Methods and compositions for treating intestinal diseases

Abstract

The presently disclosed subject matter relates to methods, compositions, and food products for improving intestinal health, treating intestinal dysbiosis, and / or treating intestinal diseases in a subject, such as a human or companion animal.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 796,021, filed on January 23, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The presently disclosed subject matter relates to methods, compositions, and food products for improving intestinal health, treating intestinal dysbiosis, and / or treating intestinal disorders in a subject, such as a human or a companion animal. Background Art

[0004] Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is a multifactorial and debilitating disorder characterized by chronic immunopathology, disruption of intestinal homeostasis, and altered composition of the gut microbiome (dysbiosis). Several lines of evidence suggest that resident gut bacteria are important players in the etiology of IBD. First, disease is often more severe in areas with the highest intestinal microbial biomass, and antibiotics are frequently used as adjunct therapy to immunosuppressants or monoclonal antibodies to manage IBD. 1,2 Second, genome-wide association studies have identified many susceptibility loci in genes responsible for recognizing or responding to bacteria. Finally, in certain mouse models of colitis, the disease can be reversed by fecal transplantation. 4-6 Transfer to the naive host, suggesting that the gut microbes play a causal role in the disease. Collectively, these findings lead to a 'two-hit' model of IBD, in which both host genetics and microbial factors influence disease manifestation, highlighting the opportunity to develop new IBD therapies targeting the microbiome. Therefore, new methods and compositions for the treatment of IBD and other intestinal diseases targeting the gut microbiome and its metabolites are needed. Summary of the invention

[0005] The subject matter disclosed herein provides pharmaceutical compositions, dietary supplements, and functional foods for use in medicine. In certain embodiments, the pharmaceutical composition, dietary supplement, or functional food comprises an effective amount of bacteria capable of producing a first bile acid for use as a medicine. In certain embodiments, the pharmaceutical composition, dietary supplement, or functional food further comprises an effective amount of a second bile acid. In certain embodiments, the pharmaceutical composition, dietary supplement, or functional food is used to treat an intestinal disease in a subject in need thereof.

[0006] In certain embodiments, the bacteria include a bile acid-inducible operon (bai operon). In certain embodiments, the bile acid-inducible operon (bai operon) comprises a nucleotide sequence that is at least about 90% homologous or identical to SEQ ID NO: 1 or SEQ ID NO: 3 or any functional fragment thereof. In certain embodiments, the bile acid-inducible operon (bai operon) comprises a nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

[0007] In certain embodiments, the bacteria comprises a 16s rRNA comprising a nucleotide sequence that is at least about 90% homologous to or identical to SEQ ID NO: 2 or SEQ ID NO: 4. In certain embodiments, the bacteria comprises a 16s rRNA comprising a nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

[0008] In certain embodiments, the bacterium is C. hiranonis, C. scindens, or a combination thereof. In certain embodiments, the bacterium is C. hiranonis.

[0009] In certain embodiments, the first bile acid and / or the second bile acid is selected from the group consisting of chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, alpha-muricholic acid, deoxycholic acid, gamma-muricholic acid, glycolithocholic acid, taurolithocholic acid acid), lithocholic acid, omega-muricholic acid, and any combination thereof.

[0010] In certain embodiments, the first bile acid and / or the second bile acid are secondary bile acids. In certain embodiments, the secondary bile acid is selected from the group consisting of taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid, and any combination thereof. In certain embodiments, the secondary bile acid is deoxycholic acid and / or lithocholic acid.

[0011] In some embodiments, the subject is a dog. In some embodiments, the intestinal disease is an acute intestinal disease or a chronic intestinal disease. In some embodiments, the chronic intestinal disease is selected from the group consisting of food-responsive intestinal disease, antibiotic-responsive intestinal disease, and idiopathic inflammatory bowel disease (IBD). In some embodiments, the intestinal disease is idiopathic inflammatory bowel disease (IBD).

[0012] In certain embodiments, the bacteria are transformed with a vector comprising a bile acid inducible operon (bai operon). In certain embodiments, the bacteria are selected from the genus Clostridium.

[0013] In certain embodiments, the amount of bacteria is between about 10,000 CFU and about 100 trillion CFU. In certain embodiments, the second bile acid is between about 10 mg / unit dose and about 500 mg / unit dose.

[0014] In certain embodiments, the first bile acid and the second bile acid are the same. In certain embodiments, the first bile acid and the second bile acid are different.

[0015] The presently disclosed subject matter provides Clostridium hirano for use as a functional food or supplement to prevent the onset of GI disorders or as a medicament. In certain embodiments, Clostridium hirano is used to treat intestinal diseases in a subject in need thereof. The presently disclosed subject matter provides Clostridium scindapsus functional food or supplement to prevent the onset of GI disorders or as a medicament. In certain embodiments, Clostridium scindapsus is used to treat intestinal diseases in a subject in need thereof.

[0016] The presently disclosed subject matter provides deoxycholic acid for use in treating inflammatory bowel disease (IBD) in a subject in need thereof. The presently disclosed subject matter provides lithocholic acid for use in treating inflammatory bowel disease (IBD) in a subject in need thereof.

[0017] The presently disclosed subject matter provides a dietary supplement or food comprising an effective amount of bacteria capable of producing a first bile acid. In certain embodiments, the dietary supplement or food further comprises an effective amount of a second bile acid. In certain embodiments, the food improves the intestinal health of the subject. In certain embodiments, the amount of bacteria is between about 10,000 CFU and about 100 trillion CFU. In certain embodiments, the second bile acid is between about 100 mg / daily dosage and about 1000 mg / daily dosage.

[0018] In certain embodiments, the food product is a pet food product. In certain embodiments, the food product is a dog food product.

[0019] The subject matter of the present disclosure provides a method for treating an intestinal disease in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition disclosed herein, a dietary supplement or a functional food, an effective amount of a food disclosed herein, or a combination thereof. In certain embodiments, the method further comprises monitoring the intestinal microorganisms of the subject. In certain embodiments, the intestinal microorganisms are sampled from a fecal sample of the subject.

[0020] The presently disclosed subject matter provides a method for determining susceptibility to intestinal diseases in companion animals. In certain embodiments, the method comprises:

[0021] a) measuring a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism in a companion animal;

[0022] b) comparing a first amount of a first intestinal microorganism with a first reference amount of the first intestinal microorganism, and / or comparing a second amount of a second intestinal microorganism with a second reference amount of the second intestinal microorganism, wherein the reference amounts of the intestinal microorganisms are determined based on amounts of intestinal microorganisms of a plurality of healthy companion animals; and

[0023] c) determining that the companion animal is susceptible to an intestinal disease when the first amount of the intestinal microorganism is higher than a first reference amount of the first intestinal microorganism, and / or when the second amount of the second intestinal microorganism is lower than a second reference amount of the second intestinal microorganism.

[0024] In certain embodiments, the first intestinal microorganism comprises one or more bacteria comprising 16S rRNA, wherein the 16S rRNA comprises HQ802983.1.1440, GQ449092.1.1375, GQ448744.1.1393, KF842598.1.1394, HG798451.1.1400, New Reference (New Reference)OTU52, HK555938.1.1357, FJ957494.1.1454, FN667392.1.1495, New reference OTU54, HQ760911.1.1437, GQ006324.1.1342, FJ950694.1.1472, FM865905.1.1392, FJ506371.1.1371, FJ957528.1.1445, JF712 675.1.1540, new reference OTU82, AB009242.1.1451, HQ751549.1.1448, AB506370.1.1516, DQ057365.1.1393, FN667422.1.1495, AJ270486.1.1241, FN668375.4306350.4307737, GQ867426.1.1494, GX182404.8.1529, JF 224013.1.1362, GQ448246.1.1389, KC245406.1.1465, FN667084.1.1493, EU470512.1.1400, EU768569.1.1352, AY239462.1.1500, KC504009.1.1465, FM179752.1.1686, new reference OTU114, HK557089.3.1395, JQ2081 81.1.1352, HQ803964.1.1435, AM276759.1.1484, JN387556.1.1324, GQ448486.1.1387, HK694029.9.1487, HQ754680.1.1441, FN563300.1.1447, FP929060.3837.5503, GQ448506.1.1374, 16S rRNA nucleotide sequence of Enterococcus durans, C. perfringens or Escherichia coli (E. coli) is at least about 90% homologous or identical to the 16S rRNA nucleotide sequence.

[0025] In some embodiments, the first intestinal microorganism is selected from the group consisting of: HQ802983.1.1440, GQ449092.1.1375, GQ448744.1.1393, KF842598.1.1394, HG798451.1.1400, new reference OTU52, HK555938.1.1357, FJ957494.1.1454, FN667392.1.1495, new reference OTU54, HQ760911.1.1437, GQ006324.1.1342, F J950694.1.1472, FM865905.1.1392, FJ506371.1.1371, FJ957528.1.1445, JF712675.1.1540, new reference OTU82, AB009242.1.1451, HQ751549.1.1448, AB506370.1.1516, DQ057365.1.1393, FN667422.1.1495, AJ270486.1.1241, FN668375.4306350.43077 37, GQ867426.1.1494, GX182404.8.1529, JF224013.1.1362, GQ448246.1.1389, JF807116.1.1260, KC245406.1.1465, FN667084.1.1493, EU470512.1.1400, EU768569.1.1352, AY239462.1.1500, KC504009.1.1465, FM179752.1.1686, new reference OTU114, H K557089.3.1395, JQ208181.1.1352, HQ803964.1.1435, AM276759.1.1484, JN387556.1.1324, GQ448486.1.1387, HK694029.9.1487, HQ754680.1.1441, FN563300.1.1447, FP929060.3837.5503, GQ448506.1.1374, Enterococcus durabilis, Clostridium perfringens, Escherichia coli, and any combination thereof. In certain embodiments, the first intestinal microorganism is Clostridium perfringens, Escherichia coli, and any combination thereof.

[0026] In certain embodiments, the second intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence that is at least about 90% homologous to or identical to a 16S rRNA nucleotide sequence of EU774020.1.1361, HQ793763.1.1451, HQ792787.1.1438, the new reference OTU109, HQ792778.1.1436, or DQ113765.1.1450.

[0027] In certain embodiments, the second intestinal microorganism is selected from the group consisting of EU774020.1.1361, HQ793763.1.1451, HQ792787.1.1438, new reference OTU109, HQ792778.1.1436, DQ113765.1.1450, and any combination thereof.

[0028] In certain embodiments, the method further comprises providing a customized recommendation for a treatment regimen when the first amount of the first intestinal microorganism is lower than a first reference amount of the first intestinal microorganism and / or when the second amount of the second intestinal microorganism is higher than a second reference amount of the second intestinal microorganism, and / or further monitoring the intestinal microorganism.

[0029] The presently disclosed subject matter provides a method for determining the responsiveness of a companion animal with an intestinal disease to a diet. In certain embodiments, the method comprises:

[0030] a) measuring a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism in a companion animal;

[0031] b) comparing a first amount of a first intestinal microorganism with a first reference amount of the first intestinal microorganism, and / or comparing a second amount of a second intestinal microorganism with a second reference amount of the second intestinal microorganism, wherein the reference amounts of the intestinal microorganisms are determined based on amounts of intestinal microorganisms of a plurality of healthy companion animals; and

[0032] c) determining that the companion animal is responsive to the diet when the first amount of the intestinal microorganism is above a first reference amount of the first intestinal microorganism and / or when the second amount of the second intestinal microorganism is below a second reference amount of the second intestinal microorganism, or determining that the companion animal is not responsive to the diet when the first amount of the intestinal microorganism is below a first reference amount of the first intestinal microorganism and / or when the second amount of the second intestinal microorganism is above a second reference amount of the second intestinal microorganism.

[0033] In certain embodiments, the first intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a 16S rRNA nucleotide sequence that is at least about 90% homologous to or identical to a 16S rRNA nucleotide sequence of reference OTU45, JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, new reference OTU54, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, new reference OTU52, or JQ208053.1.1336. The nucleotide sequence is identical to the rRNA nucleotide sequence.

[0034] In certain embodiments, the first intestinal microorganism is selected from the group consisting of: new reference OTU45, JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, new reference OTU54, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, new reference OTU52, JQ208053.1.1336, and any combination thereof.

[0035] In certain embodiments, the second intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence that is at least about 90% homologous or identical to a 16S rRNA nucleotide sequence of HK693629.1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456, or HK555938.1.1357.

[0036] In certain embodiments, the second intestinal microorganism is selected from the group consisting of: HK693629.1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456, HK555938.1.1357, and any combination thereof.

[0037] In certain embodiments, the method further comprises administering the diet to the companion animal when the companion animal is determined to be responsive to the diet. In certain embodiments, the method further comprises administering the diet, steroids, and optionally antibiotics to the companion animal when the companion animal is determined to be unresponsive to the diet.

[0038] In certain embodiments, the determining in step c) occurs prior to administering the diet or the diet, steroids, and optionally antibiotics to the companion animal.

[0039] The presently disclosed subject matter provides a method for determining the effectiveness of a diet in treating an intestinal disease in a companion animal. In certain embodiments, the method comprises:

[0040] a) measuring a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism in a companion animal after administering a diet to treat an intestinal disease to the companion animal;

[0041] b) comparing a first amount of a first intestinal microorganism with a first reference amount of the first intestinal microorganism, and / or comparing a second amount of a second intestinal microorganism with a second reference amount of the second intestinal microorganism, wherein the reference amount of the intestinal microorganism is determined based on amounts of intestinal microorganisms of a plurality of healthy companion animals; and

[0042] c) when the first amount of intestinal microorganisms is higher than a first reference amount of the first intestinal microorganisms, and / or when the second amount of the second intestinal microorganisms is lower than a second reference amount of the second intestinal microorganisms, determining that the diet is effective for treating the intestinal disease, or when the first amount of intestinal microorganisms is lower than the first reference amount of the first intestinal microorganisms, and / or when the second amount of the second intestinal microorganisms is higher than the second reference amount of the second intestinal microorganisms, determining that the diet is ineffective for treating the intestinal disease.

[0043] In certain embodiments, the first intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence that is at least about 90% homologous or identical to the 16S rRNA nucleotide sequence of HK557089.3.1395 or GQ448336.1.1418.

[0044] In certain embodiments, the first intestinal microorganism is selected from the group consisting of HK557089.3.1395, GQ448336.1.1418, and combinations thereof.

[0045] In certain embodiments, the second intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence that is at least about 90% homologous or identical to a 16S rRNA nucleotide sequence of KF842598.1.1394, GQ006324.1.1342, HQ802983.1.1440, JN387556.1.1324, FJ950694.1.1472, HG798451.1.1400, the new reference OTU52, or GQ448468.1.1366.

[0046] In certain embodiments, the second intestinal microorganism is selected from the group consisting of KF842598.1.1394, GQ006324.1.1342, HQ802983.1.1440, JN387556.1.1324, FJ950694.1.1472, HG798451.1.1400, new reference OTU52, GQ448468.1.1366, and any combination thereof.

[0047] In certain embodiments, the method further comprises administering the diet to the companion animal when the companion animal is determined to be responsive to the diet. In certain embodiments, the method further comprises administering the diet, steroids, and optionally antibiotics to the companion animal when the companion animal is determined to be unresponsive to the diet.

[0048] In certain embodiments, the determining in step c) occurs prior to administering the diet or the diet, steroids, and optionally antibiotics to the companion animal.

[0049] In certain embodiments, the reference amount of intestinal microorganisms is derived from the average amount of intestinal microorganisms in a plurality of healthy companion animals. In certain embodiments, the amount of intestinal bacteria is measured from a fecal sample of a subject.

[0050] The presently disclosed subject matter provides a diet for increasing the bacterial population capable of producing bile acids in companion animals. In certain embodiments, the diet comprises protein, fat, crude fiber, total dietary fiber, carbohydrates, calcium, phosphorus, sodium, chloride, potassium, magnesium, iron, copper, manganese, zinc, iodine, selenium, vitamin A, vitamin D3, vitamin E, vitamin C, thiamine (vitamin B1), riboflavin (vitamin B2), pantothenic acid, niacin, pyridoxine (vitamin B6), folic acid, biotin, cobalamin (vitamin B12), choline, arginine, lysine, methionine, cystine, taurine, linoleic acid, arachidonic acid, omega-6 fatty acids, omega-3 fatty acids, EPA and / or DHA.

[0051] In certain embodiments, the subject is a dog. In certain embodiments, the diet is a Royal Canin Veterinary Diet. In certain embodiments, the diet is selected from the group consisting of Ultramino, Hydrolyzed Protein Adult HP Dry, Hydrolyzed Protein Wet, Hydrolyzed Protein Adult PS Dry, Hydrolyzed Protein Moderate Calorie Dry, Hydrolyzed Protein Small Dog Dry, Hydrolyzed protein Treats, and any combination thereof.

[0052] In certain embodiments, the bacterium comprises a bile acid inducible operon. In certain embodiments, the bacterium is Clostridium hirano, Clostridium fulgidus, or a combination thereof. In certain embodiments, the bacterium is Clostridium hirano.

[0053] The presently disclosed subject matter provides a Royal Canin veterinary diet for treating an intestinal disease in a dog, wherein the dog comprises a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism, and wherein the first amount of the first intestinal microorganism is higher than a first reference amount of the first intestinal microorganism, and / or the second amount of the second intestinal microorganism is lower than a second reference amount of the second intestinal microorganism.

[0054] In certain embodiments, the first intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a 16S rRNA nucleotide sequence that is at least about 90% homologous to or identical to a 16S rRNA nucleotide sequence of reference OTU45, JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, new reference OTU54, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, new reference OTU52, or JQ208053.1.1336. The nucleotide sequence is identical to the rRNA nucleotide sequence.

[0055] In certain embodiments, the first intestinal microorganism is selected from the group consisting of: new reference OTU45, JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, new reference OTU54, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, new reference OTU52, JQ208053.1.1336, and any combination thereof.

[0056] In certain embodiments, the second intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence that is at least about 90% homologous or identical to a 16S rRNA nucleotide sequence of HK693629.1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456, or HK555938.1.1357.

[0057] In certain embodiments, the second intestinal microorganism is selected from the group consisting of: HK693629.1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456, HK555938.1.1357, and any combination thereof.

[0058] In certain embodiments, the Royal Canin veterinary diet is selected from the group consisting of Ultamino, Hydrolyzed Protein Adult HP Dry, Hydrolyzed Protein Wet, Hydrolyzed Protein Adult PS Dry, Hydrolyzed Protein Mid Calorie Dry, Hydrolyzed Protein Small Breed Dry, Hydrolyzed Protein Treats, and any combination thereof.

[0059] The presently disclosed subject matter provides bile acids for treating intestinal diseases in dogs. In certain embodiments, the bile acid is selected from the group consisting of chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid and any combination thereof. In certain embodiments, the bile acid is a secondary bile acid. In certain embodiments, the secondary bile acid is selected from the group consisting of taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, omega-muricholic acid, and any combination thereof. In certain embodiments, the secondary bile acid is deoxycholic acid and / or lithocholic acid.

[0060] The presently disclosed subject matter provides a kit comprising the presently disclosed pharmaceutical composition, dietary supplement, functional food, food, diet or bile acid. In certain embodiments, the kit further comprises written instructions for treating and / or preventing intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figures 1A-1C depict that dietary therapy induces rapid and durable remission in a canine model of chronic enteritis. Figure 1A is a schematic diagram showing the clinical study design for identifying diet-responsive (DR) and non-diet-responsive (NDR) dogs. Antibiotics (Antiboitics, Abtx) and prednisone (Pred) treatment are recommended. The abbreviated Canine Chronic Enteropathy Clinical Activity Index (CCECAI) score was assessed at four different time points in DR (n=20) (Figure 1B) and NDR (n=9) (Figure 1C) animals, ns=not significant, **p<0.01, ***p<0.0001 using the Wilcoxon rank sum test.

[0062] Figure 2A - Figure 2F depicts the identification of microbial community profiles associated with treatment outcomes. Figure 2A is a ternary plot of phylum-level OTUs of the top 5 most abundant phyla in healthy (right), DR (left), and NDR (top) animals. Bubble size represents log2 OTU abundance. Relative abundance of E. coli (Figure 2B) and C. perfringens (Figure 2D) in animals with active disease (day 0) and healthy dogs. Spearman correlation between log10 abundance of E. coli (Figure 2C) or species of Clostridium (Figure 2E) and CCECAI disease score. Figure 2F depicts differentially abundant OTUs between DR and DNR animals on day 0. Y-axis values ​​represent log2 fold change of DR versus NDR. Arrows mark OTUs corresponding to C. perfringens. *p<0.05, **p<0.01 using Wilcoxon rank sum test (or Wilcoxon signed rank test, if available). The Spearman correlations in panels C and E are significant (p<0.05), with correlation coefficients of 0.2109 and 0.2324, respectively.

[0063] Figures 3A-3F depict that therapeutic diet improves dysbiosis associated with chronic enteritis. Figure 3A depicts the Pielou evenness index of DR animals at different time points in the study. Figure 3B depicts the principal coordinate analysis (PCoA) of DR based on unweighted Unifrac distance. Figure 3C depicts the phylogenetic distance of DR animals from healthy controls (unweighted Unifrac). Figure 3D depicts a flow chart showing the phylum-level dynamics of the microbiota structure of DR animals throughout the study. Figure 3E depicts a volcano plot showing differentially abundant OTUs enriched in DR dogs during disease activity (day 0, red dots) or in remission after dietary treatment (day 14, blue dots). Selected taxa (e.g., Escherichia-Shigella spp., Clostridium spp.) are labeled. Relative abundance of Escherichia coli (Fig. 3F) and Clostridium perfringens (Fig. 3G) in DR animals throughout the study and compared with healthy controls, ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.0001, using Wilcoxon rank sum test (or Wilcoxon signed rank test if paired data were available).

[0064] Figures 4A-4F depict dietary-induced changes in the microbiome associated with remission. Figure 4A depicts Pielou's uniformity index in NDR animals, and Figure 4B shows their phylogenetic distances from healthy dogs (unweighted Unifrac). Figure 4C depicts a flow chart showing the phylum-level dynamics of the microbiota structure of NDR animals throughout the study. Dietary treatment began on day 0, metronidazole was taken on day 14, and prednisone was taken on day 28 (see methods). Figure 4D depicts a bubble chart showing the abundant genera that differed between DR (left) and NDR (right) animals on day 14 and day 0 (fold change>2 and P<0.05). The bubble size represents the absolute log fold change between day 14 and day 0, and the color reflects the direction of change. Figures 4E and 4F describe the relative abundance of Escherichia coli (Figure 4E) and Clostridium perfringens (Figure 4F) in NDR animals throughout the study, compared with healthy controls. ns = not significant, *p<0.05, **p<0.01, ***p<0.0001, using Wilcoxon rank-sum test (or Wilcoxon signed-rank test if paired data were available).

[0065] Figures 5A-5H depict that diet-induced remission is associated with metabolic reprogramming and increased levels of secondary bile acids. Figure 5A depicts a PCA analysis of KEGG pathways based on the results of Tax4Fun analysis. Figure 5B depicts the first principal component (Dim 1) for all time points in panel A. Figure 5C depicts a heat map showing the shift in metabolic potential from fat / lipid metabolism to carbohydrate / sugar metabolism as DR animals undergo dietary treatment. Figure 5D depicts the relative abundance of KEGG pathways for secondary bile acid biosynthesis predicted based on 16S sequence data. Figures 5E-5H depict the levels of deoxycholic acid (Figure 5E) and lithocholic acid (Figure 5F) measured in the feces of DR animals and NDR animals (Figures 5G and 5H). ns = not significant, *p<0.05, **p<0.01, ***p<0.0001, using Wilcoxon rank sum test (or Wilcoxon signed rank test if paired data are available).

[0066] Figures 6A-6J describe that Clostridium hirano is a diet-responsive species with the ability to produce secondary bile acids, which can inhibit the expansion of potential pathogens in vitro and in vivo. Figure 6A depicts the Spearman correlation between the abundance of bacterial genera and bile acid levels. Only genera with significant (P<0.05) correlation with bile acids are shown. Figures 6B-6E depict the in vitro growth of canine clinical isolates of Escherichia coli (Figures 6B and 6C) or Clostridium perfringens (Figures 6D and 6E) in the presence of different concentrations of lithocholic acid or deoxycholic acid (mean ± standard deviation shown). The in vitro inhibition test was biologically repeated 2 times. Each point in the figure represents a replicate well in the assay. Figure 6F depicts the relative abundance of OTUs corresponding to Clostridium hirano (FJ957494.1.1454) in 16S rRNA sequencing data of DR and NDR animals. Figure 6G depicts the coverage of the bile acid operon (bai) from the Clostridium hirano reference (ASM15605vl), and whole genome sequencing reads produced Clostridium hirano (cyan) and Clostridium perfringens (red) canine clinical isolates. Figure 6H is a schematic diagram showing the experimental design of the mouse experiment. Figure 6I depicts the colon length on day 8. Figure 6J depicts the Escherichia coli Nissle strain CFU measured in colon contents on day 8 (shown as the mean ± sd of n = 5 mice). The experiment was repeated 3 times with similar results. The data shown are from a representative experiment, ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.0001, relative abundance comparisons were performed using the Wilcoxon signed rank test or in vitro culture experiment t test.

[0067] Figure 7A-7E depicts that the bile acid producer Clostridium fulvicidium is associated with diet-induced remission in human pediatric Crohn's disease. Analysis of public data from human pediatric Crohn's disease patients treated with exclusive enteral nutrition (EEN) 23 . Relative abundance reads (map ratios) from 20 patients aligned to the C. scintillans reference (Figure 7A) or the bai operon (Figure 7B) before treatment and 1, 4, and 8 weeks after EEN administration. Patients who responded to treatment and entered remission (n=10, red) and those who failed treatment (n=10, green) are shown. Figures 7C and 7D depict the Spearman correlation between log10 transformed fecal calprotectin levels (FCP) and the relative abundance of C. scintillans (Figure 7C) (for 'responsive' R=-0.3515, P=0.0328; for 'non-responsive' R=-0.0267, P=0.8770) or the bai operon (Figure 7D) (for 'responsive' R=-0.3944, P=0.0157; for 'non-responsive' R=0.0490, P=0.7766). Figure 7E is a schematic diagram showing the proposed model for diet-induced remission. ns = not significant, *p<0.05, **p<0.01, ***p<0.0001, relative abundance comparisons were performed using the Wilcoxon rank sum test.

[0068] Figure 8 The detailed clinical design of the canine chronic enteritis study is described.

[0069] Figures 9A-9D depict the microbial community structure of dogs with CE and healthy dogs. Faith's phylogenetic diversity (Figure 9A) and Shannon index (Figure 9B) were compared between samples from dogs with CE (day 0) and samples from healthy dogs. Figure 9C depicts the ratio of phylum-level microbiota composition. Figure 9D depicts the Unifrac (unweighted) distance within the microbiome of dogs with CE or within the microbiome of healthy dogs. ns = not significant, *p<0.05, **p<0.01, ***p<0.0001, using Wilcoxon rank sum test.

[0070] Figures 10A-10C depict changes in microbial community structure caused by dietary treatment in dogs in response to diet. Figure 10A depicts the phylogenetic diversity of Faith. Figure 10B depicts the Shannon index diversity. Figure 10C depicts the principal coordinate analysis (PCoA) based on the weighted Unifrac distance of the microbiome. ns = not significant, *p<0.05, **p<0.01, ***p<0.0001, using Wilcoxon rank sum test.

[0071] Fig.11The dynamic changes of the microbiome at the phylum level in diet-responsive dogs (DR) and non-diet-responsive dogs (NDR) were depicted.

[0072] Fig.12 Principal component analysis based on KO (KEGG Orthology) abundance of day 0 and day 14 samples is depicted.

[0073] Fig.13 Depicted are the concentrations of bile acids detected in fecal samples of diet-responsive dogs. NS = not significant, *p<0.05, **p<0.01, ***p<0.0001 using Wilcoxon signed-rank test.

[0074] Fig.14 Depicted are the relative abundances of C. hirnoides in dogs in response to diet calculated from metagenomic data. DETAILED DESCRIPTION

[0075] To date, there is still a need for new methods and compositions for treating IBD and other intestinal diseases that target the intestinal microbiome and its metabolites. The present application relates to methods, compositions and foods for improving intestinal health of a subject (e.g., a human or companion animal), treating intestinal dysbiosis in a subject (e.g., a human or companion animal), and / or treating intestinal diseases in a subject (e.g., a human or companion animal), which are based at least in part on the discovery that intestinal microbes that produce bile acids can promote intestinal health and / or are associated with relief of intestinal diseases after treatment, and that changes in intestinal microbial populations are associated with intestinal health status.

[0076] For purposes of clarity and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0077] 1. Definition;

[0078] 2. Gut bacteria and related health assessment tools;

[0079] 3. Pharmaceutical composition;

[0080] 4. Food;

[0081] 5. Methods of treatment; and

[0082] 6 test kits.

[0083] 1. Definition

[0084] The terms used in this specification generally have their ordinary meanings in the art, in the context of the present invention, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners when describing the compositions and methods of the present invention and how to make and use them.

[0085] As used in this application, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification can mean one, but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one". In addition, the terms "having", "including", "containing" and "comprising" are interchangeable, and those skilled in the art recognize that these terms are open-ended terms.

[0086] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, e.g., within 5-fold, or within 2-fold.

[0087] The term "effective treatment" or "effective amount" of a substance refers to the amount of treatment or substance sufficient to produce a beneficial or desired result, including clinical results, and therefore, "effective treatment" or "effective amount" depends on the context of its application. In the case of applying a composition to improve immunity, digestive function and / or reduce inflammation, the effective amount of the composition described in the present application is enough to improve immunity, digestive function and / or reduce inflammation and relieve symptoms and / or reduce inflammation / or reduce the amount of the possibility of digestive system diseases and / or inflammation. Effective treatment described in the present application is enough to improve immunity, digestive function and / or reduce inflammation and relieve symptoms and / or reduce the possibility of digestive system diseases and / or inflammation. Reduction can be the severity of symptoms of digestive system diseases or inflammation, or the possibility of digestive system diseases or inflammation, reducing 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%. The effective amount can be administered once or multiple times. The potential for effective treatment described herein is the potential for effective treatment, that is, sufficient to treat or improve digestive system diseases and / or inflammation, and alleviate symptoms.

[0088] As used in the present application, and as known in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For the purpose of this theme, beneficial or desired clinical results include but are not limited to the alleviation or improvement of one or more symptoms, the alleviation of the degree of illness, the stable (that is, not deteriorating) state of the condition state, the prevention of illness, the delay or slowing of the progression of illness, and / or the improvement or relief of the condition state. Reduction can be a complication or symptom severity reduction of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%. "Treatment" can also mean to extend the survival period compared with the expected survival period not receiving treatment.

[0089] As used herein and as is known in the art, a "probiotic" is a preparation or composition comprising a microorganism that can provide a health benefit when consumed. Microorganisms include, but are not limited to, bacteria, fungi, yeasts, and archaea. In certain embodiments, the probiotic can modify the microbiome in the GI system to enhance the balance of the microbiome in the GI system, for example, by acting as an inoculum for an increased population of beneficial microorganisms, and / or by antagonizing the growth of harmful microorganisms. In certain embodiments, the probiotic is an animal probiotic, such as a feline probiotic or a canine probiotic.

[0090] As used herein, and as is well known in the art, a "prebiotic" is a substance or composition that can induce the growth or activity of one or more beneficial microorganisms (e.g., one or more probiotics, such as bacteria, fungi, yeasts, and archaea). In certain embodiments, prebiotics can modify the microbiome in the GI system to enhance the balance of the microbiome in the GI system. In certain embodiments, prebiotics are indigestible to animals. In certain embodiments, prebiotics can induce the growth or activity of one or more animal probiotics, such as feline probiotics or canine probiotics.

[0091] The term "pet food" or "pet food composition" or "pet food" or "final pet food" refers to a product or composition intended for consumption by a companion animal, such as a cat, dog, guinea pig, rabbit, bird, or horse. For example, but not by way of limitation, the companion animal may be a "domestic" dog, such as Canis lupus familiaris. In certain embodiments, the companion animal may be a "domestic" cat, such as Felis domesticus. "Pet food" or "pet food composition" or "pet food" or "final pet food" includes any food, feed, treat, food supplement, liquid, beverage, snack, toy (chewable and / or consumable toy), meal replacement, or meal substitute.

[0092] An "individual" or "subject" in this application is a vertebrate, such as a human or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sports animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cows; horses; and non-human primates, such as apes and monkeys.

[0093] As used in this application, the term "in vitro" refers to an artificial environment and processes or reactions that occur within an artificial environment. In vitro environments are exemplified, but not limited to, test tubes and cell cultures.

[0094] As used herein, the term "in vivo" refers to the natural environment (eg, an animal or a cell) and processes or reactions that occur in the natural environment, such as embryonic development, cell differentiation, neural tube formation, and the like.

[0095] As used herein, "pharmaceutical composition" and "pharmaceutical formulation" refer to a composition that is in a form that permits the biological activity of the active ingredients contained therein to be effective, and that contains no unacceptable additional ingredients that would be toxic to a patient to whom the formulation would be administered.

[0096] As used herein, "pharmaceutically acceptable", for example, with respect to a "pharmaceutically acceptable excipient", refers to the property of being non-toxic to a subject. A pharmaceutically acceptable ingredient in a pharmaceutical formulation may be a non-toxic ingredient other than an active ingredient. A pharmaceutically acceptable excipient may include a buffer, a carrier, a stabilizer and / or a preservative.

[0097] As used herein, the term "pharmaceutically acceptable salt" refers to any salt of a compound provided herein that retains its biological properties and is non-toxic or otherwise not undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counterions known in the art. Pharmaceutically acceptable salts also include, by way of example only and not limitation, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and when the compound contains a basic functional group, include salts of non-toxic organic or inorganic acids.

[0098] 2. Gut microbiota and related health assessment tools

[0099] The presently disclosed subject matter provides gut microbes and combinations thereof based at least in part on the discovery that bile acid-producing gut microbes can promote gut health and / or are associated with remission of gut disease after treatment, and that changes in gut microbe numbers are associated with gut health status.

[0100] Gut microbes that produce bile acids

[0101] In certain embodiments, the intestinal microorganism is used as a medicament. In certain embodiments, the intestinal microorganism is used to treat an intestinal disease in a subject in need thereof.

[0102] In certain embodiments, the intestinal microorganism is a bacterium capable of producing bile acids. In certain embodiments, the bile acid is chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, omega-muricholic acid, or any combination thereof.

[0103] In certain embodiments, the bile acid is a primary bile acid. In certain embodiments, the primary bile acid is chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, or any combination thereof.

[0104] In certain embodiments, the bile acid is a secondary bile acid. In certain embodiments, the secondary bile acid is taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, omega-muricholic acid, or any combination thereof. In certain embodiments, the secondary bile acid is deoxycholic acid and / or lithocholic acid.

[0105] In certain embodiments, the bacteria include a bile acid inducible operon (bai operon). In certain embodiments, the bacteria include an enzyme having 7-dehydroxylation activity. In certain embodiments, the bai operon comprises a nucleotide sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 1 or SEQ ID NO: 3 or any functional fragment thereof. In certain embodiments, the bai operon comprises a nucleotide sequence listed in SEQ ID NO: 1 or SEQ ID NO: 3. SEQ ID NO: 1 represents an exemplary sequence of a bile acid inducible operon of Clostridium hirsutum. SEQ ID NO: 3 represents an exemplary sequence of a bile acid inducible operon of Clostridium fulgidus.

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119] In some embodiments, the bacteria are transformed with a vector comprising a bile acid inducible operon (bai operon). In some embodiments, the bacteria stably express an enzyme with 7-dehydroxylation activity. In some embodiments, the enzyme is a bile acid 7-dehydroxylase. In some embodiments, the enzyme is selected from the group consisting of bile acid 7-dehydroxylase, bile acid 7-α-dehydroxylase, 7-α-dehydratase, bile acid CoA ligase, 3α-HSDH, CoA transferase, 3-dehydrogenation-4-7-α-oxidoreductase, 3-dehydrogenation-4-7-β-oxidoreductase, CA / CDCA transporter, 7-β-dehydratase, and AraC / XyIS. In certain embodiments, the enzyme comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acids of bile acid 7-dehydroxylase, bile acid 7-α-dehydroxylase, 7-α-dehydratase, bile acid CoA ligase, 3α-HSDH, CoA transferase, 3-dehydrogen-4-7-α-oxidoreductase, 3-dehydrogen-4-7-β-oxidoreductase, CA / CDCA transporter, 7-β-dehydratase__, or AraC / XyIS.

[0120] In some embodiments, the bacteria is selected from the group consisting of Ruminococcus, Alloprevotella, Allisonella, Anaerostipes, Anaerobiospirillum, Bacteroides, Blautia, Clostridium sensu stricto1, Collinsella, Coprococcus 1, Corynebacterium 1. 1), Campylobacter, Enterococcus, Erysipelatoclostridium, Escherichia-Shigella, Faecalitalea, Fusobacterium, Clostridium, Helicobacter, Intestinibacter, Lachnoclostridium, Lactobacillus, Megasphaera, Methanobrevibacter, Parabacteroides, Porphyromonas, Phascolarctobacterium, Peptoclostridium, Prevotellaceae UCG-001 In some embodiments, the bacteria are selected from the genus Clostridium.

[0121] In certain embodiments, the intestinal microorganism comprises a 16s rRNA comprising a nucleotide sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 2 or identical to SEQ ID NO: 2. In certain embodiments, the intestinal microorganism comprises a 16s rRNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. SEQ ID NO: 2 represents an exemplary sequence of a 16S rRNA gene in Clostridium plannosum. SEQ ID NO: 4 represents an exemplary sequence of a 16S rRNA gene in Clostridium fulgidus.

[0122]

[0123]

[0124] In certain embodiments, the intestinal microbes include Clostridium hiranosum, Clostridium scindens, or a combination thereof. In certain embodiments, the intestinal microbes include Clostridium hiranosum. In certain embodiments, the intestinal microbes include Clostridium scindens.

[0125] In the sense of the present disclosure, the "percentage of identity" between two nucleic acid or amino acid sequences is intended to represent the percentage of nucleotides or identical amino acid residues between the two sequences to be compared obtained after optimal alignment (optimal alignment), this percentage being purely statistical, and the differences between the two sequences being randomly distributed over their entire length. Traditionally, sequence comparisons between two nucleic acid or amino acid sequences are performed by comparing these sequences after optimal alignment, which comparison can be performed by segments or "comparison windows". In addition to manual, it can also be performed by the local homology algorithm Smith and Waterman (1981) [Ad. App. Math. 2:482], by the local homology algorithm Neddleman and Wunsch (1970) [J. Mol. Biol. 48:443], by the local homology algorithm Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), by computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI or BLAST N or BLAST P comparison software).

[0126] The percent identity between two nucleic acid or amino acid sequences is determined by comparing the two sequences aligned in an optimal manner, wherein the nucleic acid or amino acid sequence to be compared may contain additions or deletions relative to the reference sequence in order to achieve optimal alignment between the two sequences. The percent identity is calculated by determining the number of identical positions at which the nucleotide or amino acid residue is identical between the two sequences, dividing the number of identical positions by the total number of positions in the comparison window and multiplying the result obtained by 100 to obtain the percent identity between the two sequences.

[0127] For example, the BLAST program, "BLAST2 sequences" (Tatusova et al., "Blast 2 sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250), can be used, which can be found on the website www.ncbi.nlm.nih.gov, and the parameters used are the parameters given by default (especially for the parameters "open gap penalty": 5 and "extended gap penalty": 2; the matrix selected is, for example, the matrix "BLOSUM 62" proposed by the program), and the percentage of identity between the two sequences to be compared is calculated directly by the program. Other programs, such as "ALIGN" or "Megalign" (DNASTAR) software, can also be used.

[0128] By having an amino acid sequence with at least about 80%, such as at least about 85%, at least about 90%, at least about 95% and at least about 98% homology with a reference amino acid sequence, those with certain modifications relative to the reference sequence, particularly deletions, additions or substitutions, truncations or extensions of at least one amino acid are preferred. Where one or more continuous or non-continuous amino acids are replaced, a preferred replacement is one in which the replaced amino acid is replaced by an "equivalent" amino acid. The expression "equivalent amino acid" is intended to mean any amino acid that can be replaced by one of the amino acids of the basic structure without substantially changing the biological activity of the corresponding antibody, such as will be defined later, especially in the examples. These equivalent amino acids can be determined by their structural homology to the amino acids replaced by them, or can be determined based on the results of comparative tests of biological activity between different antibodies that can be performed.

[0129] As a non-limiting example, Table 1 represents the possibility of substitutions that can be made without causing a profound change in the biological activity of the corresponding modified antibody, the reverse substitutions being naturally conceivable under the same conditions.

[0130] Table 1.

[0131]

[0132]

[0133] Gut Microbes That Indicate Gut Health

[0134] In some embodiments, the intestinal microorganisms can be used to indicate the intestinal health of a subject. In some embodiments, the intestinal microorganisms are associated with intestinal diseases. In some embodiments, the intestinal microorganisms are associated with a healthy intestinal state. In some embodiments, the intestinal microorganisms of healthy subjects are more abundant than those of subjects with intestinal diseases. In some embodiments, the content of intestinal microorganisms in healthy subjects is less than that in subjects with intestinal diseases.

[0135] In certain embodiments, the intestinal microbiota comprises one or more bacteria and / or archaea from one or more phyla selected from the group consisting of Actinobacteria, Bacteroidetes, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, and Spirochaetae.

[0136] In certain embodiments, the intestinal microbes comprise one or more bacteria and / or archaea from one or more classes selected from the group consisting of Actinobacteria, Bacilli, Bacteroidi, Clostridia, Coriobacteria, Erysipelotrichia, Fusobacteria, Gammaproteobacteria, Methanobacteria, and Spirochaetes.

[0137] In certain embodiments, the intestinal microbes comprise one or more bacteria and / or archaea from one or more orders selected from the group consisting of Bacteriodales, Clostridiales, Coriobacteriales, Corynebacteriales, Enterobacteriales, Erysipelotrichales, Fusobacteriales, Lactobacillaes, Methanobacteriales, and Spirochaetales.

[0138] In certain embodiments, the intestinal microbes comprise one or more bacteria and / or archaea from one or more families selected from the group consisting of Bacteroidaceae, Clostridiaceae 1, Coriobacteriaceae, Corynebacteriaceae, Enterobacteriaceae, Erysipelotrichaceae, Fusobacteriaceae, Lachnospiraceae, Methanobacteriaceae, Peptostreptococcaceae, Porphyromonadaceae, Prevotellaceae, Ruminococcaceae, Spirochaetaceae, and Streptococcaceae.

[0139] In some embodiments, the intestinal microorganisms include Ruminococcus, Alloprevotella, Allisonella, Anaerostipes, Anaerobiospirillum, Bacteroides, Blautia, Clostridium sensu stricto 1, Collinsella, Coprococcus 1, Corynebacterium 1, 1), Campylobacter, Enterococcus, Erysipelatoclostridium, Escherichia-Shigella, Faecalitalea, Fusobacterium, Helicobacter, Intestinibacter, Lachnoclostridium, Lactobacillus, Megasphaera, Methanobrevibacter, Parabacteroides, Porphyromonas, Phascolarctobacterium, Peptoclostridium, Prevotellaceae UCG-001 One or more bacteria and / or archaea of ​​one or more genera in the group consisting of UCG-001, Pseudocitrobacter, Ruminiclostridium 9, Sarcina, Streptococcus, Succinivibrio, Treponema 2, Turicibacter, Tyzzerella and Tyzzerella 4.

[0140] In certain embodiments, the intestinal microorganisms include one or more bacteria and / or archaea selected from one or more species of the group consisting of Enterococcus durans, Escherichia coli, and Clostridium perfringens. In certain embodiments, the intestinal microorganisms include Escherichia coli and Clostridium perfringens.

[0141] In certain embodiments, the intestinal microorganism is selected from the group consisting of Clostridium plano, Clostridium scintillans, Veillonellaceae, Streptococcaceae, Bacteroides, Fusobacterium, Collinsella, Sarcina, Clostridium sensu stricto 1, Faecalitalea, Streptococcus, Erysipelatoclostridium, Megasphaera, Blautia, Alloprevotella, Peptoclostridium, and any combination thereof. In certain embodiments, the intestinal microorganism is Clostridium plano, Clostridium scintillans, or a combination thereof.

[0142] In certain embodiments, the intestinal microbes comprise one or more bacteria comprising a 16S rRNA comprising a sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homologous or identical to any of the sequences in Table 11.

[0143] In some embodiments, the intestinal microorganisms include one or more bacteria comprising 16S rRNA, wherein the 16S rRNA comprises HQ802983.1.1440, FJ950694.1.1472, HG798451.1.1400, new reference OTU52, new reference OTU82, GQ449092.1.1375, FJ506371.1.1371, GQ448744.1.1393, FJ957494.1.1454, HQ760911.1.1437, GQ006324.1.1342, GQ448246.1.1373, 1389, KC245406.1.1465, new reference OTU54, HQ751549.1.1448, JF712675.1.1540, JQ208181.1.1352, GX182404.8.1529, FP929060.3837.5503, FN667392.1.1495, FN667422.1.1495, HK557089.3.1395, HQ803964.1.1435, AM276759.1.1484, HK555938.1.1357, KF842598.1.1394, HQ792778.1.1436, FM865905.1.1392, FN563300.1.1447, HQ754680.1.1441, GQ867426.1.1494, EU470512.1.1400, AY239462.1.1500, new reference OTU114, FN668375.4306350.4307737, AB009242.1.1451, HQ792787.1.1438, AB506370.1.1516, DQ057365.1.1393, FN667084.1.14 93, DQ113765.1.1450, HK694029.9.1487, AJ270486.1.1241, EU768569.1.1352, FM179752.1.1686, FJ957528.1.1445, KC504009.1.1465, GQ448506.1.1374, JF224013.1.1362, EU774020.1.1361, GQ448486.1.1387, HQ793763.1.1451, JN387556.1.1324 or 16S of the new reference OTU109 rRNA nucleotide sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homologous or identical thereto.

[0144] In certain embodiments, the intestinal microorganisms comprise one or more bacteria and / or archaea of ​​one or more operational taxonomic units (OTUs) selected from the group consisting of: HQ802983.1.1440, FJ950694.1.1472, HG798451.1.1400, new reference OTU52, new reference OTU82, GQ449092.1.1375, FJ506371.1.1371, GQ448744.1.1393, FJ957494.1.1454, HQ760911.1.1437, GQ006324.1.1342, GQ4 48246.1.1389, KC245406.1.1465, new reference OTU54, HQ751549.1.1448, JF712675.1.1540, JQ208181.1.1352, GX182404.8.1529, FP929060.3837.5503, FN667392.1.1495, FN667422.1.1495, HK557089.3.1395, HQ803964.1.1435, AM276759.1.1484, HK555938.1.1357, KF842598.1.139 4. HQ792778.1.1436, FM865905.1.1392, FN563300.1.1447, HQ754680.1.1441, GQ867426.1.1494, EU470512.1.1400, AY239462.1.1500, new reference OTU114, FN668375.4306350.4307737, AB009242.1.1451, HQ792787.1.1438, AB506370.1.1516, DQ057365.1.1393, FN667084.1.1493, DQ 113765.1.1450, HK694029.9.1487, AJ270486.1.1241, EU768569.1.1352, FM179752.1.1686, JF807116.1.1260, FJ957528.1.1445, KC504009.1.1465, GQ448506.1.1374, JF224013.1.1362, EU774020.1.1361, GQ448486.1.1387, HQ793763.1.1451, JN387556.1.1324, and the new reference OTU109.

[0145] In some embodiments, the intestinal microorganisms include one or more bacteria comprising 16S rRNA, wherein the 16S rRNA comprises JRPJ01000002.1034290.1035971, JF920309.1.1340, FJ978526.1.1378, new reference OTU45, HK555938.1.1357, FJ957494.1.1454, new reference OTU52, DQ797046.1.1403, GQ449092.1.1375, AMC I01001631.34.1456, KF842598.1.1394, HQ793763.1.1451, DQ113765.1.1450, ACBW01000012.3536.5054, HK693629.1.1491, JQ208053.1.1336, GQ493166.1.1359, GQ448486.1.1387, GQ491426.1.1332, 16S of the new reference OTU54 or JN387556.1.1324 The rRNA nucleotide sequence is at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homologous or identical thereto.

[0146] In certain embodiments, the intestinal microorganisms comprise one or more bacteria and / or archaea of ​​one or more operational taxonomic units (OTUs) selected from the group consisting of: JRPJ01000002.1034290.1035971, JF920309.1.1340, FJ978526.1.1378, new reference OTU45, HK555938.1.1357, FJ957494.1.1454, new reference OTU52, DQ797046.1.1403, GQ449092.1 .1375, AMCI01001631.34.1456, KF842598.1.1394, HQ793763.1.1451, DQ113765.1.1450, ACBW01000012.3536.5054, HK693629.1.1491, JQ208053.1.1336, GQ493166.1.1359, GQ448486.1.1387, GQ491426.1.1332, new reference OTU54, and JN387556.1.1324.

[0147] In certain embodiments, the intestinal microorganisms comprise one or more bacteria comprising 16S rRNA comprising 16S rRNA corresponding to GQ006324.1.1342, new reference OTU52, HG798451.1.1400, HK557089.3.1395, GQ448336.1.1418, KF842598.1.1394, FJ950694.1.1472, HQ802983.1.1440, GQ448468.1.1366 or JN387556.1.1324 in Table 11. rRNA nucleotide sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homologous or identical thereto.

[0148] In certain embodiments, the intestinal microbes comprise one or more bacteria and / or archaea of ​​one or more operational taxonomic units (OTUs) selected from GQ006324.1.1342, new reference OTU52, HG798451.1.1400, HK557089.3.1395, GQ448336.1.1418, KF842598.1.1394, FJ950694.1.1472, HQ802983.1.1440, GQ448468.1.1366, and JN387556.1.1324.

[0149] In certain embodiments, the intestinal microorganisms include one or more bacteria comprising 16S rRNA, wherein the 16S rRNA comprises JRPJ01000002.1034290.1035971, new reference OTU45, GQ006324.1.1342, HK555938.1.1357, FJ957551.1.1489, FJ957494.1.1454 ... U52, FM865905.1.1392, GQ016239.1.1362, HG798451.1.1400, EU461791.1.1414, GU303759.1.1517, new reference OTU114, AB506154.1.1541, EU774370.1.1398, HK557089.3.1395, HQ807346.1.1456, HQ748204.1.1442, GU179917.1.1382, GQ448336.1.1418, DQ804865.1.1390, GQ491757.1.1361, new reference OTU56, KF842598.1.1394, HQ802052.1.1445, GX182404.8.1529, FJ950694.1.1472, GQ448 1.1374, HQ802983.1.1440, DQ793824.1.1370, GQ448468.1.1366, EU774020.1.1361, GQ491183.1.1360, GQ491426.1.1332, GQ493039.1.1311, JN387556.1.1324, and EU775983.1.1288 are nucleotide sequences that are at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homologous or identical thereto.

[0150] In certain embodiments, the intestinal microorganisms comprise one or more bacteria and / or archaea of ​​one or more operational taxonomic units (OTUs) selected from the group consisting of: JRPJ01000002.1034290.1035971, new reference OTU45, GQ006324.1.1342, HK555938.1.1357, FJ957551.1.1489, FJ957494. 1.1454, new reference OTU52, FM865905.1.1392, GQ016239.1.1362, HG798451.1.1400, EU461791.1.1414, GU303759.1.1517, new reference OTU114, AB506154.1.1541, EU774370.1.1398, HK557089.3.1395, HQ80734 6.1.1456, HQ748204.1.1442, GU179917.1.1382, GQ448336.1.1418, DQ804865.1.1390, GQ491757.1.1361, new reference OTU56, KF842598.1.1394, HQ802052.1.1445, GX182404.8.1529, FJ950694.1.1472 , GQ448506.1.1374, HQ802983.1.1440, DQ793824.1.1370, GQ448468.1.1366, EU774020.1.1361, GQ491183.1.1360, GQ491426.1.1332, GQ493039.1.1311, JN387556.1.1324 and EU775983.1.1288.

[0151] In some embodiments, the intestinal microorganisms include one or more bacteria comprising 16S rRNA, wherein the 16S rRNA comprises GQ449137.1.1391, HK555938.1.1357, GQ358246.1.1466, new reference OTU82, new reference OTU52, GQ138615.1.1402, JN681884.1.1409, GU303759.1.1517, new reference OTU114, EU774881.1.1422, AB469559.1.1551, HK55708 in Table 11 9.3.1395, EU358719.1.1513, HQ748204.1.1442, GQ338727.1.1397, HQ803964.1.1435, FJ951866.1.1493, EU772870.1.1289, GQ448468.1.1366, EU774020.1.1361, HQ782658.1.1415, DQ794633.1.1395, FN668375.4306350.4307737, or GQ867445.1.1457 16S The nucleotide sequence of an rRNA is at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homologous or identical thereto.

[0152] In certain embodiments, the intestinal microorganisms comprise one or more bacteria and / or archaea of ​​one or more operational taxonomic units (OTUs) selected from the group consisting of GQ449137.1.1391, HK555938.1.1357, GQ358246.1.1466, new reference OTU82, new reference OTU52, GQ138615.1.1402, JN681884.1.1409, GU303759.1.1517, new reference OTU114, EU774881.1.1422, AB469559.1.15 51. HK557089.3.1395, EU358719.1.1513, HQ748204.1.1442, GQ338727.1.1397, HQ803964.1.1435, FJ951866.1.1493, EU772870.1.1289, GQ448468.1.1366, EU774020.1.1361, HQ782658.1.1415, DQ794633.1.1395, FN668375.4306350.4307737 and GQ867445.1.1457.

[0153] Health Assessment Tools

[0154] The presently disclosed subject matter further provides health assessment tools associated with the microorganisms disclosed in the present application. In certain embodiments, the health assessment tool is used to monitor intestinal health status or dysbiosis. In certain embodiments, the health assessment tool comprises one or more probes for detecting the amount of one or more microorganisms disclosed in the present application. In certain embodiments, the health assessment tool comprises a microarray of one or more probes for detecting the amount of one or more microorganisms disclosed in the present application. In certain embodiments, the probe comprises a nucleic acid probe for detecting a characteristic gene of a microorganism disclosed in the present application. In certain embodiments, the probe detects the 16S rRNA sequence of a microorganism disclosed in the present application. In certain embodiments, the probe comprises an antibody. In certain embodiments, the antibody binds to a surface protein / antigen of a microorganism disclosed in the present application.

[0155] In certain embodiments, the amount of microorganisms is measured from a stool sample of the subject.In certain embodiments, the health assessment tool monitors intestinal health status or dysbiosis by comparing the amount of one or more microorganisms to a reference amount of one or more microorganisms.

[0156] In certain embodiments, the health assessment tool comprises probes for detecting at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 14, at least about 26 or more microorganisms disclosed in the present application. In certain embodiments, the health assessment tool comprises probes for detecting about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, or about 26 microorganisms disclosed in the present application. In certain embodiments, the health assessment tool comprises probes for detecting between about 1 to about 500, about 1 to about 100, about 1 to about 26, about 5 to about 100, about 5 to about 26, about 10 to about 26, about 15 to about 50, or about 50 to about 100 microorganisms disclosed herein.

[0157] In certain embodiments, the one or more microorganisms include bacteria comprising a 16S rRNA comprising a nucleotide sequence at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9%) homologous or identical to any sequence in Table 11.

[0158] 3. Pharmaceutical Composition

[0159] The presently disclosed subject matter provides a pharmaceutical composition for use as a medicament. In certain embodiments, the pharmaceutical composition comprises an effective amount of bacteria capable of producing a first bile acid. In certain embodiments, the pharmaceutical composition further comprises an effective amount of a second bile acid. In certain embodiments, the bacteria is any of the bacteria disclosed in the above sections. In certain embodiments, the first bile acid and / or the second bile acid is any bile acid disclosed in the above sections or a pharmaceutically acceptable salt thereof. In certain embodiments, the first bile acid and the second bile acid are the same. In certain embodiments, the first bile acid and the second bile acid are different.

[0160] In certain embodiments, the bacteria contained in the pharmaceutical composition are between about 1000 CFU and about 100 trillion CFU. In certain embodiments, the bacteria are between about 1000 CFU and about 1 trillion CFU, between about 1 million CFU and about 1 trillion CFU, between about 100 million CFU and about 100 billion CFU, between about 1 billion CFU and about 1 trillion CFU, between about 1 billion CFU and about 100 billion CFU, between about 1 billion CFU and about 100 billion CFU, between about 100 million CFU and about 100 billion CFU, between about 1 billion CFU and about 50 billion CFU, between about 100 million CFU and about 50 billion CFU, or between about 1 billion CFU and about 10 billion CFU. In certain embodiments, the bacteria contained in the pharmaceutical composition are at least about 1000 CFU, at least about 1 million CFU, at least about 10 million CFU, at least about 100 million CFU, at least about 100 million CFU, at least about 1 billion CFU, at least about 10 billion CFU, at least about 10 billion CFU, at least about 100 billion CFU, or more.

[0161] In certain embodiments, the second bile acid contained in the pharmaceutical composition is between about 1 μg / unit dose and about 1 g / unit dose. In certain embodiments, the second bile acid contained in the pharmaceutical composition is between about 10 μg / unit dose and about 1 g / unit dose, between about 10 μg / unit dose and about 500 mg / unit dose, between about 100 μg / unit dose and about 500 mg / unit dose, between about 1 mg / unit dose and about 500 mg / unit dose, between about 10 mg / unit dose and about 500 mg / unit dose, between about 100 mg / unit dose and about 500 mg / unit dose, between about 100 mg / unit dose and about 500 mg / unit dose, between about 10 mg / unit dose and about 100 mg / unit dose, between about 50 mg / unit dose and about 300 mg / unit dose. In certain embodiments, the second bile acid contained in the pharmaceutical composition is at least about 1 μg / unit dose, at least about 10 μg / unit dose, at least about 100 μg / unit dose, at least about 1 mg / unit dose, at least about 10 mg / unit dose, at least about 100 mg / unit dose, at least about 1 g / unit dose, or more.

[0162] The presently disclosed subject matter provides bile acids for treating intestinal diseases in dogs. In certain embodiments, the bile acid is selected from the group consisting of chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid and any combination thereof. In certain embodiments, the bile acid is a secondary bile acid. In certain embodiments, the secondary bile acid is selected from taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, taurolithocholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, omega-muricholic acid, and any combination thereof. In certain embodiments, the secondary bile acid is deoxycholic acid and / or lithocholic acid.

[0163] In certain embodiments, the pharmaceutical composition is used to treat an intestinal disorder in a subject in need thereof. In certain embodiments, the intestinal disorder is selected from irritable bowel syndrome, constipation, gastritis, colitis, inflammatory bowel disease (IBD), gastrointestinal ulcers, hemorrhagic gastroenteritis, diarrhea, Crohn's disease, ulcerative colitis, enteritis, antibiotic-associated diarrhea, acute or chronic enteropathy, necrotizing enterocolitis, and any combination thereof.

[0164] In certain embodiments, the subject is a dog. In certain embodiments, the intestinal disease is an acute intestinal disease or a chronic intestinal disease. In certain embodiments, the intestinal disease is a chronic intestinal disease selected from food-responsive intestinal disease, antibiotic-responsive intestinal disease, and idiopathic inflammatory bowel disease (IBD).

[0165] In certain non-limiting embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a companion animal of the family Felidae (e.g., domestic cat) or the family Canidae (e.g., domestic dog).

[0166] The exact dosage and frequency of administration depend on the specific disease treated, the age of specific patient, body weight and general physical condition and other drugs that individual can take, as known to those skilled in the art.Usually, the daily dose of pharmaceutical composition disclosed in the present application can be in the range of about 0.01mg to about 1000mg / days.In some embodiments, pharmaceutical composition can be about 0.05mg to about 1000mg / days, about 0.1mg to about 1000mg / days, about 1mg to about 500mg / days, about 0.01mg to about 500mg / days, about 0.05mg to about 200mg / days, about 1mg to about 500mg / days, about 1mg to about 200mg / days, about 5mg to about 500mg / days, about 50mg to about 200mg / days, about 100mg to about 200mg / days, about 100mg to about 1000mg / days, about 20mg to about 50mg / days or about 20mg to about 100mg / days.

[0167] In certain embodiments, the pharmaceutical composition disclosed herein can be administered from about 10 times a day to about once a day, from about 5 times a day to about once a day, or from about three times a day to about once a day. In certain embodiments, the pharmaceutical composition disclosed herein can be administered once a day. In certain embodiments, the pharmaceutical composition disclosed herein can be administered once a day. In certain embodiments, the pharmaceutical composition disclosed herein can be administered once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, or once a month.

[0168] The pharmaceutical composition disclosed in the present application can be administered in various forms. In certain embodiments, the pharmaceutical composition disclosed in the present application can be administered orally, parenterally, and rectally. In certain embodiments, the pharmaceutical composition for oral administration in solid dosage form can be administered as capsules, dragees, granules, pills, powders, and tablets. In certain embodiments, the pharmaceutical composition can be administered in liquid form as elixir, emulsion, microemulsion, solution, suspension, and syrup. In certain embodiments, the pharmaceutical composition for parenteral administration can be administered as an aqueous or oily solution or an aqueous or oily suspension, and the suspension comprises crystals, amorphous, or other insoluble forms of the pharmaceutical composition. In certain embodiments, the pharmaceutical composition for rectal administration can be administered as a cream, gel, lotion, ointment, and paste.

[0169] Depending on the form of administration, pharmaceutical compositions disclosed herein can be prepared or administered with or without a pharmaceutically acceptable excipient. In some embodiments, excipients include encapsulating materials or formulation additives, such as absorption enhancers, antioxidants, adhesives, buffers, coating agents, coloring agents, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, wetting agents, lubricants, spices, preservatives, propellants, release agents, bactericides, sweeteners, solubilizers, wetting agents, cosolvents and any combination thereof. In some embodiments, pharmaceutical compositions disclosed herein are used without a solubilizing aid.

[0170] In certain embodiments, the pharmaceutical composition may be provided individually or packaged as a kit.

[0171] 4. Food products

[0172] The presently disclosed subject matter provides a food for improving intestinal health. In certain embodiments, the food comprises an effective amount of bacteria capable of producing a first bile acid. In certain embodiments, the food further comprises an effective amount of a second bile acid. In certain embodiments, the bacteria are any bacteria disclosed in the above sections. In certain embodiments, the first bile acid and / or the second bile acid are any bile acids disclosed in the above sections or their edible salts. In certain embodiments, the first bile acid and the second bile acid are the same. In certain embodiments, the first bile acid and the second bile acid are different.

[0173] In certain embodiments, the food is a dietary supplement. In certain embodiments, the food is a human food. In certain embodiments, the food is a pet food, such as a cat food or a dog food. In certain embodiments, the food is a dog food product. In certain embodiments, the food is a pet dietary supplement.

[0174] In certain embodiments, the bacteria contained in the pharmaceutical composition are between about 10,000 CFU and about 100 trillion CFU. In certain embodiments, the bacteria are between about 1,000 CFU and about 1 trillion CFU, between about 1 million CFU and about 1 trillion CFU, between about 100 million CFU and about 100 billion CFU, between about 1 billion CFU and about 1 trillion CFU, between about 1 billion CFU and about 1 trillion CFU, between about 1 billion CFU and about 100 billion CFU, between about 100 million CFU and about 100 billion CFU, between about 1 billion CFU and about 50 billion CFU, between about 100 million CFU and about 50 billion CFU, or between about 1 billion CFU and about 10 billion CFU. In certain embodiments, the bacteria contained in the pharmaceutical composition is at least about 1000 CFU, at least about 1 million CFU, at least about 10 million CFU, at least about 100 million CFU, at least about 100 million CFU, at least about 1 billion CFU, at least about 10 billion CFU, at least about 10 billion CFU, at least about 100 billion CFU, or more.

[0175] In certain embodiments, the second bile acid contained in the pharmaceutical composition is between about 1 μg / daily dosage and about 1 g / daily dosage. In certain embodiments, the second bile acid contained in the pharmaceutical composition is between about 10 μg / daily dosage and about 1 g / daily dosage, between about 10 μg / daily dosage and about 500 mg / daily dosage, between about 100 μg / daily dosage and about 500 mg / daily dosage, between about 1 mg / daily dosage and about 500 mg / daily dosage, between about 10 mg / daily dosage and about 500 mg / daily dosage, between about 100 mg / daily dosage and about 500 mg / daily dosage, between about 10 mg / daily dosage and about 100 mg / daily dosage, and between about 50 mg / daily dosage and about 300 mg / daily dosage. In certain embodiments, the second bile acid contained in the pharmaceutical composition is at least about 1 μg / daily dose, at least about 10 μg / daily dose, at least about 100 μg / daily dose, at least about 1 mg / daily dose, at least about 10 mg / daily serving dose, at least about 100 mg / daily serving dose, at least about 1 g / daily serving dose, or more.

[0176] In certain embodiments, the formulations of the present disclosure may further comprise additional active agents. Non-limiting examples of additional active agents that may be present in the formulations of the present disclosure include nutrients (e.g., amino acids, peptides, proteins, fatty acids, carbohydrates, sugars, nucleic acids, nucleotides, vitamins, minerals, etc.), prebiotics, probiotics, antioxidants, and / or agents that improve animal health.

[0177] In certain embodiments, the food comprises one or more probiotics. In certain embodiments, the probiotic is a human probiotic. In certain embodiments, the probiotic is an animal probiotic. In certain embodiments, the animal probiotic is a feline probiotic. In certain embodiments, the animal probiotic is a canine probiotic. In certain embodiments, the probiotic is a bifidobacterium, a lactic acid bacterium, and / or an enterococcus. In certain embodiments, the probiotic is selected from any organism from the group consisting of lactic acid bacteria, and more specifically from the following bacterial genera: Lactococcus spp., Pediococcus spp., Bifidobacterium spp. (e.g., B. longum, B. bifidum, B. pseudolongum, B. animalis), Lactobacillus spp. (e.g., L. bulgaricus, L. acidophilus, L. brevis, L. casei, L. casei), Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, Enterococcus spp. (such as E. faecium), Prevotella spp., Fusobacteria spp) and any combination thereof. In certain embodiments, the probiotic is administered to a companion animal in an amount of about 1 colony forming unit (CFU) to about 100 billion CFU per day to maintain the GI flora. In certain embodiments, the probiotic is administered to a companion animal in an amount of about 1 colony forming unit (CFU) to about 20 billion CFU per day to maintain the gastrointestinal microbial community. In certain embodiments, the probiotic is administered to a companion animal in an amount of about 1 billion CFU to about 20 billion CFU per day to maintain the GI microbial community. In certain embodiments, the probiotic is administered to a companion animal in an amount of about 100 million to about 100 billion live bacteria per day. In certain embodiments, the probiotic is administered to a companion animal in an amount of about 100 million to about 10 billion live bacteria per day.

[0178] In certain embodiments, additional prebiotics may be included, such as fructooligosaccharides (FOS), xylooligosaccharides (XOS), galacto-oligosaccharides (GOS), glucans, galactans, arabinogalactans, inulin and / or oligomannans. In certain embodiments, the additional prebiotics are administered in an amount sufficient to actively stimulate the GI microbiota and / or cause proliferation of one or more probiotics.

[0179] In some embodiments, companion animal food may also include additives known in the art. In some embodiments, such additives exist in an amount that does not damage the purpose and effect provided by the currently disclosed theme. The example of expected additives includes but is not limited to functionally beneficial substances that improve health, substances with stabilizing effects, organoleptic substances, processing aids, substances that enhance palatability, coloring substances, and substances that provide nutritional benefits. In some embodiments, stabilizing substances include but are not limited to substances that tend to increase the shelf life of products. In some embodiments, such substances include but are not limited to preservatives, synergists and chelating agents, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and wetting agents. In some embodiments, emulsifiers and / or thickeners include, for example, gelatin, cellulose ethers, starch, starch esters, starch ethers, and modified starches.

[0180] In certain embodiments, additives for coloring, palatability and nutritional purposes include, for example, colorants, iron oxide, sodium chloride, potassium citrate, potassium chloride and other edible salts, vitamins, minerals and flavors. The amount of such additives in the product is typically up to about 5% (dry basis of the product).

[0181] In some embodiments, companion animal food is a dietary supplement. In some embodiments, dietary supplements include, for example, feeds used together with another feed to improve overall nutritional balance or performance. In some embodiments, supplements include compositions fed undiluted as supplements to other feeds, providing free choice with other parts of animal rations that can be provided separately, or diluting and mixing with the conventional feed of the animal to produce a complete feed. For example, AAFCO provides a discussion about supplements in the American Feed Control Officials, Incorp. Official Publication, p. 220 (2003). Supplements can be in various forms, including, for example, powders, liquids, syrups, pills, tablets, encapsulated compositions, etc.

[0182] In certain embodiments, companion animal foods are treats. In certain embodiments, treats include, for example, compositions given to animals to induce the animal to eat at non-meal times. In certain embodiments, companion animal foods are treats for canines, including, for example, dog bones. Treats can be nutritional, wherein the product contains one or more nutrients, and can, for example, have a composition as described above for food. Non-nutritional treats include any other non-toxic treats.

[0183] In certain embodiments, the bacteria and / or bile acids of the presently disclosed subject matter can be incorporated into the composition during processing of the formulation, for example during and / or after mixing of the other components of the product. These components can be dispensed into the product by conventional means.

[0184] In certain embodiments, the companion animal food of the disclosed subject matter can be prepared in canned or wet form using conventional companion animal food processes. In certain embodiments, ground animal (e.g., mammal, poultry and / or fish) protein tissue is mixed with other ingredients such as milk fish oil, grains, other nutritional balance ingredients, special purpose additives (e.g., vitamin and mineral mixtures, inorganic salts, cellulose and beet pulp, fillers, etc.); and enough water for treatment is added. These ingredients are mixed in a container suitable for heating while mixing the components. The mixture can be heated in any suitable manner, such as by direct steam injection or by using a container equipped with a heat exchanger. After adding the last ingredient, the mixture is heated to a temperature range of about 50°F to about 212°F. Temperatures beyond this range are acceptable, but commercially impractical without the use of other processing aids. When heated to a suitable temperature, the material is generally in the form of a viscous liquid. The viscous liquid is loaded into a can. Cover the lid and seal the container airtightly. The sealed can is then placed in a conventional device designed to sterilize the contents. This is typically accomplished by heating to a temperature above about 230° F. for an appropriate time, depending upon, for example, the temperature and composition used.

[0185] In certain embodiments, companion animal food of the disclosed subject matter can be prepared in dry form using conventional methods. In certain embodiments, dry ingredients, including, for example, animal protein sources, plant protein sources, grains, etc., are ground and mixed together. In certain embodiments, wet or liquid ingredients, including fat, oil, animal protein sources, water, etc., are then added to the dry mixture and mixed with it. In certain embodiments, the mixture is then processed into coarse grains or similar dry pieces. In certain embodiments, companion animal food is coarsely ground food. In certain embodiments, coarse grains are formed using an extrusion process, wherein a mixture of dry and wet ingredients is subjected to mechanical work under high pressure and high temperature and is forced through a small opening and is cut into coarse grains by a rotating knife. In certain embodiments, the wet coarsely ground powder is then dried and optionally coated with one or more topical coatings, which may include, for example, spices, fats, oils, powders, etc. In certain embodiments, coarsely ground food can also be made from dough using a baking process rather than an extrusion process, wherein the dough is placed in a mold before dry heat processing.

[0186] In certain embodiments, the snack foods of the presently disclosed subject matter can be prepared by, for example, extrusion or baking processes similar to those described above for dry foods.

[0187] The presently disclosed subject matter provides a diet for increasing the bacterial population capable of producing bile acids in companion animals. In certain embodiments, the diet comprises protein, fat, crude fiber, total dietary fiber, carbohydrates, calcium, phosphorus, sodium, chloride, potassium, magnesium, iron, copper, manganese, zinc, iodine, selenium, vitamin A, vitamin D3, vitamin E, vitamin C, thiamine (vitamin B1), riboflavin (vitamin B2), pantothenic acid, niacin, pyridoxine (vitamin B6), folic acid, biotin, cobalamin (vitamin B12), choline, arginine, lysine, methionine, cystine, taurine, linoleic acid, arachidonic acid, omega-6 fatty acids, omega-3 fatty acids, eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA).

[0188] In certain embodiments, the subject is a dog. In certain embodiments, the diet is a Royal Canin veterinary diet. In certain embodiments, the diet is selected from the group consisting of Ultramino, Hydrolyzed Protein Adult HP Dry, Hydrolyzed Protein Wet, Hydrolyzed Protein Adult PS Dry, Hydrolyzed Protein Mid Calorie Dry, Hydrolyzed Protein Small Breed Dry, Hydrolyzed Protein Treats, and any combination thereof.

[0189] In certain embodiments, the bacterium comprises a bile acid inducible operon. In certain embodiments, the bacterium is Clostridium hirano, Clostridium fulgidus, or a combination thereof. In certain embodiments, the bacterium is Clostridium hirano.

[0190] The presently disclosed subject matter provides a Royal Canin veterinary diet for treating an intestinal disease in a dog, wherein the dog comprises a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism, and wherein the first amount of the first intestinal microorganism is higher than a first reference amount of the first intestinal microorganism, and / or the second amount of the second intestinal microorganism is lower than a second reference amount of the second intestinal microorganism.

[0191] In certain embodiments, the first intestinal microorganism is selected from the group consisting of: new reference OTU45, JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, new reference OTU54, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, new reference OTU52, JQ208053.1.1336, and any combination thereof. In certain embodiments, the second intestinal microorganism is selected from the group consisting of: HK693629.1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456, HK555938.1.1357 and any combination thereof.

[0192] 5. Treatment Methods

[0193] In certain non-limiting embodiments, the presently disclosed subject matter provides a method for improving intestinal health and / or treating intestinal diseases in a subject in need thereof. In certain embodiments, the method can improve immunity, digestive function and / or reduce inflammation in companion animals.

[0194] In certain non-limiting embodiments, the presently disclosed subject matter provides a method for determining susceptibility to intestinal disease in a companion animal. In certain embodiments, the method comprises:

[0195] a) measuring a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism in a companion animal;

[0196] b) comparing a first amount of a first intestinal microorganism with a first reference amount of the first intestinal microorganism, and / or comparing a second amount of a second intestinal microorganism with a second reference amount of the second intestinal microorganism, wherein the reference amounts of the intestinal microorganisms are determined based on amounts of intestinal microorganisms of a plurality of healthy companion animals; and

[0197] c) determining that the companion animal is susceptible to an intestinal disease when the first amount of the intestinal microorganism is higher than a first reference amount of the first intestinal microorganism and / or when the second amount of the second intestinal microorganism is lower than a second reference amount of the second intestinal microorganism.

[0198] In certain embodiments, the first intestinal microorganism comprises one or more bacteria comprising 16S rRNA comprising a 16S rRNA sequence similar to HQ802983.1.1440, GQ449092.1.1375, GQ448744.1.1393, KF842598.1.1394, HG798451.1.1400, new reference OTU52, HK555938.1.1357, FJ957494.1.1454, FN667392.1.1495, new reference OTU54, HQ760911.1.1437, GQ006324.1.1342, FJ950694.1.1472, FM86 5905.1.1392、FJ506371.1.1371、FJ957528.1.1445、JF712675.1.1540、New reference OTU82、AB009242.1.1451、HQ751549.1.1448、AB506370.1.1516、DQ057365.1.1393、FN667422.1.1495、AJ270486.1.1241、FN668375.4306350.4307737、GQ867426.1.1494、 GX182404.8.1529, JF224013.1.1362, GQ448246.1.1389, JF807116.1.1260, KC245406.1.1465, FN667084.1.1493, EU470512.1.1400, EU768569.1.1352, AY239462.1.1500, KC504009.1.1465, FM179752.1.1686, new reference OTU114, HK557089.3.1395, JQ20 8181.1.1352, HQ803964.1.1435, AM276759.1.1484, JN387556.1.1324, GQ448486.1.1387, HK694029.9.1487, HQ754680.1.1441, FN563300.1.1447, FP929060.3837.5503, GQ448506.1.1374, a 16S rRNA nucleotide sequence of Enterococcus aeruginosa, Clostridium perfringens, or Escherichia coli, or a nucleotide sequence that is at least about 90% homologous to or identical to the 16S rRNA nucleotide sequence.

[0199] In some embodiments, the first intestinal microorganism is selected from the group consisting of: HQ802983.1.1440, GQ449092.1.1375, GQ448744.1.1393, KF842598.1.1394, HG798451.1.1400, new reference OTU52, HK555938.1.1357, FJ957494.1.1454, FN667392.1.1495, new reference OTU54, HQ760911.1.1437, GQ006324.1.1342, F J950694.1.1472, FM865905.1.1392, FJ506371.1.1371, FJ957528.1.1445, JF712675.1.1540, new reference OTU82, AB009242.1.1451, HQ751549.1.1448, AB506370.1.1516, DQ057365.1.1393, FN667422.1.1495, AJ270486.1.1241, FN668375.4306350.43077 37, GQ867426.1.1494, GX182404.8.1529, JF224013.1.1362, GQ448246.1.1389, JF807116.1.1260, KC245406.1.1465, FN667084.1.1493, EU470512.1.1400, EU768569.1.1352, AY239462.1.1500, KC504009.1.1465, FM179752.1.1686, new reference OTU114, H K557089.3.1395, JQ208181.1.1352, HQ803964.1.1435, AM276759.1.1484, JN387556.1.1324, GQ448486.1.1387, HK694029.9.1487, HQ754680.1.1441, FN563300.1.1447, FP929060.3837.5503, GQ448506.1.1374, Enterococcus duratus, Clostridium perfringens, Escherichia coli, and any combination thereof.

[0200] In certain embodiments, the first intestinal microorganism is Clostridium perfringens, Escherichia coli, and any combination thereof.

[0201] In certain embodiments, the second intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence that is at least about 90% homologous or identical to a 16S rRNA nucleotide sequence of EU774020.1.1361, HQ793763.1.1451, HQ792787.1.1438, the new reference OTU109, HQ792778.1.1436, or DQ113765.1.1450.

[0202] In certain embodiments, the second intestinal microorganism is selected from the group consisting of EU774020.1.1361, HQ793763.1.1451, HQ792787.1.1438, new reference OTU109, HQ792778.1.1436, DQ113765.1.1450, and any combination thereof.

[0203] In certain embodiments, the method further comprises providing a customized recommendation for a treatment regimen, and / or further monitoring the intestinal microorganisms when the first amount of the first intestinal microorganism is lower than a first reference amount of the first intestinal microorganism, and / or when the second amount of the second intestinal microorganism is higher than a second reference amount of the second intestinal microorganism.

[0204] In certain non-limiting embodiments, the presently disclosed subject matter provides a method for determining the responsiveness of a companion animal with an intestinal disease to a diet. In certain embodiments, the method comprises:

[0205] a) measuring a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism in a companion animal;

[0206] b) comparing a first amount of a first intestinal microorganism with a first reference amount of the first intestinal microorganism, and / or comparing a second amount of a second intestinal microorganism with a second reference amount of the second intestinal microorganism, wherein the reference amount of the intestinal microorganism is determined based on amounts of intestinal microorganisms of a plurality of healthy companion animals; and

[0207] c) determining that the companion animal is responsive to the diet when the first amount of the intestinal microorganism is above a first reference amount of the first intestinal microorganism and / or when the second amount of the second intestinal microorganism is below a second reference amount of the second intestinal microorganism, or determining that the companion animal is not responsive to the diet when the first amount of the intestinal microorganism is below a first reference amount of the first intestinal microorganism and / or when the second amount of the second intestinal microorganism is above a second reference amount of the second intestinal microorganism.

[0208] In certain embodiments, the first intestinal microorganism comprises one or more bacteria comprising 16S rRNA comprising a 16S rRNA identical to reference OTU45, JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, new reference OTU54, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, new reference OTU52, JQ208053.1.1336, and any combination thereof. rRNA nucleotide sequence is at least about 90% homologous or identical to the 16S rRNA nucleotide sequence. In certain embodiments, the second intestinal microorganism is selected from the group consisting of HK693629.1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456 or HK555938.1.1357.

[0209] In certain embodiments, the first intestinal microorganism is selected from the group consisting of new reference OTU45, JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, new reference OTU54, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, new reference OTU52, JQ208053.1.1336, and any combination thereof.

[0210] In certain embodiments, the second intestinal microorganism comprises one or more bacteria comprising 16S rRNA, wherein the 16S rRNA comprises a nucleotide sequence that is at least about 90% homologous to or identical to the 16S rRNA nucleotide sequence of HK693629.1.1491, GQ493166.1.1359, GQ4914326, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456 or HK555938.1.1357.

[0211] In certain embodiments, the second intestinal microorganism is selected from the group consisting of HK693629.1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456, HK555938.1.1357, and any combination thereof.

[0212] In certain embodiments, the method further comprises administering the diet to the companion animal when the companion animal is determined to be responsive to the diet. In certain embodiments, the method further comprises administering the diet, steroids, and optionally antibiotics to the companion animal when the companion animal is determined to be unresponsive to the diet.

[0213] In certain embodiments, the determining in step c) occurs prior to administering the diet or the diet, steroids, and optionally antibiotics to the companion animal.

[0214] In certain non-limiting embodiments, the presently disclosed subject matter provides a method for determining the effectiveness of a diet in treating an intestinal disease in a companion animal. In certain embodiments, the method comprises:

[0215] a) measuring a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism in a companion animal before or after feeding the companion animal a diet to treat an intestinal disease;

[0216] b) comparing a first amount of a first intestinal microorganism with a first reference amount of the first intestinal microorganism, and / or comparing a second amount of a second intestinal microorganism with a second reference amount of the second intestinal microorganism, wherein the reference amount of the intestinal microorganism is determined based on amounts of intestinal microorganisms of a plurality of healthy companion animals; and

[0217] c) when the first amount of intestinal microorganisms is higher than a first reference amount of the first intestinal microorganisms, and / or when the second amount of the second intestinal microorganisms is lower than a second reference amount of the second intestinal microorganisms, determining that the diet is effective for treating the intestinal disease, or when the first amount of intestinal microorganisms is lower than the first reference amount of the first intestinal microorganisms, and / or when the second amount of the second intestinal microorganisms is higher than the second reference amount of the second intestinal microorganisms, determining that the diet is ineffective for treating the intestinal disease.

[0218] In some embodiments, the first intestinal microorganism comprises one or more bacteria comprising 16S rRNA, wherein the 16S rRNA comprises a nucleotide sequence at least about 90% homologous or identical to the 16S rRNA nucleotide sequence of HK557089.3.1395 or GQ448336.1.1418. In some embodiments, the first intestinal microorganism is selected from the group consisting of HK557089.3.1395, GQ448336.1.1418 and combinations thereof.

[0219] In certain embodiments, the second intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence that is at least about 90% homologous to, or identical to, a 16S rRNA nucleotide sequence of KF842598.1.1394, GQ006324.1.1342, HQ8029403, JN387556.1.1324, FJ950694.1.1472, HG798451.1.1400, the new reference OTU52, or GQ448468.1.1366.

[0220] In certain embodiments, the second intestinal microorganism is selected from KF842598.1.1394, GQ006324.1.1342, HQ802983.1.1440, JN387556.1.1324, FJ950694.1.1472, HG798451.1.1400, new reference OTU52, GQ448468.1.1366, and any combination thereof.

[0221] In certain embodiments, the method further comprises administering the diet to the companion animal when the companion animal is determined to be responsive to the diet. In certain embodiments, the method further comprises administering the diet, steroids, and optionally antibiotics to the companion animal when the companion animal is determined to be unresponsive to the diet.

[0222] In certain embodiments, the determining in step c) occurs prior to administering the diet or the diet, steroids, and optionally antibiotics to the companion animal.

[0223] In certain embodiments, the reference amount of intestinal microorganisms is derived from the average amount of intestinal microorganisms in a plurality of healthy companion animals. In certain embodiments, the amount of intestinal bacteria is measured from a fecal sample of a subject.

[0224] In certain embodiments, the method comprises administering to the subject an effective amount of a currently disclosed pharmaceutical composition, an effective amount of a currently disclosed food, or any combination thereof. In certain embodiments, the method further comprises monitoring the intestinal microorganisms of the subject. In certain embodiments, the intestinal microorganisms are sampled from a fecal sample of the subject.

[0225] In some embodiments, the intestinal microorganism is selected from the group consisting of: Ruminococcus, Alloprevotella, Allisonella, Anaerostipes, Anaerobiospirillum, Bacteroides, Blautia, Clostridium sensu stricto 1, Collinsella, Coprococcus 1, Corynebacterium 1, 1), Campylobacter, Enterococcus, Erysipelatoclostridium, Escherichia-Shigella, Faecalitalea, Fusobacterium, Helicobacter, Intestinibacter, Lachnoclostridium, Lactobacillus, Megasphaera, Methanobrevibacter, Parabacteroides, Porphyromonas, Phascolarctobacterium, Peptoclostridium, Prevotellaceae UCG-001 The invention relates to bacteria selected from the group consisting of UCG-001, Pseudocitrobacter, Ruminiclostridium 9, Sarcina, Streptococcus, Succinivibrio, Treponema 2, Turicibacter, Tyzzerella, Tyzzerella 4, and any combination thereof.

[0226] In certain embodiments, the intestinal microorganism is selected from the group consisting of Escherichia-Shigella, Clostridium sensu stricto 1, Enterococcus, Fusobacterium, and any combination thereof. In certain embodiments, the intestinal microorganism is Escherichia coli, Clostridium perfringens, or a combination thereof.

[0227] In some embodiments, the amount of intestinal bacteria is reduced after the pharmaceutical composition is applied. In some embodiments, the amount of intestinal bacteria is reduced in about 14 days after the pharmaceutical composition is applied. In some embodiments, the amount of intestinal bacteria is reduced in about 21 days, about 14 days, about 12 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days or about 1 day after the pharmaceutical composition is applied. In some embodiments, the amount of intestinal bacteria is reduced in about 1 day to about 21 days, about 1 day to about 14 days, about 3 days to about 14 days, about 5 days to about 14 days, about 7 days to about 14 days, about 10 days to about 14 days or about 7 days to about 21 days after the pharmaceutical composition is applied.

[0228] In some embodiments, the intestinal microorganism is selected from the group consisting of: Clostridium plano, Clostridium scintillans, Veillonellaceae, Streptococcaceae, Bacteroides, Fusobacterium, Collinsella, Sarcina, Clostridium sensu stricto 1, Faecalitalea, Streptococcus, Erysipelatoclostridium, Megasphaera, Blautia, Peptoclostridium, and any combination thereof. In some embodiments, the intestinal microorganism is Clostridium plano, Clostridium scintillans, or a combination thereof.

[0229] In some embodiments, the amount of intestinal microorganism increases after applying pharmaceutical composition and / or food.In some embodiments, the amount of intestinal microorganism increases in about 14 days after applying pharmaceutical composition and / or food.In some embodiments, in about 21 days, in about 14 days, in about 12 days, in about 10 days, in about 7 days, in about 6 days, in about 5 days, in about 4 days, in about 3 days, in about 2 days or in about 1 day after applying pharmaceutical composition.In some embodiments, in about 1 day to about 21 days, in about 1 day to about 14 days, in about 3 days to about 14 days, in about 5 days to about 14 days, in about 7 days to about 14 days, in about 10 days to about 14 days or in about 7 days to about 21 days after applying pharmaceutical composition, the amount of intestinal microorganism increases.

[0230] In certain embodiments, the method comprises:

[0231] a) measuring a first amount of one or more intestinal microorganisms in a subject;

[0232] b) administering to the subject a treatment regimen for treating the intestinal disease;

[0233] c) measuring a second amount of intestinal microorganisms in the subject after step b); and

[0234] d) continuing to administer the treatment regimen when the second amount of intestinal microorganisms decreases compared to the first amount of intestinal microorganisms.

[0235] In some embodiments, the second amount of intestinal microorganisms is measured between about 7 days to about 14 days after step b). In some embodiments, the amount of intestinal microorganisms decreases within about 21 days, about 14 days, about 12 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day after step b). In some embodiments, the amount of intestinal bacteria decreases within about 1 day to about 21 days, about 1 day to about 14 days, about 3 days to about 14 days, about 5 days to about 14 days, about 7 days to about 14 days, about 10 days to about 14 days, or about 7 days to about 21 days after step b).

[0236] In certain embodiments, gut microbes are measured from a stool sample from a subject.

[0237] In certain embodiments, the method comprises:

[0238] a) measuring a first amount of one or more intestinal microorganisms in a subject;

[0239] b) comparing the first amount of intestinal microorganisms with a reference amount of intestinal microorganisms, wherein the reference amount of intestinal microorganisms is determined based on the amounts of intestinal microorganisms of a plurality of healthy subjects;

[0240] c) providing a customized recommendation for a treatment regimen and / or further monitoring the intestinal microorganisms when the first amount of the intestinal microorganisms is higher than a reference amount of the intestinal microorganisms.

[0241] In certain embodiments, the method further comprises measuring a second amount of intestinal microorganisms in the subject after step c), and continuing the treatment regimen when the second amount of intestinal microorganisms is reduced compared to the first amount of intestinal microorganisms and is above a reference amount of intestinal microorganisms.

[0242] In some embodiments, the second amount of intestinal bacteria is measured between about 7 days to about 14 days after step c). In some embodiments, the amount of intestinal microorganisms decreases within about 21 days, about 14 days, about 12 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day after step b). In some embodiments, the amount of intestinal microorganisms decreases within about 1 day to about 21 days, about 1 day to about 14 days, about 3 days to about 14 days, about 5 days to about 14 days, about 7 days to about 14 days, about 10 days to about 14 days, or about 7 days to about 21 days after step c).

[0243] In certain embodiments, gut microbes are measured from a stool sample from a subject.

[0244] In some embodiments, the intestinal microorganism is selected from the group consisting of Ruminococcus, Alloprevotella, Allisonella, Anaerostipes, Anaerobiospirillum, Bacteroides, Blautia, Clostridium sensu stricto 1, Collinsella, Coprococcus 1, Corynebacterium 1, 1), Campylobacter, Enterococcus, Erysipelatoclostridium, Escherichia-Shigella, Faecalitalea, Fusobacterium, Helicobacter, Intestinibacter, Lachnoclostridium, Lactobacillus, Megasphaera, Methanobrevibacter, Parabacteroides, Porphyromonas, Phascolarctobacterium, Peptoclostridium, Prevotellaceae UCG-001 In some embodiments, the intestinal microorganism is selected from the group consisting of Escherichia coli, Shigella spp., Clostridium spp. 1, Enterococcus, Fusobacterium spp., and any combination thereof. In some embodiments, the intestinal microorganism is selected from the group consisting of Escherichia coli, Clostridium perfringens, and any combination thereof.

[0245] In certain embodiments, the treatment regimen comprises administering an effective amount of a presently disclosed pharmaceutical composition, an effective amount of a presently disclosed food product, or any combination thereof.

[0246] In certain non-limiting embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a companion animal of a feline (e.g., a domestic cat) or a canine (e.g., a domestic dog). In certain non-limiting embodiments, the companion animal is at risk of intestinal disease and / or inflammation. In certain non-limiting embodiments, it is not known that the companion animal is at risk of intestinal disease and / or inflammation. In certain non-limiting embodiments, the companion animal suffers from intestinal disease and / or inflammation. In certain non-limiting embodiments, it is not known that the companion animal suffers from intestinal disease and / or inflammation. In certain non-limiting embodiments, the companion animal is receiving treatment for a digestive disorder and / or inflammation.

[0247] In certain non-limiting embodiments, the treatment is a diet. In certain embodiments, the companion animal is a dog. In certain embodiments, the enteropathy is an acute enteropathy or a chronic enteropathy. In certain embodiments, the enteropathy is a chronic enteropathy selected from the group consisting of food-responsive enteropathy, antibiotic-responsive enteropathy, and idiopathic inflammatory bowel disease (IBD).

[0248] In some embodiments, pharmaceutical compositions and / or food can be applied to subjects from 20 times a day to once a day, from 10 times a day to once a day, or from 5 times a day to once a day. In some embodiments, pharmaceutical compositions and / or food can be applied to subjects once a day, twice a day, three times a day, 4 times a day, 5 times a day, 6 times a day, 7 times a day, 8 times a day, 9 times a day, more than 10 times a day. In some embodiments, pharmaceutical compositions and / or food can be applied to subjects once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks, or once a month. In some embodiments, food can be applied to animals in a constant manner, for example, when animals are fed with continuously available tested food supplies.

[0249] In some embodiments, the dosage of the pharmaceutical composition is between about 1mg / kg body weight to about 5000mg / kg body weight every day. In some embodiments, the dosage of the pharmaceutical composition is between about 5mg / kg body weight and about 1000mg / kg body weight every day, between about 10mg / kg body weight and about 500mg / kg body weight every day, between about 10mg / kg body weight and about 250mg / kg body weight every day, between about 10mg / kg body weight and about 200mg / kg body weight every day, between about 20mg / kg body weight and about 100mg / kg body weight every day, between about 20mg / kg body weight and about 50mg / kg body weight every day or any intermediate range thereof. In some embodiments, the dosage of the pharmaceutical composition is at least about 1mg / kg body weight every day, at least about 5mg / kg body weight every day, at least about 10mg / kg body weight every day, at least about 20mg / kg body weight every day, at least about 50mg / kg body weight every day, at least about 100mg / kg body weight every day, at least about 200mg / kg body weight every day or more. In certain embodiments, the dosage of the pharmaceutical composition does not exceed about 5 mg / kg body weight per day, does not exceed about 10 mg / kg body weight per day, does not exceed about 20 mg / kg body weight per day, does not exceed about 50 mg / kg body weight per day, does not exceed about 100 mg / kg body weight per day, does not exceed about 200 mg / kg body weight per day, does not exceed about 500 mg / kg body weight per day, or more.

[0250] In certain embodiments, the amount of the pharmaceutical composition and / or food is reduced during feeding of the companion animal. In certain embodiments, the concentration of the pharmaceutical composition and / or food is increased during feeding of the companion animal. In certain embodiments, the concentration of the pharmaceutical composition and / or food is modified according to the age of the companion animal.

[0251] 6. Test kit

[0252] The presently disclosed subject matter provides a kit for treating and / or preventing an intestinal disease in a subject. In certain embodiments, the kit includes an effective amount of a pharmaceutical composition, dietary supplement, functional food, food, meal, or any combination thereof disclosed herein. In certain embodiments, the kit includes a sterile container; such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container forms known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding a drug.

[0253] If desired, the pharmaceutical composition, dietary supplement, functional food, food, and / or diet is provided with instructions for administering it to a subject suffering from or at risk of developing an intestinal disease. The instructions typically include information about the pharmaceutical composition, dietary supplement, functional food, food, and diet for treating and / or preventing intestinal diseases. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; dosage regimens and administration for treating or preventing intestinal diseases or their symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (if any), or as a label affixed to the container, or provided in or with the container as a separate sheet, brochure, card, or folder.

[0254] Advantageously, the kit can be packaged in groups for use, so that, for example, the daily prescription for each component can be identified to improve patient compliance. The group of pharmaceutical compositions can be identified in a variety of ways. For example, in some embodiments, a group of pharmaceutical compositions, dietary supplements, functional foods, foods, meals can be identified on the packaging containing them. In some embodiments, external instructions can be provided with one or more groups of pharmaceutical compositions, dietary supplements, functional foods, foods, meals, such as identifying the grouping and instructing the patient / animal owner to take the pharmaceutical composition, dietary supplement, functional food, food, diet of the kit at the appropriate time.

[0255] Example

[0256] The presently disclosed subject matter will be better understood with reference to the following examples, which are provided by way of illustration of the disclosure and not by way of limitation.

[0257] Example 1

[0258] introduce

[0259] Although multiple environmental factors have been shown to influence the microbiome, diet is considered one of the most potent regulators of the composition and function of the resident intestinal microbial communities in healthy humans and other mammals. 7,8 and may serve as a risk factor and treatment modality for IBD 9,10 Epidemiological data and mouse studies suggest that high-fat and / or low-fiber diets and dietary additives such as emulsifiers are either risk factors for IBD or, in some cases, directly impair gut barrier function, leading to disease. 11–13 Diet can also be used to treat IBD, with perhaps the most obvious example being the use of exclusive enteral nutrition (EEN) as first-line treatment for pediatric Crohn's disease. 14High remission rates (≥60%) were observed after EEN, and EEN achieved better patient growth compared with corticosteroids while reducing biomarkers of disease such as fecal calprotectin and C-reactive protein. 15-18 Interestingly, EEN has a significant effect on the microbiome, but the exact nature of this effect is difficult to discern, with some studies reporting reduced microbiome diversity after EEN treatment. 19-21 , while other studies have shown that 22,23 or increased diversity 24 .

[0260] The mechanisms by which diet affects the gut microbiome to improve IBD symptoms are unclear and difficult to dissect from human subject studies because diet is difficult to control and requires incorporation of extensive food intake surveys. 25 , controlled feeding studies 26 Conduct retrospective studies or focus on populations with different survival patterns 27-29 . In contrast, mouse models of colitis have yielded important insights into the pathophysiology of intestinal inflammation, but these generally involve chemical or genetic perturbations rather than spontaneous disease development. Furthermore, the widespread use of autoclaved food and acidified water in mouse husbandry, and the trend toward cage effects dominating mouse microbiome studies, raises concerns about the clinical relevance of diet-microbiome studies in these colitis models. As companion animals, dogs share the same environment as humans and spontaneously develop chronic intestinal inflammation that is clinically similar to human IBD, including similar gastrointestinal pathology, responses to similar treatments, and the ability to fight off infections.30 31 , the involvement of some of the same susceptibility gene loci 30-32 , and shared disease-associated microbial taxa 33-35 Interestingly, more than 50% of dogs with chronic enteritis go into long-term remission after being treated with dietary therapy. 36 , making the use of prescription diets a first-line treatment for IBD in companion animal medicine. A recent metagenomic study generated a catalog of more than 1 million classified and functionally annotated microbial genes from the canine gut and showed that—compared to other mammals such as mice and pigs—the dog’s microbial environment is most similar to that of humans. 37 Furthermore, the canine microbiome was significantly altered by dietary changes in a manner similar to that reported in humans. 37 Together, these data suggest that the dog is an ideal animal model to study diet-microbiome interactions in the context of intestinal disease.

[0261] Although the gut microbiome has been implicated in IBD pathogenesis and diet profoundly alters the microbiome and can be used to manage symptoms of IBD, understanding of how this occurs is limited. In this study, untreated dogs with chronic enteritis were examined and their fecal microbial community structure and metabolites were monitored in response to treatment. By comparing changes over time in dogs that responded to the diet with animals that failed dietary treatment and required subsequent combination therapy, the results suggest that the diet induces rapid remission by shaping community structure and reprogramming the metabolic function of the microbiome. Notably, secondary bile acids, likely produced by Clostridium, have been shown to be involved in diet-induced changes in the microbiome by inhibiting the growth of potential pathogens. These findings provide a general mechanism by which diet can modulate the microbiome to reduce gastrointestinal disease.

[0262] method

[0263] Diagnosis and Treatment of Canine Chronic Enteritis (CCE)

[0264] Client-owned animals showing clinical signs of CCE were screened at the Ryan Veterinary Hospital of the University of Pennsylvania. All animal work was performed in accordance with the guidelines of the University of Pennsylvania IACUC (Protocol 805283), and signed owner consent was obtained prior to enrollment. Dogs were screened if they showed any of the following clinical signs for a period of ≥3 weeks: vomiting, diarrhea, or weight loss despite adequate caloric intake. Dogs were excluded from screening if they had been treated with a hydrolyzed protein diet, antibiotics, corticosteroids, or probiotics within the previous 2 weeks. At screening, each animal underwent the following: a complete physical examination, routine fecal screening (including zinc sulfate flotation for parasite identification, Gram stain, and culture for Salmonella spp. and Campylobacter spp.), complete blood count, serum biochemistry profile, serum measurements of canine trypsin-like immunoreactivity, cobalamin, and folate, urinalysis, abdominal ultrasonography, and disease severity scoring using the Canine Chronic Enteropathy Clinical Activity Index (CCECAI) 36 If these initial screening tests failed to identify a cause for clinical signs, upper and / or lower gastrointestinal endoscopy and mucosal biopsies were performed. The biopsies were fixed in formalin, embedded in paraffin, sectioned with hematoxylin and eosin, and the slides reviewed by a board-certified veterinary pathologist. If histopathology showed intestinal inflammation with no identifiable underlying cause (eg, infectious agent), the dog was enrolled. If another histopathological diagnosis was identified, the dog was excluded.

[0265] The trial included three 14-day treatment phases (Fig. 1A and Fig. S1), and the dogs were evaluated for treatment response using the CCECAI at the end of each phase. Remission was determined using the abbreviated CCECAI, which includes scores on the first five indices (attitude / activity, appetite, vomiting, stool consistency, and stool frequency) and was defined as an abbreviated CCECAI score ≤2, with no score >1 on any of the five indices. Animals were first given a therapeutic hydrolyzed protein diet (Royal Canin HP). Dogs that entered remission after this treatment were designated as diet responders (DR) and maintained on the therapeutic diet as reminders of the trial. Animals that did not respond to the therapeutic diet (NDR) then began a two-week course of metronidazole (10 mg / kg PO q 12 h) while maintaining the therapeutic diet. Dogs that entered remission after antibiotic treatment maintained a combination of antibiotics and a therapeutic diet as reminders of the trial. Animals that still failed to show a good response continued on the diet and metronidazole, but received prednisone (1 mg / kg PO q 12 h) for the final 14 days of the trial (Tier 3). Dogs with hypoalbuminemia (protein-losing enteropathy) at initial screening were assumed to have more severe disease and a worse prognosis, so all three interventions were immediately performed and were not included in the analysis. All dogs with low serum cobalamin at screening were supplemented with cyanocobalamin (50 mcg / kg SQ q 7 days) during the study. At the end of the study, all animals returned to the clinic for the primary endpoint, which included a comprehensive re-evaluation of the dog including a complete physical examination, complete blood count, serum chemistry, serum measurements of cobalamin and folate (if low at the screening visit), urinalysis, CCECAI score, and final stool collection.

[0266] 16S rRNA gene sequencing and data analysis

[0267] Genomic DNA was extracted from feces using the PowerSoil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA) following the manufacturer’s recommendations. A mock community library containing purified genomic DNA from 12 known bacterial isolates was amplified and sequenced as a quality control. Additional controls included extraction blank treatment samples (in which the DNA extraction process was performed without the addition of input material) and water alone to determine background microbial signal. Dual-indexed amplicon sequencing was used to sequence the 16S rRNA gene. 61 The V4 region of the 1000-kb PCR was amplified. Pico-green based amplicons were sequenced on the MiSeq platform (Illumina, San Diego, CA) using 250 base pair paired-end chemistry. The Quantitative Insights into Microbial Ecology (QIIME) 62The reads were filtered with the -q20 -p 0.75 -r 3 filter options to remove sequences with an average Phred quality score ≤ 20. 63 Remove homopolymers with length > 10 bp and sequence < 248 bp and > 255 bp. 64 SILVA128 (97_otus_16S.fasta) 65,66 Representative 16S sequences were identified and chimeric sequences removed. Quality-controlled sequences were then clustered against the SILVA128 database (SILVA_128_QIIME_Release) using open reference OTU sorting implemented in QIIME using default parameters. The OTU table was streamlined to 10,600 sequences per sample. To obtain species-level taxonomic assignments of OTUs in the genus Clostridium sensu stricto 1, the NCBI 'nr' database was searched using the corresponding representative sequences in the SILVA database. Species were tentatively assigned by best hit (P<1e-5) and further confirmed by comparing the relative abundance of these species determined by metagenomic shotgun sequencing and 16S sequencing methods. OTU 'new reference OTU52' represents Clostridium perfringens, which is the most dominant OTU in some dogs, and OTU 'FJ957494.1.1454' corresponds to Clostridium hiranoensis.

[0268] The OTU table was analyzed using the R statistical environment 67 、bioconductor software suite 68 and Phyloseq2 package 69 Singletons and OTUs with ambiguous annotations were removed from the OTU table. Alpha diversity (Shannon diversity index and Faith's phylogenetic diversity) and beta diversity (weighted and unweighted UniFrac) were calculated using Phyloseq2. Pielou's evenness index was calculated according to the literature. 70 Calculates Tax4Fun with default parameters 71 The functional potential of the microbial community (KEGG pathways and KEGG orthologs) was predicted against the SILVA123 database. Wilcoxon and rank tests were used to compare KEGG pathways at different time points (FDR < 0.05). Principal component analysis of KEGG pathways and orthologs was performed by the R package factoextra. For differential abundance analysis and association analysis, filtering was performed to remove taxa with a maximum abundance of < 0.1% in all samples and < 10% presence in all samples. The resulting 381 species accounted for an average of 96.23% of the total microbial composition. DESeq2 implemented in Phyloseq2 (test = "Wald", fitType = "parametric") 72For differential abundance analysis at different taxonomic levels (fold change > 2 and P value < 0.05) using unrefined reads. Spearman correlations were calculated between the abundance of each microbial composition (log transformed) and the values ​​of the different factors (i.e. CCECAI, time point, concentration of each metabolite for each dog). To avoid recording zero values, 1 read was added to the abundance of each composition before calculating the Spearman correlation. Unless otherwise stated, all p-values ​​in the above analyses were adjusted for multiple comparisons by the FDR (Benjamini-Hochberg) method.

[0269] Metagenomic sequencing and data analysis

[0270] Sequencing libraries were prepared using Illumina Nextera XT, and 1 ng of canine feces was collected from 19 of the 20 dogs in the study that responded to the diet on days 0, 14, and 42. The size measurement and quantification of the libraries were performed using Tapestation 4200 (Agilent) and Qubit3 (Thermo Fisher), respectively. Equimolar amounts of each library were pooled and sequenced on an Illumina NextSeq 500 instrument to generate 150 bp double-end sequences. Sequencing adapters and low-quality reads were trimmed and filtered (leading: 3 trailing: 3 sliding window: 4: 15 min len: 36) by Trimmomatic (v0.36). High-quality reads were mapped to the canine reference genome (CanFam3.1) using Bowtie2 v2.3.4.1 (very sensitive), and aligned reads were removed using SamTools 73 After host filtering, the sequencing depth for each sample was >10 million paired-end reads (median depth = 35.8 million). 46 Taxonomic annotations for each sample were generated. The identified Clostridium spp. and Eubacterium spp. were further searched for the presence of genes involved in secondary bile acid production (bai operon) (p-value ≤ 1e-5) using protein sequences of genes in the 7α-dehydroxylation pathway (baiG, baiB, baiA, baiF, BACD, and baiE) against the reference genomes of these species in GeneBank using tBlastn.

[0271] Metagenomic data from pediatric Crohn's disease patients before and after exclusive enteral nutrition (EEN) have been described previously. 23, and downloaded from the European Nucleotide Archive (ENA) (SRP057027). The same filtering steps and settings for the metagenomic data analysis described above in this study were used for these datasets. After filtering out human reads, taxonomic annotation of each sample using Metaphlan2 showed the presence of Clostridium. Among them, Clostridium scindens is known for its secondary BA production capacity. 73 Paired reads from which PCR duplicates were removed were aligned to the C. fulgidus reference genome (ASM15450v1, strain ATCC 35704) and strain VE202-05 (ASM47184v1), and bacterial abundance (proportion of mapped reads to total reads) in different samples was estimated using bwa mem (v0.7.17-r1188)74 with default settings. Wilcoxon sum rank tests were used to test for significant differences in read mapping and Spearman correlations were used to compare to log-transformed fecal calprotectin (FCP) levels. 23 Number of mapped reads.

[0272] Identification of bacterial isolates by anaerobic culture and whole genome sequencing

[0273] Freshly collected rectal swabs from dogs with active disease (Day 0) and / or in remission at the end of the study (Day 42) were transferred to an anaerobic chamber (97.5% nitrogen, 2.5% hydrogen; Coy Labs, Grass Lake, MI) within one hour of collection. The tip of the swab was homogenized in 1 mL of pre-reduced PBS with 1% cysteine ​​(PBSc). Serial dilutions (down to 10 μl) made in PBSc were homogenized. -5 ) were inoculated into brain heart infusion (BHI), yeast tyrosine fatty acid and carbohydrate (YCFAC) 75 , Gut Microbiota Medium (GMM) 76 and De Man, Rogosa and Sharpe (MRS) 77Agar (Anaerobe Systems, Morgan Hill, CA). After incubation at 37 ° C for 1-3 days, single colonies were picked from the plates and cultured overnight in BHI, YCFAC, GMM or MRS broth (Anaerobe Systems, Morgan Hill, CA). Overnight cultures were stored as glycerol stocks (25% glycerol) and frozen neatly for DNA extraction. DNA was purified from bacterial isolates using a high-purity PCR template kit (Roche), and PCR was performed using specific primers for the bacterial 16S rRNA gene, including 27F (5'-AGAGTTTGATCMTGGCTCAG-3'), 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3'). PCR products were purified using QiaQuick PCR purification kit (Qiagen), Sanger sequenced, and assembled using Geneious software v11.1.5 (Biomatters Inc.). The longest high-quality assembled sequence segment (at least 800 bp) was used for BLAST to find the closest match in Genbank. In addition, for selected isolates of Clostridium hirano, Clostridium perfringens, and Escherichia coli, 1 ng DNA was used to construct sequencing libraries using Illumina Nextera XT. The size measurement and quantification of the library were as described above for metagenomic sequencing. For each sample, an Illumina NextSeq 500 instrument was used to generate at least about 10 million single-end reads of 150 bp. The quality control steps were the same as those for the above-mentioned metagenomic analysis. High-quality reads were mapped to the genome of Clostridium hirano (ASM15605v1) using Stampy 78 (-substitutionrate = 0.1), which allowed mapping of reads that were highly different from the reference genome. PCR duplications were removed by Samtools. For each isolate, coverage of the genomic region representing the bai operon was calculated to show the presence of genes in the 7α-dehydroxylation pathway. Metabolomics and in vitro bacterial growth inhibition assay

[0274] Bile acids in feces were quantified using a WatersAcquity uPLC system with a QDa single quadrupole mass detector and autosampler (192 sample capacity) as previously described. 79. Briefly, fecal samples were suspended in methanol (5 μL / mg feces), vortexed for 1 min, and centrifuged at 13000g for 5 min. The supernatant was transferred to a new vial and analyzed on an Acquity uPLC equipped with a CortecsUPLC C-18+1.6mm 2.1x50mm column. All chemicals and reagents were mass spectrometry grade. Canine isolates of Clostridium perfringens (n=3) and Escherichia coli were recovered from glycerol stocks in modified fortified Clostridium broth (MRCB, Fisher Scientific) or Luria broth (LB, Fisher Scientific), respectively, and cultured overnight at 37°C in an anaerobic chamber. Lithocholic acid and deoxycholic acid (Sigma) were dissolved in 100% ethanol (30 mg / mL). After overnight growth, inhibition of growth by deoxycholic acid was determined by microbroth dilution and measured by OD 630 Due to the low solubility (<1 mg / L), the inhibitory effect of lithocholic acid was evaluated by counting colonies on agar plates with LCA (0, 0.1, 0.25, 0.5, 0.75 or 1 mg / mL, and LB plates for E. coli, and 0, 0.01, 0.025, 0.05, 0.075 or 0.01 mg / mL, and Columbia blood agar supplemented with 5% defibrinated sheep blood for anaerobically cultured C. perfringens, cultured at 37°C for 24 (E. coli) or 48 (C. perfringens) hours).

[0275] Mouse experiments

[0276] Four days before the start of dextran sulfate sodium (DSS) treatment, female C57BL / 6 (7 weeks old) (Jackson Laboratory) were inoculated with a kanamycin-resistant Escherichia coli strain (Nissle 1917) (1×10 9 CFU / mouse) were orally pre-implanted. Animals were randomly assigned to groups (cages) at baseline, and drinking water was replaced with either filter-sterilized water (mock treatment) or a filter-sterilized aqueous solution of 2.5% (w / v) DSS (relative molecular weight ~40,000; Sigma-Aldrich). From day 0 to day 4, mock- or DSS-treated mice were orally gavaged with C. hiranoensis (1 × 10 8 CFU / mouse in anaerobic PBS) or PBS (control). C. hiranoensis was grown anaerobically overnight at 37°C in MRCB. Culture density was assessed by optical density (630 nm) and the required volume of culture was spun at 10,000 g for 15 minutes. The bacterial pellet was resuspended in PBS to obtain 1x10 8CFU / mouse dose. All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Pennsylvania. Mice were then euthanized on day 8, and colon contents and tissues were collected. Colon contents were weighed and cultured on LB agar plates containing kanamycin (100 μg / mL) for 16 hours. Fecal samples from baseline and colon contents on day 8 were collected and stored at -80°C for testing bile acid levels. The colon was fixed in formalin and sections were stained with hematoxylin and eosin (H&E). Pathology was blindly evaluated by a board-certified veterinary pathologist (CB) according to standard criteria for DSS colitis.

[0277] Data availability

[0278] The original 16S rRNA gene sequences of canine fecal samples have been deposited in the Sequence Read Archive (SRA 80 ; accession number to be determined). The processed OTU table and metadata can be accessed through MicrobiomeDB 56 Access. Metagenomic and whole-genome sequence data are also available at SRA (accession numbers pending).

[0279] result

[0280] Dietary therapy induces rapid and durable remission

[0281] To investigate the effects of therapeutic diets on disease and the microbiome, untreated dogs (n = 29) with chronic enteritis (CE) were enrolled in a study to assess the effects of diet on disease and the microbiome. Dogs with active disease were switched from their current diet to a commercially available therapeutic hydrolyzed protein diet ( Figure 1A ). The effects of treatment on disease were monitored using the Canine Chronic Enteropathy Clinical Activity Index (CCECAI; hereafter referred to as the 'disease score'), which is positively correlated with adverse clinical outcomes. 36. After two weeks on the therapeutic diet, 69% (20 / 29) of the animals entered remission, characterized by a decrease in mean disease score from 4.1 (95% CI = 4.8-3.3) to 1.3 (95% CI = 1.8-0.7). These diet-responsive (DR) animals remained on the diet for the remainder of the study with no additional intervention (Figure 1B). At the end of the study (day 42), the mean disease score of DR animals was 0.9 (95% CI = 1.3-0.6), a >4-fold decrease in disease severity compared to day 0 (Figure 1B). In contrast, 31% (9 / 29) of the animals did not have a significant decrease in disease score after two weeks on the therapeutic diet (Figure 1C). These non-diet-responsive (NDR) animals developed more severe disease scores than DR animals (mean score = 6.1; 95% CI = 7.4-4.7) (P < 0.05 on day 0) and did not show a significant decrease after 2 weeks of dietary therapy (Figure 1C). NDR animals maintained dietary treatment to remind the study but also received combination therapy including antibiotics (day 14) and prednisone (day 28) ( Figure 1A and Figure 8 , see Methods), but only showed a gradual improvement in disease scores (Fig. 1C). These data highlight the rapid clinical response of most dogs with chronic enteritis to the hydrolyzed diet.

[0282] Identification of microbiome profiles associated with treatment outcomes

[0283] To determine whether treatment with a hydrolyzed diet alone was sufficient to alter the microbial community in the gut, fecal samples collected from DR, NDR, and healthy control animals (n = 11) were subjected to 16S rRNA gene analysis. 38 Consistently, we found that the diversity of the canine fecal microbiome was not significantly altered in dogs with CE compared to healthy controls (Fig. 9A-B), and the communities in both groups were primarily composed of Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria (Fig. 9C). However, animals with CE showed greater inter-individual distances in microbial community structure compared to healthy dogs by unweighted Unifrac (Fig. 9D). Using ternary plot visualization, enrichment of operational taxonomic units (OTUs) was observed from Firmicutes and Proteobacteria in animals with active disease, whereas Bacteroidetes were enriched in healthy animals (Fig. Figure 2A Interestingly, a subset of Proteobacterial OTUs were highly enriched in DR animals compared to NDR and healthy controls ( Figure 2A ), tan dot in lower left corner).

[0284] These differences prompted us to perform a formal differential abundance analysis, which identified 55 OTUs that distinguished healthy from diseased animals (Table 2). For example, Escherichia coli, which is commonly associated with enteric disease, was overrepresented in animals with CE (Figure 2B) and showed a significant (albeit weak) positive correlation with disease score (R = 0.2109, P = 0.02626) (Figure 2C). OTUs from the genus Clostridium sensu stricto 1 were also enriched in CE, including Clostridium perfringens (Figure 2D), which also showed a positive correlation with disease score (Figure 2E) (R = 0.2324, P = 0.01412). These bacteria have been associated with large intestinal diarrhea / colitis in dogs. 39 . In line with previously published work 40 Together, these data suggest that dysbiosis during CE is marked by the presence of pathogens. Next, we investigated whether the microbiomes of DR and NDR animals differed before treatment initiation (day 0). Although no differences were observed between the groups in terms of community diversity, evenness, or distance to healthy controls (unweighted or weighted Unifrac), 21 OTUs that were differentially abundant between DR and NDR animals were identified, 13 of which were enriched in animals that ultimately responded to dietary treatment (Figure 2F and Table 3). Interestingly, Proteobacteria and Clostridium perfringens were found to be more abundant in DR animals (Figure 2F). Overall, these results highlight distinct microbial signatures during illness that are associated with different clinical outcomes following dietary treatment.

[0285] Therapeutic diet improves dysbiosis associated with chronic intestinal inflammation

[0286] To assess whether diet-induced remission was accompanied by changes in dysbiosis, we compared microbial community structure before and after administration of the therapeutic diet in DR animals. No significant changes were observed in phylogenetic distance or Shannon diversity (Fig. 10A-B), but a significant increase in community evenness following diet administration was indeed seen when focusing on the top 40 most abundant OTUs in the sample (Fig. 3A), which accounted for 83% of the total reads. Principal coordinates analysis based on unweighted (Fig. 3B) or weighted (Fig. 10C) UniFrac revealed clear separation between dogs even on day 0, before the dietary treatment was administered, highlighting the heterogeneity of dysbiosis associated with clinical disease. Despite this baseline difference between animals, a significant shift in community structure away from the disease state occurred at 14 and 42 days after dietary treatment (Fig. 3B). Comparing the unweighted Unifrac distances between DR and healthy animals at each time point, it was observed that diet-induced remission was marked by a decrease in phylogenetic distance relative to healthy controls, a trend that persisted until day 42, when phylogenetic similarity to day 0 was lowest and similarity to healthy dogs was highest ( Fig. 3C ).

[0287] Given that the therapeutic diet altered the microbiome structure in DR animals, it is reasonable to assume that the dietary intervention would rapidly alter the composition of the fecal microbiota. Administration of the therapeutic diet was broadly characterized by an increase in Firmicutes and a decrease in Proteobacteria (Figure 3D). Fourteen days after the start of dietary treatment, 10 genera were differentially abundant in DR animals compared with before treatment (day 0) (Table 4). Potential pathogenic genera associated with IBD were found to be underrepresented after dietary treatment. For example, Escherichia-Shigella, Clostridium sensu lato 1, Enterococcus, and Fusobacterium had high relative abundance on day 0 and were significantly reduced after 14 days of the therapeutic diet. When evaluated at the species level, 36 OTUs were significantly differentially abundant in samples collected on day 0 compared with day 14 (Figure 3E) (Table 5). In the present study, Escherichia coli was typically enriched in animals on day 0 (Figure 3E), and its relative abundance dropped dramatically after two weeks on the therapeutic diet, ultimately reaching nearly undetectable levels on day 42 that were also indistinguishable from those observed in healthy dogs (Figure 3F). Clostridium perfringens was also significantly less prevalent in day 14 samples and in healthy dogs compared with day 0 samples (Figure 3G). In turn, the presence of genera from human studies that are considered beneficial commensals, such as Blautia, was increased. 41 Several OTUs were identified in (Table 5). Taken together, these results suggest that dietary therapy improves dysbiosis by reducing pathogens and increasing beneficial commensal taxa as hallmarks of the treatment.

[0288] Remission-specific changes in the microbiome following dietary treatment

[0289] It was hypothesized that the changes observed after dietary treatment in DR animals were related to remission and not simply a response to the diet that was unrelated to clinical outcome. To test this hypothesis, the effects of the therapeutic diet on dogs that entered remission after dietary treatment alone (DR) were compared with changes observed in non-diet responders (NDR) animals that failed to enter remission after dietary treatment and required additional treatment after day 14. While dietary treatment of DR animals was associated with increased community evenness (Figure 3A) and decreased phylogenetic distance from healthy dogs (Figure 3C), the same treatment of NDR dogs did not significantly affect the evenness or Unifrac distance of the microbial community from healthy dogs (Figures 4A and 4B). As observed in DR animals (Figure 3D), the diet also altered the gut microbiota composition of NDR animals (Figure 4C). Differential abundance analysis comparing NDR animals on day 0 versus day 14 (when they received the therapeutic diet alone) identified 24 OTUs (Table 6). However, this shift was different from that observed in DR animals (Figures 4D and Fig.11). For example, dietary treatment was associated with a decrease in the relative abundance of Fusobacterium and Pseudomonas in NDR animals on day 14 compared to day 0, while these taxa were unchanged or more mildly altered by dietary treatment in DR animals. In contrast, Escherichia coli-Shigella, Enterococcus, and some Clostridium sensu stricto 1 species were reduced only in animals that entered remission after dietary treatment (Figure 4D). The disease-associated bacteria Escherichia coli and Clostridium perfringens did not change significantly in NDR animals after dietary treatment (Figures 4E and 4F). After 14 days on the therapeutic diet, NDR dogs continued on the diet but were also given metronidazole, an antibiotic that primarily targets anaerobic bacteria. Interestingly, antibiotic treatment exacerbated dysbiosis, resulting in a sharp decrease in community evenness (Figure 4A), an increased distance from healthy controls (Figure 4B), and an increase in the relative abundance of potential pathogens (Figures 4E and 4F).

[0290] Diet-induced remission is associated with metabolic reprogramming and increased levels of secondary bile acids.

[0291] To determine whether diet-induced changes in microbial community structure translated into alterations in microbial metabolism, 16S rRNA gene sequencing data were used to assess the relative abundance of predicted KEGG pathways. Principal component analysis of metabolic pathway abundance data revealed a separation between samples from animals with active disease (day 0) and those in remission (day 14). Fig.12 , Figure 5A and Figure 5B). Differential abundance analysis identified 36 pathways whose relative abundance was increased as a result of dietary treatment in DR animals (Table 7), including several involved in carbohydrate metabolism and secondary bile acid synthesis (Figure 5C and Figure 5D). In contrast, 50 pathways were decreased, including fatty acid and steroid biosynthesis (Figure 5C). This shift in metabolic potential from lipid biosynthesis to carbohydrate and bile acid synthesis as animals enter remission prompted us to quantify bile acids in feces. Using targeted metabolomics, levels of 15 bile acids were measured in feces of healthy dogs compared to feces collected on days 0, 14, and 42 of the study ( Fig.13 , Table 10). Consistent with the 16S data, the secondary bile acids deoxycholic acid (Fig. 5E) and lithocholic acid (Fig. 5F) were high in healthy controls but low in animals with active disease (day 0) and significantly increased in DR animals at days 14 and 42 after dietary treatment (Fig. 5E and 5F, and Table 8). Notably, the levels of these secondary bile acids were not increased by dietary treatment in NDR animals (Fig. 5G and 5H), suggesting that this metabolic shift is associated with diet-induced remission.

[0292] Lithocholic acid and deoxycholic acid inhibit the growth of Escherichia coli and Clostridium perfringens in vitro.

[0293] Diet-induced microbiome remodeling may be due, at least in part, to the inhibitory effects of secondary bile acids on harmful bacteria. Correlation analysis of metabolomics and microbiome data identified 13 genera that were significantly correlated with at least one bile acid (Spearman, R > 0.04 or < −0.04) ( Figure 6A ). The primary bile acid, cholic acid, was negatively correlated with 11 OTUs, consistent with the reported ability of this bile acid to negatively regulate bacterial growth. 42 consistent. It was also observed that the increase in secondary bile acids after dietary treatment was associated with a decrease in the relative abundance of certain bacteria (e.g., OTUs from the genera Escherichia-Shigella, Clostridium, and Fusobacterium) (Table 9). To directly test this hypothesis, the ability of lithocholic acid or deoxycholic acid to inhibit the in vitro growth of Escherichia coli (n=1) or Clostridium perfringens (n=3) isolates from dogs with active disease was assessed, as these species or their genera have been implicated in disease in animal models. Deoxycholic acid prevented the growth of both species at concentrations comparable to those detected in fecal samples (Figures 6C and 6E), while lithocholic acid prevented the growth of Escherichia coli but not Clostridium perfringens (Figures 6B and 6D, respectively). Overall, these results indicate that the inhibitory activity of these bile acids varies with different bacteria and suggest that the increase in secondary bile acids observed after dietary treatment helps reduce potentially harmful bacteria. Clostridium hiranoensis is a diet-responsive species capable of producing secondary bile acids.

[0294] Next, we determined the sources of lithocholic and deoxycholic acid after dietary treatment. Their production from primary bile acids requires 7-dehydroxylation activity conferred by the bile acid-inducible (bai) operon—an activity unique to a limited number of anaerobic bacteria that represent a small fraction of the microbiome, including some Clostridium and eubacterial species. 43 . Given that the levels of certain Clostridial OTUs and lithocholic and deoxycholic acids were found to increase after diet-induced remission (Figure 3G and Figure 5E-F, respectively), potential bile acid producers in DR animals were identified. Fecal samples collected on days 0, 14, and 42 after the start of dietary treatment were subjected to metagenomic sequencing. Taxonomic assignment of reads identified six Clostridial species (C. perfringens, C. hiranosum, C. nexile, C. colicanis, C. glycolicum, and C. ramosum) present in these samples and two Eubacterium species (Eubacterium biforme and E. dolichum) at a relative abundance ≥ 0.01% in at least 10% of the samples. Among these species, only C. hiranosum has been reported to have a bai operon 44 , which was confirmed by BLAST of the reference genomes of these species in GenBank. In addition, metagenomic data ( Fig.14 ) and 16S sequencing data showed that the relative abundance of C. plannosum increased significantly in DR animals after dietary treatment (Figure 6F, left), but not in NDR animals that failed dietary treatment (Figure 6F, right). Because the genus Fusobacterium exhibits a high degree of genetic divergence, even at the species level, it was confirmed that canine C. plannosum has the bai operon. Rectal swabs collected during the study were anaerobic cultured, followed by isolate selection and Sanger sequencing of the full-length 16S rRNA gene, for canine cultures collected from seven dogs with chronic enteritis before and / or after treatment. A total of 49 Clostridium isolates belonging to five species (C. baratii, C. perfringens, C. sartagoforme, C. plannosum, and C. lactatifermentans) were identified. 82% (31 / 39) of Clostridium isolates from animals with active disease were Clostridium perfringens, consistent with reported involvement of this organism in canine 39 and humans 45 6G ). Taken together, these data suggest that C. hirano, a species originally isolated from human feces, 44 , may be a bile acid producer associated with diet-induced remission in dogs.

[0295] Clostridium hirano inhibits inflammation-induced expansion of Escherichia coli in a mouse model of DSS colitis

[0296] The ability of C. hirano to produce secondary bile acids, combined with the observation that lithocholic acid and deoxycholic acid are potent inhibitors of E. coli and C. perfringens growth in vitro, prompted us to test whether C. hirano could limit the expansion of potential pathogens in vivo during inflammation. Mice were first colonized with drug-selective E. coli (Nissle 1917 strain), inflammation was initiated by administration of dextran sodium sulfate (DSS) in the drinking water, and animals were orally treated daily with PBS (mock) or C. hirano (Figure 6H). DSS treatment resulted in a reduction in colon length (Figure 6I) and a proliferation of E. coli Nissle strain (Figure 6J). In contrast, mice that received daily DSS treatment of C. hirano exhibited a significant reduction in colon shortening and almost complete abrogation of E. coli expansion. Combined with the findings that lithocholic acid and deoxycholic acid can inhibit pathogen growth, these data suggest that C. hirano or secondary bile acids produced by this species mediate colonization resistance during enteritis.

[0297] Clostridium fulgidus is associated with diet-induced remission in pediatric Crohn's disease

[0298] Given the high remission rates observed in dogs and humans following dietary treatment, it was hypothesized that similar induction of C. bai-containing operons would occur in pediatric Crohn's disease patients treated with exclusive enteral nutrition (EEN). To test this, published data from a recent study examining approximately 20 patients before and after EEN treatment were analyzed. 23 Half of the patients responded to treatment, while the other half did not respond to EEN treatment. 46 Classification of the bacterial taxa present in each sample revealed the presence of Clostridium fulgidus, which is known to have high 7-dehydroxylation activity44, 47 . Relative abundance was further estimated using the proportion of total reads mapping to the C. scintillans reference genome. As shown in Figure 7A, this bacterium increased significantly from pretreatment to 8 weeks after EEN, as did the number of reads mapping to the bai operon (Figure 7B). Notably, this increase was only observed in patients who entered remission after EEN (responders, n = 10) but not in those who failed treatment (non-responders, n = 10) (Figures 7A and 7B). Furthermore, the relative abundance of C. scintillans was correlated with fecal calprotectin (FCP), a biomarker of disease activity in IBD. 23 ) showed a significant negative correlation in EEN "responsive" patients (Figure 7C) (R = -0.3515, P = 0.03287), but not for EEN "non-responsive" patients (R = -0.0267, P = 0.877). Similarly, a significant negative correlation between the bai operon and FCP was observed in diet responders (R = -0.3944, P = 0.0157), but not in non-responders (R = 0.0490, P = 0.7766) (Figure 7D). These results collectively point to bile acid-producing Clostridia as a key feature of diet-induced remission and an effective inhibitor of colonization by animal and human pathogens (Figure 7E).

[0299] discuss

[0300] Using animal models of diet response to investigate the role of the microbiome in chronic enteritis, we identified remission-specific changes in microbiome composition and function. All animals included in the study had active disease, but their baseline microbiome composition varied widely (Figure 3), likely reflecting differences in their environment, genetic background (breed), age, and body weight. This variation in composition supports the idea that enteric disease is not driven by a single state of dysbiosis, but rather that dysbiosis reflects a loss of community stability.48 Rather than dramatic changes in microbial community structure following dietary treatment, a shift from lipid metabolism to carbohydrate and bile acid synthesis was observed. Although the metabolomic analysis focused on bile acids, a broader understanding of the metabolites produced before and after dietary treatment, as well as the macro- and micronutrients present in the diet itself, could improve understanding of the mechanisms by which remission is achieved with the diet.

[0301] An important unanswered question is exactly how therapeutic diets such as EEN or prescription pet foods alter the microbiome and whether there are general principles that can be used to guide the development of better dietary therapies. Studies in pediatric Crohn's disease have reported higher remission rates with EEN compared with partial enteral nutrition (PEN) that includes some table foods. These observations have led some to hypothesize that highly monotonous diets with reduced complexity could constitute an important component of nutritional therapy for IBD. Consistent with this idea, mice fed a monotonous diet exhibited lower microbial diversity and were more susceptible to DSS colitis than mice fed an alternating diet. 49 However, the prevalence and treatment of chronic enteritis in veterinary medicine highlights that disease often recurs even in the setting of a monotonous diet and that rapid and robust relief can be achieved with solid food. Hydrolyzed protein diets, such as the one used in the study, have been shown to be effective in treating chronic enteritis in canines50, 51 , and it has been previously demonstrated that hydrolyzed protein diets are more effective for long-term management compared with highly digestible diets formulated with non-hydrolyzed protein sources. 50,51 While it is uncertain what features of these hydrolyzed formulas are driving the response, the low molecular weight of the hydrolyzed proteins should reduce their ability to be recognized by the immune system while providing better digestibility. Altogether, the results suggest that dogs will be a useful model to dissect the beneficial and detrimental effects of different diets, especially since formulated diets have long been the standard of care for treating a wide range of diseases in companion animals.

[0302] Secondary bile acids and bile acid-producing Clostridial species were identified as key features of diet-induced remission in humans and dogs. These findings complement recent studies examining the mechanisms by which fecal microbiota transplantation (FMT) can cure Clostridium difficile (C. difficile) infections. 52 Buffy et al. identified Clostridium scindens as being involved in resistance to C. difficile infection in humans and mice, and they showed that transfer of C. scindens or consortia containing this organism protected mice from C. difficile challenge. Furthermore, inhibition of C. difficile growth in vitro by C. scindens was associated with production of secondary bile acids. These data are consistent with microbiological studies showing that primary bile acids induce germination of C. difficile, whereas certain secondary bile acids can prevent vegetative growth. 53. Although C. difficile has not been observed in animals, C. perfringens and E. coli have been identified as major disease-associated taxa, and physiological levels of secondary bile acids have been shown to effectively prevent the in vitro growth of these organisms. It is currently unknown whether bile acids can limit these organisms in vivo in a canine model, but elucidation of these mechanisms may have important health implications beyond veterinary medicine. Although C. difficile is the leading cause of nosocomial diarrhea in humans, both C. perfringens and E. coli are common human commensals and have been implicated in diarrheal disease and colitis in both humans and dogs. In addition, the ability of C. perfringens to produce large amounts of toxins makes it a major cause of foodborne illness and soft tissue infections. Interestingly, when data from a cohort of pediatric Crohn's disease patients were examined before and after dietary treatment, C. scintillans was found to be associated with diet-induced remission (Figure 7), and related studies have shown that sustained remission after EEN is characterized by low levels of Proteobacteria, while relapsing patients show a significant increase in Proteobacteria. 54 These data, together with previous studies in dogs33, 35 Highlights the importance of utilizing animal models and advocates for the use of newly developed analytical methods 55 and database methods 56,57 Used to compare multiple microbiome studies to take a One Health approach that can identify conserved themes in host-microbiome interactions.

[0303] Clostridium difficile infection often develops after antibiotic treatment, a phenomenon attributed to the effect of antibiotics on secondary bile acid levels 58 . Interestingly, antibiotics were also observed to antagonize diet-induced changes in microbiome composition and function, promoting a more dysregulated state consistent with significantly reduced lithocholic and deoxycholic acid levels (Figures 4 and 5). Taken together, these data support a more general model of microbe-microbe interactions in the gut, in which bile acid-producing Clostridium restrict the growth of a range of bile acid-sensitive pathogens to limit disease, and highlight that these processes are exquisitely sensitive to antimicrobial agents. Similarities between the findings and those reported for FMT and C. fulgidus suggest that FMT may also be beneficial in the treatment of enteritis. Clinical trials testing this hypothesis in patients with IBD have shown modest success, in stark contrast to C. difficile infection, where FMT cured the vast majority of patients. 59 This difference may be related to different pathogens that contribute to the pathogenesis of IBD. Interestingly, colitis is a common side effect observed in cancer patients undergoing immune checkpoint blockade, and a recent study showed that FMT 60 The colitis resolved completely, raising the possibility that bile acid producers may be important in treating certain types of colitis.

[0304] Table 2. OTUs with differential abundance between healthy and CCE dog samples on day 0 (fold change > 2 and P value < 0.05)

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[0309] Table 3. OTUs with differential abundance between day 0 samples from dogs that responded to the diet and dogs that did not respond to the diet.

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[0312] Table 4. Genera with differential abundance between day 14 and day 0 (day 14 vs. day 0) dog samples in dogs that responded to the diet.

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[0315] Table 5. OTUs with differential abundance between day 14 and day 0 in dog (day 14 vs. day 0) samples that responded to the diet.

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[0319] Table 6. OTUs with differential abundance (fold change >2 and P value <0.05) between day 0 and day 14 (day 0 vs. day 14) samples in dogs that did not respond to the diet.

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[0322] Table 7. Comparison of KEGG pathways between different time points in the dog study in response to diet

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[0326] Table 8. Comparison of bile acids between samples at different time points in dogs responding to diet

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[0328] Table 9. Spearma correlations between OTU abundance and bile acid concentrations in dogs responding to diet.

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[0332] Table 10. Comparison of Bile Acid Amounts in Fecal Samples of Healthy Dogs, Dogs with CCE that Responded to Diet, and Dogs with CCE that Did Not Respond to Diet

[0333]

[0334] Note: Values ​​<1 nmol / g are below the detection limit; therefore an appropriate value of 0.5 was used in the calculations.

[0335] Table 11. 16S of OTUs in Tables 2-5 rRNA sequences

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[0458] 79. Friedman, ES et al. FXR-Dependent Modulation of the Human Small Intestinal Microbiome by the Bile Acid Derivative Obeticholic Acid. Gastroenterology (2018). doi: 10.1053 / j.gastro.2018.08.022

[0459] 80. Leinonen, R., Sugawara, H., Shumway, M. & International Nucleotide Sequence Database Collaboration. The sequence read archive. Nucleic Acids Res. 39, D19-21 (2011).

[0460] ***

[0461] Although the subject matter disclosed herein and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made in the present application without departing from the spirit and scope of the present invention as defined by the appended claims. In addition, the scope of the present application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, material compositions, means, methods and steps described in the specification. One of ordinary skill in the art will understand from the subject matter disclosed herein that the processes, machines, manufactures, compositions, means, methods or steps that currently exist or will be developed later and perform substantially the same functions or achieve substantially the same results as the corresponding embodiments disclosed herein may be utilized according to the subject matter disclosed herein. Therefore, the appended claims are intended to include such processes, machines, manufactures, material compositions, means, methods or steps within their scope.

[0462] Throughout this application, patents, patent applications, publications, product descriptions, and protocols are cited, the disclosures of which are incorporated by reference in their entireties into this application for all purposes.

Claims

1. A pharmaceutical composition comprising an effective amount of a bacterium capable of producing a first bile acid for use as a medicament, wherein the bacterium comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

2. The pharmaceutical composition of claim 1, further comprising an effective amount of a second bile acid.

3. A pharmaceutical composition comprising an effective amount of bacteria capable of producing a first bile acid for treating an intestinal disease in a subject in need thereof, wherein the bacteria comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

4. The pharmaceutical composition of claim 3, further comprising an effective amount of a second bile acid.

5. A pharmaceutical composition as described in claim 3, wherein the bile acid inducible operon (bai operon) comprises the nucleotide sequence listed in SEQ ID NO:

1.

6. A pharmaceutical composition as described in any one of claims 1 to 5, wherein the bacterium comprises 16s rRNA, and the 16s rRNA comprises a nucleotide sequence identical to SEQ ID NO: 2 or SEQ ID NO:

4.

7. A pharmaceutical composition as described in claim 6, wherein the bacterium comprises 16s rRNA, and the 16s rRNA comprises the nucleotide sequence listed in SEQ ID NO:

2.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the bacterium is Clostridium hiranoensis ( C. hiranonis ), C. scindens, or a combination thereof.

9. The pharmaceutical composition of claim 8, wherein the bacterium is Clostridium hiranoensis.

10. The pharmaceutical composition of any one of claims 1, 3 and 5, wherein the first bile acid is selected from the group consisting of chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid and any combination thereof.

11. The pharmaceutical composition of any one of claims 2, 4 and 6-9, wherein the first bile acid and / or the second bile acid is selected from the group consisting of chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid and any combination thereof.

12. The pharmaceutical composition of any one of claims 1, 3, 5 and 10, wherein the first bile acid is a secondary bile acid.

13. The pharmaceutical composition of claim 12, wherein the secondary bile acid is selected from the group consisting of taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid, and any combination thereof.

14. The pharmaceutical composition of claim 13, wherein the secondary bile acid is deoxycholic acid and / or lithocholic acid.

15. The pharmaceutical composition of any one of claims 2, 4, 6-9 and 11, wherein the first bile acid and / or the second bile acid is a secondary bile acid.

16. The pharmaceutical composition of claim 15, wherein the secondary bile acid is selected from the group consisting of taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid, and any combination thereof.

17. The pharmaceutical composition of claim 16, wherein the secondary bile acid is deoxycholic acid and / or lithocholic acid.

18. The pharmaceutical composition of any one of claims 3-17, wherein the subject is a dog.

19. The pharmaceutical composition according to any one of claims 3 to 17, wherein the intestinal disease is an acute intestinal disease or a chronic intestinal disease.

20. The pharmaceutical composition of any one of claims 3-17, wherein the intestinal disease is a chronic intestinal disease selected from the group consisting of food-responsive enteropathy, antibiotic-responsive enteropathy, and idiopathic inflammatory bowel disease (IBD).

21. The pharmaceutical composition of any one of claims 3-17, wherein the intestinal disease is idiopathic inflammatory bowel disease (IBD).

22. A pharmaceutical composition as described in any one of claims 1-21, wherein the bacteria are transformed with a vector comprising a bile acid inducible operon (bai operon).

23. The pharmaceutical composition of claim 22, wherein the bacteria is selected from the genus Clostridium.

24. A pharmaceutical composition as described in any one of claims 1-23, wherein the amount of bacteria is 10,000 CFU to 100 trillion CFU.

25. The pharmaceutical composition of any one of claims 2, 4, 6-9, 11, and 15-24, wherein the second bile acid is between 10 mg / unit dose and 500 mg / unit dose.

26. The pharmaceutical composition of any one of claims 2, 4, 6-9, 11, and 15-25, wherein the first bile acid and the second bile acid are the same.

27. The pharmaceutical composition of any one of claims 2, 4, 6-9, 11, 15-25, wherein the first bile acid and the second bile acid are different.

28. Clostridium hirano for use as a medicament, wherein the Clostridium hirano comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

29. Use of Clostridium hirano in the preparation of a medicament for treating an intestinal disease in a subject in need thereof, wherein the Clostridium hirano comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

30. Clostridium scintillans for use as a medicament, wherein the Clostridium scintillans comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

31. Use of Clostridium scintillans in the preparation of a medicament for treating an intestinal disease in a subject in need thereof, wherein the Clostridium scintillans comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

32. A food product comprising an effective amount of bacteria capable of producing a first bile acid, wherein the bacteria comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

33. The food product of claim 32, further comprising an effective amount of a second bile acid.

34. The food product of claim 32, wherein the bile acid inducible operon (bai operon) comprises the nucleotide sequence set forth in SEQ ID NO:

1.

35. The food of claim 32, wherein the bacterium comprises 16s rRNA comprising the nucleotide sequence set forth in SEQ ID NO:

2.

36. The food product of any one of claims 32-35, wherein the bacteria is Clostridium plannosum, Clostridium fulgidus, or a combination thereof.

37. The food product of claim 36, wherein the bacterium is Clostridium hiranoensis.

38. The food of any one of claims 32 and 34-35, wherein the first bile acid is selected from the group consisting of chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid, and any combination thereof.

39. The food product of any one of claims 32 and 34-35, wherein the first bile acid is a secondary bile acid.

40. The food of claim 39, wherein the secondary bile acid is selected from the group consisting of taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, omega-muricholic acid, and any combination thereof.

41. The food product of claim 40, wherein the secondary bile acid is deoxycholic acid and / or lithocholic acid.

42. The food of any one of claims 33 and 36-37, wherein the first bile acid and / or the second bile acid is selected from the group consisting of chenodeoxycholic acid, cholic acid, glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, Ω-muricholic acid, and any combination thereof.

43. The food product of any one of claims 33 and 36-37, wherein the first bile acid and / or the second bile acid is a secondary bile acid.

44. The food of claim 43, wherein the secondary bile acid is selected from the group consisting of taurodeoxycholic acid, glycodeoxycholic acid, ursodeoxycholic acid, glycoursodeoxycholic acid, tauroursodeoxycholic acid, α-muricholic acid, deoxycholic acid, γ-muricholic acid, glycolithocholic acid, taurolithocholic acid, lithocholic acid, omega-muricholic acid, and any combination thereof.

45. The food product of claim 44, wherein the secondary bile acid is deoxycholic acid and / or lithocholic acid.

46. ​​The food product of any one of claims 32-45, wherein the food product improves the intestinal health of a subject.

47. The food product of any one of claims 32-46, wherein the bacteria is transformed with a vector comprising a bile acid inducible operon (bai operon).

48. The food product of any one of claims 32-33 and 38-47, wherein the bacteria is selected from the genus Clostridium.

49. The food of any one of claims 32-48, wherein the amount of the bacteria is between 10,000 CFU and 100 trillion CFU.

50. The food of any one of claims 32-49, wherein the second bile acid is between 100 mg / daily dose and 1000 mg / daily dose.

51. The food product of any one of claims 33, 36-37, and 42-50, wherein the first bile acid and the second bile acid are the same.

52. The food product of any one of claims 33, 36-37, and 42-50, wherein the first bile acid and the second bile acid are different.

53. The food product of any one of claims 32-52, wherein the food product is a pet food.

54. The food product of any one of claims 32-53, wherein the food product is a dog food product.

55. A diet for increasing the population of bacteria capable of producing bile acid in a companion animal, wherein the bacteria comprises a bile acid-inducible operon (bai operon), wherein the bile acid-inducible operon (bai operon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO:

3.

56. The diet of claim 55, comprising protein, fat, crude fiber, total dietary fiber, carbohydrates, calcium, phosphorus, sodium, chloride, potassium, magnesium, iron, copper, manganese, zinc, iodine, selenium, vitamin A, vitamin D3, vitamin E, vitamin C, thiamine (vitamin B1), riboflavin (vitamin B2), pantothenic acid, niacin, pyridoxine (vitamin B6), folic acid, biotin, cobalamin (vitamin B12), choline, arginine, lysine, methionine, cystine, taurine, linoleic acid, arachidonic acid, omega-6 fatty acids, omega-3 fatty acids, EPA and / or DHA.

57. The diet of claim 55 or 56, wherein the companion animal is a dog.

58. The diet of claim 57, wherein the diet is a Royal Canin veterinary diet.

59. The diet of any one of claims 55-58, wherein the bacteria is Clostridium plannosum, Clostridium fulgidus, or a combination thereof.

60. The diet of claim 59, wherein the bacteria is Clostridium plannosum.

61. A Royal Canin veterinary diet for increasing the population of bacteria capable of producing bile acid in a companion animal for treating intestinal disease in dogs, wherein the bacteria comprises a bile acid-inducible operon (baioperon), wherein the bile acid-inducible operon (baioperon) comprises a nucleotide sequence identical to SEQ ID NO: 1 or SEQ ID NO: 3, wherein the dog comprises a first amount of a first intestinal microorganism and / or a second amount of a second intestinal microorganism, and The first amount of the first intestinal microorganism is higher than a first reference amount of the first intestinal microorganism, and / or the second amount of the second intestinal microorganism is lower than a second reference amount of the second intestinal microorganism.

62. The Royal Canin veterinary diet of claim 61, wherein the first intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence identical to the 16S rRNA nucleotide sequence of JRPJ01000002.1034290.1035971, KF842598.1.1394, JF920309.1.1340, FJ978526.1.1378, HQ793763.1.1451, DQ113765.1.1450, DQ797046.1.1403, ACBW01000012.3536.5054, JN387556.1.1324, or JQ208053.1.1336.

63. The Royal Canin veterinary diet of claim 61 or 62, wherein the second intestinal microorganism comprises one or more bacteria comprising a 16S rRNA comprising a nucleotide sequence identical to the 16S rRNA nucleotide sequence of HK693629 1.1491, GQ493166.1.1359, GQ491426.1.1332, FJ957494.1.1454, GQ449092.1.1375, GQ448486.1.1387, AMCI01001631.34.1456, or HK55598.1.1357.

64. A kit comprising the pharmaceutical composition of any one of claims 1-27, the food of any one of claims 32-54, or the diet of any one of claims 55-63.

65. The kit of claim 64, wherein the kit further comprises written instructions for treating and / or preventing intestinal diseases.

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