Compositions and methods for treating gastrointestinal disorders

Abstract

Aspects of the disclosure encompass compositions and therapeutic methods for treating subjects having gastrointestinal disorders. The compositions provided herein can be formulated as live biotherapeutic products (LBP), fecal microbiota transplant (FMT) products and FMT enrichment compositions, probiotics and food products, and comprise one or more of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or combinations thereof. In certain aspects, an individual is determined to be a suitable recipient for these therapies based on classification of the taxa profile of their microbiome.

Description

PATENTBAYM.P0441WO / 1001315844PATENT COOPERATION TREATY (PCT) APPLICATIONFORCOMPOSITIONS AND METHODS FOR TREATING GASTROINTESTINALDISORDERSBYTOR SAVIDGE,SIL YL SO.YUNXILIU,ANDSHYAMBADU299756710.1COMPOSITIONS AND METHODS FOR TREATING GASTROINTESTINAL DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 702,432, filed October 2, 2024, hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Number(s) AU 52999, AI124290 and NR013497 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] This disclosure relates at least to the fields of gastroenterology, bacteriology, cell biology, physiology, molecular biology, bioinformatics, diagnostics, and medicine.BACKGROUND

[0004] Inflammatory Bowel Diseases (IBD) are common, costly, and cause substantial morbidity starting in childhood. Although their causes are largely unknown, IBD are chronic inflammatory disorders characterized by an excessive gastrointestinal (GI) immune response in a genetically susceptible host. The two main IBD subtypes are Crohn’s disease (CD) and ulcerative colitis (UC). Therapeutics such as small molecules and more recently immunotherapies are used as front-line therapies but their cost, side effects, development of resistance, and limited efficacy highlight the need for improved understanding and management paradigms.

[0005] Like IBDs, Irritable Bowel Syndrome (IBS), an abdominal-related chronic disorder of gut-brain interaction (DGBI), affects approximately 10-15% of adults and imposes a significant economic, social, and emotional burden on the U.S. population. Characterized primarily by abdominal pain and altered bowel habits, IBS is a heterogeneous condition frequently accompanied by a range of somatic symptoms and psychological distress. Despite the availability of various treatment options, therapeutic response remains highly variable. Meta-analyses consistently support cognitive behavioral therapy (CBT) as an effective treatment strategy for IBS, though only 50-60% of patients experience meaningful improvement in abdominal pain — a critical gap in optimizing care for this prevalent condition. It remains unclear which patients are most likely to respond, particularly given limited access to CBT due to insurance constraints and a shortage of trained providers.- 2 -299756710.1

[0006] The present disclosure provides solutions to long-felt needs in the art at least of diagnosing and treating gut-related issues.SUMMARY

[0007] In some aspects, the current disclosure encompasses a composition comprising at least one of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an excipient. In some aspects, the composition comprises the bacterial species Faecalibacterium prausnitzii and at least one, at least two, or all of, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects, the composition comprises at least two, at least three, or all of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects, the composition comprises, consists of, or consists essentially of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale,' and comprises suitable excipients.

[0008] In some aspects, the composition has a greater relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale, compared to a reference microbiome profile microbiome sample. In some aspects, the composition has a reduced relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale compared to a reference gut microbiome profile sample. In some aspects, the one or more strains of Faecalibacterium prausnitzii belong to sub-clades comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof. In some aspects, the one or more strains of Faecalibacterium prausnitzii belong to sub-clades comprising SGB 15316. In some aspects, the composition is for administration into a subject in need thereof, wherein the subject has an altered microbiome enterocluster profile relative to a reference microbiome profile. In some aspects, the composition the subject has reduced levels of any one or more of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale in the microbiome enterocluster profile relative to the reference microbiome profile. In some aspects, the subject has a reduced relative abundance of any one or more of a specific strain or strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof in the microbiome enterocluster profile relative to the reference microbiome profile. In some aspects, the subject has a greater relative abundance of any one or more of a specific strain or strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof in the- 3 -299756710.1microbiome enterocluster profile relative to the reference microbiome profile. In some aspects the composition is for administration into the subject, from whom the greater or the reduced abundance of the one or more strains is determined based on the subjects microbiome enterocluster profile.

[0009] In some aspects, the composition is for administration into a subject in need thereof, wherein the subject has an altered microbiota diversity relative to a healthy subject. In some aspects, the subject has reduced levels of any one or more of Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale in the microbiota relative to a healthy subject. In some aspects, the subject has enriched levels of one or more species of genera Bacteroides, Blautia, Lachnospiraceae, Clostridioides, Enterob acteriaceae, Streptococcus, or an unclassified taxa.

[0010] In some aspects, the subject has, or is at a risk of having a gastrointestinal (GI) disease or disorder, non-limiting examples of which include of Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD). In some aspects, the subject may have a bacterial taxa profile in a fecal matter sample that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the taxa identified in Cluster 1 or Cluster 3, wherein: Cluster 1 taxa profile comprises Bacteroides, Blautia, Unclassified_071, Faecalibacterium, Unclassified_075, Unclassified_072, Anaerostipes, Lachnospiraceae incertae sedis, Parabacteroides, Roseburia, Alistipes, Streptococcus, Ruminococcus, Dorea, Bifidobacterium, Fusicatenibacter, Unclassified_087, Ruminococcus2, Coprococcus, Flavonifractor, Eubacterium, Lachnoclostridium, Clostridium XlVa, Dialister, Lactobacillus, Erysipelatoclostridium, Unclassified_083, Unclassified_037, Intestinibacter, Veillonella, Unclassified_129, Romboutsia, Ruthenibacterium, Collinsella, Unclassified_123, Unclassified_074, Not_Available_2, Prevotella, Parasutterella, and Clostridium; Cluster 3 taxa profile comprises Bacteroides, Blautia, Streptococcus, Unclassified_123, Unclassified_071, Unclassified_129, Veillonella, Lactobacillus, Erysipelatoclostridium, Unclassified_072, Lachnoclostridium, Bifidobacterium, Enterococcus, Anaerostipes, Lachnospiraceae incertae sedis, Clostridium XlVa, Unclassified_087, Clostridium, Faecalibacterium, Parabacteroides, Unclassified_074, Unclassified_075, Flavonifractor, Unclassified_064, Roseburia, Dialister, Unclassified_132, Intestinibacter, Prevotella, Clostridium XVIII, Alistipes, Not_Available_2, Unclassified_083, Akkermansia, Haemophilus, Fusobacterium, Romboutsia, Ruminococcus2, Fusicatenibacter, and Clostridium sensu stricto; and wherein the Cluster 1 or Cluster 3 have a reduced level of Faecalibacterium prausnitzii relative to a healthy cluster, wherein the healthy correspond to any one of clusters 2, 4, or 5, wherein: Cluster 2 taxa profile comprises 1 or more- 4 -299756710.1of the following listed bacterial taxa: Bacteroides, Blautia, Faecalibacterium, Unclassified_071, Alistipes, Ruminococcus, Parabacteroides, Unclassified_072, Unclassified_075, Lachnospiraceae incertae sedis, Roseburia, Anaerostipes, Not_Available_2, Fusicatenibacter, Dorea, Coprococcus, Bifidobacterium, Eubacterium, Streptococcus, Ruminococcus2, Lactobacillus, Gemmiger, Romboutsia, Unclassified_087, Odoribacter, Akkermansia, Unclassified_123, Bilophila, Flavonifractor, Ruthenibacterium, Barnesiella, Parasutterella, Lachnoclostridium, Agathobaculum, Intestinibacter, Erysipelatoclostridium, Prevotella, Phascolarctobacterium, Clostridium lV, and Unclassified_037; Cluster 4 taxa profile comprises 1 or more of the following listed bacterial taxa: Blautia, Unclassified_071, Faecalibacterium, Bifidobacterium, Ruminococcus, Lachnospiraceae incertae sedis, Unclassified_075, Bacteroides, Dorea, Unclassified_072, Anaerostipes, Not_Available_2, Fusicatenibacter, Coprococcus, Streptococcus, Romboutsia, Roseburia, Gemmiger, Alistipes, Collinsella, Agathobaculum, Ruminococcus2, Intestinibacter, Unclassified_087, Parabacteroides, Unclassified_074, Clostridium, Eubacterium, Lactobacillus, Unclassified_083, Turicibacter, Akkermansia, Dialister, Adlercreutzia, Schaalia, Erysipelatoclostridium, Clostridium sensu stricto, Not_Available_5, Ruthenibacterium, and Clostridium IV; and Cluster 5 taxa profile comprises 1 or more of the following listed bacterial taxa: Prevotella, Bacteroides, Faecalibacterium, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae incertae sedis, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Bifidobacterium, Unclassified_087, Eubacterium, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

[0011] In some aspects, the composition comprises about, or more than about, 106to 1012cfu of each of the at least one, at least two, at least three, or at least four bacterial species. In some aspects, the composition comprises about 106to 107, or about 107to 108, about 108to 109, about 109to IO10, about IO10to 1011, or about 1011to 1012cfu or more of each of the at least one, at least two, at least three, or at least four bacterial species. In some aspects, the composition comprises about, or more than about, 106to 1012cfu of each of the at least one, at least two, at least three, or at least four bacterial species. In some aspects, the composition comprises about 106to 107, or about 107to 108, about 108to 109, about 109to IO10, about IO10to 1011, or about 1011to 1012cfu / unit dose, or more of each of the at least one, at least two, at least three, or at least four bacterial species. In some aspects, the composition can comprise one or more additional bacterial species of the genera Faecalibacterium, Bifidobacterium, or Eubacterium. In some aspects, the- 5 -299756710.1composition comprises one or more additional bacterial species. In some aspects, the additional bacterial species are from the genus Faecalibacterium, Bifidobacterium, Eubacterium, Prevotella, Bacteroides, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

[0012] In some aspects, the bacterial species have been isolated and / or otherwise purified from a natural source material. In some aspects, the bacterial species are obtained from a repository of bacterial species. In some aspects, the one or more bacterial species of the disclosed composition are genetically engineered. In some aspects, the one or more bacterial species are wild-type. In some aspects, the one or more bacterial species are isolated and cultured from fecal samples.

[0013] In some aspects, a composition is formulated as a Live Biotherapeutic Product (LBP). In some aspects, the composition is formulated as a probiotic. In some aspects, the composition is formulated as a food product, for example, without limitation, a beverage, a drink, a bar, a snack, a dairy product, a confectionery product, a cereal product, a ready-to-eat product, a nutritional formula, such as a nutritional supplementary formulation, a food or beverage additive. In some aspects, the composition is formulated as an additive to a donor fecal microbiota for a FMT procedure.

[0014] In some aspects, the composition is for oral, buccal, orogastric, nasogastric, sublingual, topical, rectal, vaginal, colonoscopy administration, and / or flexible sigmoidoscopic administration. In some aspects, the composition is for orogastric, nasogastric, oral or rectal administration.

[0015] In some aspects, the composition is in the form of a powder, a freeze dried formulation, a lyophilized formulation, a capsule, a gummy, a tablet, a sachet, a liquid, an emulsion, a suspension, fecal matter, and / or a food product.

[0016] In some aspects, the composition comprises an excipient, non-limiting examples of which include a carrier, buffer, preservative, flavoring agent, antimicrobial agent, antibiotic agent, antifungal agent, antioxidant, preservative, electrolyte, antifoaming agent, oil, stabilizer, enhancing agent, or any combination thereof.

[0017] In some aspects, the current disclose also encompasses the compositions disclosed herein, formulated for administration in combination with one or more other therapies comprising- 6 -299756710.1a fecal microbiota transplant (FMT) procedure, administration of low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols (FODMAP) diet, cognitive behavioral therapy (CBT), pain relievers, antibiotics, antispasmodic agents, antiacids, anti- diarrheal, proton-pump inhibitors, anti-inflammatory, corticosteroids, immune system suppressors, biologies, vitamins and / or supplements, surgery, nutritional support, one or more small molecules, or any combination thereof.

[0018] In some aspects, the current disclosure encompasses a composition comprising a greater or reduced relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale, for administration into a subject with a gastrointestinal disease or disorder, wherein the greater or the reduced relative abundance of the one or more strains in the composition is determined based on a comparison of the subjects microbiome enterocluster profile with a reference microbiome profile. In some aspects, the subjects microbiome enterocluster profile is determined using the steps of: (i) obtaining nucleic acids from the biological sample; (ii) determining the relative abundance of the one or more strains present in the sample based on a nucleic acid based assay; (iii) determining the biological sample’s microbiome enteroclusters profile based on the relative abundance. In some aspects, the nucleic acid based assay uses 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), and / or a bioinformatics pipeline. In some aspects, the bioinformatics pipeline comprises the Taxa4Meta, and / or BIOFIRE® SPOTFIRE® System. In some aspects, the biological sample comprises fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation.

[0019] In some aspects, the current disclosure also encompasses a method of treating a disease or a disorder in a subject in need thereof, comprising administering to the subject an effective amount of the composition disclosed herein. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising at least one of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an excipient. In some aspects, the composition for use in the method comprises, consists of, or consist essentially of the bacterial species Faecalibacterium prausnitzii and at least one, at least two, or at least 3 of Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects, the method comprises administering to a subject a composition comprising, consisting of, or consisting essentially of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects of the method disclosed herein, the subject has reduced microbiota diversity relative to a reference microbiome profile. In some aspects, the- 7 -299756710.1subject has reduced levels of any one or more of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, or Eubacterium rectale in the microbiota relative to a reference microbiome profile. In some aspects of the method, the composition has a greater relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale, compared to a reference microbiome profile. In some aspects of the method, the composition for administration has a reduced relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale compared to a reference microbiome profile. In some aspects, composition for administration is determined based on the subjects microbiome enteroclusters profile. In some aspects, the composition has greater relative abundance of one or more strains of Faecalibacterium prausnitzii compared to a reference microbiome profile. In some aspects, the composition has reduced relative abundance of one or more strains of Faecalibacterium prausnitzii compared to a reference microbiome profile. In some aspects, the one or more strains of Faecalibacterium prausnitzii belong to subclades comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof. In some aspects, the one or more strains of Faecalibacterium prausnitzii belong to subclades comprising SGB 15316.

[0020] In some aspects of the disclosed method, the subject has reduced microbiota diversity in a biological sample from the subject relative to a reference microbiome profile. In some aspects, the subject has an altered microbiome enterocluster profile in a biological sample from the subject. In some aspects, the biological sample is a fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation. In some aspects, subject has reduced levels of any one or more of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale in the microbiota relative to a reference microbiome profile. In some aspects, the subject has enriched levels of one or more species of genera Bacteroides, Blautia, Lachnospiraceae, Clostridioides, Enterob acteriaceae, Streptococcus, or an unclassified taxa.

[0021] In some aspects, the subject has or is at a risk of having a gastrointestinal disorder. In some aspects, the gastrointestinal disorder comprises one or more of Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD). In some aspects, the subject has or is at a risk of having, antibiotic associated diarrhea. In some aspects, the subject has a bacterial taxa profile in a fecal matter sample that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the taxa identified in Cluster 1 or Cluster 3, wherein: Cluster 1 taxa profile comprises Bacteroides, Blautia, Unclassified_071, Faecalibacterium, Unclassified_075, Unclassified_072, Anaerostipes,- 8 -299756710.1Lachnospiraceae incertae sedis, Parabacteroides, Roseburia, Alistipes, Streptococcus, Ruminococcus, Dorea, Bifidobacterium, Fusicatenibacter, Unclassified_087, Ruminococcus2, Coprococcus, Flavonifractor, Eubacterium, Lachnoclostridium, Clostridium XlVa, Dialister, Lactobacillus, Erysipelatoclostridium, Unclassified_083, Unclassified_037, Intestinibacter, Veillonella, Unclassified_129, Romboutsia, Ruthenibacterium, Collinsella, Unclassified_123, Unclassified_074, Not_Available_2, Prevotella, Parasutterella, and Clostridium; Cluster 3 taxa profile comprises Bacteroides, Blautia, Streptococcus, Unclassified_123, Unclassified_071, Unclassified_129, Veillonella, Lactobacillus, Erysipelatoclostridium, Unclassified_072, Lachnoclostridium, Bifidobacterium, Enterococcus, Anaerostipes, Lachnospiraceae incertae sedis, Clostridium XlVa, Unclassified_087, Clostridium, Faecalibacterium, Parabacteroides, Unclassified_074, Unclassified_075, Flavonifractor, Unclassified_064, Roseburia, Dialister, Unclassified_132, Intestinibacter, Prevotella, Clostridium XVIII, Alistipes, Not_Available_2, Unclassified_083, Akkermansia, Haemophilus, Fusobacterium, Romboutsia, Ruminococcus2, Fusicatenibacter, and Clostridium sensu stricto; and wherein the Cluster 1 or Cluster 3 has a reduced level of Faecalibacterium prausnitzii relative to a healthy clusters 2, 4 or 5, wherein: Cluster 2 taxa profile comprises 1 or more of the following listed bacterial taxa: Bacteroides, Blautia, Faecalibacterium, Unclassified_071, Alistipes, Ruminococcus, Parabacteroides, Unclassified_072, Unclassified_075, Lachnospiraceae incertae sedis, Roseburia, Anaerostipes, Not_Available_2, Fusicatenibacter, Dorea, Coprococcus, Bifidobacterium, Eubacterium, Streptococcus, Ruminococcus2, Lactobacillus, Gemmiger, Romboutsia, Unclassified_087, Odoribacter, Akkermansia, Unclassified_123, Bilophila, Flavonifractor, Ruthenibacterium, Barnesiella, Parasutterella, Lachnoclostridium, Agathobaculum, Intestinibacter, Erysipelatoclostridium, Prevotella, Phascolarctobacterium, Clostridium IV, and Unclassified_037; Cluster 4 taxa profile comprises 1 or more of the following listed bacterial taxa: Blautia, Unclassified_071, Faecalibacterium, Bifidobacterium, Ruminococcus, Lachnospiraceae incertae sedis, Unclassified_075, Bacteroides, Dorea, Unclassified_072, Anaerostipes, Not_Available_2, Fusicatenibacter, Coprococcus, Streptococcus, Romboutsia, Roseburia, Gemmiger, Alistipes, Collinsella, Agathobaculum, Ruminococcus2, Intestinibacter, Unclassified_087, Parabacteroides, Unclassified_074, Clostridium, Eubacterium, Lactobacillus, Unclassified_083, Turicibacter, Akkermansia, Dialister, Adlercreutzia, Schaalia, Erysipelatoclostridium, Clostridium sensu stricto, Not_Available_5, Ruthenibacterium, and Clostridium IV; and Cluster 5 taxa profile comprises 1 or more of the following listed bacterial taxa: Prevotella, Bacteroides, Faecalibacterium, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae incertae- 9 -299756710.1sedis, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Bifidobacterium, Unclassified_087, Eubacterium, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

[0022] In some aspects of the methods provided herein, a composition comprises about, or more than about, 106to 1012cfu of each of the at least one, at least two, at least three, or at least four bacterial species. In some aspects, the composition comprises 106to 107, or about 107to 108, about 108to 109, about 109to IO10, about IO10to 1011, or about 1011to 1012cfu or more of each of the at least one, at least two, at least three, or at least four bacterial species, optionally wherein the bacterial species have been isolated and / or otherwise purified from a source material. In some aspects, the composition comprises about, or more than about, 106to 1012cfu of each of the at least one, at least two, at least three, or at least four bacterial species. In some aspects, the composition comprises 106to 107, or about 107to 108, about 108to 109, about 109to IO10, about IO10to 1011, or about 1011to 1012cfu / unit dose, or more of each of the at least one, at least two, at least three, or at least four bacterial species, optionally wherein the bacterial species have been isolated and / or otherwise purified from a source material. In some aspects, methods comprise administering one or more additional bacterial species of the genera Faecalibacterium, Bifidobacterium, or Eubacterium. In some aspects, methods comprise administering the composition that further comprises one or more additional bacterial species of the genera Faecalibacterium, Bifidobacterium, Eubacterium, Prevotella, Bacteroides, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

[0023] In some aspects, methods comprise administering a compositions disclosed herein, wherein the composition is formulated as a Live Biotherapeutic Product (LBP), a FMT product, a probiotic, or a food product. Non-limiting examples of food products include, a beverage, a drink, a bar, a snack, a dairy product, a confectionery product, a cereal product, a ready-to-eat product, a nutritional formula, such as a nutritional supplementary formulation, a food or beverage additive. In some aspects, a composition is formulated as an additive to a donor fecal microbiota for a FMT procedure.- 10 -299756710.1

[0024] In some aspects of the methods described herein, a composition is administered by any suitable route, for example, via an oral, buccal, orogastric, nasogastric, sublingual, topical, rectal, vaginal, colonoscopy administration, and / or flexible sigmoidoscopic route. In some aspects, the composition is administered via a orogastric route or nasogastric route.

[0025] In some aspects of the methods described herein, a subject is administered one or more additional therapies. In some aspects, the additional therapy comprises FMT. In some aspects, the fecal matter for the FMT is from a donor subject having a bacterial taxa profile comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the taxa identified in Cluster 2, Cluster 4, or Cluster 5, or from a donor subject whose fecal microbiota has been characterized. In some aspects, the additional therapy comprises, for example, administration of a therapeutic comprising one or more of low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols (FODMAP) diet, cognitive behavioral therapy (CBT), pain relievers, , antibiotics, antispasmodic agents, antiacids, anti -diarrheal agents, proton-pump inhibitors, anti-inflammatory agents, corticosteroids, immune system suppressors, biologies, supplements, nutritional support, nutraceuticals, vaccines, vitamins, one or more small molecules, or any combination thereof. In some aspects, the one or more anti-inflammatory drugs is an aminosalicylate. In some aspects, the aminosalicylate comprises, consists essentially of, or consists of mesalamine, balsalazide, and / or olsalazine. In some aspects, the immune system suppressors comprises, consists essentially of, or consist of azathioprine, mercaptopurine, and / or methotrexate. In some aspects, the small molecules comprises, consists essentially of, or consist of tofacitinib, upadacitinib, and / or ozanimod. In some aspects, the biologies comprise, consist essentially of, or consist of infliximab, adalimumab, golimumab, certolizumab, vedolizumab, ustekinumab, and / or risankizumab. In some aspects, the antibiotics comprises, consists essentially of, or consists of ciprofloxacin, metronidazole, vancomycin, fidaxomicin, and / or bezlotoxumab. In some aspects, the anti- diarrheal medication comprises, consists essentially of, or consists of psyllium powder, methylcellulose, and / or loperamide. In some aspects, the one or more pain killer comprises, consists essentially of, or consists of Piroxicam. In some aspects, the Piroxicam is administered at 100 ppm with drinking water. In some aspects, the additional therapy is co-administered, or is administered in a staggered format.

[0026] In some aspects, disclosed methods can be repeated periodically, for example, at least once in one week, two weeks, three weeks, monthly, 6 monthly, or yearly over a period of time.

[0027] In some aspects, disclosed compositions are anti-inflammatory compositions. In some aspects, the subject is a mammal. In some aspects, the subject is a human.- 11 -299756710.1

[0028] In some aspects, the current disclosure also encompasses a method of restoring the gut microbiome of a subject in need thereof, the method comprising administering to the subject a composition comprising at least one of bacterial species Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof; and an excipient. In some aspecs, the method comprises administering to the subject an effective amount of a composition comprising at least one of the bacterial species Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an excipient. In some aspects, the composition for use in the method may comprise, consists of, or consist essentially of the bacterial species Faecalibacterium prausnitzii and at least one, at least two, or at least 3 of Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects, the method comprises administering to a subject a composition comprising, consisting of, or consisting essentially of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale.

[0029] In some aspects the current disclosure also encompasses a kit comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an excipient. In some aspects, the kit comprises a composition comprising, consisting of, or consisting essentially of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects, the composition comprises one or more strains from the bacterial species Faecalibacterium prausnitzii comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereofIn some aspects, the current disclosure also encompasses food products and formulations comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an ingestible additive. In some aspects, the food product comprises, consists of, or consists essentially of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale, and additional ingestible ingredients.

[0030]

[0031] In some aspects, disclosed herein is a food formulation comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an ingestible additive. In some aspects, the food formulation comprises the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale,' and an- 12 -299756710.1excipient. In some aspects, the bacterial species Faecalibacterium prausnitzii comprises strains from one or more sub-clades comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.

[0032] Also disclosed herein is a Live Biotherapeutic Product (LBP) comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an ingestible additive. In some aspects, the composition comprises the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale,' and an excipient. In some aspects, the bacterial species Faecalibacterium prausnitzii comprises one or more sub-clades of the bacterial species Faecalibacterium prausnitzii comprising SGB 15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.

[0033] Disclosed herein is a method of predicting response to a gastrointestinal disorder therapy in a subject diagnosed with or is suspected of having a gastrointestinal disorder, comprising: (a) obtaining or having obtained a biological sample from the subject; (b) identifying a microbiome enterocluster profile of the biological sample; and (c) selecting a therapy based on the microbiome enterocluster profile. In some aspects, the microbiome enterocluster profile has a reduced abundance of one or more bacterial species comprising Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or a strain thereof, or any combination thereof. In some aspects, the microbiome enterocluster profile has reduced abundance of Faecalibacterium prausnitzii belonging to sub-clades comprising SGB 15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof. In some aspects, the microbiome enteroclusters profile has increased abundance of one or more strains of Faecalibacterium prausnitzii belonging to sub-clades comprising SGB 15316. In some aspects, the therapy is a low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols (FODMAP) diet, cognitive behavioral therapy (CBT), or a microbiome-targeted composition. In some aspects, microbiome-targeted composition is a composition disclosed herein.

[0034] Disclosed herein is a method of monitoring response to a gastrointestinal disorder therapy in a subject diagnosed with a gastrointestinal disorder, comprising: (a) obtaining or having obtained a biological sample from the subject; (b) assessing a gut microbiome enterocluster profile of the biological sample; and (c) correlating changes with clinical symptom improvement.

[0035] Disclosed herein is a method of treating IBS in a subject, comprising: (a) assessing baseline psychological distress in the subject; (b) assessing baseline dietary fat intake; (c) assessing a gut microbiome enterocluster profile from a biological sample; and (d) administering a therapy selected based on the combined assessment.- 13 -299756710.1

[0036] In any of the methods disclosed herein above, assessing the gut microbiome enterocluster profile may comprise: (i) obtaining nucleic acids from the biological sample; (ii) identifying the relative abundance of bacterial taxa present in the sample based on a nucleic acid based assay; (iii) determining the sample’s microbiome enteroclusters profile based on the relative abundance profile of the bacterial taxa. In some aspects, the nucleic acid based assay uses one or more of 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), and / or a bioinformatics pipeline. In some aspects, the bioinformatics pipeline comprises the Taxa4Meta, and / or BIOFIRE® SPOTFIRE® System. In some aspects, the biological sample comprises fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation.

[0037] Also disclosed herein is a method of assessing a microbiome enterocluster profile of a subject with a gastrointestinal disorder, comprising: (i) obtaining nucleic acids from the biological sample; (ii) identifying the relative abundance of bacterial taxa present in the sample based on a nucleic acid-based assay; (iii) assessing the microbiome enteroclusters profile based on the relative abundance profile of the bacterial taxa. In some aspects, nucleic acid-based assay uses 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), and / or a bioinformatics pipeline. In some aspects, the bioinformatics pipeline comprises the Taxa4Meta, and / or BIOFIRE® SPOTFIRE® System. In some aspects, biological sample comprises fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation. In some aspects, the method further comprises use of the microbiome enteroclusters profile assessment in diagnosis, prognosis, or selection of a therapeutic intervention. In some aspects, the gastrointestinal disorder is Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD). In some aspects, therapeutic intervention comprises administering to the subject a disclosed composition. In some aspects, the therapeutic intervention comprises administering a cognitive behavioral therapy (CBT). In some aspects, the therapeutic intervention comprises administering a low FODMAP diet.

[0038] Disclosed herein is a method of stratifying IBS patients for dietary intervention, comprising: (a) determining the patients baseline dietary fat intake; (b) determining the patients microbiome enteroclusters profile from a biological sample from the patients; and (c) predicting likelihood of response to a low FODMAP diet based on the combination of (a) and (b).

[0039] Disclosed herein is a kit for the diagnosis and management of IBS, CDI and IBD, comprising: (a) reagents for multiplexed qPCR detection of microbiome enteroclusters; (b)- 14 -299756710.1instructions for interpreting a microbiome enteroclusters profile to guide dietary or behavioral therapy selection.

[0040] Disclosed herein is a method of integrating psychological, dietary, and microbiome data to guide IBS, CDI and IBD therapy, comprising: (a) collecting psychological assessment data; (b) collecting dietary intake data; (c) collecting microbiome enteroclusters profile data; and (d) using a multivariate model to predict optimal therapy. In some aspects, the multivariate model comprises a DMM Clustering (Dirichlet Multinomial Mixture). In some aspects, the multivariate model is a latent factor or machine learning model trained on clinical outcomes.

[0041] Disclosed herein is a method of predicting non-responsiveness of a subject to a low FODMAP diet, comprising: analyzing the subject’s microbiome for a shift from a Blautia / Bifidobacteria-dominated cluster to a Bacteroides-dominated cluster after dietary intervention, wherein the shift indicates that the subject will be non-responsive to the low FODMAP diet. Also disclosed herein is a method of using enterocluster profile as precision strainlevel microbial and probiotic and / or synbiotic therapy for gastrointestinal diseases selected from IBS, CDI, and IBD.

[0042] In some aspects, disclosed herein is a method comprising administering to a subject a composition comprising Faecalibacterium prausnilzii, Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale after the relative abundance of Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale, or a strain thereof, in the subject’s microbiome enterocluster profile has been assessed. In some aspects, the subject’s microbiome enterocluster profile is compared to a reference microbiome profile, to determine the relative amounts of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale, or a strain thereof to be included in the composition. In some aspects, composition is administered to provide a level of one or more strains that is within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% (or any range derivable therein) of the reference microbiome profile for that strain.

[0043] Disclosed herein are compositions, including bacterial compositions, isolated bacterial compositions, pharmaceutical compositions, non-natural compositions, therapeutic bacterial compositions, synthetic formulations, live biotherapeutic products, probiotic products, FMT products, enriched FMT products, for treatment, or prevention of gastrointestinal disorders, or for improving the general health and well-being of a subject in need thereof, wherein the composition comprises at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof. Also- 15 -299756710.1provided herein are methods of using these compositions for the treatment or prevention of gastrointestinal disorders and diseases, or for general health, or well-being of a subject in need thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0045] FIGs. 1A-1B: Gut microbiome structure and composition in diarrheal patients and controls. (FIG. 1A) Beta-diversity plot shows that there are significant compositional shifts in disease groups, especially in patients with CDI and CD (ANOSIM p=0.001); (FIG. IB) Family abundance plot showing compositional differences across disease groups.

[0046] FIG. 2: Compositional bias in human IBD. Weighted Jaccard abundance distance plots show expansion of Pathobiome (top) and Bacteroidaceae (bottom paget) in CD and UC patients compared with controls, with loss of for example Bifidobacteriaceae (bottom FIG. 2 Continued). The abundance of specific taxa is weighted (size of circle).

[0047] FIG. 3. Beta-diversity plot showing Enterbacteriaceae abundance as an example of the Pathobiome expansion that occurs in human IBD and CDI patients.

[0048] FIGs. 4A-4B 16S rRNA sequencing based profiling of microbial community structure illustrated by Dirichlet multinomial mixtures (DMM) clustering. DMM clustering is a probabilitybased model in which the samples are categorized based on the frequency of the appearance of each taxa in that sample. In a comprehensive meta-analysis, DMM clustering is a method that classifies samples by cluster or enterotype associated with a specific disease type. (FIG. 4A) DMM identified 5 major clusters in our adult training 16S dataset. Top drivers include: Cluster 1 - Bacteroides, Blautia, Lachnospiraceae; Other; Cluster 2 - Bacteroides, Blautia, Faecalibacterium; Cluster 3 - Bacteroides, Blautia, Enterobacteriaceae;Other, Streptococcus; Cluster 4 - Blautia, Lachnospiraceae; Other , Faecalibacterium, Bifidobacterium and Cluster 5 - Prevotella. (FIG. 4B) Microbiome cluster representation in different human diarrheal diseases.

[0049] FIG. 5A-5B. DMM clusters significantly differentiate different diarrheal disease specimens. FIG. 5A shows IBD and CDI data. FIG. 5B shows UC, CD, and CDI data.

[0050] Chi-squared analysis shows a significant cluster difference (Chi square p-value < 2.2e- 16) between disease groups in a combined analysis of 16S data using the Taxa4Meta profiler.- 16 -299756710.1FIG. 6. Microbiome features associated with CDI patients (i.e. a high CDI risk score) are significantly associated with dysbiotic cluster 3.

[0051] FIG. 7. Top microbiome features that drive Cluster 1.

[0052] FIG. 8. Top microbiome features that drive Cluster 2.

[0053] FIG. 9. Top microbiome features that drive Cluster 3.

[0054] FIG. 10. Top microbiome features that drive Cluster 4.

[0055] FIG. 11. Top microbiome features that drive Cluster 5.

[0056] FIG. 12. Loss of Fecalibacteria in IBD-clusters 1 (and 3) demonstrated by 16S sequencing (left). Metaproteome analysis confirmed the cluster distribution of metabolically active Fecalibacteria in fecal samples of patients (right).

[0057] FIG. 13. Loss of Fecalibacteria in IBD and CDI patients. Shotgun metaproteome analysis confirmed the disease associated clustering by demonstrating loss of metabolically active Fecalibacteria in fecal samples of IBD and CDI patients.

[0058] FIG. 14. Clinical symptoms (PCD Al score) and fecal calprotectin levels are elevated in subjects with an IBD cluster.

[0059] FIG. 15. Independent cohort validation of fecal calprotectin2 abundance by cluster and IBD subtype.

[0060] FIG. 16. DIABIMMUNE: longitudinal infant cohort. Early life human gut microbiome study- 1 shows development of microbiome clusters modeled to the adult training set. Inter-enterotype transition probability at 1 and 3 years of age analyzed and visualized using the Markov chain-based approach. Transition probably matrix for 25-30M (prob<0.2 excluded) is provided on the right, where only transition probabilities greater than 0.2 are shown .

[0061] FIG. 17. TEDDY: longitudinal infant cohort. Early life human gut microbiome study - 2 shows development of microbiome clusters modeled to the adult training set. Inter-enterotype transition probability at 1 and 3 years of age analyzed and visualized using the Markov chainbased approach. Transition probably matrix for 25-30M (prob<0.2 excluded) is provided on the right, where only transition probabilities greater than 0.2 are shown .

[0062] FIG. 18. RNASeq volcano plot (top) of genes expressed in blood specimens from age and sex matched TEDDY infants with a healthy versus IBD microbiome cluster. (Bottom) Summary of RNA SEQ data shows that transition from the IBD to healthy-cluster is associated with significant development of T cell differentiation and immune tolerance, which is lacking in IBD-associated cluster 1.

[0063] FIG. 19. Treatment / therapeutics, FMT considerations for treatment in IBD. FMT donor preparations reported in the literature demonstrate a microbiome community composition- 17 -299756710.1that is significantly different from healthy controls. Generally, FMT donor preparations lack key taxa present in the healthy gut microbiome cluster 4, notably Fecalibacteria and Bifidobacteria (Top). Donor preparations used to treat IBD patients are biased towards IBD risk clusters 1 and 3.

[0064] FIG. 20. Clinical FMT outcomes in UC patients stratified by donor cluster.

[0065] FIGs. 21A-21B. Disease-specific microbiome composition using Dirichlet multinomial mixtures (DMM) analysis of 16S amplicon data from diarrheal patients. (FIG. 21 A) Healthy human gut microbiome clusters (green) are distinguishable from dysbiotic clusters (red). Feces pelleted from conventional mice (blue) and humanized microbiota mice (purple) demonstrate compositional similarity to clinically dysbiotic fecal specimens. (FIG. 21B) Comparison of the 100 most abundant taxa in the healthy human gut microbiome versus humanized microbiota gnotobiotic mice receiving a fecal transplant with healthy human gut bacteria.

[0066] FIGs. 22A-22B. Characterization of the immune response generated after microbiota humanization of germfree C57B1 mice. (FIG. 22A) Methodology used to characterize immune responses generated after microbiota humanization of germfree C57B1 mice for 4 weeks. Flow cytometry was used to quantity T regulatory (reg) cell induction, as well as characterize Thl (proinflammatory) versus Th2 (anti-inflammatory) immune profiling in colon, mesenteric lymph node, liver and spleen. Controls studies were performed using fecal transfer from conventional mouse microbiota into gnotobiotic C57B1 animals. (FIG. 22B) Organ distribution of Fox3- positive T reg cells show that mouse microbiota are significantly more potent inducers of colonic and mesenteric lymph node responses compared with human-derived microbiota (Left). This contributes to significantly enhanced proinflammatory responses (T-Bet+ in CD4 cells) in humanized microbiota animals (Right), since these lack several anti-inflammatory taxa such as Faecalibacterium prausnitzii (*,***; p<0.05 and p<0.01, respectively; Mann Whitney U-test).

[0067] FIG. 23. Experimental outline of testing a rationally designed 4-species mix including Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum and Eubacterium rectale to restore anti-inflammatory properties in a humanized microbiota IL10- / - deficient model of inflammatory bowel disease.

[0068] FIGs. 24A-24D Clinical and immune outcomes after oral supplementation with Faecalibacterium prausnitzii consortia in humanized IL10 - / - mice. (FIG. 24A) Significantly less weight loss was seen in supplemented mice. (FIG. 24B) Animals receiving oral Faecalibacterium prausnitzii consortia showed lower inflammatory fecal lipocailin concentrations compared with human FMT alone. (FIG. 24C) Significant reduction in proinflammatory cytokines measured in colonic CD4+ cells were recorded by flow cytometry. (FIG. 24D) Significant reductions in- 18 -299756710.1proinflammatory cytokines measured in colonic CD8+ cells were also recorded by flow cytometry. CD8+ (* ***>■ p<0.05 and p<0.01, respectively; Mann Whitney U-test).

[0069] FIG. 25A-25K show strain prediction for Faecalibacterium prausnitzii in biomarker. FIG. 25 A shows a heat map of pairwise dissimilarity among 361 Faecalibacterium prausnitzii genomes from the NCBI database, revealing a clear sub-clade structure. The observed between- clade dissimilarity is greater than 0.05 across comparisons, supporting the use of sub-clade resolution for strain prediction. FIG. 25B shows the diversity and coverage of clade-specific marker genes across 362 known F. prausnitzii assemblies. Each strain is represented by most, but not all, marker genes in a clade, enabling the use of marker presence / absence to narrow down possible strains in a sample prior to k-mer-based strain assignment. FIG. 25C shows a principal coordinate analysis (PCoA) plot of 16S-based enterotype clusters, with samples positioned according to the first two principal coordinates (PCol and PCo2). The plot illustrates the separation of microbiome clusters, with Cluster 2 and Cluster 4 associated with healthy states and Cluster 3 associated with dysbiosis, including both IBD and Clostridioides difficile infection (CDIF) cases. FIG. 25D shows the distribution of F. prausnitzii strain predictions by cluster in the Biomarker / Seattle dataset. The size of each dot represents the number of samples assigned to a strain, normalized by the number of samples per cluster, and the background color indicates the F. prausnitzii sub-clades. Cluster 4 displays a broader sub-clade representation, while Cluster 2 is more restricted but remains enriched for health-associated sub-clades. FIG. 25E shows the distribution of F. prausnitzii strain predictions by cluster in the CCF dataset. Cluster 3, which is associated with dysbiosis, exhibits reduced sub-clade diversity and altered strain representation compared with Clusters 2 and 4. FIG. 25F shows the combined distribution of F. prausnitzii strain predictions across the Biomarker / Seattle and CCF datasets. Healthy-enriched clusters (2 and 4) retain multiple sub-clades, while the dysbiotic Cluster 3 shows contraction of specific species level genome bins (SGBs). FIG. 25G is a closeup of FIG. 25F and shows that in the combined datasets, Cluster 3 has an underrepresentation of SGB 15322 relative to Clusters 2 and 4, indicating a dysbiosis-associated loss of specific F. prausnitzii lineages. FIG. 25H shows an additional loss of SGB 15318 in Cluster 3 in the combined datasets, reinforcing the selective depletion of key F. prausnitzii sub-clades in dysbiosis. FIG. 251 shows the normalized abundance of F. prausnitzii SGBs across the Biomarker, CCF, and Seattle datasets. Per-sample SGB abundance is normalized by the total F. prausnitzii abundance, highlighting differential SGB composition across cohorts and clusters. Healthy-associated samples retain a richer mixture of SGBs, while dysbiotic samples concentrate in fewer SGBs with lowered evenness. FIG. 25J shows the normalized abundance of F. prausnitzii SGBs among healthy controls (HC), IBS, and- 19 -299756710.1FAP groups within the Biomarker / Seattle dataset. HC samples retain broader SGB diversity, while IBS and FAP samples show sub-clade shifts but less profound depletion than IBD, consistent with milder dysbiosis. FIG. 25K shows the normalized abundance of F. prausnitzii SGBs between HC samples from Biomarker / Seattle and IBD samples from CCF. The IBD group shows selective loss of SGBs, notably SGB 15318, and reduced representation of certain health- associated sub-clades compared to healthy controls.

[0070] FIG. 26 provides an overview of study aims and interventions.

[0071] FIG. 27 shows a volcano plot showing significant taxa in IBS vs healthy controls after False Discovery Rate correction (FDR) correction (p<0.05).

[0072] FIGs. 28A-28D show FODMAP Non-Responsive Score. FIG. 28A shows Random Forest (RF) classifier trained using three independent LFD studies. FIG. 28B shows RF Model validation showing significant (*; p<0.05) increase in non-responsive score after LDF but not CBT. FIG. 28C shows Cluster-assigned non-responsive score in IBS subjects (****;p<0.0001). FIG. 28D shows Cluster assigned responders (%) in pediatric intervention trial.

[0073] FIGs. 29A-28B show data confirming dietary intake modification can be a potential modulator of IBS treatment. FIG. 29A shows total sugar intake at baseline (BL) and posttreatment (PT) in responders (R) vs. non-responders. FIG. 29B shows total fat intake at baseline (BL) and post-treatment (PT) in responders (R) vs. non-responders (NR) (>30% reduction in MeanMax Pain; *, ****; p<0.05, <0.0001) (unpublished data).DETAILED DESCRIPTION

[0074] Described herein are methods and compositions suitable for the treatment of disorders associated with dysbiosis of the microbiome. Use of the one or more compositions described herein can be employed based on methods described herein. Methods and / or compositions described herein can be included as components of one or more kits suitable for treatment of disorders associated with dysbiosis of the microbiome.

[0075] Also described herein are methods and compositions suitable for the treatment of disorders associated with dysbiosis of the microbiome, including but not limited to irritable bowel syndrome (IBS) and related gastrointestinal conditions. The disclosure encompasses not only the use of specific bacterial consortia and live biotherapeutic products, but also integrates personalized approaches that account for individual differences in psychological distress, diet intake (such as FODMAP and fat consumption), and baseline gut microbiome composition. The methods described herein include the use of advanced bioinformatics pipelines (e.g., Taxa4Meta)- 20 -299756710.1to identify microbiome enteroclusters and other biomarkers that can predict and monitor treatment response. These approaches enable the stratification of patients and the tailoring of interventions — such as dietary modification, cognitive behavioral therapy, or microbiome- targeted therapeutics — to maximize clinical benefit. The compositions and methods can be provided as part of kits for the diagnosis, risk assessment, and treatment of microbiome-associated disorders, supporting a precision medicine framework.

[0076] Other aspects are discussed throughout this application. Any aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. The aspects in the Example section are understood to be aspects that are applicable to all aspects of the technology described herein.

[0077] This application incorporates by reference herein in its entirety PCT Patent Application Serial. No. PCT / US23 / 64978, filed March 27, 2023 and Application Serial No. PCT / US2024 / 021476, filed 26 March, 2024.

[0078] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0079] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0080] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” It is also contemplated that anything listed using the term “or” may also be specifically excluded.

[0081] As used in this specification and claim(s), 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.

[0082] In this specification, where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each sub combination of the elements of that combination is also specifically disclosed and is within the scope of the invention. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an invention is disclosed as having a plurality of alternatives, examples of that invention in which each alternative is excluded singly or in any- 21 -299756710.1combination with the other alternatives are also hereby disclosed; more than one element of an invention can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0083] The term “Antimicrobial” as used herein is a general term for drugs, chemicals, or other substances that either kill or slow the growth of microbes. Among the antimicrobial agents are antibacterial drugs, antiviral agents, antifungal agents, and antiparasitic drugs. In patients this includes drugs and / or treatment that impacts microbiome community composition.

[0084] As used herein, the terms “arrays”, “microarrays”, and “DNA chips” refer to an array of distinct oligonucleotides affixed to a substrate, such as glass, plastic, paper, nylon or other type of membrane, filter, chip, or any other suitable solid support. The polynucleotides can be synthesized directly on the substrate, or synthesized separate from the substrate and then affixed to the substrate. The oligonucleotides on the array can be designed to bind or hybridize to specific nucleic acids, such as a specific SNP or a specific CNV, for example.

[0085] The terms “Clostridioides difficile infection” “C. difficile infection” or “CDI” as used herein refers to an individual that has presence of Clostridioides difficile in their body to an extent and under conditions in which a sufficient level of toxins from the Clostridioides difficile results in diarrhea. This is in contrast to presence of Clostridioides difficile in an individual that is considered a carrier for the bacteria and that has no diarrhea.

[0086] The term “classifier” as used herein refers to an algorithm that implements a disease classification, notably CDI, IBS, IBD UC, and / or IBD CD diagnosis, or CDI, IBS, IBD UC, and / or IBD CD risk, or risk of C. difficile colonization. In other aspects, the term refers to an algorithm that implements a disease classification for diagnosis or risk or risk of colonization for one or more pathogens other than C. difficile.

[0087] As used herein, the term “enterotype” refers to classification of living organisms (including mammals, such as primates, and including humans, for example) based on the bacteriological composition of their gut microbiota.

[0088] As used herein, a "microbiome enteroclusters profile" or “bacterial taxa profile” refers to the classification and characterization of a subject’s gut microbiota into distinct community types or clusters, each defined by the relative abundance and composition of specific bacterial taxa, as determined by statistical modeling approaches such as, but not limited to Dirichlet multinomial mixtures (DMM). These enteroclusters represent reproducible, disease- or health- associated microbiome configurations, enabling stratification of individuals based on their gut microbial community structure. The profile is typically derived from high-throughput sequencing data (e.g., 16S rRNA gene amplicon or metagenomic sequencing) and incorporates both- 22 -299756710.1taxonomic and, optionally, functional features, facilitating diagnosis, prognosis, and personalized therapeutic interventions for gastrointestinal and related disorders.

[0089] As used herein the term “relative abundance” in the context of microbiome enteroclusters profiling, refers to the proportion of a specific microbial taxon (such as a genus, species, or strain) within the total microbial community present in a biological sample (e.g., fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation). It is typically expressed as a percentage or fraction, representing how much of the total microbiome is comprised by that particular taxon. In enterocluster analysis, relative abundance is used to characterize the composition of gut microbiota clusters (enterotypes) by quantifying the presence of key taxa that define each cluster. When profiling enteroclusters, the relative abundances of multiple taxa are compared to identify dominant and depleted members that distinguish healthy versus disease-associated microbiome configurations. Comparisons can be made with a reference sample.

[0090] As used herein, a "reference microbiome profile" or “reference sample” refers to a biological specimen, such as fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation, that is collected from an individual or a population of individuals and is used as a standard or comparator for assessing gut microbiome composition, diversity, or function. The reference sample may represent a healthy gut microbiome from one or more individuals without gastrointestinal disease or may comprise samples from individuals with a defined disease state or condition, including disease-matched controls. In some aspects, reference gut microbial samples are utilized to establish baseline or comparative microbial profiles for the purposes of diagnosis, prognosis, classification, or evaluation of microbiome-associated features in test subjects, and may be selected based on demographic, clinical, or technical criteria relevant to the intended analysis.

[0091] An “increased relative abundance” or “greater relative abundance” is used interchangeably to mean that the proportion of a specific taxon within the total microbiome is higher compared to the reference sample. This increase can be expressed as a fold-change (e.g., 2-fold, 10-fold) or as a percentage increase (e.g., 50% higher than control). In the context of enterocluster profiles, increased relative abundance of certain taxa may indicate a shift toward a disease-associated microbiome (dysbiosis) or, conversely, enrichment of beneficial taxa in response to treatment. For instance, if Bacteroides species are found at 20% relative abundance in a patient sample compared to 10% in healthy controls, this represents a 2-fold increase.

[0092] A “reduced relative abundance” refers to a lower proportion of a specific taxon within the total microbiome compared to a reference sample. This decrease can also be expressed as a- 23 -299756710.1fold-change (e.g., 0.5-fold, indicating a 50% reduction) or as a percentage decrease. In enterocluster profiling, reduced relative abundance of protective or beneficial taxa (such as Faecalibacterium prausnitzii or Bifidobacterium spp.) is commonly associated with microbiome immaturity, dysbiosis, or increased susceptibility to gastrointestinal disorders. For example, if Faecalibacterium prausnitzii is present at 1% relative abundance in a patient sample versus 5% in healthy controls, this is a 5-fold reduction. Such reductions are key indicators in disease classification and can guide targeted microbiome therapies as disclosed herein.

[0093] As used herein, a "pan-microbiome profiling pipeline" refers to an integrated bioinformatics framework designed to analyze, harmonize, and interpret gut microbiome data across diverse sequencing platforms, variable regions, and populations. This pipeline employs advanced computational methods — including quality control, sequence clustering, taxonomic annotation, and statistical modeling — to minimize technical and demographic biases inherent in microbiome research. By leveraging strategies such as variable read-length analysis and unified taxonomic binning, the pipeline enables robust meta-analysis of heterogeneous datasets, facilitating the identification of core microbial features and community structures that are consistent across studies, populations, and sequencing modalities.

[0094] The pan-microbiome profiling pipeline incorporates statistical approaches such as Dirichlet multinomial mixtures (DMM) to classify gut microbiota into distinct community types, or "enteroclusters," based on the relative abundance and composition of bacterial taxa. These enteroclusters are reproducible microbiome configurations that can be associated with health, disease, or treatment responsiveness. The output of the pan-microbiome profiling pipeline is the Microbiome Enteroclusters Profile, which represents the stratification of an individual’s or population’s gut microbiota into defined enteroclusters. In some aspects, this profile serves as a diagnostic, prognostic, or therapeutic tool, enabling precision medicine approaches in gastrointestinal and multisystem disorders by providing a standardized, scalable method for microbiome analysis and interpretation. In certain aspects, the pan-microbiome profiling pipeline comprises or incorporates the Taxa4Meta software, a specialized bioinformatics tool developed to address technical and demographic variability in clinical metagenomic analysis. Taxa4Meta utilizes a variable read-length strategy, region-specific taxonomic thresholds, and a novel binning approach to consolidate taxonomic annotations from different 16S rRNA variable regions and sequencing platforms. This enables accurate, cross-cohort taxonomic profiling and supports the integration of data from multiple sequencing modalities, thereby enhancing the precision and reproducibility of microbiome community classification.- 24 -299756710.1

[0095] The term “feature” as used herein refers to a microbe, biological molecule, and / or metabolic pathway that is representative of a detectable difference between a control or reference standard and the corresponding microbe, biological molecule, and / or metabolic pathway in an individual with or at risk of developing CDI, IBS, IBD UC, and / or IBD CD. A feature can be the presence, absence, relative abundance, and / or levels of a microbe, nucleic acid sequence (such as 16S rRNA), protein, small molecule, metabolic pathway, and / or a combination thereof.

[0096] The term “pan-microbiome” as used herein refers to a composit of two or more microbiomes, for example, a composit of two or more data sets reflective of two or more microbiomes. A pan-microbiome is generally larger than any single microbial community of an individual or a group. In some aspects, a pan-microbiome includes two or more populations, two or more demographics, and / or data collected through two or more acquisition methodologies.

[0097] As used herein, the term “oligonucleotide” refers to a short chain of nucleic acids, either RNA, DNA, and / or PNA. The length of the oligonucleotide could be less than 10 base pairs, or at minimum or no more than 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, or 75 base pairs. The oligonucleotide can be synthesized using by methods including phosphodiester synthesis, phosphotriester synthesis, phosphite triester synthesis, phosphoramidite synthesis, solid support synthesis, in vitro transcription, or any other method known in the art.

[0098] As used herein, the term “PCR primer” refers to an oligonucleotide that is used to amplify a strand of nucleic acid in a polymerase chain reaction (PCR). Primers may have 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the template the primers hybridize to, wherein the 3’ nucleotide of the primer is complementary to the template. In some aspects, lower annealing temperatures are used for initial cycles, for example cycles 1, 2, 3, 4, and / or 5, of the reaction.

[0099] Treatment,” “treat,” or “treating” means a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms. The treatment can be any reduction from pre-treatment levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition. Therefore, in the disclosed methods, treatment” can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or the disease progression, including reduction in the severity of at least one symptom of the disease.- 25 -299756710.1For example, a disclosed method for reducing the immunogenicity of cells is considered to be a treatment if there is a detectable reduction in the immunogenicity of cells when compared to pretreatment levels in the same subject or control subjects. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. It is understood and herein contemplated that “treatment” does not necessarily refer to a cure of the disease or condition, but an improvement in the outlook of a disease or condition. In specific aspects, treatment refers to the lessening in severity or extent of at least one symptom and may alternatively or in addition refer to a delay in the onset of at least one symptom.

[0100] “Subject” may refer to an organism that comprises a microbiome. In certain aspects, it refers to a human patient. In certain aspects, it refers to an animal.

[0101] As will be understood from context, “risk” of a disease, disorder, and / or condition refers to a likelihood that a particular individual will develop the disease, disorder, and / or condition. In some aspects, risk is expressed as a percentage. In some aspects, risk is at least, at most, exactly, or between (inclusive or exclusive) any two of from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some aspects risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some aspects, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and / or event. In some aspects a reference sample or group of reference samples are from individuals comparable to a particular individual. In some aspects, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and / or condition. In some aspects, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0102] Gastrointestinal disease” or “gastrointestinal disorders” refer to a wide range of conditions that affect the digestive system, including the esophagus, stomach, intestines, liver, pancreas, and gallbladder. These disorders can interfere with digestion, absorption of nutrients, and elimination of waste. In certain aspects, the gastrointestinal disorder is associated with microbiome dysbiosis. Non-limiting examples of gastrointestinal disorders include Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD). A “subject having a gastrointestinal (GI) disease” or gastrointestinal disorder is a subject diagnosed with, suffering from or showing symptoms of a gastrointestinal disease or disorder. A “subject at risk of having a gastrointestinal (GI) disease” is an individual who has an increased likelihood of developing a disease or a disorders that affect the digestive tract, which includes the esophagus, stomach, intestines, liver, pancreas, and gallbladder. Risk factors for GI diseases can vary depending on the specific- 26 -299756710.1condition, but common factors include family history of GI diseases like Crohn’s disease, ulcerative colitis, or colorectal cancer; dietary habits; lifestyle factors; obesity; use of certain medications, for example, nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, or proton pump inhibitors; and infections like Helicobacter pylori, which can lead to ulcers, or Clostridioides difficile, which causes severe colitis. In some aspects, the subject with a GI disease or disorder is diagnosed using a method disclosed herein. For example, the subject may have a bacterial taxa profile in a fecal matter sample that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the taxa identified in Cluster 1 or Cluster 3. In some aspects, the subject may have reduced levels of Fecaelibacteria in the gut.

[0103] As used herein, the term “Live Biotherapeutic Products” or “LBPs” are a class of biological therapeutics that comprise living microorganisms, typically bacteria, which are designed to provide therapeutic benefits when administered to a patient. LBPs differ from “probiotics”, in that probiotics are usually formulated for healthy individuals as dietary supplements or functional foods, whereas LBPs are usually formulated for delivery as therapeutics. LBPs are generally designed to treat, prevent, and / or cure specific diseases and / or medical conditions. LBPs are regulated as biological drugs by authorities like the U.S. FDA or European Medicines Agency (EMA), meaning they must undergo rigorous clinical trials to demonstrate safety, efficacy, and quality. Thus, the regulatory guidelines for the formulation of probiotics and LBPs can be different. Unlike most traditional drugs, LBPs utilize the beneficial properties of live microbes to target specific physiological conditions and / or diseases. Many LBPs are based on one or more types of bacteria that are naturally found in the human body, as disclosed herein, for example bacteria found in Clusters 2, 4, and / or 5. LBPs can be administered by any suitable route including oral, buccal, orogastric, nasogastric, sublingual, topical, rectal, vaginal, colonoscopy administration, and / or flexible sigmoidoscopic route. LBPs may also be used to enrich Fecal Microbiota Transplant (FMT) products. In certain aspects, FMT can be classified under LBPs if they conform to the guidelines provided by regulatory agencies, for instance REBYOTA® is an FDA-approved microbiome based therapy.

[0104] As used herein “Fecal Microbiota Transplant” (FMT) procedure refers to a medical procedure in which stool from a healthy donor is transplanted into the gastrointestinal (GI) tract of a patient, typically to restore a balanced microbial community. FMT has typically been utilized to treat recurrent Clostridium difficile infections, which occur when harmful bacteria overgrow in the gut after antibiotic use. The process involves collecting stool from a screened donor, processing it to extract the microbiota, and then introducing it into the patient's colon. This can be done via colonoscopy, enema, or through the upper GI tract using a nasogastric tube or capsules.- 27 -299756710.1The goal of FMT is typically to repopulate the patient’s gut with beneficial bacteria, thereby improving the balance of the gut microbiome and promoting health.

[0105] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects, are given by way of illustration only, since various changes and modifications within the spirit and scope of the immediate disclosure will become apparent to those skilled in the art from this detailed description.

[0106] Targeted metagenomic sequencing is an emerging strategy to survey disease-specific microbiome biomarkers for clinical diagnosis and prognosis. However, this approach often yields inconsistent or conflicting results due to inadequate study power and experimental bias. To address this problem, the inventors developed Taxa4Meta, a bioinformatics pipeline explicitly designed to compensate for technical and demographic bias. The inventors designed and validated Taxa4Meta for accurate taxonomic profiling of 16S amplicon data acquired from different sequencing strategies. Taxa4Meta offers tremendous potential in identifying clinical dysbiotic features that can reliably predict human disease (e.g. gastrointestinal disorders associated with microbial dysbiosis), validated comprehensively via re-analysis of individual patient 16S datasets. Specifically, Taxa4Meta facilitates pan-microbiome profiling of 16S features with excellent utility for stratification of IBD patients from diarrheal cases with Clostridioides difficile infection (CDI) and irritable bowel syndrome (IBS), who share common symptoms that are difficult to diagnose and manage. Thus, Taxa4Meta represents a novel approach to individual microbiome surveys to define dysbiosis at a population-scale level.

[0107] The new bioinformatics strategy was applied to develop microbiome biomarkers to assist in the clinical diagnosis and management of patients with IBD. A comprehensive re-analysis of >35,000 fecal 16S profiles generated across four continents covering the entire human life span (from infants to centenarians) was performed. This work enabled characterization of human gut microbiome development using pan-microbiome profiling as a strategy to minimize technological bias and identify core microbiome features. Using Taxa4Meta to investigate dysbiosis in IBD patients, the following significant discoveries were made: (1) in a detailed re-analysis of 4,491 IBD and matched control subjects from 36 clinical cohorts spanning North America, Europe and Australasia an IBD-dominant microbiome cluster that are largely absent in healthy individuals and in patients with IBS or CDI serving as diarrheal disease controls were identified; (2) the inventors demonstrated that this unique IBD-cluster can differentiate IBD from CDI and IBS patients, offering diagnostic utility; (3) the IBD-cluster is associated with microbiome immaturity- 28 -299756710.1associated with early childhood. Comprehensive RNAseq studies in matched blood specimens during infant development showed that the IBD-associated cluster is associated with immunological immaturity and lacks immune tolerance induced during normal microbiota development. Thus, the data show that IBD patients are associated with a gut microbiome immaturity that lack sufficient immune tolerance to microbial inflammatory triggers; (4) notably, by considering patient-donor microbiome cluster mismatching, the studies also indicate that Taxa4Meta can predict treatment outcomes in IBD patients who receive fecal microbiota transplants (FMT) and as such are a guide for precision microbiota therapy. The findings help disambiguate the role of the gut microbiota in the development of IBD during adolescence and adulthood. Moreover, these studies provide information for biomarker and therapeutic target discovery.I. Diseases associated with microbiome dysbiosis

[0108] The human gut microbiome comprises bacteria, viruses, and fungi ideally living symbiotically with their human host. Individual species and collective bacterial functions within the gut microbiome confer many benefits throughout life including metabolizing dietary contributions, educating the immune system, defending against pathogens, and contributing to overall health and optimal growth. The gut microbiome is affected by and influences pathologies including but not limited to inflammatory bowel disease (IBD; both ulcerative colitis (UC) and Crohn’s disease (CD)), Clostridium difficile infection (CDI), and irritable bowel syndrome (IBS).A. Clostridioides (previously termed Clostridium) difficile infection (CDI)

[0109] Clostridiodes difficile is a bacterium that causes an infection (CDI) of the large intestine (colon). Symptoms can range from diarrhea to life-threatening damage to the colon. The bacterium is often referred to as C. difficile or C. diff. Illness from C. difficile typically occurs after use of antibiotic medications. It most commonly affects older adults in hospitals or in long-term care facilities. In the United States, about 200,000 people are infected annually with C. difficile in a hospital or care setting. Thankfully these numbers are trending lower than in previous years because of improved prevention measures. People not in care settings or hospitals also can develop C. difficile infection. Some strains of the C. difficile bacterium in the general population may cause serious infections or are more likely to affect younger people. In the United States, about 170,000 infections occur annually outside of health care settings, and worryingly these numbers are increasing. Some people carry C. difficile bacteria in their intestines but never become sick. These individuals are carriers of the bacteria and may spread infections. Signs and symptoms usually develop within 5 to 10 days after starting a course of antibiotics. However, they may occur as soon- 29 -299756710.1as the first day or up to three months later. The most common signs and symptoms of mild to moderate C. difficile infection are: watery diarrhea three or more times a day for more than one day and / or mild abdominal cramping and tenderness. People who have a severe C. difficile infection tend to become dehydrated and may need to be hospitalized. C. difficile can cause the colon to become inflamed and sometimes form patches of raw tissue that can bleed or produce pus. Signs and symptoms of severe infection include: watery diarrhea as often as 10 to 15 times a day, abdominal cramping and pain (which may be severe), rapid heart rate, dehydration, fever, nausea, increased white blood cell count, kidney failure, loss of appetite, swollen abdomen, weight loss, and / or blood or pus in the stool. C. difficile infection that is severe and sudden, an uncommon condition, may also cause intestinal inflammation leading to enlargement of the colon (also called toxic megacolon) and sepsis. Sepsis is a life-threatening condition that occurs when the body's response to an infection damages its own tissues. People who have these conditions are generally admitted to an intensive care unit.

[0110] C. difficile bacteria enter the body through the mouth. They can begin reproducing in the small intestine. When they reach the large intestine (colon), they can release tissue-damaging toxins. These toxins destroy cells, produce patches of inflammatory cells and cellular debris, and cause watery diarrhea. When the bacteria are outside the colon, virtually anywhere in the environment, they are in a dormant state, or essentially quiescent. This enables them to survive for a long time in any number of places, including but not limited to human or animal feces, surfaces in a room, unwashed hands, soil, water, and / or food. When bacteria once again find their way into a person's digestive system, they begin to produce infection again. The ability of dormant C. difficile to survive outside the body enables the generally easy transmission of the bacterium, particularly in the absence of thorough hand-washing and cleaning.[OHl] Risk factors associated with developing a C. difficile infection include but are not limited to, taking antibiotics or other medications such as Clindamycin, Cephalosporins, Penicillin’s, Fluoroquinolones, and / or potentially certain proton pump inhibitors. The majority of C. difficile infections occur in people who are or who have recently been in a health care setting, including hospitals, nursing homes and long-term care facilities, where germs spread easily, antibiotic use is common and people are especially vulnerable to infection. Additionally, certain medical conditions or procedures may increase an individual’s susceptibility to a C. difficile infection, such as IBS, a weakened immune system from a medical condition or treatment (e.g., chemotherapy), chronic kidney disease, a gastrointestinal procedure, and / or other abdominal surgery. Additionally, age is a major risk factor for CDI infection.- 30 -299756710.1

[0112] Complications associated with C. difficile infection include but are not limited to: dehydration, kidney failure, toxic megacolon, bowel perforation, and / or death. The necessity for swift and correct diagnosis and initiation of appropriate therapeutic interventions are key steps in limiting the impact of this potentially devastating disease. Methods and compositions disclosed facilitate this process.B. Irritable bowel syndrome (IBS)

[0113] Irritable bowel syndrome (IBS) is a common disorder that affects the large intestine. Signs and symptoms include cramping, abdominal pain, bloating, gas, and diarrhea or constipation, or both. IBS is a chronic condition that will require long term management. Only a small number of people with IBS have severe signs and symptoms. Some people can control their symptoms by managing diet, lifestyle and stress. More-severe symptoms can be treated with medication and counseling.

[0114] The signs and symptoms of IBS vary but are usually present for a long time. The most common include: abdominal pain, cramping or bloating that is related to passing a bowel movement, changes in appearance of bowel movement, changes in how often you are having a bowel movement, and / or other symptoms that are often related include bloating, increased gas or mucus in the stool. Certain severe symptoms associated with IBS may include: weight loss, diarrhea at night, rectal bleeding, iron deficiency anemia, unexplained vomiting, difficulty swallowing, and / or persistent paint hat isn’t relieved by passing gas or a bowel movement.

[0115] The precise cause of IBS is not yet known. But factors that appear to play a role in IBS disease progression include: muscle contractions of the intestine that are stronger and / or last longer than normal and / or weaker than normal, poor nervous system signaling, severe infection, early life stress, and / or changes in the gut microbiome. IBS symptom “flares” can be triggered by certain foods such as beverages, wheat, dairy, citrus fruits, beans, cabbage, milk and / or carbonated drinks, or stress.

[0116] Risk factors associated with IBS include being young, being female, having a family history of IBS, and / or having anxiety, depression and / or other mental health issues.

[0117] Major complications associated with IBS include chronic constipation or diarrhea that can cause hemorrhoids, a reduction in the quality of life, and exacerbation of mood disorders.

[0118] Correct diagnosis and management of IBS is an essential step in long term management of the disease. Methods and compositions disclosed facilitate this process.C. Inflammatory bowel disease (IBD)

[0119] Inflammatory bowel disease (IBD) is an umbrella term used to describe disorders that involve chronic inflammation of your digestive tract. The two major types of IBD include- 31 -299756710.1Ulcerative colitis (UC) which involves inflammation and development of ulcers along the superficial lining of the large intestine and rectum; and Crohn’s disease (CD) which is characterized by inflammation of the lining of the digestive tract which can also involve the deeper layers of the digestive tract. Both ulcerative colitis and Crohn's disease usually are characterized by diarrhea, rectal bleeding, abdominal pain, fatigue and weight loss. IBD can be debilitating, and can sometimes lead to life-threatening complications.

[0120] Symptoms of IBD vary depending on the severity of the associated inflammation, and where in the digestive tract it occurs. Symptoms may range from mild to severe and may be interrupted by periods of remission. Symptoms common to both IBD UC and IBD CD include but are not limited to diarrhea, fatigue, abdominal pain and cramping, blood in the stool, reduced appetite, and / or unintended weight loss.

[0121] The exact causes of IBD remain elusive. However, it is thought that diet and stress may be involved, but perhaps these factors just aggravate the disease and are not the root cause. It is also thought that an immune system malfunction may also contribute to IBD development. When the immune system tries to fight off an invading virus or bacterium, an abnormal immune response causes the immune system to attack the cells in the digestive tract, too. Heredity also may play a role in that IBD is more common in people who have family members with the disease. However, most people with IBD don't have this family history.

[0122] Risk factors for development of IBD include but are not limited to age, race and / or ethnicity, family history, cigarette smoking and / or nonsteroidal anti-inflammatory medications (e.g., ibuprofen, naproxen sodium, etc.).

[0123] Complications associated with UC and / or CD include colon cancer, skin / eye / joint inflammation, medication side effects, primary sclerosing cholangitis, blood clots, bowel obstruction, malnutrition, fistulas, anal fissures, toxic megacolon, severe dehydration and / or perforation of the colon.

[0124] Correct diagnosis and management of IBD be it UC or CD is an essential step in long term management of the disease. Methods and compositions disclosed facilitate this process.II. Methods of identifying features, evaluating levels of features, and classifying disease state

[0125] Disclosed herein are methods of identifying features, and evaluating the presence, absence, or levels of said features in a sample. In certain aspects, a feature may also be described as a biomarker. In certain aspects, one or more features are used to classify (e.g., diagnose) a disease state and / or identify one or more effective treatment options for a patient with an intestinal- 32 -299756710.1disorder characterized by microbiome dysbiosis (e.g., CDI, IBS, IBD UC, and / or IBD CD). In some aspects, one or more features are used to diagnose a disease state and / or identify one or more effective treatment options for a patient with an intestinal disorder characterized by diarrhea.

[0126] In certain aspects, a feature is a taxonomical classification. In certain aspects, a feature is the presence, absence, or level of one or more microbial taxonomic units (e.g., genera, species, etc.). In certain aspects, a feature is a metabolic pathway. In some aspects, disclosed herein are methods of using a pan-microbiome profiling pipeline as a method suitable for identification of certain core features that can be used for accurate downstream diagnosis, accurate method of treatment prescription, and / or treatment composition determination.

[0127] It is contemplated that features can be evaluated based on one or more associated gene products. In some aspects, a gene product is an amplicon complementary to at least a portion of a gene. In some aspects, a gene product is an RNA transcript. In some aspects, a gene product is a structural and / or functional RNA transcript. In some aspects, a gene product is a protein expressed by an RNA transcript. In some aspects, a gene product is a metabolic pathway associated with expression of a number of gene products. In some aspects, a gene product is a metabolic pathway associated with expression of a number of gene products from a number of different species.

[0128] In some aspects, features are identified using a pan-microbiome approach. In some aspects, utilization of a pan-microbiome approach to identify features can reduce technical and / or demographic bias. In some aspects, a pan-microbiome approach is a method that identifies and selects classifier features by analysis of microbiome data generated from two or more different sequencing strategies (e.g., 16S sequencing strategies) and / or two or more populations (e.g., two or more demographically distinct populations).

[0129] In certain aspects, a meta-analysis to determine the presence, absence, levels, expression, and / or activity of one or more features disclosed herein for correlation with a disease state can be performed. In statistics, a meta-analysis combines the results of several studies that address a set of related research hypotheses. This is normally done by identification of a common measure of effect size, which is modeled using a form of meta-regression. Generally, three types of models can be distinguished in the literature on meta-analysis: simple regression, fixed effects meta-regression and random effects meta-regression. Resulting overall averages when controlling for study characteristics can be considered meta-effect sizes, which are more powerful estimates of the true effect size than those derived in a single study under a given single set of assumptions and conditions. A meta-gene expression value, in this context, is to be understood as being the median of the normalized expression of a marker gene or activity. Normalization of the expression of a marker gene is preferably achieved by dividing the expression level of the individual marker- 33 -299756710.1gene to be normalized by the respective individual median expression of this marker genes, wherein said median expression is preferably calculated from multiple measurements of the respective gene in a sufficiently large cohort of test individuals. In some aspects, a test cohort comprises at least 3, 10, 100, 200, 1000 individuals or more including all values and ranges thereof. In some aspects, dataset-specific bias can be removed or minimized allowing multiple datasets to be combined for meta-analyses (See Sims et al. BMC Medical Genomics (1 :42), 1-14, 2008, which is incorporated herein by reference in its entirety). In some aspects, a meta-analysis cohort comprises the combination of 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 test cohorts or more including all values and ranges thereof.A. Identification of features suitable for classification of disease state

[0130] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises simulation of full-length and / or region-specific 16S amplicon data. In certain aspects, simulation of full-length and / or region-specific 16S amplicon data can be based on reference data bases (e.g., NCBI 16S rRNA RefSeq database (downloaded in July 2019), Ribosomal Database Project (RDP) database (release 11.5) (Cole et al., 2014), etc.). In certain aspects, bioinformatics tools such as cutadapt (version 2.4)(Martin, 201 l)d can be used to extract sequence fragments as full-length amplicons of targeted 16S variable regions (V1-V3, V3-V5, V4 and V6-V9) based on the forward and reverse primers (e.g., primers as listed in Table 22). In some aspects, an error rate is permitted during sequence extraction, for example, an error rate of 0.05, 0.1, 0.15, 0.2, 0.25, etc. In certain aspects, an error rate of 0.2 is permitted during sequence extraction. In certain aspects, sequence length trimming and / or random simulation of sequence abundance and quality scores are performed for specific benchmarking purposes.

[0131] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises benchmarking of sequence clustering and denoising using simulated amplicons, optionally with variable length. In certain aspects, when benchmarking the accuracy of clustering or denoising for amplicon data using variable sequence lengths, random count ranging from 1 to 50 (e.g., 1, 2, 3, 4, 5.... 45, 46, 47, 48, 49, or 50) can be assigned for one or more or all parent full-length amplicons extracted from a reference database (e.g., NCBI 16S rRNA RefSeq sequences). In some aspects, sequencing data can be generated in the reverse orientation and / or the forward orientation. In some aspects, traditional 454 data is generated from reverse orientation, and length trimming from either forward or reverse orientation is applied to one or more, or each type of amplicon data. In some aspects, length trimming results- 34 -299756710.1in 100, 150, 170, 200, 250, 300, 350, 400 and / or 450 bases for variable regions, e.g., V1-V3, V3- V5 and V6-V9 amplicon data. In some aspects, length trimming results in 100, 150, 170, 200 and / or 250 bases for variable regions, e.g., V4 amplicon data. In some aspects, random phred quality score (ASCII_BASE=33) ranging from 30 to 42 can be assigned to each base for sequencing denoising. In some aspects, simulated amplicons of each sequence length represents one sample. In some aspects, one or more or all samples with the same sequence orientation from the same 16S region can then be included for closed-reference or de novo clustering (e.g., using UCLUST (vl.2.22)Edgar, 2010) or VSEARCH (v2.9) (Rognes et al., 2016) or denoising using DADA2 (vl.8) (Callahan et al., 2016)). In some aspects, sequence similarity thresholds including 0.97, 0.99 and 1.00 can be evaluated for each clustering strategy. In some aspects, databases (e.g., the SILVA database (release 132)) can be used for closed-reference OTU picking. In some aspects, simulated amplicons of variable length originating from the same parent full-length amplicon have the same sequence counts, in such situations, pairwise Spearman correlation analysis can be performed for sequence counts of any two sequence lengths (as two independent samples) in one or more OTU count tables

[0132] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises benchmarking of taxonomic over-classification. In some aspects, taxonomic over-classification for short amplicon data represents an important criteria for controlling false positives. In some aspects, using default parameters in the Bayesian- based Lowest Common Ancestor (BLCA) tool (Gao et al., 2017) and its default database of NCBI 16S rRNA RefSeq can be used to annotate random and repeat sequences that were previously generated for benchmarking IDTAXA and other annotation tools (Murali et al., 2018). In some aspects, full-length 16S amplicons of unannotated sequences (e.g., at least down to family rank) are extracted from a suitable database (e.g., RDP database (release 11.5)) and are used for testing BLCA. In certain aspects, BLASTN search of unannotated sequences against a suitable reference database (e.g., NCBI 16S rRNA RefSeq database) can be used to confirm that no best hits are identified at 97% threshold applied to either or both sequence identity and coverage. In some aspects, simulated amplicons of unannotated RDP sequences are tested using different thresholds of sequence coverage and identity (e.g., ranging from 0.85 to 1.00 in BLCA). In some aspects, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more iterations of random sub-sampling (e.g., 1%, 2%, 3%, 4%, 5%) and BLCA annotation on those unannotated amplicons are performed for statistical determination of optimal sequence coverage and identity required for BLCA. In some aspects, ten iterations of random sub-sampling (1%) and BLCA annotation on those unannotated amplicons- 35 -299756710.1are performed for statistical determination of optimal sequence coverage and identity required for BLCA. In some aspects, taxonomic over-classification rate is defined as the classifiable proportion of unannotated amplicons at species level. In some aspects, the confidence score of taxonomic assignment is not considered at this stage.

[0133] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises benchmarking of taxonomic accuracy using simulated amplicons of variable length. In some aspects, benchmarking taxonomic accuracy of BLCA, simulated amplicons of variable length are generated by trimming full-length amplicons derived from a suitable database (e.g., NCBI 16S RefSeq) from either forward or reverse orientation. In some aspects, trimming of full-length amplicons results in 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, and / or 460 bases. In some aspects, trimming of full-length amplicons results in 100, 150, 170, 200, 250, 300, 350, 400 and 450 bases for VI- V3, V3-V5 and V6-V9 amplicon data, and 100, 150, 170, 200 and 250 bases for V4 amplicon data. In some aspects, in addition to the known taxonomic lineage, the parent 16S sequences of simulated amplicons are also present in the BLCA default reference database using NCBI 16S RefSeq, thus taxonomic misclassification can be evaluated. In some aspects, misclassification rate is defined as the proportion of incorrect annotations for simulated amplicons. In some aspects, to further determine the optimal confidence threshold of BLCA for mitigating misclassification, amplicons with a selected sequence length range are combined to calculate the proportion of correct versus incorrect annotations using defined thresholds. In some aspects, the already known taxonomic lineage, true positive (TP) and false negative (FN) hits are correct annotations, whereas true negative (TN) and false positive (FP) hits are incorrect annotations.

[0134] In most aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises design of a data analysis pipeline. In certain aspects, the data analysis pipeline is the Taxa4Meta pipeline. In certain aspects, data analysis pipelines are generated as a function of benchmarking results. In certain aspects, a new computational pipeline “Taxa4Meta” can be used to analyze 16S amplicon data with an optimal range of variable sequence lengths. In some aspects, such a pipeline implements several open-source programs, such as VSEARCH (Rognes et al., 2016) for stringent clustering with a known identity range (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity; preferably 99% identity). In some aspects, open-source programs such as VSEARCH can be optimized for 16S amplicon data with the selected variable lengths after quality trimming. In some aspects, BLCA (Gao et al.,- 36 -299756710.12017) can be used with optimal region-specific confidence thresholds for stringent species annotation of OTUs. In certain aspects, IDTAXA (Murali et al., 2018) can be utilized for annotating OTUs that cannot be annotated down to species resolution. In certain aspects, collapsed taxonomic profiles from OTU tables are used for downstream analyses during 16S meta-analysis.

[0135] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises benchmarking of taxonomic profiling accuracy comparing new data analysis pipelines (e.g., Taxa4Meta) with other standard 16S data analysis pipelines. In some aspects, the feasibility and / or accuracy of different 16S pipelines are tested using the simulated and experimental datasets (McIntyre et al., 2017; Saulnier et al., 2011), and optionally the tests are designed to retain reads for accurate sequence clustering and for improved taxonomic accuracy. In some aspects, simulated datasets are prepared from a suitable data base (e.g., NCBI 16S RefSeq as indicated above). In some aspects, full-length amplicons of V1-V3, V3-V5, V4 and V6-V9 are simulated, and random sequence count ranging from 1 to 50 and random phred quality score (ASCII_BASE=33) ranging from 30 to 42 are generated for each full- length amplicon. In some aspects, further length trimming is performed for one or more or each of the full-length amplicons, for example but not limited to, VI -V3 forward amplicons (200, 250, 300, 350, 400 and 450 bases), V1-V3 reverse amplicons (300, 350, 400 and 450 bases), V3-V5 forward amplicons (250, 300, 350, 400 and 450 bases), V3-V5 reverse amplicons (300, 350, 400 and 450 bases), both forward and reverse amplicons of V4 (200 and 250 bases), V6-V9 forward amplicons (300, 350, 400 and 450 bases), V6-V9 reverse amplicons (250, 300, 350, 400 and 450 bases). In some aspects, trimmed amplicons from the same sequence orientation of the same 16S variable region are combined for benchmarking different 16S pipelines. In some aspects, NCBI 16S taxonomic lineage of NCBI 16S RefSeq is used as the ground truth (reference annotations) for comparison. In some aspects, a cohort (e.g., a Korean stool microbiome dataset (Whon et al.,2018) with the same DNA extracts used for 454 V1-V4, Illumina V1-V3, Illumina V3-V4, Illumina V4, and Illumina shotgun metagenomic sequencing is used as the real human microbiome dataset for benchmarking different 16S pipelines. In some aspects, primers retained in the sequence reads are removed by positional trimming. In some aspects, Illumina paired-end reads are merged (e.g., using USEARCH (version 8.1.1831)) with certain parameters (e.g., default parameters) prior to benchmarking 16S pipelines. In certain aspects, key 16S analysis pipelines can include DADA2-IDTAXA, DADA2-RDP, UCLUST-UCLUST, USEARCH-RDP, Taxa4Meta, Kraken2 and / or MetaPhlAn2. In some aspects, key 16S analysis pipelines DADA2- IDTAXA, DADA2-RDP, UCLUST-UCLUST, USEARCH-RDP, Taxa4Meta, Kraken2 and / or- 37 -299756710.1MetaPhlAn2 are benchmarked with simulated amplicons and / or ground truth datasets (e.g., Korean human microbiome dataset).

[0136] In some aspects, an analysis procedure for a DADA2-IDTAXA pipeline can be performed. In some aspects, DADA2 (version 1.8) is used for denoising amplicon data after quality filtering with a maximum expected error (e.g., of 2) and a minimum base length (e.g., of 200 bases). In some aspects, IDT AXA together with its pre-built RDP training set (version 16) is used for taxonomic annotation with the confidence threshold (e.g., of 70) using a number of bootstraps (e.g., 100 bootstraps). In some aspects, IDTAXA based analysis can only go down to genus level. In some aspects, an analysis procedure for DAD A2 -RDP pipeline can be performed. In some aspects, DADA2 (version 1.8) is used for denoising amplicon data after quality filtering with a maximum expected error (e.g., of 2) and minimum base length (e.g., of 200 bases). In some aspects, RDP Naive Bayesian Classifier algorithm implemented in DADA2’s assignTaxonomy function together with its pre-formatted RDP training set (version 16) is used for taxonomic annotation using a minimum bootstrap confidence (e.g., a minimum bootstrap confidence of 50). In some aspects, a DADA2-RDP analysis can go down to species level. In some aspects, an analysis procedure for a UCLUST-UCLUST pipeline can be performed. In some aspects, UCLUST (version 1.2.22q) is used for clustering amplicon data with known sequence similarity (e.g., of 97%) after quality filtering with the minimum quality threshold (e.g., of 20) and a minimum base length (e.g., of 140 bases). In some aspects, representative sequence(s) of OTUs are selected (e.g., with pick rep set.py script) with default parameters. In some aspects, UCLUST implemented in assign taxonomy.py script together with SILVA database (release 123; choice of silva_132_97_16S.fna) is used for taxonomic annotation, which can be down to species level using a minimum bootstrap confidence (e.g., of 0.5). In some aspects, one or more or all procedures are completed in the QIIME platform (version 1.9.1). In some aspects, such a pipeline is similar to the meta-analysis method used by Mancabelli et al. (2017). In some aspects, an analysis procedure for USEARCH-RDP pipeline can be performed. In some aspects, USEARCH is used for clustering amplicon data with known sequence similarity (e.g., 100% sequence similarity) after quality filtering with a maximum expected error (e.g., of 2) and a minimum base length (e.g., of 200 bases). In some aspects, RDP classifier (version 2.12) together with RDP training set (version 16) is used for taxonomic annotation, which can be down to species level using a minimum bootstrap confidence (e.g., of 0.5). In some aspects, such a pipeline is similar to the meta-analysis method used by Duvallet et al. (2017)). In some aspects, an analysis procedure for the Taxa4meta pipeline can be performed. In some aspects, Taxa4Meta (e.g., version 1.22) is used for clustering amplicon data after quality filtering with a maximum expected error (e.g., of- 38 -299756710.12) and a selected range of variable lengths, optionally as suggested by Taxa4Meta itself. In some aspects, taxonomic annotation by Taxa4Meta binary classifier can be down to species level. In some aspects, an analysis procedure for Metagenomic classifiers can be performed. In some aspects, Paired-end sequences are trimmed and filtered to meet a maximum expected error (e.g., of 2) with a minimum read length (e.g., of 50). In some aspects, Kraken2 (version 2.0.8) with its pre-built database (minikraken2_v2_8GB_201904_UPDATE) with default parameters is used for taxonomic profiling for shotgun metagenomic data. In some aspects, MetaPhlAn2 (version 2.7.7) with it default database (mpa_v20_m200) with default parameters is used for taxonomic profiling for shotgun metagenomic data. In some aspects, Kraken2 family-level abundance results are used as the reference for comparisons across different 16S pipelines. In some aspects, given the high precision on species identification, MetaPhlAn2 species-level abundance results are used as the reference for evaluating species calls of different 16S pipelines. In some aspects, a pseudo sample is created by averaging each family-level abundance of all WGS samples (e.g., 27 WGS samples), then the abundance-weighted Jaccard distance is calculated between the pseudo sample and any real sample analyzed by different pipelines.

[0137] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises microbiome meta-analysis of diarrheal microbiome datasets. In some aspects, one or more diarrheal datasets are run on the Taxa4Meta pipeline adopted optimal taxonomic thresholds for each 16S variable region. In some aspects, relative abundance of collapsed species profiles generated from Taxa4Meta OTU count tables are used with or without rarefaction. In some aspects, relative abundance of collapsed species profiles generated from Taxa4Meta OTU count tables require a minimum number of reads per sample. In some aspects, a minimum number of reads per sample is 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 or more or any range derivable therein. In some aspects, a minimum number of reads per sample is 1,000 reads per sample. In some aspects, if species is assigned by Taxa4Meta-BLCA, the taxonomic lineage from NCBI 16S RefSeq is adopted for that species to avoid inconsistency in taxonomic lineage. In some aspects, merging of Taxa4Meta collapsed species of is based on taxonomic lineages.

[0138] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises determining predictive metagenome functions. In some aspects, predictive metagenome functions can be determined using open source software, e.g.,- 39 -299756710.1PICRUSt2 (see e.g., Holmes et al., Generative models for microbial metagenomics. PLoS One 7, (2012)). In some aspects, default Taxa4Meta parameters, OTU count tables, and / or OTU sequences are used to infer metabolic pathway abundance profiles for one or more datasets. In some aspects, merging of PICRUSt2 pathway profiles is based on MetaCyc pathway IDs. In some aspects, either or both LEfSe analysis (one-against-one test mode; version 1.0) and random forest (RF)-based feature ranking (default parameters in Orange version 3.20) are performed using pathway abundance profiles for diseased (e.g., CDI, IBD CD, IBD UC, and / or IBS) and / or control subjects. In some aspects, mean decrease accuracy (MDA) score from RF-based analysis is used to rank pathways. In some aspects, the top 20 pathways must be listed by both RF-based feature ranking result and LEfSe analysis result. In some aspects, the top 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 pathways are listed by both RF-based feature ranking results and LEFSe analysis results. In some aspects, the top ranked pathways are selected for subsequent analysis. In some aspects, the top ranked pathways are the top 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 pathways or any range derivable therein. In some aspects, the top ranked pathways are features indicative of a disease state and / or suitable for binary classification of disease state.

[0139] In some aspects, data (e.g., relative abundances, associations, metabolic pathways, etc.) associated with a collection of one or more OTUs is collaposed into an enterotype. In some aspects, an enterotype encompasses two or more OTUs. In some aspects, one or more OTUs are collapsed into a simplified genera designation. In some aspects, an OTU is not collapsed into a simplified genera designation.

[0140] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises determining a-Diversity and / or P-diversity. In some aspects, one or more a-diversity indices are calculated at OTU levels. In some aspects, a-diversity indices are the Shannon index (e.g., alpha diversity.py in QIIME vl.9.1) and / or the richness index (e.g., breakaway package version 4.7.5). In some aspects, QIIME vl.9.1, principal coordinate analysis (PCoA) with abundance-weighted Jaccard distance metric is applied for P-diversity analysis using combined collapsed species profile. In some aspects, ANOSIM test for group comparison is performed using the beta-diversity distance profile and the permutations of 999.

[0141] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises fitting factors onto P-diversity ordination plot. In some aspects, fitting factors (e.g., taxa) onto a two-dimensional ordination plot (e.g., first two- 40 -299756710.1coordinates) is performed using the envfit function in vegan package (version 2.5-7) or a suitable alternative program. In some aspects, taxonomic abundance profile at family level is used as one of or the only factor in this analysis. In some aspects, significance of fitted factors is established using the permutation of 999 in the envfit run.

[0142] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises microbiome enterotyping. In some aspects, microbiome enterotyping is performed with family abundance profiles of one or more or all meta-analysis training sets. In some aspects, Dirichlet multinomial mixtures (DMM) algorithm, a classical method for clustering community profile data, is used for microbiome enterotyping.

[0143] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises supervised classification and / or independent cohort validation. In some aspects, supervised classification procedures are performed using Orange software (Demsar et al., 2013) (e.g., version 3.20) or a suitable alternative thereof, and applied to the reported cohorts with clinical definitions. In some aspects, an original sample grouping information from each cohort is adopted. In some aspects, such an adoption is done so the gold standard definition is clear for each sample. In some aspects, random forest-based feature ranking was used as a first pass to select the top 100 input features (e.g., taxa, or biochemical pathways) for downstream supervised learning. In some aspects, unless performing sub-sampling, input samples are used for training procedure. In some aspects, supervised classification is performed using individual learning algorithms including but not limited to Random Forest (RF), Support Vector Machine (SVM), Naive Bayes (NB), and / or Neural Network (NN). In some aspects, a Stack model as an aggregated meta-learner of RF, SVM and NB is assessed. In some aspects, a 5- fold cross-validation method is applied for sub-sampling of training and test data during a training procedure. In some aspects, receiver-operating-characteristic (ROC) analysis is performed using the training results. In some aspects, values of area-under-the curve (AUC) and classification accuracy (CA) are calculated to evaluate the performance of each classification model. In some aspects, a suitable AUC value is more than 0.80, 0.81. 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99 or any range derivable therein. In some aspects, a preferred AUC value is more than 0.95, 0.96, 0.97, 0.98, or 0.99 or any range derivable therein. In some aspects, CA refers to the proportion of correct predicted samples from the classification model compared to the original clinical diagnosis. In some aspects, a suitable CA value is more than 0.80, 0.81. 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92,- 41 -299756710.10.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99 or any range derivable therein. In some aspects, a preferred CA value is more than 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99 or any range derivable therein. In some aspects, independent validation of classification models is performed using datasets of recently reported microbiome surveys of human diarrheal diseases that were not included in the training set. In some aspects, one or more validation datasets are analyzed individually using the Taxa4Meta pipeline to generate taxonomic profile data for validating classification models. In some aspects, CDI and IBD scores refer to the predicted scores of each sample as the class of CDI and IBD, respectively.

[0144] In some aspects, a step in determining features suitable for classification of microbiome dysbiosis associated disease state, enteroclusters profiling, and / or determination of appropriate treatment methods comprises statistical analysis. In some aspects, comparisons between two groups are made using non-parametric Mann-Whitney -Wilcoxon two-tailed test or a suitable alternative thereof, and comparisons for more than two groups are made using nonparametric Kruskal-Wallis two-tailed test or a suitable alternative thereof. In some aspects, multiple comparisons and pairwise Spearman or Pearson correlations are adjusted using the Benjamini -Hochberg (BH) false discovery rate (p<0.05, regarded as statistically significant), or a suitable alternative thereof.

[0145] In some aspects, calculation of a meta-feature value is performed by: (i) determining the feature value of at least two, preferably more features, (ii) "normalizing" the feature value of each individual feature by dividing the value with a coefficient which is approximately the median value of the respective feature in a representative cohort, and (iii) calculating the median of the group of normalized gene expression values. In some aspects, meta-feature analysis is performed as described herein.

[0146] As disclosed herein, in some aspects, a feature shall be understood to be specifically increased in presence if the abundance level of the feature is at least about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100- fold, 1000-fold, or 10000-fold higher (or any range derivable therein) than in a reference, or in a mixture of references. References include but are not limited to, biological samples from one or more otherwise healthy individuals, biological samples from one or more individuals diagnosed with a different disease, and / or non-diarrheal biological samples from one or more individuals. In some aspects, references can include normalized values across a cohort.

[0147] In certain algorithms a suitable threshold level is first determined for a feature. The suitable threshold level can be determined from measurements of feature presence, absence, relative abundance, and / or levels (e.g., quantity, activity, etc.) in one or more individuals from a- 42 -299756710.1test cohort. In some aspects, median feature values in a multiple expression measurement is taken as a suitable threshold value. In some aspects, mean feature values in a multiple expression measurement is taken as a suitable threshold value. In some aspects, mode feature values in a multiple expression measurement is taken as a suitable threshold value. Comparison of multiple features with a threshold level can be performed as follows: 1) The individual features are compared to their respective threshold levels, 2) The number of features, the level of which is above and / or below their respective threshold level, is determined, 3) If a feature value is above its respective threshold level, then the feature level of is taken to be "above the threshold level", 4) If a feature value is below its respective threshold level, then the feature level is taken to be “below the threshold level”.

[0148] In some aspects, a disease classification can be determined from analysis of a sufficiently large number of features. In this context, a sufficiently large number of features means at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% (or any range derivable therein) of the features described by one or more binary tests.

[0149] In certain aspects, the determination of feature presence, absence, relative abundance, and / or levels is on a substrate that allows evaluation of RNA molecule levels from a given sample, such as a gene chip, for example but not limited to Affymetrix™ gene chip, NanoString nCounter™, Illlumina BeadChip™, etc.

[0150] Commonly used methods for determining feature presence, absence, relative abundance, and / or levels include:

[0151] In some aspects, the determination of feature presence, absence, relative abundance, and / or levels is by 16S rRNA sequencing.

[0152] In some aspects, the determination of feature presence, absence, relative abundance, and / or levels is by RNA sequencing.

[0153] In some aspects, the determination of feature presence, absence, relative abundance, and / or levels is by whole genome sequencing, for example but not limited to, whole genome shotgun sequencing.

[0154] In other aspects, the determination of feature presence, absence, relative abundance, and / or levels is done by polymerase chain reaction (PCR), for example but not limited to, realtime PCR, quantitative real time PCR, reverse transcriptase PCR, multiplexed PCR, nested PCR, long-range PCR, single-cell PCR, fast-cycling PCR, methylation-specific PCR, hot start PCR, high-fidelity PCR, in situ PCR, etc.- 43 -299756710.1

[0155] In some aspects, the determination of feature presence, absence, relative abundance, and / or levels is performed by measuring proteins, polypeptides, metabolites, small molecules, etc. instead of nucleic based analyses (e.g., RNA and / or DNA based analyses). In some aspects, techniques suitable for measuring the same include but are not limited to methods such as western blotting, IP-MS / MS, LC-MS / MS, NMR, PQN, ELISAs, HPLC, etc.

[0156] In some aspects, the feature is a relative abundance of microbial taxa. In some aspects, commonly used methods for determining relative abundance include, but are not limited to 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), metaproteomics and metabolomics, bioinformatics Pipelines (e.g., Taxa4Meta).B. Nucleic acid based assays

[0157] Screening methods based on differential levels of genes and / or gene products are common place in the art. In accordance with one aspect of the present invention, the differential patterns of features can be determined by measuring the levels of RNA transcripts indicative of these features, or genes whose expression is modulated by the presence or absence of one or more of these features, present in a patient’s biological sample (e.g., a fecal sample, swab, irrigation, mucosal biopsy, etc.). Suitable methods for this purpose include, but are not limited to, DNA sequencing, RNA sequencing, RT-PCR, Northern Blot, in situ hybridization, Southern Blot, slotblotting, nuclease protection assay, and oligonucleotide arrays.

[0158] In some aspects, feature absence, presence, relative abundance and / or levels are determined from a biological sample obtained from a fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation. In certain aspects, feature absence, presence, relative abundance, and / or levels are preferably determined from a biological sample obtained from a fecal sample. In some aspects, a biological sample can be a fixated samples (e.g., those fixed using formalin, paraformaldehyde, paraffin, etc.), blood, tears, semen, saliva, urine, feces, tissue, breast milk, lymph fluid, stool, sputum, cerebrospinal fluid, and / or supernatant from cell lysate.

[0159] In certain aspects, RNA isolated from a biological sample can be amplified to cDNA or cRNA before detection and / or quantitation. In some aspects, isolated RNA can be either total RNA or mRNA. In some aspects, RNA amplification can be specific or non-specific. In some aspects, suitable amplification methods include, but are not limited to, reverse transcriptase PCR, isothermal amplification, ligase chain reaction, and Qbeta replicase. In some aspects, amplified nucleic acid products can be detected and / or quantitated through hybridization to labeled probes. In some aspects, detection may involve fluorescence resonance energy transfer (FRET) or some other kind of quantum dots.- 44 -299756710.1

[0160] In some aspects, amplification primers or hybridization probes for detection of presence, absence, relative abundance, and / or levels of a feature can be prepared from a gene sequence or obtained through commercial sources, such as Affymetrix, NanoString, Illumina BeadChip, etc. In certain aspects a gene sequence is identical or complementary to at least 8, 10, 12, 14, 16, 18, or 20 contiguous nucleotides of a coding sequence.

[0161] In some aspects, sequences suitable for making probes / primers for detection of a corresponding feature includes those that are identical or complementary to all or part of one or more genes specific to taxonomic units described herein. In some aspects, sequences suitable for making probes / primers for detection of a corresponding feature includes those that are unique to one or more genes specific to taxonomic units described herein.

[0162] In some aspects, use of a probe or primer of between 13 and 100 nucleotides, preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and / or selectivity of the hybrid molecules obtained. One will generally prefer to design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments can be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

[0163] In some aspects, each probe / primer comprises at least 15 nucleotides. For instance, each probe can comprise at least or at most 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein). They may have these lengths and have a sequence that is identical or complementary to a gene or portion of a genome of a taxonomic unit described herein. Preferably, each probe / primer has relatively high sequence complexity and does not have any ambiguous residue (undetermined "n" residues). In some aspects, probes / primers can hybridize to a target gene, including its RNA transcripts, under stringent or highly stringent conditions. In some aspects, because each of the features has more gene sequences, it is contemplated that probes and primers can be designed for use with any one or more of these gene sequences. For example, inosine is a nucleotide frequently used in probes or primers to hybridize to more than one sequence. It is contemplated that probes or primers may have inosine or other design implementations that accommodate recognition of more than one sequence for a particular feature.

[0164] For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and / or- 45 -299756710.1high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0165] In some aspects, probes / primers for a gene are selected from regions which significantly diverge from the sequences of other genes. Such regions can be determined by checking the probe / primer sequences against relevant genome sequence databases. One algorithm suitable for this purpose is the BLAST algorithm. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length (W) in the query sequence, which either match or satisfy some positive-valued threshold score (T) when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence to increase the cumulative alignment score. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. These parameters can be adjusted for different purposes, as appreciated by one of ordinary skill in the art.

[0166] In some aspects, quantitative RT-PCR (such as TaqMan, AB I) is used for detecting and comparing the levels of RNA transcripts in biological samples. Quantitative RT-PCR involves reverse transcription (RT) of RNA to cDNA followed by relative quantitative PCR (RT-PCR). The concentration of the target DNA in the linear portion of the PCR process is proportional to the starting concentration of the target before the PCR was begun. By determining the concentration of the PCR products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific transcripts from which the target sequence was derived can be determined for the respective cells. This direct proportionality between the concentration of the PCR products and the relative transcript abundances is true in the linear range portion of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of- 46 -299756710.1target DNA. Therefore, the sampling and quantifying of the amplified PCR products preferably are carried out when the PCR reactions are in the linear portion of their curves. In addition, relative concentrations of the amplifiable cDNAs preferably are normalized to some independent standard, which can be based on either internally existing RNA species or externally introduced RNA species. The abundance of a particular transcript or DNA species may also be determined relative to the average abundance of all transcript or DNA species in the sample.

[0167] In some aspects, PCR amplification utilizes one or more internal PCR standards. The internal standard can be an abundant housekeeping gene in a cell. These standards can be used to normalize expression and / or abundance levels so that the expression and / or abundance levels of different features can be compared directly. A person of ordinary skill in the art would know how to use an internal standard to normalize expression and / or abundance levels.

[0168] A problem inherent in clinical samples is that they are generally of variable quantity and / or quality. In some aspects, this problem can be overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable nucleic acid fragment that is similar or larger than the target nucleic acid fragment and in which the abundance of the nucleic acid fragment encoding the internal standard is roughly 5-100 fold higher than the nucleic acid fragment encoding the target. This assay measures relative abundance, not absolute abundance of the respective nucleic acid species.

[0169] In another aspect, the relative quantitative RT-PCR uses an external standard protocol. Under this protocol, the PCR products are sampled in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling can be empirically determined for each target nucleic acid fragment.

[0170] Nucleic acid arrays can also be used to detect and compare the differential presence, absence, or levels of microbiome dysbiosis features. Probes suitable for detecting the corresponding features can be stably attached to known discrete regions on a solid substrate. As used herein, a probe is "stably attached" to a discrete region if the probe maintains its position relative to the discrete region during the hybridization and the subsequent washes. Construction of nucleic acid arrays is well known in the art. Suitable substrates for making polynucleotide arrays include, but are not limited to, membranes, films, plastics and quartz wafers.

[0171] A nucleic acid array can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more different polynucleotide probes, which may hybridize to different and / or the same targets representative of one or more features. Multiple probes for the same feature can be used on a single nucleic acid array. Probes for other features can also be included in the nucleic acid array. Probe combinations suitable for delineation of- 47 -299756710.1healthy, CDI, IBS, IBD UC, and / or IBD CD can be included on a nucleic acid array. The probe density on the array can be in any range. In some aspects, the density can be 50, 100, 200, 300, 400, 500 (or any range derivable therein) or more probes / cm2.

[0172] Specifically contemplated by the present inventors are chip-based nucleic acid technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high-density arrays and screen these molecules on the basis of hybridization (see also, Pease etal., 1994; and Fodor et al, 1991). It is contemplated that this technology can be used in conjunction with evaluating the presence, absence, relative abundance, and / or levels of one or more features with respect to diagnostic, prognostic, and treatment methods of the disclosure.

[0173] The present disclosure may involve the use of arrays or data generated from an array. Data can be readily available and / or an array may be prepared in order to generate data that can then be used in correlation studies. An array generally refers to ordered macroarrays or microarrays of nucleic acid molecules (probes) that are fully or nearly complementary or identical to a plurality genes and / or gene products and that are positioned on a support material in a spatially separated organization. Macroarrays are typically sheets of nitrocellulose or nylon upon which probes have been spotted. Microarrays position the nucleic acid probes more densely such that up to 10,000 nucleic acid molecules can be fit into a region typically 1 to 4 square centimeters. Microarrays can be fabricated by spotting nucleic acid molecules, e.g., genes, oligonucleotides, etc., onto substrates or fabricating oligonucleotide sequences in situ on a substrate. Spotted or fabricated nucleic acid molecules can be applied in a high-density matrix pattern of up to about 30 non-identical nucleic acid molecules per square centimeter or higher, e.g. up to about 100 or even 1000 per square centimeter. Microarrays typically use coated glass as the solid support, in contrast to the nitrocellulose-based material of filter arrays. By having an ordered array of complementing nucleic acid samples, the position of each sample can be tracked and linked to the original sample. A variety of different array devices in which a plurality of distinct nucleic acid probes are stably associated with the surface of a solid support are known to those of skill in the art. Useful substrates for arrays include nylon, glass and silicon. Such arrays may vary in a number of different ways, including average probe length, sequence or types of probes, nature of bond between the probe and the array surface, e.g. covalent or non-covalent, and the like. The labeling and screening methods of the present invention and the arrays are not limited in its utility with respect to any parameter except that the probes detect absence, presence, or levels of one or more- 48 -299756710.1features; consequently, methods and compositions may be used with a variety of different types of genes and / or gene products.

[0174] Representative methods and apparatus for preparing a microarray have been described, for example, in U.S. Patent Nos. 5,143,854; 5,202,231; 5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,432,049; 5,436,327; 5,445,934;5,468,613; 5,470,710; 5,472,672; 5,492,806; 5,525,464; 5,503,980; 5,510,270; 5,525,464;5,527,681; 5,529,756; 5,532,128; 5,545,531; 5,547,839; 5,554,501; 5,556,752; 5,561,071;5,571,639; 5,580,726; 5,580,732; 5,593,839; 5,599,695; 5,599,672; 5,610;287; 5,624,711;5,631,134; 5,639,603; 5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972; 5,695,940;5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; 5,837,196; 5,871,928; 5,847,219;5,876,932; 5,919,626; 6,004,755; 6,087,102; 6,368,799; 6,383,749; 6,617,112; 6,638,717;6,720, 138, as well as WO 93 / 17126; WO 95 / 11995; WO 95 / 21265; WO 95 / 21944; WO 95 / 35505; WO 96 / 31622; WO 97 / 10365; WO 97 / 27317; WO 99 / 35505; WO 09923256; WO 09936760; WO 0138580; WO 0168255; WO 03020898; WO 03040410; WO 03053586; WO 03087297; WO 03091426; WO 03100012; WO 04020085; WO 04027093; EP 373 203; EP 785 280; EP 799 897 and UK 8 803 000; the disclosures of which are all herein incorporated by reference. In some aspects, the use of 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), metaproteomics and metabolomics, bioinformatics Pipelines (e.g., Taxa4Meta). BIOFIRE® SPOTFIRE® System is contemplated.

[0175] It is contemplated that the arrays can be high density arrays, such that they contain 100 or more different probes. It is contemplated that they may contain 1000, 16,000, 65,000, 250,000 or 1,000,000 or more different probes. The probes can be directed to targets in one or more different organisms. The oligonucleotide probes range from 5 to 50, 5 to 45, 10 to 40, or 15 to 40 nucleotides in length in some aspects. In certain aspects, the oligonucleotide probes are 20 to 25 nucleotides in length.

[0176] The location and sequence of each different probe sequence in the array are generally known. Moreover, the large number of different probes can occupy a relatively small area providing a high-density array having a probe density of generally greater than about 60, 100, 600, 1000, 5,000, 10,000, 40,000, 100,000, or 400,000 different oligonucleotide probes per cm2. The surface area of the array can be about or less than about 1, 1.6, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm2.

[0177] Moreover, a person of ordinary skill in the art could readily analyze data generated using an array. Such protocols include information found in WO 9743450; WO 03023058; WO 03022421; WO 03029485; WO 03067217; WO 03066906; WO 03076928; WO 03093810; WO 03100448, all of which are specifically incorporated by reference.- 49 -299756710.1

[0178] In some aspects, nuclease protection assays are used to quantify RNAs derived from a biological sample. There are many different versions of nuclease protection assays known to those practiced in the art. The common characteristic that these nuclease protection assays have is that they involve hybridization of an antisense nucleic acid with the RNA to be quantified. The resulting hybrid double-stranded molecule is then digested with a nuclease that digests singlestranded nucleic acids more efficiently than double-stranded molecules. The amount of antisense nucleic acid that survives digestion is a measure of the amount of the target RNA species to be quantified. An example of a nuclease protection assay that is commercially available is the RNase protection assay manufactured by Ambion, Inc. (Austin, Tex.).

[0179] In some aspects, the presence, absence, relative abundance, and / or levels of one or more features are determined from a biological sample using 3' RNA sequencing, using products such as Lexogen QuantSeq™, QioSeq UPX 3' Transcriptome, etc. In some aspects, 3' RNA sequencing does not require transcripts to be fragmented before reverse transcription, and cDNAs are reverse transcribed only from the 3' RNA sequencing end of the transcripts, resulting in only one copy of cDNA for each transcript, resulting in a direct 1 : 1 ratio between RNA and cDNA copy numbers.

[0180] In some aspects, gene expression is determined from a biological sample using specific targeted sequencing, using products such as BioSpyder TempO-Seq®, Ion Ampliseq™ Transcriptome, etc. In some aspects, specific targeted sequencing targets RNA sequences by hybridization to DNA oligos followed by removal of unhybridized oligos and amplification of remaining products.

[0181] C. Proteins, polypeptide, metabolites, etc. based assays

[0182] In other aspects, the differential features (e.g., taxonomic and / or metabolic pathway biomarkers) can be determined by measuring levels of polypeptides encoded by components of the microbiome in a biological sample (e.g., a fecal sample, intestinal swap, intestinal biopsy, intestinal irrigation sample, etc.). Methods suitable for this purpose include, but are not limited to, immunoassays such as ELISA, RIA, FACS, dot blot, Western Blot, immunohistochemistry, and antibody-based radioimaging. Protocols for carrying out these immunoassays are well known in the art. Other methods such as 2-dimensional SDS-polyacrylamide gel electrophoresis can also be used. These procedures can be used to recognize any of the polypeptides encoded or implicated by one or more features described herein.

[0183] One example of a method suitable for detecting the levels of target proteins in biological samples is ELISA. In an exemplifying ELISA, antibodies capable of binding to the- 50 -299756710.1target proteins encoded by the genome of one or more features are immobilized onto a selected surface exhibiting protein affinity, such as wells in a polystyrene or polyvinylchloride microtiter plate. Then, samples to be tested are added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen(s) can be detected. Detection can be achieved by the addition of a second antibody which is specific for the target proteins and is linked to a detectable label. Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label. Proper extraction procedures can be used to separate the target proteins from potentially interfering substances.

[0184] In another ELISA aspect, one or more samples containing the target proteins reflective of one or more features are immobilized onto the well surface and then contacted with antibodies. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen is detected. Where the initial antibodies are linked to a detectable label, the immunocomplexes can be detected directly. The immunocomplexes can also be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.

[0185] Another typical ELISA involves the use of antibody competition in the detection. In this ELISA, the target proteins are immobilized on the well surface. The labeled antibodies are added to the well, allowed to bind to the target proteins, and detected by means of their labels. The amount of the target proteins in an unknown sample is then determined by mixing the sample with the labeled antibodies before or during incubation with coated wells. The presence of the target proteins in the unknown sample acts to reduce the amount of antibody available for binding to the well and thus reduces the ultimate signal.

[0186] Different ELISA formats can have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunocomplexes. For instance, in coating a plate with either antigen or antibody, the wells of the plate can be incubated with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test samples. Non-limiting examples of these nonspecific proteins include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.- 51 -299756710.1

[0187] In ELISAs, a secondary or tertiary detection means can also be used. After binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control and / or clinical or biological sample to be tested under conditions effective to allow immunocomplex (antigen / antibody) formation. These conditions may include, for example, diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween and incubating the antibodies and antigens at room temperature for about 1 to 4 hours or at 4 °C overnight. Detection of the immunocomplex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

[0188] After all of the incubation steps in an ELISA, the contacted surface can be washed so as to remove non-complexed material. For instance, the surface can be washed with a solution such as PBS / Tween, or borate buffer. Following the formation of specific immunocomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of the amount of immunocomplexes can be determined.

[0189] To provide a detecting means, the second or third antibody can have an associated label to allow detection. In some aspects, a label is an enzyme that generates color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one may contact and incubate the first or second immunocomplex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0190] After incubation with a labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-(3-ethyl)-benzhiazoline-6-sulfonic acid (ABTS) and hydrogen peroxide, in the case of peroxidase as the enzyme label. Quantitation can be achieved by measuring the degree of color generation, e.g., using a spectrophotometer.

[0191] In some aspects, another suitable method is RIA (radioimmunoassay). An example of RIA is based on the competition between radiolabeled-polypeptides and unlabeled polypeptides for binding to a limited quantity of antibodies. Suitable radiolabels include, but are not limited to, 1125. In some aspects, a fixed concentration of 1125-labeled polypeptide is incubated with a series of dilution of an antibody specific to the polypeptide. When the unlabeled polypeptide is added to the system, the amount of the 1125-polypeptide that binds to the antibody is decreased. A standard curve can therefore be constructed to represent the amount of antibody-bound 1125-polypeptide- 52 -299756710.1as a function of the concentration of the unlabeled polypeptide. From this standard curve, the concentration of the polypeptide in unknown samples can be determined. Various protocols for conducting RIA to measure the levels of polypeptides in a sample are well known in the art.

[0192] In some aspects, suitable antibodies for biomarker detection include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library.

[0193] In some aspects, antibodies can be labeled with one or more detectable moieties to allow for detection of antibody-antigen complexes. In some aspects, detectable moieties can include compositions detectable by spectroscopic, enzymatic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. In some aspects, detectable moieties include, but are not limited to, radioisotopes, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like.

[0194] Protein array technology is discussed in detail in Pandey and Mann (2000) and MacBeath and Schreiber (2000), each of which is herein specifically incorporated by reference. These arrays typically contain thousands of different proteins or antibodies spotted onto glass slides or immobilized in tiny wells and allow one to examine the biochemical activities and binding profiles of a large number of proteins at once. To examine protein interactions with such an array, a labeled protein is incubated with each of the target proteins immobilized on the slide, and then one determines which of the many proteins the labeled molecule binds. In certain aspects such technology can be used to quantitate a number of proteins in a sample, such as a sample comprising a representative population of a microbiome.

[0195] The basic construction of protein chips has some similarities to DNA chips, such as the use of a glass or plastic surface dotted with an array of molecules. These molecules can be DNA or antibodies that are designed to capture proteins. Defined quantities of proteins are immobilized on each spot, while retaining some activity of the protein. With fluorescent markers or other methods of detection revealing the spots that have captured these proteins, protein microarrays are being used as powerful tools in high-throughput proteomics and drug discovery.

[0196] The earliest and best-known protein chip is the ProteinChip by Ciphergen Biosystems Inc. (Fremont, Calif.). The ProteinChip is based on the surface-enhanced laser desorption and ionization (SELDI) process. Known proteins are analyzed using functional assays that are on the chip. For example, chip surfaces can contain enzymes, receptor proteins, or antibodies that enable researchers to conduct protein-protein interaction studies, ligand binding studies, or- 53 -299756710.1immunoassays. With state-of-the-art ion optic and laser optic technologies, the ProteinChip system detects proteins ranging from small peptides of less than 1000 Da up to proteins of 300 kDa and calculates the mass based on time-of-flight (TOF).

[0197] The ProteinChip biomarker system is the first protein biochip-based system that enables biomarker pattern recognition analysis to be done. This system allows researchers to address important clinical questions by investigating the proteome from a range of crude clinical samples (i.e., laser capture microdissected cells, biopsies, tissue, urine, and serum). The system also utilizes biomarker pattern software that automates pattern recognition-based statistical analysis methods to correlate protein expression patterns from clinical samples with disease phenotypes.

[0198] In some aspects, the levels of polypeptides in a biological sample can be determined by detecting the biological activities associated with the polypeptides. If a biological function / activity of a polypeptide is known, suitable in vitro bioassays can be designed to evaluate the biological function / activity, thereby determining the amount of the polypeptide in the sample.

[0199] In some aspects, the levels of polypeptides and / or metabolites in a biological sample can be determined by IP-MS / MS and / or HPLC.D. Methods of delineation between disease classification states.

[0200] In certain aspects, one or more features identified herein can be used to delineate between disease classification states, and / or to provide stake holders with a basis for prescribing one or more appropriate methods of treatment.

[0201] In certain aspects, one or more features are the presence, absence, and / or level of one or more a metabolic pathways.

[0202] In certain aspects, one or more features are the presence, absence, and / or level of one or more taxonomic unit.

[0203] In certain aspects, one or more features are the presence, absence, and / or level of one or more taxonomic units represented by one or more particular bacteria.

[0204] In certain aspects, one or more features associated with the presence, absence, and / or level of one or more a metabolic pathways is used in conjunction with one or more features associated with the presence, absence, and / or level of one or more taxonomic units. In certain aspects, one or more features associated with a feature are described in any one of tables 1-19.

[0205] In certain aspects, a disease classification can be determined by the presence, absence, or relative level of at least one of AST-PWY (L-arginine degradation II (AST pathway)), ECASYN-PWY (enterobacterial common antigen biosynthesis), THREOCAT-PWY (superpathway of L-threonine metabolism), PPGPPMET-PWY (ppGpp biosynthesis), PWY0-- 54 -299756710.11338 (polymyxin resistance), PWY-6263 (superpathway of menaquinol-8 biosynthesis II), PWY- 7371 (l,4-dihydroxy-6-naphthoate biosynthesis II), PWY-7374 (l,4-dihydroxy-6-naphthoate biosynthesis I), P221-PWY (octane oxidation), PWY-6749 (CMP-legionaminate biosynthesis I), PWY-7456 (mannan degradation), NONMEVIPP-PWY (methylerythritol phosphate pathway I), PWY-5097 (L-lysine biosynthesis VI), PWY-5505 (L-glutamate and L-glutamine biosynthesis), PWY-6122 (5-aminoimidazole ribonucleotide biosynthesis II), PWY-7663 (gondoate biosynthesis (anaerobic)), THRESYN-PWY (superpathway of L-threonine biosynthesis), HEMESYN2-PWY (heme biosynthesis II (anaerobic)), PWY-5304 (superpathway of sulfur oxidation (archaea), PWY-6478 (GDP-D-glycero-alpha-D-manno-heptose biosynthesis), PWY- 7198 (pyrimidine deoxyribonucleotides de novo biosynthesis IV), and / or PWY-7210 (pyrimidine deoxyribonucleotides biosynthesis from CTP).

[0206] In certain aspects, an increased abundance relative to an appropriate control of at least one of or all of AST-PWY (L-arginine degradation II (AST pathway)), ECASYN-PWY (enterobacterial common antigen biosynthesis), THREOCAT-PWY (superpathway of L- threonine metabolism), PPGPPMET-PWY (ppGpp biosynthesis), and / or PWY0-1338 (polymyxin resistance) is associated with CDI causative diarrhea. In certain aspects, following detection of one or more of the indicative features, an individual is then treated accordingly.

[0207] In certain aspects, an increased abundance relative to an appropriate control of at least one of or all of PWY-6263 (superpathway of menaquinol-8 biosynthesis II), PWY-7371 (1,4- dihydroxy-6-naphthoate biosynthesis II), PWY-7374 (l,4-dihydroxy-6-naphthoate biosynthesis I), P221-PWY (octane oxidation), PWY-6749 (CMP-legionaminate biosynthesis I), and / or PWY- 7456 (mannan degradation) is associated with IBD UC causative diarrhea. In certain aspects, following detection of one or more of the indicative features, an individual is then treated accordingly.

[0208] In certain aspects, an increased abundance relative to an appropriate control of at least one of or all of NONMEVIPP-PWY (methylerythritol phosphate pathway I), PWY-5097 (L-lysine biosynthesis VI), PWY-5505 (L-glutamate and L-glutamine biosynthesis), PWY-6122 (5- aminoimidazole ribonucleotide biosynthesis II), PWY-7663 (gondoate biosynthesis (anaerobic)), THRESYN-PWY (superpathway of L-threonine biosynthesis), HEMESYN2-PWY (heme biosynthesis II (anaerobic)), PWY-5304 (superpathway of sulfur oxidation (archaea), PWY-6478 (GDP-D-glycero-alpha-D-manno-heptose biosynthesis), PWY-7198 (pyrimidine deoxyribonucleotides de novo biosynthesis IV), and / or PWY-7210 (pyrimidine deoxyribonucleotides biosynthesis from CTP) is associated with IBD causative. In certain aspects,- 55 -299756710.1following detection of one or more of the indicative features, an individual is then treated accordingly.

[0209] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Adlercreutzia, or a metabolic pathway associated therewith.

[0210] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Agathobaculum, or a metabolic pathway associated therewith.

[0211] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Akkermansia, or a metabolic pathway associated therewith.

[0212] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Alistipes, or a metabolic pathway associated therewith.

[0213] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Anaerostipes, or a metabolic pathway associated therewith.

[0214] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Bacteroides, or a metabolic pathway associated therewith.

[0215] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Barnesiella, or a metabolic pathway associated therewith.

[0216] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Bifidobacterium, or a metabolic pathway associated therewith.

[0217] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Bilophila, or a metabolic pathway associated therewith.

[0218] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Blautia, or a metabolic pathway associated therewith.- 56 -299756710.1

[0219] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Butyricimonas, or a metabolic pathway associated therewith.

[0220] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Clostridium, or a metabolic pathway associated therewith.

[0221] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Clostridium IV, or a metabolic pathway associated therewith.

[0222] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Clostridium XlVa, or a metabolic pathway associated therewith.

[0223] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Clostridium XVIII, or a metabolic pathway associated therewith.

[0224] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Clostridium sensu stricto, or a metabolic pathway associated therewith.

[0225] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Collinsella, or a metabolic pathway associated therewith.

[0226] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Coprococcus, or a metabolic pathway associated therewith.

[0227] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Dialister, or a metabolic pathway associated therewith.

[0228] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Dorea, or a metabolic pathway associated therewith.

[0229] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Enterococcus, or a metabolic pathway associated therewith.- 57 -299756710.1

[0230] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Erysipelatoclostridium, or a metabolic pathway associated therewith.

[0231] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Eubacterium, or a metabolic pathway associated therewith.

[0232] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Faecalibacterium, or a metabolic pathway associated therewith.

[0233] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Flavonifractor, or a metabolic pathway associated therewith.

[0234] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Fusicatenibacter, or a metabolic pathway associated therewith.

[0235] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Fusobacterium, or a metabolic pathway associated therewith.

[0236] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Gemmiger, or a metabolic pathway associated therewith.

[0237] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Haemophilus, or a metabolic pathway associated therewith.

[0238] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Intestinibacter, or a metabolic pathway associated therewith.

[0239] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Lachnoclostridium, or a metabolic pathway associated therewith.

[0240] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Lachnospiraceae, or a metabolic pathway associated therewith.- 58 -299756710.1

[0241] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Lachnospiraceae incertae sedis, or a metabolic pathway associated therewith.

[0242] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Lactobacillus, or a metabolic pathway associated therewith.

[0243] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Odoribacter, or a metabolic pathway associated therewith.

[0244] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Parabacteroides, or a metabolic pathway associated therewith.

[0245] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Paraprevotella, or a metabolic pathway associated therewith.

[0246] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Parasutterella, or a metabolic pathway associated therewith.

[0247] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Phascolarctobacterium, or a metabolic pathway associated therewith.

[0248] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Prevotella, or a metabolic pathway associated therewith.

[0249] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Romboutsia, or a metabolic pathway associated therewith.

[0250] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Roseburia, or a metabolic pathway associated therewith.

[0251] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Ruminococcus, or a metabolic pathway associated therewith.- 59 -299756710.1

[0252] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Ruminococcus2, or a metabolic pathway associated therewith.

[0253] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Ruthenibacterium, or a metabolic pathway associated therewith.

[0254] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Ruminococcus, or a metabolic pathway associated therewith.

[0255] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Schaalia, or a metabolic pathway associated therewith.

[0256] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Streptococcus, or a metabolic pathway associated therewith.

[0257] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Sutterella, or a metabolic pathway associated therewith.

[0258] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Turicibacter, or a metabolic pathway associated therewith.

[0259] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of Veillonella, or a metabolic pathway associated therewith.

[0260] In certain aspects, methods described herein for classification of a disease state may comprise, or expressly does not comprise, detection and / or quantification of strains of any of the genera disclosed herein.

[0261] In certain aspects, methods disclosed herein can relate to a system for performing such methods, the system comprising (a) apparatus or device for storing data regarding feature levels of one or more microbiome components; (b) apparatus or device for determining feature levels of at least one feature; (c) apparatus or device for comparing feature levels of a first feature with a predetermined first threshold value and / or test value; (d) apparatus or device for determining feature level of at least one second or more features; and (e) computing apparatus or device programmed to provide treatment with an appropriate methodology if the data indicates altered- 60 -299756710.1feature levels or activity of said first feature as compared to the predetermined first threshold value and / or test value, and, alternatively or in concert, expression level and / or activity of said second or more features as compared to the predetermined second or more feature threshold level and / or test value.

[0262] A person skilled in the art readily appreciates that an accurate prognosis can be given or determined if a sufficiently large number of feature levels are analyzed and compared to an appropriate control. In some aspects, accurate prognosis can facilitate determination of disease recurrence and / or appropriate therapies to provide, including a particular therapy of any kind, such as an antibiotic therapy.

[0263] In some aspects, feature levels and / or patterns can also be compared by using one or more ratios between feature abundance levels associated with an otherwise healthy microbiome and / or one or more dysbiosed microbiomes. Other suitable measures or indicators can also be employed for assessing the relationship or difference between different feature patterns.

[0264] In some aspects, one or more of the features can be used to determine whether a patient with a diarrheal disorder should be treated with antimicrobials and / or antibiotics. In certain aspects, a pattern of features in a patient fecal sample and / or other microbiome samples can be used to evaluate a patient to determine whether they are likely to respond to one or more therapeutic interventions. In some aspects, likeliness of a therapeutic response for the patient can be considered with respect to an individual that lacks the particular feature pattern of the patient.

[0265] In some aspects, a subject’s (e.g., a patient’s) feature levels can be compared to reference feature levels using various methods. In some aspects, reference levels can be determined using expression levels of a reference based on otherwise healthy patients, all types of FGID patients, and / or all types of CDI, IBS, and / or IBD patients. In some aspects, reference levels can be based on an internal reference such as a gene, metabolic pathway, and / or microbe that is present ubiquitously. In some aspects, comparison can be performed using the fold change or the absolute difference between the feature levels to be compared. In some aspects, one or more taxonomic and / or metabolic features can be used in the comparison.

[0266] In some aspects, it is contemplated that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and / or 25 features can be compared to each other and / or to a reference that is internal or external. In some aspects, it is contemplated that 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, and / or 100 features can be compared to each- 61 -299756710.1other and / or to a reference that is internal or external. In some aspects, it is contemplated that 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, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 1443, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 82, 183,184, 185, 186, 187, 188, 189, 190, 191, 191, 192, 193, 194, 195, 196, 197, 198, 199, and / or 200 features can be compared to each other and / or to a reference that is internal or external.

[0267] In certain aspects, comparisons or results from comparisons may reveal or be expressed as x-fold increase or decrease in expression relative to a standard or relative to another feature or relative to the same feature but in a different patient cohort (e.g., a disease patient and / or cohort compared to an appropriate health control). In some aspects, patients with a particular disease diagnosis may have a relatively high level of feature presentation (e.g., over representation) or relatively low level of feature presentation (e.g., under representation) when compared to patients with a different disease diagnosis and / or otherwise healthy patients, or vice versa.

[0268] Fold increases or decreases can be, be at least, or be at most 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- , 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 55-, 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100- or more, or any range derivable therein. Alternatively, differences in expression can be expressed as a percent decrease or increase, such as at least or at most 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, 300, 400, 500, 600, 700, 800, 900, 1000, or greater than 1000% difference, or any range derivable therein. In some aspects, a fold level change for one or more features may not be calculatable, as one or more features can be absent in one or more disease and / or control patients and / or cohorts (e.g., dividing by zero).

[0269] Other ways to express relative expression levels are by normalized or relative numbers such as 0, 0.00001, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03. 0.04, 0.05, 0.06,0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,- 62 -299756710.16.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or any range derivable therein.

[0270] In certain aspects, a feature can be ranked in importance and / or otherwise identified according to a random forest feature rank mean decrease in accuracy. For example, a feature can be considered more integral for appropriate disease classification as a function of the random forest feature rank mean decrease in accuracy. In some aspects, a higher random forest feature mean decrease in value means the feature has a greater potential disease classification value when compared to a feature with a lower value. In certain aspects, a feature random forest feature rank mean decrease in accuracy can be 0.00001, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03. 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, or any range derivable therein.

[0271] In some aspects, algorithms, such as the weighted voting programs, are used to facilitate the evaluation of feature levels. In addition, in some aspects, other clinical evidence are combined with a feature-based test to reduce the risk of false evaluations. In some aspects, other molecular based evaluations are considered. In some aspects, patient questionnaires are considered. In some aspects, patient medical histories can be considered. In some aspects, patient endoscopy results are considered.

[0272] In some aspects, any biological sample from a patient that accurately represents the microbiome, is used to evaluate the presence, absence, and / or level of any feature discussed herein. In some aspects, a biological sample from a fecal sample is used. In some aspects, a biological sample from an endoscopy is used. In some aspects, a biological sample from a mucosal biopsy is used. In some aspects, a biological sample from intestinal fluid is used. Evaluation of a biological sample may involve, though it need not involve, panning (enriching) for microbiome components or isolation of specific microbes.III. Compositions

[0273] In some aspects, the current disclosure encompasses compositions comprising bacterial species as provided herein, for use to prevent or treat a disease or disorder in a subject in need thereof. The provided compositions are based on the unexpected result that one or more bacterial species depleted in the taxa profile Cluster 1 or Cluster 3, relative to a healthy cluster (clusters 2, 4, or 5), can be used to restore microbiome health, thereby reducing the symptoms, and / or preventing gastrointestinal disoders associated with microbiome dysbiosis.

[0274] In some aspects, provided herein are compositions comprising at least one of bacterial species Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum,- 63 -299756710.1Eubacterium rectale, or any combination thereof; and an excipient. In some aspects, the composition comprises the bacterial species Faecalibacterium prausnitzii and at least one, at least two, or all of Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects, the composition comprises at least two, at least three, or all of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale. In some aspects, the composition comprises all of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale.

[0275] In some aspects, the composition may further comprise additional bacterial species. Non-limiting examples of bacterial genera whose species can be included in the disclosed compositions, or that are expressly excluded from the disclosed compositions, include Faecalibacterium, Bifidobacterium, Eubacterium, Prevotella, Bacteroides, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor. In some aspects, some additional bacterial species that can be included in, or can be expressly excluded from, the disclosed compositions, include but are not limited to Eubacterium limosum, Roseburia spp., B.ovatus, P. distasonis, Eubacterium eligens, Eubacterium ventriosum, Roseburia spp., Blautia spp., Blautia producta, Dorea spp., R. torques, Eubacterium hadrum, Anaerostipes coli, Clostridium aldenense, Clostridium hathewayi, Clostridium symbiosum, Clostridium orbiscindens, Clostridium citroniae, Clostridium thermocellum, Ruminococcus obeum, Ruminococcus productus, Ruminococcus torques, Roseburia inulinovorans, Blautia coccoides, Dorea sp., Sutterella sp., Dialister invisus, and Bifidobacterium pseudocatenulatum. The bacterial species of the compositions provided herein can be aerobes, anaerobes, or facultative anaerobes. The species may be sporulating or non-sporulating. In some aspects, specific strains of the disclosed bacterial species may be included or specifically excluded in the composition. In some aspects, the decision to include or exclude one or more strain may be based on a subjects microbiome profile as determined by a method disclosed herein.

[0276] In some aspects, the compositions disclosed herein are formulated for administering to a subject after the relative abundance of one or more bacterial taxa in the subject’s microbiome enterocluster profile has been assessed. In some aspects, the subject’s microbiome enterocluster- 64 -299756710.1profile is compared to a reference microbiome profile, to determine the relative amounts of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale, or a strain thereof to be included in the composition. In some aspects, the composition is administered to restore the subject’s microbiome profile to match the reference microbiome profile. In some aspects, the composition provides a level of one or more strains that is within 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% (or any range derivable therein) of the reference microbiome profile for that strain.

[0277] Strain-level Faecalibacterium yrausnitzH compositions: In certain aspects, the compositions disclosed herein are configured to modulate the relative abundance of strain-level populations of Faecalibacterium prausnitzii. As used herein, “relative abundance” refers to the proportion of reads, molecules, or colony forming units attributed to a taxon or strain in a sample, normalized to the total microbial signal. In some aspects, the use of the compositions may be informed by comparison of the relative abundance from a subject suffering from gut dysbiosis to a reference microbiome profile, for example fecal or intestinal biopsy from an individual or a population, including healthy subjects, disease-matched subjects, and / or demographically matched controls, used as a comparator for establishing baseline or target abundances. The reference may be study-specific, registry-based, or predefined from cohorts exemplified herein (e.g., healthy clusters 2, 4, or 5; dysbiotic clusters 1 or 3).

[0278] In some aspects, the composition has a greater relative abundance of one or more F. prausnitzii strains than a reference microbiome profile. In other aspects, the composition has a reduced relative abundance of one or more F. prausnitzii strains than a reference microbiome profile. Without limitation, such modulation can be achieved by: (i) enriching or depleting cultured strains during manufacturing; (ii) selecting donors or inputs with desired strain profiles; (iii) blending isolated strains with background consortia; and / or (iv) adjusting dosing ratios of species disclosed herein (e.g., F. prausnitzii, B. adolescentis, B. longum, E. rectale) to shift F. prausnitzii strain proportions upon engraftment. In some aspects the strain level F. prausnitzii may be combined with one or more strains of one or more of B. adolescentis, B. longum, E. rectale. In some aspects, the actual composition may be fixed or personalized based on the subjects microbial profiling information.

[0279] In some aspects, one or more strains of F. prausnitzii may be specifically included or excluded depending on the subjects condition. In some aspects, the strains to be included or specifically excluded may be determined by the microbiome enteroclusters profile of the subject. In certain aspects, the one or more F. prausnitzii strains to be included in the composition may comprise sub-clades identified by species-genome bins (SGBs, see Example 5), including but not- 65 -299756710.1limited to SGB15318, SGB 15342, SGB 15326, and / or SGB 15322, individually or in combination, when the composition increases relative abundance versus a reference. In other aspects, the one or more F. prausnitzii strains does not comprise or has a low relative abundance of sub-clade SGB15316 (see Example 5). Strain / sub-clade assignment may be performed using metagenomic read mapping to MAGs (Metagenome- Assembled Genome), marker gene presence / absence, average nucleotide identity thresholds (e.g., >95-97% ANI), or targeted / qPCR assays specific to clade-defining markers. In some aspects, the composition is formulated so that the relative abundance of strains associated with sub-clades SGB 15318, SGB 15342, SGB 15326, and / or SGB 15322 within F. prausnitzii is at least 1.2X, 1.5X, 2X, or 3X (or any range derivable therein) or more than that of a reference. In some aspects, the relative abundance of strains associated with sub-clades SGB 15318, SGB 15342, SGB 15326, and / or SGB 15322 within F. prausnitzii is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more (or any range derivable therein), versus a reference. Conversely, the relative abundance of SGB 15316 within F. prausnitzii is reduced by at least 10%, 20%, 30%, 40% , 50%, 60%, 70%, 80%, 90% or more (or any range derivable therein), versus a reference. These targets may be specified at the time of manufacture (pre-administration) and / or as observed in stool following administration (post- engraftment), measured at predefined windows (e.g., 7-42 days).

[0280] Quantification of strain-level relative abundance can be performed by 16S rRNA gene sequencing with clade-informative variable regions, shallow or deep shotgun metagenomics, targeted amplicon panels, digital PCR, or culture-based CFU normalization, optionally harmonized using the Taxa4Meta pan-microbiome profiling pipeline to minimize technical and demographic biases across cohorts, variable regions, and platforms. Comparisons to reference microbiome profiles may be expressed as fold-change, percentage point difference, z-scores relative to a matched reference distribution, or ratios of strain-abundance to total F. prausnitzii abundance.

[0281] In some aspects, the disclosed compositions are synthetic compositions. In some aspects, synthetic compositions comprise a mixture of isolated or purified bacterial species as provided herein. In some aspects, synthetic compositions may comprise enriched levels of one or more bacterial species relative to the levels of the same one or more bacterial species in a natural source, for example, a fecal sample. Additionally, as further elaborated below, the disclosed compositions may comprise altered ratios of the bacterial species relative to each other and / or other bacterial species, ratios that are different from and / or not found in a natural source.

[0282] In some aspects, compositions provided herein comprise a mixture of isolated bacterial species. As used herein, the term “isolated” bacteria encompasses bacteria that have been- 66 -299756710.1separated from one or more undesired component, such as other bacterium or bacterial species, one or more component of a growth medium, and / or one or more component of a sample, such as a fecal sample. In some aspects, the bacteria are substantially isolated from a source such that other components of the source are not detected (e.g., other bacteria, microbes, and / or macromolecules). As also used herein, the term “purified” refers to a bacterial species or composition that has been separated from one or more components, such as contaminants (e.g., other bacteria, microbes, and / or macromolecules). In some aspects, the bacteria are substantially free of contaminants. In some aspects, one or more bacterial species of a composition are independently purified from one or more other bacteria produced and / or present in a culture or a sample containing the bacterial species. In some aspects, a bacterial species is isolated or purified from a sample and then cultured under the appropriate conditions for bacterial replication, e.g., under anaerobic culture conditions. The bacteria that are grown under appropriate conditions for bacterial replication can subsequently be isolated / purified from the culture in which it is grown.

[0283] In some aspects, the one or more bacterial species of the composition are isolated from a natural or a laboratory source. In some aspects, one or more bacterial species of the composition are isolated from a natural source, for example, fecal samples, intestinal biopsies, endoscopic samples, surgical or autopsy samples of an animal, environmental samples, non-limiting examples of which include soil, water, and food samples. In some aspects, the one or more bacterial species are isolated from an artificial source, for example, microbial culture libraries, engineered animal models, gnotobiotic or germ-free mice, or in vitro gut models (e.g. Simulator of the Human Intestinal Microbial Ecosystem). Method of isolating bacterial species are well known in the art. In some aspects, bacterial species are isolated from a source using: selective culturing wherein specific media is used to select for certain types of microbes; anaerobic culturing mainly used for anaerobic bacteria, using anaerobic chambers or anaerobe specific media; enrichment cultures wherein samples are enriched with nutrients that promote the growth of target microbial species; single-cell sorting using techniques like flow cytometry to isolate individual microbial cells for culturing and further culturing them; serial dilution culturing wherein the samples are diluted serially to obtain single cell isolated; or co-culturing or growing microbes in association with other microbes or host cells to mimic natural environments, or any combination of these techniques.

[0284] Methods of identifying bacterial species in natural or isolated samples are also well established in the art. Identification of a bacterial species can be based on many factors, including cell and colony morphology, chemical composition of cell walls (e.g., Gram-negative vs. Grampositive, cell wall fatty acid make-up), biochemical activities, nutritional requirements, motility, presence or absence of structures external to the cell wall (e.g., flagella, pili), endospore formation,- 67 -299756710.1genomic sequence (including 16S rRNA gene sequence), etc. It should be understood therefore that many different factors can be used to identify a bacterial species and that an exact identification may not always be feasible. Accordingly, as used herein, reference to a certain bacterial species includes a species having identifying characteristics of the bacterial species. Identifying characteristics used to identify bacterial species may include factors listed above, such as cell morphology, colony morphology, Gram staining reaction, biochemical activities (e.g., aerobic or anaerobic), nutritional requirements, 16S rRNA sequence, or a subset or combination thereof. The particular identifying characteristics used will depend on the type of bacteria and are determined by the skilled artisan. In some aspects, bacterial species are identified by 16S rRNA sequence, e.g., sequence of V6 region of 16S rRNA. In some aspects, two bacterial species are considered to be the same, or to share identifying characteristics, if they share at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) sequence identity in their 16S rRNA sequences or in the V6 region of their 16S rRNA. sequences. In some aspects, two bacterial species are considered to be the same, or to share identifying characteristics, if they share at least 95%, 96%, 97%, 98%, 99%, or 100% (or any range derivable therein) sequence identity in their 16S rRNA sequences or in the V6 region of their 16S rRNA.

[0285] In some aspects, the one or more bacterial species in the composition are engineered to provide any number of desirable characteristics to the bacterial species, or the therapeutic composition. Desirable characteristics may include improved physical characteristics, fitness, survival, therapeutic, probiotic, and / or metabolic characteristics. In some aspects, bacteria are engineered to comprises at least one, at least two, at least three, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 20, at least 30 (or any range derivable therein) or more insertions, deletions and / or substitutions in the genome. In some aspects, one or more bacterial species are engineered to encode functional polypeptides and / or functional RNAs that provide one or more of these desirable characteristics.

[0286] In some aspects, the composition may comprise each of the one or more bacterial species in any amount that would provide a therapeutic benefit to subject. The amount of bacteria in the compositions can be expressed in weight, number of bacteria, and / or CFUs (colony forming units). In some aspects, compositions disclosed herein contain, contain at least, or contain at most, about 10, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, about 1013or more, or any range derivable therein, of each of the bacteria of the composition per dosage amount. In some aspects, the compositions disclosed herein contain, contain at least, or contain at most, about 10, about 102, about 103, about 104, about- 68 -299756710.1IO5, about 106, about 107, about 108, about 109, about IO10, about 1011, about 1012, about 1013, about 1014, about 1015, about 1016, about IO17, about 1018, or more, or any range derivable therein, total bacteria per dosage amount. It should further be appreciated that the bacteria of the compositions can be present in different amounts. Thus, for instance, as a non-limiting example, a composition may include 103of bacteria A, 104of bacteria B, and 106of bacteria C. In some aspects, a composition provided herein comprises 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10 of one bacteria species relative to another bacteria species. In some aspects, compositions disclosed herein contain, contain at least, or contain at most, about 10, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about IO10, about 1011, about 1012, about 1013, or more, or any range derivable therein, CFUs of each of the bacteria in the composition per dosage amount. In some aspects, compositions disclosed herein contain, contain at least, or contain at most, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about IO10, about 1011, about 1012, about 1013, about 1014, about 1015, about 1016, about 1017, about 1018, or more, or any range derivable therein, CFUs in total for all of the bacteria combined per dosage amount. As discussed above, bacteria of the compositions can be present in different amounts. In some aspects, compositions disclosed herein contain about 107, about 106, about I O5, about I O4, about 103, about I O2, about 101or more grams of each of the bacteria in the composition per dosage amount. In some aspects, compositions (including but not limited to pharmaceutical compositions) disclosed herein contain about 107, about 106, about 105, about I O4, about 103, about I O2, about 101or more grams in total for all of the bacteria combined per dosage amount. In some aspect, the dosage amount is one administration device (for example but not limited to, one table, pill, or capsule). In some aspect, the dosage amount is the amount that is administered in a particular period (for example but not limited to, the amount administered in one day or one week).

[0287] In some aspects, compositions disclosed herein contain between 10 and 1013, between 102and 1013, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1013, between 109and 1013, between 1010and 1013, between 1011and 1013, between 1012and 1013, between 10 and 1012, between 102and 1012, between 103and 1012, between 104and 1012, between 105and 1012, between 106and 1012, between 107and 1012, between 108and 1012, between 109and 1012, between 1010and 1012, between 10nand 1012, between 10 and 1011, between 102and 1011, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1011, between 108and 1011, between 109and 1011, between 1010and 1011, between 10 and 1010, between 102and 1010, between 103and 1010, between 104and 1010, between 105and 1010, between 106and 1010, between 107and 1010, between- 69 -299756710.1108and IO10, between 109and IO10, between 10 and 109, between 102and 109, between 103and 109, between 104and 109, between IO5and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 108, between 10 and IO7, between 102and 107, between 103and 107, between 104and 107, between IO5and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and IO5, between 102and IO5, between 103and IO5, between 104and IO5, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102of each of the bacteria of the composition per dosage amount. In some aspects, the compositions disclosed herein contain between 10 and 1013, between 102and 1013, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1013, between 109and 1013, between IO10and 1013, between IO11and 1013, between 1012and 1013, between 10 and 1012, between 102and 1012, between 103and1012, between 104and 1012, between 105and 1012, between 106and 1012, between 107and 1012, between 108and 1012, between 109and 1012, between 1010and 1012, between 10nand 1012, between 10 and IO11, between 102and IO11, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and IO11, between 108and IO11, between 109and1011, between 1010and IO11, between 10 and IO10, between 102and IO10, between 103and IO10, between 104and IO10, between 105and IO10, between 106and IO10, between 107and IO10, between 108and IO10, between 109and IO10, between 10 and 109, between 102and 109, between 103and 109, between 104and 109, between IO5and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 108, between 10 and 107, between 102and 107, between 103and 107, between 104and 107, between IO5and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and IO5, between 102and IO5, between 103and IO5, between 104and IO5, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102total bacteria per dosage amount.

[0288] In some aspects, compositions disclosed herein contain between 10 and 1013, between 102and 1013, between 103and 1013, between 104and 1013, between 105and 1013, between 106and1013, between 107and 1013, between 108and 1013, between 109and 1013, between 1010and 1013, between IO11and 1013, between 1012and 1013, between 10 and 1012, between 102and 1012, between 103and 1012, between 104and 1012, between 105and 1012, between 106and 1012, between 107and1012, between 108and 1012, between 109and 1012, between 1010and 1012, between 10nand 1012,-70-299756710.1between 10 and 1011, between 102and IO11, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and IO11, between 108and IO11, between 109and1011, between 1010and IO11, between 10 and IO10, between 102and IO10, between 103and IO10, between 104and IO10, between 105and IO10, between 106and IO10, between 107and IO10, between 108and IO10, between 109and IO10, between 10 and 109, between 102and 109, between 103and 109, between 104and 109, between IO5and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 108, between 10 and IO7, between 102and 107, between 103and 107, between 104and 107, between IO5and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and IO5, between 102and IO5, between 103and IO5, between 104and IO5, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102CFUs of each of the bacteria of the composition per dosage amount.

[0289] In some aspects, compositions disclosed herein contain between 10 and 1013, between 102and 1013, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1013, between 109and 1013, between 1010and 1013, between IO11and 1013, between 1012and 1013, between 10 and 1012, between 102and 1012, between 103and 1012, between 104and 1012, between 105and 1012, between 106and 1012, between 107and1012, between 108and 1012, between 109and 1012, between 1010and 1012, between 10nand 1012, between 10 and IO11, between 102and IO11, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and IO11, between 108and IO11, between 109and IO11, between 1010and IO11, between 10 and IO10, between 102and IO10, between 103and IO10, between 104and IO10, between 105and IO10, between 106and IO10, between 107and IO10, between 108and IO10, between 109and IO10, between 10 and 109, between 102and 109, between 103and 109, between 104and 109, between IO5and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 108, between 10 and 107, between 102and 107, between 103and 107, between 104and 107, between IO5and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and IO5, between 102and IO5, between 103and IO5, between 104and IO5, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102total CFUs per dosage amount.-71 -299756710.1

[0290] In some aspects, compositions may comprise about 105to 1012cfu / unit dose of the composition. In some aspects, composition comprises about 106to 107, or about 107to 108, or about 108to 109, or about 109to IO10, or about 1010to 1012cfu / unit dose of each of the at least one, at least two, at least three, or at least four bacterial species. In some aspects, the relative ratios of the various bacterial species may vary based on the disease condition and the formulation.

[0291] In some aspects, any two bacterial species in the composition may comprise a mutual weight ratio of at least or at most 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, 1:4.5, 1:4.6, 1:4.7, 1:4.8, 1:4.9, 1:5, 1:5.1, 1:5.2, 1:5.3, 1:5.4, 1:5.5, 1:5.6, 1:5.7, 1:5.8, 1:5.9, 1:6, 1:6.1, 1:6.2, 1:6.3, 1:6.4, 1:6.5, 1:6.6, 1:6.7, 1:6.8, 1:6.9, 1:7, 1:7.1, 1:7.2, 1:7.3, 1:7.4, 1:7.5, 1:7.6, 1:7.7, 1:7.8, 1:7.9, 1:8, 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9, 1:9, 1:9.1, 1:9.2, 1:9.3, 1:9.4, 1:9.5, 1:9.6, 1:9.7, 1:9.8, 1:9.9, 1:10, 1:10.5, 1:11, 1:11.5, 1:12, 1:12.5, 1:13, 1:13.5, 1:14, 1:14.5, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50 (or any range derivable therein) relative to each other. In some aspects, the relative ratio of the any two bacterial species may mimic the relative ratios of the two bacterial species in the gut microbiome of a healthy individual, exemplified by a taxonomic profile of any one of Clusters 2, 4 or 5. In some aspects, the relative ratio of the any two bacterial species may mimic the relative ratios of the two bacterial species in the fecal matter of a healthy individual, exemplified by a taxonomic profile of any one of Clusters 2, 4 or 5. In some aspects, the relative ratio of the any two bacterial species are different from the ratios of the bacterial species found in their natural habitat, for example, the gut.

[0292] It is envisioned that at least a portion, such as at least a majority in some embodiments, of the bacterial species of the compositions provided herein are alive and will be alive when they reach the target area (e.g., the intestines). Bacterial spores are considered to be alive in this regards. In some aspects, bacteria that are administered as spores may germinate in the target area (e.g., the intestines). It should further be appreciated that not all of the bacteria are alive and the compositions can include a percentage (e.g., by weight) of bacteria that is not alive. In some aspects, a composition comprises less than or equal to 0.2, 0.4, 0.6, 0.8, 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 percentage by weight, or any range derivable therein, dead bacteria. In addition, in some aspects, the compositions include bacterial species that are not alive when administered or at the time when the composition reaches the target area (e.g.,-72-299756710.1the intestines). It is envisioned that non-living bacteria may still be useful by providing some nutrients and / or metabolites for the other bacterial species in the composition.

[0293] In some aspects, the compositions of the current disclosure may be pharmaceutical compositions. In some aspects, the compositions of the current disclosure may not be pharmaceutical compositions (for example, probiotics and food formulations). In some aspects, compositions of the current disclosure are formulated in any suitable form of delivery, nonlimiting examples of which include live biotherapeutic product (LBP) preparations, fecal microbiota transplants, probiotics, or food formulations. In some aspects, the composition may be formulated with one or more ingestible additives providing one or more benefits to the formulation. Ingestible additives are incorporated into the formulation to enhance their properties, improve stability, or deliver specific health benefits. These additives can serve a variety of functions, such as preserving freshness, improving taste, texture, or appearance, and facilitating the delivery of active ingredients like probiotics or medications. In the context of microbiomebased therapies and functional foods, ingestible additives are particularly important for ensuring the viability and efficacy of live microorganisms, as well as for optimizing the sensory and nutritional qualities of the final product. Some of the most common ingestible additives include stabilizers, preservatives, flavoring agents, sweeteners, emulsifiers, and carriers. Stabilizers such as hydroxypropyl methylcellulose, xanthan gum, and pectin are frequently used to maintain the consistency and suspension of active ingredients in liquids or semi-solids. Preservatives like ascorbic acid (vitamin C), sodium benzoate, and potassium sorbate help extend shelf life by inhibiting the growth of spoilage organisms and pathogens. Flavoring agents and sweeteners, including natural extracts (vanilla, citrus oils), sucralose, and stevia, are added to improve palatability, especially in products containing live biotherapeutics or nutraceuticals that may have off-putting tastes. Emulsifiers such as lecithin, mono- and diglycerides, and polysorbates are used to blend ingredients that would otherwise separate, ensuring a uniform product. Carriers and bulking agents, like maltodextrin, cellulose, and various starches, provide structure and facilitate the even distribution of active components. In probiotic and microbiome-based formulations, prebiotic fibers such as inulin, fructooligosaccharides (FOS), and galactooligosaccharides (GOS) are often included as ingestible additives to support the growth and activity of beneficial bacteria in the gut. In the development of live biotherapeutic products, probiotics, and functional foods, the choice of ingestible additives is critical. These additives not only protect the viability of sensitive bacterial strains during processing and storage but also ensure their effective delivery to the target site in the gastrointestinal tract. For example, enteric coatings made from pH-sensitive polymers can be used as ingestible additives to shield probiotics from stomach acid, releasing- 73 -299756710.1them only when they reach the intestines. Overall, ingestible additives play a vital role in the safety, stability, and effectiveness of a wide range of consumable products, particularly those designed to modulate or support the human microbiome.

[0294] In some aspects, the bacterial portion of a LBP formulation of the current disclosure may comprise, consist essentially of, or consist of, at least one of bacterial species Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof. In some aspects, disclosed compositions are formulated as LBPs. LBPs can come in various forms, depending on the target condition, the type of microorganisms used, and the method of delivery to ensure the viability of the microbes and effectiveness of the treatment. Common forms include:

[0295] Oral Capsules: Oral capsules are one of the most common forms of LBPs. These capsules contain live microorganisms that are often freeze-dried to maintain their viability during storage and during transit through the stomach’s acidic environment.

[0296] Enema or Rectal Suspensions: Some LBPs are formulated to be administered rectally, typically for gastrointestinal conditions that affect the lower part of the gut or colon. This method bypasses the stomach and delivers live microorganisms directly to the site of inflammation or imbalance, ensuring that the bacteria remain viable. In some aspects, these formulations can be similar to fecal microbiota transplant and can be delivered using enema or colonoscopy.

[0297] Topical Applications: For skin or mucosal conditions, LBPs can be formulated as topical creams, gels, or sprays, delivering live microorganisms directly to the affected area. These are particularly useful for dermatological conditions where the skin microbiome plays a role in health or disease.

[0298] Lyophilized powders: Some LBPs are available as freeze-dried (lyophilized) powders that can be reconstituted with water or other liquids before administration. This format is often used to ensure the long-term stability of the microorganisms during storage and transportation.

[0299] Sublingual or Buccal Tablets: LBPs can also be formulated as sublingual or buccal tablets, which dissolve in the mouth and release live microorganisms to the oral or gastrointestinal tract. This approach is typically used for targeting the oral microbiome or for patients who cannot tolerate capsules or other delivery methods.

[0300] In some aspects, compositions of the current disclosure are formulated as a probiotic formulation. Probiotic formulations are dietary supplements or foods that contain live microorganisms, primarily bacteria, which aim to maintain or restore healthy gut flora. These formulations comprise one or more bacterial species, each of which plays a role in supporting digestive health, improving immune function, and / or promoting overall well-being. Probiotics are- 74 -299756710.1thought to work by enhancing the gut's balance of good bacteria, which can be disrupted by factors such as antibiotic use, poor diet, or illness. These formulations can come in various forms like capsules, tablets, powders, drinks, yogurts, etc., providing a convenient way to incorporate beneficial microbes into daily nutrition. Probiotic products may be tailored to target specific health conditions or may be generic and be thought to provide widespread health benefits. For instance, certain formulations may focus on reducing symptoms of irritable bowel syndrome (IBS), preventing antibiotic-associated diarrhea, and / or supporting immune health. In some aspects, bacterial components of a probiotic formulations of the current disclosure may comprise, consist essentially of, or consist of at least one of bacterial species Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof.

[0301] In some aspects, the compositions of the current disclosure are formulated as additive to fecal matter for a fecal matter transplant therapy. A Fecal Microbiota Transplant (FMT) is a medical procedure in which stool from a healthy donor is transplanted into the intestines of a patient to restore healthy gut bacteria. It is primarily used to treat recurrent Clostridioides difficile (CD) infections, which can cause severe gastrointestinal issues. FMT can be administered in several ways, for example, colonoscopic infusion, Enema, nasogastric or nasojejunal tube or capsules - oral ingestion of freeze-dried stool encapsulated in pill form. In some aspects, fecal matter for the procedure is obtained from any suitable donor subject. In some aspects, the donor subject is a healthy human. In some aspects, the fecal microbiota is from a donor subject having a bacterial taxa profile comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the taxa identified in Cluster 2, Cluster 4, or Cluster 5, or from a donor subject whose fecal microbiota has been characterized and can be enriched with the disclosed composition. In some aspects, the fecal matter is obtained from the subject in need for therapy (recipient) and then enriched with the compositions of the current disclosure before retransplant. In some aspects, the recipient and the donor re the same subject or are different subjects.

[0302] In some aspects, compositions of the current disclosure are formulated in unit dosage form as a solid, semi-solid, lyophilized, freeze dried, liquid, concentrate, capsule, powder, emulsions, suspensions, and / or tablets, and suitably packaged. In some aspects, the species of the disclosure, or combination of species and formulations disclosed herein are encapsulated. These formulations are a further aspect of this disclosure. In some aspects, formulations are mixed with liquids suitable for orogastric and / or nasogastric delivery. In some aspects, the amount of a species, or a combination of species, is between 0.1-95% by weight of the formulation, or between 0.1-1% or 1 %-10% or 10%-20%, or 20%-30%, or 30%-40%, or 40%- 50%, or 50%-60%, or- 75 -299756710.160%-70%, or 70%-80% or 80%-90% or 90%-99% by weight of the formulation. Methods of formulating compositions are discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. (1980).

[0303] Compositions described herein may further comprise any suitable (e.g., dietarily acceptable, pharmaceutically acceptable, etc.) excipients, carriers, and / or stabilizers. Acceptable excipients, carriers, or stabilizers may include, for example, buffers, antioxidants, preservatives, polymers, chelating reagents, and / or surfactants. In some aspects, the compositions are pharmaceutical compositions. Pharmaceutical compositions (for example LBPs) are preferably manufactured under GMP conditions. In some aspects, compositions can be used orally, nasally or parenterally, for instance, in the form of capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewables, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, cataplasms, transdermal absorption systems, lotions, inhalations, aerosols, injections, suppositories, and the like. In certain aspects, any one or more of the aforementioned agents are expressly excluded from a composition.

[0304] Pharmaceutically acceptable carriers and excipients. The phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are interchangeably used herein to refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0305] In certain aspects, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), and / or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer's solution, phosphate solution or buffer, buffered saline, and other carriers known in the art

[0306] In some aspects, compositions herein may also include stabilizers, anti-oxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. Herein, the term "excipient" refers to an inert substance added to a composition to further facilitate- 76 -299756710.1administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0307] In certain aspects, compositions described herein are formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In some aspects, any of the well-known techniques, carriers, and excipients are used as suitable and / or as understood in the art.

[0308] In certain aspects, compositions described herein are an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymetllylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In certain aspects, any one or more of the aforementioned agents are expressly excluded from a composition. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of polymers as suspending agent(s) by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of polymers as suspending agent(s) by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of polymers as suspending agent(s) by total weight of the composition. In some aspects, compositions disclosed herein may not comprise polymers or suspending agents.

[0309] In certain aspects, compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of a composition described herein are increased by the addition of one or more gelling and / or thickening agents. In some aspects, compositions disclosed herein may comprise one or more gelling and / or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In some aspects, compositions disclosed herein- 77 -299756710.1may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethylmethacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or any combination thereof. In certain aspects, any one or more of the aforementioned agents are expressly excluded from a composition.

[0310] In certain aspects, compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, or at least about 45%, or at least about 50% total- 78 -299756710.1amount of one or more agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more agents by total weight of the composition. In some aspects, one or more of these agents are added to improve the performance, efficacy, safety, shelf-life and / or other property of the muscarinic antagonist composition of the present disclosure. In some aspects, additives are biocompatible, without being harsh, abrasive, and / or allergenic. In certain aspects, any one or more of the aforementioned agents are expressly excluded from a composition.

[0311] In certain aspects, compositions disclosed herein may comprise one or more acidifying agents. As used herein, "acidifying agents" refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid is used. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more acidifying agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more acidifying agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more acidifying agents by total weight of the composition. In some aspects, compositions disclosed herein may not comprise acidifying agents.

[0312] In certain aspects, compositions disclosed herein may comprise one or more alkalizing agents. As used herein, "alkalizing agents" are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In some aspects, compositions disclosed herein may- 79 -299756710.1comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more alkalizing agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more alkalizing agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more alkalizing agents by total weight of the composition. In some aspects, compositions disclosed herein may not comprise alkalizing agents.

[0313] In certain aspects, compositions disclosed herein may comprise one or more antioxidants. As used herein, "antioxidants" are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more antioxidants by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more antioxidants by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more antioxidants by total weight of the composition. In some aspects, compositions disclosed herein may not comprise antioxidant agents.

[0314] In certain aspects, compositions disclosed herein may comprise a buffer system. As used herein, a "buffer system" is a composition comprised of one or more buffering agents wherein "buffering agents" are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any- 80 -299756710.1pharmaceutically acceptable organic or inorganic buffer can be used. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more buffering agents by total weight of the composition. In some aspects, compositions disclosed herein comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more buffering agents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition. In some aspects, compositions disclosed herein do not comprise buffering agents.

[0315] In some aspects, the amount of one or more buffering agents can depend on the desired pH level of a composition. In some aspects, compositions disclosed herein have a pH of about 6 to about 9. In some aspects, compositions disclosed herein have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0316] In certain aspects, compositions disclosed herein comprise one or more preservatives. As used herein, "preservatives" refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more preservatives by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more preservatives by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition. In some aspects, compositions disclosed herein do not comprise preservatives agents.

[0317] In certain aspects, compositions disclosed herein comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents are synthetic, natural, and / or semi-synthetic. In some aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholy tic detergents,- 81 -299756710.1amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more surfaceacting reagents or detergents by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some aspects, compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some aspects, compositions disclosed herein do not comprise surface-acting reagents or detergents.

[0318] In certain aspects, compositions disclosed herein may comprise one or more stabilizers. As used herein, a "stabilizer" refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more stabilizers by total weight of the composition. In some aspects, compositions disclosed herein may comprise at most about 1%, at most about 5%, at most about 10%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, or at most about 50% total amount of one or more stabilizers by total weight of the composition. In some aspects, compositions disclosed herein comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition. In some aspects, compositions disclosed herein do not comprise stabilizers.

[0319] In some aspects, compositions disclosed herein comprise one or more tonicity agents. As used herein, a "tonicity agents" refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those- 82 -299756710.1or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art In some aspects, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a composition disclosed herein results in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L. In some aspects, a composition herein has an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg or from about 280 mOsm / kg to about 320 mOsm / kg. In some aspects, a composition described herein has an osmolarity of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L. In some aspects, a composition disclosed herein comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, a composition disclosed herein comprises at most 1%, at most 5%, at most 10%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, or at most 50% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, compositions disclosed herein comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, compositions disclosed herein do not comprise tonicity agents.

[0320] In some aspects, bacterial species described herein are formulated for delivery to the intestines (e.g., the small intestine and / or the colon). In some aspects, the bacteria are formulated with an enteric coating that increases the survival of the bacteria through the harsh environment in the stomach. In some aspects, the enteric coating is one which resists the action of gastric juices in the stomach so that the bacteria which are incorporated therein will pass through the stomach and into the intestines. The enteric coating may readily dissolve when in contact with intestinal fluids, so that the bacteria enclosed in the coating will be released in the intestinal tract. Enteric coatings can comprise, consist essentially of, or consist of polymer and copolymers well known in the art, such as but not limited to commercially available EUDRAGIT (Evonik Industries). (See e.g., Zhang, AAPS PharmSciTech, (2016) 17 (1), 56-67).- 83 -299756710.1

[0321] In some aspects, bacterial species described herein are formulated for rectal delivery to the intestine (e.g., the colon). Thus, in some aspects, bacterial compositions disclosed herein are formulated for delivery by suppository, colonoscopy, endoscopy, sigmoidoscopy, and / or enema.

[0322] A composition, for example, a pharmaceutical preparation and particularly a pharmaceutical preparation for oral administration, can include an additional component that enables efficient delivery of the compositions of the disclosure to the intestine (e.g., the colon). A variety of pharmaceutical preparations that allow for the delivery of the compositions to the intestine (e.g., the colon) can be used. Examples thereof include pH sensitive compositions, more specifically, buffered sachet formulations or enteric polymers that release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach. When a pH sensitive composition is used for formulating the pharmaceutical preparation, the pH sensitive composition is preferably a polymer whose pH threshold of the decomposition of the composition is between about 6.8 and about 7.5. Such a numeric value range is a range in which the pH shifts toward the alkaline side at a distal portion of the stomach, and hence is a suitable range for use in the delivery to the colon. It should further be appreciated that each part of the intestine (e.g., the duodenum, jejunum, ileum, cecum, colon and rectum), has different biochemical and chemical environment. For instance, parts of the intestines have different pHs, allowing for targeted delivery by compositions that have a specific pH sensitivity. Thus, in some aspects, the compositions provided herein is formulated for delivery to the intestine or specific parts of the intestine (e.g., the duodenum, jejunum, ileum, cecum, colon and rectum) by providing formulations with the appropriate pH sensitivity. (See e.g., Villena et al., Int J Pharm 2015, 487 (1-2): 314-9).

[0323] Another aspect of a pharmaceutical compositions useful for delivery of the compositions to the intestine (e.g., the colon) is one that ensures the delivery to the colon by delaying the release of the contents (e.g., the bacterial species) by approximately 3 to 5 hours, which corresponds to the small intestinal transit time. In some aspects of a pharmaceutical preparation for delayed release, a hydrogel is used as a shell. The hydrogel is hydrated and swells upon contact with gastrointestinal fluid, with the result that the contents are effectively released (released predominantly in the colon). Delayed release dosage units include drug-containing compositions having a material which coats or selectively coats a drug or active ingredient to be administered. Examples of such selective coating material include in vivo degradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and / or enzyme degradable polymers. A wide variety of coating materials for efficiently delaying the release is available and includes, for example, cellulose-based polymers such as hydroxypropyl cellulose, acrylic acid- 84 -299756710.1polymers and copolymers such as methacrylic acid polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.

[0324] Additional examples of pharmaceutical compositions that allow for the delivery to the intestine (e.g., the colon) include bioadhesive compositions which specifically adhere to the colonic mucosal membrane (for example, a polymer described in the specification of U.S. Pat. No. 6,368,586) and compositions into which a protease inhibitor is incorporated for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease.

[0325] Another example of a system enabling the delivery to the intestine (e.g., the colon) is a system of delivering a composition to the colon by pressure change in such a way that the contents are released by utilizing pressure change caused by generation of gas in bacterial fermentation at a distal portion of the stomach. Such a system is not particularly limited, and a more specific example thereof is a capsule which has contents dispersed in a suppository base and which is coated with a hydrophobic polymer (for example, ethyl cellulose).

[0326] A further example of a system enabling the delivery of a composition to the intestine (e.g., the colon), is a composition that includes a coating that can be removed by an enzyme present in the gut (e.g., the colon), such as, for example, a carbohydrate hydrolase or a carbohydrate reductase. Such a system is not particularly limited, and more specific examples thereof include systems which use food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.

[0327] The compositions provided herein can also be delivered to specific target areas, such as the intestine, by delivery through an orifice (e.g., a nasal tube) or through surgery. In addition, the compositions provided herein that are formulated for delivery to a specific area (e.g., the cecum or the colon), can be administered by a tube (e.g., directly into the small intestine). Combining mechanical delivery methods such as tubes with chemical delivery methods such as pH specific coatings, allow for the delivery of the compositions provided herein to a desired target area (e.g., the cecum or the colon).

[0328] In some aspects, compositions comprising bacterial species are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. In some aspects, dosage regimens are adjusted to provide the optimum desired response (e.g., the prophylactic or therapeutic effect). In some aspects, the dosage form of the composition is a tablet, pill, capsule, powder, granules, solution, or suppository. In some aspects, the composition is formulated for oral administration. In some aspects, the composition is formulated such that the bacteria of the composition, or a portion thereof, remain viable after passage through the stomach- 85 -299756710.1of the subject. In some aspects, the composition is formulated for rectal administration, e.g. as a suppository. In some aspects, the composition is formulated for delivery to the intestine or a specific area of the intestine (e.g., the colon) by providing an appropriate coating (e.g., a pH specific coating, a coating that can be degraded by target area specific enzymes, or a coating that can bind to receptors that are present in a target area).

[0329] Dosages of the active ingredients in the compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired pharmaceutical response for a particular subject, composition, and mode of administration, without being toxic or having an adverse effect on the subject. The selected dosage level can depend upon a variety of factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors.

[0330] A physician, veterinarian or other trained practitioner, can start doses of the composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect (e.g., treatment of a pathogenic infection, reduction of bacterial burden of pathogenic infection, reduction or inhibition of toxin production) is achieved. In general, effective doses of the compositions of the present invention, for the prophylactic treatment of groups of people as described herein vary depending upon many different factors, including routes of administration, physiological state of the subject, whether the subject is human or an animal, other medications administered, and the therapeutic effect desired. Dosages need to be titrated to optimize safety and efficacy. In some aspects, the dosing regimen entails oral administration of a dose of any of the compositions described herein. In some aspects, the dosing regimen entails oral administration of multiple doses of any of the compositions described herein. In some aspects, the composition is administered orally the subject once, twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or at least 10 times.

[0331] In some aspects, administration of the compositions comprising at least one bacterial species as disclosed herein, can be combined with simultaneous, or staggered administration of other bacterial / probiotic species. Dosage and forms of such formulations can be empirically determined by a person of skill in the art.

[0332] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, in some aspects, a single bolus is administered, several divided doses are administered overtime, or the doses may be proportionally- 86 -299756710.1reduced or increased as indicated by the exigencies of the therapeutic situation. In some aspects, it is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active composition and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0333] Also within the scope of the present disclosure are food products comprising any of the bacterial species described herein, and a nutrient. Food products are, in general, intended for the consumption of a human or an animal. In some aspects, any of the bacterial species described herein can be formulated as a food product. In some aspects, the bacterial species are formulated as a food product in spore form. In some aspects, the bacterial species are formulated as a food product in vegetative form. In some aspects, the food product comprises both vegetative bacteria and bacteria in spore form. The compositions disclosed herein can be used in a food or beverage, such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed. Non-limiting examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; bars, paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products such as western confectionery products including biscuits, cookies, and the like, Japanese confectionery products including steamed bean-jam buns, soft adzuki -bean jellies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soybean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.- 87 -299756710.1

[0334] Food products containing bacterial species described herein can be produced using methods known in the art and may contain the same amount of bacteria (e.g., by weight, amount, or CFU) as the compositions provided herein. Selection of an appropriate amount of bacteria in the food product may depend on various factors, including for example, the serving size of the food product, the frequency of consumption of the food product, the specific bacterial species contained in the food product, the amount of water in the food product, and / or additional conditions for survival of the bacteria in the food product.

[0335] Examples of food products which can be formulated to contain any of the bacterial species described herein include, without limitation, a beverage, a drink, a bar, a snack, a dairy product, a confectionery product, a cereal product, a ready-to-eat product, a nutritional formula, such as a nutritional supplementary formulation, a food or beverage additive.IV. Methods of treatment and prophylaxis

[0336] In some aspects, the current disclosure encompasses methods of treatment, the methods comprising administering to a subject in need thereof, a therapeutically effective quantity of a composition as disclosed herein. In some aspects, the methods disclosed herein are used in the prevention and / or treatment of a diseases and / or disorders. In some aspects, these diseases or disorders are associated with microbiome dysbiosis. In some aspects, microbiome dysbiosis can be diagnosed by any method known in the art and / or disclosed herein. In some aspects, methods of diagnosis include symptomatic determination, biomarker test, genetic test, imaging, culturing of gut microbiome, 16S rRNA analysis, or any combination thereof. In some aspects, the disease or disorder is diagnosed by a method disclosed herein. In certain aspect, methods of treatment of intestinal disorders are based on features (e.g., taxa and / or metabolic pathways) identified by Taxa4Meta mediated diverse 16S data analysis.

[0337] In some aspects, the current disclosure encompasses methods of treating or preventing gastrointestinal disorders by administering: (i) an effective amount of one or more of the disclosed bacterial compositions (e.g., Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale), individually or in combination; and / or (ii) a behavioral or dietary intervention comprising a low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols (lowFODMAP) diet (LFD) or cognitive behavioral therapy (CBT), or combinations thereof. In some aspects, these inventions may be further combined with one or more additional therapies disclosed herein (e.g., FMT, antibiotics, biologies).

[0338] In some aspects, the disclosed methods comprise administering the bacterial composition(s) alone or together with an LFD and / or CBT. In certain aspects, the order of- 88 -299756710.1administration is selected to optimize engraftment and symptom control, and may be simultaneous, sequential, or staggered over a defined interval (e.g., 1-12 weeks), with repetition as clinically indicated. In some aspects, the defined interval may be at least, at most, or about 1- 4 weeks, 4-8 weeks, 8-12 weeks, 12-16 weeks, 16-20 weeks, 20-24 weeks, 24-28 weeks, 28- 32 weeks, 32-36 weeks, 36-40 weeks, 40-44 weeks, 44-48 weeks, 48-52 weeks (or any range derivable therein).

[0339] In some aspects, disclosed herein are methods of determining patient populations suffering from gut dysbiosis that will respond well to various therapies, including administration of the compositions disclosed herein, LFD and / or CDT.

[0340] Personalized treatments: In some aspects, the disclosed methods of treatment are personalized using baseline clinical, dietary, and microbiome features identified by, for example, pan-microbiome profiling (e.g., Taxa4Meta) and enterocluster assignment. For example, in certain aspects, subjects with a baseline microbiome enterocluster enriched for Blautia / Bifidobacteria / Lachnoclostridia (Cluster 4) may be preferentially selected for LFD and / or administration of one or more disclosed compositions containing Bifidobacterium and / or Eubacterium rectale. Subjects with a baseline Bacteroides-dominant enterocluster (Cluster 2) and / or elevated gut-directed anxiety may be selected for CBT and / or administration of one or more disclosed compositions. In some aspects, baseline dietary fat intake is used as a moderator; higher baseline fat intake can identify subjects more likely to respond to LFD, while lower baseline fat intake can identify subjects more likely to respond to CBT.

[0341] In some aspects, methods comprise a series of steps. Step 1 involves baseline assessment of symptoms (such as IBS-SSS), quality of life, dietary intake (for example, a 3-day food record), psychological distress (including visceral sensitivity and anxiety), and stool-based microbiome profiling (such as 16S and / or point-of-care qPCR enterocluster assignment). Step 2 consists of initiation of therapy according to a decision algorithm that assigns subjects to one of the following: bacterial composition(s) alone, LFD alone, CBT alone, bacterial composition(s) plus LFD, bacterial composition(s) plus CBT, or LFD plus CBT. Any one or more of these therapies may be specifically excluded. This may be optionally followed by add-on or switch therapy based on interim response. Additional therapies as disclosed herein may be included, or specifically excluded as necessary. Step 3 is reassessment at 4-12 weeks and iterative adjustment, which may include reintroduction or personalization phase of LFD, CBT booster sessions, composition dose titration, or addition of, for example, FMT, LBPs or pharmacotherapy.

[0342] Bacterial compositions: In some aspects, the disclosed method comprises administering about 106to 1012cfu per species per unit dose, once daily to once weekly, for 2-12- 89 -299756710.1weeks or longer, via oral, buccal, orogastric, nasogastric, rectal, colonoscopic, or sigmoidoscopic routes, in dosage forms disclosed herein (e.g., lyophilized capsules, suspensions) as disclosed herein above. In certain aspects, compositions comprising, consisting essentially of, or consisting of Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale are administered as a 2-4 species consortium. In some aspects, compositions are co-administered with prebiotic-controlled diets (e.g., personalized LFD reintroduction) to support engraftment. In some aspects, compositions are administered before, during, or after LFD and / or CBT. Methods of administration of disclosed bacterial compositions are further described above, and / or are well established in the art.

[0343] Low FODMAP diet (LFD). As used herein a “low FODMAP (Fermentable Oligosaccharides, Disaccharides, Monosaccharides, and Polyols) diet” is a dietary intervention designed to reduce the intake of certain types of carbohydrates that are poorly absorbed in the small intestine and rapidly fermented by gut bacteria, which can trigger gastrointestinal symptoms in sensitive individuals, particularly those with irritable bowel syndrome (IBS)

[0344] In some aspects, a low FODMAP diet (LFD) comprises a structured nutritional intervention that limits short-chain, rapidly fermentable oligosaccharides, di saccharides, monosaccharides, and polyols to reduce luminal osmotic load and gas production associated with abdominal pain and other gastrointestinal symptoms in irritable bowel syndrome (IBS). In some aspects, the LFD includes an initial restriction phase of about, at least, or at most 3, 4, 5, 6, 7 or 8 weeks, followed by a systematic reintroduction phase over about 1-2 months to identify individual FODMAP intolerances while maintaining dietary adequacy.

[0345] In some aspects, the LFD is delivered by a trained dietitian across the duration of the treatment, with education on FODMAP identification, food label interpretation, menu planning, recipes, and problem-solving to enhance adherence. In some aspects, adherence is assessed using food journals obtained at baseline and during the intervention and analyzed with nutrition software to quantify intake patterns, including sugars, carbohydrates, fiber, and dietary fat.

[0346] In some aspects, the LFD improves IBS outcomes in a subset of subjects, with clinically meaningful reductions in abdominal pain severity and enhancements in health-related quality of life. In some aspects, treatment response is moderated by baseline microbiome enteroclusters and dietary factors, where an enterocluster enriched in Blautia, Bifidobacterium, and Lachnoclostridia (Cluster 4) is associated with greater LFD responsiveness relative to a Bacteroides-dominant enterocluster (Cluster 2). In some aspects, higher baseline dietary fat intake is associated with superior response to the LFD, whereas lower baseline fat intake may favor response to non-dietary interventions such as cognitive behavioral therapy (CBT).- 90 -299756710.1

[0347] In some aspects, patient selection and real-time risk stratification for LFD responsiveness are facilitated by microbiome profiling using pan-microbiome analytics (e.g., Taxa4Meta) and / or using multiplexed qPCR assays that assigns enterocluster composition. In some aspects, supervised machine learning models generate a FODMAP non-responsiveness score from pre- and post-intervention fecal profiles, where a shift away from Cluster 4 features toward Cluster 2 features indicates reduced likelihood of clinical benefit from the LFD.

[0348] In some aspects, clinical implementation of the LFD is integrated into a prospective protocol that collects stool specimens and symptom assessments at baseline and at approximately 4 weeks, with follow-up at about 3 and 6 months to evaluate durability of response. In some aspects, primary outcomes include changes in abdominal pain and quality of life, with secondary outcomes including stool form, dietary adherence, and shifts in microbiome community structure. In some aspects, statistical analysis employs mixed-effects models to test moderation by baseline psychological distress, dietary fat intake, and enterocluster classification.

[0349] In some aspects, the LFD is combined with adjunctive or alternative therapies, including CBT, live biotherapeutic products (LBPs), probiotics, fecal microbiota transplant (FMT), or pharmaceuticals, administered simultaneously, sequentially, or staggered based on clinical judgment. In some aspects, for subjects identified as LFD non-responders by baseline moderators or interim non-responsiveness scoring, alternative pathways prioritize centrally acting therapies (e.g., CBT) or microbiota-directed therapeutics to avoid unnecessary dietary restriction.

[0350] In some aspects, safety and nutritional adequacy are maintained by limiting the restriction phase to about 4-8 weeks, followed by targeted reintroduction to preserve beneficial taxa and metabolites while mitigating symptom provocation. In some aspects, the LFD protocol includes monitoring for risks associated with overly restrictive eating patterns, with dietitian- guided diversification to sustain gut microbiome health alongside symptom control.

[0351] In some aspects, the disclosed method comprises prescribing an LFD that includes an elimination phase of 2-10 weeks (e.g., at least, at most, or about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks, or any range derivable therein) during which high-FODMAP foods are restricted. In some aspects, this phase may be delivered by a trained dietitian who provides educational materials, label-reading instruction, recipe plans, and / or adherence tracking such as 3-day food records. The next phase is a reintroduction phase, which involves a systematic, stepwise challenge over approximately 2 months to identify individual FODMAP tolerances by subgroup, including fructans, galacto-oligosaccharides, lactose, excess fructose, and polyols. The final phase is personalization, in which the long-term diet is tailored to symptom control while maximizing overall nutritional quality and microbiome- 91 -299756710.1health, such as by promoting fiber diversity and short chain fatty acid (SCFA)-supporting foods. In certain aspects, LFD is combined with the disclosed bacterial compositions to mitigate unintended losses of beneficial taxa, in non-responders and to enhance clinical response in predicted responders, such as those in Cluster 4.

[0352] Cognitive behavioral therapy (CBT): In some aspects, cognitive behavioral therapy (CBT) comprises a structured psychological intervention designed to modify maladaptive thoughts, behaviors, and emotional responses associated with gastrointestinal symptoms in irritable bowel syndrome (IBS) and related disorders. In some aspects, CBT is delivered over a range of about 3, 4, 5, 6, 7, 8, 9, 10 or more sessions, and may be administered in-person, by telephone, or via digital platforms. In some aspects, the duration of CBT intervention may span about 1-12 weeks, depending on the protocol and patient needs.

[0353] In some aspects, CBT includes components such as cognitive restructuring, stress management, relaxation training, behavioral activation, and problem-solving strategies. In some aspects, CBT protocols may incorporate education about IBS, symptom monitoring, and coping skills to address illness-related anxiety, catastrophizing, and avoidance behaviors. In some aspects, CBT may be delivered individually, in group settings, or as part of a comprehensive selfmanagement program that integrates dietary and lifestyle guidance.

[0354] In some aspects, adherence to CBT is monitored through homework assignments, selfreport diaries, or digital tracking tools, with adherence rates varying depending on delivery format and patient engagement. In some aspects, CBT is provided by licensed psychologists, therapists, or other trained healthcare professionals with expertise in behavioral interventions for gastrointestinal disorders. In some aspects, treatment fidelity is maintained through standardized protocols, session checklists, and periodic supervision or fidelity assessment.

[0355] In some aspects, CBT is associated with clinically meaningful improvements in abdominal pain, psychological distress, and health-related quality of life in a subset of subjects with IBS. In some aspects, treatment response to CBT may be moderated by baseline psychological distress, gut-directed anxiety, coping style, or microbiome composition. In some aspects, CBT may be combined with adjunctive therapies, including dietary interventions, pharmacological agents, probiotics, or live biotherapeutic products, administered simultaneously, sequentially, or staggered over a defined interval (e.g., about 1-52 weeks).

[0356] In some aspects, CBT protocols are adapted to accommodate patient preferences, comorbidities, and access to care, with flexible delivery formats and session schedules. In some aspects, safety and efficacy of CBT are evaluated through standardized outcome measures,- 92 -299756710.1including symptom severity scales, quality of life assessments, and psychological inventories, collected at baseline and at intervals ranging from about 4 weeks to 12 months post-intervention.

[0357] In some aspects, methods comprise administering a manualized CBT program focused on gut-brain interaction, delivered in-person, via telehealth, or by telephone, over at least, 3, 4, 5, 6, 7, 8, 9, 10, or more sessions across at least, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks, or more. In some aspects, the program includes relaxation training and stress management, cognitive restructuring such as reducing catastrophizing and threat appraisal, exposure-based techniques for Gl-directed anxiety and avoidance behaviors, and behavioral self-management and maintenance strategies. In certain aspects, CBT is selected as first-line when baseline gut-directed anxiety is elevated and / or enterocluster assignment favors CBT response, such as Cluster 2.

[0358] Combination regimens. In some aspects, methods include administering bacterial composition(s) together with LFD by starting the composition(s) during the LFD elimination phase and continuing through reintroduction, with dose adjustment based on symptoms and microbiome monitoring. Alternatively, bacterial composition(s) may be initiated within 1-2 weeks of starting CBT to synergize symptom reduction and microbiome remodeling. LFD and CBT may be implemented concurrently where both psychological distress and diet are predicted moderators, with sequencing tailored to adherence considerations. In refractory cases or where multi-domain moderators are present, a triple modality approach may be used, combining composition(s), LFD, and CBT, and optionally including FMT and / or pharmacotherapies disclosed herein.

[0359] Monitoring and response-guided adaptation. In some aspects, methods comprise periodic evaluation (e.g., every 1-4 weeks, 4-8 weeks, 8-12 weeks, 12-16 weeks, 16-20 weeks, 20-24 weeks, 24-28 weeks, 28-32 weeks, 32-36 weeks, 36-40 weeks, 40-44 weeks, 44-48 weeks, 48-52 weeks, or any range derivable therein) of IBS-SSS (Irritable Bowel Syndrome Symptom Severity Scale), IBS-QoL (Irritable Bowel Syndrome Quality of Life), pain behaviors, stool form, adherence, and microbiome enterocluster status. In certain aspects, failure to achieve predefined response thresholds (e.g., >30% reduction in abdominal pain or >50-point reduction in IBS-SSS) triggers protocolized adaptations (e.g., switch from LFD to CBT, add bacterial composition(s), adjust diet reintroduction, or add approved pharmacologic agents).

[0360] Safety and nutritional adequacy. In some aspects, dietitian-guided LFD ensures adequate macronutrient and micronutrient intake, mitigates unnecessary long-term restriction, and screens for disordered eating risk; CBT protocols include monitoring for psychological safety;- 93 -299756710.1bacterial composition(s) are administered per applicable LBP / probiotic quality and viability standards.

[0361] In some aspects, disclosed compositions and methods are used for the treatment of any disease and / or disorder responsive or preventable by the administration of the disclosed compositions and practice of the disclosed methods.

[0362] In some aspects, the disease is a gastrointestinal disease or disorder, necrotizing enterocolitis, nosocomial infections, enteric inflammation, inflammatory disorders, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancer (particularly of the gastrointestinal and immune systems), diarrheal disease, antibiotic associated diarrhea, pediatric diarrhea, appendicitis, allergies, autoimmune disorders, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, coeliac disease, diabetes mellitus, organ transplantation, bacterial infections, viral infections, fungal infections, periodontal disease, urogenital disease, sexually transmitted disease, HIV infection, HIV replication, HIV associated diarrhea, surgical associated trauma, surgical-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia, wound healing, ulcers, gut barrier function, allergy, asthma, respiratory disorders, circulatory disorders, coronary heart disease, anemia, disorders of the blood coagulation system, renal disease, disorders of the central nervous system, hepatic disease, ischemia, nutritional disorders, osteoporosis, endocrine disorders, epidermal disorders, psoriasis, acne vulgaris, panic disorder, behavioral disorder and / or post- traumatic stress disorders. In some aspects, the disease or a disorder is a gastrointestinal disease, non-limiting examples of which include Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD). In some aspects, the compositions provided herein are administered to a subject if the subject has a dysbiosis (e.g., has as microbiome associated with a disease state). In some aspects, treatment with the compositions provided herein results in the change in the microbiome of the subject. In some aspects, treatment with the compositions provided herein removes the dysbiosis in the subject resulting in a healthy microbiome. In some aspects, treatment with the compositions provided herein removes the dysbiosis in the subject resulting in microbiome refractory and / or less susceptible to infection by a pathogen. In some aspects, the disease or disease state of the subject includes symptoms such as colitis, diarrhea, watery diarrhea, abdominal cramping, fever, blood or pus in the stool, nausea, dehydration, loss of appetite, chills, weight loss, and / or kidney failure. In some aspects, the composition and / or methods disclosed herein are used for CDI, IBS, UC, IBD, Crohn’s disease, and antibiotic-associated diarrhea; in certain aspects, IBS patients are prioritized for LFD and / or CBT according to moderator profiles based on the microbiome enterocluster definitions as above.- 94 -299756710.1In some aspects, subjects with dysbiosis characterized by reduced Faecalibacterium prausnilzii. Bifidobacterium adolescentisUongum, or Eubacterium rectale, and / or expansion of Bacteroides are treated using the compositions disclosed herein.

[0363] In some aspects, the current disclosure also encompasses methods for modifying, repairing, and / or improving the gut microbiota of a subject in need thereof by administration of a therapeutically effective quantity of the compositions provided, to a subject in need thereof. In some aspects, the current disclosure also encompasses administration of a therapeutically effective quantity of the disclosed compositions to a subject in need thereof, to enhance the uptake and / or utilization of metabolites.

[0364] As used herein the term “therapeutically effective quantity” refers to an amount of the formulation that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms or prevents or provides prophylaxis for the disorder or condition. An “effective amount’ refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of compositions of the invention are outweighed by the therapeutically beneficial effects. In some aspects, the therapeutically effective quantity is a quantity that results in reduction in biomarkers of enteric inflammation in the subject. In some aspects, the therapeutically effective quantity is an amount that results in increases in the levels of beneficial plasma protein biomarkers. In some aspects the therapeutically effective quantity can be a quantity that results in significant improvement in symptoms associated with the disease or disorder. In some aspects the therapeutically effective quantity is an amount that results in enhanced colonization of the beneficial probiotic populations in the gut as demonstrated by various objective means used in the art, including but not limited, to fecal cultures, genomic analysis of fecal, and / or intestinal swabs. In some aspects, the therapeutically effective quantity can be an amount of the formulation that when administered in conjunction with an additional therapy, improves efficacy and long term impact of the therapy for the subject. In some aspects, suitable therapy can include a fecal microbiota transplant (FMT) procedure, administration of pain relievers, antibiotics, antispasmodic agents, antiacids, anti -diarrheal, proton-pump inhibitors, anti-inflammatory, corticosteroids, immune system suppressors, biologies, vitamins and / or supplements, surgery, nutritional support, one or more small molecules, or any combination thereof.

[0365] In some aspects, the therapeutically effective quantity is an amount of the formulation that improves the overall health of the subject, as measured by objective measures known in the art. In some aspects, the subject has not received a dose of an antibiotic prior to administration of- 95 -299756710.1the bacterial composition. In some aspects, the subject has not been administered an antibiotic at least 1, at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 60, at least 90, at least 120, at least 180 or at least 360 days prior to administration of the compositions provided herein. In some aspects, the person has not been administered antibiotics to treat the pathogenic infection. In some aspects, the compositions provided herein comprise a first treatment of the pathogenic infection. In some aspects, the subject is administered one or more doses of an antibiotic prior to or concurrently with a bacterial composition. Generally, the first line of defense in the treatment of a pathogenic infection is the administration of an antibiotic. In some aspects, the subject is administered a single dose of an antibiotic prior to the bacterial composition. In some aspects, the subject is administered multiple doses of an antibiotic prior to the bacterial composition. In some aspects, the subject is administered at least 2, 3, 4, 5 or more doses of an antibiotic prior to the bacterial composition. In some aspects, the subject is administered a dose of an antibiotic at substantially the same time as the bacterial composition. Examples of antibiotics that can be administered include, without limitation, kanamycin, gentamicin, colistin, metronidazole, vancomycin, clindamycin, fidaxomicin, and / or cefoperazone.

[0366] In some aspects, the amount of a composition administered to a subject and the frequency of administration may vary depending upon the subject or host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0367] Additionally, in some aspects, compositions as disclosed herein can be combined with food formulations as described herein or additional probiotic species or both. In some aspects, formulations is administered together, or the administration may be staggered. In some aspects, amounts of food formulations or probiotic formulations or both can vary and are determined by a person of skill in the art.

[0368] As discussed above, in some aspects, administration of compositions provided herein can be oral, gastric, orogastric, nasogastric, implanted, buccal, and rectal. In some aspects, compositions provided herein can be administered orally as any one of, but not limited to, a solid, semi-solid, liquid, capsule, powder, emulsions, suspensions and tablet, or combinations thereof. In some aspects compositions are administered and / or mixed with any one of, but not limited to, water, juice, gruel, milk, breast milk, baby food, or any other commercially available formula, beverage, food products, fruits and vegetables, raw foods and cooked foods. In some aspects, the compositions are administered once daily. In some aspects the compositions are administered- 96 -299756710.1more than once daily. In some aspects, the compositions are administered sequentially over more than one day, such as over 2, 3, 4, 5, 10, 15, 20, 30 days, or more days, or any range derivable herein. In some aspects, compositions are administered at least once a month for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or longer. In some aspects, compositions are administered orogastrically. In some aspects, compositions are administered nasogastrically.

[0369] Compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0370] In some aspects, the present disclosure also encompasses methods for modifying, repairing or improving the health of the gut microbiota of a subject in need thereof. As used herein the term “modifying the gut microbiota” means any intervention that results in change in the gut microbiome as measured by one of many methods available in the art. The change can be a decrease or an increase in the presence of a particular microbial strains, species, genus, family, order, or class. These methods to monitor gut microbiota are well known in the art and may include but are not restricted to fecal cultures, genomic analysis of the feces, or analysis of fecal or intestinal swabs. In some aspects, the present disclosure encompasses methods for repairing or improving the health of the gut microbiota of a subject in need thereof. The “health” of a subject's gut microbiota may be defined by relative abundances of microbial community members, expression of microbial genes, biomarkers, mediators of gut barrier function. To “repair the gut microbiota of a subject,” which is synonymous with “improve gut microbiota health,” means to change the microbiota of a subject, in particular the relative abundances of age- and health- discriminatory taxa, in a statistically significant manner towards chronologically-age matched reference healthy subjects. The term encompasses complete repair (i.e., the measure of gut microbiota health does not deviate by 1 .5 standard deviation or more) and levels of repair that are less than complete. The term also encompasses preventing or lessening a change in the relative abundances of age-and health-discriminatory taxa, wherein the change would have been significantly greater absent intervention. A subject with a gut microbiota in need of repair (e.g., a- 97 -299756710.1microbiota in “disrepair”, an “immature” gut microbiota, etc.) has a measure of gut microbiota health that deviates by 1.5 standard deviation or more (e.g., 2 std. deviation, 2.5 std. deviation, 3 std. deviation, etc.) from that of chronologically-age matched subjects, wherein the term “chronological age” means the amount of time a subject has lived from birth.

[0371] In some aspects, the subject is a human. In some aspects the subject is an individual clinically diagnosed with a disease or disorder or syndrome or exhibiting symptoms of disease or disorder or syndrome. In some aspects the subject is a healthy individual.

[0372] The aforementioned methods are not limited to subjects of a particular age. In some aspects, a subject is less than six months of age. In some aspects, a subject is at least six months of age. In some aspects, a subject is at least six months of age. In another aspect, a subject is one hundred years old, or younger. In some aspects, a subject is < 150 years, < 140 years, < 130 years,< 120 years, < 110 years, < 100 years, < 90 years, < 80 years, < 70 years, < 60 years, < 50 years, < 40 years, < 30 years, < 20 years < 15 years, < 14 years, < 13 years, < 12 years, < 11 years, < 10 years, < 9 years, < 8 years, < 7 years, < 6 years, < 5 years, < 4 years, < 3 years, < 2 years< 15 years, < 14 years, < 13 years, < 12 years, < 11 years, < 10 years, < 9 years, < 8 years, < 7 years, < 6 years,< 5 years, < 4 years, < 3 years, < 2 years< 15 years, < 14 years, < 13 years, < 12 years, < 11 years, < 10 years, < 9 years, < 8 years, < 7 years, < 6 years, < 5 years, < 4 years, < 3 years, < 2 years, or any range derivable therein. In still other aspects, a subject is a newborn to six months of age, six months to five years of age, six months to 2 years of age, or six months to 18 months of age. In some aspects the subject is a pre-term baby.

[0373] In some aspects, the subject is an animal. In some aspects, the subject is a mammal. In some aspects, the subject is a laboratory animal. In some aspects, the animal is a mouse model. In some aspects, the animal is a dog. In some aspects, the animal is a pig. In some aspects, the subject is a human.

[0374] Certain features of the microbiome can be transfer from mother to infant. In some aspects of the disclosure, compositions are administered to women during pregnancy to facilitate colonization of the probiotic in the infant gut.

[0375] In some aspects, effective amounts of the compositions detailed herein are administered prophylactically to reduce the the incidence of one or more diseases or disorders. In some aspects, the compositions disclosed herein are administered to “improve a subject’s health". To “improve a subject’s health” means to change one or more aspects of a subject’s health in a statistically significant manner towards chronologically-age matched reference healthy subjects, as well as to prevent or lessen a change in one or more aspects of the subject’s health wherein the- 98 -299756710.1change would have been significantly greater absent intervention. The improved aspect of the subject’s health can be growth or rate of growth, for example as measured by a score on an anthropometric index; signs or symptoms of disease; relative abundances of health discriminatory plasma proteins, including but not limited to biomarkers, mediators of gut barrier function, bone growth, neurodevelopment, acute and inflammation, and the like. Those in need of treatment to improve their health include those already with a disease, condition, or disorder as well as those prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented. In some aspects, methods described herein are not limited to intestinal disorders, but are applicable to other microbiome dysbiosis associated disorders.

[0376] In certain aspects, methods of treatment of intestinal disorders are based on features (e.g., taxa and / or metabolic pathways) identified by Taxa4Meta mediated diverse 16S data analysis.

[0377] In certain aspects, provided herein are methods for treating a subject determined to have CDI, IBS, IBD UC, and / or IBD CD based on a predetermined profile of one or more taxonomic and / or metabolic pathway features disclosed herein.

[0378] In certain aspects, provided herein are methods for identifying features associated with diseases associated with microbiome dysbiosis, for example but not limited to CDI, IBS, IBD UC, IBD CD, antibiotic-associated diarrhea (AAD), celiac disease, food allergies, autoimmune disease, cancer, and / or graft versus host disease.

[0379] Therapy provided herein can comprise administration of administration of one or more additional therapeutic agents, such as for example, a first therapy (e.g., compositions disclosed herein) and a second therapy (e.g., antibiotics, etc.). The additional therapies can be administered in any suitable manner known in the art. For example, the treatment may be administered sequentially (at different times) or concurrently (at the same time).

[0380] In some aspects, the one or more additional therapies are administered substantially simultaneously. In some aspects, the one or more additional therapies are administered sequentially. In some aspects, the first therapy, the second therapy, and a third therapy are administered sequentially. In some aspects, the first therapy is administered before administering the second therapy. In some aspects, the first therapy is administered after administering the second therapy.

[0381] Other combination of temporal administration may be easily envisaged by a person with ordinary skill in the art.

[0382] In some aspects, the one or more additional therapies may comprise administration of a therapeutic comprising one or more of pain relievers, antibiotics, antispasmodic agents,- 99 -299756710.1antiacids, anti -diarrheal agents, proton-pump inhibitors, anti-inflammatory agents, corticosteroids, immune system suppressors, biologies, supplements, nutritional support, nutraceuticals, vaccines, vitamins, one or more small molecules, or any combination thereof. Nonlimiting examples of anti-inflammatory drugs include ibuprofen, aspirin, naproxen, diclofenac, celecoxib, indomethacin, meloxicam, and aminosalicylate. In some aspects, the aminosalicylate may comprise, consist essentially of, or consist of mesalamine, balsalazide, and / or olsalazine. Non-limiting examples of suitable immunosupressors include cyclosporine, tacrolimus, azathioprine, mycophenolate mofetil, mercaptopurine, methotrexate, sirolimus, everolimus, and corticosteroids (like prednisone). Non-limiting examples of suitable small molecule drugs include tofacitinib, upadacitinib, and / or ozanimod. In some aspects, the one or more small molecules comprises, consists essentially of, or consists of tofacitinib, upadacitinib, and / or ozanimod. Nonlimiting examples of biologies comprises, consists essentially of, or consists of infliximab, adalimumab, golimumab, certolizumab, vedolizumab, ustekinumab, and / or risankizumab. Nonlimiting examples of antibiotics comprises, consists essentially of, or consists of ciprofloxacin, metronidazole, vancomycin, fidaxomicin, and / or bezlotoxumab. Non-limiting examples of anti- diarrheal medication comprises, consists essentially of, or consists of psyllium powder, methylcellulose, and / or loperamide. Non-limiting examples of pain killers comprises, consists essentially of, or consists of piroxicam, acetaminophen (paracetamol), ibuprofen, aspirin, naproxen, diclofenac, tramadol, codeine, and morphine. In some aspects, the additional therapy is or comprises FMT therapy. In some aspects, the additional FMT therapy may compirse FMT with fecal microbiota from a donor subject having a bacterial taxa profile comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100%, of the taxa identified in Cluster 2, Cluster 4, or Cluster 5, or from a donor subject whose fecal microbiota has been characterized.

[0383] 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 can be employed.

[0384] Additional therapeutic agents of the disclosure can be administered by the same route of administration or by different routes of administration. In some aspects, the additional therapy is administered 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 can be- 100 -299756710.1determined based on the type of disease to be treated, severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.

[0385] 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 dos...

Claims

CLAIMSWhat is claimed is:

1. A composition comprising at least one of bacterial species Faecalibacterium prausnitzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an excipient.

2. The composition of claim 1, comprising the bacterial species Faecalibacterium prausnitzii and at least two of the bacterial species Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale.

3. The composition of claim 1, comprising at at least three of the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale.

4. The composition of claim 1, comprising Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale.

5. The composition of any one of claims 1-4, wherein the composition has a greater relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale, compared to a reference microbiome profile.

6. The composition of any one of claims 1-4, wherein the composition has a reduced relative abundance of one or more strains of Faecalibacterium prausnitzii Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale compared to a reference microbiome profile.

7. The composition of claim 5, wherein the one or more strains of Faecalibacterium prausnitzii belong to a sub-clade comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.

8. The composition of claim 6, wherein the one or more strains of Faecalibacterium prausnitzii belong to a sub-clade comprising SGB 15316.

9. The composition of any one of claims 1-8, for administration into a subject in need thereof, wherein the subject has an altered microbiome enterocluster profile relative to a reference microbiome profile.- 136 -299756710.

110. The composition of claim 9, wherein the subject has reduced levels of any one or more of Faecalibacterium prausnilzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale in the microbiome enterocluster profile relative to the reference microbiome profile.

11. The composition of claim 9, wherein the subject has a reduced relative abundance of any one or more of a specific strain or strains of Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof in the microbiome enterocluster profile relative to the reference microbiome profile.

12. The composition of claim 9, wherein the subject has a greater relative abundance of any one or more of a specific strain or strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof in the microbiome enterocluster profile relative to the reference microbiome profile.

13. The composition of any one of claims 5 or 6, for administration into the subject as in claim 9-12, wherein the greater or the reduced abundance of the one or more strains is determined based on the subjects microbiome enterocluster profile.

14. The composition of claim 13, wherein the composition for administration is determined based on the subjects microbiome enteroclusters profile.

15. The composition of any one of claims 9-14, wherein the subject has enriched levels of one or more species of genera Bacteroides, Blautia, Lachnospiraceae, Clostridioides, Enterob acteriaceae, Streptococcus, or an unclassified taxa, or a strain thereof.

16. The composition of any one of claims 9-15, wherein the subject has, or is at a risk of having a gastrointestinal disorder.

17. The composition claim 16, wherein the gastrointestinal disorder comprises one or more of Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD).

18. The composition of any one of claims 9-17, wherein the subject has a bacterial taxa profile in a fecal matter sample that comprises at least 70% of the taxa identified in Cluster 1 or Cluster 3, wherein:- 137 -299756710.1Cluster 1 taxa profile comprises Bacteroides, Blautia, Unclassified_071, Faecalibacterium, Unclassified_075, Unclassified_072, Anaerostipes, Lachnospiraceae incertae sedis, Parabacteroides, Roseburia, Alistipes, Streptococcus, Ruminococcus, Dorea, Bifidobacterium, Fusicatenibacter, Unclassified_087, Ruminococcus2, Coprococcus, Flavonifractor, Eubacterium, Lachnoclostridium, Clostridium XlVa, Dialister, Lactobacillus, Erysipelatoclostridium, Unclassified_083, Unclassified_037, Intestinibacter, Veillonella, Unclassified_129, Romboutsia, Ruthenibacterium, Collinsella, Unclassified_123, Unclassified_074, Not_Available_2, Prevotella, Parasutterella, and Clostridium;Cluster 3 taxa profile comprises Bacteroides, Blautia, Streptococcus, Unclassified_123, Unclassified_071, Unclassified_129, Veillonella, Lactobacillus, Erysipelatoclostridium, Unclassified_072, Lachnoclostridium, Bifidobacterium, Enterococcus, Anaerostipes, Lachnospiraceae incertae sedis, Clostridium XlVa, Unclassified_087, Clostridium, Faecalibacterium, Parabacteroides, Unclassified_074, Unclassified_075, Flavonifractor, Unclassified_064, Roseburia, Dialister, Unclassified_132, Intestinibacter, Prevotella, Clostridium XVIII, Alistipes, Not_Available_2, Unclassified_083, Akkermansia, Haemophilus, Fusobacterium, Romboutsia, Ruminococcus2, Fusicatenibacter, and Clostridium sensu stricto; and wherein the Cluster 1 or Cluster 3 has a reduced level of Faecalibacterium prausnitzii relative to a healthy cluster, wherein the healthy correspond to any one of clusters 2, 4, or 5, wherein:Cluster 2 taxa profile comprises 1 or more of the following listed bacterial taxa: Bacteroides, Blautia, Faecalibacterium, Unclassified_071, Alistipes, Ruminococcus, Parabacteroides, Unclassified_072, Unclassified_075, Lachnospiraceae incertae sedis, Roseburia, Anaerostipes, Not_Available_2, Fusicatenibacter, Dorea, Coprococcus, Bifidobacterium, Eubacterium, Streptococcus, Ruminococcus2, Lactobacillus, Gemmiger, Romboutsia, Unclassified_087, Odoribacter, Akkermansia, Unclassified_123, Bilophila, Flavonifractor, Ruthenibacterium, Barnesiella, Parasutterella, Lachnoclostridium, Agathobaculum, Intestinibacter, Erysipelatoclostridium, Prevotella, Phascolarctobacterium, Clostridium IV, and Unclassified_037;- 138 -299756710.1Cluster 4 taxa profile comprises 1 or more of the following listed bacterial taxa: Blautia, Unclassified_071, Faecalibacterium, Bifidobacterium, Ruminococcus, Lachnospiraceae incertae sedis, Unclassified_075, Bacteroides, Dorea, Unclassified_072, Anaerostipes, Not_Available_2, Fusicatenibacter, Coprococcus, Streptococcus, Romboutsia, Roseburia, Gemmiger, Alistipes, Collinsella, Agathobaculum, Ruminococcus2, Intestinibacter, Unclassified_087, Parabacteroides, Unclassified_074, Clostridium, Eubacterium, Lactobacillus, Unclassified_083, Turicibacter, Akkermansia, Dialister, Adlercreutzia, Schaalia, Erysipelatoclostridium, Clostridium sensu stricto, Not_Available_5, Ruthenibacterium, and Clostridium IV; andCluster 5 taxa profile comprises 1 or more of the following listed bacterial taxa: Prevotella, Bacteroides, Faecalibacterium, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae incertae sedis, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Bifidobacterium, Unclassified_087, Eubacterium, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

19. The composition of claim 18, wherein the reduced level of Faecalibacterium prausnitzii relative to a healthy cluster is for one or more strains belonging to the subclades comprising SGB15318, SGB15342, SGB15326, SGB15322, or any combination thereof20. The composition of any one of claims 1-19, wherein the composition comprises about, or more than about, 106to 1012colony forming units (cfu) of each of the at least one, at least two, at least three, or at least four of the bacterial species.

21. The composition of claim 20, wherein the composition comprises about 106to 107, or about 107to 108, or about 108to 109, or about 109to IO10, or about 1010to 1012cfu / unit dose or more of each of the at least one, at least two, at least three, or at least four bacterial species.

22. The composition of any one of claim 1-21, further comprising one or more additional bacterial species of the genera Faecalibacterium, Bifidobacterium, Eubacterium, Prevotella,- 139 -299756710.1Bacteroides, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

23. The composition of any one of claims 1-22, wherein the composition is formulated as a Live Biotherapeutic Product (LBP).

24. The composition of any one of claims 1-22, wherein the composition is formulated as a probiotic.

25. The composition of any one of claims 1-22, wherein the composition is formulated as a food formulation.

26. The composition of any one of claims 1-22, wherein the composition is formulated as a fecal microbiota transplant (FMT) product or an additive to a FMT procedure.

27. The composition of any one of claims 1-26, wherein the composition is for oral, buccal, orogastric, nasogastric, sublingual, topical, rectal, vaginal, colonoscopy administration, and / or flexible sigmoidoscopic administration.

28. The composition of claim 27, wherein the composition is for orogastric, nasogastric, oral or rectal administration.

29. The composition of claim 27 or claim 28, wherein the composition is in the form of a powder, a freeze dried formulation, a lyophilized formulation, a capsule, a gummy, a tablet, a sachet, a liquid, an emulsion, a suspension, fecal matter, and / or a food formulation.

30. The composition of any one of claims 1-29, wherein the excipient comprises one or more of a carrier, buffer, preservative, flavoring agent, antimicrobial agent, antibiotic agent, antifungal agent, antioxidant, preservative, electrolyte, antifoaming agent, oil, stabilizer, enhancing agent, or any combination thereof.

31. The composition of claim 1-30, wherein the composition is for administration in combination with one or more other therapies comprising a fecal microbiota transplant (FMT) procedure, a low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols- 140 -299756710.1(FODMAP) diet, cognitive behavioral therapy (CBT), administration of pain relievers, antibiotics, antispasmodic agents, antiacids, anti -diarrheal, proton-pump inhibitors, antiinflammatory, corticosteroids, immune system suppressors, biologies, vitamins and / or supplements, surgery, nutritional support, one or more small molecules, or any combination thereof.

32. The composition of any one of claims 1-31, wherein the one or more bacterial species are genetically engineered.

33. The composition of any one of claims 1-31, wherein the one or more bacterial species are wild-type.

34. The composition of claim 1-33 wherein the one or more bacterial species are isolated and cultured from fecal samples.

35. A composition comprising a greater or reduced relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale, for administration into a subject with a gastrointestinal disease or disorder, wherein the greater or the reduced relative abundance of the one or more strains in the composition is determined based on a comparison of the subjects microbiome enterocluster profile with a reference microbiome profile.

36. The composition of claim 35, wherein the subjects microbiome enterocluster profile is determined using the steps of:(i) obtaining nucleic acids from the biological sample;(ii) determining the relative abundance of the one or more strains present in the sample based on a nucleic acid based assay;(iii) determining the biological sample’s microbiome enteroclusters profile based on the relative abundance.

37. The composition of claim 36, wherein the nucleic acid based assay comprises 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), and / or a bioinformatics pipelines.

38. The composition of claim 37, wherein the bioinformatics pipeline comprises the Taxa4Meta, and / or BIOFIRE® SPOTFIRE® System.- 141 -299756710.

139. The method of claim 36, wherein the biological sample comprises fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation.

40. A method of treating a gastrointestinal disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of claims 1-39.

41. A method of treating a gastrointestinal disease or disorder a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an excipient.

42. The method of claim 41, wherein the composition comprises the bacterial species Faecalibacterium prausnitzii and at least one of Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale.

43. The method of any one of claims 41-42, wherein the composition has a greater relative abundance of one or more strains of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale, compared to a reference microbiome profile.

44. The method of any one of claims 41-43, wherein the composition has a reduced relative abundance of one or more strains of Faecalibacterium prausnitzii Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale compared to a reference microbiome profile.

45. The method of any one of claims 41-44, wherein the composition for administration is determined based on the subjects microbiome enteroclusters profile.

46. The method of any one of claims 40-42, wherein the composition has greater relative abundance of one or more strains of Faecalibacterium prausnitzii compared to a reference microbiome profile.

47. The method of any one of claims 40-42, wherein the composition has reduced relative abundance of one or more strains of Faecalibacterium prausnitzii compared to a reference gut microbiome sample.

48. The method of claim 46, wherein the one or more strains of Faecalibacterium prausnitzii belong to sub-clades comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.- 142 -299756710.

149. The method of claim 47, wherein the one or more strains of Faecalibacterium prausnitzii belong to sub-clades comprising SGB15316.

50. The method of claim 41 or claim 42, wherein the composition comprises Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale.

51. The method of claim 40-50, wherein the subject has reduced microbiota diversity in a biological sample from the subject relative to a reference microbiome profile.

52. The method of claim 40-50, wherein the subject has an altered microbiome enterocluster profile in a biological sample from the subject.

53. The method of claim 51 or 52, wherein the biological sample is a fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation.

54. The method of claim 51 or 52, wherein the subject has reduced levels of any one or more of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale in the microbiota relative to a reference microbiome profile.

55. The method of any one of claims 50-54, wherein the subject has increased levels of one or more species of genera Bacteroides, Blautia, Lachnospiraceae, Clostridioides,Enterob acteriaceae, Streptococcus, or an unclassified taxa.

56. The method of any one of claims 41-55, wherein the subject has or is at a risk of having a gastrointestinal disorder.

57. The method of claim 56, wherein the gastrointestinal disease or disorder comprises one or more of Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD).

58. The method of any one of claims 41-55, wherein the subject has, or is at a risk of having antibiotic associated diarrhea.

59. The method of any one of claims 51-58, wherein the subject has a bacterial taxa profile in a fecal matter sample that comprises at least 70%, of the taxa identified in Cluster 1 or Cluster 3, wherein:- 143 -299756710.1Cluster 1 taxa profile comprises Bacteroides, Blautia, Unclassified_071, Faecalibacterium, Unclassified_075, Unclassified_072, Anaerostipes, Lachnospiraceae incertae sedis, Parabacteroides, Roseburia, Alistipes, Streptococcus, Ruminococcus, Dorea, Bifidobacterium, Fusicatenibacter, Unclassified_087, Ruminococcus2, Coprococcus, Flavonifractor, Eubacterium, Lachnoclostridium, Clostridium XlVa, Dialister, Lactobacillus, Erysipelatoclostridium, Unclassified_083, Unclassified_037, Intestinibacter, Veillonella, Unclassified_129, Romboutsia, Ruthenibacterium, Collinsella, Unclassified_123, Unclassified_074, Not_Available_2, Prevotella, Parasutterella, and Clostridium;Cluster 3 taxa profile comprises Bacteroides, Blautia, Streptococcus, Unclassified_123, Unclassified_071, Unclassified_129, Veillonella, Lactobacillus, Erysipelatoclostridium, Unclassified_072, Lachnoclostridium, Bifidobacterium, Enterococcus, Anaerostipes, Lachnospiraceae incertae sedis, Clostridium XlVa, Unclassified_087, Clostridium, Faecalibacterium, Parabacteroides, Unclassified_074, Unclassified_075, Flavonifractor, Unclassified_064, Roseburia, Dialister, Unclassified_132, Intestinibacter, Prevotella, Clostridium XVIII, Alistipes, Not_Available_2, Unclassified_083, Akkermansia, Haemophilus, Fusobacterium, Romboutsia, Ruminococcus2, Fusicatenibacter, and Clostridium sensu stricto; and wherein the Cluster 1 or Cluster 3 has a reduced level of Faecalibacterium prausnitzii relative to a healthy clusters 2, 4 or 5, wherein:Cluster 2 taxa profile comprises 1 or more of the following listed bacterial taxa: Bacteroides, Blautia, Faecalibacterium, Unclassified_071, Alistipes, Ruminococcus, Parabacteroides, Unclassified_072, Unclassified_075, Lachnospiraceae incertae sedis, Roseburia, Anaerostipes, Not_Available_2, Fusicatenibacter, Dorea, Coprococcus, Bifidobacterium, Eubacterium, Streptococcus, Ruminococcus2, Lactobacillus, Gemmiger, Romboutsia, Unclassified_087, Odoribacter, Akkermansia, Unclassified_123, Bilophila, Flavonifractor, Ruthenibacterium, Barnesiella, Parasutterella, Lachnoclostridium, Agathobaculum, Intestinibacter, Erysipelatoclostridium, Prevotella, Phascolarctobacterium, Clostridium IV, and Unclassified_037;Cluster 4 taxa profile comprises 1 or more of the following listed bacterial taxa: Blautia, Unclassified_071, Faecalibacterium, Bifidobacterium, Ruminococcus, Lachnospiraceae- 144 -299756710.1incertae sedis, Unclassified_075, Bacteroides, Dorea, Unclassified_072, Anaerostipes, Not_Available_2, Fusicatenibacter, Coprococcus, Streptococcus, Romboutsia, Roseburia, Gemmiger, Alistipes, Collinsella, Agathobaculum, Ruminococcus2, Intestinibacter, Unclassified_087, Parabacteroides, Unclassified_074, Clostridium, Eubacterium, Lactobacillus, Unclassified_083, Turicibacter, Akkermansia, Dialister, Adlercreutzia, Schaalia, Erysipelatoclostridium, Clostridium sensu stricto, Not_Available_5, Ruthenibacterium, and Clostridium IV; andCluster 5 taxa profile comprises 1 or more of the following listed bacterial taxa: Prevotella, Bacteroides, Faecalibacterium, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae incertae sedis, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Bifidobacterium, Unclassified_087, Eubacterium, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

60. The method of claim 59, wherein the reduced level of Faecalibacterium prausnitzii relative to a healthy cluster is for one or more strains belonging to the subclades comprising SGB15318, SGB15342, SGB15326, SGB15322, or any combination thereof61. The method of any one of claims 41-51, wherein the composition comprises about, or more than about, 106to 1012cfu of each of the at least one, at least two, at least three, or at least four bacterial species.

62. The method of claim 61, wherein the composition comprises about 106to 107, or about 107to 108, about 108to 109, about 109to IO10, about IO10to 1011, or about 1011to 1012, cfu / unit dose or more of each of the at least one, at least two, at least three, or at least four bacterial species, optionally wherein the bacterial species have been isolated and / or otherwise purified from a source material.

63. The method of any one of claim 41-62, further comprising administering one or more additional bacterial species of the genera Faecalibacterium, Bifidobacterium, o Eubacterium.- 145 -299756710.

164. The method of claim 63, wherein the composition comprises the one or more additional bacterial species of the genera Faecalibacterium, Bifidobacterium, Eubacterium, Prevotella, Bacteroides, Unclassified_071, Blautia, Roseburia, Unclassified_072, Unclassified_075, Ruminococcus, Parabacteroides, Lachnospiraceae, Coprococcus, Dorea, Alistipes, Fusicatenibacter, Anaerostipes, Lactobacillus, Gemmiger, Streptococcus, Unclassified_037, Romboutsia, Ruminococcus2, Unclassified_083, Odoribacter, Sutterella, Not_Available_2, Bilophila, Barnesiella, Dialister, Butyricimonas, Clostridium, Intestinibacter, Paraprevotella, Haemophilus, Clostridium IV, Phascolarctobacterium, and Flavonifractor.

65. The method of any one of claims 40-64, wherein the composition is formulated as a Live Biotherapeutic Product (LBP).

66. The method of any one of claims 40-64, wherein the composition is formulated as a probiotic.

67. The method of any one of claims 40-64, wherein the composition is formulated as a food formulation.

68. The method of any one of claims 40-64, wherein the composition is formulated as an FMT product, or an additive to a donor fecal microbiota for a FMT procedure.

69. The method of claim 68, wherein the composition is administered via an oral, buccal, orogastric, nasogastric, sublingual, topical, rectal, vaginal, colonoscopy administration, and / or flexible sigmoidoscopic route.

70. The method of claim 69, wherein the composition is administered via an orogastric route or nasogastric route.

71. The method of claim 70, further comprising administering to the subject one or more additional therapies.

72. The method of claim 71, wherein the additional therapy comprises a FMT procedure.

73. The method of claim 72, wherein the fecal microbiota is from a donor subject having a bacterial taxa profile comprising at least 70%, of the taxa identified in Cluster 2, Cluster 4, or Cluster 5, or from a donor subject whose fecal microbiota has been characterized.- 146 -299756710.

174. The method of any one of claims 70-73, wherein the additional therapy comprises administration of a therapeutic comprising one or more of a low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols (FODMAP) diet, cognitive behavioral therapy (CBT), pain relievers, antibiotics, antispasmodic agents, antiacids, anti -diarrheal agents, protonpump inhibitors, anti-inflammatory agents, corticosteroids, immune system suppressors, biologies, supplements, nutritional support, nutraceuticals, vaccines, vitamins, one or more small molecules, or any combination thereof.

75. The method of claim 74, wherein the one or more anti-inflammatory drugs is an aminosalicylate.

76. The method of claim 75, wherein the aminosalicylate comprises, consists essentially of, or consists of mesalamine, balsalazide, and / or olsalazine.

77. The method of claim 74, wherein the one or more immune system suppressors comprises, consists essentially of, or consists of azathioprine, mercaptopurine, and / or methotrexate.

78. The method of claim 74, wherein the one or more small molecules comprises, consists essentially of, or consists of tofacitinib, upadacitinib, and / or ozanimod.

79. The method of claim 74, wherein the one or more biologies comprises, consists essentially of, or consists of infliximab, adalimumab, golimumab, certolizumab, vedolizumab, ustekinumab, and / or risankizumab.

80. The method of claim 74, wherein the one or more antibiotics comprises, consists essentially of, or consists of ciprofloxacin, metronidazole, vancomycin, fidaxomicin, and / or bezlotoxumab.

81. The method of claim 74, wherein the anti -diarrheal medication comprises, consists essentially of, or consists of psyllium powder, methylcellulose, and / or loperamide.

82. The method of claim 74, wherein the one or more pain killer comprises, consists essentially of, or consists of Piroxicam.

83. The method of claim 82, wherein the Piroxicam is administered at 100 ppm with drinking water.- 147 -299756710.

184. The method of any one of claims 71-83, wherein the method comprises coadministration of the additional therapy with the method.

85. The method of any one of claims 71-83, wherein the method comprises staggered administration of the additional therapy with the method.

86. The method of any one of claims 41-85 wherein the method is repeated periodically.

87. The method of claim 86, wherein the method is repeated at least once in one week, two weeks, three weeks, monthly, 6 monthly, or yearly over a period of time.

88. The method of claim any one of claims 41-87, wherein the composition is an antiinflammatory composition.

89. The method of any one of claims 41-88, wherein the subject is a mammal.

90. The method of claim 89, wherein the subject is a human.

91. A method of restoring the gut microbiome of a subject in need thereof, the method comprising administering to the subject a composition comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or any combination thereof; and an excipient.

92. The method of claim 91, comprising administering to the subject a composition comprising the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale,' and an excipient.

93. A kit comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an excipient.

94. The kit of 86, comprising the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale,' and an excipient.

95. The kit of claim 94, wherein the bacterial species comprises one or more strains from the bacterial species Faecalibacterium prausnitzii comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.- 148 -299756710.

196. A food formulation comprising at least one of bacterial species Faecalibacterium prausnilzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an ingestible additive.

97. The food formulation of claim 96, comprising the bacterial species Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale,' and an excipient.

98. The food formulation of claims 96 or 97, wherein the bacterial species Faecalibacterium prausnitzii comprises one or more sub-clades of the bacterial species Faecalibacterium prausnitzii comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.

99. A Live Biotherapeutic Product (LBP) comprising at least one of bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale or any combination thereof; and an ingestible additive.

100. The LBP of claim 99, comprising the bacterial species Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and Eubacterium rectale,' and an excipient.

101. The LBP of claims 99 or 100, wherein the bacterial species Faecalibacterium prausnitzii comprises one or more sub-clades of the bacterial species Faecalibacterium prausnitzii comprising SGB 15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.

102. A method of predicting response to a gastrointestinal disorder therapy in a subject diagnosed with or is suspected of having a gastrointestinal disorder, comprising:(a) obtaining or having obtained a biological sample from the subject;(b) identifying a microbiome enterocluster profile of the biological sample; and(c) selecting a therapy based on the microbiome enterocluster profile.

103. The method of claim 102, wherein the microbiome enterocluster profile has a reduced abundance of one or more bacterial species comprising Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, Eubacterium rectale, or a strain thereof, or any combination thereof.- 149 -299756710.1104. The method of claim 103, wherein the microbiome enterocluster profile has reduced abundance of Faecalibacterium prausnitzii belonging to sub-clades comprising SGB15318, SGB 15342, SGB 15326, or SGB 15322, or any combinations thereof.

105. The method of claim 103, wherein the microbiome enteroclusters profile has increased abundance of one or more strains of Faecalibacterium prausnitzii belonging to sub-clades comprising SGB 15316.

106. The method of claim 102, wherein the therapy is a low Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols (FODMAP) diet, cognitive behavioral therapy (CBT), or a microbiome-targeted composition.

107. The method of claim 106, wherein the microbiome-targeted composition is a composition of any one of claims 1-34.

108. A method of monitoring response to a gastrointestinal disorder therapy in a subject diagnosed with a gastrointestinal disorder, comprising:(a) obtaining or having obtained a biological sample from the subject;(b) assessing a gut microbiome enterocluster profile of the biological sample; and(c) correlating changes with clinical symptom improvement.

109. A method of treating IBS in a subject, comprising:(a) assessing baseline psychological distress in the subject;(b) assessing baseline dietary fat intake;(c) assessing a gut microbiome enterocluster profile from a biological sample; and(d) administering a therapy selected based on the combined assessment.

110. The method of any one of claims 108 or 109, wherein assessing the gut microbiome enterocluster profile comprises:(i) obtaining nucleic acids from the biological sample;- 150 -299756710.1(ii) identifying the relative abundance of bacterial taxa present in the sample based on a nucleic acid based assay;(iii) determining the sample’s microbiome enteroclusters profile based on the relative abundance profile of the bacterial taxa.

111. The method of claim 110, wherein the nucleic acid based assay uses 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), and / or a bioinformatics Pipelines.

112. The method of claim 111, wherein the bioinformatics pipeline comprises the Taxa4Meta, and / or BIOFIRE® SPOTFIRE® System.

113. The method of claim 110, wherein the biological sample comprises fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation.

114. A method of assessing the gut microbiome enterocluster profile of a subject with a gastrointestinal disorder, comprising:(i) obtaining nucleic acids from the biological sample;(ii) identifying the relative abundance of bacterial taxa present in the sample based on a nucleic acid-based assay;(iii) assessing the microbiome enteroclusters profile based on the relative abundance profile of the bacterial taxa.

115. The method of claim 110 or 114, wherein the nucleic acid-based assay uses 16S rRNA gene sequencing, whole genome shotgun (WGS) metagenomic sequencing, quantitative PCR (qPCR), and / or a bioinformatics pipelines.

116. The method of claim 115, wherein the bioinformatics pipeline comprises the Taxa4Meta, and / or BIOFIRE® SPOTFIRE® System.

117. The method of claim 110 or 114, wherein the biological sample comprises fecal sample, intestinal biopsy, intestinal swab, mucosal biopsy, and / or intestinal irrigation.- 151 -299756710.1118. The method of any one of claims 114-117, further comprising use of the microbiome enteroclusters profile assessment in diagnosis, prognosis, or selection of a therapeutic intervention.

119. The method of any one of claims 101 or 114, wherein the gastrointestinal disorder is Clostridioides difficile infection (CDI), Irritable bowel syndrome (IBS), Ulcerative colitis (UC), Inflammatory Bowel Disease (IBD), or Crohn’s disease (IBD CD).

120. The method of claim 118, wherein the therapeutic intervention comprises administering to the subject a composition of any one of claims 1-34.

121. The method of any one of claims 118-120, wherein the therapeutic intervention comprises administering a cognitive behavioral therapy (CBT).

122. The method of any one of claims 118-121, wherein the therapeutic intervention comprises administering a low FODMAP diet.

123. A method of stratifying IBS patients for dietary intervention, comprising:(a) determining the patients baseline dietary fat intake;(b) determining the patients microbiome enteroclusters profile from a biological sample from the patients;(c) predicting likelihood of response to a low FODMAP diet based on the combination of(a) and (b).

124. A kit for the diagnosis and management of IBS, CDI and IBD, comprising:(a) reagents for multiplexed qPCR detection of microbiome enteroclusters;(b) instructions for interpreting a microbiome enteroclusters profile to guide dietary or behavioral therapy selection.

125. A method of integrating psychological, dietary, and microbiome data to guide IBS, CDI and IBD therapy, comprising:(a) collecting psychological assessment data;(b) collecting dietary intake data;(c) collecting microbiome enteroclusters profile data;- 152 -299756710.1(d) using a multivariate model to predict optimal therapy.

126. The method of claim 125, wherein the multivariate model comprises a DMM Clustering (Dirichlet Multinomial Mixture).

127. The method of claim 125 or 126, wherein the multivariate model is a latent factor or machine learning model trained on clinical outcomes.

128. A method of predicting non-responsiveness of a subject to a low FODMAP diet, comprising: analyzing the subject’s microbiome for a shift from a Blautia / Bifidobacteria- dominated cluster to a Bacteroides-dominated cluster after dietary intervention, wherein the shift indicates that the subject will be non-responsive to the low FODMAP diet.

129. A method of using enterocluster profile as precision strain-level microbial and probiotic and / or synbiotic therapy for gastrointestinal diseases selected from IBS, CDI, and IBD.

130. A method comprising administering to a subject a composition comprising Faecalibacterium prausnilzii, Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale after the relative abundance of Faecalibacterium prausnilzii. Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale, or a strain thereof, in the subject’s microbiome enterocluster profile has been assessed.

131. The method of claim 130, wherein the subject’s microbiome enterocluster profile is compared to a reference microbiome profile, to determine the relative amounts of Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Bifidobacterium longum, and / or Eubacterium rectale, or a strain thereof to be included in the composition.

132. The method of claims 130 or 131, wherein the composition is administered to provide a level of one or more strains that is within 10% of the reference microbiome profile for that strain.- 153 -299756710.1

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