Methods of treatment of alcohol associated hepatitis

Abstract

Alcohol-associated liver disease, particularly alcohol-associated hepatitis, constitutes a significant global health burden with high mortality rates. Imbalances in the gut microbiota are important for the progression of alcohol-associated liver disease, a condition classified as non-infectious. Disclosed herein are methods of treating or ameliorating a symptom of alcohol-associated hepatitis and methods of reducing or preventing mortality associated with alcohol-associated hepatitis. Also disclosed herein are methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis. Further disclosed are methods of determining responsiveness of a subject to treatment with the small molecule inhibitor C7, methods of identifying a subject for a clinical trial for treatment for alcohol-associated hepatitis and methods of screening for candidate compounds for treating alcohol-associated hepatitis.

Description

37759.0643P1 METHODS OF TREATMENT OF ALCOHOL ASSOCIATED HEPATITIS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant Number BX 004594-01 awarded by the Department of Veteran’s Affairs. The government has certain rights in this invention. BACKGROUND

[0002] Alcohol-associated liver disease is the most prevalent liver disease worldwide and the main cause of liver-related mortality1-4. Alcohol-associated liver disease has recently become the leading cause of liver transplantation in the United States5.

[0003] Patients may develop hepatic inflammation, fibrosis, and liver cirrhosis with an increased risk for hepatocellular carcinoma6,7. Alcohol-associated hepatitis represents a severe form within this spectrum with 90-day mortality rates of 20%-50%8. The global incidence of alcohol-associated hepatitis is on the rise, particularly among young adults and women10. Although early liver transplantation is the only curative therapy, it is available at select centers to a limited group of patients11-13. Therefore, there is an urgent need for new therapeutic targets for this severe and life-threating disease. BRIEF SUMMARY

[0004] Disclosed herein are methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby treating or ameliorating a symptom of alcohol-associated hepatitis in the subject.

[0005] Disclosed herein are methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol-associated hepatitis comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby reducing or preventing mortality in the subject.

[0006] Disclosed herein are methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby treating or ameliorating a symptom of alcohol-associated hepatitis in the subject.

[0007] Disclosed herein are methods of determining responsiveness of a subject to37759.0643P1 treatment with the small molecule inhibitor C7 comprising: determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample from said subject, wherein when the patient has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus, the subject will be responsive to treatment with the small molecule inhibitor C7.

[0008] Disclosed herein are methods of identifying a subject for a clinical study for a treatment for alcohol-associated hepatitis with composition comprising small molecule inhibitor C7, comprising determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample from said subject, wherein when the patient has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus, the subject is appropriate for the clinical study for a treatment for alcohol-associated hepatitis.

[0009] Disclosed herein are methods for screening for a candidate compound for treating alcohol-associated hepatitis comprising: (1) contacting the candidate compound with a sample comprising a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus; and (2) determining that the bacteria is reduced / killed, thereby (3) identifying a compound for treating alcohol-associated hepatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0011] FIGS.1A-1F show increased carriage of virulence gene kpsM in E. coli associates with mortality in patients with alcohol-associated hepatitis. FIG.1A, shows relative taxa abundance by shotgun metagenomic sequencing is shown for each stool sample. FIG.1B shows the presence of 350 different virulence-related core genes in fecal samples from 81 patients with alcohol-associated hepatitis (AH), 41 patients with alcohol use disorder (AUD), and 9 non-alcoholic controls (Ctrl). P values were determined by Kruskal-Wallis test with Dunn’s post-hoc test followed by false discovery rate (FDR) procedures. FIG.1C shows the relative abundance of virulence factors found in fecal samples from 81 patients with alcohol-associated hepatitis, 41 patients with alcohol use disorder, and 9 non-alcoholic controls; factors are color-coded by genus of bacteria that produce them. FIG.1D shows the Kaplan-Meier curve of survival of patients with alcohol-associated hepatitis whose fecal37759.0643P1 samples were virulence factor (VF)-positive (n=55) or VF-negative (n=26). P=.008. For the only patient who underwent liver transplantation, the transplantation date was considered as date of death. Patients were censored at the time point they were last seen alive. P values were determined by two-sided Log-rank (Mantel-Cox) test. FIG.1E shows Multivariate Cox regression adjusted for treatment with antibiotics and steroids. Only significant virulence factors are shown. Squares identify hazard ratios, with lower and upper 95% confidence intervals (CI) values shown as lines. Colors indicate the genus of bacteria that produces each specific virulence factor. P values were determined by two-side Wald test. FIG.1F shows the Kaplan-Meier survival curve for alcohol-associated hepatitis patients in the validation cohort. The patients were grouped into kpsM-positive (n=20) and kpsM-negative (n=29). P values were determined by two-sided Log-rank (Mantel-Cox) test.

[0012] FIGS.2A-2H show fecal transplantation from kpsM-positive AH patients exacerbates ethanol-induced liver injury in gnotobiotic mice. FIG.2A shows serum levels of ALT. FIG.2B shows hepatic triglyceride content. FIG.2C shows representative images of liver sections stained with hematoxylin and eosin. FIG.2D shows representative liver sections stained with Oil Red O. FIG.2E, FIG.2F, and FIG.2G show mRNA expression of inflammatory genes (Il1b, Cxcl1, and Cxcl2). FIG.2H shows hepatic E. coli levels in ethanol-fed mice. Data are normalized to 16S. The results are expressed as mean±SEM and generated from at least three independent replicates. Significance for (FIG. 2A, FIG.2B, FIG.2E - FIG.2G) was evaluated by one-way ANOVA with Tukey’s post- hoc test. Significance for (FIG.2H) was determined using Kruskal-Wallis test with Dunn’s post-hoc test followed by false discovery rate (FDR) procedures. *p<0.05. ALT, alanine transaminase. AH, alcohol-associated hepatitis. Scale bars = 100 μm.

[0013] FIGS.3A-3J show marco protects from kpsM-positive E. coli-exacerbated ethanol-induced liver disease. FIG.3A shows macrophage phagocytosis assessed by intracellular survival of E. coli, with bacterial CFU normalized per Kupffer cell. P values were determined by the Mann-Whitney-Wilcoxon rank-sum test. FIG.3B shows E. coli load in different tissues in conventional mice that were fed the chronic-binge ethanol diet. FIG. 3C shows intracellular E. coli (kpsM-positive and kpsM-knockout) in Kupffer cells with or without Marco knockdown. P values were determined by Kruskal-Wallis test with Dunn’s post-hoc test. FIG.3D shows serum levels of ALT. FIG.3E shows hepatic triglyceride content. FIG.3F shows representative images of liver sections stained with hematoxylin and eosin and Oil Red O. FIG.3G, FIG.3H, and FIG.3I show mRNA expression of inflammatory genes (Il1b, Cxcl1, and Cxcl2). FIG.3J shows hepatic E. coli levels in ethanol-37759.0643P1 fed mice. Data are normalized to 16S. The results are expressed as mean±SEM and generated from at least three independent replicates. Significance for (a, b) was evaluated by Mann- Whitney-Wilcoxon rank-sum test. Significance for (c-e, g-j) was evaluated by Kruskal-Wallis test with Dunn’s post-hoc test followed by false discovery rate (FDR) procedures. *p<0.05, **p<0.01, ***p<0.001. ALT, alanine transaminase. Scale bars = 100 μm.

[0014] FIGS.4A-4I show inhibitor C7 targeting kpsM-positive E. coli reduced ethanol-induced liver injury in gnotobiotic mice. FIG.4A shows confocal microscopy images depicting Kupffer cell phagocytosis of E. coli. The cytoplasmic membrane of Kupffer cell is in red and E. coli in green. FIG.4B shows TEM images of E. coli with the treatment of inhibitor C7. Scale bars = 200 nm. FIG.4C shows serum levels of ALT. FIG.4D shows Hepatic triglycerides content. FIG.4E shows representative images showing liver sections after hematoxylin and eosin staining. Scale bars = 100 μm. FIG.4F shows representative liver sections after Oil Red O staining. Scale bars = 100 μm. FIG.4G and FIG.4H show gene expressions of inflammatory factors (Il1b, Cxcl1) in mice. FIG.4I shows expression of kpsM in the liver normalized to 16S. The results are expressed as mean±SEM and generated from at least three independent replicates. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test. *p<0.05, **p<0.01. ALT, alanine transaminase. AH, alcohol-associated hepatitis.

[0015] FIGS.5A-5F show distribution of virulence genes and associations with clinical data in patients with alcohol-associated hepatitis. FIG.5A shows the association between virulence-related genes and time (days) between admission to the hospital and collection of fecal samples from 80 patients with alcohol-associated hepatitis; P=.78. P values were determined by two-tailed Spearman´s rank correlation coefficient. FIG.5B shows hospitalization time (days) for patients with alcohol-associated hepatitis either positive (n=55) or negative (n=26) for any virulence factor; P=.48. P values were determined by two- sided Mann-Whitney-Wilcoxon rank-sum test. FIG.5C shows distribution of virulence factors across countries. Each bar presents data from one virulence factor-positive patient with alcohol-associated hepatitis (n=55). The x-axis reports the total number of virulence factor genes detected in fecal samples from each patient; patients are grouped by region (top of graph). The y-axis presents the relative abundance of virulence factors in each patient; factors are color-coded by genus of bacteria that produce them. FIG.5D shows associations of virulence-related genes with region of origin; P=.95. P values were determined by Kruskal-Wallis test. FIG.5E shows virulence factor positivity in fecal samples from 81 patients with alcohol-associated hepatitis, by region of origin; P=.52. P values were37759.0643P1 determined by two-sided Fisher’s exact test. FIG.5F shows the percentage of patients with alcohol-associated hepatitis (n=81) positive for specific single virulence factors. The graph shows the top 60 most frequently detected virulence-related genes, color coded by genus of bacteria that expressed them.

[0016] FIGS.6A-6H show diversity and relative abundance of E. coli strains isolated from patients with alcohol-associated hepatitis. FIG.6A and FIG.6B show Maximum likelihood phylogeny of E. coli strains (n=45, from 81 alcohol-associated hepatitis patients), showing phylogenetic distribution of ecpR and kpsM positive E.coli (n=25 and n=18, respectively) across the commensal (blue branches; Escherichia coli and Escherichia sp000208585) and pathogenic (black branches; Shigella flexneri and uropathogenic E. coli) lineages. FIG.6C shows the proportions of patients with alcohol-associated hepatitis (n=68), with (n=13) or without (n=55) infection, whose fecal samples were positive (n=19) or negative (n=49) for ecpR; P=.49. P values were determined by two-sided Fisher’s exact test. FIG.6D shows proportions of patients with alcohol-associated hepatitis (n=68), with (n=13) or without (n=55) infection, whose fecal samples were positive (n=13) or negative (n=55) for kpsM; P=.25. P values were determined by two-sided Fisher’s exact test. FIG.6E shows principal component analysis (PCA), based on 1137 different taxa on genus level. P=.001; data from 81 patients with alcohol-associated hepatitis were included. P values were determined by permutational multivariate analysis of variance (PERMANOVA). FIG.6F shows relative E. coli abundance in fecal samples from 81 patients with alcohol-associated hepatitis positive (n=26) or negative (n=55) for ecpR and / or kpsM. P values were determined by two-sided Mann-Whitney-Wilcoxon rank-sum test. FIG.6G shows phylogenetic tree of isolated E. coli strains from alcohol-associated hepatitis patients. The name of each strain was annotated alongside its sequence types (STs). The presence of kpsM and / or ecpR was denoted by circles adjacent to the nodes. Squares indicated the antibiotic resistance genes carried by each strain. FIG.6H shows the prevalence of kpsM in ExPEC strains from publicly available databases. ExPEC, Extraintestinal pathogenic E. coli.

[0017] FIGS.7A-7F show associations of virulence genes with liver cirrhosis and intestinal permeability in patients with alcohol-associated hepatitis. FIG.7A shows the number of virulence-related genes in fecal samples from patients with biopsy-proven alcohol- associated hepatitis, with (n=32) or without (n=13) liver cirrhosis; P=.66. P values were determined by two-sided Mann-Whitney-Wilcoxon rank-sum test. FIG.7B shows positivity for any virulence factor in fecal samples from patients with (n=32) or without (n=13) liver cirrhosis. P=0.49. P values were determined by two-sided Fisher’s exact test. FIG.7C shows37759.0643P1 the correlation between the number of virulence-related genes and levels of zonulin in serum from 57 patients with alcohol-associated hepatitis; P=0.84. P values were determined by two- tailed Spearman´s rank correlation coefficient. FIG.7D shows levels of zonulin in serum from virulence factor positive-patients (n=40) and virulence factor negative-patients (n=17); P=.76. P values were determined by two-sided Mann-Whitney-Wilcoxon rank-sum test. FIG. 7E shows the correlation between the number of virulence-related genes and levels of lipopolysaccharide binding protein (LPS-BP) in serum from 56 patients with alcohol- associated hepatitis; P=.47. P values were determined by two-tailed Spearman´s rank correlation coefficient. FIG.7F shows levels of LPS-BP in serum from virulence factor- positive patients (n=39) and virulence factor-negative patients (n=17); P=.38. P values were determined by two-sided Mann-Whitney-Wilcoxon rank-sum test.

[0018] FIGS.8A-8H show fecal transplantation from kpsM-positive AH patients exacerbates ethanol-induced liver injury in gnotobiotic mice. FIG.8A shows a schematic overview of a chronic-binge ethanol feeding study in female germ-free mice. FIG.8B shows mRNA expression of F4 / 80 in the liver of mice. FIG.8C shows serum levels of ethanol in ethanol-fed mice. FIG.8D and FIG.8E show mRNA expression of ethanol metabolism genes (Adh1, Cyp2e1). FIG.8F, FIG.8G, and FIG.8H show the relative abundance of E. coli, kpsM, and ecpR levels in fecal samples of ethanol-fed mice. Data are normalized to 16S. The results are expressed as mean±SEM and generated from at least three independent replicates. Significance for (b, c, f-h) was determined using Kruskal-Wallis test with Dunn’s post-hoc test followed by false discovery rate (FDR) procedures. Significance for (d, e) was evaluated by one-way ANOVA with Tukey’s post-hoc test. *p<0.05. AH, alcohol-associated hepatitis. FMT, Fecal microbiota transplantation.

[0019] FIGS.9A-9N show monocolonization of kpsM-positive E. coli caused more severe ethanol-induced liver injury in mice. FIG.9A shows the Study design involving a chronic-binge ethanol feeding study in conventional mice alongside gavage of a single bacterial strain. FIG.9B shows serum levels of ALT. FIG.9C shows hepatic triglycerides content. FIG.9D shows representative images showing liver sections after hematoxylin and eosin staining. FIG.9E shows representative liver sections after Oil Red O staining. FIG. 9F, FIG.9G, and FIG.9H show gene expressions of inflammatory factors (Il1b, Cxcl1, and Cxcl2). FIG.9I shows hepatic E. coli in ethanol-fed mice. Data are normalized to 16S. j, Serum levels of ethanol. FIG.9K and FIG.9L show mRNA expression of genes in ethanol metabolism (Adh1, Cyp2e1). FIG.9M and FIG.9N show relative abundance of kpsM and E. coli levels in fecal samples of ethanol-fed mice. The results are expressed as mean±SEM and37759.0643P1 generated from at least three independent replicates. For FIG.9B, FIG.9C, FIG.9F- FIG. 9I, FIG.9K, and FIG.9L, significance was evaluated by one-way ANOVA with Tukey’s post-hoc test. For FIG.9J, FIG.9M, and FIG.9N, significance was determined using Kruskal-Wallis test with Dunn’s post-hoc test followed by false discovery rate (FDR) procedures. *p<0.05, **p<0.01, ***p<0.001. ALT, alanine transaminase. Scale bars = 100 μm.

[0020] FIGS.10A-10H show Marco protects from kpsM-positive E. coli-exacerbated ethanol-induced liver disease. FIG.10A shows the study design detailing the phagocytosis of E. coli by Kupffer cells from conventional mice. FIG.10B shows mRNA expression of Marco which was knockdown in vitro by siRNA. FIG.10C shows flow cytometry to detect the binding of recombinant mouse MARCO with E. coli, represented as a percentage of AF647 labeled cells. FIG.10D shows a schematic overview of a chronic-binge ethanol feeding study in female Clec4f-Cre mice. FIG.10E shows mRNA expression of Marco which was knockdown in vivo of the Clec4f-Cre mice. FIG.10F shows serum levels of ethanol in ethanol-fed mice. FIG.10G and FIG.10H show mRNA expression of ethanol metabolism genes (Adh1, Cyp2e1). The results are expressed as mean±SEM and generated from at least three independent replicates. Significance was evaluated by Kruskal-Wallis test with Dunn’s post-hoc test followed by false discovery rate (FDR) procedures. *p<0.05, **p<0.01, ****p<0.0001.

[0021] FIGS.11A-11M show inhibitor C7 targeting kpsM-positive E. coli reduced ethanol-induced liver injury in gnotobiotic mice. FIG.11A shows intracellular E. coli (kpsM-positive and kpsM-knockout) in Kupffer cells treated with the inhibitor C7. FIG.11B shows the growth curve of E. coli strains with the treatment of the inhibitor C7. FIG.11C, FIG.11D, and FIG.11E show gene expression of inflammatory factors (Il1b, Cxcl1, Cxcl2) in Kupffer cells treated with E. coli. FIG.11F shows the study design involving a chronic- binge ethanol feeding study in germ-free mice treated with the inhibitor C7. FIG.11G shows mRNA expression of F4 / 80 in the liver of mice. FIG.11H mRNA expression of Marco in the liver of mice. FIG.11I and FIG.11J, Relative abundance of kpsM and E. coli levels in fecal samples of ethanol-fed mice. FIG.11K shows serum levels of ethanol. FIG.11L and FIG.11M show mRNA expression of ethanol metabolism genes (Adh1, Cyp2e1). The results are expressed as mean±SEM and generated from at least three independent replicates. For FIG.11A, FIG.11C - FIG.11E, FIG.11G, FIG.11I, FIG.11J, and FIG.11K, significance was determined using Kruskal-Wallis test with Dunn’s post-hoc test followed by false discovery rate (FDR) procedures. For FIG.11H, FIG.11L, and FIG.11M,37759.0643P1 significance was evaluated by one-way ANOVA with Tukey’s post-hoc test. *p<0.05, **p<0.01. AH, alcohol-associated hepatitis. FMT, Fecal microbiota transplantation. DETAILED DESCRIPTION

[0022] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0023] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0024] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C- D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed. A. Definitions

[0025] It is understood that the disclosed method and compositions are not limited to the37759.0643P1 particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0026] 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. Thus, for example, reference to "an exosome" includes a plurality of such exosomes, reference to "the exosome" is a reference to one or more exosomes and equivalents thereof known to those skilled in the art, and so forth.

[0027] By a “therapeutically effective amount” of a composition as provided herein is meant a sufficient amount of the composition to provide the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of disease (or underlying genetic defect) that is being treated, the particular composition used, its mode of administration, and the like. Thus, it is not possible to specify an exact “therapeutically effective amount.” However, an appropriate “therapeutically effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

[0028] The term “therapeutic” refers to a composition that treats a disease, including a composition comprising the small molecule inhibitor C7. As used herein, the small molecule inhibitor C7 refers to 2-(4-phenylphenyl)-benzo[g]quinoline-4-carboxylic acid (NSC136469) having the structure set forth as:a therapeutic or composition of the invention (e.g. the small molecule inhibitor C7) to a subject, such as a human or other mammal (for example, an animal model), that has an increased susceptibility for developing alcohol- associated hepatitis, or that has alcohol-associated hepatitis, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects37759.0643P1 of the disease. By “treat” is also meant to administer a therapeutic or composition of the invention to a subject, such as a human or other mammal (for example, an animal model), that has an increased susceptibility for developing sepsis, urinary tract infection, pneumonia, intra-abdominal infection and / or endocarditis, or that has sepsis, urinary tract infection, pneumonia, intra-abdominal infection and / or endocarditis, in order to prevent or delay a worsening of the effects of the disease or condition, or to partially or fully reverse the effects of the disease.

[0030] By “prevent” is meant to minimize the chance that a subject who has an increased susceptibility for developing alcohol-associated hepatitis will end up with alcohol-associated hepatitis. With relation to mortality, “prevent” is meant to minimize the chance that a subject who has an increased risk for mortality associated with alcohol-associated hepatitis will die within 30 days, 60 days, 90 days, or 180 days.

[0031] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. Thus, the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. The term "subject" also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). In one aspect, a subject is a mammal. In another aspect, a subject is a human. The term does not denote a particular age or sex.

[0032] An “effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. The phrase “therapeutically effective amount”, as used herein, refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

[0033] “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0034] Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to37759.0643P1 the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub- ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0036] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step. B. Methods37759.0643P1 1. Methods of Treating i. Methods of Treating or ameliorating a symptom of alcohol-associated hepatitis

[0037] Disclosed are methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby treating or ameliorating a symptom of alcohol-associated hepatitis in the subject.

[0038] As used herein, the small molecule inhibitor C7 refers to 2-(4-phenylphenyl)- benzo[g]quinoline-4-carboxylic acid (NSC136469).

[0039] In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, C7 is administered in the acute phase of alcohol-associated hepatitis for about one week, about two weeks, about three weeks or about four weeks.

[0040] In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, the subject has or has been diagnosed with alcohol- associated hepatitis.

[0041] In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

[0042] In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, the subject was identified as being in need thereof by determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

[0043] In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, the bacterial virulence factor is kpsM (Escherichia coli) or ecpR (Escherichia coli). In some aspects of the methods of treating or ameliorating a symptom of alcohol-associated hepatitis in a subject, the bacterial virulence factor is ecpR (Escherichia coli). In some aspects of the methods of treating or ameliorating a symptom of alcohol-associated hepatitis in a subject, the bacterial virulence factor is kpsM (Escherichia coli). In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, the kpsM (Escherichia coli) is multi-drug resistant.

[0044] In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, the method further comprises administering to the subject a therapeutically effective amount of an antibiotic. In some aspects of the methods of treating or ameliorating a symptom of alcohol-associated hepatitis in a subject, the antibiotic is co-37759.0643P1 formulated with the small molecule inhibitor C7. In some aspects of the methods of treating or ameliorating a symptom of alcohol-associated hepatitis in a subject, the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin, doxycycline, amoxicillin-clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin, azithromycin, sulfamethoxazole-trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

[0045] In some aspects of the methods of treating or ameliorating a symptom of alcohol- associated hepatitis in a subject, the symptom is liver injury, steatosis, and / or inflammation. ii. Methods of reducing or preventing mortality associated with alcohol- associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis

[0046] Disclosed are methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol-associated hepatitis comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby reducing or preventing mortality in the subject.

[0047] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, the subject has or has been diagnosed with alcohol-associated hepatitis.

[0048] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, C7 is administered in the acute phase of alcohol-associated hepatitis for about one week, about two weeks, about three weeks or about four weeks.

[0049] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

[0050] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, the subject was identified as being in need thereof by determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

[0051] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, the bacterial virulence factor is kpsM (Escherichia coli) or ecpR37759.0643P1 (Escherichia coli). In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol-associated hepatitis, the bacterial virulence factor is ecpR (Escherichia coli). In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol-associated hepatitis, the bacterial virulence factor is kpsM (Escherichia coli). In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol-associated hepatitis, the kpsM (Escherichia coli) is multi-drug resistant.

[0052] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, the methods further comprise administering to the subject a therapeutically effective amount of an antibiotic.

[0053] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, the antibiotic is co-formulated with the small molecule inhibitor C7.

[0054] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin, doxycycline, amoxicillin- clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin, azithromycin, sulfamethoxazole-trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

[0055] In some aspects of the methods of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol- associated hepatitis, mortality is reduced at 30 days, 60 days, 90 days, and / or 180 days. iii. Methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis

[0056] Disclosed are methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby treating or ameliorating a symptom of alcohol-associated hepatitis in the subject.

[0057] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a37759.0643P1 subject, the subject has or has been diagnosed with sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis.

[0058] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, C7 is administered in the acute phase of alcohol-associated hepatitis for about one week, about two weeks, about three weeks or about four weeks.

[0059] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

[0060] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the subject was identified as being in need thereof by determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

[0061] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the bacterial virulence factor is kpsM (Escherichia coli) or ecpR (Escherichia coli). In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the bacterial virulence factor is ecpR (Escherichia coli). In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra- abdominal infections, and / or endocarditis in a subject, the bacterial virulence factor is kpsM (Escherichia coli). In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the kpsM (Escherichia coli) is multi-drug resistant.

[0062] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the methods further comprise administering to the subject a therapeutically effective amount of an antibiotic.

[0063] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the antibiotic is co-formulated with the small molecule inhibitor C7.

[0064] In some aspects of the methods of treating or ameliorating a symptom of sepsis,37759.0643P1 urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin, doxycycline, amoxicillin-clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin, azithromycin, sulfamethoxazole- trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

[0065] In some aspects of the methods of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject, wherein the symptom is fever, swelling, pain, and / or systemic and organ-specific inflammation. 2. Methods of Screening i. Methods of determining responsiveness of a subject to treatment with the small molecule inhibitor C7

[0066] Disclosed are methods of determining responsiveness of a subject to treatment with the small molecule inhibitor C7 comprising: determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample from said subject, wherein when the patient has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus, the subject will be responsive to treatment with the small molecule inhibitor C7.

[0067] In some aspects of the methods of determining responsiveness of a subject to treatment with the small molecule inhibitor C7, the subject has alcohol-associated hepatitis.

[0068] In some aspects of the methods of determining responsiveness of a subject to treatment with the small molecule inhibitor C7, the bacterial virulence factor is ecpR (Escherichia coli). In some aspects of the methods of determining responsiveness of a subject to treatment with the small molecule inhibitor C7, the bacterial virulence factor is kpsM (Escherichia coli). In some aspects of the methods of determining responsiveness of a subject to treatment with the small molecule inhibitor C7, the kpsM (Escherichia coli) is multi-drug resistant. ii. Methods of identifying a subject for a clinical study for a treatment for alcohol-associated hepatitis with composition comprising small molecule inhibitor C7

[0069] Disclosed are methods of identifying a subject for a clinical study for a treatment for alcohol-associated hepatitis with composition comprising small molecule inhibitor C7, comprising determining that the subject has a bacterial virulence factor from Escherichia,37759.0643P1 Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample from said subject, wherein when the patient has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus, the subject is appropriate for the clinical study for a treatment for alcohol-associated hepatitis.

[0070] In some aspects of the methods of identifying a subject for a clinical study for a treatment for alcohol-associated hepatitis with composition comprising small molecule inhibitor C7, the bacterial virulence factor is kpsM (Escherichia coli) or ecpR (Escherichia coli).

[0071] In some aspects of the methods of identifying a subject for a clinical study for a treatment for alcohol-associated hepatitis with composition comprising small molecule inhibitor C7, the bacterial virulence factor is ecpR (Escherichia coli). In some aspects of the methods of identifying a subject for a clinical study for a treatment for alcohol-associated hepatitis with composition comprising small molecule inhibitor C7, the bacterial virulence factor is kpsM (Escherichia coli). In some aspects of the methods of identifying a subject for a clinical study for a treatment for alcohol-associated hepatitis with composition comprising small molecule inhibitor C7, the kpsM (Escherichia coli) is multi-drug resistant. iii. Methods for screening for a candidate compound

[0072] Disclosed are methods for screening for a candidate compound for treating alcohol-associated hepatitis comprising: (1) contacting the candidate compound with a sample comprising a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus; and (2) determining that the bacteria is reduced / killed, thereby (3) identifying a compound for treating alcohol-associated hepatitis.

[0073] In some aspects of the disclosed methods for screening for a candidate compound for treating alcohol-associated hepatitis, the bacterial virulence factor is ecpR (Escherichia coli).

[0074] In some aspects of the disclosed methods for screening for a candidate compound for treating alcohol-associated hepatitis, the bacterial virulence factor is kpsM (Escherichia coli). 3. Compounds

[0075] Disclosed are compositions comprising the small molecule inhibitor C7. Disclosed are compositions comprising the small molecule inhibitor C7 and an antibiotic.

[0076] In some aspects of the compositions, the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin, doxycycline, amoxicillin-clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin,37759.0643P1 azithromycin, sulfamethoxazole-trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

[0077] Pharmaceutical Compositions

[0078] Disclosed herein are pharmaceutical compositions, comprising C7. As disclosed herein, are pharmaceutical compositions, comprising C7 and a pharmaceutical acceptable carrier described herein. In some aspects, the C7 can be formulated for intranasal administration. In some aspects, the C7 can be formulated for systemic administration (infusion or injection). In some aspects, the C7 can be formulated for intravenous, intraperitoneal, or oral administration. In some aspects, the C7 can be formulated for oral or parental administration. In some aspects, the parental administration can be intravenous, subcutaneous, intramuscular or direct injection. In some aspects, the C7 can be administered intramuscularly, intravenously, subcutaneously, orally, topically, transdermally, or sublingually. In some aspects, the C7 can be administered both orally and systemically. The compositions can be formulated for administration by any of a variety of routes of administration, and can include one or more physiologically acceptable excipients, which can vary depending on the route of administration. As used herein, the term “excipient” means any compound or substance, including those that can also be referred to as “carriers” or “diluents.” Preparing pharmaceutical and physiologically acceptable compositions is considered routine in the art, and thus, one of ordinary skill in the art can consult numerous authorities for guidance if needed.

[0079] The compositions can be administered directly to a subject. Generally, the compositions can be suspended in a pharmaceutically acceptable carrier (e.g., physiological saline or a buffered saline solution) to facilitate their delivery. Encapsulation of the compositions in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery.

[0080] The compositions can be formulated in various ways for parenteral or nonparenteral administration. Where suitable, oral formulations can take the form of tablets, pills, capsules, or powders, which may be enterically coated or otherwise protected. Sustained release formulations, suspensions, elixirs, aerosols, and the like can also be used.

[0081] Pharmaceutically acceptable carriers and excipients can be incorporated (e.g., water, saline, aqueous dextrose, and glycols, oils (including those of petroleum, animal, vegetable or synthetic origin), starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monosterate, sodium chloride, dried skim milk, glycerol, propylene glycol, ethanol, and the like). The compositions may be subjected to conventional pharmaceutical expedients such as37759.0643P1 sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceutical carriers and their formulations are described in “Remington's Pharmaceutical Sciences” by E.W. Martin, which is herein incorporated by reference. Such compositions will, in any event, contain an effective amount of the compositions together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the patient.

[0082] The pharmaceutical compositions as disclosed herein can be prepared for oral or parenteral administration. Pharmaceutical compositions prepared for parenteral administration include those prepared for intravenous (or intra-arterial), intramuscular, subcutaneous, intraperitoneal, transmucosal (e.g., intranasal, intravaginal, or rectal), or transdermal (e.g., topical) administration. Aerosol inhalation can also be used. Thus, compositions can be prepared for parenteral administration that includes C7 dissolved or suspended in an acceptable carrier, including but not limited to an aqueous carrier, such as water, buffered water, saline, buffered saline (e.g., PBS), and the like. One or more of the excipients included can help approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like. Where the compositions include a solid component (as they may for oral administration), one or more of the excipients can act as a binder or filler (e.g., for the formulation of a tablet, a capsule, and the like).

[0083] The pharmaceutical compositions can be sterile and sterilized by conventional sterilization techniques or sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation, which is encompassed by the present disclosure, can be combined with a sterile aqueous carrier prior to administration. The pH of the pharmaceutical compositions typically will be between 3 and 11 (e.g., between about 5 and 9) or between 6 and 8 (e.g., between about 7 and 8). The resulting compositions in solid form can be packaged in multiple single dose units, each containing a fixed amount of the above- mentioned agent or agents, such as in a sealed package of tablets or capsules.

[0084] Articles of Manufacture

[0085] The compositions described herein can be packaged in a suitable container labeled, for example, for use as a therapy to treating or preventing rheumatoid arthritis or any of the methods disclosed herein. Accordingly, packaged products (e.g., sterile containers containing the composition described herein and packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations) and kits, including C7 as described herein and37759.0643P1 instructions for use, are also within the scope of the disclosure. A product can include a container (e.g., a vial, jar, bottle, bag, or the like) containing the composition described herein. In addition, an article of manufacture further may include, for example, packaging materials, instructions for use, syringes, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required. The product may also include a legend (e.g., a printed label or insert or other medium describing the product's use (e.g., an audio- or videotape)). The legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the compound therein should be administered (e.g., the frequency and route of administration), indications therefor, and other uses. The compositions can be ready for administration (e.g., present in dose- appropriate units), and may include a pharmaceutically acceptable adjuvant, carrier or other diluent. Alternatively, the compositions can be provided in a concentrated form with a diluent and instructions for dilution. 4. Administration

[0086] The disclosed methods can include one or more of the types of administration disclosed herein.

[0087] In the methods described herein, administration or delivery of the therapeutics to a subject can be via a variety of mechanisms. For example, the therapeutic can be formulated as a pharmaceutical composition.

[0088] Pharmaceutical compositions can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.

[0089] Preparations of parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride, lactated Ringer’s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer’s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0090] Formulations for optical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.37759.0643P1

[0091] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. Some of the compositions can be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mon-, di-, trialkyl and aryl amines and substituted ethanolamines. C. Kits

[0092] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits comprising compositions and instructions for carrying out the claimed methods. Examples A. Example 1: Targeted Inhibition of Pathobiont Virulence Factor KpsM Mitigates Alcohol-Associated Liver Disease

[0093] Gut microbial dysbiosis has been linked to the development and progression of several chronic noncommunicable diseases including alcohol-associated liver disease14. Chronic alcohol consumption alters the gut microbiota composition15-18. Transplantation of intestinal microbiota from patients with alcohol-associated hepatitis promotes ethanol- induced liver disease in mice23.

[0094] While many studies have explored the association between the gut microbiota and diseases, focusing on diversity, relative abundance, and metabolic profiles of specific microorganisms25-28, little is known about specific pathobionts and their contributing factors to diseases. Bacterial virulence factors are proteins or peptides encoded by bacterial genes that help the organisms colonize the intestine or mediate disease. Recent findings highlighted the role of cytolysin, a virulence factor expressed by Enterococcus faecalis (E. faecalis), in causing direct lysis of hepatocytes and liver damage29.

[0095] In this study, intestinal bacterial metagenomes and genes encoding virulence factors in fecal samples from a well-characterized, multi-center cohort of patients with alcohol-associated hepatitis were analyzed.37759.0643P1 RESULTS

[0096] Increased carriage of virulence-related genes associates with mortality in patients with alcohol-associated hepatitis

[0097] Shotgun metagenomic sequencing was performed on fecal samples from 9 individuals who were not alcoholics (controls), 41 patients with alcohol use disorder and 81 patients with alcohol-associated hepatitis, who were enrolled in a multi-center, international observational study (Table 1 and 2); and aligned sequences with those in the virulence factor gene database (VFDB)58. Patients with alcohol-associated hepatitis displayed distinctive changes in microbial composition, with an enrichment of Proteobacteria at the phylum level and pathobionts such as Enterococcus faecium, Escherichia coli, and Enterococcus faecalis at the species level in patients with alcohol-associated hepatitis (FIG.1A). Significantly more virulence factors were found in fecal metagenomes from patients with alcohol-associated hepatitis than patients with alcohol use disorder or healthy controls (FIG.1B). The increased copies of virulence factor genes were not associated with the duration of hospitalization, suggesting they were not acquired during hospital stays (FIG.5A–5B). Overall, 68% of patients with alcohol-associated hepatitis had at least one virulence gene, contrasting with 29% in patients with alcohol use disorder and 11% in controls (P=2.75e-04 and 2.27e-03, respectively, by Fisher’s exact test; FIG.1C). There was heterogeneity in the host origin of virulence factor genes among the study subjects; the carriers usually carry several virulence factor genes originated from different host bacterial genera, independent from the center where the patient was enrolled (FIGs.5C–5E). The most frequently observed virulence factors were derived from Escherichia, followed by Klebsiella, Shigella, Klebsiella / Yersinia, and then Enterococcus (FIG.1C; FIG.5F). Table 1: Demographic and laboratory parameters of the study cohort Variables Controls Alcohol use Alcohol-associated (n=9) disorder hepatitis 41 8137759.0643P1r number and percentage in parentheses for categorical variables. Percentages are calculated based on the actual number of patients in each group where the respective data was available. The number of subjects for which the respective data was available is indicated in the first column. BMI, body mass index; AST, aspartate aminotransferase; ALT, alanine aminotransferase; INR, international normalized ratio; GGT, gamma-glutamyl transferase; FIB-4, fibrosis-4 index Table 2: Characteristics of patients with alcohol-associated hepatitis (n=81) Treatment at admission Steroids n (%) n=79 31 (392)37759.0643P1antibiotics. Values are presented as median (range) for continuous variables or number (percentage) for categorical variables. Percentages are calculated based on the actual number of patients in each group where the respective data was available. Individual persons can have more than one reason for infections. The number of subjects for which the respective data was available is indicated in the first column. MELD, model for end-stage liver disease; DF, discriminant function; ABIC, Age, serum bilirubin, INR, and serum creatinine score

[0098] In patients with alcohol-associated hepatitis, intestinal carriage of virulence factors was associated with decreased probability of surviving 180 days (FIG.1D); 82% of virulence factor-negative patients were alive after 180 days following enrollment, compared with 18% of virulence factor-positive patients (P=.008). In the univariate Cox regression analysis, a fecal sample with any virulence factor gene, with any Escherichia virulence factor gene, or with 1 of 6 specific virulence factor genes was associated with 180-day mortality (Table 3). After antibiotic treatment was adjusted for in the multivariate Cox regression analysis as well as the sequence batch, fecal samples with any virulence factor gene remained associated with increased risk of death, with an adjusted hazard ratio of 5.67 (95% CI 1.26– 25.52; P=.024; FIG.1E). Specifically, among all 350 core virulence factor genes, fecal samples containing kpsM (E. coli), ecpR (E. coli), or gspC (Shigella dysenteriae) were independently associated with mortality (FIG.1E).

[0099] Table 3: Univariate Cox regression analysis of virulence factors associated with 180-day mortality in patients with alcohol-associated hepatitis Univariate Cox regression Organism HR 95% CI P value Any virulence factor positivity 5.9 1.3–25.7 0.019 1 9 137759.0643P1 7 nedby two-sided Wald test. HR, hazard ratio; CI, confidence interval

[0100] Since genomes of Shigella and Escherichia are very similar and patients did not exhibit clinical signs of dysenteric diarrhea, the Shigella association is most likely a misalignment. Therefore, E. coli virulence factors were studied. The strain-level phylogeny of ecpR- and kpsM-positive E. coli revealed high diversity which grouped into distinct lineages (FIG.6A–6B; P=.035 and .013, respectively, by Fisher’s exact test). These virulence factor genes were not associated with infection immediately before or during hospitalization (FIG.6C–6D). Patients positive for ecpR and / or kpsM (32% of patients with alcohol-associated hepatitis) had a microbiome that differed significantly from that of non- carriers, when the relative abundance of fecal bacterial populations were analyzed in the metagenomic dataset (FIG.6E). In particular, the presence of genes encoding virulence factors from E. coli was associated with the relative abundance of E. coli (FIG.6F). Culture- based whole genome sequencing further validated that kpsM-positive E. coli strains exhibited high diversity and belonged to different sequence types, indicating that the prevalence of kpsM was not due to clonal dissemination (FIG.6G). Colonies from the same patient displayed identical similarities, indicating that kpsM-positive strains had outgrown and dominated the E. coli population. Notably, most kpsM-positive E. coli strains are multidrug resistant (FIG.6G). Analysis of publicly available E. coli genomes revealed that kpsM was more prevalent in extraintestinal pathogenic E. coli (ExPEC) lineage, including the globally dominant multidrug resistant ESBL-producing E. coli ST13159(FIG.6H).

[0101] Higher E. coli abundance itself was independently associated with 180-day mortality; however, when the detection of ecpR and / or kpsM was adjusted for, this association was no longer statistically significant (Table 4). This indicates that the abundance of E. coli and the carriage of genes encoding virulence factors each are associated with an increased risk of death in these patients. The presence of underlying cirrhosis was not associated with higher numbers of virulence factor genes (FIG.7A–7B). Furthermore, levels of zonulin (a marker for gut barrier dysfunction) and lipopolysaccharide binding protein (LPS-BP) (a marker of the systemic immune response to LPS) in serum samples were not associated with the number or presence of any virulence factor genes (FIG.7C–7F).37759.0643P1 Virulence factors therefore do not seem to be associated with increased intestinal permeability or translocation of LPS, a hallmark in patients with alcohol-associated hepatitis.

[0102] Table 4: Multivariate Cox regression analysis of virulence factors associated with 180-day mortality in patients with alcohol-associated hepatitis Multivariate Cox re ression HR 95% CI P value*s and the sequence batch (2 different runs). In the second row, E. coli abundance was additionally adjusted for the presence of the VFs ecpR and / or kpsM. Patients were censored at the time point they were last seen alive. P values were determined by two-sided Wald test. HR, hazard ratio; CI, confidence interval.

[0103] Given the greater importance of kpsM compared with ecpR in our mouse studies (refer to the details below), the results were validated in an independent cohort using qPCR analyses to detect the presence of kpsM in fecal samples from patients with alcohol- associated hepatitis (Table 5).20 (40.81%) out of 49 patients were positive for kpsM. Patients who were positive for kpsM had a higher risk to mortality within 180 days with a hazard ratio of 4.10 (95% CI 1.11–15.19; P=.035; FIG.1E), which remained significant after adjustment for antibiotic use with an adjusted hazard ratio of 3.86 (95% CI 1.04–14.32; P=.044).

[0104] Table 5: Characteristics of patients with alcohol-associated hepatitis in the validation cohort (n=49) Demographic and laboratory parameters Sex (male), n (%), n=47 26 (55.3)37759.0643P1

[0106] To study the contribution of virulence factor-positive bacteria to liver disease, germ-free C57BL / 6 mice were colonized with stool from E. coli kpsM-positive and ecpR- positive, E. coli kpsM-negative and ecpR-negative, and E. coli kpsM-positive and ecpR- negative patients with alcohol-associated hepatitis and placed on a chronic-binge ethanol diet (FIG.8A). Of note, not any patient with alcohol-associated hepatitis was positive for E. coli ecpR alone in our cohort. All fecal samples were selected to be negative for E. faecalis cytolysin. Compared with mice colonized with feces from kpsM-negative and ecpR-negative37759.0643P1 patients with alcohol-associated hepatitis, mice fed ethanol after they were colonized with kpsM-positive and ecpR-positive feces developed more severe liver injury, indicated by higher level of alanine amino-transferase (ALT) (FIG.2A) and increased hepatic steatosis (FIG.2B–2D). Mice fed ethanol after they were colonized with kpsM-positive and ecpR- positive feces also showed more liver inflammation with increased expression levels of mRNAs encoding inflammatory cytokines and chemokines (Interleukin-1 beta (Il1b), chemokine (C-X-C motif) ligand-1 (Cxcl1), and Cxcl2) (FIG.2E–2G) compared with mice colonized with kpsM-negative and ecpR-negative feces. Mice fed ethanol after they were colonized with kpsM-positive and ecpR-negative feces had significantly more ethanol- induced liver injury, steatosis, and inflammation (FIG.2A–2G) as compared with mice fed ethanol after they were colonized with kpsM-negative and ecpR-negative feces, indicating that kpsM rather than ecpR is responsible for the disease exacerbating effect. F4 / 80 as marker for Kupffer cells was not significantly different between ethanol-fed groups (FIG.8B). KpsM was only detectable in livers of mice colonized with kpsM-positive feces following chronic ethanol administration, but not when fed an isocaloric (control) diet (FIG.2H), indicating that ethanol-induced changes in the gut barrier are necessary for translocation of kpsM- positive E. coli from the intestine to the liver. No significant differences were observed for serum ethanol and mRNAs encoding the two primary enzymes that metabolize ethanol in the liver, alcohol dehydrogenase 1 (Adh1) and cytochrome P450 family 2 subfamily E polypeptide 1 (Cyp2e1) (FIG.8C–8E), indicating that colonization did not affect ethanol metabolism. Fecal E. coli was similar in all three groups following ethanol feeding (FIG. 8F), while increased fecal kpsM and ecpR was confirmed in microbiota humanized mice (FIG.8G–8H), indicating that kpsM or ecpR positivity does not result in a colonization advantage of E. coli. These findings suggest that E. coli positive for kpsM rather than for ecpR are associated with exacerbated ethanol-induced liver disease.

[0107] To validate our findings from microbiota humanized mice, conventional C57BL / 6 mice were gavaged with kpsM-positive E. coli or isogenic E. coli with a kpsM mutation (E. coli ^kpsM) (Zong B, Liu W, Zhang Y, Wang X, Chen H, Tan C. Effect of kpsM on the virulence of porcine extraintestinal pathogenic Escherichia coli. FEMS Microbiol Lett.2016 Nov 1;363(21):fnw232. doi: 10.1093 / femsle / fnw232. PMID: 27737948) and subjected them to a chronic-binge ethanol diet (FIG.9A). Mice gavaged with kpsM-positive E. coli and fed ethanol showed more liver injury, steatosis and inflammation as compared with mice given phosphate-buffered saline (PBS) (FIG.9B–9H). Mice fed ethanol after they were gavaged with E. coli ^kpsM had significantly less ethanol-induced liver disease as compared with37759.0643P1 mice fed ethanol after they were administered kpsM-positive E. coli (FIG.9B–9H). KpsM was detected in livers of ethanol-fed mice gavaged with kpsM-positive E. coli (FIG.9I). No significant differences were observed in ethanol metabolism (FIG.9J–9L). Abundance of fecal E. coli was similar in mice gavaged with kpsM-positive or ^kpsM E. coli (FIG.9M). Presence of fecal kpsM was confirmed in ethanol-fed mice gavaged with kpsM-positive E. coli (FIG.9N). These results indicate that kpsM promotes ethanol-induced liver disease.

[0108] Marco protects from kpsM-positive E. coli-exacerbated ethanol-induced liver disease

[0109] KpsM is an ABC transporter facilitating translocation of the host-mimicking polysialic acid across the periplasmic space and onto the cell surface of E. coli60, which causes invasion and protection from elimination by phagocytosis62. When bacteria breach the intestinal barrier, they move into the portal vein and reach the liver, which acts as a secondary barrier to curb their dissemination. Kupffer cells, the resident macrophage population in the liver, capture bacteria that have escaped from the gut, phagocytose and kill them REF. Kupffer cells phagocytosed kpsM-positive E. coli much less efficiently than ^kpsM E. coli in culture (FIG.3A; FIG.10A). To determine whether phagocytosis of bacteria translocated from gut to the liver is also different in vivo, ethanol-fed mice were gavaged with kpsM- positive and ^kpsM E. coli. The number of translocated kpsM-positive E. coli recovered in the liver was significantly lower, but significantly higher in spleen, lung and heart as compared with ^kpsM E. coli (FIG.3B). These data indicate that fewer translocated kpsM- positive E. coli are captured in the liver, resulting in increased numbers in other organs.

[0110] The scavenger receptor Macrophage Receptor with Collagenous structure (Marco) is primarily expressed on periportal Kupffer cells (Miyamoto Y, Kikuta J, Matsui T, Hasegawa T, Fujii K, Okuzaki D, Liu YC, Yoshioka T, Seno S, Motooka D, Uchida Y, Yamashita E, Kobayashi S, Eguchi H, Morii E, Tryggvason K, Shichita T, Kayama H, Atarashi K, Kunisawa J, Honda K, Takeda K, Ishii M. Periportal macrophages protect against commensal-driven liver inflammation. Nature.2024 May;629(8013):901-909. doi: 10.1038 / s41586-024-07372-6. Epub 2024 Apr 24. PMID: 38658756). Marco+ Kupffer cells exert anti-inflammatory effects through sequestration of bacteria such as E. coli and through production of anti-inflammatory cytokines (Miyamoto Y, Kikuta J, Matsui T, Hasegawa T, Fujii K, Okuzaki D, Liu YC, Yoshioka T, Seno S, Motooka D, Uchida Y, Yamashita E, Kobayashi S, Eguchi H, Morii E, Tryggvason K, Shichita T, Kayama H, Atarashi K, Kunisawa J, Honda K, Takeda K, Ishii M. Periportal macrophages protect against37759.0643P1 commensal-driven liver inflammation. Nature.2024 May;629(8013):901-909. doi: 10.1038 / s41586-024-07372-6. Epub 2024 Apr 24. PMID: 38658756). Following knockdown of Marco in Kupffer cells (FIG.10B), phagocytosis of ^kpsM E. coli was reduced to the level of kpsM-positive E. coli (FIG.3C). Recombinant Marco binds ^kpsM E. coli more efficiently than kpsM-positive E. coli (FIG.10C). These results indicate that Marco is important for binding and uptake of E. coli by Kupffer cells, which is inhibited in the presence of kpsM. To investigate the contribution of Marco-mediated phagocytosis of kpsM- positive E. coli to ethanol-induced liver injury, in vivo knockdown of Marco was performed selectively in Kupffer cells. Mice were subjected to chronic–binge ethanol diet and gavaged with kpsM-positive and ^kpsM E. coli (FIG.10D–10E). AAV8-mediated transgene expression of the double-floxed inverse ORF (DIO) shRNA against Marco in Clec4f-Cre mice reduced Marco expression as compared with mice injected with the control vector expressing scramble shRNA (FIG.11D). Ethanol-induced liver injury, steatosis and inflammation were increased following knockdown of Marco in Kupffer cells in mice gavaged with kpsM-positive E. coli and fed ethanol as compared with control mice (FIG. 3G–I). Mice with knockdown of Marco on Kupffer cells also showed increased liver disease when gavaged with ^kpsM E. coli (FIG.3G–I) indicating that E. coli lacking kpsM can induce disease if they evade phagocytosis. Hepatic kpsM was reduced after knockdown of Marco indicating reduced phagocytosis in Kupffer cells of mice gavaged with kpsM-positive E. coli (FIG.3J). No significant differences were observed in ethanol metabolism (FIG. 10F–10H). Taken together, these results indicate that Marco on Kupffer cells limits ethanol- induced liver disease by elimination of translocated kpsM-positive E. coli. E. coli lacking kpsM do not exacerbate ethanol-induced liver disease due to effective phagocytosis, thereby preventing disease exacerbation. Consequently, kpsM is a significant virulence factor in E. coli, crucial for evading phagocytosis and promoting pathogenicity.

[0111] Targeted inhibition of kpsM attenuates ethanol-induced liver disease

[0112] To further demonstrate a causative role of kpsM for the development of ethanol- induced steatohepatitis, the effects of treatment with the small molecule inhibitor C7 were investigated. C7 inhibits polysaccharide translocation to the capsule in kpsM-positive E. coli61. C7 increased phagocytosis of kpsM-positive E. coli and eliminated the difference in phagocytosis rate as compared with ^kpsM E. coli in cultured Kupffer cells (FIG.4A) without affecting growth rate of E. coli. kpsM-positive E. coli-induced gene expression of Il1b, Cxcl1, and Cxcl2 in Kupffer cells as compared with ^kpsM E. coli. C7 reduced kpsM-37759.0643P1 positive E. coli-induced gene expression of Il1b, Cxcl1, and Cxcl2 in Kupffer cells, likely through increased phagocytosis and a subsequent reduced presence of E. coli in the supernatant binding to pathogen recognition receptors on the surface of Kupffer cells. C7 inhibited capsule formation in kpsM-positive E. coli. Both ^kpsM E. coli and C7-treated kpsM-positive E. coli lacked an intact capsule structure, displaying a capsule-deficient phenotype (FIG.4B).

[0113] To develop a novel therapeutic approach to precisely inhibit pathobiontic E. coli, gnotobiotic mice were colonized with feces from kpsM-positive and kpsM-negative E. coli patients with alcohol-associated hepatitis and treated with C7 in the liquid diet. C7 reduced ethanol-induced steatohepatitis in mice colonized with feces from kpsM-positive patients with alcohol-associated hepatitis, based on lower levels of ALT, decreased hepatic triglycerides, decreased hepatic levels of Il1b and Cxcl1 mRNAs, and reduced hepatic levels of kpsM, compared with mice given vehicle (FIG.4C–4I). Intestinal absorption of ethanol and hepatic metabolism were similar in all groups. C7 did not decrease ethanol-induced steatohepatitis in mice colonized with feces from kpsM-negative patients with alcohol-associated hepatitis (FIG.4C–4I). Hepatic F4 / 80 and Marco mRNA were not significantly different in ethanol- fed mice between the groups. Our findings indicate that a small molecule inhibitor can selectively attenuate ethanol-induced liver disease caused by kpsM-positive E. coli in microbiota humanized mice.

[0114] Discussion

[0115] The presence of virulence-related genes in the gut metagenome, and more specifically of the virulence factor kpsM encoded in the genome of Escherichia coli, is independently associated with mortality in patients with alcohol-associated hepatitis, a non- communicable and non-infectious disease. The presence of kpsM in the gut microbiota was not associated with increased rates of infection. The broad phylogenetic distribution indicates that kpsM presence is a variable trait among E. coli isolates, and kpsM-positive E. coli are largely non-epidemic commensal strains. This is also consistent with the low frequency of kpsM detection in fecal samples, as epidemic isolates have typically higher frequencies of virulence genes64. Liver failure was the main cause of death in our cohort. Therefore, mechanisms beyond infection such as liver disease progression contribute to the association between mortality and the presence of these virulence factors in patients with alcohol- associated hepatitis. kpsM facilitates the transport of sialic acid to the capsule, resembling host sugar molecules. This enables translocated kpsM-positive E. coli to evade phagocytosis by Kupffer cells. Escaped bacteria are now able to bind to pattern recognition receptors on37759.0643P1 various cell types in the liver and thereby contribute to the progression of liver disease.

[0116] The discovery that precise interventions targeting specific bacterial virulence factors, such as the small molecule C7, can provide therapeutic benefits in alcohol-associated liver disease highlights a novel approach to treating a non-infectious condition. This approach is especially significant given the limited and often inadequate traditional treatment options for alcohol-associated liver disease, which frequently fail to address the underlying causes of disease progression. This new generation of antimicrobials offers distinct advantages over conventional antibiotics by preserving the commensal microbiota, thus reducing the selection pressure for multidrug resistance and the emergence of virulence factors.

[0117] The strength of this study is inclusion of patients through an international multi- center observational trial, which reduced the chances that the virulence-related genes studied are region specific. Limited by sequencing depth and thus probably less sensitive than qPCR, metagenomic sequencing might not fully capture the presence of virulence factors among all bacterial populations in the gut; increased cytolysin in fecal samples from patients with alcohol-associated hepatitis, which was detected by targeted quantitative PCR29, was not observed by shotgun sequencing. Therefore, a combination of different techniques can be used to detect genes that encode virulence factors in bacteria of low abundance.

[0118] This data shows the efficacy of a small molecule inhibitor against multidrug- resistant and virulent bacteria in a disease not typically classified as an infectious disease. However, C7 may also be beneficial for treating infectious diseases such as urinary tract infections caused by extraintestinal pathogenic E. coli. To assess this novel treatment strategy for patients with alcohol-associated hepatitis, a clinical trial is required.

[0119] Experimental Procedures

[0120] Human Subjects

[0121] A total of nine individuals without alcohol use disorder (controls), 41 patients with alcohol use disorder, and 81 patients with alcohol-associated hepatitis were included in the metagenomic analysis. Patient cohorts have been described in details30-32. Patients were identified as having alcohol use disorder if they fulfilled the DSM IV criteria33. Controls were social drinkers who consumed less than 20 g alcohol per day. Neither controls nor patients with alcohol use disorder took antibiotics or immunosuppressive medication during the 2 months preceding enrollment. Besides this cohort, 49 new subjects with alcohol- associated hepatitis were used as a validation cohort for qPCR analyses. Inclusion and exclusion criteria of patients with alcohol-associated hepatitis are described in our previous publication29. Patients with alcohol-associated hepatitis had clinical features of this disease.37759.0643P1 Liver biopsies were collected only if indicated as part of routine clinical care for the purpose of alcohol-associated hepatitis diagnosis. Histologic features of biopsies supported diagnoses of alcohol-associated hepatitis.

[0122] Shotgun metagenomic sequencing and analysis

[0123] DNA was extracted from human stool samples using FastDNA Spin Kit for Soil (MP-Biomedicals). Whole-genome shotgun metagenomic sequencing was performed on Illumina HiSeq 4000 generating 150 bp pair-end reads. Quality control of shotgun metagenomics reads was performed conducted as described previously34using a combination of KneadData v0.7.5. Taxonomic classification of processed metagenomics reads were performed using MetaPhIAn v3.135and Kraken2 v2.1.236against genomes of the Human Gastrointestinal Bacteria Genome Collection37. The maximum-likelihood phylogenetic trees of Escherichia coli were built by applying RAxML v8.2.1038(internally in StrainPhlAn39) on the alignment of E. coli marker genes, using default parameters and with the options ‘– alignment_program mafft’ and ‘–relaxed_parameters3’. The major lineages were taxonomically assigned to GTDB40species clusters using GTDB r89 representative genomes. HUMAnN 3.035was used to perform functional profiling of microbial metabolic pathways. Quality-filtered metagenomic reads were screened for the presence of genes encoding for bacterial virulence factors using ARIBA41with “--min_scaff_depth 1”, and otherwise default parameters. As previously described34, VFDB database42core virulence genes were grouped into clusters based on 90% nucleotide identity, and then included to build a BLAST database for virulence-factor screening. The detected virulence genes were grouped by their host genera as found in VFDB. All potential host genera were shown (e.g. Klebsiella / Yersinia) if the virulence factor gene cluster was found in multiple genera.

[0124] Whole-genome sequencing and genotyping

[0125] E. coli strains were isolated from patients’ stool samples using CHROMagarTME. coli selective medium (CHROMagar) and cultured in LB broth medium (Sigma). Genomic DNA was extracted using the QIAamp DNA microbiome Kit (QIAGEN). DNA libraries were constructed with 100-bp paired-end fragments and sequenced using an Illumina NovaSeq S4 platform. The quality of the original reads was evaluated using FASTQC. FASTX-trimmer and Trimmomatic were used for trimming sequencing reads43. Short-read sequence data were de novo assembled using SPAdes v3.15.344. Acquired antibiotic resistance genes (ARGs) and virulence genes were identified using ABRicate version 0.5 (https: / / github.com / tseemann / abricate) by aligning genome sequences to the ResFinder database45and VFDB database42with 80% coverage and 80% identity. For each de novo37759.0643P1 assembly, coding sequences were predicted using Prodigal v2.646and annotated using Prokka v1.13.347. The core genes were identified and used to build the core genome using Roary v3.1248with the –e –mafft setting to create a concatenated alignment of core genomic CDS. SNP-sites (https: / / github.com / sanger-pathogens / snp-sites) was used to extract the core genomic SNPs49. To construct a maximum likelihood phylogeny of the sequencing isolates, RAxML v8.2.10 was used with the generalized time-reversible model and a GTRGAMMA distribution to model site-specific rate variation50. Support for the ML phylogeny was assessed by 1000 bootstrap pseudo-analyses of the alignment data. iTOL51was used to visualize and edit the phylogenetic tree. To explore the prevalence of virulence factors in E. coli, available E. coli assembled genomes from RefSeq databases were retrieved as of January 1, 2022. Exclusion criteria for all the data were as follows: sequencing performed on non-Illumina platforms; single-end reads; sequencing depth of <30×; >300 assembled contigs; abnormal genome lengths.

[0126] Data availability

[0127] Sequence data were deposited in the European Nucleotide Archive under accession numbers ERP106878.

[0128] Fecal qPCR for kpsM

[0129] For DNA extraction, 250 mg stool were placed in 2 mL screw-cap tubes.500 μL of InhibitEX Buffer were added to each stool sample and maintained on ice. Samples were homogenized using a bead beater. Subsequently, genomic DNA from human stool was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen) following the provided instructions52. kpsM-positive E. coli and ^kpsM E. coli strains served as positive and negative controls, respectively, while nuclease-free water served as a no-template control. qPCR was performed as described above. Once amplification was complete, the system automatically generated Ct (cycle threshold) values for each reaction. A cut-off Ct value of <30 was applied to indicate the presence of kpsM.

[0130] Mice

[0131] Germ-free, female C57BL / 6 mice, bred at UC San Diego, were colonized twice with feces from patients diagnosed with alcohol-associated hepatitis. In brief, 1 g stool samples were dissolved in 30 mL LB broth containing 15% glycerol under anaerobic conditions. Subsequently, mice were orally administered 100 µl of the stool dilutions, initiating at 4-5 weeks of age and repeating the process two weeks later. After the second gavage, mice were subjected to a chronic-binge ethanol diet (NIAAA model) or an isocaloric control diet. Mice were fed with Lieber DeCarli diet, and the caloric intake from ethanol was37759.0643P1 0% on days 1–5 and 36% (v / v) from day 6 until the end of the study period. At day 16, mice were gavaged with a single dose of ethanol (5 g / kg body weight) in the early morning, followed by sacrifice 9 hours later. Pair-fed control mice received a diet with an isocaloric substitution of maltodextrin. For inhibiting kpsM in E. coli, mice were gavaged with the small molecule inhibitor C761(25 µM) every third day and 1 hour before the binge.

[0132] To colonize mice with ^kpsM or kpsM-positive E. coli, wild-type C57BL / 6 mice (female, age 8-10 weeks, Charles River) were subjected to the chronic-binge ethanol diet (NIAAA model) or an isocaloric control diet and gavaged with 1x109CFU ^kpsM or kpsM- positive E. coli every third day and 1 hour before the binge.

[0133] To knockdown Marco in Kupffer cells in vivo, mice with Cre expression under the Kupffer cell specific promoter Clec4f (Clec4f-Cre mice; female, age 8-10 weeks) were injected with AAV8-CAG-DIO-eGFP-mMarco-shRNAmir (Vector Biolabs; 1×1011Gc / mouse in 100 µL) or AAV8-CAG-DIO-eGFP-scrmb-shRNAmir as control (Vector Biolabs; 1×1011Gc / mouse in 100 µL) into the tail vein. AAV8 expresses double-floxed inverse ORF (DIO)-eGFP-shRNA driven by an ubiquitous CAG promoter. In the DIO scenario, scrambled shRNA or shRNA for silencing mouse Marco, is expressed in the presence of Cre in Kupffer cells. The transgene will not be expressed in the absence of Cre. After two weeks, mice were subjected to the chronic-binge ethanol diet as described above. Mice were gavaged with 1x109CFU E. coli every three days and 1 hour before the binge.

[0134] Biochemical and histological analyses

[0135] Blood was collected from mice in 2 mL collection tubes (Greiner Bio-One, Germany) and serum was obtained through centrifugation at 3000xg for 20 min. Serum levels of alanine transaminase (ALT) were determined using Infinity ALT kit (Thermo Scientific) to assess hepatic injury. Lipids were extracted from the liver. Hepatic triglyceride levels were measured according to manufacturer’s instructions using Triglyceride Liquid Reagents kit (Pointe Scientific). To detect absorption and hepatic metabolism of alcohol in mice, serum ethanol levels were determined using the Ethanol Colorimetric Assay kit (Abcam) according to manufacturer’s protocol.

[0136] Formalin-fixed liver samples were embedded in paraffin (Paraplast plus, McCornick) and 5 µm sections were stained with hematoxylin-eosin (H&E) (Surgipath) for liver morphology. Mice liver sections were embedded in OCT compound, and 5 µm frozen sections were stained with Oil Red O (Sigma-Aldrich) to visualize lipid accumulation.

[0137] RNA extraction and RT-qPCR

[0138] To isolate liver RNA, approximately 50 mg tissue pieces from the left lateral liver37759.0643P1 lobes were snap frozen in liquid nitrogen. Frozen tissue was homogenized mechanically using stainless steel beads in a Tissue Lyser II (Qiagen). RNA extraction was performed using TRIzol reagent (Life Technologies). For RNA isolation from Kupffer cells, cells were harvested by adding 1 mL TRIzol reagent to each well and employing the same procedure as for liver RNA isolation. RNA content and quality were assessed using Nanodrop (Peqlab). For quantitative real-time PCR, up to 1 µg of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) to generate cDNA. Real- time PCR was performed on an ABI StepOnePlus real-time PCR system (Bio-Rad Laboratories) using the SYBR mix kit (Bio-Rad Laboratories). Gene expression was normalized to mouse 18S rRNA. Expression of target genes in relation to reference gene was determined using the 2−∆∆CT method. Primer sequences for mouse genes were originally obtained from the NIH qPrimerDepot.

[0139] DNA extraction from sterile mouse liver and qPCR

[0140] For bacterial DNA extraction from sterile liver, right lobes of the mouse liver were carefully dissected under sterile conditions. Genomic DNA was isolated to quantify bacterial 16S as previously described with slight modifications53. Briefly, the tissue was weighed and homogenized using 1.0 mm Zirconia / Silica beads in PBST supplemented with Proteinase K, and digested with RNaseA and 10% SDS. The suspensions were then transferred to Qbiogene lysing matrix B tubes and homogenized with UltraPure Buffer- Saturated Phenol (Thermo Fisher Scientific) and lysate was extracted twice with UltraPure Phenol: Chloroform:isoamyl alcohol (Thermo Fisher Scientific) and once with chloroform and sodium acetate buffer. DNA was then precipitated, washed with ethanol, and resuspended in sterile water. Relative expression of kpsM was normalized to host 18S rRNA.

[0141] Isolation of primary mouse Kupffer cells

[0142] Kupffer cells were isolated as described. In brief, the liver of C57BL / 6 mice (age 12 weeks; Charles River) was perfused for 5 min at 10 mL / min with perfusion buffer SC1, followed by a two-step collagenase-pronase perfusion (Roche Diagnostics) at 10 mL / min for 5 min. The liver was dissected from the mouse and ruptured with forceps in buffer D containing collagenase D and pronase, followed by a 20-min incubation at 37 ºC. The cells were filtered through a 70 µm cell strainer and centrifuged at 800xg for 7 min. Supernatant was resuspended with GBSS-B. Kupffer cells were obtained by a three-layer discontinuous density gradient centrifugation with 8.2% (w / v) and 14.5% (w / v) Nycodenz (Accurate Chemical and Scientific Corporation) centrifugation at 2,000xg for 20 min without a brake. Isolated cells were counted and plated in 6-well or 12-well plates in RPMI 1640 (Thermo37759.0643P1 Fisher Scientific) containing 10% (v / v) FBS and 1% P / S. After 30 min attachment, the cell medium was changed and incubated at 37 ºC.

[0143] Phagocytosis and intracellular survival of E. coli within Kupffer cells

[0144] The intracellular survival of bacteria within macrophages was assessed following a previously described method with slight modifications54. Kupffer cells were plated at a density of 5 x 105cells per well in RPMI 1640 medium. On the day of co-culture, the medium was replaced with antibiotic-free RPMI 1640, and cells were infected with freshly cultured E. coli strains at a multiplicity of infection (MOI) of 30:1 for 2 h at 37 ºC. Then a mixture of 200 µg / ml of gentamicin, 500 µg / ml streptomycin, and 100 µg / ml chloramphenicol were added to the medium to eliminate extracellular bacteria for 1 h, followed by three PBS washes. Cells were then lysed with 0.1% Triton X-100 (Sigma) and cell lysates were diluted and plated on LB agar plates. Bacterial colonies were counted on the next day.

[0145] siRNA mediated in vitro knockdown of Marco

[0146] Three hours after isolation, primary mouse Kupffer cells were transfected with either control siRNA (ON-TARGETplus Non-targeting Control Pool, Horizon) or mouse Marco siRNA (ON-TARGETplus siRNA, Horizon) using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions55. After 24 h, cells were harvested for RNA extraction and qPCR to detection the gene expression of Marco.

[0147] Flow cytometry of E. coli binding to Marco

[0148] Freshly cultured and pelleted E. coli were resuspended in 1 mL of PBS with 1x 488 CellBrite Fix Membrane Dye (Biotium, green) to stain bacterial XYZ and incubated for 30 min at room temperature. After centrifugation at 12,000xg for 5 min, the pellet was washed with PBS three times. Recombinant mouse Marco (Sino Biological) was labeled with AF 647 using a protein labeling kit according to the manufacturer’s instructions. AF 647- labeled recombinant Marco protein (20 µg / ml) was incubated with 1x106CFU E. coli at room temperature for 2 h. Bacterial cells were then washed three times, resuspended in fixation buffer (1% PFA) for 30 min, and subjected to flow cytometry56and analyzed with FlowJo.

[0149] Transmission electron microscopy (TEM)

[0150] E. coli strains were culture overnight in LB broth with or without adding the small molecule inhibitor C7 by selecting a single colony from the LB agar plates. Then the culture was subjected to centrifugation at 12,000xg for 5 min and the supernatant was discarded. The bacterial pellets were fixed with 2% Glutaraldehyde in 0.1 M Sodium Cacodylate Buffer (pH 7.4) followed by post-fixation with 1% OsO4 in 0.1M cacodylate buffer for 1 h on ice. The37759.0643P1 cells were stained all at once with 2% uranyl acetate for 1 h and dehydrated in ethanol (50- 100%) on ice. The cells were washed for 10 min each once with 100% ethanol and two times with acetone and embedded with Durcupan. Sections were cut at 60 nm on a Leica UCT ultramicrotome, and picked up on 300 mesh copper grids. Sections were post-stained with 2% uranyl acetate for 5 min and Sato's lead stain for 1 min. Images were obtained by using Jeol 1400 plus Transmission Electron Microscope equipped with a bottom-mount Gatan digital camera at the University of California, San Diego-Cellular and Molecular Medicine Electron Microscopy Core.

[0151] Confocal and super-resolution imaging

[0152] Approximately 5 x 104primary Kupffer cells were plated in µ-slide 8 well glass bottom (ibidi). Kupffer cells were labeled 30 min at 37 ºC with a CellBrite intracellular cytoplasmic membrane dye (Biotium, red, Ex / Em: 644 / 665 nm), a lipophilic carbocyanine dye based on DiD. The cells were then washed three times with PBS. E. coli strains were freshly cultured overnight, centrifuged and the pellet was resuspended in PBS. Then the bacteria were stained with 1x 488 CellBrite Fix Membrane Dye (Biotium, green, Ex / Em: 480 / 513 nm) for 30 min at room temperature. After staining, the bacteria were washed 3 times by PBS. Then the bacteria were co-cultured with Kupffer cells for one hour. After that, the extracellular bacteria were removed by washing three times with PBS. Both live or fixed (with 4% PFA) cells were subjected to imaging. Images were acquired with a Leica SP8 confocal microscope and analysis using ImageJ57. For super-resolution imaging, images were collected with a Zeiss Elyra 7 Lattice SIM super-resolution instrument using a Plan Apochromatic 63× (1.40 NA) oil objective lens. Z-stack images were collected in Leap-mode with 10 phase positions of the lattice SIM pattern. Images were processed with Zeiss Zen Black SIM2 processing software.

[0153] Statistical analysis

[0154] Three or more groups were compared using the Kruskal-Wallis test with Dunn’s post-hoc test for continuous and Fisher’s exact test for categorical variables, each followed by false discovery rate (FDR) procedures to correct for multiple comparisons. Univariate and multivariate Cox regression analysis were used to assess associations of virulence factors with 180-day mortality. The multivariate model was adjusted for treatment with antibiotics. Kaplan-Meier curves were used to compare survival between virulence factor-positive and virulence factor-negative patients with alcoholic hepatitis. Two patients who underwent liver transplantation, the transplantation date was considered as the date of death. Patients were censored at the time point they were last seen alive. Spearman´s rank correlation coefficient37759.0643P1 was used to investigate associations between 2 continuous variables. To investigate gut microbiota profiles among patients with alcoholic hepatitis that were either positive or negative for specific virulence factors, the logarithmic relative abundance of all intestinal bacteria were calculated on the genus level. Jaccard dissimilarity matrices were determined for the principal component analysis (PCA) and P values were determined by permutational multivariate analysis of variance (PERMANOVA). Statistical analyses were performed using R statistical software, R version 3.5.1, 2018 the R Foundation for Statistical Computing. A P<.05 was considered to be statistically significant (adjusted for multiple comparison when performing multiple tests by False Discovery Rate (FDR)). All statistical tests were two- sided. For mouse or cell culture experiments, results are expressed as median and range unless stated otherwise. Two groups were compared using the Mann-Whitney-Wilcoxon rank-sum test for continuous and Fisher’s exact test for categorical variables.

[0155] 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 method and compositions described herein. Such equivalents are intended to be encompassed by the following claims. References

[0156] 1. Collaborators, G. B. D. C. The global, regional, and national burden of cirrhosis by cause in 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterol Hepatol 5, 245-266 (2020).

[0157] 2. Singal, A. K. & Mathurin, P. Diagnosis and Treatment of Alcohol-Associated Liver Disease: A Review. JAMA : the journal of the American Medical Association 326, 165- 176 (2021).

[0158] 3. Rehm, J., Samokhvalov, A. V. & Shield, K. D. Global burden of alcoholic liver diseases. J Hepatol 59, 160-168 (2013).

[0159] 4. Lozano, R. et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380, 2095-2128 (2012).

[0160] 5. Lee, B. P., Vittinghoff, E., Dodge, J. L., Cullaro, G. & Terrault, N. A. National Trends and Long-term Outcomes of Liver Transplant for Alcohol-Associated Liver Disease in the United States. JAMA Internal Medicine 179, 340-348 (2019).

[0161] 6. Seitz, H. K. et al. Alcoholic liver disease. Nature Reviews Disease Primers 4, 16 (2018).

[0162] 7. Seki, E. & Schwabe, R. F. Hepatic inflammation and fibrosis: Functional links37759.0643P1 and key pathways. Hepatology 61, 1066-1079 (2015).

[0163] 8. Bataller, R., Arab, J. P. & Shah, V. H. Alcohol-Associated Hepatitis. The New England journal of medicine 387, 2436-2448 (2022).

[0164] 9. Singal, A. K. et al. Hospitalizations for Acute on Chronic Liver Failure at Academic Compared to Non-academic Centers Have Higher Mortality. Digestive diseases and sciences 66, 1306-1314 (2021).

[0165] 10. Singal, A. K. et al. Alcohol-associated liver disease in the United States is associated with severe forms of disease among young, females and Hispanics. Aliment Pharmacol Ther 54, 451-461 (2021).

[0166] 11. Thursz, M. R. et al. Prednisolone or Pentoxifylline for Alcoholic Hepatitis. New England Journal of Medicine 372, 1619-1628 (2015).

[0167] 12. Mathurin, P. & Lucey, M. R. Management of alcoholic hepatitis. Journal of Hepatology 56, S39-S45 (2012).

[0168] 13. Lucey, M. R., Mathurin, P. & Morgan, T. R. Alcoholic hepatitis. New England Journal of Medicine 360, 2758-2769 (2009).

[0169] 14. Hsu, C. L. & Schnabl, B. The gut-liver axis and gut microbiota in health and liver disease. Nature reviews. Microbiology (2023).

[0170] 15. Chen, P., Stärkel, P., Turner, J. R., Ho, S. B. & Schnabl, B. Dysbiosis-induced intestinal inflammation activates tumor necrosis factor receptor I and mediates alcoholic liver disease in mice. Hepatology 61, 883-894 (2015).

[0171] 16. Stärkel, P. & Schnabl, B. Bidirectional Communication between Liver and Gut during Alcoholic Liver Disease. Semin Liver Dis 36, 331-339 (2016).

[0172] 17. Lang, S. & Schnabl, B. Microbiota and Fatty Liver Disease-the Known, the Unknown, and the Future. Cell host & microbe 28, 233-244 (2020).

[0173] 18. Leclercq, S. et al. Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity. Proceedings of the National Academy of Sciences of the United States of America 111, E4485-4493 (2014).

[0174] 19. Puri, P. et al. The circulating microbiome signature and inferred functional metagenomics in alcoholic hepatitis. Hepatology 67, 1284-1302 (2018).

[0175] 20. Perlemuter, G. Circulating bugs in blood in alcoholic liver disease! Hepatology 67, 1207-1209 (2018).

[0176] 21. Zhong, W. et al. Paneth cell dysfunction mediates alcoholic steatohepatitis through promoting bacterial translocation in mice: role of zinc deficiency. Hepatology n / a (2019).37759.0643P1

[0177] 22. Uesugi, T., Froh, M., Arteel, G. E., Bradford, B. U. & Thurman, R. G. Toll- like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice. 108 (2001).M. et al. Intestinal microbiota contributes to individual susceptibility to alcoholic liver disease. Gut 65, 830 (2016).

[0179] 24. Philips, C. A. et al. Healthy Donor Fecal Microbiota Transplantation in Steroid-Ineligible Severe Alcoholic Hepatitis: A Pilot Study. Clinical Gastroenterology and Hepatology 15, 600-602 (2017).

[0180] 25. Chen, Y. et al. Characterization of fecal microbial communities in patients with liver cirrhosis. Hepatology 54, 562-572 (2011).

[0181] 26. Zhu, L., Baker, R. D., Zhu, R. & Baker, S. S. Sequencing the gut metagenome as a noninvasive diagnosis for advanced nonalcoholic steatohepatitis. Hepatology 66, 2080- 2083 (2017).

[0182] 27. Caussy, C. et al. Link between gut-microbiome derived metabolite and shared gene-effects with hepatic steatosis and fibrosis in NAFLD. Hepatology 68, 918-932 (2018).

[0183] 28. Ponziani, F. R. et al. Hepatocellular Carcinoma Is Associated With Gut Microbiota Profile and Inflammation in Nonalcoholic Fatty Liver Disease. Hepatology 69, 107-120 (2019).

[0184] 29. Duan, Y. et al. Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease. Nature 575, 505-511 (2019).

[0185] 30. Chu, H. et al. The Candida albicans exotoxin Candidalysin promotes alcohol- associated liver disease. Journal of Hepatology (2019).

[0186] 31. Lang, S. et al. Intestinal fungal dysbiosis and systemic immune response to fungi in patients with alcoholic hepatitis. Hepatology n / a (2019).

[0187] 32. Gao, B. et al. Serum and fecal oxylipins in patients with alcohol-related liver disease. Digestive Diseases and Sciences 64, 1878-1892 (2019).

[0188] 33. Ball, S. A., Tennen, H., Poling, J. C., Kranzler, H. R. & Rounsaville, B. J. Personality, temperament, and character dimensions and the DSM-IV personality disorders in substance abusers. Journal of Abnormal Psychology 106, 545-553 (1997).

[0189] 34. Shao, Y. et al. Stunted microbiota and opportunistic pathogen colonization in caesarean-section birth. Nature 574, 117-121 (2019).

[0190] 35. Beghini, F. et al. Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3. Elife 10 (2021).

[0191] 36. Wood, D. E., Lu, J. & Langmead, B. Improved metagenomic analysis with37759.0643P1 Kraken 2. Genome biology 20 (2019).

[0192] 37. Forster, S. C. et al. A human gut bacterial genome and culture collection for improved metagenomic analyses. Nature Biotechnology 37, 186-192 (2019).

[0193] 38. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post- analysis of large phylogenies. Bioinformatics 30, 1312-1313 (2014).

[0194] 39. Truong, D. T., Tett, A., Pasolli, E., Huttenhower, C. & Segata, N. Microbial strain-level population structure and genetic diversity from metagenomes. Genome Research 27, 626-638 (2017).

[0195] 40. Parks, D. H. et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nature Biotechnology 36, 996-1004 (2018).

[0196] 41. Hunt, M. et al. ARIBA: rapid antimicrobial resistance genotyping directly from sequencing reads. Microbial Genomics 3, e000131 (2017).

[0197] 42. Chen, L., Zheng, D., Liu, B., Yang, J. & Jin, Q. VFDB 2016: hierarchical and refined dataset for big data analysis--10 years on. Nucleic acids research 44, D694-697 (2016).

[0198] 43. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014).

[0199] 44. Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of computational biology 19, 455-477 (2012).

[0200] 45. Zankari, E. et al. Identification of acquired antimicrobial resistance genes. Journal of Antimicrobial Chemotherapy 67, 2640-2644 (2012).

[0201] 46. Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC bioinformatics 11, 119 (2010).

[0202] 47. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068-2069 (2014).

[0203] 48. Page, A. J. et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31, 3691-3693 (2015).

[0204] 49. Page, A. J. et al. SNP-sites: rapid efficient extraction of SNPs from multi- FASTA alignments. Microb Genom 2, e000056 (2016).

[0205] 50. Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688-2690 (2006).

[0206] 51. Letunic, I. & Bork, P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic acids research 44, W242-245 (2016).37759.0643P1

[0207] 52. Cabre, N., Yang, Y., Wang, Y. & Schnabl, B. Development of a Quantitative PCR Method for Detecting Enterococcus faecalis Cytolysin in Human Stool Samples. Methods Protoc 6 (2023).

[0208] 53. Llorente, C. et al. Gastric acid suppression promotes alcoholic liver disease by inducing overgrowth of intestinal Enterococcus. Nature communications 8, 837 (2017).

[0209] 54. Hoh, C. H., Tan, Y. H. & Gan, Y. H. Protective Role of Kupffer Cells and Macrophages in Klebsiella pneumoniae-Induced Liver Abscess Disease. Infection and immunity 87 (2019).

[0210] 55. Kouno, T. et al. Selective PPARdelta agonist seladelpar suppresses bile acid synthesis by reducing hepatocyte CYP7A1 via the fibroblast growth factor 21 signaling pathway. The Journal of biological chemistry 298, 102056 (2022).

[0211] 56. Zeng, Z. T. et al. CRIg Functions as a Macrophage Pattern Recognition Receptor to Directly Bind and Capture Blood-Borne Gram-Positive Bacteria. Cell host & microbe 20, 99-106 (2016).

[0212] 57. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676-682 (2012).

[0213] 58. Chen, L., Zheng, D., Liu, B., Yang, J. & Jin, Q. VFDB 2016: hierarchical and refined dataset for big data analysis—10 years on. Nucleic Acids Research 44, D694-D697 (2015).

[0214] 59. Pitout, J. D. & DeVinney, R. Escherichia coli ST131: a multidrug-resistant clone primed for global domination. F1000Res 6 (2017).

[0215] 60. Lin, B. X., Qiao, Y., Shi, B. & Tao, Y. Polysialic acid biosynthesis and production in Escherichia coli: current state and perspectives. Applied microbiology and biotechnology 100, 1-8 (2016).

[0216] 61. Goller, C. C. & Seed, P. C. High-throughput identification of chemical inhibitors of E. coli Group 2 capsule biogenesis as anti-virulence agents. PloS one 5, e11642 (2010).

[0217] 62. Lehti, T. A., Pajunen, M. I., Skog, M. S. & Finne, J. Internalization of a polysialic acid-binding Escherichia coli bacteriophage into eukaryotic neuroblastoma cells. Nature communications 8, 1915 (2017).

[0218] 63. Arvaniti, V. et al. Infections in Patients With Cirrhosis Increase Mortality Four-Fold and Should Be Used in Determining Prognosis. Gastroenterology 139, 1246- 1256.e1245 (2010).

[0219] 64. Bert, F. et al. Genetic diversity and virulence profiles of Escherichia coli37759.0643P1 isolates causing spontaneous bacterial peritonitis and bacteremia in patients with cirrhosis. Journal of Clinical Microbiology 48, 2709-2714 (2010).

[0220] 65. Brandl, K. et al. Dysregulation of serum bile acids and FGF19 in alcoholic hepatitis. Journal of Hepatology 69, 396-405 (2018).

Claims

37759.0643P1 CLAIMS We claim:

1. A method of treating or ameliorating a symptom of alcohol-associated hepatitis in a subject comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby treating or ameliorating a symptom of alcohol-associated hepatitis in the subject.

2. The method of claim 1, wherein the subject has or has been diagnosed with alcohol- associated hepatitis.

3. The method of claim 1 or claim 2, wherein the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

4. The method of any one of claims 1 to 3, wherein the subject was identified as being in need thereof by determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

5. The method of claim 3 or claim 4, wherein the bacterial virulence factor is kpsM (Escherichia coli) or ecpR (Escherichia coli).

6. The method of claim 5, wherein the bacterial virulence factor is ecpR (Escherichia coli).

7. The method of claim 5, wherein the bacterial virulence factor is kpsM (Escherichia coli).

8. The method of claim 7, wherein the kpsM (Escherichia coli) is multi-drug resistant.

9. The method of any one of claims 1 to 8, further comprising administering to the subject a therapeutically effective amount of an antibiotic.

10. The method of claim 9, wherein the antibiotic is co-formulated with the small molecule inhibitor C7.

11. The method of claim 9 or claim 10, wherein the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin,37759.0643P1 doxycycline, amoxicillin-clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin, azithromycin, sulfamethoxazole-trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

12. The method of any one of claims 1 to 11, wherein the symptom is liver injury, steatosis, and / or inflammation.

13. A method of reducing or preventing mortality associated with alcohol-associated hepatitis in a subject at risk of mortality associated with alcohol-associated hepatitis comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby reducing or preventing mortality in the subject.

14. The method of claim 13, wherein the subject has or has been diagnosed with alcohol- associated hepatitis.

15. The method of claim 13 or claim 14, wherein the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

16. The method of any one of claims 13 to 15, wherein the subject was identified as being in need thereof by determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

17. The method of claim 15 or claim 16, wherein the bacterial virulence factor is kpsM (Escherichia coli) or ecpR (Escherichia coli).

18. The method of claim 17, wherein the bacterial virulence factor is ecpR (Escherichia coli).

19. The method of claim 17, wherein the bacterial virulence factor is kpsM (Escherichia coli).

20. The method of claim 19, wherein the kpsM (Escherichia coli) is multi-drug resistant.

21. The method of any one of claims 13 to 20, further comprising administering to the subject a therapeutically effective amount of an antibiotic.

22. The method of claim 21, wherein the antibiotic is co-formulated with the small molecule inhibitor C7.37759.0643P1 23. The method of claim 21 or claim 22, wherein the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin, doxycycline, amoxicillin-clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin, azithromycin, sulfamethoxazole-trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

24. A method of treating or ameliorating a symptom of sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis in a subject comprising: administering a therapeutically effective amount of the small molecule inhibitor C7 to the subject, thereby treating or ameliorating a symptom of alcohol-associated hepatitis in the subject.

25. The method of claim 24, wherein the subject has or has been diagnosed with sepsis, urinary tract infection, pneumonia, intra-abdominal infections, and / or endocarditis.

26. The method of claim 24 or claim 25, wherein the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

27. The method of any one of claims 24 to 26, wherein the subject was identified as being in need thereof by determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample.

28. The method of claim 26 or claim 27, wherein the bacterial virulence factor is kpsM (Escherichia coli) or ecpR (Escherichia coli).

29. The method of claim 28, wherein the bacterial virulence factor is ecpR (Escherichia coli).

30. The method of claim 28, wherein the bacterial virulence factor is kpsM (Escherichia coli).

31. The method of claim 30, wherein the kpsM (Escherichia coli) is multi-drug resistant.

32. The method of any one of claims 24 to 31, further comprising administering to the subject a therapeutically effective amount of an antibiotic.37759.0643P1 33. The method of claim 32, wherein the antibiotic is co-formulated with the small molecule inhibitor C7.

34. The method of claim 32 or claim 33, wherein the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin, doxycycline, amoxicillin-clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin, azithromycin, sulfamethoxazole-trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

35. The method of any one of claims 24 to 34, wherein the symptom is fever, swelling, pain, and / or systemic and organ-specific inflammation.

36. A method of determining responsiveness of a subject to treatment with the small molecule inhibitor C7 comprising: determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample from said subject, wherein when the patient has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus, the subject will be responsive to treatment with the small molecule inhibitor C7.

37. The method of claim 36, wherein the subject has alcohol-associated hepatitis.

38. The method of claim 36 or claim 37, wherein the bacterial virulence factor is ecpR (Escherichia coli).

39. The method of claim 36 or claim 37, wherein the bacterial virulence factor is kpsM (Escherichia coli).

40. The method of claim 39, wherein the kpsM (Escherichia coli) is multi-drug resistant.

41. A method of identifying a subject for a clinical study for a treatment for alcohol- associated hepatitis with composition comprising small molecule inhibitor C7, comprising determining that the subject has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus in a fecal sample from said subject, wherein when the patient has a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus, the subject is appropriate for the clinical study for a treatment for alcohol-associated hepatitis.37759.0643P1 42. The method of claim 41, wherein the bacterial virulence factor is kpsM (Escherichia coli) or ecpR (Escherichia coli).

43. The method of claim 42, wherein the bacterial virulence factor is ecpR (Escherichia coli).

44. The method of claim 42, wherein the bacterial virulence factor is kpsM (Escherichia coli).

45. The method of claim 44, wherein the kpsM (Escherichia coli) is multi-drug resistant.

46. A method for screening for a candidate compound for treating alcohol-associated hepatitis comprising: (1) contacting the candidate compound with a sample comprising a bacterial virulence factor from Escherichia, Klebseilla, Shigella, Klebsiella / Yersinia, and / or Enterococcus; and (2) determining that the bacteria is reduced / killed, thereby (3) identifying a compound for treating alcohol-associated hepatitis.

47. The method of claim 46, wherein the bacterial virulence factor is ecpR (Escherichia coli).

48. The method of claim 46, wherein the bacterial virulence factor is kpsM (Escherichia coli).

49. A composition comprising: the small molecule inhibitor C7 and an antibiotic.

50. The composition of claim 49, wherein the antibiotic is piperacillin / tazobactam, ceftriaxone, cotrimoxazole, cefepime, metronidazole, meropenem, imipenem, oxacillin, doxycycline, amoxicillin-clavulanate, rifaximin, ciprofloxacin, levofloxacin, norfloxacin, azithromycin, sulfamethoxazole-trimethoprim, ceftazidime, tazocilline, and / or vancomycin.

Images