0257] Appendix Table 7.5: Liquid YT Media Stock Components (Mineral I Stock Solution) 1L of 10X Mineral I Solutionk Solution) 1L of 3X Mineral II Solutionn Stock Solution) 100mL of Porcine Hemin Solution[ ] ppen x a e .: qu e a oc omponens ( amn ock Solution) 500mL of Vitamin Solution I201720021.2 - 67 -Folic acid 25.0 mg Pridoxine·HCl 750 m kSolution) 500mL of Vitamin Solution II[ ] ppen x a e . : qu e a omponents Liquid Stocks for 1L of TPY ASolids for 1L of TPY201720021.2 - 68 -Zinc Sulfate 0.25gFeSO4 Stockppe a e . : e a qu oc o po e s oc e e n) 100mL of Porcine Hemin Solution201720021.2 - 69 -Example 6 - Pharmacology and Toxicology Information Introduction

[0265] The drug product will contain 8 capsules of bacterial strains that have been lyophilized in a substrate of sucrose or sucrose and trehalose. Each capsule will contain a single bacterial species. Structural Formula of the Drug

[0266] Consortia Strains: Below is a list of the strains included with their strain label and the link to their NCBI whole genome sequence. Bile acid deconjugators: Bifidobacterium longum DFI.7.60 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143939.1) Bifidobacterium pseudocatenulatum DFI.7.61 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143938) Bifidobacterium bifidum DFI.7.51 (https: / / www.ncbi.nlm.nih.gov / nuccore / JAKNHM000000000) Secondary bile acid producer: Clostridium scindens SL.1.22 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143935) Polysaccharide metabolizer and secondary bile acid modifier: Bacteroides thetaiotaomicron DFI.6.40 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143941.1) Bacteroides ovatus MSK.18.37 (https: / / www.ncbi.nlm.nih.gov / nuccore / JAHOIB000000000) Bacteroides cellulosilyticus DFI.5.60 (https: / / www.ncbi.nlm.nih.gov / nuccore / JAJCMC000000000) Phocaeicola vulgatus DFI.4.81 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143952.1) Parabacteroides distasonis DFI.5.23 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143949.1) Parabacteroides merdae DFI.4.73 (https: / / www.ncbi.nlm.nih.gov / nuccore / JANGCV000000000)201720021.2 - 70 -Secondary bile acid modifier: Eggerthella lenta DFI.5.72 (https: / / www.ncbi.nlm.nih.gov / nuccore / JANGCM000000000) Butyrate producer: Anaerostipes hadrus DFI.4.30 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143954) Anaerostipes caccae DFI.7.76 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143937) Clostridium symbiosum DFI.5.64 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143946) Coprococcus comes DFI.3.84 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143955) Anaerobutyricum hallii DFI.9.38 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143936) Eubacterium rectale DFI.5.28 (https: / / www.ncbi.nlm.nih.gov / nuccore / CP143947.1) Consortia Strain Composition:

[0267] Consortia were designed to 1) produce butyrate and 2) facilitate bile acid metabolism from conjugated primary bile acids (which are secreted by the liver) to bioactive secondary and modified secondary bile acids. Every consortium contains 8 different bacterial strains, including two different organisms that produce butyrate, at least one organism that can deconjugate conjugated primary bile acids, one that can convert primary to secondary bile acids, and one that can modify secondary bile acids. Strains that metabolize a wide range of polysaccharides are also included in each consortium. Strain engraftment is expected to be variable, so the inventors have included functional redundancy into the consortia (e.g. 2 butyrate producers per consortium) for a total of 8 bacterial strains per consortium. Unique consortia were assembled to change the members based on the metabolic capacity of consortium A according to the adaptive trial design. The composition of each of the consortia (A through E.2) are shown in Table 8.1.201720021.2 - 71 -

[0268] Table 8.1: Consortia Compositions Consortia Function Organism A B1 B2 C1 C2 D1 D2 E1 E2 B. longum DFI.7.60 x x x x x x x x x x x x

[0269] Table 8.1: Consortia Compositions. Table of composition of each consortium. Columns corresponds to a consortium to be used in the adaptive trial design. Rows correspond to each organism. Cells are shaded blue if the consortium contains that organism and are white if not included. C. scindens, B. ovatus, and E. lenta are present in all consortia. Strain and Consortia Capacity to Perform Metabolic Functions:

[0270] The ability of strains to perform specific metabolic functions was determined in vitro as individual strains and within assembled consortia. To test the ability of strains to catalyze a specific bile acid conversion, they were grown in media supplemented with different bile acids. For example, Bifidobacterium species, which are included for their ability to deconjugate bile acids, were grown in media enriched with conjugated primary bile acids (e.g. taurocholic acid) to test their ability to convert these to unconjugated primary bile acids (e.g. cholic acid). Similarly, C. scindens was grown in the presence of primary bile acids to201720021.2 - 72 -determine the capacity to make secondary bile acids, and E. lenta was grown in lithocholic- acid spiked media to determine modification to 3-oxo-lithocholic acid. All media contained a carbon source that butyrate producers could use for butyrate production. This experimental design and results are shown in FIGs.13A-13D.

[0271] The ability of each consortium (A through E.2) to catalyze multiple steps of bile acid metabolism from conjugated primary bile acids to secondary and modified secondary bile acids was tested in vitro by growing each consortium in media containing either taurocholic acid (TCA) or taurochenodeoxycholic acid (TCDCA). Each consortium grew efficiently with final relative abundances shown in FIGs. 14A-14E. Consortia also efficiently deconjugated TCA and TCDCA and generated both secondary and modified secondary bile acids. This experimental design and results are depicted in FIGs.14A-14E.

[0272] Finally, each consortium was also administered to germ-free (GF) mice to evaluate in vivo colonization of the gut and metabolite production by MARCO Trial commensal bacteria consortia A, B1, B2, C1, C2, D1, D2, E1 and E2 (FIGs. 15A-15E) and to determine whether these consortia damage the ileal or colonic epithelial barrier or lead to bowel inflammation (toxicology studies). All mouse studies were approved by The University of Chicago Institutional Animal Care and Use Committee (IACUC, Protocol 72599) and began with colonization of germ-free mice. Germ-free C57BL / 6 mice were 6-18 week-old wild type (WT) and had been initially obtained from The Jackson Laboratory and subsequently bred and raised in a germ-free isolators. After removal from the germ-free isolator, mice were handled in a sterile manner and individually housed in sealed negative pressure (BCU) isolators. Throughout breeding and experimental conditions, mice were housed within the University of Chicago Gnotobiotic Research Animal Facility (GRAF). The GRAF is operated with adherence to the Guide for Care and Use of Laboratory Animals, 8th edition, 2011, National Academy of Sciences. This includes standardized 12-hour light / dark cycles (light from 6AM - 6PM), centrally monitored temperature (maintained between 68°F and 79°F) and centrally monitored humidity (maintained between 30-70%). Gnotobiotic mice were fed an ad libitum diet of autoclaved Teklad Global 18% Protein Rodent Diet (Sterilizable) (2018S / 2018SC).

[0273] Commensal bacterial strains were grown to early steady state as measured by OD600and normalized to OD600 = 0.3. Depending on individual strain requirements, they were grown in BHIS, Wilkins-Chalgren (Fisher), de Man, Regosa, and Sharpe (MRS) broth (Fisher), or modified Yeast Casitone Fatty Acids (YCFA) medium. Stocks of the consortia were stored at -80°C in 20% glycerol, 0.1% cysteine until ready for use. For each consortium, 12 germ-free201720021.2 - 73 -mice were gavaged with 200mL of consortium mix constituted with equal volumes of each of the 8 consortium bacterial strains for 3 consecutive days. Four mice were given regular sterile water and 4 mice each were given sterile water supplemented with filter sterilized lactulose or inulin at a final concentration of 20g / L of water. Mice were monitored daily for signs of illness, including scruffy fur, lethargy and diarrhea. Fecal pellets were collected on days 7 and 14 for microbiome and metabolome analyses. On day 15, mice were euthanized and cecal contents were taken for microbiome and metabolome analyses and tissue from the ileum and colon was harvested for histologic analysis. Mice were grouped to equalize the number of male and female mice in each group, but no formal randomization was done due to scarcity of gnotobiotic animals. While there was no formal blinding, microbiome data was collected and analyzed by core facilities with staff that were blinded to the experimental design (commensal exposure and treatments) that microbiome data was linked to.

[0274] To determine intestinal colonization by the 9 MARCO Trial consortia, fecal pellets and cecal contents were extracted and subjected to 16S rRNA gene sequencing. FIG. 15B shows aggregate microbiome compositional data (by 16S rRNA analysis) for each of the 9 commensal consortia in mice treated with water. Mice were stably colonized with between 6 to 8 of the donor strains, that colonization was similar between mice treated with water, lactulose or inulin, but that lactulose consistently increased representation of Bifidobacteria.

[0275] Fecal samples were also subjected to targeted metabolomics to determine fecal concentrations of short chain fatty acids and specific bile acids (FIGs.15A-15E). GF mice have low levels of short chain fatty acids, including absent butyrate concentrations. The bile acid pool is also heavily shifted towards conjugated primary bile acids (i.e. taurocholic and taurochenodeoxycholic acid), and GF mice have absent secondary and modified secondary bile acids. After colonization with consortia, the primary, secondary, and modified secondary bile acids are generated. Colonization of mice intestines with human-derived organisms that produce butyrate was variable, but mice that were colonized with butyrate producers had significantly increased fecal butyrate concentrations. Formulation of the Dosage Forms

[0276] Each microbe will be lyophilized in a substrate of either 10% sucrose or 10% sucrose with 5% trehalose, milled into a fine powder and packed into an enteric, time release capsule. Each capsule will contain a concentration of live bacterial cells between 1x105and 1x1010total organisms per capsule.201720021.2 - 74 -201720021.2 - 75 -Route of Administration

[0277] Oral Pharmacology Pharmacological Effects

[0278] The pharmacological effect of the drug is to reestablish specific metabolic functions of the gut microbiota through production of butyrate and secondary and modified secondary bile acids. These metabolites are associated with improved intestinal epithelial barrier function and mucosal immune function as well as reduced intestinal burdens of pathogenic bacteria. Clinically, losses of these metabolites have been associated with poor outcomes in patients with liver disease, including systemic bacterial infections. Mechanism of Action

[0279] Each drug consortium is assembled with the goal of colonizing the lower intestinal tract and cooperatively metabolizing enteric contents and producing short chain fatty acids and secondary bile acids (FIG. 16 Mechanism of Action). Butyrate is produced by bacterial fermentation of carbohydrates in a stepwise fashion that includes breakdown of dietary complex polysaccharides and fibers into simpler sugars that are fermented to produce short chain fatty acids, including butyrate. Microbial metabolism of bile acids also involves multiple steps that begin with conjugated primary bile acids (taurocholic acid, taurochenodeoxycholic acid, glycocholic acid, and glycochenodeoxycholic acid) that are produced by the liver and delivered into the intestinal lumen via bile ducts and the gall bladder. Commensal bacteria, including Bifidobacteria species, mediate bile salt deconjugation by expressing bile salt hydrolases. After deconjugation, primary bile acids are dehydroxylated at carbon 7 by C. scindens, producing the secondary bile acids deoxycholic and lithocholic acid. Additionally, some commensal bacterial species encode reductases and hydroxysteroid dehydrogenase genes that further modify secondary bile acids to other forms such as 3-oxolithocholic acid. These short chain fatty acids and secondary bile acids can inhibit growth of pathogenic bacteria, promote gut barrier integrity, and modulate the immune system.1–10201720021.2 - 76 -Absorption, Distribution, Metabolism, and Excretion

[0280] The drug product contains live bacterial strains that reside in the intestinal lumen. The bacterial strains do not encode genes associated with mucosal or tissue invasion and the inventors are excluding patients with known intestinal barrier dysfunction. Thus, the administered strains are not expected to traverse the epithelial barrier and access sterile tissues. The bacterial strains are expected to colonize the GI tract and will be expelled in feces without traversing the epithelial barrier and accessing sterile tissues.

[0281] The metabolic products of commensal bacteria (e.g. butyrate and secondary bile acids) can be absorbed by intestinal epithelial cells and gain access to the portal circulation. Butyrate is a nutrient used by enterocytes and colonocytes, and little is systemically absorbed.11While some studies suggest that butyrate is distributed systemically (e.g. in liver, adipose tissue and in the central nervous system),11the inventors have detected negligible serum butyrate concentrations even in patients with high fecal butyrate concentrations. Because secondary bile acids are chemically diverse, their absorption, distribution, metabolism, and excretion cannot be generalized. Of the secondary bile acids that are absorbed, they flow via the portal vein to the liver and can be reprocessed by the liver and excreted back into the bile via enterohepatic circulation.12Consistent with this, the inventors also detect negligible levels of bile acids in the serum of patients with liver disease, except for patients with significant cholestasis, who have high serum levels of conjugated primary bile acids, which are produced by the liver. Because the MARCO Trial strains are derived from and normally reside in the intestines of healthy humans, the inventors anticipate absorption, distribution, metabolism, and excretion of their metabolites to approximate that in detected in healthy humans. Toxicology Introduction

[0282] Bacterial strains may cause infection and / or transmit pathogenicity genes to other bacteria. To mitigate the risk of infection following commensal strain administration, each of the bacterial species was derived from a healthy donor / A comprehensive literature review was performed to determine the potential for pathogenicity of each organism. Each species has been whole genome sequenced to assess for virulence factors as well as antibiotic resistance genes. Mouse toxicity studies of each of the bacteria as well as combinations of bacteria were performed (FIG. 16). Finally, to ensure any product derived infections can be treated,201720021.2 - 77 -antimicrobial sensitivity testing has been performed for each strain in accordance with CLSI. The literature review, genetic analysis, and antimicrobial sensitivity testing is described below. Integrated Summary of the Toxicity Studies

[0283] Infection is a concern associated with administration of LBPs. Human infections can result when microbial organisms that inhabit the intestinal lumen gain access to sterile body sites following compromise of tissue barriers. Thus, commensal organisms that provide health benefits when they are confined to the gut lumen have been implicated as causes of infection following, for example, bowel perforation. To address the virulence potential of the strains included in the MARCO trial, the inventors have searched the published literature for associations between specific commensal bacterial species and systemic infections. The inventors have categorized MARCO strains according to their anticipated metabolic function within each consortium. Whole Genome Sequencing

[0284] Each strain has been whole genome sequenced and the sequences have been uploaded to NCBI.gov. To screen for possible virulence factors or antimicrobial resistance genes, each organism sequence was compared to Virulence Factor Database (VFDB)61and Comprehensive Antibiotic Resistance Database (CARD).62Additional analysis for antimicrobial resistance genes not indexed in the CARD database was performed using Pathosystems Resource Integration Center (PATRIC).63Genotypic Analysis for Virulence Factors

[0285] The whole genome sequence of each strain was compared to VFDB, a publicly available database of confirmed and putative virulence factor genes. Homologous genes were considered present if any sequence with ≥70% sequence identity was identified. No sequences with homology to genes known to mediate pathogen invasion, effector delivery systems, biofilm, exotoxin, or exoenzymes were identified. Phenotypic Antimicrobial Sensitivity Testing

[0286] Each bacterial strain was tested for sensitivity to clinically relevant antibiotics for treatment of anaerobic infection in accordance with CLSI standards (Table 8.3).71Each organism has at least 5 antibiotics with reliable clinical activity as defined by CLSI201720021.2 - 78 -breakpoints.72Two drugs, Ampicillin / Sulbactam and Meropenem, have activity against all 17 strains and could be administered empirically if an infection was suspected. Each organism has at least one non-b-lactam antibiotic with activity in the event a patient has a penicillin, cephalosporin, and / or carbapenem allergy. Patients will be regularly screened for symptoms of infection as outlined in the study protocol. If a drug derived infection is identified, antibiotics will be given in accordance with the sensitivity data. Table 8.2 Antibiotic Sensitivities: Metro. Amp. Amp. / Sulb. Pip. / Tazo. Mero. Clinda. Cefox. CLSI Breakpoint ug / mL 16 1 16 / 8 32-64 / 4 16 4 32 2. g g g n this clinical study against commonly used antibiotics. Sensitivities were determined using agar dilution assays in accordance with CLSI.71Interpretive categories were color coded green- sensitive, yellow-intermediate, red-resistant.72Each isolate has at least 5 active agents. Ampicillin / Sulbactam and Meropenem are active against every isolate. E. lenta was inhibited at 32 / 4 ug / mL and 64 / 4ug / mL of Pip. / Tazo. both being intermediate. C. comes was inhibited at 16ug / ml and 32ug / ml of cefoxitin, deference was given to the higher MIC and categorized as intermediate. Abbreviations: Metro. – Metronidazole; Amp. – Ampicillin; Sulb. –201720021.2 - 79 -Sulbactam; Pip. - Piperacillin; Tazo. – Tazobactam; Mero. – Meropenem; Clinda. – Clindamycin; Cefox. – Cefoxitin. Genotypic Analysis for Antibiotic Resistance

[0288] The whole genome sequences of each organism were compared to CARD, the comprehensive database of curated antimicrobial resistance genes.62The inventors used their resistance gene identifier tool (RGI;CARD 3.2.8) to screen each genome for perfect and strict hits on established antimicrobial resistance genes. Perfect hits have 100% homology and likely result in resistance. Strict hits indicate a gene with partial homology to an established gene but its spectrum has not been demonstrated phenotypically (FIG.17).64Among the 17 strains, one perfect hit was identified for a folate metabolism gene that causes sulfonamide resistance was detected in C. scindens.65Thirteen strict hits were identified for additional genes. The gene adeF encodes a single protein in a multiple protein efflux pump that causes fluoroquinolone and tetracycline resistance, but none of the other necessary genes were detected.66Tetracycline resistance genes were common and not unexpected given their ubiquity in gut anaerobes.67B. thetaiotaomicron had an Erm gene that causes clindamycin and erythromycin resistance.68Seven genes homologous to those involved in vancomycin resistance were identified and present in many of the strains. Vancomycin resistance requires a functioning cluster of genes including regulatory, core, and accessory genes.69Of the organisms containing partial hits for Van genes, no complete gene cluster was present in any one organism. Two Van gene clusters, VanA and VanB, exist on mobile plasmids.70Only one gene from either of these clusters was identified, VanY in the VanB cluster in B. bifidum. It is an accessory gene that does not result in resistance without the other VanB genes products. None of the other VanY genes were present in B. bifidum or any of the other consortia members. Given the absence of complete Van gene clusters in the proposed consortia, the likelihood of vancomycin resistance or horizontal transfer of resistance is negligible. Finally, all these vancomycin genes were only partially homologous to the known Van genes and likely represent peptidoglycan synthesis / crosslinking enzymes that are unique to the consortia strains and are unlikely to be functional in phylogenetically distinct pathogens such as Enterococcus or Staphylococcus.

[0289] The CARD database did not identify genes that explain phenotypic resistance profiles identified by phenotypic testing in a few of the strains (Table 8.3). Specifically, no b- lactamase genes were identified in organisms with phenotypic evidence of b-lactamase production (B. thetaiotaomicron, B. ovatus, B. cellusilyticus, P. vulgatus, P. distasonis, and E.201720021.2 - 80 -rectale), metronidazole resistance genes were not detected in B. pseudocatenulatum, and clindamycin resistance genes were not identified in P. distasonis. To assess this gap, WGSs of these organisms were compared to another antimicrobial resistance database with less stringent criteria for gene homology: Pathosystems Resource Integration Center (PATRIC)63specifically looking for gene homologs that are associated with resistance to the antibiotics identified in phenotypic testing. Five of the six organisms with phenotypic b-lactamases had a b-lactamase gene identified. An Erm gene conferring clindamycin resistance in P. distasonis was identified. The b-lactamase gene expressed by E. rectale was not identified. This likely represents inadequate characterization of b-lactamase in this non-pathogenic organism. The gene causing metronidazole resistance in B. pseudocatenulatum was also not identified. The genotypic basis for metronidazole resistance in Bifidobacteria is not well studied and not included in both CARD and PATRIC databases. Table 8.3: PATRIC Organism Additional Genes Detected. tibiotic resistance for select organisms using PATRIC. Table depicting antimicrobial resistance genes identified using the PATRIC database.63WGS for each organism with phenotypic resistance to an antimicrobial with activity against anaerobes but no corresponding resistance gene in the CARD database was compared to the PATRIC database for genes causing resistance to that antibiotic. B. thetaiotaomicron, B. ovatus, B. cellusilyticus, P. vulgatus, and E. rectale were searched for b-lactamase genes, P. distasonis was searched for b-lactamase and clindamycin resistance genes, and B. pseudocatenulatum was searched for metronidazole resistance genes. At least one gene corresponding to phenotypic resistance was identified in all cases but B. pseudocatenulatum and E. rectale, likely reflecting incomplete databases for non-pathogenic organisms.201720021.2 - 81 -Murine toxicology studies

[0291] Murine toxicology studies are described in detail in a prior section. Along with testing for intestinal colonization and metabolite production (FIGs. 15A-15E), mice were observed for survival and signs of clinical illness after colonization with each of the 9 MARCO Trial commensal consortia. The inventors did not detect signs of illness, i.e. listlessness, diarrhea or scruffy fur. There was one case of mortality in a mouse colonized with consortium D1 that occurred shortly after gavage that was deemed to have resulted from trauma caused by the gavage procedure.

[0292] Finally, to determine whether MARCO Trial consortia administration is associated with intestinal toxicity or inflammatory responses, the inventors conducted histologic analyses on ileum and colon samples obtained from mice euthanized on day 15 following consortium inoculation (FIG. 18). Tissues were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin in the University of Chicago Histology Core facility. Slides obtained from mice that had received each of the 8-member consortia were examined by Christopher R. Weber, MD, PhD, Associate Professor of Pathology at the University of Chicago. There were no signs of histologic disease in any section. Specifically, there were no signs of submucosal inflammation and the epithelial barrier appeared intact and orderly without signs of neoplasia. Testing Facility for the Nonclinical Toxicity Study

[0293] All non-clinical toxicity studies were performed in mice, and all mouse studies were approved by The University of Chicago Institutional Animal Care and Use Committee (IACUC, Protocol #72599). Throughout breeding and experimental conditions, mice were housed within separate areas of the University of Chicago Gnotobiotic Research Animal Facility (GRAF). During experimental conditions, mice were individually housed in biocontainment units (BCU). The GRAF is operated under and adheres to the Guide for Care and Use of Laboratory Animals, 8th edition, 2011, National Academy of Sciences. This includes standardized light / dark cycles, centrally monitored and controlled temperature, and centrally monitored and controlled humidity.201720021.2 - 82 -Declaration of GLP Compliance

[0294] All animal and non-clinical toxicity studies were conducted in accordance with Good Laboratory Practices as outlined in 21 CFR. 58 and described at https: / / www.ecfr.gov / current / title-21 / chapter-I / subchapter-A / part-58. * * *

[0295] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. References

[0296] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0297] References for Examples 1-3: 1. Taur, Y. et al. Intestinal Domination and the Risk of Bacteremia in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation. Clin Infect Dis 55, 905–914 (2012). 2. Shono, Y. et al. Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice. Sci. Transl. Med.8, 339ra71 (2016). 3. Peled, J. U. et al. Microbiota as Predictor of Mortality in Allogeneic Hematopoietic-Cell Transplantation. New Engl J Med 382, 822–834 (2020). 4. Lloyd-Price, J. et al. Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. Nature 569, 655–662 (2019). 5. Odenwald, M. A. et al. Bifidobacteria metabolize lactulose to optimize gut metabolites and prevent systemic infection in patients with liver disease. Nat. Microbiol. 1–17 (2023) doi:10.1038 / s41564-023-01493-w.201720021.2 - 83 -6. Lehmann, C. J. et al. Fecal metabolite profiling identifies liver transplant recipients at risk for postoperative infection. Cell Host Microbe 32, 117-130.e4 (2024). 7. Stutz, M. R. et al. Immunomodulatory fecal metabolites are associated with mortality in COVID-19 patients with respiratory failure. Nat Commun 13, 6615 (2022). 8. Stoma, I. et al. Compositional Flux Within the Intestinal Microbiota and Risk for Bloodstream Infection With Gram-negative Bacteria. Clin. Infect. Dis.73, ciaa068 (2020). 9. Dsouza, M. et al. Colonization of the live biotherapeutic product VE303 and modulation of the microbiota and metabolites in healthy volunteers. Cell Host Microbe 30, 583-598.e8 (2022). 10. Kakihana, K. et al. Fecal microbiota transplantation for patients with steroid-resistant acute graft-versus-host disease of the gut. Blood 128, 2083–2088 (2016). 11. Tariq, R. et al. Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection Reduces Recurrent Urinary Tract Infection Frequency. Clin. Infect. Dis. 65, 1745– 1747 (2017). 12. Bloom, P. P. et al. Fecal microbiota transplant improves cognition in hepatic encephalopathy and its effect varies by donor and recipient. Hepatol. Commun. 6, 2079–2089 (2022). 13. Bajaj, J. S. et al. Fecal microbiota transplant from a rational stool donor improves hepatic encephalopathy: A randomized clinical trial. Hepatology 66, 1727–1738 (2017). 14. Philips, C. A. et al. Healthy Donor Fecal Microbiota Transplantation in Steroid-Ineligible Severe Alcoholic Hepatitis: A Pilot Study. Clin Gastroenterol H 15, 600–602 (2017). 15. Bajaj, J. S. et al. A Randomized Clinical Trial of Fecal Microbiota Transplant for Alcohol Use Disorder. Hepatology 73, 1688–1700 (2021). 16. Wolstenholme, J. T. et al. Reduced alcohol preference and intake after fecal transplant in patients with alcohol use disorder is transmissible to germ-free mice. Nat. Commun.13, 6198 (2022). 17. Sorbara, M. T. & Pamer, E. G. Microbiome-based therapeutics. Nat Rev Microbiol 20, 365– 380 (2022). 18. DeFilipp, Z. et al. Drug-Resistant E. coli Bacteremia Transmitted by Fecal Microbiota Transplant. New Engl J Med 381, 2043–2050 (2019). 19. Feuerstadt, P. et al. SER-109, an Oral Microbiome Therapy for Recurrent Clostridioides difficile Infection. New Engl J Med 386, 220–229 (2022).201720021.2 - 84 -20. Lee, C. et al. Safety of fecal microbiota, live-jslm (REBYOTATM) in individuals with recurrent Clostridioides difficile infection: data from five prospective clinical trials. Ther. Adv. Gastroenterol.16, 17562848231174276 (2023). 21. Louie, T. et al. VE303, a Defined Bacterial Consortium, for Prevention of Recurrent Clostridioides difficile Infection. JAMA 329, 1356–1366 (2023). 22. Dsouza, M. et al. Colonization of the live biotherapeutic product VE303 and modulation of the microbiota and metabolites in healthy volunteers. Cell Host Microbe 30, 583-598.e8 (2022).

[0298] References for Examples 4 and 5: 1. Moon, A. M., Singal, A. G. & Tapper, E. B. Contemporary Epidemiology of Chronic Liver Disease and Cirrhosis. Clin Gastroenterol H 18, 2650–2666 (2019). 2. Younossi, Z. M. et al. Epidemiology of chronic liver diseases in the USA in the past three decades. Gut 69, 564 (2020). 3. Termeie, O. et al. Alarming Trends: Mortality from Alcoholic Cirrhosis in the United States. Am J Medicine (2022) doi:10.1016 / j.amjmed.2022.05.015. 4. Collaborators, G. 2017 C. et al. The global, regional, and national burden of cirrhosis by cause in 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterology Hepatology 5, 245–266 (2020). 5. Franchis, R. de et al. BAVENO VII - RENEWING CONSENSUS IN PORTAL HYPERTENSION Report of the Baveno VII Consensus Workshop: personalized care in portal hypertension. J Hepatol 76, 959–974 (2021). 6. Ge, P. S. & Runyon, B. A. Treatment of Patients with Cirrhosis. New Engl J Medicine 375, 767–777 (2016). 7. Biggins, S. W. et al. Diagnosis, Evaluation, and Management of Ascites, Spontaneous Bacterial Peritonitis and Hepatorenal Syndrome: 2021 Practice Guidance by the American Association for the Study of Liver Diseases. Hepatology 74, 1014–1048 (2021). 8. Wiest, R., Krag, A. & Gerbes, A. Spontaneous bacterial peritonitis: recent guidelines and beyond. Gut 61, 297 (2012). 9. Piano, S. et al. Epidemiology and Effects of Bacterial Infections in Patients With Cirrhosis Worldwide. Gastroenterology 156, 1368-1380.e10 (2019). 10. Shamsaddini, A. et al. Impact of Antibiotic Resistance Genes in Gut Microbiome of Patients With Cirrhosis. Gastroenterology 161, 508-521.e7 (2021).201720021.2 - 85 -11. Odenwald, M. A. et al. Bifidobacteria metabolize lactulose to optimize gut metabolites and prevent systemic infection in patients with liver disease. Nat. Microbiol. 1–17 (2023) doi:10.1038 / s41564-023-01493-w. 12. 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Bifidobacterium pseudocatenulatum Ameliorates DSS-Induced Colitis by Maintaining Intestinal Mechanical Barrier, Blocking Proinflammatory Cytokines, Inhibiting TLR4 / NF-κB Signaling, and Altering Gut Microbiota. J. Agric. Food Chem. 69, 1496–1512 (2021). 18. Esaiassen, E. et al. Bifidobacterium Bacteremia: Clinical Characteristics and a Genomic Approach To Assess Pathogenicity. J. Clin. Microbiol.55, 2234–2248 (2017). 19. Bertelli, C. et al. Bifidobacterium longum Bacteremia in Preterm Infants Receiving Probiotics. Clin. Infect. Dis.60, 924–927 (2015). 20. Weber, E. et al. Bifidobacterium Species Bacteremia: Risk Factors in Adults and Infants. Clin. Infect. Dis.61, 482–484 (2015). 21. Funabashi, M. et al. A metabolic pathway for bile acid dehydroxylation by the gut microbiome. Nature 582, 566–570 (2020). 22. Wang, M. et al. Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome. Cell 186, 2839-2852.e21 (2023). 23. Wexler, A. G. & Goodman, A. L. 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Novel strains of Bacteroides fragilis and Bacteroides ovatus alleviate the LPS-induced inflammation in mice. Appl. Microbiol. Biotechnol.103, 2353–2365 (2019).201720021.2 - 89 -30. Li, H., Sheng, D., Jin, C., Zhao, G. & Zhang, L. Identifying and ranking causal microbial biomarkers for colorectal cancer at different cancer subsites and stages: a Mendelian randomization study. Front. Oncol.13, 1224705 (2023). 31. Mandal, R. K. et al. Gut Bacteroides act in a microbial consortium to cause susceptibility to severe malaria. Nat. Commun.14, 6465 (2023). 32. Lin, X. et al. Gut microbiota impacts bone via Bacteroides vulgatus-valeric acid-related pathways. Nat. Commun.14, 6853 (2023). 33. Ali, R. O. et al. Longitudinal multi-omics analyses of the gut–liver axis reveals metabolic dysregulation in hepatitis C infection and cirrhosis. Nat. Microbiol.8, 12–27 (2023). 34. Sun, H. et al. Gut commensal Parabacteroides distasonis alleviates inflammatory arthritis. Gut 72, 1664–1677 (2023). 35. 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Claims

WHAT IS CLAIMED IS:

1. A composition comprising a collection of bacteria from one or more of DFI Consortia A, DFI Consortia B1, DFI Consortia B2, DFI Consortia C1, DFI Consortia C2, DFI Consortia D1, DFI Consortia D2, DFI Consortia E1, DFI Consortia E2, and DFI Commensal Consortia, or a combination thereof.

2. The composition of claim 1, wherein one or more bacteria of DFI Consortia A, one or more bacteria of DFI Consortia B1, one or more bacteria of DFI Consortia B2, one or more bacteria of DFI Consortia C1, one or more bacteria of DFI Consortia C2, one or more bacteria of DFI Consortia D1, one or more bacteria of DFI Consortia D2, one or more bacteria of DFI Consortia E1, one or more bacteria of DFI Consortia E2, and / or one or more bacteria of DFI Commensal Consortia are excluded from the collection.

3. The composition of claim 1 or 2, wherein the collection of bacteria comprises at least two bacteria that produce butyrate, at least one bacteria that can deconjugate conjugated primary bile acids, at least one bacteria that can convert primary to secondary bile acids, and / or at least one bacteria that can modify secondary bile acids.

4. The composition of claim 3, wherein the bacteria that produce butyrate are selected from Anaerostipes hadrus DFI.4.30, Anaerostipes caccae DFI.7.76, Clostridium symbiosum DFI.5.64, Coprococcus comes DFI.3.84, Anaerobutyricum hallii DFI.9.38, and / or Eubacterium rectale DFI.5.

28.

5. The composition of claim 3 or 4, wherein the bacteria that can deconjugate conjugated primary bile acids are selected from Bifidobacterium longum DFI.7.60, Bifidobacterium pseudocatenulatum DFI.7.61, and / or Bifidobacterium bifidum DFI.7.

51.

6. The composition of any one of claims 3 to 5, wherein the bacteria that can convert primary to secondary bile acids is Clostridium scindens SL.1.

22.

7. The composition of any one of claims 3 to 6, wherein the bacteria that modify secondary bile acids are selected from Bacteroides thetaiotaomicron DFI.6.40, Bacteroides ovatus MSK.18.37, Bacteroides cellulosilyticus DFI.5.60, Phocaeicola vulgatus DFI.4.81, Parabacteroides distasonis DFI.5.23, Parabacteroides merdae DFI.4.73, and / or Eggerthella lenta DFI.5.

72.

8. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia A.

9. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia B1.

10. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia B2.

11. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia C1.201720021.2 - 94 -12. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia C2.

13. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia D1.

14. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia D2.

15. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia E1.

16. A composition comprising 2, 3, 4, 5, 6, 7, 8 bacteria of DFI Consortia E2.

17. A composition comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 bacteria of DFI Commensal Consortia.

18. The composition of any one of claims 1 to 16, wherein the composition further comprises a metabolite, an antibiotic, and / or an immunosuppressant.

19. The composition of claim 18, wherein the metabolite comprises a fatty acid, an amino acid, a primary bile acid, a secondary bile acid, a sugar, or a combination thereof.

20. The composition of any one of claims 1 to 19, wherein the composition comprises 103, 104, 105, 106, 107,108, 109, 1010, 1011, 1012colony forming units of at least one bacteria.

21. A method of reestablishing commensal organisms in a patient’s microbiome, the method comprising administering an effective amount of the composition of any one of claims 1 to 20 to the patient.

22. A method of treating liver disease in a patient, the method comprising administering to the patient an effective amount of the composition of any one of claims 1 to 20 to the patient.

23. The method of claim 22, wherein the patient has or will receive a liver transplant.

24. The method of claim 22 or 23, wherein the patient has, has been diagnosed with, or is suspected of having, chronic liver disease.

25. A method of treating a patient, the method comprising administering to the patient an effective amount of the composition of any one of claims 1 to 20 to the patient.

26. The method of claim 25, wherein the patient has received a cell transplant.

27. The method of claim 26, wherein the stem cell transplant comprises an allogeneic hematopoietic cell transplantation.

28. The method of any one of claims 21 to 27, wherein a stool sample from the patient has been measured to have low fecal taxonomic alpha diversity.

29. The method of any one of claims 21 to 28, wherein a stool sample from the patient has or has been determined to have less than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 µM or mM, or any range derivable therein, of butyrate.201720021.2 - 95 -30. The method of any one of claims 21 to 29, wherein a stool sample from the patient has or has been determined to have more than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or any range derivable therein, µM of taurocholic acid.

31. The method of any one of claims 21 to 30, wherein a stool sample from the patient has or has been determined to have less than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or any range derivable therein, µM of deoxycholic acid.

32. The method of any one of claims 21 to 31, wherein a stool sample from the patient has or has been determined to have less than or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or any range derivable therein, µM of alloisolithocholic acid.

33. The method of any one of claims 21 to 28, wherein a stool sample from the patient has or has been determined to have less than 700 µM butyrate and less than 10 µM deoxycholate.

34. The method of any one of claims 21 to 28, wherein a stool sample from the patient has or has been determined to have reduced butyrate and / or deoxycholic acid levels compared to a standard.

35. The method of any one of claims 21 to 28, wherein a stool sample from the patient is or has been determined to be substantially free of butyrate and / or deoxycholic acid.

36. The method of any one of claims 21 to 35, wherein the composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 times per day.

37. The method of any one of claims 21 to 36, wherein the composition is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

38. The method of claim 37, wherein the days are consecutive.

39. The method of claim 37, wherein the days are every other day.

40. The method of any one of claims 21 to 39, the method further comprising measuring one or more bacteria and / or one or more metabolites in a sample from the patient.

41. The method of claim 40, wherein the sample comprises a stool sample.

42. The method of claim 40 or 41, wherein the bacteria and / or metabolite comprise one or more bacteria and / or metabolites disclosed in the Appendix.

43. A method of treating a disease in a patient, the method comprising administering to the patient an effective amount of one or more of DFI Commensal Consortia bacteria.201720021.2 - 96 -44. The method of claim 43, wherein the disease comprises a liver disease and / or an infection.

45. The method of claim 43 or 44, wherein the patient has or will receive a transplant.

46. The method of claim 45, wherein the transplant comprises a liver transplant or cell transplant.

47. A method of detecting microbiome dysbiosis in a patient, the method comprising measuring a level of one or more DFI Commensal Consortia bacteria in a sample from the patient.

48. The method of claim 47, wherein one or more bacteria of the DFI Commensal Consortia are not measured.

49. The method of claim 47 or 48, wherein the patient has, has been diagnosed with, or is suspected of having, liver disease or COVID-19.201720021.2 - 97 -

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