Treatment of sorbitol intolerance after antibiotic treatment with sorbitol dehydrogenase

Abstract

Inflammatory bowel disease (IBD) is the general name for diseases that cause inflammation in the small intestine and colon. Disclosed herein is a method for treating an irritable bowel syndrome (IBS) or IBD in a subject that involves administering to the subject a composition comprising an effective amount of a sorbitol dehydrogenase enzyme and a composition comprising an effective amount of an aminosalicylate.

Description

TREATMENT OF SORBITOL INTOLERANCE AFTER ANTIBIOTIC TREATMENT WITH SORBITOL DEHYDROGENASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 626,348, filed January 29, 2024, which is hereby incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0002] Inflammatory bowel disease (IBD) is the general name for diseases that cause inflammation in the small intestine and colon. Ulcerative colitis is the most common inflammatory bowel disease and it affects various portions of the gastrointestinal (Gl) tract, particularly the lower Gl tract, and more particularly the colon and / or rectum. A second IBD is Crohn's disease, which predominates in the small intestine (ileum) and the large intestine (colon).SUMMARY OF THE INVENTION

[0003] Disclosed herein is a method for treating gastrointestinal symptoms (e.g. diarrhea and abdominal pain) in irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), or other conditions in a subject that involves administering to the subject a composition comprising an effective amount of a sorbitol dehydrogenase enzyme and a composition comprising an effective amount of an aminosalicylate.

[0004] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0005] FIGs. 1A to 1F shows a mouse model of prolonged sorbitol intolerance. Mice maintained on a low-fat diet (LF) or a high-fat diet (HF) for 14 days were mock-treated or received a single dose of streptomycin (Str) by oral gavage. After maintaining mice for four more weeks on the same diet, mice received drinking water supplemented with 5 % sorbitol for three days. FIG. 1 A shows change in body weight for each group (n = 14) over time. FIG. 1 B shows fecal water content after 3 days of sorbitol supplementation. FIG. 1C shows sorbitol concentration after 3 days of sorbitol supplementation. LOD, limit of detection. FIG. 1D shows sorbitol dehydrogenase activity in cecal contents. FIGs. 1B-1 D show group sizes (n) are indicated by the number of symbols. *, P < 0.05; **, P < 0.01 ; ***, P < 0.005; ****, P < 0.001. Fig. 1A-1 B, 1 F: analysis with two-way (Fig. 1A) or one-way (Figs. 1 B-1 E) ANOVA followed by Tukey’s multiple-comparison test (Fig. 1A), or student’s t test (Fig. 1 F).

[0006] Figs. 2A to 2F show Clostridia are a main source of sorbitol dehydrogenase genes during homeostasis. Mice were maintained on a low-fat diet (LF) or a high-fat diet (HF) for 14 days. Fecal samples were collected (“LF” or “HF before Str”) and mice weremock-treated or received a single dose of streptomycin (Str) by oral gavage. After maintaining mice for four more weeks on the same diet, a second fecal sample was collected for analysis (“LF + 4 weeks” or “HF 4 weeks after Str”). FIGs. 2A-2C shows microbiota profiling of fecal DNA collected at the indicated time points. FIG. 2A and 2B show relative abundance of amplicon sequence variants (ASVs) belonging to the class Clostridia (Fig. 2A) or the genus Enterococcus (Fig. 2B). Box plots represent the first to third quartiles, and lines indicates median values. *, P < 0.05. Analysis with Kruskal-Wallis test. FIG. 2C shows the cladogram shows differences in taxa composition between samples collected before streptomycin treatment (HF before Str) and four weeks after streptomycin treatment (HF 4 weeks after Str). Taxa that are significantly (Kruskal-Wallis test, LEfSe) more abundant or less abundant before compared to 4 weeks after streptomycin treatment are shown. Fig. 2D-2F shows DNA isolated from the indicated samples were subjected to metagenomic analysis. Fig. 2D shows volcano plot of genes involved in carbohydrate metabolism. Negative values indicate genes with decreased abundance four weeks after streptomycin treatment compared to prior to streptomycin treatment. FIG. 2E and 2F show abundance of genes encoding sorbitol dehydrogenase (Fig. 2E) or sorbitol-6-phosphate 2- dehydrogenase (Fig. 2F) in samples collected prior to or four weeks after streptomycin treatment.

[0007] Figs. 3A to 3K show abundant probiotics protect against transient sorbitol intolerance. Figs. 3A-3D and 3F-3K show mice mock-treated or treated with a single dose of streptomycin. One day later, drinking water was supplemented with 5% sorbitol and mice were inoculated with different doses of E. coli Nissle 1917 (EcN WT), a E. coli Nissle 1917 srIABE mutant (EcN srIABE) or A. caccae. Samples were collected after 2 days of sorbitol supplementation. Fig. 3A shows clony-forming units (cfu) recovered from feces of animals (y axis) 2 days after inoculation with the indicated doses of EcN WT (x axes). Fig. 3B shows the abundance of E. coli in fecal samples was determined by real-time PCR using Enterobacterales-specitic primers. Fig. 3C and 3J show fecal water content in fecal pellets (y axis) collected from animals two days after inoculation with the indicated doses (x axes) of E. coli Nissle 1917 (Fig. 3C) or A. caccae (Fig. 3J). Fig. 3D shows sorbitol dehydrogenase activity in cecal contents of mice inoculated with the indicated doses of E. coli Nissle 1917. Fig. 3E shows minimal medium containing sorbitol as a sole carbon source was inoculated with the indicated E. co / / Nissle 1917 strains carrying either no plasmid, a plasmid encoding the srIAEB genes (pAWLR169) or the empty plasmid vector (pWSK29). After overnight culture, the sorbitol concentration in culture supernatants was measured. Fig. 3F shows fecal water content in fecal pellets (y axis) collected from animals two days after inoculation with the indicated doses of EcN srIABE (x axes). Fig. 3G shows Cfu recovered from feces of animals (y axis) 2 days after inoculation with the indicated doses of EcN srIABE (x axes).Fig. 3H shows in vitro anaerobic growth of A. caccae in no-carbon defined medium (NCDM) supplemented with glucose, sorbitol or without supplementation (no sugar). Enterocloster asparagiformis, a Clostridia species that does not ferment sorbitol35, was used as a negative control. FIG. 31 shows the abundance of A. caccae in fecal samples was determined by realtime PCR using genus specific primers. Fig. 3K shows sorbitol dehydrogenase activity in cecal contents of mice inoculated with the indicated doses of A caccae. Figs. 3A, 3B and 31: a grey bar indicates the threshold of colonization required for protection against sorbitol- induced diarrhea. Figs. 3A, 3B, 3G and 31: LOD, limit of detection. *, P < 0.05; ", P < 0.01 ; ***, P < 0.005; P < 0.001. Figs. 3C-3F, 3H, 3J, 3K: analysis by one-way ANOVA followed by Tukey’s multiple-comparison tests.

[0008] Figs. 4A to 4G show sorbitol-catabolizing probiotics protect against prolonged sorbitol intolerance. Mice reared and maintained throughout the experiment on a low-fat or a high-fat (HF) diet were mock-treated or received a single dose of streptomycin (Str), respectively. Four weeks later, mice received drinking water supplemented with 5 % sorbitol and were inoculated with 109colony-forming units (cfu) of E. coll Nissle 1917 (EcN), A. caccae (AC), or Lactiplantibacillus plantarum (LP). Samples were collected after three or seven days of sorbitol supplementation. Fig. 4A is a schematic of experimental groups and time points. Fig. 4B shows fecal water content in fecal pellets. Fig. 4C shows sorbitol dehydrogenase activity in cecal contents. Fig. 4D shows Cfu of EcN or LP. Fig. 4E shows absolute abundance of AC in feces was determined by real-time PCR using genus specific primers. Fig. 4F shows absolute abundance of Clostridia in feces determined by real-time PCR using class specific primers. Fig. 4G shows numbers of amplified sequence variants (ASVs) belonging to the class Clostridia at the indicated time points. *, P < 0.05; **, P < 0.01 ; ***, P < 0.005; ****, P < 0.001 . Analisis by one-way ANOVA followed by Tukey’s multiplecomparison tests (Figs. 4B-4D, 4F) or student’s t test (Fig. 4E). Figs. 4C, 4E, and 4F: LOD, limit of detection.

[0009] Figs. 5A to 5D show A. caccae restores butyrate levels and epithelial hypoxia in mice with prolonged sorbitol intolerance. Mice reared and maintained throughout the experiment on a low-fat or a high-fat (HF) diet were mock-treated or received a single dose of streptomycin (Str), respectively. Four weeks later, mice received drinking water supplemented with 5 % sorbitol and were inoculated with 109colony-forming units (cfu) of E. coli Nissle 1917 (EcN), A. caccae (AC), or Lactiplantibacillus plantarum (LP). Samples were collected after three or seven days of sorbitol supplementation. Fig. 5A shows butyrate concentrations in cecal contents. Fig. 5B shows relative abundance of Clostridia families containing gene sequence involved in butyrate metabolism. Figs. 5C-5D show mice injected with pimonidazole (PMDZ) before euthanasia. PMDZ was detected using hypoxyprobe-1 primary antibody and a Cy-3 conjugated goat anti-mouse secondary antibody in colonicsections counter stained with nuclear stain. Fig. 5C contains representative images for each group seven day after inoculation with probiotics. L, intestinal lumen. The graph shows PMDZ intensity from the lumen across the epithelial layer (distance in arbitrary units). Fig. 5D is a graph shows the average peak PMDZ intensity. Fig. 5A and 5D: each symbol represent data from one animal. **, P < 0.01 ; ***, P < 0.005. Analysis by one-way ANOVA followed by Tukey’s multiple-comparison tests (Fig. 5A) or Kruskal-Wallis test (Fig. 5D).

[0010] Figs. 6A to 6J show A. caccae and butyrate stimulate epithelial PPAR-y signaling to promote microbiota recovery. Mice reared and maintained throughout the experiment on a low-fat or a high-fat (HF) diet were mock-treated or received a single dose of streptomycin (Str), respectively. Four weeks later, mice received drinking water supplemented with 5 % sorbitol and were inoculated with A. caccae (AC) or received supplementation with 5 aminosalicylic acid (5-ASA) or tributyrin (TB). Samples were collected after three or seven days of sorbitol supplementation. Figs. 6A-6F show experiments with C57BL / 6J mice. Fig. 6A shows butyrate concentrations in cecal contents. Fig. 6B shows fecal water content in fecal pellets. Fig. 6C shows sorbitol concentration. Figs. 6D and 6I show mice injected with pimonidazole before euthanasia. Binding of pimonidazole (PMDZ) was detected using hypoxyprobe-1 primary antibody and a Cy-3 conjugated goat anti-mouse secondary antibody. Fig. 6D shows average PMDZ peak intensity. Fig. 6E shows absolute abundance of Clostridia in fecal samples was determined by real-time PCR using class specific primers. Fig. 6F shows relative abundance of Clostridia families containing gene sequence involved in butyrate metabolism. Figs. 6G-6J shows experiments performed with PpargmVillincrel- mice (Pparg) or Pparg^'Villin1- littermate controls (WT). Fig. 6G shows fecal water content. Fig. 6H shows sorbitol concentration. Fig. 61 shows average PMDZ peak intensity. Fig. 6J shows butyrate concentrations in cecal contents. *, P < 0.05; **, P < 0.01 ; ***, P < 0.005; ****, P < 0.001. Analysis by one-way ANOVA followed by Tukey’s multiplecomparison tests (Fig. 6A-6C, 6E 6G-6J) or Kruskal-Wallis test (Fig. 6D). Figs. 6C and 6H: LOD, limit of detection.

[0011] Figs. 7A to 7F show 5-ASA treatment prevents development of prolonged sorbitol intolerance. Mice maintained on a low-fat diet (LF) or a high-fat diet (HF) for 14 days were mock-treated or received a single dose of streptomycin (Str) by oral gavage. FIG. 7A- 7B shows four weeks later, mice received drinking water supplemented with 5 % sorbitol and were treated by supplementing chow with 5-ASA. Fig. 7A shows the relative abundance of Clostridia in fecal samples collected from C57BL / 6J mice. Fig. 7B shows relative abundance of Clostridia families containing gene sequence involved in butyrate metabolism. Figs. 7C-7F shows at the time of streptomycin treatment mice were switched to chow supplemented with 5-ASA. Four weeks later, mice received drinking water supplemented with 5 % sorbitol to assess sorbitol tolerance. Fig. 7C and 7D show absolute abundance of Clostridia in fecalsamples of C57BL / 6J mice (Fig. 7C), Ppargfl / flVillincre / - mice (Pparg) or Ppargfl / flVillin / - littermate control mice (WT) (Fig. 7D) was determined by real-time PCR using class specific primers. Fig. 7E shows sorbitol concentration in C57BL / 6J mice. LOD, limit of detection. Fig. 7F shows fecal water content in samples from C57BL / 6J mice. *, P < 0.05; **, P < 0.01 ; ***, P < 0.005; ****, P < 0.001 . Figs. 7C-7F shows analysis by one-way ANOVA followed by Tukey’s multiple-comparison tests.

[0012] Figs. 8A to 8J show streptomycin treatment causes transient sorbitol intolerance, whereas high fat intake does not induce sorbitol intolerance in the absence of antibiotic treatment, related to Figure 1. Mice fed a low-fat diet (10 % fat) were mock-treated or treated with a single dose of streptomycin (Str) to generate sorbitol intolerance. Supplementation of drinking water with 5 % sorbitol was started one day after streptomycin treatment (Fig. 8A and 8B) or at the indicated later time points after streptomycin treatment (Fig. 8C, 8D, and 8E). Fig. 8A shows fecal water content was determined after 2 days of sorbitol supplementation. Fig. 8B shows change in body weight was monitored overtime. Stars indicate significate decrease in weight compared to sorbitol only group. Fig. 8C shows fecal water content was determined after 2 days of sorbitol supplementation. Fig. 8D shows change in body weight after the beginning of sorbitol supplementation was monitored over time. Stars indicate significate decrease in weight compared to sorbitol only group. Fig. 8E shows the sorbitol concentration in cecal contents was measured by a colorimetric assay after 2 days of sorbitol supplementation. Figs. 8F and 8G shows mice maintained on a low- fat diet (LF) or a high-fat diet (HF) for 14 days were mock-treated or received a single dose of streptomycin (Str) by oral gavage. After maintaining mice for four more weeks on the same diet, mice received drinking water supplemented with 5 % sorbitol for three days. Fig. 8F shows water intake per cage was determined for each group during the period of sorbitol supplementation. Fig. 8G shows colon length was determined at necropsy. Figs. 8H-8J shows mice maintained on a low-fat diet (LF) or a high-fat diet (HF) for 42 days received normal drinking water or drinking water supplemented with 5 % sorbitol for three days. Fig. 8H shows change in body weight was monitored during sorbitol supplementation. Fig. 8I shows fecal water content determined after 3 days of sorbitol supplementation. Fig. 8J shows the sorbitol concentration in cecal contents measured by a colorimetric assay after 3 days of sorbitol supplementation. Fig. 8E and J8 shows a grey dashed line indicates the limit of detection (LOD). *, P < 0.05; ***, P < 0.005; P < 0.001. R values were calculated by one-way ANOVA followed by Tukey’s multiple-comparison tests (Figs. 8A, 8C, 8E, 8F, 8G, 8I-8J) or two-way ANOVA followed by Tukey’s multiple-comparison tests (Figs. 8B, 8D, 8H).

[0013] Figs. 9A to 9E show compositional and functional changes in the microbiota of mice with prolonged sorbitol intolerance, related to Figure 2. Mice were maintained on a low-fat diet (LF) or a high-fat diet (HF) for 14 days. Fecal samples were collected (“LF” or“HF before Str”) and mice were mock-treated or received a single dose of streptomycin (Str) by oral gavage. After maintaining mice for four more weeks on the same diet, a second fecal sample was collected for analysis (“LF + 4 weeks” or “HF 4 weeks after Str”). Fig. 9A is a schematic showing experimental groups and time points of sample collection. Fig. 9B shows relative abundance of taxa (class level) in fecal samples collected from individual mice at the indicated time points. Fig. 9C contains box blots showing the relative abundance of amplicon sequence variants (ASVs) belonging to the class Bacilli. The box plots represent the first to third quartiles, and the line indicates the median value. Fig. 9D, left panel, shows the normalized abundance of sorbitol dehydrogenase or sorbitol-6-phosphate 2-dehydrogenase encoding sequences in samples collected prior to streptomycin treatment or four weeks after streptomycin treatment. Each circle denotes data collected from one animal. Fig. 9D, right panel, shows the 95 % confidence interval for data displayed in the left panel. Fig. 9E shows abundance of genes encoding sorbitol dehydrogenase in samples collected prior to streptomycin treatment (HF before Str, left panels) or four weeks after streptomycin treatment (HF 4 weeks after Str, right panels). Colors denote the taxa (order level) to which gene sequences were assigned. *, P < 0.05; ", P < 0.01 . P values were calculated by Kruskal- Wallis test (Fig. 9C) or by paired DESeq2 wald test (Fig. 9E).

[0014] Fig. 10A and 10B show a history of antibiotic treatment and high fat intake reduces the abundance of genes involved in polyol catabolism and butyrate metabolism in the fecal microbiota, related to Figure 2. Feces were collected from mice maintained on a high-fat diet (HF) for 14 days. Mice then received a single dose of streptomycin (Str) by oral gavage. After maintaining mice on a HF diet for four more weeks, feces were collected for analysis. DNA isolated from fecal samples collected before streptomycin treatment (HF before Str) and four weeks after streptomycin treatment (HF 4 weeks after Str) were subjected to metagenomic analysis. Figs. 10A-10B, left panels, show the normalized abundance of genes encoding the indicated enzymes involved in polyol catabolism (Fig. 10A) or butyrate metabolism (Fig. 10B) in samples collected prior to streptomycin treatment or four weeks after streptomycin treatment. Each circle denotes data collected from one animal. Figs. 10A-10B, right panels, show the 95 % confidence interval for data displayed in the left panels. *, P < 0.05 by paired DESeq2 wald test.

[0015] Figs. 11A to 11C show the ability to catabolize sorbitol is required by E. coli Nissle 1917 to protect against prolonged sorbitol intolerance, related to Figure 4. Mice reared and maintained throughout the experiment on a low-fat or a high-fat (HF) diet were mock-treated or received a single dose of streptomycin (Str), respectively. Four weeks later, mice received drinking water supplemented with 5 % sorbitol and were inoculated with 109colony-forming units (cfu) of E. coli Nissle 1917 (EcN) or a E. coli Nissle 1917 srIAEB mutant (EcN srlABE). Fig. 11 A shows colony-forming units (cfu) of E. coli were determined in thefeces. Fig. 11 B shows the sorbitol concentration in cecal contents was measured by a colorimetric assay. Fig. 11 C shows fecal water content was determined in feces. *, P < 0.05; **, P < 0.01 ; ***, P < 0.005; ****, P < 0.001 . P values were calculated by student’s t test (Fig. 11A) or one-way ANOVA followed by Tukey’s multiple-comparison tests (Figs. 11 B-11C). A grey dashed line denotes the limit of detection (LOD).

[0016] Fig. 12 shows butyrate and A. caccae restore epithelial hypoxia by stimulating epithelial PPAR-y signaling, related to Figure 6I. PpargfmVillincrel- mice (Pparg) or PpargfmVillin-'- littermate control mice (WT) reared and maintained throughout the experiment on a low-fat (LF) or a high-fat (HF) diet received a single dose of streptomycin (Str). Four weeks later, mice received drinking water supplemented with 5 % sorbitol and were inoculated with A. caccae (AC) or received supplementation with or tributyrin (TB). Samples were collected after seven days of sorbitol supplementation. Mice were injected with pimonidazole before euthanasia. To stain hypoxic tissue, binding of pimonidazole (PMDZ) was detected using hypoxyprobe-1 primary antibody and a Cy-3 conjugated goat anti-mouse secondary antibody (red fluorescence) in sections of the colon that were counter stained with DAPI nuclear stain (blue fluorescence). Representative images for each group are shown. DETAILED DESCRIPTION

[0017] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0020] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0021] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0022] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0023] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0024] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.Definitions

[0025] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0026] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subjectcan be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0027] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0028] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0029] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0030] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0031] The term “prevent” refers to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent that disease in a subject who has yet to suffer some or all of the symptoms.Sorbitol Dehydrogenase Enzyme

[0032] Sorbitol dehydrogenase (or SDH) is a cytosolic enzyme. In humans this protein is encoded by the SORD gene. Sequences are described in Accession Nos.NM_003104 and Q00796, which are incorporated by reference in their entireties for these sequences.Aminosalicylates

[0033] Aminosalicylates, which are drugs that contain 5-aminosalicylic acid (5-ASA; mesalamine) or 4-aminosalicylic acid (4-ASA), help to control the inflammation. However, both mesalamine and 4-ASA may be absorbed as it passes through the Gl tract and may adversely affect the amount of mesalamine that reaches the lower Gl tract, particularly the colon and rectum. Thus, various mesalamine formulations have been introduced in an attempt to protect mesalamine as it passes through the gut and upper Gl tract.

[0034] In addition, several pro-drugs of mesalamine have been introduced which can aid in colon-specific delivery of mesalamine. These pro-drugs are generally less readily absorbed in the gut and upper Gl tract and thus can more easily reach the colon.

[0035] Sulfasalazine is a combination of sulfapyridine and 5-ASA and is employed to induce and maintain remission. Sulfasalazine is metabolized in the body to form 5-ASA and sulfapyridine. The sulfapyridine component carries the anti-inflammatory 5-ASA to the intestine.

[0036] Other 5-ASA agents such as olsalazine, ipsalazide and balsalazide, each of which have a different carrier, offer fewer side effects, and may be used by people who cannot take sulfasalazine. Unlike sulfasalazine, the breakdown of these 5-ASA compounds in the intestinal tract may not give rise to undesirable metabolic products.

[0037] In general, 5-ASA compounds are given orally, through an enema, or in a suppository, depending on the location of the inflammation in the colon. Most people with mild or moderate ulcerative colitis are treated with this group of drugs first. However, in general, this therapy cannot be considered optimal, mainly because of the poor potency of the drug that causes also a poor compliance for the patient. Also 5-ASA is subject to microbial metabolism, which may reduce its potency (Mehta, R. S. et al. Gut microbial metabolism of 5-ASA diminishes its clinical efficacy in inflammatory bowel disease (Nature medicine 29, 700-709 (2023).)

[0038] Other drugs that are used are corticosteroids such as prednisone, hydrocortisone, budesonide etc. and immunomodulators such as azathioprine and 6- mercaptopurine (6-MP). These drugs can cause side effects such as hypertension, increased risk of infections etc.

[0039] Sulfasalazine, olsalazide and balsalazide are mesalamine derivatives where the non-mesalamine carrier is linked to mesalamine via a diazo bond. These pro-drugs are not as readily absorbed in the gut and upper Gl tract and thus can reach the colon vhere they are split by azo-reductases of the colonic microflora to release the mesalamine and carrier directly in the colon.

[0040] Other derivatives of mesalamine comprise a carrier attached to mesalamine via the carboxylic and hydroxyl functional groups of the molecule. Among these, the preparation of esters or amides with amino acids such as L-serine and L-glycine or the addition of other biological compound such as taurine has been reported. These pro-drugs base their activity on the action of carboxypeptidases and aminopeptidases A for releasing mesalamine. (R. Pellicciari et al. (1993) Journal of Medicinal Chemistry, 36, pg. 4201-7).Compositions and Methods

[0041] Compounds of the present invention may be utilized for the prophylaxis or treatment of gastrointestinal symptoms in various diseases, particularly inflammatory conditions of the Gl tract including, but not limited to, inflammatory conditions of the mouth such as mucositis, infectious diseases (e.g., viral, bacterial and fungal diseases), and Crohn's disease; inflammatory conditions of the esophagus such as esophagitis, Crohn's disease, and esophageal stricture; inflammatory conditions such as gastritis (e.g., Helicobacter pylori, acid-peptic disease and atrophic gastritis), celiac disease, peptic ulcer disease, functional gastrointestinal diseases such as irritable bowel syndrome (IBS), non-ulcer dyspepsia; inflammatory conditions of the stomach such as Crohn's disease, bacterial overgrowth, peptic ulcer disease, and fissures of the intestine; inflammatory conditions of the colon such as Crohn's disease, ulcerative colitis, infectious colitis (e.g., pseudomembranous colitis such as Clostridium difficile colitis, salmonella enteritis, shigella infections, yersiniosis, cryptospiridiosis, microspridial infections, and viral infections), antibiotics-induced colitis, radiation-induced colitis, colitis in the immunocompromised host (e.g., typhlitis), proctitis, inflammation associated with hemorrhoids, proctalgia fugax, and rectal fissures;

[0042] Depending on the specific condition or disease state to be treated, subjects may be administered compounds of the present invention at any suitable therapeutically effective and safe dosage, as may be readily determined within the skill of the art. These compounds are, most desirably, administered in dosages ranging from about 1 to about 2000 mg per day, in a single or divided doses, although variations will necessarily occur depending upon the weight and condition of the subject being treated and the particular route of administration chosen. However, a dosage level that is in the range of about 0.1 to about 10 g / kg, preferably between about 5 and 90 mg / kg, and more preferably between about 5 and 50 mg / kg, is most desirable. Variations may nevertheless occur depending upon the weight and conditions of the persons being treated and their individual responses to said medicament, as well as on the type of pharmaceutical formulation chosen and the time period and interval during which such administration is carried out. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effects,provided that such large doses are first divided into several small doses for administration throughout the day.

[0043] The compounds of the present invention can be administered in the form of any pharmaceutical formulation, the nature of which will depend upon the route of administration. These pharmaceutical compositions can be prepared by conventional methods, using compatible, pharmaceutically acceptable excipients or vehicles. Examples of such compositions include capsules, tablets, transdermal patches, lozenges, troches, sprays, syrups, powders, granulates, gels, elixirs, suppositories, and the like, for the preparation of extemporaneous solutions, injectable preparations, rectal, nasal, ocular, vaginal etc. A preferred route of administration is the oral and rectal route.

[0044] For oral administration, tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc can be used for tabletting purposes. Solid compositions of similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar, as well as high molecular weight polyethylene glycols. When aqueous suspensions and / or elixirs are desired for oral administration the active ingredient may be combined with sweetening or flavoring agents, coloring matter and, if so desired, emulsifying and / or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

[0045] The dosage form can be designed for immediate release, controlled release, extended release, delayed release or targeted delayed release. The definitions of these terms are known to those skilled in the art. Furthermore, the dosage form release profile can be effected by a polymeric mixture composition, a coated matrix composition, a multiparticulate composition, a coated multiparticulate composition, an ion-exchange resinbased composition, an osmosis-based composition, or a biodegradable polymeric composition. Without wishing to be bound by theory, it is believed that the release may be effected through favorable diffusion, dissolution, erosion, ion-exchange, osmosis or combinations thereof.

[0046] For parenteral administration, a solution of an active compound in either sesame or peanut oil or in aqueous propylene glycol can be employed. The aqueous solutions should be suitably buffered (preferably pH greater than 8), if necessary, and the liquid diluent first rendered isotonic. The aqueous solutions are suitable for intravenousinjection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.Embdiments

[0047] Embodiment 1. A method for treating an irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD) in a subject, comprising administering to the subject a composition comprising an effective amount of a sorbitol dehydrogenase enzyme and a composition comprising an effective amount of an aminosalicylate.

[0048] Embodiment 2. The method of embodiment 1 , wherein the aminosalicylate is 5-aminosalicylic acid (5-ASA) or a prodrug or derivative thereof.

[0049] Embodiment 3. The method of embodiment 2, wherein the 5-ASA derivative or prodrug is mesalamine, mesalazine, olsalazine, sulfasalazine, or balsalazide.

[0050] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the sorbitol dehydrogenase enzyme and aminosalicylate are in the same composition.

[0051] Embodiment 5. The method of any one of embodiments 1 to 4, further comprising one or more additional sugar dehydrogenases.

[0052] Embodiment 6. The method of embodiment 5, wherein the one or more additional sugar dehydrogenases are selected from the group consisting of lactase, lactate dehydrogenase, fructose dehydrogenase, and malate dehydrogenase.

[0053] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the IBD is ulcerative colitis.

[0054] Embodiment 8. The method of any one of embodiments 1 to 6, wherein the IBD is Chron’s disease.

[0055] Embodiment 9. A composition comprising a sorbitol dehydrogenase enzyme and aminosalicylate in a pharmaceutically acceptable carrier.

[0056] Embodiment 10. The composition of embodiment 9, wherein the aminosalicylate is 5-aminosalicylic acid (5-ASA) or a prodrug or derivative thereof.

[0057] Embodiment 11. The composition of embodiment 10, wherein the 5-ASA derivative or prodrug is mesalamine, mesalazine, olsalazine, sulfasalazine, or balsalazide.

[0058] Embodiment 12. The composition of any one of embodiments 9 to 11 , wherein the sorbitol dehydrogenase enzyme and aminosalicylate are in the same composition.

[0059] Embodiment 13. The composition of any one of embodiments 9 to 12, further comprising one or more additional sugar dehydrogenases.

[0060] Embodiment 14. The composition of embodiment 13, wherein the one or more additional sugar dehydrogenases are selected from the group consisting of glucose dehydrogenase, lactate dehydrogenase, fructose dehydrogenase, and malate dehydrogenase.

[0061] Embodiment 15. The composition of any one of embodiments 9 to 14, formulated as a pill, capsule, or tablet for oral administration.

[0062] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1 : High fat intake sustains sorbitol intolerance after antibiotic- mediated Clostridia depletion from the gut microbiotaIntroduction

[0063] Sorbitol is a naturally occurring polyol that is poorly absorbed by the small intestine, resulting in a low caloric content. Therefore, sorbitol is used as low-calorie sweetener in “sugar-free” foods, such as sugar-free chewing gum, candy, mints, jam, diet drinks and chocolate (Lenhart, A., et al. Adv Nutr 2017 8:587-596). The estimated daily sorbitol intake in the UK averages 3.5 g, which comes mostly from its use as sweetener, but sorbitol is also naturally present at low concentrations in some fruits of the Rosaceae family, such as apples, pears and apricots (Wallaart, R.A.M. Phytochemistry 1980 19:2603-2610). Excessive consumption of polyols can trap fluid in the colonic lumen to trigger osmotic diarrhea (Reele, S.B., et al. Int J Clin Pharmacol Ther Toxicol 1985 23:403-405). For example, ingestion of 20 g sorbitol can induce symptoms of carbohydrate intolerance in healthy volunteers, including diarrhea, abdominal distention, and flatulence, but most volunteers ingesting 5 g sorbitol do not develop such symptoms (Corazza, G.R., et al. Gut 198829:44-48). Susceptibility to polyol-induced diarrhea varies among individuals, resulting in heightened intolerance in patients with irritable bowel syndrome (IBS) (Magge, S., et al. Gastroenterol Hepatol 2012 8:39-745; de Roest, R.H., et al. Int J Clin Pract 2013 67:895- 903) or quiescent inflammatory bowel disease (IBD) (Gearry, R.B., et al. J Crohns Colitis 2009 3:8-14; Gibson, P.R. J Gastroenterol Hepatol 2017 32 Suppl 1 :40-42). For example, ingesting 5 g of sorbitol can intensify gastrointestinal symptoms in individuals with IBS (Rumessen, J. J., et al. Gastroenterology 1988 95:694-700), whereas consuming 3 g of sorbitol can exacerbate gastrointestinal symptoms in IBD patients (Cox, S.R., et al. J Crohns Colitis 2017 11 :1420-1429).

[0064] Antibiotic treatment can transiently heighten polyol intolerance by disrupting the gut microbiota, which can impair metabolic functions that remove osmotically active solutes (Hammer, H.F., et al. Gastroenterol Clin North Am 2012 41 :611-627; Rao, S.S., et al. Gastroenterology 1988 94:928-932). In a mouse model of antibiotic-induced sorbitol intolerance, the addition of 5 % sorbitol to the drinking water increases fecal water content during treatment with ampicillin or streptomycin, but not in the absence of antibiotictreatment (Hattori, K., et al. Nutrients 2021 13). However, antibiotic-induced changes in the microbiota composition are short lived, as the gut microbiota regains its normal composition within five days after withdrawing streptomycin (Stecher, B., et al. PLoS Biol 2007 5: 2177- 2189; Rivera-Chavez, F„ et al. Cell Host Microbe 2016 19:443-454; Gillis, C.C., et al. Cell Host Microbe 2018 23:54-64 e56). Since polyol intolerance resolves as the microbiota recovers after antibiotic treatment (Chassany, O., et al. Drug Saf 200022:53-72), a transient disruption of the microbiota by antibiotics does not explain the prolonged carbohydrate intolerance in patients with IBS or quiescent IBD (Magge, S., et al. Gastroenterol Hepatol 2012 8:39-745; de Roest, R.H., et al. Int J Clin Pract 2013 67:895-903; Gearry, R.B., et al. J Crohns Colitis 2009 3:8-14; Gibson, P.R. J Gastroenterol Hepatol 2017 32 Suppl 1 :40-42). Treatment of prolonged polyol intolerance therefore relies on dietary interventions that reduce the intake of polyols and other poorly absorbed mono-, di- and oligosaccharides (Fernandez, K., et al. Mutat Res 2020 821 :111702).

[0065] A recent history of antibiotic usage (between 4 and 56 weeks prior to enrollment) in combination with high fat intake is an environmental risk factor in adult patients for developing diarrhea, abdominal distention, and flatulence (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284). These individuals can be differentiated from IBS patients by their elevated fecal calprotectin levels (between 50 and 200 j-tg / g feces), which is a marker of intestinal inflammation. However, intestinal inflammation in these individuals does not rise to the level of IBD, which is characterized by fecal calprotectin levels exceeding 250 pg / g feces during active disease (D'Haens, G., et al. Inflamm Bowel Dis 2012 18:2218-2224). Patients with low grade mucosal inflammation that is associated with a history of antibiotics and high fat intake thus represents a syndrome located at the intersection of the clinical spectra of IBS and IBD (Spiller, R., et al. Nat Rev Gastroenterol Hepatol 2016 13:613-621 ; Spiller, R., and Lam, C. Curr Opin Pharmacol 2011 11 :586-592; Colombel, J.F., et al. Clin Gastroenterol Hepatol 2019 17:380-390 e381). Elevated fecal calprotectin levels and diarrhea observed in these patients can be recapitulated in mice exposed to high fat intake in combination with a history of streptomycin treatment (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284). When antibiotics are combined with maintaining mice on a high-fat diet, microbiota recovery is impaired even 4 weeks after streptomycin treatment, as indicated by increased Enterobacterales (phylum Proteobacteria) and reduced Clostridia (phylum Firmicutes) abundance. These changes in the microbiota composition match those in the feces of patients with a history of antibiotics and high fat intake (Lee, J.Y., et al. Cell Host Microbe 202028:273-284), and in IBD patients (Rigottier-Gois, L. ISME J 2013 7:1256-1261 ; Rizzatti, G., et al. Biomed Res Int 2017 2017:9351507; Shin, N.R., et al. Trends Biotechnol 2015 33:496-503). As mice with a history of antibiotic treatment and high fat intake recapitulatesigns of disease seen in patients with diarrhea, abdominal distention, and flatulence, we wanted to investigate whether exposure to these environmental risk factors creates a mouse model for prolonged sorbitol intolerance that can be used to explore approaches for diagnosis, treatment, and prevention.ResultsStreptomycin induces sorbitol intolerance that resolves within five days after treatment

[0066] First, we wanted to investigate how quickly sorbitol intolerance resolves after cessation of antibiotic treatment. Mice (C57BL / 6J) fed a low-fat diet (10 % fat) were mock- treated or treated with a single dose of streptomycin. Subsequently, mice received drinking water without supplementation or drinking water supplemented with 5 % sorbitol for 2 days to assess sorbitol tolerance. When supplementation of drinking water with 5 % sorbitol was started one day after streptomycin treatment, it resulted in increased fecal water content in streptomycin-treated mice, but not in mock-treated control animals (Fig. 8A). Furthermore, antibiotic treatment exacerbated weight loss during sorbitol supplementation and a humane endpoint (20 % weight loss) was reached for some animals by the second day of sorbitol supplementation (Fig. 8B). However, when supplementation with 5 % sorbitol was started five days after streptomycin treatment, signs of sorbitol intolerance were no longer observed (Fig. 8C and 8D). Concentrations of sorbitol in the cecal contents were elevated when sorbitol supplementation was started one day after streptomycin treatment, which indicated that the solute accumulates in the lumen of the large intestine when the microbiota is disrupted. However, concentrations of sorbitol in the cecal contents were no longer elevated when sorbitol supplementation commenced five days after streptomycin treatment (Fig. 8E), when the microbiota regains a normal composition (Stecher, B., et al. PLoS Biol 2007 5: 2177-2189; Rivera-Chavez, F„ et al. Cell Host Microbe 2016 19: 443-454; Gillis, C.C., et al. Cell Host Microbe 201823:54-64 e56). Thus, our data suggested that transient sorbitol intolerance was correlated with an antibiotic-mediated disruption of the gut microbiota.Combining antibiotic exposure with high-fat diet drives prolonged sorbitol intolerance

[0067] Our previous work shows that microbiota recovery is impaired when exposure to streptomycin is combined with high fat intake (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284). We thus wanted to determine whether exposure to this combination of environmental factors would result in prolonged sorbitol intolerance. Mice (C57BL / 6J) reared and maintained throughout the experiment on a low-fat diet (10 % fat) or a high-fat diet (45 % fat) were mock-treated or received a single dose of streptomycin by oral gavage to generate a history of antibiotic usage, respectively. Four weeks later, mice were considered to have a “history” of antibiotic treatment and received drinking water supplemented with 5 % sorbitol for three days. Combining streptomycin treatment with high fat intake produced signsof sorbitol intolerance, including weight loss (Fig. 1 A), increased fecal water content (Fig. 1 B) and elevated sorbitol levels in cecal contents (Fig. 1C), even four weeks after antibiotic exposure. Supplementation with sorbitol for three days did not alter water intake (Fig. 8F) or signs of inflammation, as assessed by measuring colon length (Fig. 8G). Importantly, high- fat diet alone did not produce signs of sorbitol intolerance, such as weight loss (Fig. 8H), increased fecal water content (Fig. 8I) or elevated sorbitol levels in cecal contents (Fig. 8J).Reduced sorbitol dehydrogenase activity is a potential biomarker for sorbitol intolerance

[0068] We wanted to determine whether sorbitol intolerance is associated with reduced activity of sorbitol dehydrogenase (officially designated L-iditol 2-dehydrogenase), the enzyme catalyzing the conversion of sorbitol into sorbose or fructose. Mice with a history of antibiotic treatment and high fat intake featured reduced sorbitol dehydrogenase activity in cecal extracts (Fig. 1D).

[0069] Next, we explored whether reduced fecal sorbitol dehydrogenase activity is also a biomarker observed in patients. Combining a history of streptomycin treatment with high fat intake provides an animal model for patients complaining about diarrhea, abdominal distention, and flatulence, who have a recent history of antibiotic usage and report higher fat intake than healthy controls and patients with IBS (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284). Fecal samples banked from this previous study were analyzed for fecal sorbitol dehydrogenase activity. Notably, patients with elevated fecal calprotectin had decreased fecal sorbitol dehydrogenase activity compared to IBS patients or healthy controls (Fig. 1 E). In a questionnaire, patients with low fecal sorbitol dehydrogenase activity were more likely to indicate that they experienced gastrointestinal symptoms associated with consumption of sugar-free food or beverages (Fig. 1F). Collectively, these data identify low fecal sorbitol dehydrogenase activity as a potential biomarker for prolonged sorbitol intolerance.Streptomycin exposure combined with high fat intake depletes the classes Clostridia, Betaproteobacteria and Actinobacteria

[0070] Next, we wanted to determine which bacterial taxa protect against sorbitol intolerance during homeostasis. To analyze changes in the microbiota composition, fecal samples collected from the experiment shown in Figures 1A-1 D prior to antibiotic treatment (day 0; homeostasis) and 4 weeks after antibiotic treatment (day 28; sorbitol intolerance) (Fig. 9A) were analyzed by 16S ribosomal RNA gene amplicon sequencing (microbiota profiling) (Fig. 9B). Consistent with previous results (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284), prior streptomycin treatment and high fat intake significantly reduced the relative abundance of amplicon sequence variants (ASVs) belonging to the class Clostridia (Fig. 2A). A history of antibiotic treatment and high fat intake increased the abundance of ASVs belonging to the classes Bacilli, Bacteroidia or Verrucomicrobiae in some animals (Fig.9B and 9C). In mice with a history of antibiotic exposure and high fat intake, the most notable increase in the relative abundance was observed for ASVs belonging to the genus Enterococcus (class Bacilli). The microbiota composition varied between animals from this group (Fig. 2B), but variability could not be explained by cage effects. Linear discriminant analysis of fecal samples prior to, and 4 weeks after streptomycin treatment revealed that prior streptomycin treatment combined with high fat intake significantly increased several ASVs within the class Bacilli and significantly reduced the relative abundances of the classes Clostridia, Betaproteobacteria, and Actinobacteria (Fig. 2C).Metagenomic analysis identifies Clostridia as the main source of genes involved in sorbitol catabolism during homeostasis

[0071] We next wanted to determine which of the taxa that were depleted in mice with sorbitol intolerance were likely to prevent sorbitol intolerance during homeostasis. To analyze the metabolic capacity of the microbiota, we performed shotgun metagenomic sequencing on fecal samples collected from the experiment shown in Figures 1A-1 D prior to antibiotic treatment (homeostasis) or 4 weeks after antibiotic treatment (sorbitol intolerance) (Fig. 9A).

[0072] Metagenomic analysis identified a total of 11 ,979 unique genes from medium to high quality binned genomes (bins), which were annotated using KEGGs brite orthology database (Kanehisa, M., et al. Nucleic Acids Res 200028:27-30). Since this study focused on carbohydrate metabolism, our analysis remained focused on a small subset of unique genes. However, to allow readers to explore data regarding genes not related to carbohydrate catabolism, we developed a web application that allows interactive analysis of all KEGG orthology pathways within the dataset. Metagenomic analysis identified 421 genes involved in carbohydrate catabolism. Hierarchical clustering of samples using the 421 genes belonging to carbohydrate metabolism revealed 3 distinct clusters. One cluster contained samples collected before streptomycin treatment, while the other 2 clusters contained samples 4 weeks after streptomycin treatment. Differential abundance analysis of genes involved in carbohydrate metabolism revealed more significantly decreased genes than increased genes four weeks after streptomycin treatment (Fig. 2D). Among the genes with significantly reduced abundance after exposure to environmental risk factors were those involved in the catabolism of polyols, including mannitol, galactitol, erythritol, ribitol and arabinitol (Fig. 10A).

[0073] Two pathways for sorbitol catabolism have been described in bacteria. The first pathway involves transport across the bacterial cytoplasmic membrane through an ATP binding cassette [ABC] transporter (Carvajal, A.S., et al. Bmc Evol Biol 2012 12:Artn 147) or a transport system of the major facilitator superfamily (MFS) (Soemphol, W., et al. Biosci Biotech Bioch 2012 76:1497-1505). Transport of sorbitol into the cytosol is then followed byan NAD+-dependent conversion into either sorbose or fructose by sorbitol dehydrogenase. Genes encoding sorbitol dehydrogenase activity form a distinct subfamily within the polyol dehydrogenase family (Riveros-Rosas, H., et al. Eur J Biochem 2003 270:3309-3334). The majority of sorbitol dehydrogenase encoding reads from medium and high-quality bins in samples collected prior to antibiotic treatment were derived from bins belonging to the class Clostridia (Fig. 2E). A history of antibiotic treatment and high fat intake significantly reduced the abundance of sorbitol dehydrogenase encoding reads (Fig. 2E and 8D).

[0074] The second pathway for sorbitol utilization involves transport across the bacterial cytoplasmic membrane through a phosphotransferase (PTS) system, which is coupled to a phosphoenolpyruvate-dependent phosphorylation of sorbitol to yield sorbitol-6- phosphate. The enzyme sorbitol-6-phosphate 2-dehydrogenase then catalyzes the cytosolic conversion of sorbitol-6-phosphate and NAD+to fructose-6-phosphate, NADH and H+. The main source of sorbitol-6-phosphate 2-dehydrogenase encoding reads in samples collected prior to antibiotic treatment were from bins belonging to the class Clostridia (Fig. 2F). In contrast, the majority of sorbitol-6-phosphate 2-dehydrogenase encoding reads collected four weeks after streptomycin treatment of mice on a high fat diet were derived from bins belonging to the class Bacilli. However, the overall relative abundance of sorbitol-6- phosphate 2-dehydrogenase encoding reads did not significantly change between treatment groups (Fig. 8D). Overall, sorbitol dehydrogenase encoding reads were more abundant than sorbitol-6-phosphate 2-dehydrogenase encoding reads (Fig. 2E and 2F), suggesting that the former might be the dominate pathway for removal of sorbitol from the intestinal lumen during homeostasis.

[0075] The majority of sorbitol dehydrogenase and sorbitol-6-phosphate 2- dehydrogenase encoding reads in samples collected prior to antibiotic exposure were derived from bins belonging to the class Clostridia (Fig. 2E and 2F) and the order Lachnospirales (class Clostridia) (Fig. 8E). A history of antibiotic treatment and high fat intake reduced the abundance of Clostridia, which made Bacilli and Verrucomicrobiae the most prominent sources of genes involved in sorbitol catabolism. However, an increased abundance of sorbitol dehydrogenase encoding reads from Bacilli and Verrucomicrobiae did not fully compensate for the loss of sorbitol dehydrogenase encoding reads from Clostridia, thus reducing the overall metabolic potential to catabolize sorbitol in microbial communities from mice with a history of antibiotic treatment and high fat intake (Fig. 2E).Diminished microbial sorbitol catabolism causes sorbitol intolerance

[0076] To directly test whether impairment of microbial sorbitol catabolism causes sorbitol intolerance, we used Escherichia coli strain Nissle 1917 (family Enterobacteriaceae), a genetically tractable probiotic that catabolizes sorbitol (Nissle, A. DMW-Deutsche Medizinische Wochenschrift 1925 51 :1809-1813). To facilitate engraftment of the probiotic,we initially used a model of transient sorbitol intolerance (Fig. 8) in which disruption of the gut microbiota with streptomycin ensures efficient colonization. Mice reared and maintained throughout the experiment on a low-fat diet were inoculated one day after streptomycin treatment with different doses (between 10° to 109colony-forming units [cfu] per animal) of E. coli strain Nissle 1917 to generate mice that differed in their absolute E. coli abundance in the feces (Fig. 3A and 3B). At the day of inoculation with E. coli Nissle 1917, supplementation of drinking water with 5 % sorbitol was started. Supplementation of drinking water with 5 % sorbitol for 2 days produced sorbitol-induced diarrhea (Fig. 3C) in streptomycin-treated mice that carried E. coli Nissle 1917 below a threshold of 108cfu / g feces (Fig. 3A) or 10s16S rRNA gene copies / 20 ng DNA (Fig. 3B). In contrast, mock-treated mice or streptomycin-treated mice that carried E. coli Nissle 1917 above threshold levels were protected from sorbitol-induced diarrhea (Fig. 3A-3C). Cecal sorbitol dehydrogenase activity was reduced by treatment with streptomycin but was restored when mice were colonized with E. coli at levels above the threshold (Fig. 3D).

[0077] To test whether the ability of E. coli to protect against sorbitol intolerance was dependent on its ability to ferment sorbitol, we constructed a mutant deficient for sorbitol catabolism (srIAEB mutant). Whereas E. coli Nissle 1917 was able to deplete sorbitol in aerobic broth culture, a srIAEB mutant was no longer able to catabolize this sugar in vitro. Sorbitol fermentation could be restored in a srIAEB mutant by introducing the srIAEB genes on a plasmid (pAWLR196) but not by introducing an empty vector control (pWSK29) (Fig. 3E). Notably, the ability of E. coli to protect against sorbitol-induced diarrhea in vivo was dependent on its ability to ferment sorbitol, because protection was no longer observed (Fig. 3F) in streptomycin-treated mice colonized above threshold levels with an E. coli strain deficient for sorbitol catabolism (srIAEB mutant) (Fig. 3G). These data provided genetic evidence to causatively linked sorbitol intolerance to an impairment of microbial sorbitol catabolism.

[0078] Assignment of most reads involved in sorbitol catabolism to Clostridia bins during homeostasis (Fig. 2E and 2F) suggested that a lasting depletion of this taxon (Fig. 2A) triggers sorbitol intolerance. We thus explored whether a sorbitol-consuming member of the class Clostridia could serve as a second-generation probiotic (O'Toole, P.W., et al. Nat Microbiol 20172:17057) to treat sorbitol intolerance. Anaerostipes caccae is a commensal, butyrate-producing Clostridia isolate (Narushima, S., et al. Gut Microbes 2014 5:333-339) that can utilize sorbitol as a sole carbon source in vitro (Fig. 3H) (Tiffany, C.R., et al. Microbiome 2021 9:174). A. caccae was neither detected by microbiota profiling nor by metagenomic analysis in microbiota of mice analyzed in this study but this species is prevalent and abundant in the human fecal microbiota (Walker, A.W., et al. ISME J 2011 5:220-230; Arumugam, M., et al. Nature 2011 473:174-180). In a proof-of-conceptexperiment, mice (C57BL / 6J) reared and maintained throughout the experiment on a low-fat diet were mock-treated or treated with a single dose of streptomycin to generate transient sorbitol intolerance and to facilitate colonization. One day later, drinking water was supplemented with 5 % sorbitol and groups of streptomycin-treated mice were inoculated with different doses of A. caccae. The resulting groups of streptomycin-treated mice differed in their absolute abundance of A caccae in the feces (Fig. 31). Supplementation of drinking water with 5 % sorbitol for 2 days produced signs of sorbitol-induced diarrhea (Fig. 3J) in streptomycin-treated mice that carried A. caccae below a threshold of approximately 10616S rRNA gene copies / 20 ng DNA (Fig. 3I), which was similar to the threshold determined for E. coli (Fig. 3B). The sorbitol dehydrogenase activity of the microbiota was reduced by treatment with streptomycin but was restored when mice were colonized with A. caccae at levels above the threshold (Fig. 3K).Treatment of prolonged sorbitol intolerance with sorbitol-catabolizing probiotics

[0079] We next wanted to test whether treatment with sorbitol-consuming probiotics would protect against prolonged sorbitol intolerance. Mice reared and maintained throughout the experiment on a low-fat or a high-fat diet were mock-treated or received a single dose of streptomycin, respectively. Four weeks later, mice received drinking water supplemented with 5 % sorbitol and were inoculated with 109colony-forming units (cfu) of E. coli Nissle 1917, A. caccae, or Lactiplantibacillus plantarum strain NICMB8826-R (class Bacilli, phylum Firmicutes), a probiotic strain that can catabolize sorbitol (Fig. 4A). After three days of sorbitol supplementation, all three probiotics conferred protection against sorbitol-induced diarrhea (Fig. 4B). Whereas the degree of protection against sorbitol-induced diarrhea afforded by A. caccae and E. coli Nissle 1917 did not change overtime, L. plantarum no longer conferred protection after seven days of sorbitol supplementation. All three probiotics prevented accumulation of sorbitol in cecal contents after three days of supplementation, but sorbitol started to accumulate in ceca of mice inoculated with L. plantarum after seven days (Fig. 4C). Whereas all three probiotics provided some degree of protection against sorbitol- induced diarrhea, only A. caccae lowered fecal water content to levels seen in controls without sorbitol intolerance (Fig. 4B).

[0080] Since protection against transient sorbitol intolerance requires colonization above a certain threshold (Fig. 3A, 3B, 3C, 3I, and 3J), we first considered that sorbitol- consuming probiotics might differ in their ability to protect against prolonged sorbitol intolerance (Fig. 4B) because they differed in their ability to colonize mice. Whereas E. coli was recovered from feces in similar numbers at three and seven days after inoculation, both L plantarum and A. caccae colonization levels dropped by several orders of magnitude between days three and seven after inoculation (Fig. 4D and 4E). Thus, a possibleexplanation for a loss of protection conferred by L. plantarum seven days after inoculation (Fig. 4B) was that the probiotic was beginning to be cleared (Fig. 4D).

[0081] The consistently high colonization levels of E. coll Nissle 1917 throughout the experiment (Fig. 4D) suggested that the probiotic was able to catabolize sorbitol to confer protection throughout the seven-day exposure to sorbitol (Fig. 4B). To test whether protection by E. coll Nissle 1917 required its ability to catabolize sorbitol, the experiment was repeated with mice inoculated with E. coll Nissle 1917 wild type or an isogenic sorbitol fermentation-deficient strain (srIAEB mutant). Three days after inoculation, wild type and srIAEB mutant were recovered in similar numbers from the feces (Fig. 11 A), but only the sorbitol fermentation-proficient wild-type depleted sorbitol in cecal contents (Fig. 11 B) and prevented a rise in fecal water content during sorbitol exposure (Fig 11C). These data suggested that sorbitol catabolism of E. coll Nissle 1917 was causatively linked to its ability to protect against prolonged sorbitol intolerance.

[0082] Colonization above threshold levels at 3 days after inoculation (Fig. 3I and 4E) suggested that A. caccae might protect against sorbitol intolerance at this time point (Fig. 4B) by depleting sorbitol (Fig. 4C). However, the probiotic still normalized fecal water content after seven days of sorbitol exposure (Fig. 4B) even though A. caccae was being cleared by this time point (Fig. 4E). Thus, protection conferred by A. caccae at seven days after inoculation (Fig. 4B) could not be attributed to its ability to catabolize sorbitol.Treatment with a butyrate-producing probiotic promotes microbiota recovery

[0083] We next investigated whether A. caccae protected mice from sorbitol intolerance at seven days after inoculation because this probiotic promoted an increase in the abundance of other Clostridia species, thereby restoring sorbitol catabolism of the microbiota to normal levels. Consistent with this idea, inoculation with A. caccae restored a normal absolute abundance of Clostridia as indicated by real-time PCR using class-specific primers (Fig. 4F), which persisted even after the probiotic had been cleared seven days after inoculation (Fig. 4E). Furthermore, A. caccae promoted a rise in Clostridia richness, as indicated by detecting increasing numbers of distinct ASVs belonging to the class Clostridia during microbiota profiling at seven days after inoculation with the probiotic (Fig. 4G). In contrast, the absolute abundance and richness of the Clostridia population remained low in mice treated with E. coll Nissle 1917 or L. plantarum (Fig. 4F and 4G).

[0084] One feature that distinguishes A. caccae from E. coll or L. plantarum is its ability to produce the short-chain fatty acid butyrate (Narushima, S., et al. Gut Microbes 2014 5:333-339). We thus wanted to determine whether butyrate production is linked to microbiota recovery. Our metagenomic analysis predicted that sorbitol intolerance was linked to a reduced capacity of the microbiota to produce butyrate (Fig 10B). Consistent with thisprediction, a history of streptomycin treatment and high fat intake was associated with a marked reduction in the cecal butyrate concentration (Fig. 5A). Microbiota profiling revealed that this drop in cecal butyrate levels was linked to a depletion of Lachnospiraceae and Ruminococcaceae (Fig 5B), two Clostridia families that harbor butyrate producers (Vital, M., et al. MBio 2014 5:e00889). Treatment with A. caccae increased cecal butyrate levels, whereas inoculation with E. coli Nissle 1917 or L. plantarum did not restore production of this short-chain fatty acid (Fig. 5A). Clearance of A. caccae by day seven after inoculation (Fig. 4E) suggested that restoration of butyrate levels at this time point (Fig. 5A) was likely attributable to an increased abundance of other butyrate-producing species as suggested by an increased abundance of Lachnospiraceae (Fig. 5B).

[0085] Butyrate is a PPAR-y (Peroxisome Proliferator-activated Receptor-gamma) agonists that maintains epithelial hypoxia in the colon to limit the diffusion of oxygen into the intestinal lumen, which helps maintain anaerobiosis in the lumen (Byndloss, M.X., et al. Science 2017 357:570-575). In support of increased oxygen availability in the gut lumen, metagenomic analysis revealed that microbial genes involved in oxidative phosphorylation exhibited an increased abundance after exposure to antibiotics and high fat intake. Consistent with a previous report (Lee, J.Y., et al. Cell Host Microbe 202028:273-284), a history of streptomycin treatment and high-fat intake was associated with a loss of epithelial hypoxia (Fig. 5C and 5D). Notably, a rise in cecal butyrate levels during A. caccae treatment (Fig. 5A) was correlated with a restoration of epithelial hypoxia (Fig. 5C and 5D). In contrast, when mice with a history of streptomycin treatment and high-fat intake were treated with E. coli Nissle 1917 or L. plantarum, neither cecal butyrate levels (Fig. 5A) nor epithelial hypoxia (Fig. 5C and 5D) were restored. Collectively, these data supported the hypothesis that butyrate production by A. caccae activates epithelial PPAR-y signaling to restore epithelial hypoxia, which in turn promotes microbiota recovery.

[0086] To test this hypothesis, we determined whether butyrate activates epithelial PPAR-y signaling to promote microbiota recovery in mice with prolonged sorbitol intolerance. Since butyrate is absorbed in the small intestine, we inoculated mice with tributyrin, a triglyceride that is poorly absorbed in the small intestine and is cleaved by host enzymes in the large intestine to release glycerol and butyrate. Mice reared and maintained throughout the experiment on a high-fat diet received a single dose of streptomycin. Four weeks later, mice received drinking water supplemented with 5 % sorbitol and were mock-treated or treated with tributyrin. Three days of tributyrin treatment resulted in a small but significant increase in the cecal butyrate concentration (Fig. 6A). By seven days of tributyrin treatment, cecal butyrate levels were markedly increased. Tributyrin treatment protected against sorbitol-induced diarrhea (Fig. 6B) which was linked to depletion of sorbitol in cecal contentsafter seven days of sorbitol supplementation (Fig. 6C). Consistent with our hypothesis, tributyrin restored epithelial hypoxia in the colon (Fig. 6D) and enlarged the absolute Clostridia abundance (Fig. 6E), which included a rise in the relative abundance of Lachnospiraceae (Fig. 6F), after seven days of treatment. These data suggested that butyrate restores hypoxia to promote microbiota recovery, a process that confers resistance to sorbitol-induced diarrhea within a week of tributyrin supplementation.

[0087] Next, we wanted to determine whether PPAR-y signaling in the host epithelium was required for maintaining protection against sorbitol intolerance even after A. caccae has been cleared. Mice lacking PPAR-y synthesis specifically in the intestinal epithelium (Pparg^'Vlllin^'- mice) and wild-type littermate controls (Pparg^Villin1- mice) reared and maintained throughout the experiment on a low-fat or a high-fat diet were mock- treated or received a single dose of streptomycin, respectively. Four weeks later, mice received drinking water supplemented with 5 % sorbitol (Fig. 4A). At the time of sorbitol exposure, mice were either mock inoculated, inoculated with A. caccae or received tributyrin treatment. Notably, in mice lacking epithelial PPAR-y signaling, treatment with A. caccae or tributyrin no longer protected against sorbitol-induced diarrhea (Fig. 6G) and the accumulation of sorbitol in cecal contents (Fig. 6H). Furthermore, treatment with A. caccae or tributyrin no longer restored epithelial hypoxia in mice lacking epithelial PPAR-y signaling (Figs. 61 and 12). Consistent with a role of PPAR-y signaling in promoting the recovery of butyrate-producing members of the microbiota, butyrate levels increased one week after treatment with A. caccae in littermate control mice but not in mice lacking epithelial PPAR-y signaling (Fig. 6J).5-ASA promotes microbiota recovery to prevent sorbitol intolerance

[0088] The finding that butyrate production by A. caccae promoted microbiota recovery within seven days after inoculation suggested that restoration of epithelial hypoxia could be a potential strategy for promoting microbiota recovery after antibiotic exposure. We reasoned that PPAR-y agonists that target the host to restore epithelial hypoxia would reestablish anaerobiosis, thereby normalizing growth of obligately anaerobic Clostridia to prevent sorbitol intolerance. Previous work shows that the PPAR-y agonist 5-amino salicylic acid (5-ASA) (Rousseaux, C., et al. J Exp Med 2005 201 :1205-1215) restores epithelial hypoxia in mice with a history of antibiotic treatment and high fat intake (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284). We thus explored whether 5-ASA could be used for treatment or prophylaxis of sorbitol intolerance.

[0089] Mice with a history of antibiotic treatment and high-fat intake (Fig. 4A) received chow supplemented with 5-ASA and drinking water supplemented with 5 % sorbitol. Supplementation with 5-ASA for three days did not alleviate signs of sorbitol intolerance.However, by seven days of 5-ASA treatment, cecal butyrate levels were markedly increased (Fig. 6A). Seven days of 5-ASA treatment protected mice against sorbitol-induced diarrhea (Fig. 6B), which was linked to a depletion of sorbitol in cecal contents (Fig. 6C), a restoration of epithelial hypoxia in the colon (Fig. 6D), a greater absolute Clostridia abundance (Fig. 6E), an increased richness of the Clostridia population (Fig. 7 A) and a rise in the relative abundance of Lachnospiraceae (Fig. 7B). Collectively, these data suggested that activation of epithelial PPAR-y signaling with 5-ASA facilitated microbiota recovery by activating epithelial PPAR-y signaling, but this process did not restore homeostasis fast enough to provide immediate relieve from sorbitol intolerance.

[0090] To test whether 5-ASA could be used for prophylaxis, mice reared and maintained throughout the experiment on a low-fat or a high-fat diet were mock-treated or received a single dose of streptomycin, respectively, and received prophylaxis by supplementing chow with 5-ASA starting on the day of streptomycin treatment. Four weeks later, mice received drinking water supplemented with 5 % sorbitol to assess sorbitol tolerance. Prophylaxis with 5-ASA restored a normal absolute abundance of Clostridia in streptomycin-treated mice on a high-fat diet (Fig. 7C). 5-ASA drove this change in the microbiota composition by activating PPAR-y in the intestinal epithelium, because 5-ASA prophylaxis no longer increased Clostridia levels in mice lacking PPAR-y synthesis specifically in the intestinal epithelium (PpargmVillincrel- mice) (Fig. 7D). 5-ASA prophylaxis prevented accumulation of sorbitol in cecal contents during sorbitol supplementation (Fig. 7E) and reduced fecal water content (Fig. 7F). Thus, prophylactic activation of epithelial PPAR-y signaling with 5-ASA prevented development of prolonged sorbitol intolerance after exposure to antibiotics and high fat intake by promoting microbiota recovery, thereby normalizing microbial sorbitol catabolism.Discussion

[0091] Up to 30 % of the population in high-income countries experience episodes of carbohydrate intolerance, with abdominal complaints being associated most commonly with lactose, fructose or sorbitol (Born, P. World J Gastroenterol 2007 13:5687-5691). Lactose and fructose intolerance can be explained by malabsorption in the ileum due to congenital or acquired defects in specific host enzymes or transport systems (Fernandez-Banares, F. Nutrients 2022 14). Sorbitol intolerance is commonly included in the category of carbohydrate malabsorption (Born, P. World J Gastroenterol 2007 13:5687-5691). However, since humans lack a specific sorbitol transporter, its uptake relies on passive diffusion, resulting in poor absorption in the small intestine even in healthy individuals. There is no experimental evidence to suggest that patients with sorbitol intolerance exhibit reduced uptake of sorbitol by passive diffusion (Fernandez-Banares, F., et al. Gastroenterology 1991101 :1453-1454). Instead, our results suggest that in healthy individuals, microbial sorbitol catabolism protects against symptoms of sorbitol intolerance arising from an inherently poor absorption of this polyol. Here we show that sorbitol intolerance arises from an impairment of microbial sorbitol catabolism, a finding with implications for diagnosis, pathophysiology, treatment, and prevention.

[0092] The development of carbohydrate intolerance tests that reliably predict the outcome of dietary management remains a clinical challenge in the field of functional bowel disease (Fernandez-Banares, F. Nutrients 2022 14). Here we show that measurements of microbiota-derived sorbitol dehydrogenase levels in the feces and the abundance of sorbitol dehydrogenase gene sequences in the metagenome predict sorbitol intolerance in a mouse model. Furthermore, the analysis of human fecal samples suggested that low sorbitol dehydrogenase levels correlate with patients exhibiting intolerance to sugar-free food. Thus, it may be possible to develop microbiota-based diagnostic tests for sorbitol intolerance in high-risk groups, which represents a promising area for future research.

[0093] By developing an animal model for prolonged sorbitol intolerance, our results provide insights into the pathophysiology of this condition. The observation that exposure to antibiotics in combination with high fat intake impairs microbial sorbitol catabolism by lowering the abundance of Clostridia challenges conventional wisdom that prolonged sorbitol intolerance is caused by malabsorption (Hammer, H.F., et al. Gastroenterol Clin North Am 201241 :611-627; Born, P. World J Gastroenterol 2007 13:5687-5691). Exposure to antibiotics and high fat intake are also environmental risk factors involved in the pathophysiology of IBD (Albenberg, L.G., et al. Curr Opin Gastroenterol 2012 28:314-320; Frolkis, A., et al. Can J Gastroenterol 2013 27:e18-24; Hildebrand, H., et al. Scand J Gastroenterol 2008 43:961-966; Hviid, A, et al. Gut 2011 60:49-54; Lewis, J.D. Nestle Nutr Inst Workshop Ser 2014 79:1-18; Zou, Y., et al. Scand J Gastroenterol 2020 55:301-311). Epidemiological studies from Western countries, where these risk factors are more prevalent, report a higher incidence of carbohydrate intolerance compared to Asian countries (Born, P. World J Gastroenterol 2007 13:5687-5691) and feces of East Asian subjects exhibit increased levels of short-chain fatty acids and greater abundance of reads encoding short-chain fatty acid-related metabolic pathways binned to Clostridia compared to white subjects (Ang, Q.Y., et al. Elife 2021 10). A reduced relative abundance of Clostridia in IBD patients is well documented (Alam, M.T., et al. Gut Pathog 2020 12:1 ; Lepage, P., et al. Gastroenterology 2011 141 :227-236; Machiels, K., et al. Gut 201463:1275-1283; Sartor, R.B. Gastroenterology 2008 134:577-594). Our results show that a combination of environmental risk factors can give rise to prolonged sorbitol intolerance, which is observed in some patients with quiescent IBD (Gearry, R.B., et al. J Crohns Colitis 2009 3:8-14; Gibson, P.R. J Gastroenterol Hepatol 2017 32 Suppl 1 :40-42). Clinical trials show acorrelation between carbohydrate intake (including polyols) and gastrointestinal symptoms in IBD patients (Cox, S.R., et al. J Crohns Colitis 2017 11 :1420-1429; Zhan, Y.L., et al. Clin Nutr 2018 37:123-129; Bodini, G., et al. Nutrition 2019 67-68:110542). These clinical observations align well with our observation that diminished butyrate production and a reduced abundance of Clostridia in the fecal microbiota are functionally linked to prolonged sorbitol intolerance in a mouse model.

[0094] Epithelial hypoxia limits the diffusion of oxygen into the intestinal lumen to maintain anaerobiosis (Litvak, Y., et al. Science 2018 362:eaat9076), thereby providing a host environment that favors growth of obligately anaerobic bacteria, such as Clostridia (Miller, B.M., et al. Curr Opin Microbiol 2021 63:221-230). Antibiotic-mediated disruption of the colonic microbiota depletes short-chain fatty acids, which in turn triggers a shift in epithelial metabolism to increase the availability of host-derived oxygen (Byndloss, M.X., et al. Science 2017 357:570-575). A high-fat diet rich in saturated fatty acids triggers oxidative stress in host cells (Gulhane, M., et al. Sci Rep 20166:28990) by inducing elevated mitochondrial hydrogen peroxide production (Cardoso, A.R., et al. PLoS One 2013 8:e77088; Kakimoto, P.A., et al. Redox Biol 20154:375-380). Through this mechanism, a prolonged high-fat diet impairs recovery of mitochondrial bioenergetics after antibiotic treatment, thereby interfering with microbiota recovery (Lee, J.Y., et al. Cell Host Microbe202028:273-284). Since increased epithelial oxygenation is ultimately responsible for an impaired microbiota recovery after antibiotic exposure, it should be possible to prevent the development of prolonged sorbitol intolerance after exposure to antibiotics and high fat intake by restoring host functions that maintain the colonic epithelium in a state of physiological hypoxia. Our results suggest that prophylaxis with 5-ASA, a drug that activates epithelial PPAR-y signaling to stimulate mitochondrial activity (Cevallos, S.A., et al. mBio2021 12), can reestablish epithelial hypoxia and promote microbiota recovery after antibiotic treatment even in the face of high fat intake. These data identify the intestinal epithelium as a potential treatment target to prevent the development of prolonged sorbitol intolerance.

[0095] The finding that prolonged sorbitol intolerance is due to impaired microbial sorbitol catabolism suggests that the microbiota represents a second potential treatment target. One treatment option might be the use of sorbitol-consuming probiotics, such as E. coii Nissle 1917 or L plantarum, to augment microbial sorbitol catabolism in patients with sorbitol intolerance. However, the protective effect of probiotics was dependent on reaching high numbers in the feces. Such high colonization levels might be difficult to accomplish with some probiotics. Furthermore, high colonization levels of E. coii Nissle 1917 might not be desirable, since intestinal domination by Enterobacterales is considered a microbial signature of dysbiosis2864. Interestingly, our data identify A. caccae as a second-generation probiotic that targets both the host and the microbes to protect against sorbitol intolerance.The ability of A caccae to catabolize sorbitol resulted in a depletion of this polyol from cecal contents when the probiotic was abundant, which conferred protection against transient sorbitol intolerance after antibiotic exposure. Importantly, A. caccae still conferred protection against prolonged sorbitol intolerance even when the abundance of the probiotic was low, which was linked to its ability to produce butyrate. Butyrate stimulates PPAR-y signaling in the host to restore epithelial hypoxia (Byndloss, M.X., et al. Science 2017 357:570-575), which promoted microbiota recovery within a week after inoculating mice with A. caccae. Once microbiota recovery was complete, a high abundance of A. caccae was no longer required to confer protection against sorbitol intolerance. Thus, the appeal of using this second-generation probiotic is that the sorbitol catabolism of A. caccae provides immediate relief from sorbitol intolerance, whereas its ability to produce butyrate promotes microbiota recovery within a week, thereby allowing other bacteria that metabolize sorbitol to grow to numbers that are high enough to deplete this polyol.MethodsMouse Models

[0096] The Institutional Animal Care and Use Committee at the University of California at Davis approved all animal experiments in his study. Male C57BL6 / J mice, aged 6 weeks were obtained from The Jackson Laboratory. C57BL / 6 Ppargfl / flVillincre / _and littermate Ppargfl / flVillin_ / _mice were generated at UC Davis by mating Ppargfl / flmice with Villincre / _mice (The Jackson Laboratory). Animals were either fed a (sorbitol-free) 10 % control (LF) diet (Teklad Diet, #TD 11065) or (sorbitol-free) 45% fat (HF) diet (Teklad Diet, #TD06415) from weaning at the age of 6 weeks until the end of the experiment.

[0097] For experiments using a model of antibiotic-induced transient sorbitol intolerance, mice were maintained on a sorbitol-free low-fat diet and mock-treated or treated with single dose of 20 mg / animal streptomycin via oral gavage. One day or five days later, drinking water was supplemented with 5 % (w / v) D-sorbitol (Sigma Aldrich) solution for three days. In some experiments, mice were orally inoculated with different doses (between 10° and 109cfu in a volume 100 pL) of A.caccae one day after streptomycin treatment. In some experiments, mice were orally inoculated with different doses (between 10° and 109cfu in a volume of 100 pL) of either wild-type Escherichia coli strain Nissle 1917 or an isogenic strain deficient for sorbitol utilization (srIAEB mutant) one day after streptomycin treatment. After starting sorbitol supplementation in drinking water, body weights of mice were monitored daily, and the experiment was terminated 2 days after starting sorbitol supplementation. Fecal colonization of bacteria was determined by homogenizing feces in 1 mL of sterile PBS, followed by serially diluting the samples and plating on LB plates containing appropriate antibiotics for E. coli. The colonization levels of A. caccae and were ascertained using qRT-PCR. For comparison, the colonization levels of E. coli were also ascertained using qRT- PCR.

[0098] For experiments using a model of prolonged sorbitol intolerance, mice maintained on a sorbitol free low-fat or high fat diet for 14 days were mock-treated or treated with a single dose of 20 mg / animal streptomycin via oral gavage. After maintaining mice for four more weeks on the same diet, drinking water was supplemented with 5 % D-sorbitol solution for three days. For some experiments, mice were inoculated with 109cfu of Anaerostipes caccae, E. coli Nissle 1917 or Lactiplantibacillus plantarum strain NCIMB8826- R in volume of 100 L at the beginning of seven days of sorbitol exposure. After starting sorbitol supplementation in drinking water, body weights of mice were monitored daily, and the experiment was terminated three or seven days after starting sorbitol supplementation. For some experiments, mice received supplements of 5-aminosalicylic acid (5-ASA) at a dose of 1650 mg / kg / day (Teklad Diet, #TD 180827) mixed into high-fat diet chow or tributyrin (5 g / kg), administered via oral gavage over a seven-day period. For 5-ASA prophylaxis, mice were maintained on a low-fat or a high-fat diet for 14 days and were mock-treated or received a single dose of streptomycin (20 mg / mouse) via oral gavage, and high-fat diet chow was switched to a HF diet chow supplemented with 5-ASA for the remainder of the experiment.Bacterial strains and culture conditions

[0099] The E. coli strains used in this study were routinely grown in LB broth (BD Bioscience) or on LB plates. The L. plantarum strain was grown in MRS (BD Bioscience) broth or on MRS plates. For animal experiments, bacterial cultures were grown overnight at 37°C in LB broth or MRS broth under aerobic conditions. 0.1 mg / mL of carbenici I lin or 0.05 mg / mL of kanamycin or 0.05 mg / mL of rifampicin were added as required. For inoculating mice with different does of A. caccae, A.caccae strain was grown anaerobically in EG broth for 48 hours and 10 ml of overnight culture were spun down and pelleted at 4°C, then resuspended to a final concentration between 1x101and 1010cfu / ml in fresh media.Human studies

[0100] Banked fecal samples from a previous study were utilized to assess sorbitol dehydrogenase levels in human feces (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284). The study conformed to the Declaration of Helsinki's guidelines and received the necessary approvals from the Institutional Review Board of CHA Bundang Medical Center (protocol number: 2016-06-055) and Yonsei University College of Medicine (protocol number: 4-2015- 0608). Subjects who met the ROME III criteria for IBS were enrolled between June 2016 and July 2017 at the Department of Family Medicine, CHAUM Hospital, Seoul, Korea. Healthy controls were recruited through advertisements and were required to complete a questionnaire to ensure they did not have any gastrointestinal symptoms. To further classifyIBS patients, calprotectin levels in feces were determined using an ELISA. A questionnaire was used to determine intolerance to sugar-free foods or beverages, asking participants, "Does consuming sugar-free food or drinks with substitute / artificial sugar exacerbate your gastrointestinal symptoms?" Sorbitol dehydrogenase in feces was quantified using a sorbitol dehydrogenase assay kit, following the provided instructions.Construction of E. coli Nissle 1917 AsrIAEB

[0101] A srIAEB mutant of E. coli Nissle 1917 was generated by allelic exchange. Approximately 500 bp upstream of srIA and 500 bp downstream of srIB (the flanking regions of the srIAEB operon) were amplified from genomic DNA using primers srlAEB_EcN_AB_F / R and srlAEB_EcN_CD_F / R (Key Resources Table), respectively. PCR using genomic DNA as the template was carried out using Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and amplification products were purified using the QIAquick PCR Purification Kit (Qiagen). The suicide vector pRE112 (Edwards, R.A., et al. Gene 1998 207:149-157) was linearized by PCR using the primers pRE_linear_F / R (Key Resources Table). Primers used to amplify the flanking regions were designed to allow assembly of amplification products together with linearized pRE112 using the NEBuilder Assembly Tool (NEB). Linearized pRE112 and the amplified flanking regions of the srIAEB operon were assembled into a circularized plasmid using NEBuilder HiFi DNA Assembly Master Mix (NEB). Assembled candidate plasmids were transformed into E. coli strain S17-1 pir (Simon, R., et al. 1983 Bio-Technology 1 :784-791), screened for correct insert size by colony PCR using MyTaq Red Mix (Meridian Bioscience). Assembled candidate plasmids were visualized by agarose gel electrophoresis, extracted using the QIAprep Spin Miniprep Kit (Qiagen), and submitted to the UC Davis DNA Sequencing Core for insert sequence verification. A clone with a sequence-verified insert was termed pAWLR80.

[0102] Plasmid pAWLR80 was conjugated into E. coli Nissle 1917 carrying the temperature-sensitive plasmid pSW172 (Lopez, C.A., et al. MBio 2012 3:e00143-00112) for counter selection. Mating was performed overnight at 30°C and transconjugants were selected by plating on LB agar containing carbenicillin and chloramphenicol. Sucrose selection was then performed on agar plates containing 8 g / L nutrient broth base (Difco) and 5 % sucrose. Sucrose-resistant, chloramphenicol-sensitive clones were screened by colony PCR for the shortened allele of the srIAEB operon. Plasmid pSW172 was cured from resulting srIAEB deletion mutants by cultivating clones at 37°C and selecting for a carbenicillin-sensitive clone, which was termed AWLR133. Plasmid pCAL61 (Spees, A.M., et al. MBio 2013 4:e00935-00919) was electroporated into AWLR133 to introduce a selectable kanamycin marker.

[0103] Complementation of the srIAEB genes was accomplished by cloning them into the low-copy vector pWSK29 (Wang, R.F., et al. Gene 1991 100:195-199). The native promoter of the genes was maintained in the expression construct. The NEBuilder Assembly Tool (New England Biolabs) web application was used to generate primers for the amplification of the locus containing srIAEB, including 500 bp upstream of the srIA start codon, and 50 bp downstream of the srIB stop codon. To facilitate efficient amplification by PCR, the srIAEB locus was amplified in two fragments of equal length using genomic DNA as the template with the primers srlAEB_pcomp_up_F, srlAEB_pcomp_up_R, srlAEB_pcomp_down_F, and srlAEB_pcomp_down_R. Q5 Hot Start High-Fidelity 2X Master Mix (NEB) and amplification products were purified using the QIAquick PCR Purification Kit (Qiagen). Linearization of pWSK29 was carried out by restriction-digestion using EcoRI-HF (New England Biolabs) and Sacl-HF (New England Biolabs). Linearized pWSK29 and the amplified srIAEB fragments were assembled into a circularized plasmid using NEBuilder HiFi DNA Assembly Master Mix (NEB). Assembled candidate plasmids were transformed into E. coll strain DH5ot pir (Pal, D., et al. J Bacteriol 2005 187:7167-7175), and screened for correct insert size by colony PCR using MyTaq Red Mix (Meridian Bioscience). Assembled candidate plasmids were extracted using the QIAprep Spin Miniprep Kit (Qiagen), and submitted to Genewiz (Azenta Life Sciences) for whole plasmid sequencing. A sequence- verified clone was termed pAWLR169 and electroporated into E. coll Nissle 1917 slrAEB.16S rRNA gene amplicon sequencing

[0104] For 16S rRNA amplicon library preparation and sequencing, primers were used to amplify the V3-V4 domain of the 16S rRNA using a two-step PCR procedure. In step one of the amplification procedures, both forward and reverse primers contained an Illumina tag sequence (bold), a variable length spacer (no spacer, C, TC, or ATC for 319F; no spacer, G, TG, ATG for 806R) to increase diversity and improve the quality of the sequencing run, a linker sequence (italicized), and the 16S target sequence (underlined). Each 25 pl PCR reaction contained 1 Unit Kapa2G Robust Hot Start Polymerase (Kapa Biosystems), 1.5 mM MgCh, 0.2 mM final concentration dNTP mix, 0.2 pM final concentration of each primer and 1 pl of DNA for each sample. PCR conditions were; an initial incubation at 95°C for 3 min, followed by 25 cycles of 95°C for 45 seconds, 50°C for 30 seconds, 72°C for 30 seconds and a final extension of 72°C for 3 minutes. In step two, each sample was barcoded with a unique forward and reverse barcode combination using forward primers with an Illumina P5 adapter sequence (bold), a unique 8 nt barcode (N), a partial matching sequence of the forward adapter used in step one, and reverse primers with an Illumina P7 adapter sequence (bold), unique 8 nt barcode (N), and a partial matching sequence of the reverse adapter used in step one. The PCR reaction in step two contained 1 Unit Kapa2G Robust Hot Start Polymerase (Kapa Biosystems), 1.5 mM MgCh, 0.2 mMfinal concentration dNTP mix, 0.2 pM final concentration of each uniquely barcoded primer and 1 ul of the product from the PCR reaction in step one diluted at a 10:1 ratio in water. PCR conditions were; an initial incubation at 95°C for 3 min, followed by 8 cycles of 95°C for 30 seconds, 58°C for 20 seconds, 72°C for 20 seconds and a final extension of 72°C for 3 minutes. The final product was quantified on the Qubit instrument using the Qubit Broad Range DNA kit (Invitrogen) and individual amplicons were pooled in equal concentrations. The pooled library was cleaned utilizing Ampure XP beads (Beckman Coulter) then the band of interest was further subjected to isolation via gel electrophoresis on a 1 .5 % Blue Pippin HT gel (Sage Science). The library was quantified via qPCR followed by 300-bp paired- endvsequencing using an Illumina MiSeq instrument in the Genome Center DNA Technologies Core, University of California, Davis.16S rRNA gene amplicon sequencing analysis

[0105] Sequencing reads were demultiplexed using QIIME 1.877, and non-biological nucleotides were trimmed using Trimmomatic (Bolger, A.M., et al. Bioinformatics 2014 30:2114-2120). 16S rRNA sequencing reads were then processed and assembled into amplicon sequence variants (ASV) using dada2 (Callahan, B.J., et al. Nat Methods 2016 13:581-583) in R. First, reads with more than 2 expected errors were removed. Dereplication and sample inferences were then performed on forward and reverse reads, prior to merging. A sequence table was constructed from merged reads, and chimeric reads were subsequently removed. Taxonomy was assigned to reads to the species level using the dada2 formatted rdp training dataset 14. The R package phyloseq (McMurdie, P.J., et al. PLoS One 2013 8:e61217) was then used in downstream analysis of the data, including the generation of a phyloseq object, relative abundance bar plots. Relative abundance boxplots were generated using ggplot2. For linear discriminant analysis, data transformed using the DESeq2 (Love, M.I., et al. Genome Biol 2014 15:550) median of ratios method to account for differences in reads between samples, parsed, written to a tab separated text file, and then uploaded to the LEfSe (Segata, N., et al. Genome Biol. 2011 12(6):R60) galaxy server where the default statistical parameters were used in the analysis to generate LDA scores and the LDA cladogram.Shotgun Metagenome Data Analysis

[0106] Assembly, Annotation, and Binning-. Metagenome QC and assembly was performed (Clum et al., 2021) with modifications for co-assembly to yield higher quality bins. Metaspades V3.15.2 (Nurk et al., 2017) was used to create a co-assembly. Once coassembled, each of the sequencing FASTQ reads files from the individual samples were mapped to the co-assembly contigs with BBMap. The contigs and coverage information were then submitted to IMG / M for annotation and binning (Clum et al., 2021).

[0107] Generation of count matrices of genes'. For the gene count matrices, a custom script was used to parse out the contigJD, gene_start, gene_stop (gene positions on contig), and gene_attributes data for each gene in the GFF files that were created during annotation. The custom script then parsed out the contigJD and read_start (read positions on contig) from each of the BBMap mapping files. For each mapping file, if the read_ start position was within any gene_start and gene_stop positions in the GFF files, then the read was counted as mapping to that gene and the gene attributes (cathjunfam, COG, EC, hypothetical, KO, pfam, SMART, superfamily, tigrfam, and / or tRNA IDs) from the GFF file were tallied. All gene counts were derived using medium and high-quality bins.

[0108] Clustering and Plots: Gene names and KEGG (Kanehisa, M., et al. Nucleic Acids Res 2000 28:27-30) orthology brite hierarchy metadata were assigned using the R package omu (Tiffany, C.R., et al. Microbiol Resour Announc 2019 8). Heatmaps were constructed using the function heatmap.2 from the R package gplots. Read counts were normalized using the median of ratios method in DESeq2, and then transformed using the function ln(1+x) before clustering. The sample dendrogram was created using hierarchical clustering on Euclidean distances calculated between sample vectors, with a complete linkage approach.

[0109] Bar plots of gene counts by taxonomic group were generated using the raw counts and the R package ggplot2. The following R packages were used in order to develop the web application featuring the shotgun metagenomic data: shiny, bslib, shinywidgets, ggplot2, officer, thematic, colourpicker, reshape2, RColorBrewer, gplots, gridextra, cowplot, ggrepel, and ggplotify.D-Sorbitol Measurements

[0110] To measure D-sorbitol levels in the murine cecum, cecal contents were homogenized in 1 mL of sterile PBS, centrifuged at 300 g for 10 min at 4 °C, and then the supernatants were filtered through a 10-kDa spin column (Biovision). D-Sorbitol concentrations were determined using a D-sorbitol assay kit (Biovision) according to the manufacturer’s instruction. Spike / Recovery assay was performed for validating cecal contents in this assay and the calculated recovery range was 117.60 %.Sorbitol Dehydrogenase Activity Measurements

[0111] To measure sorbitol dehydrogenase activity in the murine cecum, cecal contents were homogenized 1 mL of sterile PBS, centrifuged at 300 g for 10 minutes at 4°C. Sorbitol Dehydrogenase activity was determined using a Sorbitol Dehydrogenase (SDH) Assay Kit (LSBio) according to the manufacturer’s instructions.Fecal water content

[0112] To measure the fecal water content, fecal pellets were collected and weighed. Then, fecal pellets were dried in a 50°C oven for 24 hours to remove water contents and were then weighed again. The percentage of water contents was calculated.Determining bacterial absolute abundance using quantitative real-time PCR

[0113] For measuring absolute copy numbers of Clostridia, Enterobacterales, or Anaerostipes 16S rRNA genes in the feces, fecal DNA was extracted using the DNeasy PowerSoil Pro kit (Qiagen) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR-Green (Applied Biosystems) and class-specific (for Clostridia) (Croswell, A., et al. Infect Immun 2009 77:2741-2753), Enterobacterales-spectfic primers (Resendiz-Nava, C.N., et al. Pathogens 2021 11) or genus-specific (for Anaerostipes) primers at a final concentration of 0.25 mM. To create Genus specific primers to target Anaerostipes spp., a custom script was written to gather all 16S genes from Anaerostipes isolates on the NCBI. Entrez direct was used to gather accessions from all Anaerostipes isolates on NCBI into a text file which was then parsed using a custom script, and NCBI datasets was then used to gather the genes associated with those accessions. A multiple sequence alignment was then created using clustal omega (Sievers, F., et al. Curr Protoc Bioinformatics. 201448:3.13.1-3.13.16). The alignment was then used to generate primers with the software DEGEPRIME (Hugerth, L.W., et al. Appl Environ Microbiol 2014 80:5116- 5123), with a degeneracy score of 56 and length of 24 as parameters. The forward and reverse primers were chosen from hypervariable region 7 of the 16S gene to avoid hybridization to other Lachnospiraceae bacteria. We downloaded all Clostridia genomes from the NCBI, made a local BLAST database, and verified in silico that the primers do not hybridize to other Clostridia. I n-sillico hybridization from DEGEPRIME showed 98.3 % and 100 % hybridization across Anaerostipes 16S rRNA genes for the forward and reverse primers, respectively. The amplicon generated by the primers has a length of 221 base pairs.

[0114] To generate plasmid standards, PCR was performed using a thermocycler with class-specific (for Clostridia) or genus-specific (for Anaerostipes) primers (Key Resources Table). The PCR was confirmed by gel electrophoresis, and the PCR product was then purified using a DNA clean-up kit (Zymo Research) according to the standard kit protocol with no modifications. Following purification, the PCR product was then ligated into a linear TOPO-TA cloning vector, using a TOPO-TA cloning kit (Invitrogen). After ligation, the construct was transformed into chemically competent Escherichia coli TOP10 cells provided with the kit and cultured for approximately 1 hour in LB broth at 37°C. The cells were then plated on LB Agar plates with X-Gal (5-Bromo-4-Chloro-3-lndolyl [3-D- Galactopyranoside) and Kanamycin to select colonies which kept the plasmid. A miniprepusing a Qiaprep spin mini-prep kit was performed on a pure liquid culture from a single colony isolate to extract plasmid DNA. The plasmid DNA product was then linearized using the restriction enzyme BamHI (New England Biolab) according to manufacturer protocol. The linear plasmid DNA was quantified using a nanodrop and diluted to create a stock standard with 1010copies of the 16S rRNA amplicon insert for use in qRT-PCR.

[0115] To calculate absolute copy numbers, a standard curve ranging from 101to 1010copies / mL of plasmid carrying a cloned 16S rRNA gene diluted in a 0.02 mg / mL of yeast RNA (Sigma Aldrich) was generated.In vitro measurement of sorbitol in E. coli culture supernatants

[0116] The in vitro utilization of sorbitol by E. coli strains was assessed using M9 minimal medium (12.8 g / L Na2HPO47H2O, 3 g / L KH2PO4, 0.5 g / L NaCI, and 1 g / L NH4CI), supplemented with 1 mM MgSO4, 0.1 mM CaCI2, 0.1% casamino acids, and 0.5% (w / v) D- sorbitol. 20 pL of an overnight culture of each E. coli strain containing 1O10CFU / mL were used to inoculate into 2 mL of M9 minimal medium containing D-sorbitol. The cultures were then incubated at 37°C overnight. Sorbitol concentrations were quantified in the cell culture supernatant using a D-sorbitol colorimetric assay kit.In vitro growth assay of Clostridia strains

[0117] To determine the ability of A. caccae and E. asparagiforme to utilize sorbitol as a carbon source in vitro, no carbon defined media (NCDM), which is a modified form of non-carbon minimal media (Theriot, C.M., et al. Nat Commun 2014 5:3114) supplemented with 5 % (v / v) ATCC Vitamin Supplement (ATCC), 2 % (v / v) ATCC Trace Mineral Supplement (ATCC), Bacto Casamino acids (4.575 g / L) (ThermoFisher), cysteine (400 mg / L) (Sigma-Aldrich), methionine (27 mg / L) (Sigma-Aldrich), alanine (72 mg / L) (Sigma- Aldrich), tryptophan (30 mg / L) (Sigma-Aldrich) and Vitamin K2 (72 mg / L) (Sigma-Aldrich) was used. Glucose or D-sorbitol was used as a carbon source at a final concentration of 0.5% (w / v). Individual strains were grown in the anaerobic chamber in 2 mL of NCDM containing glucose for 24 hours. Then, the culture was spun down at 12,500 rpm for 3 minutes and the pellets were resuspended in 750 pL of reduced sterile PBS. 25 pL of resuspended pellet was inoculated into 2mL of NCDM containing glucose, D-sorbitol, or no added carbohydrates and incubated at 37°C for 72 hours. The growth of bacteria was determined by serially diluting the cultures and plating on plates of EG.Hypoxia staining and imaging

[0118] Mice were injected intraperitoneally with 100 mg / kg of pimonidazole HCI (Hypoxyprobe) in PBS sixty minutes before necropsy. The staining procedure with the Hypoxyprobe kit was conducted as detailed in prior studies (Lee, J.Y., et al. Cell Host Microbe 2020 28:273-284). Paraffin-embedded tissues were mounted on slides and prepared for staining: they were treated with xylene twice for 10 minutes each and then withethanol for 3 minutes in sequences of 95 %, 80 %, and 70 %. Samples were treated with 20 mg / mL Proteinase K in TE buffer at 37°C for 15 minutes. Nonspecific binding sites were blocked using serum at room temperature for an hour, followed by an overnight stain at 4°C using the mouse lgG1 anti-PMDZ monoclonal antibody 4.3.11 .3 (Hypoxyprobe). The slides were then subjected to a 90-minute stain at room temperature with Cyanine3-labeled goat anti-mouse IgG (Jackson ImmunoResearch). Between each staining phase, slides were washed thrice in PBS for 5 minutes each. After the final wash, slides were briefly air-dried and mounted using Shandon Immu-Mount (Thermo Scientific). For imaging, the image numbers were randomized and blinded. Three representative images from each sample were captured using a Carl Zeiss AxioVision microscope equipped with AxioVision 4.8.1 software (Zeiss) at 20X for scoring and 63X for detailed imagery. Using Imaged (NIH), the Texas Red channel (linked to Cyanine3) was singled out. From each image, three representative slices, each of the same size and containing the epithelial-lumen border, were saved. The Plot Profile for each slice was then determined. After unblinding, PMDZ peaks from each image were aligned, and the profiles for the 9 slices associated with each mouse were averaged, producing an average PMDZ Plot Profile and PMDZ Peak for every mouse.Butyrate analysis

[0119] Approximately 100mg of cecal contents per mouse were collected in 200 pL PBS. Samples were vortexed to disrupt particulate matter and then centrifuged at 6,000 g for 10 min to pellet any remaining debris. For each sample, 100 pL of supernatant was combined with 10 pL of a solution containing deuterated acetate, propionate, and butyrate so that each deuterated metabolite was at a final concentration of 100 pM. Samples were dried without heat in a vacuum dryer and then stored at -80°C until use. Dried extracts were then solubilized by sonication in 0.1 ml anhydrous pyridine and then incubated for 20 min at 80°C. An equal amount of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert- butyldimethylchlorosilate (Sigma-Aldrich) was added, and the samples were incubated for 1 h at 80°C. Samples were centrifuged at 20,000 g for 1 min to remove leftover particles. One hundred microliters of the supernatant were transferred to an autosampler vial and analyzed by gas chromatography-mass spectrometry (Agilent 8890 Gas Chromatograph and Agilent 7000D Mass spectrometer). 1 pL of the sample was injected with a 1 :50 split ratio at an injection temperature of 250°C on an HP 5ms Ultra Inert (2x15-m-length, 0.25-mm diameter, 0.25 pm film thickness) fused silica capillary column. Helium was used as the carrier gas with a constant flow of 1 .2 mL / min. The gas chromatograph (GC) oven temperature started at 50°C for 20 min, rising to 90°C at 10°C / min and holding for 1 min, then raised to 310°C at 40°C / min with a final hold for 2 min. The interface was heated to 300°C. The ion source was used in electron ionization (El) mode (70 V, 150 pA, 200°C). The dwell time for selected ion monitoring (SIM) events was 50 ms. Both acetate, propionate and butyrate were quantifiedusing SIM. Efficient recovery of target metabolites was determined using deuterated compounds as internal standards. Quantification was based on external standards comprised of a series of dilutions of pure compounds, derivatized as described above at the same time as the samples.Quantification and Statistical Analysis

[0120] Ratio’s (fold increases or percentages) were transformed logarithmically before analysis to normalize the data. Bacterial numbers were transformed logarithmically before analysis. An unpaired Student’s t test (for comparing two groups) or one-way ANOVA followed by Tukey’s multiple-comparison test (for comparing more than two groups) were used to determine the differences among groups and two-way ANOVA followed by Tukey’s multiple-comparison test were performed to determine the differences among the groups and period. To compare peak pimonidazole staining intensities, a Kruskal-Wallis test was performed. We performed all statistical analyses using the GraphPad Prism 8.0 Software. Statistical significance was defined as P < 0.05.

[0121] Microbiota profiling and metagenomic analysis: For linear discriminant analysis, data transformed using the DESeq2 (Love, M.I., et al. Genome Biol 2014 15:550) median of ratios method to account for differences in reads between samples, parsed, written to a tab separated text file, and then uploaded to the LEfSe (Segata, N., et al. Genome Biol. 2011 12(6):R60) galaxy server where the default statistical parameters were used in the analysis to generate LDA scores and the LDA cladogram.

[0122] Differential gene abundances were calculated using DESeq2 on reads normalized by the median of ratios method. A paired-sample wald test with a parametric fit was performed to obtain P values, which were then corrected to account for the false discovery rate using the Benjamini-Hochberg procedure. A significance threshold of 0.05 on the FDR corrected P values was used to determine significance. Confidence intervals were calculated using the following formula in R log2(fold-change) + qnorm(0.025)*lfcSE and Iog2(fold-change) - qnorm(0.025)*lfcSE, for upper and lower bounds respectively, where Iog2(fold-change) is the log base 2 of the fold change between groups, qnorm is a function to compute probabilities from known bounding values, and IfcSE is the log fold change standard error calculated when performing the DESeq2 model. Volcano plots, dot plots, and confidence interval of the Iog2(fold-change) plots were generated using the R package ggplot2.

[0123] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0124] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A method for treating gastrointestinal symptoms (e.g. diarrhea and abdominal pain) in an irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD) in a subject, comprising administering to the subject a composition comprising an effective amount of a sorbitol dehydrogenase enzyme and a composition comprising an effective amount of an aminosalicylate.

2. The method of claim 1 , wherein the aminosalicylate is 5-aminosalicylic acid (5-ASA) or a prodrug or derivative thereof.

3. The method of claim 2, wherein the 5-ASA derivative or prodrug is mesalamine, mesalazine, olsalazine, sulfasalazine, or balsalazide.

4. The method of claim 1 , wherein the sorbitol dehydrogenase enzyme and aminosalicylate are in the same composition.

5. The method of claim 1 , further comprising one or more additional sugar dehydrogenases.

6. The method of claim 5, wherein the one or more additional sugar dehydrogenases are selected from the group consisting of glucose dehydrogenase, lactate dehydrogenase, fructose dehydrogenase, and malate dehydrogenase.

7. The method of claim 1 , wherein the IBD is ulcerative colitis.

8. The method of claim 1 , wherein the IBD is Chron’s disease.

9. A composition comprising a sorbitol dehydrogenase enzyme and aminosalicylate in a pharmaceutically acceptable carrier.

10. The composition of claim 9, wherein the aminosalicylate is 5-aminosalicylic acid (5- ASA) or a prodrug or derivative thereof.11 . The composition of claim 10, wherein the 5-ASA derivative or prodrug is mesalamine, mesalazine, olsalazine, sulfasalazine, or balsalazide.

12. The composition of claim 9, wherein the sorbitol dehydrogenase enzyme and aminosalicylate are in the same composition.

13. The composition of claim 9, further comprising one or more additional sugar dehydrogenases.

14. The composition of claim 13, wherein the one or more additional sugar dehydrogenases are selected from the group consisting of lactase, lactate dehydrogenase, fructose dehydrogenase, and malate dehydrogenase.

15. The composition of claim 9, formulated as a pill, capsule, or tablet for oral administration.

Images