Methods and probiotic compositions for treating metabolic diseases and disorders

Abstract

Methods and probiotic compositions for treating metabolic diseases or disorders, such as, for example, obesity, type 2 diabetes, or fatty liver, are disclosed. In some embodiments, heat-killed Parabacteroides goldsteinii is administered enterally to a subject (e.g., a human patient) to treat a metabolic disease or disorder, or to promote the development of a warm microbiota to treat a metabolic disease or disorder. In some aspects, spermine or spermidine can be administered to a subject or used in vitro to promote the growth of a microbial flora that can be used to treat a metabolic disease or disorder.

Description

[0001] Priority Information

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 902,076, filed September 18, 2019, and U.S. Provisional Patent Application No. 63 / 069,458, filed August 24, 2020, the entire contents of which are incorporated herein by reference. BACKGROUND 1. TECHNICAL FIELD

[0004] The present invention relates generally to the fields of biology and medicine. More particularly, it concerns methods and compositions for treating diseases, such as metabolic diseases and disorders.

[0005] 2. DESCRIPTION OF RELATED ART

[0006] Obesity and type 2 diabetes remain serious clinical challenges. Obesity is a relatively common medical condition that can lead to very serious adverse health outcomes (e.g., heart disease, diabetes, hypertension, and certain cancers). Type 2 diabetes is characterized by resistance to insulin, and the disease can lead to several very serious health outcomes, including, for example, neuropathy, skin problems, and / or kidney damage. Postmenopausal weight gain can also lead to health problems in many individuals. The rates of obesity and type 2 diabetes are relatively high. For example, in the United States alone, the number of people with obesity or type 2 diabetes currently exceeds 70 million and exceeds 30 million, respectively.

[0007] The intestinal tract of healthy mammalian subjects harbors a microbiota that can affect human health. In some cases, the intestinal microbiome can play a role in the development of obesity, but the mechanisms by which this can occur are complex and not well understood (e.g., see Davis et al., 2016). For example, Lactobacillus reuteri has been observed to be elevated in individuals with obesity (Million et al., 2012). Clearly, new methods for treating obesity and type 2 diabetes are needed. SUMMARY

[0008] The present disclosure is based, in part, on the discovery that specific bacteria, such as Lactobacillus gasseri or Lactobacillus reuteri, and / or heat-killed Parabacteroides goldsteinii can be used to treat obesity, reduce fatty liver, and increase insulin sensitivity. In some embodiments, pharmaceutical or probiotic compositions comprising inactivated or heat-killed Parabacteroides goldsteinii are provided and can be administered to a mammalian subject for the treatment of obesity or metabolic diseases, such as type 2 diabetes or fatty liver disease. In some aspects, it has been observed that culturing or expanding a microbial consortium in polyamines, such as spermine or spermidine, can be used to treat diseases, such as metabolic diseases or disorders.

[0009] For example, as shown in the examples below, and contrary to the view that only live microbial consortium bacteria, such as Parabacteroides goldsteinii, can affect the health of a mammalian subject, Parabacteroides goldsteinii was killed with heat (heat-killed) and the resulting composition was orally administered to ovariectomized mice, which resulted in improved glucose tolerance and metabolism, increased insulin sensitivity, and reduced fatty liver in vivo. The warm exposed animals also showed improved glucose tolerance and increased insulin secretion after an oral glucose load. These results indicate that heat-killed Parabacteroides goldsteinii can be used to treat obesity or metabolic diseases, such as type 2 diabetes or fatty liver disease, in vivo in a mammalian subject. In vivo, enteral administration of Lactobacillus gasseri or Lactobacillus reuteri also improved glucose tolerance. Some of the beneficial effects of heat-killed Parabacteroides goldsteinii on blood glucose levels, oral glucose tolerance, and insulin sensitivity were observed to be more significant than those produced by Akkermansia muciniphila.

[0010] One aspect of the application relates to a method for treating a metabolic disease or disorder in a mammalian subject, comprising administering to the gastrointestinal system of the subject a composition, wherein the composition comprises inactivated P. gordonii, growth medium of P. gordonii, or vesicles from P. gordonii. In some embodiments, the inactivated P. gordonii is heat-inactivated. In some embodiments, the inactivated P. gordonii has been inactivated by exposure to a peroxide (e.g., hydrogen peroxide or hydrogen peroxide vapor). In some embodiments, the inactivated P. gordonii has been inactivated by exposure to radiation or ionizing radiation (e.g., comprising or consisting of light having a wavelength of about 400 nm to about 420 nm, more preferably about 400 nm to 410 nm, or about 405 nm). In some embodiments, the inactivated P. gordonii has been inactivated by exposure to air plasma, ultrasound under pressure, exposure to an alcohol (e.g., ethanol, isopropanol, etc., e.g., at a concentration of about 40% to 100%, more preferably about 60% to 99%, 70% to 85%, or 65%, 70%, 75%, or any range therein, in solution), high hydrostatic pressure (HHP), or pulsed electric field (PEF). The composition can comprise extracellular vesicles from P. gordonii. The composition can comprise about 1 x 10 8 cfu to about 1 x 10 13inactivated Parabacteroides goldsteinii. In some embodiments, the composition further comprises Lactobacillus gasseri, Lactobacillus reuteri, and / or Akkermansia muciniphila. In some embodiments, the composition is further defined as a pharmaceutical composition. In some embodiments, the composition is further defined as a probiotic composition. In some embodiments, the composition further comprises Lactobacillus gasseri or Lactobacillus reuteri. In some embodiments, the composition further comprises extracellular vesicles from Lactobacillus gasseri or Lactobacillus reuteri. In some embodiments, the pharmaceutical composition or probiotic composition is administered orally, colonically, by enema, by orogastric tube, or by nasogastric tube. In some embodiments, the inactivated Parabacteroides goldsteinii or vesicles from Parabacteroides goldsteinii are included in a pharmaceutical composition or probiotic composition that is resistant to degradation in the stomach but releases the bacteria in the small and / or large intestine of the subject. The pharmaceutical composition or probiotic composition can comprise an enteric coating, chitosan-alginate beads, or a hydrogel. In some embodiments, the enteric coating is a fatty acid; a wax; a shellac; a plastic such as a phthalate, CAP, CAT, PVAP, HPMCP; or a plant fiber. In some embodiments, the pharmaceutical composition or probiotic composition does not comprise an enteric coating. In some embodiments, the pharmaceutical composition or probiotic composition is a tablet or a capsule. In some embodiments, the subject is a human (e.g., a postmenopausal woman). In some embodiments, the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease (e.g., nonalcoholic fatty liver disease (NAFLD)), insulin resistance, or dyslipidemia. In some embodiments, the microbiota in the composition has been purified or cultured. In some embodiments, the inactivated Parabacteroides goldsteinii has been inactivated by heating the bacteria, for example, to about 95°C to 105°C for about 10 minutes to 20 minutes, or to about 100°C for about 15 minutes.

[0011] The methods can also include administering spermine and / or spermidine enterally to the subject. In some embodiments, the methods include administering both spermine and spermidine enterally to the subject. The methods can include administering to the subject about 1 mg / kg body weight / day to 50 mg / kg body weight / day of spermine, or any range derivable therein (e.g., 1 to 25, 2.5 to 15, 5 to 10, 5 to 25 mg spermine / kg body weight / day, etc.). In some embodiments, the methods include administering to the subject about 1 mg / kg body weight / day to 50 mg / kg body weight / day of spermidine, or any range derivable therein (e.g., 1 to 25, 2.5 to 15, 5 to 10, 5 to 25 mg spermidine / kg body weight / day, etc.). The compositions can include spermine and / or spermidine. In some embodiments, the compositions include both spermine and spermidine. In some embodiments, the Parabacteroides goldsteinii is inactivated in a culture medium comprising spermidine or spermine; for example, the Parabacteroides goldsteinii can be cultured or expanded in a culture medium comprising spermidine and / or spermine prior to inactivation by a method described herein or above (e.g., heat inactivation, exposure to peroxide, etc.). In some embodiments, the culture medium comprises about 0.1 mM to 6 mM spermidine and / or about 0.1 mM to 6 mM spermine. In some embodiments, the subject is administered an antibiotic and exposed to an environment of about 25°C to 50°C, more preferably about 32°C to 35°C, for at least about 15 minutes.

[0012] Another aspect of the application relates to a pharmaceutical or probiotic composition comprising heat-inactivated Parabacteroides goldsteinii, a growth medium of Parabacteroides goldsteinii, or a vesicle from Parabacteroides goldsteinii; wherein the composition is formulated for delivery to the gastrointestinal system. In some embodiments, the composition comprises heat-inactivated Parabacteroides goldsteinii. The composition can also comprise Lactobacillus gasseri or Lactobacillus reuteri. The composition can also comprise an extracellular vesicle from Lactobacillus gasseri or an extracellular vesicle from Lactobacillus reuteri. In some embodiments, the pharmaceutical or probiotic composition is formulated for oral, colonic, enema, oro-gastric, or naso-gastric administration. In some embodiments, the pharmaceutical or probiotic composition is resistant to degradation in the stomach but releases the bacteria in the small and / or large intestine of a subject. The pharmaceutical or probiotic composition can comprise an enteric coating, a chitosan-alginate bead, or a hydrogel. In some embodiments, the enteric coating is a fatty acid; a wax; a shellac; a plastic such as a phthalate, CAP, CAT, PVAP, HPMCP; or a plant fiber. In some embodiments, the pharmaceutical or probiotic composition does not comprise an enteric coating. In some embodiments, the pharmaceutical or probiotic composition is a tablet or a capsule. The pharmaceutical or probiotic composition can also comprise spermine and / or spermidine. The pharmaceutical or probiotic composition can comprise about 1 mg / kg body weight / day to 50 mg / kg body weight / day of spermine, or any range derivable therein (e.g., 1 to 25, 2.5 to 15, 5 to 10, 5 to 25 mg spermine / kg body weight / day, etc.). The pharmaceutical or probiotic composition can comprise about 1 mg / kg body weight / day to 50 mg / kg body weight / day of spermidine, or any range derivable therein (e.g., 1 to 25, 2.5 to 15, 5 to 10, 5 to 25 mg spermine / kg body weight / day, etc.). These amounts can be tailored for humans (e.g., weighing about 45 kg to 136 kg). In some embodiments, the pharmaceutical or probiotic composition further comprises both spermine and spermidine. In some embodiments, the Parabacteroides goldsteinii has been inactivated by exposure to peroxide, ionizing radiation, heat, air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric field (PEF). In some embodiments, the Parabacteroides goldsteinii has been inactivated by exposure to peroxide, ionizing radiation, or heat. The composition can be used to treat a metabolic disease or disorder in a mammalian subject. The metabolic disease or disorder can be obesity, type 2 diabetes, fatty liver disease (e.g., nonalcoholic fatty liver disease (NAFLD)), insulin resistance, or dyslipidemia. In some embodiments, the subject is a human (e.g., a postmenopausal woman).

[0013] Another aspect of the application relates to a method of treating a metabolic disease or disorder in a mammalian subject, comprising applying heat to the torso of the subject. The method can comprise placing the subject in a climate chamber having an ambient temperature of about 65 °C to about 95 °C for about 3 minutes to 30 minutes, about 3 minutes to 15 minutes, about 3 minutes to 10 minutes, or about 3 minutes to 5 minutes. In some embodiments, the subject is repeatedly exposed to the climate chamber, with a period of time between each exposure. In some embodiments, a heating pad is applied to the torso, stomach, abdomen, head, leg, and / or foot of the subject, wherein the heating pad is about 27 °C to about 50 °C or wherein a heating lamp is applied to the torso, stomach, abdomen, head, leg, and / or foot of the subject, wherein the heating lamp is about 60 °C to about 95 °C. The heat can be applied for a period of about 30 minutes to about 9 hours. In some embodiments, the heat is applied at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days per week for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or more. In some embodiments, the heat is repeatedly applied to the torso of the subject. In some embodiments, the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease (e.g., nonalcoholic fatty liver disease (NAFLD)), insulin resistance, or dyslipidemia. The subject can be a human (e.g., a postmenopausal woman).

[0014] Yet another aspect of the application relates to a method of treating a metabolic disease or disorder in a mammalian subject, comprising enterally administering to the subject (i) a vesicle from Lactobacillus gasseri or Lactobacillus reuteri, or (ii) a growth-conditioned medium from Lactobacillus gasseri or Lactobacillus reuteri. The method can further comprise administering to the subject about 1 x 10 8 cfu to about 1 x 10 13 cfu of Lactobacillus gasseri or Lactobacillus reuteri. The method can further comprise enterally administering to the subject spermidine and / or spermine. In some embodiments, however, the vesicle or growth-conditioned medium is enterally administered to the subject without administering Lactobacillus gasseri or Lactobacillus reuteri to the subject. The subject can be a human, e.g., a postmenopausal woman. The disease can be obesity, type 2 diabetes, fatty liver disease, insulin resistance, or dyslipidemia.

[0015] Yet another aspect of the present disclosure relates to a method of treating a disease or disorder in a mammalian subject, comprising (i) expanding a microbial consortium in a culture medium comprising spermine or spermidine, and (ii) administering the microbial consortium to the subject enterally. In some embodiments, the spermine or spermidine is present in the culture medium at a concentration of about 0.1 mM to 10 mM. The spermine can be present in the culture medium at a concentration of about 1 mM to 6 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, or any range derivable therein (e.g., 1 mM to 3 mM, 2 mM to 3 mM, etc.). The spermidine can be present in the culture medium at a concentration of about 1 mM to 6 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM (or any range derivable therein). In some embodiments, the culture medium comprises both spermine and spermidine. The microbial consortium can comprise or consist of Parabacteroides gordinii, Lactobacillus reuteri, and / or Lactobacillus gasseri. The microbial consortium can comprise or consist of Parabacteroides gordinii. In some embodiments, the Parabacteroides gordinii is inactivated prior to administration to the subject. The Parabacteroides gordinii can be inactivated by exposure to peroxide, ionizing radiation, heat, air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric field (PEF). In some embodiments, the Parabacteroides gordinii is inactivated by exposure to peroxide, ionizing radiation, or heat. The peroxide can be hydrogen peroxide. In some embodiments, the Parabacteroides gordinii is inactivated by heating to about 95°C to 105°C for about 10 minutes to 20 minutes. In some embodiments, the mammalian subject is a human. In some embodiments, the disease is a metabolic disease or disorder (e.g., obesity, type 2 diabetes, fatty liver disease, insulin resistance, or dyslipidemia). In some embodiments, the disease is a bone disease or the method comprises increasing bone strength. The bone disease can be osteoporosis, osteomalacia, osteolysis, osteochondrodysplasias, periodontitis, rheumatoid arthritis, metabolic bone disease, parathyroid disorders, steroid-induced osteoporosis, chemotherapy-induced bone loss, premenopausal bone loss, fragility and recurrent fractures, renal osteodystrophy, or Paget's disease.

[0016] Lactobacillus gasseri is a bacterial species that has been identified as part of the vaginal flora and has been found in the lower digestive system of women. Particular Lactobacillus gasseri strains that can be used to treat metabolic diseases or disorders in a mammalian subject can include DSM 20077, DSM 107525, DSM 20243, DSM 20604, 3332、 2960、 BAA-2841, PTA4483, PTA4481, PTA4484, PTA4480 and / or PTA4479. Various amounts of L. gasseri can be administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder (e.g., obesity, type 2 diabetes, fatty liver, etc.) as described herein. For example, in some embodiments, about 1 x 10 8 cfu to about 1 x 10 13 cfu of L. gasseri can be administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder.

[0017] L. reuteri is a bacterial species that has been found in the intestines of healthy mammals. Particular L. reuteri strains that can be used to treat a metabolic disease or disorder in a mammalian subject include DSM 100191, DSM 100192, DSM 17509, DSM 20015, DSM 20016, DSM 20053, DSM 20056, DSM 28673, DSM 32035, BAA-2837 TM , 55148, 53608, 23272, 23272D5 and / or PTA6475. Various amounts of L. reuteri can be administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder (e.g., obesity, type 2 diabetes, fatty liver, etc.) as described herein. For example, in some embodiments, about 1 x 10 8 cfu to about 1 x 10 13 cfu of L. reuteri can be administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder.

[0018] P. haemolyticum is a gram-negative, obligate anaerobic, non-spore-forming, and non-motile bacterium that has been isolated from human blood. Particular P. haemolyticum strains that can be used to treat a metabolic disease or disorder in a mammalian subject include DSM 19448 and / or DSM 29187. Various amounts of P. haemolyticum can be administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder (e.g., obesity, type 2 diabetes, fatty liver, etc.) as described herein. For example, in some embodiments, about 1 x 10 8 cfu to about 1 x 10 13Inactivated P. gordonii cfu are administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder. As shown in the examples below, heat-inactivated P. gordonii can be administered to treat a metabolic disease or disorder. Heat-inactivation methods that can be used to prepare heat-inactivated P. gordonii are well known and include heating the bacteria to about 100°C for about 15 minutes (Wu et al., 2019). P. gordonii can also be preserved by freezing or by desiccation. Inactivated P. gordonii can be produced using a variety of methods. For example, in some embodiments, bacteria can be irradiated or killed by radiation, exposure to ethanol, or autoclaving (e.g., as described in Lin et al., 2015). In some embodiments, P. gordonii can be inactivated by exposure to light comprising or consisting of light having a wavelength of about 405 nm (e.g., Maclean et al., 2009). Inactivated P. gordonii can be produced by exposure to air plasma, e.g., direct current, cold atmospheric pressure air plasma microjet (e.g., Tian et al., 2010). Inactivated P. gordonii can be produced by exposure to hydrogen peroxide or hydrogen peroxide vapor (e.g., Malik et al., 2013; Erttmann et al., 2019; Grigoryan et al., UDC 579.67). Inactivated P. gordonii can be produced by exposure to ionizing radiation, ultrasound under pressure, high hydrostatic pressure (HHP), and / or pulsed electric field (PEF) (e.g., Manas, et al., 2005). In some preferred embodiments, heat-inactivation is utilized to kill P. gordonii.

[0019] As used herein in the specification, a noun clause that does not specify a quantity is intended to include the singular noun or more than one of the noun. As used herein in the claims, the use of the term “or” means “and / or” unless specifically stated otherwise, but the disclosure

[0020] The use of the term “or” in the claims is used to mean “and / or” unless specifically stated otherwise, but the disclosure supports a limitation that the disjunctive word means either the conjunctive or the disjunctive sense. As used herein “additional” can mean at least a second or more.

[0021] Throughout this application the term “about” is used to mean approxi mately, as in an acceptable degree of error, with the term “substantially” useful about the meaning as “entirely or virtually” or “for all practical purposes” as in having no or virtually no relevance or impact.

[0022] The terms "comprise", "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises", "comprising", "included", "have", "has" and "having", are also open-ended. For example, any method that "comprises", "has" or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

[0023] Other objects, features, and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to one of ordinary skill in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present application. The application can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] Figures 1A-1B : Warm exposure improves tolerance to orally administered glucose. Figure 1A Oral glucose tolerance test (OGTT) in 8-week-old male mice exposed to 34°C for 1 month. Figure 1B Associated insulin release at time points 0 min, 7 min, and 15 min.

[0026] Figures 2A-2B : Warm exposure prevents ovariectomy-induced oral glucose intolerance. Figure 2A Oral glucose tolerance test in 16-week-old ovariectomized or sham-operated female mice. Figure 2B Oral glucose tolerance test in ovariectomized mice kept at room temperature or 34°C for 2 months.

[0027] Figures 3A-3C : Heat-inactivated Parabacteroides goldacus prevents ovariectomy-induced hyperglycemia and improves glucose tolerance and insulin sensitivity. Figure 3A Blood glucose levels in mice supplemented with heat-inactivated Parabacteroides goldacus (OVA Gold) after 12 hours or 6 hours. Figure 3B Oral glucose tolerance test (left) and area under the curve (right) in ovariectomized mice supplemented with heat-inactivated Parabacteroides goldacus or live Akkermansia muciniphila kept at room temperature. Figure 3C : Heat-inactivated Parabacteroides goldacus prevents ovariectomy-induced hyperglycemia and improves glucose tolerance and insulin sensitivity. Figures 3A-3BThe insulin tolerance test was performed on mice after they were injected with 0.5 U / kg insulin and their blood glucose was monitored for more than 2 hours.

[0028] Figure 4 Heat-inactivated *Pseudomonas gondii* prevented ovariectomy-induced hepatic steatosis. Liver lipid levels in ovariectomized mice supplemented with heat-inactivated *Pseudomonas gondii* at room temperature were measured.

[0029] Figure 5 Supplementation with heat-inactivated *P. glehnia littoralis* prevented ovariectomy-induced loss of endogenous *P. glehnia littoralis*. Fecal *P. glehnia littoralis* from ovariectomized mice kept at room temperature 48 hours after the last administration.

[0030] Figures 6A-6C :( Figure 6A Oral glucose tolerance test after 3 weeks of treatment. The inset on the left shows the area under the curve (AU, top) and the initial fasting blood glucose (bottom). Figure 6B Adipose tissue weight at sacrifice in C57bl / 6J mice 4 weeks after treatment (epiVAT or rpVAT: epididymal or retroperitoneal visceral adipose tissue; ingSAT: inguinal subcutaneous adipose tissue; rpVAT: retroperitoneal visceral adipose tissue; iBAT: interscapular brown adipose tissue). Deposition of [3H]-2DG glucose analogue in adipose tissue following intraperitoneal injection. Bars show mean ± SD (n = 6 to 8 per group). Statistical analysis was performed using an unpaired two-tailed Student's t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0031] Figures 7A-7B: (Figure 7A) Representative H&E staining of visceral adipose tissue from C57B1 / 6 mice after 30 days of treatment as shown. Black arrows indicate multilocular adipocytes. (Figure 7B) Oxygen consumption rate measured under basal conditions using a Seahorse analyzer after 30 days of treatment. Significance was calculated using an unpaired two-tailed Student t-test. ***P ≤ 0.001.

[0032] Figure 8A to 8E: (Figure 8A) Oral glucose tolerance test in Ucpl-KO mice after 3 weeks of treatment. (Figure 8B) Representative H&E staining of visceral adipose tissue in Ucpl-KO mice after 30 days of treatment as indicated. Black arrows indicate multilocular adipocytes. (Figure 8C to 8E) Relative gene expression of thermogenic genes in rpVAT and epiVAT of WT (Figure 8C and 8D) or epiVAT of Ucpl-KO (Figure 8E) mice, normalized to Tbp. Significance in A, C and D was calculated using unpaired two-tailed Student t test. ***P < 0.001.

[0033] Figure 9 Figure 7: Liver lipid weight of ovariectomized mice at sacrifice after oral supplementation with HI Parabacteroides distasonii or Lactobacillus reuteri. Significance was calculated based on one-way ANOVA; *P < 0.05.

[0034] Figure 10 Figure 6: Body weight of mice fed a high caloric diet after oral supplementation with HI Parabacteroides distasonii. Data show the change in body weight after the start of bacterial gavage (left), or the area under the curve from the left graph (right). Significance was calculated based on one-way ANOVA; ****P < 0.0001.

[0035] Figures 11A-11B Figure 5: Weight of subcutaneous adipose tissue (A) or liver (B) in mice fed a high caloric diet for three months followed by oral supplementation with HI Parabacteroides distasonii for 4 weeks. *P < 0.05, ***P < 0.001. Figure 11A Figure 11B Figure 4: Spermidine or spermine supplementation promotes the growth of beneficial bacteria in vitro.

[0036] Figure 12 Figure 3: Spermidine or spermine supplementation improves the growth of bacterial mixtures from mouse fecal samples in vitro.

[0037] Figure 13 Figure 2: Transplanting the polyamine-adapted microbiota to ovariectomized old female mice (blue) reduces body weight gain by limiting fat weight and liver tissue weight.

[0038] DETAILED DESCRIPTION Figure 14 I. DEFINITIONS

[0039] I. DEFINITIONS

[0040] ​A "bacterial composition" is a composition comprising one or more types of bacteria (e.g., live, dried, or heat-inactivated) or extracellular vesicles from bacteria (i.e., secreted extracellular vesicles) from bacteria. In some embodiments, the bacteria are from the Clostridiaceae, Lactobacillaceae, and / or Porphyromonadaceae families. Particular bacteria contemplated include Lactobacillus gasseri, Lactobacillus reuteri, and Parabacteroides goldsteinii (e.g., live or heat-inactivated P. goldsteinii). In some preferred embodiments, heat-, freeze-, or dry-inactivated P. goldsteinii is used.

[0041] The term "effective" as that term is used in the specification and / or claims means adequate to accomplish a desired, intended, or desired result. When used in the context of treating a patient or subject with a bacterial composition, "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means an amount of the bacterial composition that is sufficient, when administered to a subject or patient, to effect such treatment or prevention of a disease when administered to a subject or patient to treat or prevent a disease.

[0042] An "excipient" is a pharmaceutically acceptable substance formulated with the active ingredient of a drug, pharmaceutical composition, preparation, or drug delivery system. Excipients can be used, for example, to stabilize the composition, to bulk up the composition (hence, when used for this purpose, often referred to as a "bulking agent," "filler," or "diluent"), or to impart therapeutic enhancement to the active ingredient in the final dosage form, such as to facilitate drug absorption, reduce viscosity, or enhance solubility. Excipients include pharmaceutically acceptable forms of antiadherents, binders, coatings, colorants, disintegrants, flavorants, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. The primary excipient used as a medium for delivery of the active ingredient is often referred to as a vehicle. Excipients can also be used in the manufacturing process, for example, to facilitate handling of the active substance, for example, by facilitating powder flow or non-sticky properties, in addition to contributing to in vitro stability (e.g., preventing denaturation and aggregation over the intended shelf life). The suitability of an excipient will generally vary according to the route of administration, the dosage form, the active ingredient, and other factors.

[0043] The term "patient" or "subject" as used herein refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic non-human species thereof. In certain embodiments, the patient or subject is a primate. Some non-limiting examples of human patients are adults, teenagers, and infants.

[0044] A "pharmaceutically acceptable carrier," "drug carrier," or simply "carrier" is a pharmaceutically acceptable material that is formulated with an active ingredient and that participates in the carriage, delivery, and / or transport of a biopharmaceutical agent. Carriers can be used to improve the delivery and effectiveness of an active ingredient, including, for example, controlled release technology to modulate drug bioavailability, reduce drug metabolism, and / or reduce drug toxicity. Some carriers can improve the effectiveness of delivery of an active ingredient to a particular target site. Some examples of carriers include: liposomes, microspheres (e.g., made of or comprising poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, red blood cells, virosomes, hydrogels, starches, and dendrimers. In some embodiments, the carrier comprises an enteric coating (e.g., a fatty acid; a wax; a shellac; a plastic such as a phthalate, CAP, CAT, PVAP, HPMCP; or a plant fiber) to reduce or slow degradation in the stomach, chitosan-alginate beads, or hydrogels.

[0045] "Preventing" or "prevention" includes: (1) inhibiting the onset of a disease in a subject or patient that can be at risk for the disease and / or predisposed to the disease but has not yet experienced or displayed any or all of the pathology or symptoms of the disease, and / or (2) slowing the onset of the pathology or symptoms of a disease in a subject or patient that can be at risk for the disease and / or predisposed to the disease but has not yet experienced or displayed any or all of the pathology or symptoms of the disease.

[0046] "Treatment" includes: (1) inhibiting the disease (e.g., arresting further development of the pathology and / or symptoms), (2) relieving the disease (e.g., reversing the pathology and / or symptoms), and / or (3) producing a measurable reduction in the disease or its symptoms in a subject or patient that is experiencing or displaying the pathology or symptoms of the disease.

[0047] II. Mesophilic Microbial Flora

[0048] As shown in the examples below, a variety of bacteria have been observed in mesophilic microbiota or microbiota obtained from mammalian subjects living in warmer environments. Mesophilic microbiota include Clostridiale ace-assimilate spp., Lactobacillus spp. (e.g., Lactobacillus gasseri or Lactobacillus reuteri), Bifidobacteriaceae spp. (e.g., Bifidobacterium longum), Parabacteroides spp. (e.g., Parabacteroides goldsteinii), and Akkermansia spp. (e.g., Akkermansia muciniphila). In some embodiments, it is contemplated that the pharmaceutical or probiotic compositions disclosed herein can include bacteria described in any one of Tables 1-5. In some embodiments, the pharmaceutical or probiotic compositions can include Lactobacillus reuteri, Lactobacillus acidophilus, and / or Lactobacillus rhamnosus. As shown in the examples, therapeutic responses can also be observed when using heat-killed Parabacteroides goldsteinii.

[0049] In various embodiments, it is contemplated that one, two, three, four, five, six or more of the following types of bacteria can be included in the pharmaceutical or probiotic compositions disclosed herein. For example, one, two, three, four, five, six or more of the following can be included in the pharmaceutical or probiotic compositions disclosed herein and / or administered to a mammalian subject (e.g., a human patient) to treat a metabolic disease or disorder: Clostridialeace-assimilatespp., Lactobacillus spp. (e.g., Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus acidophilus, and / or Lactobacillus rhamnosus), Bifidobacterium spp. (e.g., Bifidobacterium longum), Parabacteroides spp. (e.g., Parabacteroides gottfriedii), and Akkermansia spp. (e.g., Akkermansia muciniphila). Various interactions between the gut microbiota and physiology can be used in connection with the present disclosure (e.g., as described in Ohlsson and Sjogren, 2015). Lactobacillus spp. such as Lactobacillus reuteri (Britton et al., 2014; also recently described in humans in Nilsson et al.), Lactobacillus acidophilus (Dar et al., 2018), and / or Lactobacillus rhamnosus (Li et al., 2016) can be included in the compositions to treat a metabolic disease or disorder. In other embodiments, heat (e.g., from a heating chamber, heating pad, or heating lamp) can be applied to the subject (e.g., to the entire body or to a particular region, such as the torso, stomach, limbs, and / or abdomen) to treat a metabolic disease or disorder described herein, such as obesity, type 2 diabetes, or fatty liver. In some embodiments, the applied heat can promote the growth of a mesophilic microbiota.

[0050] III. Inactivated Parabacteroides gottfriedii

[0051] Inactivated Parabacteroides gottfriedii can be produced by various methods. In some embodiments, inactivated Parabacteroides gottfriedii is inactivated by exposure to heat. Nonetheless, it is contemplated that additional methods of inactivation can be used to produce inactivated Parabacteroides gottfriedii that can perform similar functions and can be used to treat metabolic diseases or disorders as described herein (e.g., obesity, diabetes, etc.). For example, inactivated Parabacteroides gottfriedii can be produced by exposing the bacteria to light (e.g., radiation or ionizing radiation), air plasma, pressure (e.g., ultrasound under pressure, high hydrostatic pressure), peroxides (e.g., hydrogen peroxide), alcohols (e.g., ethanol), exposure to low temperatures or freezing, dehydration, lyophilization, or pulsed electric fields (PEF); in some embodiments, one or more of the foregoing methods for inactivating bacteria can be used in combination with the application of heat to produce inactivated Parabacteroides gottfriedii that can be used, for example, to treat metabolic diseases or disorders as described herein. In some embodiments, the compositions as described herein can include both live and inactivated Parabacteroides gottfriedii.

[0052] Various methods can be used to produce heat-inactivated P. gouldii. For example, the bacteria can be heated to at least 95°C, at least 100°C, or about 100°C for at least 10 minutes, 10 to 20 minutes, or about 15 minutes, e.g., as described in Wu et al. (2019). It is contemplated that autoclaving or heating in solution (e.g., boiling in water) can also be used. Generally, heat can inactivate the bacteria by one or more of the following: membrane damage, loss of nutrients and ions, ribosome aggregation, DNA strand breakage, inactivation of essential enzymes, and protein coagulation. Additional inactivation methods that can be used with the present application are described in, e.g., Lin et al. (2015). After producing inactivated (e.g., heat-inactivated) P. gouldii, the bacteria can be subsequently dried, frozen, or lyophilized, if desired. In some preferred embodiments, heat inactivation is utilized to kill P. gouldii.

[0053] Inactivated P. gouldii can also be produced based on exposure to radiation (e.g., ionizing radiation). In some embodiments, the bacteria can be inactivated by exposure to light comprising or consisting of light having a wavelength of about 405 nm. For example, a light-emitting diode (LED) array producing light having a wavelength of about 405 nm can be used to inactivate the bacteria (e.g., Maclean et al., 2009). In some embodiments, the radiation can be ultraviolet (UV) radiation having a wavelength of about 240 nm to about 280 nm. In some embodiments, the radiation is ionizing radiation, e.g., X-rays.

[0054] Inactivated P. gouldii can be produced by exposure to a peroxide, e.g., as hydrogen peroxide. The hydrogen peroxide can be contacted with the bacteria in solution. In some embodiments, the bacteria are contacted with hydrogen peroxide vapor to inactivate the bacteria. For example, the bacteria can be exposed to about 10 mg / m 3 (ppm) to 100 mg / m 3 (ppm) of hydrogen peroxide vapor for about 1.5 hours to 48 hours (e.g., Malik et al., 2013). Hydrogen peroxide can be applied as a liquid or as a vapor to inactivate the bacteria. In some cases, the mode of action of hydrogen peroxide in vapor form can result in enhanced oxidation of a range of biological macromolecules compared to aqueous hydrogen peroxide solutions (Finnegan et al., 2010). Similar to heat inactivation, using hydrogen peroxide to inactivate the bacteria has the advantage that it breaks down into non-toxic byproducts after reacting with the bacteria. In some embodiments, it is contemplated that a 1% to 2% hydrogen peroxide solution can be contacted with P. gouldii for about 5 minutes to 10 minutes to inactivate the bacteria. In some embodiments, a combination of heat and hydrogen peroxide can be used to inactivate P. gouldii.

[0055] Various other methods for inactivating P. goshidaii can also be used. For example, bacteria can be inactivated by contacting the bacteria with a specific concentration of an alcohol (e.g., ethanol, methanol, propanol, or isopropanol) (e.g., at least 70% v / v alcohol, such as 70% ethanol) to inactivate the bacteria.

[0056] Inactivation of P. goshidaii can also be produced by exposure to air plasma, such as direct current, cold atmospheric pressure air plasma microjet (e.g., Tian et al., 2010); for example, after about 10 minutes of plasma treatment, a decrease in pH can be observed due to NO x A decrease in pH can be observed as a result of the reaction with water at the gas-liquid interface.

[0057] Inactivation of P. goshidaii can be produced by exposure to ionizing radiation (e.g., gamma rays produced by cobalt 60, electron beams, or X-rays). Typically, the dose of ionizing radiation applied to the bacteria is preferably sufficient to damage the DNA of the bacteria and / or to prevent further growth of the bacteria.

[0058] Ultrasonic or high hydrostatic pressure (HHP) under pressure can also be used to inactivate the bacteria. In some embodiments, a pressure of about 100 MPa to 1000 MPa is applied to inactivate the bacteria. The effectiveness of HHP has been demonstrated in the field of food sterilization. Ultrasound is defined as sound waves with a frequency above the human threshold of hearing (> 16 kHz). In some embodiments, ultrasonic application can be used in combination with external hydrostatic pressure (e.g., pressure manosonication (MS) up to 600 kPa) and / or heat application to inactivate P. goshidaii.

[0059] Pulsed electric fields (PEF) can also be used (e.g., Manas et al., 2005). PEF methods generally involve the application of high electric field pulses (10 kV em -1 to 50 kV cm -1 ) for short durations (e.g., 1 μβ to 100 μβ) to sample between two electrodes. In various embodiments, a combination of one or more of the foregoing methods is contemplated to be used to produce inactivated P. goshidaii.

[0060] IV. Spermine and Spermidine

[0061] In some aspects, agmatine and / or spermidine can be administered enterally to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder (e.g., diabetes, obesity, etc.), or a bone disease or disorder, as described herein. For example, the bone disease or disorder can be, e.g., osteoporosis, osteomalacia, osteolysis, osteochondrodysplasia, periodontitis, rheumatoid arthritis, metabolic bone disease, parathyroid disorder, steroid-induced osteoporosis, chemotherapy-induced bone loss, premenopausal bone loss, fragility and recurrent bone fractures, renal osteodystrophy, or Paget's disease. In some embodiments, spermidine and / or spermidine can be administered in combination with an inactivated Parabacteroides goldsteinii (e.g., heat-inactivated Parabacteroides goldsteinii), as described herein. In some embodiments, agmatine or spermidine can be administered in combination with another live or inactivated microflora (e.g., Lactobacillus reuteri (L. Reuteri), Lactobacillus gasseri (L. Gasseri), and / or Akkermansia muciniphila (A. Muciniphila)), as described herein, optionally in combination with live or inactivated Parabacteroides goldsteinii. As shown in the Examples below, enteral administration of microflora cultured in spermidine or agmatine reduced obesity in animal models in vivo, and a reduction in total fat was observed (including a reduction in subcutaneous adipose tissue, visceral adipose tissue, and brown adipose tissue).

[0062] Spermidine (N-(3-(aminopropyl)-1,4-butanediamine) and agmatine (N,N'-bis(3- aminopropyl)-1,4-butanediamine) are naturally occurring polyamines and can act as modulators of a variety of cellular processes, including DNA stabilization, transcription, translation, apoptosis, and can affect cell growth and differentiation (Igarashi et al., 2010). In some studies, agmatine and spermidine inhibited experimental inflammation associated with suppressed proinflammatory cytokine expression (Soda et al., 2005). Agmatine and spermidine can affect observed osteoclast differentiation (Yamamoto et al., 2012), as well as a correlation between polyamine levels and symptoms of skeletal muscle hypertrophy (Turchanowa et al., 2000), Alzheimer's disease (Morrison et al., 1995), and ischemia (Paschen et al., 1987).

[0063] Various doses of spermine and / or spermidine can be administered enterally to a subject (preferably a human) to treat a metabolic disease or disorder as described herein. Spermine and / or spermidine can each be administered (e.g., orally, e.g., in drinking water) at a concentration of about 0.3 mM to about 3 mM, or more preferably about 0.3 mM to 1 mM, or about 0.5 mM, and these concentrations have been shown to be effective in mouse models of osteoporosis and aging. In experimental studies, 0.5 mM (equivalent to 18.2 mg / kg body weight) in drinking water also showed beneficial effects of polyamine supplementation on ovariectomy-induced bone loss and life span extension. In some embodiments, 1 to 50, 2 to 40, 5 to 25, or 15 to 20 mg / kg body weight / day, or any range derivable therein, can be used to treat a metabolic disease or disorder as described herein.

[0064] In some aspects, a microbiota is cultured in spermine and / or spermidine and then administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder as described herein, or a bone disease or disorder. For example, in some embodiments, a microbiota is cultured in 0.1 mM to 10 mM, 1 mM to 7 mM, or more preferably 1 mM to 5 mM, or 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM, or any range derivable therein, of spermine and / or spermidine and then administered to a subject to treat a metabolic disease or disorder. The microbiota can comprise or consist of Parabacteroides distasonii, Lactobacillus reuteri, and / or Lactobacillus gasseri. In some embodiments, Parabacteroides distasonii that has been cultured or expanded in spermine and / or spermidine is subsequently inactivated by a method as described herein (e.g., heat inactivation, exposure to hydrogen peroxide, etc.), and the inactivated Parabacteroides distasonii can be included in a pharmaceutical or probiotic composition or administered enterally to a human to treat a metabolic disease or disorder as described herein. In some embodiments, the microbiota includes live and / or inactivated Akkermansia muciniphila in combination with Parabacteroides distasonii (live and / or inactivated), Lactobacillus reuteri, and / or Lactobacillus gasseri; and in some embodiments, the microbiota includes both live and inactivated Akkermansia muciniphila.

[0065] V. Pharmaceutical formulations and routes of administration

[0066] In another aspect, for administration to a patient in need of such treatment, a pharmaceutical preparation (also referred to as a bacterial preparation or a pharmaceutical composition) comprises a therapeutically effective amount of a live or heat-inactivated bacterial composition disclosed herein formulated with one or more excipients and / or carriers suitable for the indicated route of administration. In some embodiments, the bacteria disclosed herein are formulated in a manner suitable for treating human and / or veterinary patients. In some embodiments, the preparation comprises one or more bacteria disclosed herein (e.g., a mesophilic microflora and / or heat-inactivated Parabacteroides goldsteinii) in admixture or combination with one or more of the following excipients: lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. In some embodiments, for example, for oral administration, the pharmaceutical preparation can be tableted or encapsulated. In some embodiments, the bacteria can be slurry in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and / or various buffers. In some embodiments, the pharmaceutical preparation can be subjected to pharmaceutical operations such as sterilization, and / or can contain carriers and / or excipients such as preservatives, stabilizers, wetting agents, emulsifying agents, encapsulating agents (e.g., lipids), dendrimers, polymers, proteins (e.g., albumin), nucleic acids, and buffers.

[0067] In some embodiments, the pharmaceutical preparation comprises inactivated (e.g., heat-inactivated) Parabacteroides goldsteinii. Various amounts of heat-inactivated Parabacteroides goldsteinii can be included, such as about 1 x 10 8 cfu to about 1 x 10 13 cfu. In some embodiments, the heat-inactivated Parabacteroides goldsteinii is included in a pharmaceutical composition or probiotic composition formulated for oral or enteral administration. For single (cultured) bacterial administration or mixed (cultured) bacterial administration, the heat-inactivated Parabacteroides goldsteinii can be administered orally (e.g., in the form of a tablet). For fecal microbiota transplantation (FMT) from a donor, the heat-inactivated Parabacteroides goldsteinii can be added, for example, to the microbiota, which is delivered to the gastrointestinal system, for example, through a nasogastric tube or colonically.

[0068] The pharmaceutical preparation can be administered by a variety of methods, such as orally, colonically, intranasally, intrarectally, by catheter, by lavage, by nasogastric tube, by local delivery, or by fecal microbiota transplant (FMT) methods. Depending on the route of administration, the bacterial compositions disclosed herein can be coated in a material to protect the bacterial compositions from the effects of acids and other natural conditions that can inactivate the bacterial compositions. To administer the bacterial compositions, it can be desirable to coat the bacterial compositions with, or co-administer with the bacterial compositions, a material that protects the bacterial compositions from inactivation. In some embodiments, the bacterial compositions can be administered to a patient in a suitable carrier, such as a polymer, hydrogel, liposome, starch, or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions, as well as conventional liposomes.

[0069] Formulations can be used to protect the bacterial compositions from the harsh stomach environment (Govander et al., 2014). Gastric resistant polymers and coatings have been shown to provide protection against the harsh stomach environment. These coatings include enteric coated tablets and capsules that deliver the administered probiotic site-specifically to the intestinal system. These enteric coatings are typically pH selective and allow for protection against harsh stomach conditions and subsequent dissolution in the alkaline media of the intestinal system (Calinescu et al., 2005 and Yang et al., 2002). Some non-limiting examples of excipients that can be used to formulate the bacterial compositions are hydroxypropyl methylcellulose phthalate and carboxymethyl high amylose starch. Excipients can be combined to enhance the delivery of the bacterial compositions to the gastrointestinal tract. For example, carboxymethyl high amylose starch can be combined with chitosan to deliver the bacterial compositions to the colon. Formulations can include different polymers with different properties, or similar polymers with different properties, depending on the intended delivery site of the bacterial compositions to different regions of the gastrointestinal tract (Yang et al., 2002).

[0070] The bacterial compositions disclosed herein can also be administered orally, colonically, intranasally, intrarectally, by catheter, by lavage, by nasogastric tube, by local delivery, or by fecal microbiota transplant (FMT) methods. The bacterial compositions can be in the form of a dispersion. The dispersion can be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils.

[0071] In some embodiments, the carrier comprises an enteric coating to reduce or slow degradation in the stomach. For example, the enteric coating can be a fatty acid; a wax; a shellac; a plastic such as a phthalate, CAP, CAT, PVAP, HPMCP; or a plant fiber (e.g., Hussan et al., 2012). In some embodiments, the pharmaceutical or probiotic composition can comprise chitosan-alginate beads or hydrogels. Nonetheless, it is contemplated that in some embodiments,

[0072] The bacterial compositions disclosed herein can be orally administered, for example, with an inert diluent or an assimilable edible carrier. The bacterial compositions and other ingredients can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the patient's diet. For oral therapeutic administration, the bacterial compositions disclosed herein can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of therapeutic agent in the compositions and preparations may, of course, be varied. The amount of therapeutic agent in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[0073] In some embodiments, it can be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the size of the dosage unit form of the present application is dictated by and directly depends on (a) the unique characteristics of the therapeutic agent and the particular therapeutic effect to be achieved, and (b) the inherent

[0074] In some embodiments, the effective dosage range of the therapeutic agent can be extrapolated from the effective dosage for a variety of different animals as determined in animal studies. The precise amount of the therapeutic composition required can depend on the judgment of the practitioner and can be peculiar to each individual. Other factors affecting dose include the physical and clinical state of the patient, the route of administration, the intended goal of therapy, and the potency, stability and toxicity of the particular therapeutic formulation.

[0075] The actual dose of the bacterial compositions of the present disclosure administered to a patient can be determined by such factors as the type of animal being treated, age, sex, weight, severity of the condition, type of disease to be treated, prior or concurrent therapeutic interventions, idiopathy of the patient, and route of administration. These factors can be determined by the skilled artisan. The practitioner administering will generally determine the concentration of active ingredient in the composition and the appropriate dose for an individual patient. In the event an adverse or adverse reaction occurs, the dose can be adjusted by the individual physician.

[0076] Single or multiple doses of the pharmaceutical agent are contemplated. One of ordinary skill in the art can determine the desired time interval for delivery of multiple doses using only routine experimentation. As one example, the patient can be administered two doses per day at about 12 hour intervals. In some embodiments, the pharmaceutical agent is administered once per day.

[0077] A composition comprising a bacterial composition (e.g., heat-killed Parabacteroides goldsteinii) can be administered on a regular schedule. As used herein, a regular schedule refers to a predetermined specified period of time. A regular schedule can encompass time periods of the same or different lengths, so long as the schedule is predetermined. For example, a regular schedule can include administration twice per day, daily, every two days, every three days, every four days, every five days, every six days, weekly, monthly, or any set number of days or weeks therebetween. Alternatively, a predetermined regular schedule can include administration twice per day for the first week, followed by daily administration for several months, etc. In other embodiments, the present application provides a pharmaceutical agent that can be taken orally and is time-dependent or independent of food intake. Thus, for example, the pharmaceutical agent can be taken every morning and / or every night, regardless of when the patient has eaten or will eat.

[0078] VI. Extracellular vesicles of bacteria

[0079] In some embodiments, extracellular vesicles from the bacteria described herein can be administered to a subject to treat a metabolic disease or disorder. For example, extracellular vesicles (EVs) can be produced by methods such as described in Chelakkot et al., 2018 or Choi et al., 2015. EVs from Parabacteroides goldsteinii, Lactobacillus gasseri, or Lactobacillus reuteri are contemplated to be useful in treating metabolic diseases or disorders as described herein (e.g., obesity, type 2 diabetes, fatty liver).

[0080] Extracellular vesicles (EVs) are lipid bilayered structures secreted by the gut microbiota, which includes both Gram-negative and Gram-positive bacteria (Ellis and Kuehn, 2010 and Lee et al., 2009). Multiple bacteria constitutively produce EVs, defined as spherical lipid bilayers with an average diameter of 20-200 nm (Lee et al., 2007). EVs are composed of proteins, lipids, nucleic acids, lipopolysaccharides, and other virulence factors associated with pathogenesis (Horstman and Kuehn, 2002, Hong et al., 2011, and Kim et al., 2013). Bacterially released EVs can have multiple roles in the microbial community, and some data suggest that they can transfer genetic material and proteins from bacteria to the host (Kuehn and Nesty, 2005). EVs can directly interact with immune cells and epithelial cells to initiate several signaling pathways and can influence or mediate host-pathogen interactions.

[0081] For example, in some embodiments, EVs can be prepared by the following method. A bacterial species or a community of mesophilic microorganisms can be cultured under aerobic or anaerobic conditions (e.g., at 37°C under 95% N2, 5% CO2) until desired (e.g., as previously described, when the optical density at 600 nm reaches 1.5; Derrien et al., 2004). Isolation of EVs can be performed as previously described in Kang et al., 2013. More specifically, the bacterial culture can be pelleted at 10,000 g for 20 minutes, and then the supernatant is filtered through a 0.45 pm vacuum filter. The filtrate can be concentrated, for example, using a QuixStand (GE Healthcare, Little Chalfont, UK), and then filtered through a 0.22 pm bottle-top filter. The filtrate can then be pelleted by ultracentrifugation (e.g., at 45 Ti rotor at 150,000 g for 2 hours at 4°C). The final pellet can then be resuspended in phosphate-buffered saline (PBS) and stored at -80°C. If desired, EVs can be analyzed by transmission electron microscopy, dynamic light scattering, and / or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by gel staining with Coomassie Brilliant Blue R-250. The amount of protein or DNA extracted from EVs can be measured and used to assess the amount of EVs obtained.

[0082] VII. Metabolic diseases and disorders

[0083] It is anticipated that the methods and bacterial compositions described herein can be used to treat a variety of metabolic diseases or conditions. For example, bacterial compositions as described herein (e.g., live bacteria, heat-inactivated bacteria, lyophilized bacteria, bacteria in pharmaceutical compositions, or secretory extracellular vesicles of bacteria) can be administered enterically or to the gastrointestinal tract of a subject to treat metabolic diseases or conditions. In some embodiments, the bacterial composition is heat-inactivated *Pseudomonas gondii*. In some embodiments, the metabolic disease or condition is obesity, fatty liver disease, type 2 diabetes, insulin intolerance, or dyslipidemia. Not wishing to be bound by any theory, the following examples provide the methods and bacterial compositions (e.g., heat-inactivated *Pseudomonas gondii*) shown herein, which can be used to improve glucose tolerance and metabolism, increase insulin secretion, and reduce fatty liver.

[0084] The prevalence of obesity, as measured by body mass index (BMI), has risen to unacceptable levels among men and women in the United States and worldwide, causing harmful health effects. Genetic, environmental, and behavioral factors influence the development of obesity. In adults, the classification system (World Health Organization Tech Rep Ser., 2000) and obesity guidelines (Jensen et al., 2014) define healthy weight as a BMI of 18.5 kg / m². 2 Up to 24.9 kg / m 2 Overweight is defined as 25.0 kg / m 2 Up to 29.9 kg / m 2 Obesity is defined as ≥30kg / m². 2 In children and adolescents, the Centers for Disease Control and Prevention (CDC) BMI-for-age growth chart defines overweight as a BMI equal to or higher than the 90th percentile of ideal weight, and obesity as a BMI higher than the 95th percentile of ideal weight. Obesity is associated with and promotes the following: shortened lifespan, type 2 diabetes, cardiovascular disease, some cancers, kidney disease, obstructive sleep apnea, gout, osteoarthritis, and hepatobiliary disease (Bray et al., 2018). Weight loss can reduce these diseases in a dose-related manner, and the greater the weight loss, the better the outcome.

[0085] Fatty liver disease, also known as hepatic steatosis, is a condition in which there is an excessive accumulation of fat in the liver. Fatty liver disease can be nonalcoholic fatty liver disease (NAFLD) or alcoholic liver disease. Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease and its prevalence is increasing worldwide with the growing epidemic of obesity. Nonalcoholic fatty liver disease is the most common cause of elevated liver enzymes. Within the NAFLD spectrum, usually only nonalcoholic steatohepatitis progresses to cirrhosis and hepatocellular carcinoma (Vernon et al., 2011). With the growing epidemic of obesity, the prevalence and impact of NAFLD is increasing.

[0086] Type 2 diabetes (T2D), formerly called adult-onset diabetes, is a form of diabetes mellitus characterized by high blood sugar, insulin resistance, and relative lack of insulin. Type 2 diabetes can be diagnosed using a glycosylated hemoglobin (A1C) test to determine average blood sugar levels, or using a random blood sugar test, a fasting blood sugar test, or an oral glucose tolerance test. Type 2 diabetes is the most common form of diabetes and can be caused by a variety of factors, including obesity, lack of physical activity, and genetics. Insulin can help patients with type 2 diabetes achieve ideal hemoglobin A1c goals (Wallia et al., 2014). In some cases, obesity can cause insulin resistance, and this is common in patients with type 2 diabetes. In some embodiments, a bacterial composition as disclosed herein (e.g., heat-killed Parabacteroides goldsteinii) is administered to a mammalian subject, e.g., a human, in combination with another treatment for type 2 diabetes, e.g., such as metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-IV inhibitors, GLP-1 receptor agonists (e.g., exenatide), SGLT2 inhibitors, or insulin.

[0087] Dyslipidemia is characterized by abnormal levels of lipids in the blood, for example: elevated plasma cholesterol, triglycerides (TG), or both; increased low-density lipoprotein (LDL) or very-low-density lipoprotein (VLDL) levels; low high-density lipoprotein (HDL) levels; or low HDL cholesterol levels. In some embodiments, dyslipidemia is hyperlipidemia (elevated blood lipids). Dyslipidemia can contribute to the development of atherosclerosis.

[0088] VIII. Temperature and Intestinal Microbiology

[0089] As shown herein, exposure to a warm environment can alter the microbiota of a mammalian subject, and the resulting "warm microbiota" has been shown herein to produce effects including improved glucose tolerance and reduced fatty liver, which can be particularly beneficial for treating metabolic diseases or conditions, such as obesity, type 2 diabetes, or fatty liver. Some living organisms adapt to the constant changes in their surrounding environment. One such external parameter is temperature, which can vary from below -35°C to above 40°C, and depends on the season, and on the time of day.

[0090] Homeothermic animals need to maintain a constant body temperature; therefore, they have developed different strategies to adapt to these external fluctuations. In rodents, thermogenic programs are initiated upon cold stimuli, including shivering thermogenesis from muscle and non-shivering thermogenesis from adipose tissue. In contrast, during warm exposure, thermogenic programs are attenuated and energy expenditure is correspondingly reduced (Kaiyala et al., 2012). Moreover, to dissipate excess heat, rodents increase cutaneous vasodilation in specific locations where the body surface is high, to maximize heat loss. This is the case in the ear and tail (Meyer et al., 2017). Interestingly, few reports suggest that, upon longer exposure to elevated temperatures, rodents adapt to maximize their heat dissipation capacity by increasing the length / surface of their tail and ear (Alhilli and Wright, 1983, Ashoub, 1958, and Harland, 1960).

[0091] The gut microbiota has been shown to influence some aspects of host physiology. Adaptation to cold exposure was shown to be mediated in part by the gut microbiota (Chevalier et al., 2015). The present disclosure shows that warm exposure can be beneficial for metabolic features, including improved insulin sensitivity, improved glucose tolerance, and reduced fatty liver, and that gut microbiota alterations play a role in these changes. These beneficial effects can thus be used to treat metabolic diseases and conditions.

[0092] IX. Examples

[0093] The following examples are included to demonstrate preferred embodiments of the application. Those of skill in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found function well in the practice of the application, and, therefore, can be considered to be preferred modes for the practice of the application. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the particular embodiments that are disclosed while still obtaining the same or similar results of the present application, yet still be within the scope and spirit of the application.

[0094] Example 1

[0095] Warm exposure and heat-inactivated Parabacteroides goldii increased glucose metabolism and reduced fatty liver in a postmenopausal mouse model.

[0096] To assess the effect of warm temperature on glucose metabolism, 8-week-old male mice were housed at 34°C for one month. Their glucose tolerance was monitored, and animals exposed to warm showed an increased glucose tolerance after oral glucose load ( Figure 1A ), which was associated with an increased insulin secretion ( Figure 1B ). It was then investigated whether a similar effect would be observed in the context of metabolic challenges such as postmenopausal weight gain and glucose intolerance. Postmenopausal weight gain and glucose intolerance induced by ovariectomy were mimicked, and estrogen deficiency worsened glucose tolerance, as observed during oral glucose tolerance test (OGTT, Figure 2A ). Interestingly, when mice were exposed to warm temperature, the reduced glucose tolerance was restored ( Figure 2B ), indicating that warm exposure improved glucose tolerance in a postmenopausal mouse model.

[0097] As warm exposure can modify microbiota composition, the inventors investigated whether some of the most significantly changed bacteria could mimic the warm exposure phenotype. Among the most consistently modulated bacteria were Parabacteroides goldii, Akkermansia muciniphila, Lactobacillus reuteri and Lactobacillus gasseri, which were all increased during warm exposure. To assess their individual effect on the observed phenotype, bacteria were freshly cultured and gavaged every two days to ovariectomized mice.

[0098] Additional tests were performed using heat-inactivated (HI) Parabacteroides goldii. Supplementation with heat-inactivated Parabacteroides goldii (OVAGold) was able to prevent ovariectomy-induced hyperglycemia, with effects observed after 12 hours or 6 hours of fasting ( Figure 3A ). Moreover, HI Parabacteroides goldii supplementation improved oral glucose tolerance ( Figure 3B ) and partially improved insulin sensitivity ( Figure 3C ) in ovariectomized mice. The effect of HI Parabacteroides goldii was observed to be more pronounced than the effect produced by Akkermansia muciniphila, a bacterium described to improve metabolic health during obesity. HI Parabacteroides goldii was also observed to reduce the accumulation of lipids in the liver ( Figure 4 ). These data support the idea that HI Parabacteroides goldii could be used to treat or prevent fatty liver disease.

[0099] Supplementation with heat-inactivated Parabacteroides goldii also increased the levels of Parabacteroides goldii detected 48 hours after the last administration. As Figure 5As shown in the middle panel, supplemental heat-killed B. gordinii prevented ovariectomy-induced loss of endogenous B. gordinii. Without wishing to be bound by any theory, since clearance of the typically supplemented heat-killed B. gordinii should not take more than 24 hours, one possibility is that this treatment can reduce ovariectomy-induced loss of endogenous B. gordinii or can promote its growth.

[0100] To further investigate the therapeutic potential of heat-killed B. gordinii in improving metabolic outcomes, the effect of supplementing heat-killed B. gordinii can be tested in a diet-induced obese mouse model. Secreted vesicles from B. gordinii can also be obtained, and this vesicle can be used to test whether the vesicle can reproduce the effect of supplemented heat-killed B. gordinii. Since supplementing L. reuteri and L. gasseri in ovariectomized mice also improved glucose tolerance and insulin sensitivity, secreted vesicles of these bacteria can also be isolated and used to determine whether they can exhibit similar therapeutic effects. Growth media from B. gordinii, L. reuteri, and / or L. gasseri can be obtained and further tested, for example, in a diet-induced obese mouse model.

[0101] Example 2

[0102] Methods for treating obesity

[0103] Visceral adipose tissue browning. To address the importance of the microbiota during long-term cold exposure, we cold-treated mice that were depleted of the microbiota by antibiotic (Abx) treatment (referred to as “combined treatment”). Unexpectedly, according to our preliminary results, not only did mice show further improvement in their glucose tolerance compared to untreated or single-stimulus-treated mice, but cold exposure of the microbiota-depleted mice resulted in a large and preferential fat loss from the VAT ( Figures 6A-6B ) compared to the SAT. While under non-stimulated conditions, all treated groups showed similar SAT[ 3 H]-2-deoxyglucose ([ 3 H]-2-DG) glucose uptake was improved, VAT of the cold-exposed microbiota-depleted mice showed significantly higher glucose uptake ( Figure 6C ) compared to all other groups. Despite the improved glucose uptake in this tissue in the cold-exposed microbiota-depleted mice, VAT weight was reduced significantly, even under basal conditions, indicating that combined treatment resulted in remodeling and increased energy dissipation, mainly restricted to visceral fat.

[0104] As Figures 6A-6CAs shown in FIG. 7A, a significant increase in glucose uptake in epiVAT and rpVAT and a decrease in total epiVAT and rpVAT mass were observed in animals after combination treatment. To gain further insight into the morphological changes in epiVAT, H&E staining was performed on epiVAT and rpVAT sections of all mouse groups. Notably, combination treatment resulted in approximately 40% more multilocular cells within the VAT depots compared to control or single-treated cold- or abx-animals (FIG. 7A). This multilocular appearance is a classic feature of beige adipocytes in vivo. Thus, when compared to room temperature (RT) or single-treated cold-exposed mice, visceral adipocytes from cold-exposed microbiota-depleted mice showed an increased oxygen consumption rate (FIG. 7B). These data, in agreement with the gene expression data shown in FIGS. 8A-8E, strongly suggest that the combination of cold exposure and microbiota depletion results in a massive VAT remodeling and the typical multilocular cell appearance of beige fat, and that this is a phenomenon unique to the visceral adipose tissue of double-treated animals. These data support the notion that significant visceral fat browning is possible without genetic intervention and suggest that VAT browning can contribute to changes in host physiology and metabolic status.

[0105] To gain insight into the necessity of UCP1 in visceral fat browning and remodeling, we compared UCP1-KO mouse groups kept at room temperature, cold, or combination cold and abx treatment. Interestingly, Ucp1-KO mice showed similar responses to WT mice, with combination treatment significantly improving glucose tolerance and promoting the development of multilocular cells in the visceral fat depots of combination cold / abx mice (FIGS. 8A-8B). Gene expression profiling of epiVAT in WT mice showed that all thermogenic markers were significantly increased after combination treatment, except Ucp1, compared to the rest of the groups. Specifically, while cold exposure increased Ucp1 expression in pgVAT of WT mice, its levels were not different after combination cold / abx treatment (FIG. 8C). In agreement with these observations and with data from WT mice, UCP1-KO animals showed similar increases in thermogenic gene expression after combination treatment compared to WT controls, further suggesting dispensability of UCP1 in visceral fat browning. Without wishing to be bound by any theory, these data support the notion that the appearance of visceral multilocular adipocytes and the increase in thermogenic gene expression can occur in a UCP1-independent manner and highlight the importance of identifying the molecular mechanisms that coordinate this process.

[0106] Identification of bacteria with beneficial metabolic effects

[0107] Using a multi-omic approach, Parabacteroides goldsteinii was identified as consistently deregulated under a variety of conditions, which led to improved glucose tolerance and weight loss. Since P. goldsteinii is classified as a potential pathogen despite being a commensal bacterium, the inventors used heat-inactivated (HI) bacteria to test metabolic effects. Oral supplementation of HI P. goldsteinii eliminated ovariectomy-induced hepatic lipid accumulation (e.g., Figure 9 ). These data support the idea that this bacterium can have a protective effect against fatty liver disease. In addition, it also improved overall glycemic control and reduced ovariectomy-induced blood glucose levels and hyperglycemia.

[0108] To address whether oral administration of HI P. goldsteinii can treat symptoms in vivo during diet-induced obesity, the following experiment was performed. C57B1 / 6 mice were fed a high caloric diet (HCD) for over three months. Subsequently, mice were orally administered HI P. goldsteinii. Body weight measurements showed that HI P. goldsteinii reduced body weight gain despite HCD feeding to levels similar to chow diet fed controls ( Figure 10 ). These effects were consistent with reduced adipose tissue weight ( Figure 11A ), and lower liver weight consistent with reduced hepatic triglyceride content ( Figure 11B ). These results support the idea that HI P. goldsteinii can attenuate diet-induced obesity, resulting in weight loss and improved liver health. The full version of these data has been provided to the Clayton foundation.

[0109] Polyamines promote growth of beneficial commensal bacteria

[0110] The inventors next investigated whether culturing several bacteria with beneficial metabolic effects can be influenced by the presence of polyamines in the growth medium. Both ( Figure 12 ) supplementation of different concentrations of spermine and spermidine improved growth of P. goldsteinii, L. reuteri, and / or L. gasseri ( Figure 12 ), all of which were identified as beneficially metabolizing bacteria. To further address whether the general growth of bacteria present in fresh fecal samples is affected, we inoculated fresh mouse fecal content in growth medium supplemented with different concentrations of spermine or spermidine. Both polyamines improved optical density ( Figure 13 ) during the exponential growth phase, indicating that polyamines promote bacterial growth in a bacterial mixture.

[0111] Aging is associated with a decline in polyamine levels and polyamine supplementation can prevent several age-related diseases, including memory impairment, cardiovascular disease, cancer, and can extend lifespan in some organisms. To investigate whether some of these beneficial effects are mediated, to some extent, by the microbiota adaptation, we administered a mixture of spermine and spermidine in mice and transplanted the microbiota from polyamine-treated animals to ovariectomized mice. Strikingly, polyamine-adapted microbiota decreased subcutaneous adipose tissue, visceral adipose tissue and brown adipose tissue, thus reducing total fat mass. Transplantation of control warm-adapted microbiota did not affect these parameters.

[0112] In addition, polyamine-adapted microbiota also decreased total liver weight Figure 14 These data suggest that the presence of polyamines in vivo induces a microbiota adaptation that can have beneficial metabolic effects, thus supporting such therapeutic strategies.

[0113] Example 3

[0114] Materials and methods

[0115] Animals: All C57BL / 6J mice were purchased from Janvier Labs and maintained in a specific pathogen-free (SPF) facility in individual ventilated cages. All mice were on a 12-hour light / dark cycle and fed a standard chow diet (16.2 MJ / kg total energy; 9 kJ% fat, 33 kJ% protein, 58 kJ% carbohydrates, V1534-727, Ssniff, Germany). All mice used were either male and entered the experiment at 8 weeks of age or female and started at 16 weeks of age (for ovariectomy experiments). Environmentally adapted animals were grouped based on their body weight to ensure identical starting points. Warm exposure was performed in individually ventilated cages under SPF conditions in a light- and humidity-controlled climate room (TSE, Germany) at 34°C. All mice were sacrificed after a 5-hour fasting period. All animal experiments were approved by the Swiss Federal and Canton of Geneva Animal Experimentation Authorities (Office Vétérinaire Fédéral and Commission Cantonale pour les Expériences sur les animaux de Genève).

[0116] Ovariectomy: Mice are anesthetized with xylazine / ketamine (injection of 120 μl of a mixture of 120 mg / kg ketamine and 16 mg / kg xylazine) and shaved under the dorsal ribs. Betadine is applied to the area for proper disinfection. After cutting the skin and muscle layer 1 em to 2 cm below the rib, the ovaries are located, the oviducts are ligated with dissolvable suture and the ovaries are removed. The muscle layer is sutured with dissolvable suture, the wound is closed with staples and disinfected. The same procedure is performed on the other side. One dose of Tamgesic is administered 4 hours after surgery and the staples are removed under isoflurane anesthesia 7 days after surgery. The same procedure is performed on sham-operated animals, but the oviducts are not ligated and the ovaries are not removed.

[0117] Microbiota transplantation: For the ovariectomy mouse experiment (already in the presence of a regular microbiota), donor fecal pellets are freshly collected every 2 days and immediately homogenized in 1 ml of anaerobic PBS. After a brief centrifugation (300 g, 30 s), the supernatant is then immediately gavaged to the corresponding recipient. In this case, one cage of donors (one pellet from each of two mice) is used to repopulate one cage of recipients. Each recipient receives 200 μl of donor mixture every 2 days.

[0118] Single microorganism transplantation: Lactobacillus gasseri (DSM 20604) and Parabacteroides goldsteinii (DSM 19948) were purchased from DSMZ. Lactobacillus reuteri (PTA-6475) and Akkermansia muciniphila (BAA835) were purchased from ATCC. L. gasseri and L. reuteri were cultured in MRS (deMan, Rogosa and Sharpe, US biological Life Sciences, L1021-01) medium and P. goldsteinii in Anaerobic Bacterium Base Broth (Thermo Scientific Oxoid Microbiology Products, CM0957) and A. muciniphila in SCHAEDLER Broth + Vitamin K3 (Biomerieux ref 42106) in an anaerobic chamber (Coy vinyl anaerobic chamber type C) set at 37°C with a gas mixture of 5% C02, 5% H and 90% N. Freshly prepared bacteria were diluted in anaerobic PBS to a final concentration equivalent to 1 OD at 600 nm. Ovariectomized mice were gavaged with 300 μΐ of this suspension every two days, starting 3 days after surgery for 2 months until sacrifice. Bacterial suspension was gavaged to mice fed with a high caloric diet (HCD) starting one week after the beginning of the HCD to address a protective effect in diet-induced obesity or after three months of HCD to address a curative effect. P. goldsteinii preparation was heat-inactivated at 100°C for 15 min before gavage and inactivation was confirmed.

[0119] Metabolic experiments: Oral glucose tolerance test (OGTT) was performed by oral gavage of a glucose bolus (2 mg / kg body weight) after an overnight fast of 10 hours. Insulin tolerance test was performed by intraperitoneal injection of 0.5 U / kg (I9278, Sigma-Aldrich) after a 5-hour morning fast. Insulin levels during OGTT were measured with Mouse Insulin ELISA kit (ref. 10-1247-01, Mercodia) following the manufacturer's instructions.

[0120] Liver lipid measurement: Lipids were extracted from 50 mg of liver using 1 ml of hexane:isopropanol (3:2) by homogenizing the tissue in a bead-based TissueLyser apparatus (Qiagen) by shaking at 30 Hz for 30 seconds in the presence of one bead per tube. The lysate was spun in a table top centrifuge at full speed for 3 minutes. The supernatant was removed. The pellet was re-extracted with 0.5 ml of hexane:isopropanol, spun again and the supernatant was pooled. 0.5 ml of a 1 g / 15 ml Na2S04solution was added and the tubes were mixed. The samples were spun at full speed for 3 minutes and the upper organic phase was collected into pre-weighed Eppendorf tubes. After overnight evaporation, the tubes were re-weighed and the lipid weight was recorded.

[0121] In vivo polyamine supplementation and inhibitor treatment: 6-week-old C57BL / 6J female mice were given a mixture of spermine (Sigma-Aldrich) and spermidine (Sigma-Aldrich) dissolved fresh in drinking water at a concentration of 0.5 mM of each compound every two days for an additional 45 days at room temperature. 16-week-old C57BL / 6J female mice, maintained at 34°C in a temperature-controlled room in regular facilities, were supplemented with Diaminazene Acetureate (Sigma-Aldrich) at a concentration of 50 μm in drinking water every two days for a period of 45 days. Food and water were provided ad libitum.

[0122] Micro-CT analysis: Mice were scanned with a micro-CT (VivaCT 40 / Scanco system; Zurich, Switzerland). Limbs were scanned in vivo and then ovariectomized to determine the basal state. After toluenesulphetal / ketamine anesthesia, mice limbs were scanned for 18 minutes. Isolated bones were scanned for a final time post-mortem. For femur and tibia trabecular regions, we analyzed 100 slices starting 50 slices below the distal growth plate. Cortical structure of femur and tibia was assessed by 60 consecutive CT slices (600 μm) from the bone midshaft. The images were segmented using an adaptive iterative threshold method instead of a fixed threshold. Morphometric variables were calculated from binary images using direct 3D techniques that do not rely on prior assumptions about underlying structures. For trabecular bone regions, we assessed the bone volume / total volume (BV / TV). For cortical bone of femur and tibia midshaft, we measured the cortical bone volume (mm3) and the mean cortical thickness, called cortical width (μm).

[0123] Biomechanical analysis of bone: We used a 3-point bending test to measure the biomechanical parameters of the bone. The femur was placed on two supports 9.9 mm apart and a load was applied to the midpoint of the shaft (creating a 3-point bend). The resistance to failure (displacement and applied load) was measured using a servo-controlled electromechanical system (Instron 1114, Instron corp., High Wycombe, UK) with an actuator to displace at 2 mm / min. The ultimate force (maximum load, measured in Newtons [N]), the yield point (N), the stiffness (elastic energy, N / mm) and the fracture energy (plastic region curve under surface, N*mm) were calculated. The Young's modulus (MPa) was determined by the equation described previously

[0124] ***

[0125] According to the present disclosure, all of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While compositions and methods according to the present application have been described in terms of preferred embodiments, it will be apparent to those with skill in the art that variations can be applied to the methods described and / or to the steps or order of steps in the methods described without departing from the concept, spirit and scope of the application. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for the agents described herein and be equally effective. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the application as defined by the appended claims.

[0126] References

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Claims

1. Use of a composition comprising inactivated Parabacteroides goldsteinii in the manufacture of a medicament for use in a method of treating a metabolic disease or disorder in a mammalian subject, the method comprising administering the composition to the gastrointestinal system of the subject, wherein the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease, or insulin resistance.

2. The use of claim 1, wherein the composition further comprises a growth medium of Parabacteroides goldsteinii or vesicles from Parabacteroides goldsteinii.

3. The use of claim 1 or 2, wherein the inactivated Parabacteroides goldsteinii is heat inactivated.

4. The use of claim 1 or 2, wherein the inactivated Parabacteroides goldsteinii has been inactivated by exposure to a peroxide.

5. The use of claim 4, wherein the peroxide is hydrogen peroxide.

6. The use of claim 4, wherein the peroxide is hydrogen peroxide vapor.

7. The use of claim 1 or 2, wherein the inactivated Parabacteroides goldsteinii has been inactivated by exposure to radiation or ionizing radiation.

8. The use of claim 7, wherein the radiation comprises or consists of: light having a wavelength of 400 nm to 420 nm.

9. The use of claim 1 or 2, wherein the inactivated Parabacteroides goldsteinii has been inactivated by exposure to air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric field (PEF).

10. The use of claim 9, wherein the alcohol is ethanol.

11. The use of claim 1 or 2, wherein the composition comprises extracellular vesicles from Parabacteroides goldsteinii.

12. The use of claim 1 or 2, wherein the composition comprises 1 x 10 8 cfu to 1 x 10 13 cfu of the inactivated Parabacteroides goldmanii.

13. The use of claim 1 or 2, wherein the composition further comprises Lactobacillus gasseri, Lactobacillus reuteri, or Akkermansia muciniphila.

14. The use of claim 1 or 2, wherein the composition is further defined as a pharmaceutical composition.

15. The use of claim 1 or 2, wherein the composition is further defined as a probiotic composition.

16. The use of claim 1 or 2, wherein the composition further comprises Lactobacillus gasseri or Lactobacillus reuteri.

17. The use of claim 1 or 2, wherein the composition further comprises extracellular vesicles from Lactobacillus gasseri or Lactobacillus reuteri.

18. The use of claim 14, wherein the pharmaceutical composition is administered orally, colonically, by enema, by orogastric tube, or by nasogastric tube.

19. The use of claim 15, wherein the probiotic composition is administered orally, colonically, by enema, by orogastric tube, or by nasogastric tube.

20. The use of claim 1 or 2, wherein the inactivated Parabacteroides goldsteinii or vesicles from Parabacteroides goldsteinii are contained in a pharmaceutical composition or probiotic composition that is resistant to degradation in the stomach but releases the bacteria in the small and / or large intestine of the subject.

21. The use of claim 14, wherein the pharmaceutical composition comprises an enteric coating, a chitosan-alginate bead, or a hydrogel.

22. The use of claim 15, wherein the probiotic composition comprises an enteric coating, a chitosan-alginate bead, or a hydrogel.

23. The use of claim 21 or 22, wherein the enteric coating is a fatty acid, a wax, a shellac, a plastic, a CAP, a CAT, a PVAP, a HPMCP, or a plant fiber.

24. The use of claim 21 or 22, wherein the enteric coating is a phthalate.

25. The use of claim 14, wherein the pharmaceutical composition does not comprise an enteric coating.

26. The use of claim 15, wherein the probiotic composition does not comprise an enteric coating.

27. The use of claim 14, wherein the pharmaceutical composition is a tablet or a capsule.

28. The use of claim 15, wherein the probiotic composition is a tablet or a capsule.

29. The use of claim 1 or 2, wherein the subject is a human.

30. The use of claim 29, wherein the human is a postmenopausal woman.

31. The use of claim 1 or 2, wherein the metabolic disease or disorder is obesity.

32. The use of claim 1 or 2, wherein the metabolic disease or disorder is type 2 diabetes.

33. The use of claim 1 or 2, wherein the metabolic disease or disorder is fatty liver disease.

34. The use of claim 1 or 2, wherein the fatty liver disease is nonalcoholic fatty liver disease (NAFLD).

35. The use of claim 1 or 2, wherein the microflora in the composition has been purified or cultured.

36. The use of claim 1 or 2, wherein the Parabacteroides goldsteinii has been inactivated by heating to 95°C to 105°C for 10 minutes to 20 minutes.

37. The use of claim 36, wherein the Parabacteroides goldsteinii has been inactivated by heating to 100°C for 15 minutes.

38. The use of claim 1 or 2, wherein the method further comprises enterally administering spermine and / or spermidine to the subject.

39. The use of claim 38, wherein the method comprises enterally administering both spermine and spermidine to the subject.

40. The use of claim 38, wherein the method comprises administering 1 mg / kg body weight / day to 50 mg / kg body weight / day of spermine to the subject.

41. The use of claim 38, wherein the method comprises administering 1 mg / kg body weight / day to 50 mg / kg body weight / day of spermidine to the subject.

42. The use of claim 1 or 2, wherein the composition comprises spermine and / or spermidine.

43. The use of claim 42, wherein the composition comprises both spermine and spermidine.

44. The use of claim 1 or 2, wherein the inactivated Parabacteroides goldsteinii is cultured or expanded in a culture medium comprising spermidine or spermine.

45. The use of claim 44, wherein the culture medium comprises 0.1 mM to 6 mM spermidine.

46. The use of claim 44, wherein the culture medium comprises 0.1 mM to 6 mM spermine.

47. The use of claim 1 or 2, wherein the subject is administered an antibiotic and exposed to an environment of 25 °C to 50 °C for at least 15 minutes.

48. The use of claim 47, wherein the subject is exposed to an environment of 32 °C to 35 °C for at least 15 minutes.

49. Use of a microbiota comprising Parabacteroides goldsteinii in the manufacture of a medicament for use in a method of treating a metabolic disease or disorder in a mammalian subject, the method comprising: (i) expanding the microbiota in a culture medium comprising spermidine or spermine, and (ii) administering the microbiota enterally to the subject; wherein the Parabacteroides goldsteinii is inactivated prior to administration to the subject, wherein the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease, or insulin resistance.

50. The use of claim 49, wherein the spermine or spermidine is present in the culture medium at a concentration of 0.1 mM to 10 mM.

51. The use of claim 50, wherein the spermine is present in the culture medium at a concentration of 1 mM to 6 mM.

52. The use of claim 50, wherein the spermidine is present in the culture medium at a concentration of 1 mM to 6 mM.

53. The use of any one of claims 49 to 52, wherein the culture medium comprises both spermine and spermidine.

54. The use of any one of claims 49 to 52, wherein the microflora comprises or consists of: Parabacteroides goldsteinii, Lactobacillus reuteri, or Lactobacillus gasseri.

55. The use of claim 49, wherein the Parabacteroides goldsteinii is inactivated by exposure to peroxide, ionizing radiation, heat, air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric field (PEF).

56. The use of claim 49, wherein the Parabacteroides goldsteinii is inactivated by exposure to peroxide, ionizing radiation, or heat.

57. The use of claim 56, wherein the peroxide is hydrogen peroxide.

58. The use of claim 56, wherein the Parabacteroides goldsteinii is inactivated by heating to 95 °C to 105 °C for 10 minutes to 20 minutes.

59. The use of any one of claims 49 to 52, wherein the subject is a human.

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