WO2020234746A1

WO2020234746A1 - Materials and methods for microbiome modulation

Info

Publication number: WO2020234746A1
Application number: PCT/IB2020/054689
Authority: WO
Inventors: Lani R. SAN MATEO; Andrew J. BALTUS; Gerardus M. BONGERS; Ryan Balfour SARTOR; Akihiko Oka
Original assignee: Janssen Biotech, Inc.; The University Of North Carolina At Chapel Hill
Priority date: 2019-05-18
Filing date: 2020-05-18
Publication date: 2020-11-26

Abstract

Specific Enterobacteriaceae bacterial strains have been shown to induce intestinal inflammation in a mammalian subject. In particular, compositions containing these bacterial strains can be used to make an animal model for identifying an agent useful for treating an intestinal inflammation, and to treat a subject having an intestinal inflammation.

Description

MATERIALS AND METHODS FOR MICROBIOME MODULATION

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name

“JBI6095WOPCTlSequenceListing5-ll-20.txt” creation date ofMayll, 2020, and having a size of 6 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed generally to a composition comprising a purified bacterial mixture and applications thereof. It also relates to a method of inducing an intestinal inflammation in a mammalian subject comprising administering the

composition to the subject, as well as a mouse model of the intestinal inflammation produced by the method thereof. It also relates to a method of identifying an agent useful for treating an intestinal inflammation and a method of treating a subject having an intestinal inflammation.

BACKGROUND OF THE INVENTION

Hundreds of species of commensal microorganisms are harbored in the gastrointestinal tracts of mammals, where they interact with the host immune system. Commensal microorganisms have an essential role in driving immune-mediated experimental inflammation in the distal intestine. Research using germ-free (GF) animals has shown that the commensal microorganisms influence the development of the mucosal immune system, such as histogenesis of Foyer's patches (PPs) and isolated lymphoid follicles (ILFs), secretion of antimicrobial peptides from the epithelium, and

accumulation of unique lymphocytes in mucosal tissues, including immunoglobulin A- producing plasma cells, intraepithelial lymphocytes, IL-17-producing CD4-positive T cells (Thl7), and IL-22-producing NK-like cells. Consequently, the presence of intestinal bacteria enhances protective functions of the mucous membranes, enabling the host to mount robust immune responses against pathogenic microbes invading the body. On the other hand, the mucosal immune system maintains unresponsiveness to dietary antigens and harmless microbes. Abnormality in the regulation of cross-talk between commensal bacteria and the immune system (intestinal dysbiosis) may lead to overly robust immune response to environmental antigens and inflammatory bowel disease (IBD) may result.

Recent studies have shown that an altered intestinal microbial composition (dysbiosis) is associated with intestinal inflammation in human IBD and with cases of acute and chronic rodent experimental enterocolitis harboring complex enteric microbiotas, which are characterized by decreased bacterial diversity and an altered ratio of dominant bacterial species (Hansen et al., Curr. Opin. Gastroenterol. 2010, 26(6): 564- 71; Frank at al., Proc. Natl. Acad. Sci. USA. 2007, 104(34): 13780-5.). In addition, rodent models monoassociated with bacteria relevant to the dysbiosis of IBD, such as certain Escherichia coli, Enterococcus faecalis, Bacteroides vulgatus, and Bacteroides thetaiotaomicron strains, demonstrated that single intestinal bacteria can selectively induce host inflammation and also provided insights into underlying disease mechanisms (Rath et al., J. Clin. Invest., 1996, 98(4):945-53).

Nevertheless, the study of specific bacterium-host interactions by use of conventional and monoassociated rodent models has some limitations. The microbiotas of conventional animal models are too complex and variable to easily define and manipulate the functionally dominant component species and strain. In addition, monoassociated mice do not reflect the actual situation in the distal intestine, where different bacterial groups reciprocally interact with each other and the host in multiple ways. To resolve these shortcomings, new animal models with defined, interacting intestinal microbiotas need to be developed.

The foregoing discussion is presented solely to provide a better understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes“prior art” to the instant application. SUMMARY OF THE INVENTION

The present invention is generally related to a composition comprising a purified bacterial mixture and its related applications, e.g., in the treatment of intestinal inflammation.

In one general aspect, the invention relates to a composition comprising at least one purified Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3; preferably a purified bacterial mixture comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, wherein the bacterial strains are present in the composition at a ratio of 1:1:1 or other desirable ratio.

In certain embodiments, the three Enterobacteriaceae bacterial strains comprise 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

In certain embodiments, the three Enterobacteriaceae bacterial strains are

Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4.

In another general aspect, the invention relates to a method of inducing an inflammation, preferably an intestinal inflammation, in a mammalian subject, comprising administering to the subject an effective amount of a composition comprising at least one purified Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3; preferably a purified bacterial mixture comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, wherein the bacterial strains are present in the composition at a ratio of 1:1:1 or other desirable ratio.

In certain embodiments, the mammalian subject is a rodent, preferably a germ- free I110^-1- or a germ-free wild type (WT) mouse, more preferably a germ-free I110^-1- mouse.

In certain embodiments, the above methods produce a mouse model of the intestinal inflammation.

In another general aspect, the invention relates to a method of identifying an agent useful for treating an inflammation, preferably an intestinal inflammation, comprising: a. administering to a mammalian subject a composition comprising at least one purified Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3; preferably a purified bacterial mixture comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3 to induce the intestinal inflammation in the subject, wherein the bacterial strains are present in the composition at a ratio of 1:1:1 or other desirable ratio;

b. administering to the subject the agent; and

c. measuring the efficacy of the agent on the intestinal inflammation in the subject.

In certain embodiments, the composition is administered to the subject by gavage, or by oral or anal swab. In certain embodiments, the intestinal inflammation is inflammatory bowel disease (IBD), such as ulcerative colitis or Crohn's disease, or a celiac disease.

In certain embodiments, the agent is an anti-inflammatory agent, preferably a microbiome composition.

In certain embodiments, the efficacy is evaluated by an index selected from the group consisting of colonic lamina propria (LP) immune cell activation, fecal lipocalin-2 (LCN) concentration, histology score, pro-inflammatory cytokine measurements, and stool consistency score. In further embodiments, the colonic lamina propria (LP) immune cell is selected from the group consisting of IFNg⁺CD4⁺, IL-lT⁺CD4⁺ and Foxp3⁺CD4⁺ T cell. In further embodiments, the pro-inflammatory cytokine is selected from the group consisting of IL-12p40, INF, EFNy, and LL-17a.

In another general aspect, the invention relates to a method of treating a subject having an inflammation, preferably an intestinal inflammation, comprising:

a. determining the presence of at least one Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3; preferably a panel of bacterial strains comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3 in a biological sample, preferably a fecal sample, of the subject; and

b. administering a therapeutic agent to the subject if the panel of bacterial strains is detected in the subject

In certain embodiments, the intestinal inflammation is characterized by an index selected from the group consisting of colonic lamina propria (LP) immune cell activation, fecal lipocalin-2 (LCN) concentration, fecal calprotectin concentration, fecal lactoferrin concentration, histology score, stool consistency score, and pro-inflammatory cytokine measurements, wherein the pro-inflammatory cytokine is selected from the group consisting of IL-12p40, TNF, IFNy, and IL-17a. In further embodiments, the colonic lamina propria (LP) immune cell is selected from the group consisting of IFNy⁺CD4⁺, IL- irCD4⁺ and Foxp3⁺CD4⁺ T cell.

In certain embodiments, the intestinal inflammation is inflammation of the small intestine or inflammation of the large intestine. In certain embodiments, the intestinal inflammation is inflammatory bowel disease (IBD), such as ulcerative colitis or Crohn's disease, or a celiac disease, or pouchitis.

In certain embodiments, the therapeutic agent is an anti-inflammatory agent, preferably a microbiome composition.

In certain embodiments, the therapeutic agent is a microbial metabolite. In certain embodiments, the microbial metabolite is an indole. In certain embodiments, the microbial metabolite is selected from the group consisting of indole-3 -propionic acid (IP A), indole, and indole-3-acetic acid (IAA). In a specific embodiment, the microbial metabolite is indole-3 -propionic acid (IP A).

Further aspects, features and advantages of the present invention will be better appreciated upon a reading of the following detailed description of the invention and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in -onjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 demonstrates the experimental design in Example 2.

FIGs. 2A-C demonstrate corresponding colonic lamina propria (LP) immune cell activation in Example 2:

FIG 2A for IRNg⁺ CD4⁺ cells;

FIG. 2B for Foxp3⁺CD4⁺ cells; and

FIG. 2C for histology score.

FIG. 3 demonstrates the experimental design in Example 3.

FIGs. 4A-C demonstrate the inflammation induced by KEE strains in Example 3:

FIG 4A for IFNy⁺ CD4⁺ cells;

FIG. 4B for histology score; and

FIG. 4C for stool consistency score.

FIG. 5 demonstrates the experimental design in Example 4.

FIG. 6 demonstrate that KEE strains induced more severe intestinal inflammation than the donor stool.

FIG. 7 shows the protocol of KEE I110^-1- treatment experiment in Example 5.

FIG. 8 demonstrates Week 6 stool scores in control group and treatment group in Example 5. Error bar shows SEM. ****P<0.0001, unpaired t test.

FIG. 9 demonstrate representative stool at week 6 (autopsy).

FIG. 10 shows that vehicle group have thick wall and shorten colon compared to VE202 treatment group in Example 5.

FIGs. 11A-B show that VE202 treatment significantly decreased fecal lipocalin-2 levels: FIG. 11 A, two weeks after VE202 therapy started; and FIG. 1 IB, four weeks after therapy. Error bar shows SEM. Kruskal- Wallis test multiple comparisons test

FIG. 12 shows that VE202 treatment significantly reduced pro-inflammatory cytokine IL-12p40 level in supernatant of tissue fragment culture. Unpaired two tailed t test FIGs. 13A-F demonstrate histology scores of ileum-end (FIG.13 A), cecum (FIG.13B), proximal-colon (FIG.13C), distal-colon (FIG.13D), rectum (F1G.13E) and total (cecum + proximal colon + distal colon) (FTG.13F). Error bar shows SEM. Tukey’s multiple comparisons test.

FIG. 14 demonstrates representative histology pictures of cecum and distal colon. Mean ± SD, *P< 0.05, **P<0.01, ***P<0.001, ****P<0.0001, N.S.: not significant.

FIGs. 15A-C show flow date of colonic LP cells.

FIG. 15A for Foxp3⁺CD4⁺ CDS^" T cells;

FIG. 15B for IFNy⁺ CD4⁺ CDS^" T cells; and

FIG. 15C for IL-17⁺ CD4⁺ CDS^" T cells.

FIG. 16 shows correlation between fecal lipocalin-2 (week 6) and total histology score.

FIG. 17 shows the OD at 600 nm, as a percentage of optical density at 0 mM IP A. FIG. 18 shows the percentage of difference in OD as compared to vehicle.

FIG. 19 shows the OD of bacterial cultures after 15 h of incubation, as the percent change as compared to vehicle.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. It must be noted that as used herein and in the appended claims, the singular forms“a,”“an,” and“the” include plural reference unless the context clearly dictates otherwise.

Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term“about” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

Unless otherwise indicated, the term“at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

As used herein, the terms“comprises,”“comprising,”“includes,”“including,” “has,”“having,”“contains” or“containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary,“of” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

It should also be understood that the terms“about,”“approximately,”“generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension/ characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors,

manufacturing tolerances, etc.), would not vary the least significant digit

The term“biomarker” or“marker’' as used herein refers generally to a measurable substance or agent in an organism whose presence is indicative of a certain nature of the organism. For example, a biomarker can be a molecule, including a gene, protein, carbohydrate structure, or glycolipid, the expression of which in or on a mammalian tissue or cell or secreted can be detected by known methods (or methods disclosed herein). The biomarker can also be one or more bacterial strains, the existence of which can be detected in a fecal sample by known method (or methods disclosed herein), such as whole genome or 16s rDNA gene sequencing. The biomarker is indicative of the status of a disease/disorder, and/or is predictive or can be used to predict (or aid prediction) for a mammalian cell's or tissue's sensitivity to, and in some embodiments, to predict (or aid prediction) an individual's responsiveness to therapeutic agents and treatment regimens.

In certain embodiments, the biomarkers disclosed herein include 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or Enterobacteriaceae bacterial strains containing the 16S rDNA, the detection of which can be used alone or in combination with other biomarkers for intestinal inflammation. The identification of biomarkers for inflammation, such as the detection of the presence of at least one of Enterobacteriaceae bacterial strains comprising 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, also provides a tool with which the efficacy of a therapeutic agent for inflammation can be evaluated in an ongoing basis. Furthermore, the detection of the presence of one or more of these Enterobacteriaceae bacterial strains also predicts (or aids prediction) an individual's responsiveness to therapeutic agents and treatment regimens, such as an anti-inflammatory agent, preferably a microbiome composition, for inflammation.

As used herein,“probe” refers to any molecule or agent that is capable of selectively binding to an intended target biomolecule. The target molecule can be a biomarker, for example, a nucleotide tianscript or a protein encoded by or corresponding to a biomarker. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations, in view of the present disclosure. Probes can be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, peptides, antibodies, aptamers, affibodies, and organic molecules.

As used herein,“mammalian subject” means any mammal, most preferably a rodent. The term“mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, a germ-free III Or¹ or a germ-free wild type (WT) mouse, etc. , more preferably a germ-free III mouse.

As used herein,“sample” is intended to include any sampling of fecal material, biopsies of specific organ tissues, including large and small intestinal biopsies, synovial fluid, and synovial fluid biopsies. Samples can be obtained from a subject by a variety of techniques, which are known to those skilled in the art.

As used herein,“treatment” or“treating” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an inflammation. Those in need of treatment include those diagnosed with the inflammation as well as those prone to have the inflammation or those in whom the inflammation is to be prevented. The treatment for inflammation can be any treatment disclosed in the art, such as an anti-inflammatory agent. An anti-inflammatory agent is a drug or substance that reduces inflammation (redness, swelling, and pain) in the body. Anti-inflammatory agents block certain substances in the body that cause inflammation. Examples of anti-inflammatory agents include, but are not limited to, nonsteroidal antiinflammatory drugs (NSAIDs) including aspirin, ibuprofen, and naproxen;

glucocorticoids, microbial compositions, and microbial metabolites. For example, an anti-inflammatory agent can be a microbial composition for treating inflammation, such as that described in U.S. Patent No. 10,052,353, which comprises a mixture of microbial strains (also named as VE 17 or VE202 in the present application), the entire contents of which are incorporated herein by reference.

Examples of the inflammation include autoimmune diseases, allergic diseases, infectious diseases, and rejection in organ transplantations, such as inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, Type I diabetes, multiple sclerosis, graft vs. host disease following bone marrow transplantation, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondyloarthropathy, systemic lupus erythematosus, insulin dependent diabetes mellitus, thyroiditis, asthma, psoriasis, dermatitis scleroderma, atopic dermatitis, graft versus host disease, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlejn purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, polyglandular deficiency type I syndrome and polyglandular deficiency type P syndrome, Schmidt's syndrome, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthropathy, arthropathy, Reiter's disease, psoriatic arthropathy, chlamydia, yersinia and salmonella associated arthropathy, spondyloarthropathy, atheromatous disease/arteriosclerosis, allergic colitis, atopic allergy, food allergies such as peanut allergy, tree nut allergy, egg allergy, milk allergy, soy allergy, wheat allergy, seafood allergy, shellfish allergy, or sesame seed allergy, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic

encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired

Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis C, common varied immunodeficiency (common variable

hypogammaglobulinaemia), dilated cardiomyopathy, fibrotic lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung disease, ankylosing spondylitis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, idiopathic leucopenia, autoimmune neutropenia, renal disease NOS, glomemlonephritides, microscopic vasculitis of the kidneys, discoid lupus,

erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), insulindependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatic fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, allergic rhinitis (pollen allergies), anaphylaxis, pet allergies, latex allergies, drug allergies, allergic rhinoconjuctivitis, eosinophilic esophagitis,

hypereosinophilic syndrome, eosinophilic gastroenteritis cutaneous lupus erythematosus, eosinophilic esophagitis, hypereosinophilic syndrome, and eosinophilic gastroenteritis, and diarrhea.

As used herein, the term“purified” in the context of“purified bacterial strain” or “purified bacterial mixture” refers to the isolation of the bacterial strain or bacterial mixture from its natural habitat such that a composition containing a“purified bacterial strain” or a“purified bacterial mixture” is manmade and different from a naturally occurring counterpart For example, a composition comprising at least one of three purified Enterobacteriaceae bacterial strains, e.g., Klebsiella pneumoniae str3, Enterobacter ludwigii str12, and Escherichia coli str4, is different from a fecal sample, from which the Enterobacteriaceae bacterial strains are isolated. The composition can include one or more substances that are not present in the natural environment of the purified Enterobacteriaceae bacterial strain(s). The composition can also differ from a natural product, such as a fecal sample, in the number of bacterial cells and/or the ratio of different bacterial cells in the composition.

In certain embodiments, the inflammation is intestinal inflammation, including but not limited to, inflammatory bowel disease (IBD) such as ulcerative colitis, Crohn's disease, or a celiac disease.

The term“administering” with respect to the methods of the invention, means a method for therapeutically or prophylactically preventing, treating or ameliorating a syndrome, disorder or disease as described herein. Such methods include administering an effective amount of said therapeutic agent at different times during the course of a therapy or concurrently in a combination form. The methods of the invention are to be understood as embracing all known therapeutic treatment regimens.

The term“effective amount” means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human, that is being sought by a researcher, veterinarian, medical doctor, or other clinician, which includes preventing, treating or ameliorating a syndrome, disorder, or disease being treated, or the symptoms of a syndrome, disorder or disease being treated (e.g., inflammation).

The present invention relates generally to a composition comprising a purified bacterial mixture and provides applications of the composition. Provided herein is a composition comprising at least one purified Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ

ID NO:3, preferably a purified bacterial mixture of the Enterobacteriaceae bacterial strains, which does not exist naturally in nature. The composition comprises one, two, or three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, and/or SEQ ID NO:3. The identity to the 16s rDNA sequences can be at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and any percentage in between, preferably at least 95% , and more preferably 100%.

In some embodiments, the invention provides a composition comprising three purified Enterobacteriaceae bacterial strains at a desirable ratio. In certain embodiments, the ratio of the number of the three strains in the composition can be any of 1 to 5 portions of the first bacterial strain, to any of 1 to 5 portions of the second bacterial strain and any of 1 to 5 portions of the third bacterial strain. In further embodiments, the ratio of the number of the three strains in the composition is 1 :1:1. The composition can also comprise the three strains at other desirable ratios, such as 1:1:1.5, 1 :1:2, 1:1:2.5, 1:1 :3, 1:1:3.5, 1:1:4, 1:1:4.5, 1:1:5, 1:1.5:1, 1:2:1, 1:2.5:1, 1:3:1, 1:3.5:1, 1:4:1, 1:4.5: 1, 1:5:1,

1:1.5:1.5, 1:2:2, 1:3:3, 1:3.5:3.5, 1:4:4, 1:45:4.5, 1:5:5, etc., or any ratio in between.

In some embodiments, the composition comprises one Enterobacteriaceae bacterium strain which is Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, or Escherichia coli str4. In some embodiments, the composition comprises two

Enterobacteriaceae bacterial strains which are any two of Klebsiella pneumoniae str3,

Enterobacter ludwigii strl2, and Escherichia coli str4. In some embodiments, the three Enterobacteriaceae bacterial strains in the composition are Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4.

In some embodiments, the composition comprises one Enterobacteriaceae bacterium strain which comprises a 16S rDNA sequence of SEQ ID NO: 1 , SEQ ID

NO:2, or SEQ ID NO:3. In some embodiments, the composition comprises two

Enterobacteriaceae bacterial strains and each of them comprises a 16S rDNA sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the three Enterobacteriaceae bacterial strains in the composition comprise 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

Pharmaceutical preparations can be formulated from the compositions described by drug formulation methods known to those of skill in the art. For example, the composition can be used orally or parenterally 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.

For formulating these preparations, the compositions can be used in appropriate combination with carriers that are pharmacologically acceptable or acceptable for ingestion, including one or more of the following: sterile water, physiological saline, vegetable oil, solvent, a base material, an emulsifier, a suspending agent, a surfactant, a stabilizer, a flavoring agent, an aromatic, an excipient, a vehicle, a preservative, a binder, a diluent, a tonicity adjusting agent, a soothing agent, a bulking agent, a disintegrating agent, a buffer agent, a coating agent, a lubricant, a colorant, a sweetener, a thickening agent, a flavor corrigent, a solubilizer, and other additives.

The composition of the present invention can be used to induce an inflammation in a subject. Preferably, the composition can be used to induce an intestinal inflammation in a mammalian subject. More preferably, the mammalian subject is susceptible to inflammation, for example a I110^-1- mouse. Provided herein is a method of inducing an intestinal inflammation in a mammalian subject, comprising administering to the subject an effective amount of a composition comprising a purified bacterial mixture comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3.

In certain embodiments, the identity to the 16s rDNA sequences can be at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and any percentage in between, preferably at least 95% , and more preferably 100%.

In certain embodiments, the ratio of the three strains in the composition is 1 :1:1.

The composition can be administered to the subject by any methods known in the art In certain embodiments, the composition is administered to enables efficient delivery to the colon, in order to induce intestinal inflammation. A variety of pharmaceutical preparations that enable the delivery to the colon can be used. Examples thereof include pH sensitive compositions, delayed release dosage units, and bioadhesive compositions. Preferably, the composition is administered by oral or anal swab, or by gavage.

The amount of the composition to be administered can be determined empirically, taking into consideration such factors as the age, body weight, gender, symptoms, health conditions of the subject For example, the amount per administration is generally 0.01 mg/kg body weight to 100 mg/kg body weight, and, in specific embodiments, 1 mg/kg body weight to 10 mg/kg body weight

The composition may be administered to a subject once, or it may be administered more than once.

In some embodiments, the induced inflammation can be measured by any activities known in the art. Examples of these activities include, but are not limited to, weight loss, rectal bleeding, shortening of the color, gross appearance of the colon, edema, hemorrhage, stool scores and histological features. 'The stool score is defined as:

0: normal, 1: loose stool, 2: loose/some diarrhea, 3: diarrhea, 4: severe watery diarrhea. The histological features include, but are not limited to histological scores of ileum-end, cecum, proximal-colon, distal-colon, rectum and total (cecum + proximal colon + distal colon ).

In certain embodiments, the induced intestinal inflammation can be characterized by colonic lamina propria (LP) immune cell activation, fecal lipocalin-2 (LCN)

concentration, fecal calprotectin concentration, fecal lactoferrin concentration, histology score, pro-inflammatory cytokine measurements, and stool consistency score. In certain embodiments, the colonic lamina propria (LP) immune cell is selected from the group consisting of EFNy⁺CD4⁺, IL-17⁺CD4⁺ and Foxp3⁺CD4⁺ cells. In certain embodiments, the inflammatory cytokine is selected from the group consisting of interleukin 12 p40 (IL-12p40), tumor necrosis factor (TNF), interferon g (IFNg), and interleukin 17a (IL-

17a).

In certain embodiments, the subject is a rodent, preferably a germ-free I110^-1- or a germ-free wild type (WT) mouse, more preferably a germ-free I110^-1- mouse.

Provided herein is a mammalian animal model, comprising a mammalian animal produced by the above methods to induce inflammation in the animal comprising administering the «imposition of the present invention.

In certain embodiments, the animal model is a mouse model, preferably a germ- free I110^-1- or a germ-free wild type (WT) mouse model, more preferably a germ-free I110^-1- mouse model.

Enterobacteriaceae bacterial strains which are any two of Klebsiella pneumoniae str3, Enterobacter ludwigii strl 2, and Escherichia coli str4. In some embodiments, the three Enterobacteriaceae bacterial strains in the composition are Klebsiella pneumoniae str3, Enterobacter ludwigii strl 2, and Escherichia coli str4.

In some embodiments, the composition comprises one Enterobacteriaceae bacterium strain which comprises a 16S rDNA sequence of SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the composition comprises two

Enterobacteriaceae bacterial strains and each of them comprises a 16S rDNA sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the three

Enterobacteriaceae bacterial strains in the composition comprise 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

Provided herein are also methods of identifying an anti-inflammation agent using the animal mode described above. In one aspect, the application provides a method of identifying an agent useful for treating an inflammation, the method comprising:

a. administering to a mammalian subject a composition comprising a

purified bacterial mixture comprising one, two, or three

Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90%, such as at least 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO: 3 to induce the intestinal inflammation in the subject, wherein the bacterial strains are present in the composition at a ratio of 1:1:1, or other desirable ratios, such as 1 :1:1.5, 1 : 1 :2, 1:1:2.5, 1:1:3, 1:1 :3.5, 1:1 :4, 1:1 :4.5, 1:1 :5, 1 :1.5:1, 1 :2:1, 1:2.5: 1, 1:3:1 , 1 :3.5:1 , 1:4: 1, 1 :4 5:1, 1 :5:1, 1 :1.5:1.5, 1:2:2, 1 :3:3, 1:3.5:3.5, 1 :4:4, 1:4.5:4.5, 1:5:5, etc., or any ratio in between;

b. administering to the subject the agent; and

According to the present invention, the method can be used for the purpose of high throughput screening of anti-inflammation agents. In certain embodiments, the identity to the 16s rDNA sequences can be at least 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and any percentage in between, preferably at least 95% , more preferably 100%.

In certain embodiment, the subject is a rodent, preferably a germ-free I110^-1- or a germ-free wild type (WT) mouse, more preferably a germ-free III mouse.

In certain embodiment, the inflammation is inflammation of the small intestine or inflammation of the large intestine. In certain embodiment, the inflammation is an intestinal inflammation such inflammatory bowel disease (IBD), including ulcerative colitis, Crohn's disease, pouchitis, and a celiac disease.

In certain embodiments, the composition can be administered to the subject by any methods known in the art In certain embodiments, the composition is administered to enables efficient delivery to the colon, in order to induce intestinal inflammation. A variety of pharmaceutical preparations that enable the delivery to the colon can be used. Examples thereof include pH sensitive compositions, delayed release dosage units, and bioadhesive compositions. Preferably, the composition is administered by oral or anal swab, or by gavage.

The amount of the composition to be administered can be determined empirically, taking into consideration such factors as the age, body weight, gender, symptoms, health conditions of the subject For example, the amount per administration is generally 0.01 mg/kg body weight to 100 mg/kg body weight, and, in specific embodiments, 1 mg/kg body weight to 10 mg/kg body weight.

The composition may be administered to a subject once, or it may be administered more titan once.

In some embodiments, the agent is an anti-inflammatory agent, preferably a microbiome composition.

In some embodiments, the efficacy of the agent can be measured by any activities known in the art associated with the inflammation, especially intestinal inflammation. Examples of these activities include, but are not limited to, weight loss, rectal bleeding, shortening of the color, gross appearance of the colon, edema, hemorrhage, stool scores and histological features. The stool score is defined as: 0: normal, 1: loose stool, 2:

loose/some diarrhea, 3: diarrhea, 4: severe watery diarrhea. The histological features include, but are not limited to histological scores of ileum-end, cecum, proximal-colon, distal-colon, rectum and total (cecum + proximal colon + distal colon ).

In certain embodiments, die efficacy of the induced intestinal inflammation can be characterized by colonic lamina propria (LP) immune cell activation, fecal lipocalin-2 (LCN) concentration, fecal calprotectin concentration, fecal lactoferrin concentration, histology score, stool consistency score, and pro-inflammatory cytokine measurements, wherein the pro-inflammatory cytokine is selected from the group consisting of IL- 12p40, TNF, IRNg, and IL-17a. In certain embodiments, the colonic lamina propria (LP) immune cell is selected from the group consisting of IFNY⁺CD4⁺, IL-17⁺CD4⁺ and Foxp3⁺CD4⁺ cells.

Enterobacteriaceae bacterial strains which are any two of Klebsiella pneumoniae. str3, Enterobaeter ludwigii strl2, and Escherichia coli str4. In some embodiments, the three Enterobacteriaceae bacterial strains in the composition are Klebsiella pneumoniae str3, Enterobaeter ludwigii strl2, and Escherichia coli str4.

In some embodiments, the composition comprises one Enterobacteriaceae bacterium strain which comprises a 16S rDN A sequence of SEQ ID NO: 1 , SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the composition comprises two

Enterobacteriaceae bacterial strains and each of them comprises a 16S rDNA sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the three Enterobacteriaceae bacterial strains in the composition comprise 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3

Provided herein are also biomarkers which are indicative of the status of an intestinal inflammation, and predictive of an individual's responsiveness to therapeutic agents and treatment regimens for the intestinal inflammation. According to the present invention, the biomarkers are a panel of bacterial strains comprising one, two, or three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3. In certain embodiments, the identity to the 16s rDNA sequences can be 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and any percentage in between, preferably at least 95% identity, and more preferably 100%.

The biomarkers described herein can be used to predict the responsiveness to therapeutic agents for the intestinal inflammation. The panel of biomarkers can also be used for other purposes, such as for determining a treatment regimen for a subject diagnosed with intestinal inflammation, facilitating the understanding of intestinal inflammation pathogenesis prior to disease diagnosis, monitoring the responsiveness to a treatment regimen in the subject, and stratifying patients in clinical trials.

In one aspect, the invention relates to a method of treating a subject having an intestinal inflammation, comprising, the method comprising:

a. determining the presence of a panel of bacterial strains comprising one, two, or three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 90% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3 in a biological sample, preferably a fecal sample, of the subject; and

In some embodiments, the induced inflammation can be measured by any activities known in the art. Examples of these activities include, but are not limited to, weight loss, rectal bleeding, shortening of the color, gross appearance of the colon, edema, hemorrhage, stool scores and histological features. The stool score is defined as: 0: normal, 1: loose stool, 2: loose/some diarrhea, 3: diarrhea, 4: severe watery diarrhea.

The histological features include, but are not limited to histological scores of ileum-end, cecum, proximal-colon, distal-colon, rectum and total (cecum + proximal colon t distal colon ).

In certain embodiments, the induced intestinal inflammation can be characterized by colonic lamina propria (LP) immune cell activation, fecal lipocalin-2 (LCN) concentration, fecal calprotectin concentration, fecal lactoferrin concentration, histology score, stool consistency score, and pro-inflaminatory cytokine measurements, wherein the pro-inflammatory cytokine is selected from the group consisting of IL-12p40, INF,

IFNg, and IL-17a. In certain embodiments, the colonic lamina propria (LP) immune cell is selected from the group consisting of IFNy⁺CD4⁺, EL-17⁺CD4⁺ and Foxp3⁺CD4⁺ cells.

In certain embodiment, the inflammation is an intestinal inflammation such inflammatory bowel disease (IBD), including ulcerative colitis, Crohn's disease, and a celiac disease.

In some embodiments, the therapeutic agent is an anti-inflammatory agent, preferably a microbiome composition.

In some embodiments, the therapeutic agent is a microbial metabolite. In some embodiments, the microbial metabolite is an indole. In some embodiments, the microbial metabolite is selected from the group consisting of indole-3-propionic acid (IPA), indole, and indole-3-acetic acid (IAA). In a specific embodiment, is indole-3-propionic acid

(1PA).

In some embodiments, the panel comprises one Enterobacteriaceae bacterium strain which is Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, or Escherichia coli str4. In some embodiments, the panel comprises two Enterobacteriaceae bacterial strains which are any two of Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4. In some embodiments, the three Enterobacteriaceae bacterial strains in the panel are Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4.

In some embodiments, the panel comprises one Enterobacteriaceae bacterium strain which comprises a 16S rDNA sequence of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the panel comprises two Enterobacteriaceae bacterial strains and each of them comprises a 16S rDNA sequence of SEQ ID NO: 1, SEQ ID

NO:2, or SEQ ID NO:3. In some embodiments, the three Enterobacteriaceae bacterial strains in the panel comprise 16S rDNA sequences of SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3. EXAMPLES

Example 1: Isolation and Identification of Enterobacteriaceae from Human Stool

Procedure for isolation of Enterobacteriaceae from human stool

Stool material representing healthy human stool (purchased from BIOVT, Lot BRH 1042896) was provided as a frozen sample of about 200 mg in a cryogenic vial without preservatives.

The stool was allowed to thaw to room temperature, and 0.5 mL of sterile saline was added. The contents were homogenized by vortexing. The stool suspension was then serially diluted to 1 x 10^-5 in sterile saline. 0.1 mL of each dilution was spiral plated to MacConkey P agar (BD BBL). The agar plates were then incubated for 24 hours at 37°C in ambient air without humidity. 15 isolated colonies representing a variety of morphologies were picked and streaked to individual TS A + 5% sheep blood agar plates (BD BBL) for pure cultures. These plates were incubated for 24 hours at 37°C in ambient air without humidity. Glycerol stocks of each isolate were made, and kept frozen at -80°C.

16S rDNA analysis

One isolated colony from each of the 15 Enterobacteriaceae isolates was emulsified in 0.1 mL of TE buffer, and boiled for 10 minutes at 100°C. The lysates were centrifuged for 1 minute at 13.2 krpm to remove debris, and 1 mL was used as DNA template. PCR reactions were set up according to manufacturer’s instructions (please provide the manufacture of PCR).

PCR Primers to amplify full-length 16S:

a. 27F: 5’ -AGAGTTTGATCMTGGCTCAG-3’

b. 1492R: 5’ -GGTTACCTTGTTACGACTT-3’

PCR Primers to amplify V3-V4 region of 16S:

c. 319F: 5’ - ACTCCT ACGGGAGGC AGC AG-3’ d. 806R 5’ -GGACTACHVGGGTWTCTAAT-3’

Results

7 Klebsiella pneumoniae (K) strains, 4 Enterobacter ludwigii (E) strains, 4 Escherichia coli (E) strains, and 4 Bacteroidetes strains are isolated and classified from a healthy human fecal sample and classified by 16S rRNA gene sequence. <210> SEQ ID N0:1

<211> 1380

<212> DNA

<213> Klebsiella pneumoniae str3

<400> 1

ctcagattga acgctggcgg caggcctaac acatgcaagt cgagcggtag cacagagagc 60 ttgctctcgg gtgacgagcg gcggacgggt gagtaatgtc tgggaaactg cctgatggag 120 ggggataact actggaaacg gtagctaata ccgcataatg tcgcaagacc aaagtggggg 180 accttcgggc ctcatgccat cagatgtgcc cagatgggat tagctagtag gtggggtaac 240 ggctcaccta ggcgacgatc cctagctggt ctgagaggat gaccagccac actggaactg 300 agacacggtc cagactccta cgggaggcag cagtggggaa tattgcacaa tgggcgcaag 360 cctgatgcag ccatgccgcg tgtgtgaaga aggccttcgg gttgtaaagc actttcagcg 420 gggaggaagg cggtgaggtt aataaccttg tcgattgacg ttacccgcag aagaagcacc 480 ggctaactcc gtgccagcag ccgcggtaat acggagggtg caagcgttaa tcggaattac 540 tgggcgtaaa gcgcacgcag gcggtctgtc aagtcggatg tgaaatcccc gggctcaacc 600 tgggaactgc attcgaaact ggcaggctag agtcttgtag aggggggtag aattccaggt 660 gtagcggtga aatgcgtaga gatctggagg aataccggtg gcgaaggcgg ccccctggac 720 aaagactgac gctcaggtgc gaaagcgtgg ggagcaaaca ggattagata ccctggtagt 780 ccacgccgta aacgatgtcg atttggaggt tgtgcccttg aggcgtggct tccggagcta 840 acgcgttaaa tcgaccgcct ggggagtacg gccgcaaggt taaaactcaa atgaattgac 900 gggggcccgc acaagcggtg gagcatgtgg tttaattcga tgcaacgcga agaaccttac 960 ctggtcttga catccacaga actttccaga gatggattgg tgccttcggg aactgtgaga 1020 caggtgctgc atggctgtcg tcagctcgtg ttgtgaaatg ttgggttaag tcccgcaacg 1080 agcgcaaccc ttatcctttg ttgccagcgg ttaggccggg aactcaaagg agactgccag 1140 tgataaactg gaggaaggtg gggatgacgt caagtcatca tggcccttac gaccagggct 1200 acacacgtgc tacaatggca tatacaaaga gaagcgacct cgcgagagca agcggacctc 1260 ataaagtatg tcgtagtccg gattggagtc tgcaactcga ctccatgaag tcggaatcgc 1320 tagtaatcgt agatcagaat gctacggtga atacgttccc gggccttgta cacaccgccc 1380

<210> SEQ ID N0:2

<211> 1178

<212> DNA

<213> Enterobacter ludwigii strl2

<400> 2

tcagattgaa cgctggcggc aggcctaaca catgcaagtc gaacggtagc acagagagct 60 tgctctcggg tgacgagtgg cggacgggtg agtaatgtct gggaaactgc ctgatggagg 120 gggataacta ctggaaacgg tagctaatac cgcataacgt cgcaagacca aagaggggga 180 ccttcgggcc tcttgccatc agatgtgccc agatgggatt agctagtagg tggggtaacg 240 gctcacctag gcgacgatcc ctagctggtc tgagaggatg accagccaca ctggaactga 300 gacacggtcc agactcctac gggaggcagc agtggggaat attgcacaat gggcgcaagc 360 ctgatgcagc catgccgcgt gtatgaagaa ggccttcggg ttgtaaagta ctttcagcgg 420 ggaggaaggt gttgtggtta ataaccacag caattgacgt tacccgcaga agaagcaccg 480 gctaactccg tgccagcagc cgcggtaata cggagggtgc aagcgttaat cggaattact 540 gggcgtaaag cgcacgcagg cggtctgtca agtcggatgt gaaatccccg ggctcaacct 600 gggaactgca ttcgaaactg gcaggctaga gtcttgtaga ggggggtaga attccaggtg 660 tagcggtgaa atgcgtagag atctggagga ataccggtgg cgaaggcggc cccctggaca 720 aagactgacg ctcaggtgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc 780 cacgccgtaa acgatgtcga cttggaggtt gtgcccttga ggcgtggctt ccggagctaa 840 cgcgttaagt cgaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg 900 ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc 960 tactcttgac atccagagaa cttagcagag atgctttggt gccttcggga actctgagac 1020 aggtgctgca tggctgtcgt cagctcgtgt tgtgaaatgt tgggttaagt cccgcaacga 1080 gcgcaaccct tatcctttgt tgccagcggt ccggccggga actcaaagga gactgccagt 1140 gataaactgg aggaaggtgg ggatgacgtc aagtcatc 1178

<210> SEQ ID NO:3

<211> 1257

<212> DNA

<213> Escherichia coli str4

<400> 3

tcagattgaa cgctggcggc aggcctaaca catgcaagtc gaacggtaac aggaagcagc 60 ttgctgcttt gctgacgagt ggcggacggg tgagtaatgt ctgggaaact gcctgatgga 120 gggggataac tactggaaac ggtagctaat accgcataac gtcgcaagac caaagagggg 180 gaccttaggg cctcttgcca tcggatgtgc ccagatggga ttagctagta ggtggggtaa 240 cggctcacct aggcgacgat ccctagctgg tctgagagga tgaccagcca cactggaact 300 gagacacggt ccagactcct acgggaggca gcagtgggga atattgcaca atgggcgcaa 360 gcctgatgca gccatgccgc gtgtatgaag aaggccttcg ggttgtaaag tactttcagc 420 ggggaggaag ggagtaaagt taataccttt gctcattgac gttacccgca gaagaagcac 480 cggctaactc cgtgccagca gccgcggtaa tacggagggt gcaagcgtta atcggaatta 540 ctgggcgtaa agcgcacgca ggcggtttgt taagtcagat gtgaaatccc cgggctcaac 600 ctgggaactg catctgatac tggcaagctt gagtctcgta gaggggggta gaattccagg 660 tgtagcggtg aaatgcgtag agatctggag gaataccggt ggcgaaggcg gccccctgga 720 cgaagactga cgctcaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag 780 tccacgccgt aaacgatgtc gacttggagg ttgtgccctt gaggcgtggc ttccggagct 840 aacgcgttaa gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga 900 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta 960 cctggtcttg acatccacgg aagttttcag agatgagaat gtgccttcgg gaaccgtgag 1020 acaggtgctg catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac 1080 gagcgcaacc cttatccttt gttgccagcg gtccggccgg gaactcaaag gagactgcca 1140 gtgataaact ggaggaaggt ggggatgacg tcaagtcatc atggccctta cgaccagggc 1200 tacacacgtg ctacaatggc gcatacaaag agaagcgacc tcgcgagagc aagcgga 1257 Example 2: Determination of Relative Inflammatory Potential of Human-Derived

Bacterial

To determine the relative inflammatory potential of the bacterial strains obtained from Example 1, germ-free (GF) I110^-1- mice were colonized for 2 weeks with each strain, all 15 Enterobacteriaceae combined strains, donor stool or media alone (GF negative control). The 15 Enterobacteriaceae strains and 4 Bacteroides strains were cultured individually in BHI broth overnight at 37°C. The inocula were diluted with BHI broth into OD600=1. For 15 mix of Enterobacteriaceae, the same dose (based on ODeoo) of each strain was mixed and the ODeoo of the mixture was adjusted into OD600=1. For human donor inocula, the frozen stool was thawed, diluted with oxygen-reduced PBS in anaerobic chamber, and adjusted into OD600=1. These inocula were administrated to GF I110^-1- by oral/anal swab (all the mice drunk three drop of inocula which is about 100 pL). The left-over inocula were thrown into housing cages. See FIG. 1 for the

experimental design.

The colonic lamina propria (LP) immune cell activation (Thl, Thl7, Foxp3) by flow cytometry and colonic inflammation by blinded histology scoring and fecal lipocalin-2 (LCN) concentration were accessed.

Results

Different strains within the same species variably induced colonic EFNy⁺, IL-17¹ and Foxp3⁺CD4⁺ cells (FIGs.2A-B) and different histology scores (FIG. 2C). K, E, E strains activated IFNy⁺ cells, while Bacteroides strains stimulated Tregs but not Thl cells. % IFNY⁺CD4⁺ cells correlated with histology score (R2=0.57, p>.OOOl) and fecal lipocalin (R2=0.23, p=0.001) and negatively correlated with % Foxp3⁺CD4 (R2=0.37, p<0.001l).

Example 3: KEE Strains Induced Intestinal Inflammation

Based on Example 2 results, Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4 (KEE) were chosen from each Enterobacteriaceae genus

(K,E,E) with the greatest ability to induce colitis and activate colonic Thl cells. GF I110^-1- and wild type (WT) mice were colonized with the 3 pooled selected KEE strains. The KEE strains ( Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4) were cultured individually in BHI broth overnight at 37°C. Based on OD, the KEE strains were mixed with the ratio 1:1:1. The OD of inocula was adjusted to OD=l by diluting with BHI. The inocula were administrated to GF I110^-1- or WT by oral/anal swab (all the mice drunk three drop of inocula which is about 100 pL). The left-over inocula were placed into housing cages. After 6 weeks, the colitis activity by colonic LP immune cell activation, colitis, LCN and stool consistency were measured. See FIG. 3 for the experimental design.

Results These 3 pooled strains (KEE), Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4, induced more colonic IFNy⁺CD4⁺ cells in GF IUG^4' mice than in GF mice or KEE-colonized WT mice. KEE caused moderate-to-severe colitis and diarrhea but no colitis and normal stools in KEE-colonized WT mice (FIGs. 4A-C). Example 4: Comparison of Intestinal Inflammation between KEE Strains and Human Donor Stool

The intestinal inflammation between selected strains 4wk-colonized and donor fecal-inoculated ex-GF I110^-1- mice were compared. For human donor inocula, the frozen stool was thawed, diluted with oxygen-reduced PBS in anaerobic chamber, and adjusted into OD=l.The KEE strains ( Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and

Escherichia coli str4) were cultured individually in BHI broth overnight at 37°C. Based on OD, the KEE strains were mixed with the ratio 1:1:1. The OD of inocula was adjusted to OD=l by diluting with BHI. The inocula were administrated to GF I110^-1- or WT by oral/anal swab (all the mice drunk three drop of inocula which is about 100 mL). The left- over inocula were thrown into housing cages. See FIG. 5 for the experimental design. Results

KEE strains induced more severe intestinal inflammation than the donor stool as shown in FIG.6.

Conclusion: An individual harbors multiple E. coli, K. pneumoniae and Enterobacter strains with variable immunomodulatory and colitogenic activities. A novel

Enterobacteriaceae colitis model was created with the most active KEE strains.

Therefore, Analyzing KEE strain-specific function in addition to describing community structure is important to understanding LBD pathogenesis.

Example 5: KEE- I110^-1- treatment experiment

The aim of this example is to determine whether VE202 microbial consortium can treat Enterobacteriaceae-induced chronic colitis. The protocol of this treatment is demonstrated in FIG. 7.

Materials and Methods

Mice·. Nineteen GF 129S6/SvEv I110^-1- and WT control mice (7-12 weeks age) were obtained from University of North Carolina (UNC) National Gnotobiotic Rodent Resource Center and distributed to 2 groups (2 isolators), vehicle group ( I110^-1=- 10 mice, WT = 5 mice, isolator #244) and VE202 treatment group I110^-1- 9 mice, WT = 6 mice, isolator#272).

Initiation of colitis: Base on the results of Example 2, the following strain from each group with the greatest ability to induce colitis and activate Thl cells were chosen - Klebsiella pneumoniae str3, Enterobacter ludwigii str12, and Escherichia coli str4 (KEE). The KEE strains (Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4) were cultured individually in BHI broth overnight at 37°C. Based on OD, the KEE strains were mixed with the ratio 1:1:1. The OD of inocula was adjusted to OD=l by diluting with BHI. The inocula were administrated to GF I110^-1- or WT by oral/anal swab (all the mice drunk three drop of inocula which is about 100 pL) at day 0 and day 3.

Fecal collection: Fresh feces (2-5 pieces/mouse) were collected (week 0, 2, 3, 4, 5, and 6), immediately snap-frozen on dry ice and stored at -80°C. Week-0 feces were collected right before KEE inoculation.

VE202 strains preparation and gavage: VE202 strains were individually cultured for 20 hours and mixed with the same amount on the basis of the absorbance of 600 nm. The preparation of the bacteria mixture was performed inside the anaerobic chamber. Two weeks after KEE inoculation, each mouse was given either EG media as control (Vehicle group), or VE202 in cultured EG media (2 x 10⁷ CFU in 200 pL) by gavage twice a week for 4 weeks.

Stool score: Fresh stools (2-3 pieces/mouse) were evaluated by the well- established stool scoring system (Fitzpatrick LR, Gut Pathog., 2011, 3:16) at necropsy (week 6). Score 0: normal, 1: loose stool, 2: loose/some diarrhea, 3: diarrhea, 4: severe watery diarrhea.

Fecal bacteria lysate preparation: The bacteria lysate was prepared from the feces from each group. The feces were disrupted with 0.1 -mm glass microbeads by using a Mini Bead Beater (2 cycles of 45 sec, 6,500 speed) and were centrifuged at 10,000 r.p.m. for 15 min. The resulting supernatants were filtrated through 0.45-pm filter. The sterility of the bacteria lysate was confirmed by aerobic/anaerobic culture. Protein concentrations were measured according to the manufacturer’s instructions (Bio-Rad Laboratories). Necropsy: Cecal contents were harvested and immediately snap-frozen on dry ice and stored at -80°C. Tissues were harvested, incubated in RNAlater (QIAGEN) overnight and stored at -20°C. Cecal and colon tissues were harvested and put in cold PBS for the sequential lamina propria cells isolation and tissue fragment culture.

Tissue fragment culture (Gut culture ) : Colonic tissue was thoroughly irrigated with 1 xPBS, shaken at room temperature in RPMI containing SO pg/mL gentamicin for 30 min at 250 r.p.m., cut into 1cm fragments, dried up with paper, and weighed. Colonic tissue fragments were distributed (50 mg/well) into 24-well plates (Costar) and incubated in lmL of RPMI 1640 medium supplemented with 5% FBS, 50 pg/mL gentamicin and 1% antibiotic/antimycotic (penicillin/streptomycin/amphotericin B, GIBCO) for 20 hours at 37°C in CO2 incubator. Supernatants were collected and stored at -20°C before use for cytokine quantification. The data were normalized to 50 mg of tissue weight.

Cytokine measurements by ELISA : For quantification of fecal lipocalin-2 by ELISA, fecal samples (5-20 mg) were incubated overnight in PBS with 0.1% Tween 20 (Fisher Scientific) and vortexed shortly to get a homogenous fecal suspension. The fecal suspensions were centrifuged for 10 min at 12,000 r.p.m. at 4°C. Clear supernatants were collected and stored at -20 °C. ELIS As were performed according to the manufacturer’s protocols (Lcn-2: R&D Systems). Supernatants from the cell/tissue culture were analyzed by ELIS As according to the manufacturer’s protocols (IL-12p40: R&D Systems).

Histology and scoring: The intestine and liver were fixed in 10% neutral-buffered formalin. Paraffin-embedded sections (5 pm) were stained with hematoxylin and eosin by the Histology Core of the CGIBD at UNC-CH. Ileal, cecal, proximal-, distal-colon, and rectum were quantitated in a blinded fashion by well validated histological scoring system (Liu B, Gastroenterology, 2011, 141:653) (Rath HC, JCI, 1996, 98:945).

Lamina propria cell isolation: Colonic tissues were opened longitudinally, washed twice with 1 xPBS, cut into 1 cm pieces and incubated with stirrer for 250 r.p.m. in HBSS (Coming) medium containing 2.5% FBS (Sigma- Aldrich), 1% penicillin- streptomycin (Gibco), 5 mM EDTA (Coming) and 10 mM dithiothreitol (Sigma) for 20 min at 37°C to remove the epithelial layer. Denuded tissue samples were washed twice with HBSS containing 2.5% FBS and 1% penicillin-streptomycin and incubated with stirrer for 450 r.p.m. in HBSS containing 2.5% FBS, 0.5 mg/mL of collagenase (Sigma) for 20 min at 37°C. Cell preparations were filtrated through 100- mm nylon mesh to achieve single-cell suspensions. Lamina propria cells were purified using a 40-70% discontinuous Percoll gradient (GE Healthcare, 2,000 r.p.m., 20 min, room temperature) and washed with HBSS.

Staining cells for flow cytometry analysis: Single cells were stained for 20 min at 4°C after FcyRII/III blocking with anti-CD 16/CD32 monoclonal antibody. For intracellular staining, cells were re-stimulated with SO ng/mL phorbol 12-myristate 13- acetate (PMA, Sigma) and 500 ng/mL ionomycin (Sigma) for 4 hours at room temperature with 1 ml/mL protein transport inhibitor (GolgiStop, BD) during the last 3 hours. After washing, cells were first surface stained, then fixed for 5 min at 37°C using PBS containing 4% paraformaldehyde (Electron Microscopy Sciences) and 0.01% Tween20 (Fisher Scientific), permed using PBS containing 0.1% Triton X-100 (MP Biomedicals), 0.5% BSA (Sigma), 2 mM EDTA (Coming) for 45 min at room temperature, and stained overnight with indicated antibodies. The data from all samples were acquired on LSRII (BD Biosciences) and analyzed using FlowJo software

(FLOWJO, LLC).

Statistical Analysis: Data are presented ± SEM or SD. Significance was determined using 2-tailed, impaired Student t tests for 2 groups; one-way ANOVA corrected for multiple comparisons with Tukey and Dunnett’s posttests; two-way ANOVA for multiple comparisons with a Bonferroni test The statistical analysis was performed using Prism version 7.0 (GraphPad Software). P values less than .05 were considered significant

Results

VE202 treatment significantly reduced stool score as shown in FIG. 8 for Week 6 stools (at necropsy) scores at autopsy (Fitzpatrick LR, Gut. Pathog., 2011, 3:16).

Representative stool pictures at week 6 (autopsy) are shown in FIG. 9.

VE202 improved gloss appearance of colon. Representative pictures of colon showing that vehicle group have thick wall and shorten colon compared to VE202 treatment group (FIG. 10).

VE202 treatment significantly decreased fecal lipocalin-2 levels as shown in FIGs. 11 A and 11B. VE202 treatment significantly reduced pro-inflammatory cytokine IL-12p40 level in supernatant of tissue fragment culture as shown in FIG. 12.

VE202 treatment significantly attenuate colitis score. FIGs 13A-F demonstrate histology scores of ileum-end, cecum, proximal-, distal-colon, rectum and total (cecum + proximal colon + distal colon). FIG. 14 demonstrates representative histology pictures of cecum and distal colon. FIG.15 shows % colonic LP Foxp3⁺CD4⁺, IFNY⁺CD4⁺, and IL- 17⁺CD4⁺ T cells. FIG. 16 shows correlation between fecal lipocalin-2 (week 6) and total histology score. Example 6: Effect of Indole-3-propionic acid (IPA) on the growth of KEE strains.

Next, the effect of a bacterial metabolite indole-3-propionic acid (IPA) on the growth of the KEE strains was evaluated.

Materials and Methods.

Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4 were cultured anaerobically in Eggerth-Gagnon media (EG media, Atarashi et al, Nature,

2013, 500:232) at 37°C overnight. 10⁷ CFU of bacterial cells were inoculated in 3 mL of EG media with indole-3-propionic acid (20, 100, 250, 500 mM) (Acros Organics, Fair Lawn, NJ). Ethanol was used as a vehicle; final concentration in the medium was 0.1%- 2.5%. The final pH was adjusted to 7.20-7.22 with NaOH. The bacteria were cultured anaerobically at 37°C for 15 hours and the optical density at 600 nm (OD) was measured.

Results

FIG. 17 shows the OD as a percentage of initial optical density. Indole-3 - propionic acid (IPA) inhibited the growth of Klebsiella pneumoniae str3 (denoted as “Kp”, see FIG.17), Enterobacter ludwigii strl2 (denoted as“El”, see FIG.17), and Escherichia coli str4 (denoted as“Ec”, see FIG.17) in a dose dependent manner (FIG.

17).

Example 7: Effect of Indole-3-propionic acid (IPA) on the growth of additional bacterial strains.

Next, the effect of Indole-3-propionic acid (IP A) on the growth of the Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, Escherichia coli str4, as well as Enterococcus faecalis 0G1RF, Fusobacteria varium 113 (PMID: 12477765), and Ruminococcus gnavus ATCC29149, associated with the induction of colitis (DDW2018, Gastroenterology 2018;154:8-1036); Lactobacillus plantarum BAA-793 and VE202 strains was evaluated.

Materials and Methods.

Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, Escherichia coli str4, Enterococcus faecalis OG1RF, Fusobacteria varium 113 (PMID: 12477765), VE202, Ruminococcus gnavus ATCC29149, and Lactobacillus plantarum BAA-793 were cultured anaerobically in EG media at 37°C overnight and 10⁷ CFU of bacterial cells were inoculated in 3 mL of EG media with 500 mM of indole-3-propionic acid. Ethanol was used as vehicle. The final pH was adjusted to 7.20-7.22 with NaOH. The bacterial were cultured anaerobically at 37°C for 15 hours and the optical density at 600 nm (OD) was measured.

Results

FIG. 18 shows the percentage of difference in OD as compared to vehicle.

Representative data are shown from two repeated experiments with triplicated samples. IPA inhibited the growth of KEE strains and other potentially pathogenic bacteria, such as E. faecalis and F. varium (FIG. 18). Example 8: Effect of select metabolites on the growth of bacterial strains.

Next, the effect of a panel of bacterial metabolites, including indole-3-propionic acid (IPA), indole-3 -acetic acid (IAA), indole, serotonin, tryptophan, acetic acid, butyric acid, cholic acid, and histamine on the growth of several bacterial strains was analyzed. The bacterial strains included in this experiment were the KEE strains ( Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4) and additional strains that were shown to be associated with intestinal inflammation, specifically Klebsiella pneumoniae 10273, Enterococcus faecalis OGIRF, Fusobacteria varium 113 (PMID: 12477765), and Ruminococcus gnavus ATCC29149 (DDW2018,

Gastroenterology 2018; 154: S- 1036; CCC2020, Inflammatory Bowel Diseases

2020;26:836-837; Kim SC, Sartor RB, Inflammatory Bowel Diseases 2007, 13, 1457).

Materials and Methods. Klebsiella pneumoniae str3, Klebsiella pneumoniae ATCC10273, Enterobacter ludwigii strl2, Escherichia coli str4, Escherichia coli LF82, Enterococcus faecalis 0G1RF, Fusobacteria varium 113, VE202, and Ruminococcus gnavus ATCC29149 were cultured anaerobically in EG media at 37°C overnight and 10⁷ CFU of bacterial cells were inoculated in 3 mL of EG media with 20 mM of indicated individual metabolites: indole (Sigma, cat# 13408); indole-3-propionic acid (Acros Organics, cat# 204990010); indole-3 -acetic acid (Sigma, cat# 13750); serotonin (Sigma, cat# H9523); tryptophan (Sigma, cat# 193747); acetic acid (Fisher, cat# A38-212); butyric acid (Sigma, cat# B103500); cholic acid (Sigma, cat# Cl 129); histamine (Sigma, cat# 53300). Ethanol was used as vehicle for indole-3-propionic acid, indole-3-acetic acid, and indole; final concentration in the medium was 0.1%. HC1 was used as vehicle for tryptophan; final concentration in the medium was 0.1%. Water was used as vehicle for serotonin, butyric acid, acetic acid, and histamine. Methanol was used as vehicle for cholic acid; final concentration in the medium was 0.1%. The final pH was adjusted to 7.20-7.22 with NaOH. The bacterial were cultured anaerobically at 37°C for 15 hours and the optical density at 600 nm was measured.

Results

FIG.19 shows the OD of bacterial cultures 15 h post incubation, as the percent change of OD of bacterial cultures, calculated as:

(((OD of culture with metabolite) - (OD of culture with vehicle)) / (OD of culture with vehicle)) x 100%

The Indoles group of bacterial metabolites (IPA, IAA, indole) inhibited bacterial growth broadly while other metabolites tryptophan, SCFAs, bile acid, histamine had lower anti-bacterial activity (FIG. 19).

It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

All documents cited herein are incorporated by reference.

Claims

CLAIMS It is claimed:

1. A composition comprising a purified bacterial mixture comprising three

Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3, wherein the bacterial strains are present in the composition at a ratio of 1:1:1.

2. The composition of claim 1, wherein the three Enterobacteriaceae bacterial strains comprise 16S rDNA sequences of SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3.

3. The composition of claim 1, wherein the three Enterobacteriaceae bacterial strains are Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4.

4. A method of inducing an intestinal inflammation in a mammalian subject,

comprising administering to the subject an effective amount of a composition comprising a purified bacterial mixture comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, wherein the bacterial strains are present in the composition at a ratio of 1:1:1.

5. The method of claim 4, wherein the composition is administered to the subject by oral or anal swab, or by gavage.

6. The method of claim 4, wherein the intestinal inflammation is characterized by an index selected from the group consisting of fecal lipocalin-2 (LCN) concentration, histology score, colonic lamina propria (LP) immune cell activation, stool consistency score, and pro-inflammatory cytokine measurements, wherein the pro-inflammatory cytokine is selected from the group consisting of IL-12p40, TNF, IFNg, and IL-17a.

7. The method of claim 6, wherein the colonic lamina propria (LP) immune cell is selected from the group consisting of IFNy⁺CD4⁺, IL-17⁺CD4⁺ and Foxp3⁺CD4⁺ T cell.

8. The method of claim 4, wherein the intestinal inflammation is inflammation of the small intestine or inflammation of the large intestine.

9. The method of claim 4, wherein the subject is a rodent, preferably a germ-free I110^-1- or a wild type (WT) mouse, more preferably a germ-free I110^-1- mouse.

10. A mouse model of an intestinal inflammation, comprising a mouse produced by the method of any one of claims 4-9.

11. The method of any one of claims 4-9, wherein the three Enterobacteriaceae

bacterial strains comprise 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

12. The method of any one of claims 4-9, wherein the three Enterobacteriaceae

bacterial strains are Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and Escherichia coli str4.

13. The mouse model of claim 10, wherein the three Enterobacteriaceae bacterial strains comprise 16S rDNA sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.

14. The mouse model of claim 10, wherein the three Enterobacteriaceae bacterial strains are Klebsiella pneumoniae str3, Enterobacter ludwigii strl2, and

Escherichia coli str4.

15. A method of identifying an agent useful for treating an intestinal inflammation, comprising:

a. administering to a mammalian subject a composition comprising a

purified bacterial mixture comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3 to induce the intestinal inflammation in the subject, wherein the bacterial strains are present in the composition at a ratio of 1 : 1 : 1 ;

b. administering the agent to the subject; and

16. The method of claim 15, wherein the subject is a rodent, preferably a germ-free I110^-1- or a germ-free wild type (WT) mouse, more preferably a germ-free I110^-1- mouse.

17. The method of claim 15 or 16, wherein the subject is the germ-free I110^-1- mouse.

18. The method of any one of claims 15-16, wherein the composition is administered to the subject by oral or anal swab, or by gavage.

19. The method of any one of claims 15-18, wherein the intestinal inflammation is characterized by an index selected from the group consisting of colonic lamina propria (LP) immune cell activation, fecal lipocalin-2 (LCN) concentration, histology score, stool consistency score, and pro-inflammatory cytokine measurements, wherein the pro-inflammatory cytokine is selected from the group consisting of IL-12p40, TNF, IFNy, and IL-17a.

20. A method of treating a subject having an intestinal inflammation, comprising: a. determining the presence of a panel of bacterial strains comprising three Enterobacteriaceae bacterial strains having 16S rDNA sequences of at least 95% identity to SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3 in a biological sample, preferably a fecal sample, of the subject; and b. administering a therapeutic agent to the subject if the panel of bacterial strains is detected in the subject

21. The method of claim 20, wherein the intestinal inflammation is characterized by an index selected from the group consisting of colonic lamina propria (LP) immune cell activation, fecal lipocalin-2 (LCN) concentration, fecal calprotectin concentration, fecal lactoferrin concentration, histology score, stool consistency score, and pro-inflammatory cytokine measurements, wherein the pro- inflammatory cytokine is selected from the group consisting of IL-12p40, TNF, IFNg, and IL-17a.

22. The method of claim 21, wherein the colonic lamina propria (LP) immune cell is selected from the group consisting of IFNY⁺CD4⁺, IL-17⁺CD4⁺ and Foxp3⁺CD4⁺ T cell.

23. The method of any one of claims 20-22, wherein the intestinal inflammation is inflammatory bowel disease (IBD), such as ulcerative colitis or Crohn's disease, or a celiac disease.

24. The method of any one of claims 20-23, wherein the therapeutic agent is an antiinflammatory agent

25. The method of claim 24, wherein the agent is a microbial composition.

26. The method of any one of claims 20-25, wherein three Enterobacteriaceae

27. The method of any one of claims 20-26, wherein three Enterobacteriaceae

28. The method of claim 20, wherein the therapeutic agent is a microbial metabolite.

29. The method of claim 28, wherein the microbial metabolite is an indole.

30. The method of claim 28, wherein the microbial metabolite is selected from the group consisting of indole-3-propionic acid (IP A), indole, and indole-3-acetic acid (IAA).

31. The method of claim 28, wherein the microbial metabolite is indole-3 -propionic acid (IP A).