Methods and probiotic compositions for treating metabolic diseases and disorders

Abstract

For example, methods and probiotic compositions are disclosed for treating metabolic diseases or disorders, such as obesity, type 2 diabetes, or fatty liver. In some embodiments, heat-inactivated Parabacteroides gordosteinii is administered intraenterally to a subject, such as a human patient, to treat a metabolic disease or disorder or to promote the development of a temperate 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 microbiota that can be used to treat a metabolic disease or disorder.

Description

[Technical Field]

[0001] Priority information 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, which are incorporated by reference in their entireties.

[0002] 1. Field of the Invention The present invention relates generally to the fields of biology and medicine. In particular, it relates to methods and compositions for treating diseases, such as metabolic diseases and disorders. [Background technology]

[0003] 2. Description of Related Art Obesity and type 2 diabetes remain serious clinical challenges. Obesity is a relatively common medical condition that can lead to very serious adverse health consequences, including heart disease, diabetes, high blood pressure, and certain cancers. Type 2 diabetes is characterized by resistance to insulin, and this disease can lead to several very serious health consequences, including, for example, nerve damage, skin disorders, and / or kidney damage. Postmenopausal weight gain can also contribute to health problems for many people. The rates of obesity and type 2 diabetes are relatively high. For example, the number of people with obesity and type 2 diabetes in the United States alone is currently over 70 million and over 30 million, respectively.

[0004] The gut of healthy mammalian subjects harbors a microbiota that can influence human health. In some cases, the gut microbiota may be involved in the development of obesity, although the mechanisms by which this may occur are complex and not fully understood (see, for example, Davis et al., 2016). For example, Lactobacillus reuteri has been observed to be increased in obese individuals (Million et al., 2012). Clearly, new methods for treating obesity and type 2 diabetes are needed. Summary of the Invention

[0005] The present disclosure is based, in part, on the discovery that certain bacteria (e.g., Lactobacillus gasseri or Lactobacillus reuteri) and / or heat-inactivated Parabacteroides goldsteinii can be used to treat obesity, reduce liver steatosis, and improve insulin sensitivity. In some embodiments, pharmaceutical or probiotic compositions containing inactivated or heat-inactivated Parabacteroides gordosteinii are provided and can be administered to a mammalian subject to treat obesity or a metabolic disease, such as type 2 diabetes or fatty liver disease. In some aspects, it has been observed that growing or propagating a microbiota in polyamines, such as spermine or spermidine, can be used to treat diseases, such as metabolic diseases or disorders.

[0006] For example, as shown in the following examples, contrary to the general belief that only live microbiota bacteria, such as Parabacteroides gordosteinii, can affect the health of mammalian subjects, heat-killing Parabacteroides gordosteinii (heat-inactivating it) and orally administering the resulting composition to ovariectomized mice resulted in improved glucose tolerance and metabolism, increased insulin sensitivity, and reduced hepatic steatosis in vivo. Furthermore, animals exposed to warm temperatures showed improved glucose tolerance and increased insulin secretion after an oral glucose load. These results demonstrate that heat-inactivated Parabacteroides gordosteinii can be used to treat obesity or metabolic diseases, such as type 2 diabetes or fatty liver disease, in mammalian subjects in vivo. Enteral administration of Lactobacillus gasseri or Lactobacillus reuteri also improved glucose tolerance in vivo. Some of the beneficial effects of heat-inactivated Parabacteroides gordosteinii on blood glucose levels, oral glucose tolerance, and insulin sensitivity were observed to be more pronounced than those produced by Akkermansia muciniphila.

[0007] One aspect of the present invention 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 containing inactivated Parabacteroides gordosteinii, growth medium of Parabacteroides gordosteinii, or vesicles derived from Parabacteroides gordosteinii. In some embodiments, the inactivated Parabacteroides gordosteinii is heat-inactivated. In some embodiments, the inactivated Parabacteroides gordosteinii is inactivated via exposure to peroxide (e.g., hydrogen peroxide or hydrogen peroxide vapor). In some embodiments, the inactivated Parabacteroides gordosteinii is inactivated via exposure to radiation or ionizing radiation (e.g., comprising or consisting of light having a wavelength of about 400 to about 420 nm, more preferably about 400 to 410 nm, or about 405 nm). In some embodiments, the inactivated Parabacteroides gordosteinii has been inactivated via exposure to air plasma, ultrasound under pressure, alcohol (e.g., ethanol, isopropanol, etc., at a concentration of about 40-100% in solution, more preferably about 60-99%, 70-85%, or 65%, 70%, 75%, or any range therein), high hydrostatic pressure (HHP), or pulsed electric field (PEF). The composition may contain extracellular vesicles derived from Parabacteroides gordosteinii. The composition may contain about 1 x 10 8 ~Approx. 1×10 13In some embodiments, the composition may comprise inactivated Parabacteroides gordosteinii cfu. 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 derived from Lactobacillus gasseri or Lactobacillus reuteri. In some embodiments, the pharmaceutical or probiotic composition is administered orally, colonically, via enema, via an oral gavage tube, or via a nasogastric tube. In some embodiments, inactivated Parabacteroides gordosteinii or Parabacteroides gordosteinii-derived vesicles are included in a pharmaceutical or probiotic composition that is resistant to degradation in the subject's stomach but releases the bacteria in the small and / or large intestine. The pharmaceutical or probiotic composition may include an enteric coating, chitosan-alginate beads, or a hydrogel. In some embodiments, the enteric coating is a fatty acid, wax, shellac, a plastic such as phthalate, CAP, CAT, PVAP, HPMCP, or plant fiber. In some embodiments, the pharmaceutical or probiotic composition does not include an enteric coating. In some embodiments, the pharmaceutical or probiotic composition is a tablet or 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 is purified or cultured. In some embodiments, the inactivated Parabacteroides gordosteinii is inactivated by heating the bacteria to about 95-105°C for about 10-20 minutes, or to about 100°C for about 15 minutes, for example.

[0008] The method may further comprise enterally administering spermine and / or spermidine to the subject. In some embodiments, the method comprises enterally administering both spermine and spermidine to the subject. The method may comprise administering to the subject about 1 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 / kg body weight / day of spermine, etc.). In some embodiments, the method comprises administering to the subject about 1 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 / kg body weight / day of spermidine, etc.). The composition may comprise spermine and / or spermidine. In some embodiments, the composition comprises both spermine and spermidine. In some embodiments, inactivated Parabacteroides gordosteinii is cultured or grown in a medium containing spermidine or spermine; for example, Parabacteroides gordosteinii can be cultured or grown in a medium containing 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 medium contains about 0.1-6 mM spermidine and / or about 0.1-6 mM spermine. In some embodiments, the subject is administered an antibiotic and exposed to an environment at about 25-50°C, more preferably about 32-35°C, for at least about 15 minutes.

[0009] Another aspect of the present invention relates to a pharmaceutical or probiotic composition containing heat-inactivated Parabacteroides gordosteinii, growth medium of Parabacteroides gordosteinii, or vesicles derived from Parabacteroides gordosteinii; wherein the composition is formulated for delivery to the gastrointestinal system. In some embodiments, the composition comprises heat-inactivated Parabacteroides gordosteinii. The composition may further comprise Lactobacillus gasseri or Lactobacillus reuteri. The composition may further comprise extracellular vesicles derived from Lactobacillus gasseri or extracellular vesicles derived from Lactobacillus reuteri. In some embodiments, the pharmaceutical or probiotic composition is formulated for oral administration, colonic administration, enema administration, orogastric administration, or nasogastric administration. In some embodiments, the pharmaceutical or probiotic composition is resistant to degradation in the subject's stomach but releases bacteria in the small and / or large intestine. The pharmaceutical or probiotic composition may comprise an enteric coating, chitosan-alginate beads, or a hydrogel. In some embodiments, the enteric coating is a fatty acid, wax, shellac, a plastic such as phthalate, CAP, CAT, PVAP, HPMCP, or 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 capsule. The pharmaceutical or probiotic composition may further comprise spermine and / or spermidine. The pharmaceutical or probiotic composition may comprise about 1-50 mg / kg body weight / day of spermine, or any range derivable therein (e.g., 1-25, 2.5-15, 5-10, 5-25 mg / kg body weight / day of spermine, etc.). Pharmaceutical or probiotic compositions can contain about 1-50 mg spermidine per kg body weight per day, or any range derivable therein (e.g., 1-25, 2.5-15, 5-10, 5-25 mg spermine per kg body weight per day, etc.) These amounts can be adjusted for a human weighing, for example, about 45-136 kg.In some embodiments, the pharmaceutical or probiotic composition further comprises both spermine and spermidine. In some embodiments, Parabacteroides gordosteinii has been inactivated via exposure to peroxide, ionizing radiation, heat, air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric fields (PEF). In some embodiments, Parabacteroides gordosteinii has been inactivated via exposure to peroxide, ionizing radiation, or heat. The composition may be for use in treating a metabolic disease or disorder in a mammalian subject. The metabolic disease or disorder may 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).

[0010] Another aspect of the present invention 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 may include placing the subject in a climate chamber at an ambient temperature of about 65°C to about 95°C for about 3 to 30 minutes, about 3 to 15 minutes, about 3 to 10 minutes, or about 3 to 5 minutes. In some embodiments, the subject is repeatedly exposed to the climate chamber, with a time delay between each exposure. In some embodiments, a heating pad or heat lamp is applied to the torso, stomach, abdomen, head, legs, and / or feet of the subject; wherein the heating pad is about 27°C to about 50°C, or the heat lamp is about 60°C to about 95°C. Heat can be applied for about 30 minutes to about 9 hours. In some embodiments, heat is applied for at least 1, 2, 3, 4, 5, 6, or 7 days per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9 weeks, or more. In some embodiments, 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., non-alcoholic fatty liver disease (NAFLD)), insulin resistance, or dyslipidemia. The subject can be a human (e.g., a postmenopausal woman).

[0011] Yet another aspect of the present invention relates to a method of treating a metabolic disease or disorder in a mammalian subject, comprising the step of enterally administering to the subject (i) vesicles derived from Lactobacillus gasseri or Lactobacillus reuteri, or (ii) growth conditioned medium from Lactobacillus gasseri or Lactobacillus reuteri, wherein the method comprises administering to the subject about 1×10 8 ~Approx. 1×10 13 The method may further comprise administering cfu of Lactobacillus gasseri or Lactobacillus reuteri to the subject.The method may further comprise administering spermidine and / or spermine to the subject enterally.However, in some embodiments, vesicles or growth conditioned medium are administered to the subject enterally without administering Lactobacillus gasseri or Lactobacillus reuteri to the subject.The subject may be a human, such as a postmenopausal woman.The disease may be obesity, type 2 diabetes, fatty liver disease, insulin resistance, or dyslipidemia.

[0012] Yet another aspect of the present disclosure relates to a method of treating a disease or disorder in a mammalian subject, the method comprising: (i) growing a microbiota in a culture medium containing spermidine or spermine; and (ii) enterally administering the microbiota to the subject. In some embodiments, spermine or spermidine is present in the medium at a concentration of about 0.1-10 mM. Spermine can be present in the medium at a concentration of about 1-6, 1, 2, 3, 4, 5, 6 mM, or any range derivable therein (e.g., 1-3 mM, 2-3 mM, etc.). Spermidine can be present in the medium at a concentration of about 1-6, 1, 2, 3, 4, 5, 6 mM, or any range derivable therein. In some embodiments, the culture medium contains both spermine and spermidine. The microbiota may comprise or consist of Parabacteroides gordosteinii, Lactobacillus reuteri, and / or Lactobacillus gasseri. The microbiota may comprise or consist of Parabacteroides gordosteinii. In some embodiments, Parabacteroides gordosteinii is inactivated prior to administration to the subject. Parabacteroides gordosteinii may be inactivated via exposure to peroxide, ionizing radiation, heat, air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric fields (PEF). In some embodiments, Parabacteroides gordosteinii is inactivated via exposure to peroxide, ionizing radiation, or heat. The peroxide may be hydrogen peroxide. In some embodiments, Parabacteroides gordosteinii is inactivated by heating to about 95-105°C for about 10-20 minutes. In some embodiments, the mammalian subject is 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 improving bone strength. The bone disease can be 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 fractures, renal osteodystrophy, or Paget's disease.

[0013] Lactobacillus gasseri is a type of bacterium identified as part of the vaginal flora and found in the lower digestive system of women. Specific strains of Lactobacillus gasseri that can be used to treat metabolic diseases or disorders in mammalian subjects include: DSM 20077, DSM 107525, DSM 20243, DSM 20604, ATCC® 3332, ATCC® 2960, ATCC® BAA-2841, ATCC® PTA4483, ATCC® PTA4481, ATCC® PTA4484, ATCC® PTA4480, and / or ATCC® PTA4479. Various amounts of Lactobacillus gasseri can be administered to mammalian subjects (e.g., humans) to treat metabolic diseases or disorders described herein (e.g., obesity, type 2 diabetes, fatty liver, etc.). For example, in some embodiments, about 1 x 10 8 ~Approx. 1×10 13 CFU of Lactobacillus gasseri can be administered to a mammalian subject, such as a human, to treat a metabolic disease or disorder.

[0014] Lactobacillus reuteri is a type of bacterium found in the intestinal tract of healthy mammals. Specific strains of Lactobacillus reuteri that can be used to treat metabolic diseases or disorders in mammalian subjects include: DSM 100191, DSM 100192, DSM 17509, DSM 20015, DSM 20016, DSM 20053, DSM 20056, DSM 28673, DSM 32035, ATCC® BAA-2837™, ATCC® 55148, ATCC® 53608, ATCC® 23272, ATCC® 23272D5, and / or ATCC® PTA6475. Various amounts of Lactobacillus reuteri can be administered to a mammalian subject (e.g., a human) to treat a metabolic disease or disorder described herein (e.g., obesity, type 2 diabetes, fatty liver, etc.). For example, in some embodiments, about 1 x 10 8~Approx. 1×10 13 CFU of Lactobacillus reuteri can be administered to a mammalian subject, such as a human, to treat a metabolic disease or disorder.

[0015] Parabacteroides gordosteinii is a Gram-negative, obligately anaerobic, non-spore-forming, non-motile bacterium isolated from human blood. Specific strains of Parabacteroides gordosteinii that can be used to treat metabolic diseases or disorders in mammalian subjects include DSM 19448 and / or DSM 29187. Various amounts of Parabacteroides gordosteinii can be administered to mammalian subjects (e.g., humans) to treat metabolic diseases or disorders described herein (e.g., obesity, type 2 diabetes, fatty liver, etc.). For example, in some embodiments, about 1 x 10 8 ~Approx. 1×10 13Inactivated Parabacteroides gordosteinii cfu can be administered to mammalian subjects, such as humans, to treat metabolic diseases or disorders. As shown in the following examples, heat-inactivated Parabacteroides gordosteinii can be administered to treat metabolic diseases or disorders. Heat inactivation methods that can be used to prepare heat-inactivated Parabacteroides gordosteinii are well known and involve heating the bacteria to about 100°C for about 15 minutes (Wu et al., 2019). Parabacteroides gordosteinii can also be preserved by freezing or dehydration. Various methods can be used to produce inactivated Parabacteroides gordosteinii. For example, in some embodiments, the bacteria are irradiated or killed by radiation, exposure to ethanol, or autoclaving (e.g., as described in Lin et al., 2015). In some embodiments, Parabacteroides gordosteinii can be inactivated by exposure to light comprising or consisting of light having a wavelength of approximately 405 nm (e.g., Maclean et al., 2009). Inactivated Parabacteroides gordosteinii can be generated via exposure to air plasma, such as direct current, low-temperature, atmospheric-pressure air plasma microjets (e.g., Tian et al., 2010). Inactivated Parabacteroides gordosteinii can be generated via 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 Parabacteroides gordosteinii can be generated by exposure to ionizing radiation, ultrasound under pressure, high hydrostatic pressure (HHP), and / or pulsed electric fields (PEF) (e.g., Manas, et al., 2005). In some preferred embodiments, Parabacteroides gordosteinii is killed using heat inactivation.

[0016] As used herein, "a" or "an" may mean one or more. As used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0017] The use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, but in this disclosure the definition referring to alternatives only and "and / or" is supported. As used herein, "another" can mean at least a second or more.

[0018] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among study subjects.

[0019] The terms "comprise," "have," and "include" are open-ended linking verbs. Also, one or more forms or tenses of these verbs, such as "comprises," "comprising," "has," "having," "includes," "including," etc., are open-ended. For example, a method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also covers other steps not listed.

[0020] [The present invention 1001] A method for treating a metabolic disease or disorder in a mammalian subject, comprising administering a composition to the gastrointestinal system of the subject, the composition containing inactivated Parabacteroides goldsteinii, growth medium of Parabacteroides goldsteinii, or vesicles derived from Parabacteroides goldsteinii. [The present invention 1002] 1001. The method of claim 1001, wherein the inactivated Parabacteroides gordosteinii is heat-inactivated. [The present invention 1003] 1001. The method of claim 1001, wherein the inactivated Parabacteroides gordosteinii is inactivated via exposure to peroxide. [The present invention 1004] The method of claim 1003, wherein the peroxide is hydrogen peroxide. [The present invention 1005] The method of claim 1003, wherein the peroxide is hydrogen peroxide vapor. [The present invention 1006] 1001. The method of claim 1001, wherein the inactivated Parabacteroides gordosteinii is inactivated via exposure to radiation or ionizing radiation. [The present invention 1007] 1006. The method of claim 1006, wherein the radiation comprises or consists of light having a wavelength of about 400-420 nm. [The present invention 1008] 1001. The method of claim 1001, wherein the inactivated Parabacteroides gordosteinii is inactivated via exposure to air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric fields (PEF). [The present invention 1009] The method of claim 1008, wherein the alcohol is ethanol. [The present invention 1010] 1001. The method of claim 1001, wherein the composition comprises extracellular vesicles derived from Parabacteroides gordosteinii. [The present invention 1011] The composition is about 1×10 8 ~Approx. 1×10 13 Any of the methods of 1001 to 1010, comprising inactivated Parabacteroides gordosteinii cfu. [The present invention 1012] 1012. The method of any of claims 1001 to 1011, wherein the composition further comprises Lactobacillus gasseri, Lactobacillus reuteri, or Akkermansia muciniphila. [The present invention 1013] The composition of any one of claims 1001 to 1012, wherein said composition is further defined as a pharmaceutical composition. [The present invention 1014] 10. The composition of any one of claims 1001 to 1012, wherein said composition is further defined as a probiotic composition. [The present invention 1015] Any of the methods of claims 1001 to 1014, wherein the composition further comprises Lactobacillus gasseri or Lactobacillus reuteri. [The present invention 1016] The method of any one of claims 1001 to 1014, wherein the composition further comprises extracellular vesicles derived from Lactobacillus gasseri or Lactobacillus reuteri. [The present invention 1017] 1016. The method of any of claims 1001 to 1016, wherein said pharmaceutical or probiotic composition is administered orally, colonically, via an enema, via an orogastric tube, or via a nasogastric tube. [The present invention 1018] Any of the methods of inventions 1001 to 1017, wherein the inactivated Parabacteroides gordosteinii or vesicles derived from Parabacteroides gordosteinii are contained in a pharmaceutical or probiotic composition that is resistant to degradation in the stomach of a subject but releases the bacteria in the small intestine and / or large intestine. [The present invention 1019] The method of any of claims 1001 to 1018, wherein the pharmaceutical or probiotic composition comprises an enteric coating, chitosan-alginate beads, or a hydrogel. [The present invention 1020] The method of claim 1019, wherein the enteric coating is a fatty acid, a wax, a shellac, a plastic such as a phthalate, CAP, CAT, PVAP, HPMCP, or a vegetable fiber. [The present invention 1021] The method of any of claims 1001 to 1018, wherein the pharmaceutical or probiotic composition does not include an enteric coating. [The present invention 1022] The method of any of claims 1001 to 1021, wherein the pharmaceutical or probiotic composition is a tablet or capsule. [The present invention 1023] The method of any one of claims 1001 to 1022, wherein the subject is a human. [The present invention 1024] The method of claim 1023, wherein the human is a postmenopausal woman. [The present invention 1025] The method of any of claims 1001 to 1024, wherein the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease, insulin resistance, or dyslipidemia. [The present invention 1026] The method of claim 1025, wherein the metabolic disease or disorder is obesity. [The present invention 1027] The method of claim 1025, wherein the metabolic disease or disorder is type 2 diabetes. [The present invention 1028] The method of claim 1025, wherein the metabolic disease or disorder is fatty liver disease. [The present invention 1029] The method of claim 1025, wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD). [The present invention 1030] The method of any one of claims 1001 to 1029, wherein the microbial flora in said composition is purified or cultured. [The present invention 1031] The method of any of claims 1001 to 1030, wherein the Parabacteroides gordosteinii is inactivated by heating to about 95 to 105°C for about 10 to 20 minutes. [The present invention 1032] 1031. The method of claim 1031, wherein the Parabacteroides gordosteinii is inactivated by heating to about 100°C for about 15 minutes. [The present invention 1033] The method of any of claims 1001 to 1031, further comprising the step of administering spermine and / or spermidine into the enteric region of the subject. [The present invention 1034] The method of claim 1033, comprising the step of enterally administering to a subject both spermine and spermidine. [This invention 1035] The method of claim 1033, comprising administering to the subject about 1 to 50 mg / kg body weight / day of spermine. [The present invention 1036] The method of claim 1033, comprising administering to the subject about 1 to 50 mg / kg body weight / day of spermidine. [This invention 1037] The method of any one of claims 1001 to 1036, wherein the composition comprises spermine and / or spermidine. [The present invention 1038] 1037. The method of claim 1037, wherein the composition comprises both spermine and spermidine. [This invention 1039] The method of any of claims 1001 to 1038, wherein the inactivated Parabacteroides gordosteinii is cultured or grown in a medium containing spermidine or spermine. [The present invention 1040] 1039. The method of claim 1039, wherein said medium comprises about 0.1 to 6 mM spermidine. [This invention 1041] 1039. The method of claim 1039, wherein the medium comprises about 0.1 to 6 mM spermine. [The present invention 1042] The method of any of inventions 1001 to 1041, wherein the subject is administered an antibiotic and exposed to an environment of about 25 to 50°C, more preferably about 32 to 35°C, for at least about 15 minutes. [This invention 1043] A pharmaceutical or probiotic composition containing inactivated Parabacteroides gordosteinii, growth medium of Parabacteroides gordosteinii, or vesicles derived from Parabacteroides gordosteinii, and formulated for delivery to the gastrointestinal system. [This invention 1044] 1043. The composition of the present invention, comprising heat-inactivated Parabacteroides gordosteinii. [This invention 1045] Any of the compositions of claims 1043 to 1044, further comprising Lactobacillus gasseri or Lactobacillus reuteri. [The present invention 1046] Any of the compositions of claims 1043 to 1044, further comprising extracellular vesicles derived from Lactobacillus gasseri or extracellular vesicles derived from Lactobacillus reuteri. [This invention 1047] 1047. The composition of any of claims 1043 to 1046, wherein said pharmaceutical or probiotic composition is formulated for oral, colonic, enema, orogastric, or nasogastric administration. [This invention 1048] 8. The composition of any of claims 1043 to 1047, wherein said pharmaceutical or probiotic composition is resistant to degradation in the stomach of a subject but releases bacteria in the small and / or large intestine. [This invention 1049] The composition of claim 1048, wherein said pharmaceutical or probiotic composition comprises an enteric coating, chitosan-alginate beads, or a hydrogel. [The present invention 1050] The composition of claim 1049, wherein the enteric coating is a fatty acid, a wax, a shellac, a plastic such as a phthalate, CAP, CAT, PVAP, HPMCP, or a vegetable fiber. [This invention 1051] The composition of claim 1048, wherein said pharmaceutical or probiotic composition does not include an enteric coating. [This invention 1052] 1052. The composition of any one of claims 1043 to 1051, wherein said pharmaceutical or probiotic composition is a tablet or capsule. [This invention 1053] 1053. The composition of any one of claims 1043 to 1052, wherein said pharmaceutical or probiotic composition further comprises spermine or spermidine. [This invention 1054] 1053. The composition of claim 1053, wherein said pharmaceutical or probiotic composition comprises about 1 to 50 mg / kg body weight / day of spermine. [This invention 1055] 1053. The composition of claim 1053, wherein said pharmaceutical or probiotic composition comprises about 1 to 50 mg / kg body weight / day of spermidine. [The present invention 1056] 1056. The composition of any one of claims 1053 to 1055, wherein said pharmaceutical or probiotic composition further comprises both spermine and spermidine. [This invention 1057] Any of the compositions of claims 1043 to 1056, wherein the Parabacteroides gordosteinii has been inactivated via exposure to peroxide, ionizing radiation, heat, air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric fields (PEF). [This invention 1058] 1057. The composition of claim 1057, wherein the Parabacteroides gordosteinii is inactivated via exposure to peroxide, ionizing radiation, or heat. [This invention 1059] 9. The composition of any of claims 1043 to 1058 for use in treating a metabolic disease or disorder in a mammalian subject. [The present invention 1060] The composition of invention 1059, wherein the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease such as non-alcoholic fatty liver disease (NAFLD), insulin resistance, or dyslipidemia. [This invention 1061] The composition of claim 1060, wherein the subject is a human. [This invention 1062] The composition of claim 1061, wherein the human is a postmenopausal woman. [This invention 1063] 1. A method of treating a metabolic disease or disorder in a mammalian subject, the method comprising the step of applying heat to the torso of the subject. [This invention 1064] The method of claim 1063, comprising placing the object in an artificial climate chamber at an ambient temperature of about 65°C to about 95°C for about 3 to 30 minutes, about 3 to 15 minutes, about 3 to 10 minutes, or about 3 to 5 minutes. [This invention 1065] 1065. The method of any one of claims 1063 to 1064, wherein the subject is repeatedly exposed to the climate chamber, with a time interval between each exposure. [The present invention 1066] 1063. The method of claim 1063, wherein a heating pad or heating lamp is applied to the torso, stomach, abdomen, head, legs, and / or feet of the subject, wherein the heating pad is at about 27°C to about 50°C or the heating lamp is at about 60°C to about 95°C. [This invention 1067] 1067. The method of any one of claims 1063 to 1066, wherein heat is applied for a period of from about 30 minutes to about 9 hours. [The present invention 1068] The method of claim 1067, wherein heat is applied for at least 1, 2, 3, 4, 5, 6, or 7 days per week for 1, 2, 3, 4, 5, 6, 7, 8, 9, or more weeks. [The present invention 1069] Any of methods 1063 to 1067 of the present invention, wherein heat is repeatedly applied to the body of the subject. [The present invention 1070] The method of any of claims 1063 to 1067, wherein the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease (eg, non-alcoholic fatty liver disease (NAFLD)), insulin resistance, or dyslipidemia. [This invention 1071] The method of any one of claims 1063 to 1070, wherein the subject is a human. [This invention 1072] 1072. The method of claim 1071, wherein said human is a postmenopausal woman. [This invention 1073] A method for treating a disease or disorder in a mammalian subject, comprising the step of enterally administering to the subject (i) vesicles derived from Lactobacillus gasseri or Lactobacillus reuteri, or (ii) growth conditioned medium from Lactobacillus gasseri or Lactobacillus reuteri. [This invention 1074] Approximately 1×10 8 ~Approx. 1×10 13 The method of claim 1073, further comprising administering cfu of Lactobacillus gasseri or Lactobacillus reuteri to said subject. [This invention 1075] The method of claim 1073, further comprising the step of enterally administering spermidine and / or spermine to said subject. [This invention 1076] The method of any one of claims 1073 to 1074, wherein the subject is a human, for example a postmenopausal woman. [This invention 1077] The method according to any one of claims 1073 to 1076, wherein said disease is obesity, type 2 diabetes, fatty liver disease, insulin resistance, or dyslipidemia. [This invention 1078] 1. A method of treating a metabolic disease or disorder in a mammalian subject, comprising: (i) growing the microbiota in a culture medium containing spermidine or spermine; and (ii) enterally administering the microbiota to the subject. The method comprising: [This invention 1079] The method of claim 1078, wherein spermine or spermidine is present in said medium at a concentration of about 0.1 to 10 mM. [The present invention 1080] 1079. The method of claim 1079, wherein spermine is present in said medium at a concentration of about 1 to 6 mM. [This invention 1081] 1079. The method of claim 1079, wherein spermidine is present in said medium at a concentration of about 1 to 6 mM. [This invention 1082] 1082. The method of any one of claims 1078 to 1081, wherein said culture medium contains both spermine and spermidine. [This invention 1083] The method of any of claims 1078 to 1082, wherein the microbiota comprises or consists of Parabacteroides gordosteinii, Lactobacillus reuteri, or Lactobacillus gasseri. [This invention 1084] The method of claim 1083, wherein the microbiota comprises or consists of Parabacteroides gordosteinii. [This invention 1085] The method of claim 1084, wherein the Parabacteroides gordosteinii is inactivated prior to administration to said subject. [The present invention 1086] The method of the present invention 1085, wherein Parabacteroides gordosteinii is inactivated via exposure to peroxide, ionizing radiation, heat, air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric fields (PEF). [This invention 1087] The method of claim 1085, wherein Parabacteroides gordosteinii is inactivated via exposure to peroxide, ionizing radiation, or heat. [This invention 1088] 1087. The method of claim 1087, wherein the peroxide is hydrogen peroxide. [This invention 1089] 1087. The method of claim 1087, wherein Parabacteroides gordosteinii is inactivated by heating to about 95-105°C for about 10-20 minutes. [The present invention 1090] The method of any one of claims 1078 to 1089, wherein the subject is a human. [This invention 1091] The method of any one of claims 1078 to 1090, wherein the disease is a metabolic disease or disorder. [This invention 1092] The method of claim 1091, wherein the metabolic disease or disorder is obesity, type 2 diabetes, fatty liver disease, insulin resistance, or dyslipidemia. [This invention 1093] The method of any one of claims 1078 to 1090, wherein said disease is a bone disease or said method comprises the step of improving bone strength. [This invention 1094] 1093. The method of claim 1093, wherein the bone disease is osteoporosis, osteomalacia, osteolysis, osteochondrodysplasia, 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. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Figure 1] Figure 1A-B: 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) Relative insulin release at 0, 7, and 15 minutes. [Figure 2] Figure 2A-B: 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 maintained at RT or 34°C for 2 months. [Figure 3A] Heat-inactivated Parabacteroides gordosteinii prevents ovariectomy-induced hyperglycemia and improves glucose tolerance and insulin sensitivity. Blood glucose levels in mice supplemented with heat-inactivated Parabacteroides gordosteinii (OVA Gold) after 12 or 6 hours of fasting. [Figure 3B]Heat-inactivated Parabacteroides gordosteinii prevents ovariectomy-induced hyperglycemia and improves glucose tolerance and insulin sensitivity. Oral glucose tolerance test (left) and area under the curve (right) in ovariectomized mice supplemented with heat-inactivated Parabacteroides gordosteinii or live Akkermansia muciniphila and maintained at RT. [Figure 3C] Heat-inactivated Parabacteroides gordosteinii prevents ovariectomy-induced hyperglycemia and improves glucose tolerance and insulin sensitivity. Mice (similar to Figure 3A-B) were injected with 0.5 U / kg insulin, followed by monitoring blood glucose over a 2-hour insulin tolerance test. [Figure 4] Figure 4: Heat-inactivated Parabacteroides gordosteinii prevents ovariectomy-induced hepatic fat accumulation. Liver lipid content in ovariectomized mice supplemented with heat-inactivated Parabacteroides gordosteinii and maintained at RT. [Figure 5] Figure 5: Supplementation with heat-inactivated Parabacteroides gordosteinii prevents the loss of endogenous Parabacteroides gordosteinii induced by ovariectomy. Fecal Parabacteroides gordosteinii 48 hours after the last dose from ovariectomized mice maintained at RT with supplementation with heat-inactivated Parabacteroides gordosteinii. [Figure 6A] Figure 6A-C: (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 fasting starting blood glucose (bottom). (Figure 6B) Adipose tissue weight at sacrifice of C57BL / 6J mice after 4 weeks of 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 into adipose tissue after intraperitoneal injection. Bar graphs show mean ± SD (n = 6-8 per group). Statistics were performed using an unpaired two-tailed Student's t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. [Figure 6B] See the legend to Figure 6A. [Figure 6C] See the legend to Figure 6A. [Figure 7] Figure 7A-B: (Figure 7A) Representative H&E staining of visceral adipose tissue from C57Bl / 6 mice after 30 days of treatment as indicated. Black arrows indicate multilocular adipocytes. (Figure 7B) Oxygen consumption rate measured using a Seahorse analyzer in basal conditions after 30 days of treatment. Significance was calculated using an unpaired, two-tailed Student's t-test. ***P≦0.001. [Figure 8-1] Figures 8A-E: (Figure 8A) Oral glucose tolerance test in Ucp1-KO mice after 3 weeks of treatment. (Figure 8B) Representative H&E staining of visceral adipose tissue from Ucp1-KO mice after 30 days of treatment as indicated. Black arrows indicate multilocular adipocytes. (Figure 8C-E) Relative gene expression of thermogenic genes in rpVAT and epiVAT of WT (Figures 8C and 8D) or Ucp1-KO mice (Figure 8E), normalized to Tbp. Significance in A, C, and D was calculated using an unpaired, two-tailed Student's t-test. ***P ≤ 0.001. [Figure 8-2] See the description of Figure 8-1. [Figure 9] Figure 9: Liver lipid weight at sacrifice in ovariectomized mice after oral supplementation with HI Parabacteroides gordosteinii or Lactobacillus reuteri. Significance calculated based on one-way ANOVA; *P<0.05. [Figure 10] Figure 10: Body weight of mice fed a high-calorie diet after oral supplementation with Parabacteroides gordosteinii. Data show the change in body weight after the start of bacterial gavage (left) or the area under the curve from the left panel (right). Significance was calculated based on one-way ANOVA; ****P<0.0001. [Figure 11]Figures 11A-B: Subcutaneous adipose tissue (Figure 11A) or liver (Figure 11B) weights in mice fed a high-calorie diet for 3 months followed by oral supplementation with Parabacteroides gordosteinii for 4 weeks. *P<0.05, ***P<0.001. [Figure 12-1] Figure 12: Spermine or spermidine supplementation promotes the growth of beneficial bacteria in vitro. [Figure 12-2] See the description of Figure 12-1. [Figure 13] Figure 13: Supplementation with spermine or spermidine enhances the growth of a bacterial mix from mouse fecal samples in vitro. [Figure 14] Figure 14: Transplantation of polyamine-adapted microbiota into ovariectomized aged female mice (blue) suppresses weight gain by limiting adipose and liver tissue weight. DETAILED DESCRIPTION OF THE INVENTION

[0022] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Definition A "bacterial composition" is a composition containing one or more types of bacteria (e.g., live, dried, or heat-inactivated bacteria) or extracellular vesicles (i.e., secreted extracellular vesicles) derived from bacteria. In some embodiments, the bacteria are from the Clostridiaceae, Lactobacillaceae, and / or Porphyromonadaceae families. Specific bacteria contemplated include Lactobacillus gasseri, Lactobacillus reuteri, and Parabacteroides gordosteinii (e.g., live or heat-inactivated P. gordosteinii). In some preferred embodiments, inactivated Parabacteroides gordosteinii inactivated using heat, freezing, or desiccation is used.

[0023] The term "effective," as used herein and / or in the claims, means sufficient to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount," when used in the context of treating a patient or subject with a bacterial composition, refers to that amount of a bacterial composition that, when administered to a subject or patient for treating or preventing a disease, is sufficient to effect treatment or prevention of such disease.

[0024] An "excipient" is a pharmaceutically acceptable substance that is formulated with the active ingredient(s) of a drug product, pharmaceutical composition, formulation, or drug delivery system. Excipients can be used, for example, to stabilize the composition, bulk the composition (and therefore, when used for this purpose, are often also called "fillers," "fillers," or "diluents"), or to impart therapeutic benefits to the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or increasing solubility. Excipients include anti-adherents, binders, coating agents, colorants, disintegrants, flavoring agents, glidants, lubricants, preservatives, adsorbents, sweeteners, and pharmaceutically acceptable versions of vehicles. The primary excipient that serves as a vehicle for carrying the active ingredient is usually referred to as the vehicle. Excipients may also be used in manufacturing processes to aid in handling of the active agent, for example, by promoting powder flow or non-sticking, as well as aiding in in vitro stability, such as preventing denaturation or aggregation during the expected storage period. The suitability of an excipient will generally vary with the route of administration, dosage form, active ingredient, as well as other factors.

[0025] As used herein, the term "patient" or "subject" refers to a living mammal, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic non-human species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, and infants.

[0026] A "pharmaceutically acceptable carrier," "drug carrier," or simply "carrier" is a pharmaceutically acceptable substance formulated with an active ingredient that participates in the carrying, delivery, and / or transport of a biologic. Carriers can be used to improve the delivery and effectiveness of the active ingredient, including, for example, controlled-release technologies to regulate drug bioavailability, reduce drug metabolism, and / or reduce drug toxicity. Some carriers can enhance the delivery of active ingredients to specific target sites. Examples of carriers include liposomes, microspheres (e.g., made of or containing 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., fatty acids, waxes, shellac, plastics such as phthalates, CAP, CAT, PVAP, HPMCP, or vegetable fibers), chitosan-alginate beads, or hydrogels to reduce or delay degradation in the stomach.

[0027] "Prevention" or "preventing" includes: (1) arresting the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the pathology or symptoms of the disease; and / or (2) delaying the onset of a disease pathology or symptom in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the pathology or symptoms of the disease.

[0028] "Treatment" or "treating" includes: (1) inhibiting a disease (e.g., preventing further progression of pathology and / or symptoms) in a subject or patient experiencing or exhibiting pathology or symptoms of the disease; (2) ameliorating a disease (e.g., reversing pathology and / or symptoms) in a subject or patient experiencing or exhibiting pathology or symptoms of the disease; and / or (3) causing a measurable reduction in the disease or its symptoms in a subject or patient experiencing or exhibiting pathology or symptoms of the disease.

[0029] II. Warm microflora As shown in the examples below, a variety of bacteria have been observed in warm microbiota, i.e., microbiota obtained from mammalian subjects living in warmer environments. Warm microbiota include Clostridialeaceae-assimilate spp., Lactobacillus spp. (e.g., Lactobacillus gasseri or Lactobacillus reuteri), Bifidobacteriaceae spp. (e.g., Bifidobacterium longum), Parabacteroides spp. (e.g., Parabacteroides gordosteinii), and Akkermansia spp. (e.g., Akkermansia muciniphila). In some embodiments, it is contemplated that the bacteria listed in any one of Tables 1-5 can be included in the pharmaceutical or probiotic compositions disclosed herein. In some embodiments, the pharmaceutical or probiotic composition may contain Lactobacillus reuteri, Lactobacillus acidophilus, and / or Lactobacillus rhamnosus. As shown in the Examples, therapeutic effects can also be observed when using heat-inactivated Parabacteroides gordosteinii.

[0030] 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: Clostridium species, Lactobacillus species (e.g., Lactobacillus reuteri, Lactobacillus gasseri, Lactobacillus acidophilus, and / or Lactobacillus rhamnosus), Bifidobacterium species (e.g., Bifidobacterium longum), Parabacteroides species (e.g., Parabacteroides gordosteinii), and Akkermansia species (e.g., Akkermansia muciniphila), can be included in the pharmaceutical or probiotic compositions disclosed herein and / or administered to a mammalian subject, such as a human patient, to treat a metabolic disease or disorder. Various interactions between gut microbiota and physiology (e.g., as described in Ohlsson and Sjogren, 2015) can be used in conjunction with the present disclosure. Lactobacillus species, such as Lactobacillus reuteri (Britton et al., 2014; more recently described in humans in Nilsson et al., 2018), Lactobacillus acidophilus (Dar et al., 2018), and / or Lactobacillus rhamnosus (Li et al., 2016), can be included in compositions for the treatment of metabolic diseases or disorders. In other embodiments, heat (e.g., from a heating chamber, heating pad, or heat lamp) can be applied to a subject (e.g., the entire body or to specific regions such as the trunk, stomach, limbs, and / or abdomen) to treat metabolic diseases or disorders described herein, such as obesity, type 2 diabetes, and fatty liver. In some embodiments, the applied heat may promote the growth of warm microbiota.

[0031] III. Inactivated Parabacteroides gordosteinii Inactivated Parabacteroides gordosteinii can be produced via a variety of methods. In some embodiments, inactivated Parabacteroides gordosteinii is inactivated via exposure to heat. However, it is anticipated that other inactivation methods can be used to produce inactivated Parabacteroides gordosteinii that exert similar effects and can be used to treat metabolic diseases or disorders (e.g., obesity, diabetes, etc.) described herein. For example, inactivated P. gordosteinii 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), peroxide (e.g., hydrogen peroxide), alcohol (e.g., ethanol), low temperature exposure or freezing, dehydration, lyophilization, or pulsed electric fields (PEF); in some embodiments, one or more of the aforementioned bacterial inactivation methods can be used in combination with heat to create inactivated P. gordosteinii that can be used, for example, for the treatment of metabolic diseases or disorders described herein. In some embodiments, the compositions described herein can include both live and inactivated Parabacteroides gordosteinii.

[0032] Various methods can be used to produce heat-inactivated Parabacteroides gordosteinii. For example, as described in Wu et al. (2019), the bacteria can be heated to at least 95°C, at least 100°C, or up to about 100°C for at least 10 minutes, 10-20 minutes, or about 15 minutes. It is also anticipated that autoclaving or heating in solution (e.g., boiling in water) can also be used. Generally, heat can inactivate bacteria through one or more of the following: membrane damage, loss of nutrients and ions, ribosome aggregation, DNA strand scission, inactivation of essential enzymes, and protein coagulation. Additional inactivation methods that can be used with the present invention are described, for example, in Lin et al. (2015). After producing inactivated (e.g., heat-inactivated) P. gordosteinii, the bacteria can be subsequently dried, frozen, or lyophilized, if desired. In some preferred embodiments, the Parabacteroides gordosteinii is killed using heat inactivation.

[0033] Inactivated P. gordosteinii can also be produced by exposure to radiation, such as ionizing radiation. In some embodiments, the bacteria can be inactivated by exposure to a light beam comprising or consisting of light having a wavelength of about 405 nm. For example, a light-emitting diode (LED) array emitting light at a wavelength of about 405 nm can be used to inactivate bacteria (e.g., Maclean et al., 2009). In some embodiments, the radiation can be ultraviolet (UV) light having a wavelength of about 240 nm to about 280 nm. In some embodiments, the radiation is ionizing radiation, such as X-rays.

[0034] Inactivated Parabacteroides gordosteinii can be generated through exposure to a peroxide, such as hydrogen peroxide. Hydrogen peroxide can be contacted with the bacteria in a solution. In some embodiments, the bacteria are inactivated by contacting the bacteria with hydrogen peroxide vapor. For example, the bacteria can be inactivated by contacting the bacteria with about 10-100 mg / m 3Exposure to hydrogen peroxide vapor at 10 ppm for approximately 1.5 to 48 hours can be used (e.g., Malik et al., 2013). Hydrogen peroxide can be applied as both a liquid and a vapor to inactivate bacteria. In some cases, the mechanism of action of hydrogen peroxide in vapor form may result in increased and more thorough oxidation of a wider range of biopolymers than aqueous hydrogen peroxide solutions (Finnegan et al., 2010). Similar to heat inactivation, the use of hydrogen peroxide for bacterial inactivation has the advantage that it decomposes into non-toxic by-products after reacting with the bacteria. In some embodiments, it is contemplated that a 1-2% solution of hydrogen peroxide is contacted with the bacteria for approximately 5-10 minutes to inactivate P. gordosteinii. In some embodiments, a combination of heat and hydrogen peroxide can be used to inactivate P. gordosteinii.

[0035] Various other methods for inactivating P. gordosteinii can also be used, for example, by contacting the bacteria with a certain concentration of alcohol (e.g., ethanol, methanol, propanol, or isopropanol) (e.g., at least 70% v / v alcohol, e.g., 70% ethyl alcohol) to inactivate the bacteria.

[0036] Inactivated Parabacteroides gordosteinii can also be generated through exposure to air plasma, such as direct current, low-temperature, atmospheric-pressure air plasma microjets (e.g., Tian et al., 2010); for example, after approximately 10 minutes of plasma treatment, NO generated in air plasma was x A decrease in pH can be observed as the reacts with water at the gas-liquid interface.

[0037] Inactivated Parabacteroides gordosteinii can be produced through exposure to ionizing radiation (e.g., gamma rays generated by cobalt-60, electron beam, or X-rays). Generally, the amount of ionizing radiation to which the bacteria are exposed is preferably sufficient to damage the DNA of the bacteria and / or prevent further growth of the bacteria.

[0038] Ultrasound under pressure or high hydrostatic pressure (HHP) can also be used to inactivate bacteria. In some embodiments, pressures of about 100-1000 MPa are applied to inactivate bacteria. The effectiveness of HHP has been demonstrated in the field of food hygiene. Ultrasound is defined as sound waves with frequencies above the threshold of human hearing (>16 kHz). In some embodiments, the application of ultrasound can be combined with external hydrostatic pressure (e.g., pressurized sonication (manosonication) up to 600 kPa) and / or heat to inactivate P. gordosteinii.

[0039] Pulsed electric fields (PEF) can also be used (e.g., Manas et al., 2005). PEF approaches generally involve applying a short duration (e.g., 1–100 μs) high electric field pulse (10–50 kV cm) to a sample between two electrodes. -1 ) is applied. Various embodiments contemplate using a combination of one or more of the foregoing methods to produce inactivated P. gordosteinii.

[0040] IV. Spermine and Spermidine In some aspects, spermine and / or spermidine can be administered enterally to a mammalian subject, such as a human, to treat a metabolic disease or disorder (e.g., diabetes, obesity, etc.) or bone disease or disorder described herein. For example, the bone disease or disorder can be 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 fractures, renal osteodystrophy, or Paget's disease. In some embodiments, spermine and / or spermidine can be administered in combination with an inactivated P. gordosteinii (e.g., heat-inactivated P. gordosteinii) described herein. In some embodiments, spermine or spermidine can be administered in combination with another live or inactivated microbiota described herein (e.g., L. reuteri, L. gasseri, and / or A. muciniphila), optionally in combination with live or inactivated P. gordosteinii. As shown in the Examples below, enteral administration of microbiota cultured in spermidine or spermine suppressed obesity in in vivo animal models, and reductions in total fat (including reductions in subcutaneous fat, visceral fat, and brown adipose tissue) were observed.

[0041] Spermidine (N-(3-aminopropyl)-1,4-butanediamine) and spermine (N,N'-bis(3-aminopropyl)-1,4-butanediamine) are naturally occurring polyamines that can function as regulators of various cellular processes, such as DNA stability, transcription, translation, and apoptosis, and may also affect cell proliferation and differentiation (Igarashi et al., 2010). In some studies, spermine and spermidine suppressed experimental inflammation, coupled with the suppression of inflammatory cytokine expression (Soda et al., 2005). Spermine and spermidine can affect osteoclast differentiation (Yamamoto et al., 2012), and correlations have been observed 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).

[0042] Various dosages of spermine and / or spermidine can be administered enterally to a subject, preferably a human, to treat the metabolic diseases or disorders described herein. Spermine and / or spermidine can be administered (e.g., orally in drinking water) at concentrations of about 0.3 mM to about 3 mM, more preferably about 0.3-1 mM, or about 0.5 mM, respectively. These concentrations have been shown to be effective in mouse models of osteoporosis and aging. Experimental studies have also shown that 0.5 mM in drinking water (equivalent to 18.2 mg / kg body weight) has beneficial effects of polyamine supplementation on ovariectomy-induced bone loss and lifespan extension. In some embodiments, dosages ranging from 1-50, 2-40, 5-25, or 15-20 mg / kg body weight / day, or any range derivable therein, can be used to treat the metabolic diseases or disorders described herein.

[0043] In some aspects, the microbiota is cultured in spermine and / or spermidine prior to administration to a mammalian subject, such as a human, to treat a metabolic disease or disorder or a bone disease or disorder described herein. For example, in some embodiments, the microbiota is cultured in 0.1-10 mM, 1-7 mM, more preferably 1-5 mM, or 0.5, 1, 2, 3, 4, or 5 mM spermine and / or spermidine, or any range derivable therein, prior to administration to a subject to treat a metabolic disease or disorder. The microbiota can comprise or consist of Parabacteroides gordosteinii, Lactobacillus reuteri, and / or Lactobacillus gasseri. In some embodiments, Parabacteroides gordosteinii cultured or grown in spermine and / or spermidine is subsequently inactivated via methods described herein (e.g., heat inactivation, exposure to hydrogen peroxide, etc.); the inactivated P. gordosteinii can be included in pharmaceutical or probiotic compositions or administered intraenterally to humans to treat metabolic diseases or disorders described herein. In some embodiments, the microbiota includes live and / or inactivated Akkermansia muciniphila together with Parabacteroides gordosteinii (live and / or inactivated), Lactobacillus reuteri, and / or Lactobacillus gasseri; in some embodiments, the microbiota includes both live and inactivated Akkermansia muciniphila.

[0044] V. Pharmaceutical Formulations and Routes of Administration In another aspect, a pharmaceutical preparation (also referred to as a bacterial preparation or 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 appropriate for the indicated route of administration, for administration to a patient in need of said treatment. In some embodiments, the bacteria disclosed herein are formulated in a manner suitable for treating human and / or animal patients. In some embodiments, the formulation comprises blending or combining one or more of the bacteria disclosed herein (e.g., temperate microbiota and / or heat-inactivated Parabacteroides gordosteinii) with one or more of the following excipients: lactose, sucrose, starch flour, cellulose esters of alkanoic acid, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, gelatin, acacia, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol. In some embodiments, for example, for oral administration, the pharmaceutical formulation may be tableted or encapsulated. In some embodiments, the bacteria may be slurried 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 formulation may be subjected to pharmaceutical operations such as sterilization and / or may include carriers and / or excipients, for example, preservatives, stabilizers, wetting agents, emulsifiers, encapsulating agents such as lipids, dendrimers, polymers, proteins such as albumin, nucleic acids, buffers, etc.

[0045] In some embodiments, the pharmaceutical formulation comprises inactivated (e.g., heat-inactivated) Parabacteroides gordosteinii. Various amounts of heat-inactivated Parabacteroides gordosteinii, for example, about 1 x 10 8 ~Approx. 1×10 13cfu. In some embodiments, the heat-inactivated Parabacteroides gordosteinii is included in a pharmaceutical or probiotic composition formulated for oral or enteral administration. In the case of administration of a single bacterium or a mixture of bacterium (culture), the heat-inactivated Parabacteroides gordosteinii can be administered orally (e.g., in tablet form). In the case of fecal microbiota transplantation (FMT) from a donor, the heat-inactivated Parabacteroides gordosteinii can be added to the microbiota delivered to the gastrointestinal system, for example, via a nasogastric tube or intracolonically.

[0046] Bacterial preparations can be administered in a variety of ways, for example, orally, colonically, nasally, rectally, via a catheter, via lavage, via a nasogastric tube, by topical delivery, or by fecal microbiota transplantation (FMT). Depending on the route of administration, the bacterial compositions disclosed herein may be coated with a material to protect the composition from the action of acids and other natural conditions that may inactivate the bacterial composition. Administration of the bacterial composition may require coating the bacterial composition with a material to prevent its inactivation, or co-administration of the bacterial composition with the material. In some embodiments, the bacterial composition may be administered to a patient with a suitable carrier, such as a polymer, hydrogel, liposome, starch, or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffers. Liposomes include conventional liposomes as well as water-in-oil-in-water CGF emulsions.

[0047] Formulations can be employed to protect bacterial compositions from the harsh gastric environment (Govander et al., 2014). Gastro-resistant polymers and coatings have been shown to provide protection from the harsh gastric environment. These coatings include enteric-coated tablets and capsules that site-specifically deliver administered probiotic bacteria to the intestinal system. Such enteric coatings are often pH-selective, providing protection from the harsh gastric conditions and subsequently dissolving in the alkaline medium of the intestinal system (Calinescu et al., 2005 and Yang et al., 2002). Non-limiting examples of excipients that can be used in formulating bacterial compositions include hydroxypropyl methylcellulose phthalate and carboxymethyl high-amylose starch. Excipients can also be combined to enhance delivery of bacterial compositions to the gastrointestinal tract. For example, carboxymethyl high-amylose starch can be combined with chitosan to deliver bacterial compositions to the colon. To deliver bacterial compositions to different regions of the gastrointestinal tract, the formulation can contain different polymers with different properties or similar polymers with different properties, depending on the intended delivery site (Yang et al., 2002).

[0048] The bacterial compositions disclosed herein can also be administered orally, intracolonically, intranasally, intrarectally, via a catheter, via lavage, via a nasogastric tube, by topical delivery, or by fecal microbiota transplantation (FMT). The bacterial composition can be in the form of a dispersion. The dispersion can be prepared in glycerol, liquid polyethylene glycol, a mixture thereof, or in oil.

[0049] In some embodiments, the carrier comprises an enteric coating to reduce or delay degradation in the stomach. For example, the enteric coating can be fatty acid, wax, shellac, plastic such as phthalate, CAP, CAT, PVAP, HPMCP, or plant fiber (e.g., Hussan et al., 2012). In some embodiments, the pharmaceutical or probiotic composition may comprise chitosan-alginate beads or hydrogels. However, in some embodiments, the following is envisioned:

[0050] The bacterial compositions disclosed herein can be administered orally, 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 intimately mixed 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 formulations can, of course, vary. The amount of therapeutic agent in such pharmaceutical formulations is such that a suitable dosage will be obtained.

[0051] In some embodiments, it may be advantageous to formulate compositions into dosage unit forms for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for a patient to be treated; each unit contains a predetermined amount of a therapeutic agent calculated to produce a desired therapeutic effect in association with the necessary pharmaceutical carrier. In some embodiments, the specifications for the dosage unit forms of the present invention are dictated by and directly depend on (a) the unique characteristics of the therapeutic agent and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technology for formulating therapeutic agents to treat a selected condition in a patient. In some embodiments, the active agent(s) are administered in a therapeutically effective amount sufficient to treat a condition associated with the patient's condition. For example, the effectiveness of a bacterial composition can be evaluated in an animal model system that may predict efficacy in treating a disease in humans or another animal.

[0052] In some embodiments, the effective dose range of a therapeutic agent can be extrapolated from effective doses determined in animal studies involving a variety of different animals. The precise amount of a therapeutic composition is left to the discretion of the practitioner and is specific to each individual. Other factors that affect dosage include the patient's physical and clinical condition, the route of administration, the intended therapeutic goal, and the efficacy, stability, and toxicity of the particular therapeutic formulation.

[0053] The actual dosage of the bacterial composition of the present disclosure administered to a patient can be determined by physical and physiological factors, such as the species of animal being treated, its age, sex, weight, the severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, the patient's idiopathic disease, and the route of administration. These factors can be determined by a physician. Generally, the physician responsible for administration will determine the concentration of active ingredient(s) in the composition and the appropriate dose for the individual patient. In the event of any complications, the dosage can be adjusted by the individual physician.

[0054] The drug can be administered once or multiple times.The desired time interval for multiple administration can be determined by those skilled in the art by simply using routine experimentation.For example, the patient can be administered twice a day at intervals of about 12 hours.In some embodiments, the drug is administered once a day.

[0055] The composition containing the bacterial composition (e.g., heat-inactivated Parabacteroides gordosteinii) 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 include identical or varying lengths of time, as long as the schedule is predetermined. For example, a regular schedule can include administration twice daily, daily, every other day, every third day, every fourth day, every fifth day, every sixth day, every fifth day, every sixth day, once weekly, once monthly, or any number of days or weeks in between. Alternatively, a predetermined regular schedule can include twice daily administration for the first week, followed by once daily administration for several months. In other aspects, the present invention provides that the agent(s) can be taken orally, and the timing may or may not depend on food intake. Thus, for example, the agent can be taken every morning and / or every evening, regardless of when the patient has eaten or will eat.

[0056] VI. Bacterial extracellular vesicles In some embodiments, the extracellular vesicles derived from the bacteria described herein can be administered to a subject to treat metabolic diseases or disorders. For example, extracellular vesicles (EVs) can be produced by methods described in, for example, Chelakkot et al., 2018, or Choi et al., 2015. EVs derived from Parabacteroides gordosteinii, Lactobacillus gasseri, or Lactobacillus reuteri are expected to be used to treat metabolic diseases or disorders described herein (e.g., obesity, type 2 diabetes, and fatty liver).

[0057] Extracellular vesicles (EVs) are lipid bilayer structures secreted by enteric bacteria, including both Gram-negative and Gram-positive bacteria (Ellis and Kuehn, 2010, Lee et al., 2009). Various 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; Kim et al., 2013). EVs released by bacteria may play diverse roles in the microbial community, and some data suggest they can transfer genetic material and proteins from bacteria to the host (Kuehn and Nesty, 2005). EVs can directly interact with immune and epithelial cells, initiating several signaling pathways and potentially influencing or mediating host-pathogen interactions.

[0058] For example, in some embodiments, EVs can be prepared by the following approach: Bacterial species or temperate microbiota are cultured under aerobic or anaerobic conditions (e.g., 95% N, 5% CO at 37°C) until desired (e.g., until the optical density at 600 nm reaches 1.5, as previously described in Derrien et al., 2004). EV isolation is performed as previously described in Kang et al., 2013. More specifically, bacterial cultures are pelleted at 10,000g for 20 minutes, and the supernatant is filtered through a 0.45μm vacuum filter. The filtrate is concentrated, for example, using a QuixStand (GE Healthcare, Little Chalfont, UK), and then filtered through a 0.22μm bottle-top filter. The filtrate is then pelleted by ultracentrifugation (e.g., 150,000g in a 45 Ti rotor at 4°C for 2 hours). The final pellet is then resuspended in phosphate-buffered saline (PBS) and stored at -80 °C. EVs can be analyzed, if desired, 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 quantity of EVs obtained.

[0059] VII. Metabolic Diseases and Disorders It is expected that various metabolic diseases or disorders can be treated using the methods and bacterial compositions described herein. For example, the bacterial compositions described herein (e.g., live bacteria, heat-inactivated bacteria, lyophilized bacteria, bacteria in a pharmaceutical composition, or extracellular vesicles secreted from the bacteria) can be administered into the intestine or to the gastrointestinal tract of a subject to treat metabolic diseases or disorders. In some embodiments, the bacterial composition is heat-inactivated Parabacteroides gordosteinii. In some embodiments, the metabolic disease or disorder is obesity, fatty liver disease, type 2 diabetes, insulin intolerance, or dyslipidemia. Without wishing to be bound by any theory, the following examples demonstrate that the methods and bacterial compositions provided herein (e.g., heat-inactivated Parabacteroides gordosteinii) can be used to improve glucose tolerance and metabolism, increase insulin secretion, and reduce fatty liver.

[0060] The prevalence of obesity, as measured by body mass index, has risen to unacceptable levels in both men and women in the United States and worldwide, resulting in concerns about its detrimental health effects. Genetic, environmental, and behavioral factors influence the development of obesity. In adults, the classification system (World Health Organ Tech Rep Ser., 2000) and obesity guidelines (Jensen et al., 2014) define a BMI of 18.5–24.9 kg / m2 as the most common obesity risk. 2 Healthy weight: 25.0-29.9 kg / m 2 Overweight: 30kg / m 2 Obesity is defined as a BMI above the 90th percentile of ideal weight. In children and adolescents, the Centers for Disease Control and Prevention (CDC) BMI-for-Age growth chart defines a BMI above the 95th percentile of ideal weight as overweight and above the 90th percentile of ideal weight as obese. Obesity is associated with and contributes to, among other things, shorter life expectancy, type 2 diabetes mellitus, 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-dependent manner, with greater weight loss associated with better outcomes.

[0061] Fatty liver disease, also known as hepatic steatosis, is a condition in which excess fat accumulates 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 global prevalence continues to increase with the obesity epidemic. Nonalcoholic fatty liver disease is the most common cause of elevated liver enzymes. Of the NAFLD spectrum, only nonalcoholic steatohepatitis usually progresses to cirrhosis and hepatocellular carcinoma (Vernon et al., 2011). The prevalence and impact of NAFLD continue to increase with the obesity epidemic.

[0062] Type 2 diabetes (T2D), formerly known as adult-onset diabetes, is a form of diabetes characterized by hyperglycemia, insulin resistance, and a relative lack of insulin. T2D can be diagnosed using a glycosylated hemoglobin (A1C) test, which measures average blood glucose levels, as well as random blood glucose tests, fasting blood glucose tests, or oral glucose tolerance tests. T2D is the most common form of diabetes and can be caused by several factors, including obesity, physical inactivity, and genetics. Insulin can help achieve ideal hemoglobin A1C targets for T2D patients (Wallia et al., 2014). Obesity, which can sometimes lead to insulin resistance, is common in T2D patients. In some embodiments, the bacterial compositions disclosed herein (e.g., heat-inactivated Parabacteroides gordosteinii) are administered to a mammalian subject, such as a human, in combination with another therapeutic agent for type 2 diabetes, such as metformin, a sulfonylurea, a meglitinide, a thiazolidinedione, a DPP-4 inhibitor, a GLP-1 receptor agonist (e.g., exenatide), an SGLT2 inhibitor, or insulin.

[0063] Dyslipidemia is characterized by abnormal blood lipid levels, such as elevated plasma cholesterol, triglycerides (TG), or both; elevated low-density lipoprotein (LDL) or very-low-density lipoprotein (VLDL) levels; low high-density lipoprotein cholesterol (HDL) levels; or low HDL cholesterol levels.In some embodiments, dyslipidemia is hyperlipidemia (increased blood lipids).Dyslipidemia can contribute to the development of atherosclerosis.

[0064] VIII. Temperature and Intestinal Microbiology As shown herein, exposure to a warm environment can alter the microbiota of a mammalian subject; the resulting "warm microbiota" has been shown herein to produce effects including improved glucose tolerance and reduced hepatic steatosis, which may be particularly beneficial for the treatment of metabolic diseases or disorders such as obesity, type 2 diabetes, and fatty liver. Some living organisms adapt to constant changes in their surrounding environment. One such external parameter is temperature, which can vary from below -35°C to above 40°C and is dependent on seasonal periodicity and the time of day.

[0065] Warm-blooded animals must maintain a constant body temperature; as a result, they have developed various strategies to adapt to these environmental fluctuations. In rodents, cold exposure involves thermogenic programs, including shivering thermogenesis from muscle and non-shivering thermogenesis from adipose tissue. In contrast, during warm exposure, the thermogenic program is blunted, resulting in a corresponding decrease in energy expenditure (Kaiyala et al., 2012). Furthermore, to dissipate excess heat, rodents enhance cutaneous vasodilation in specific areas with a high surface-to-body ratio, maximizing heat loss, such as the ears and tail (Meyer et al., 2017). Interestingly, rare reports suggest that upon prolonged exposure to elevated temperatures, rodents adapt to maximize their heat dissipation capacity by increasing the length / surface of their tails and ears (Alhilli and Wright, 1983; Ashoub, 1958; and Harland, 1960).

[0066] The gut flora has been shown to affect several aspects of host physiology. Adaptation to cold exposure has been shown to be partially mediated by the gut microbiota (Chevalier et al., 2015). This disclosure demonstrates that warm exposure benefits metabolic characteristics (including improved insulin sensitivity, improved glucose tolerance, and reduced hepatic steatosis), and that alterations in the gut flora play a role in these changes. Therefore, these beneficial effects can be utilized in the treatment of metabolic diseases and disorders. [Example]

[0067] IX. Working Example The following examples are included to demonstrate preferred embodiments of the invention. It will be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention and, as such, are to be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0068] Example 1 Warm exposure and heat-inactivated Parabacteroides gordosteinii improve glucose metabolism and reduce hepatic steatosis in a postmenopausal mouse model. To assess the effects of warm temperatures on glucose metabolism, 8-week-old male mice were housed at 34°C for 1 month. Their glucose tolerance was monitored, and the warm-exposed animals showed improved glucose tolerance after an oral glucose load (Figure 1A), which was associated with increased insulin secretion (Figure 1B). Next, we investigated whether similar effects would be observed in metabolically impaired conditions, such as postmenopausal weight gain and glucose intolerance. When ovariectomy was used to mimic postmenopausal weight gain and glucose intolerance, estrogen deficiency worsened glucose tolerance, as observed during an oral glucose tolerance test (OGTT) (Figure 2A). Interestingly, the impaired glucose tolerance was rescued when the mice were exposed to warm temperatures (Figure 2B), indicating that warm exposure improves glucose tolerance in a postmenopausal mouse model.

[0069] Because warm exposure can alter the composition of the microbiota, we investigated whether some of the most predominantly altered bacteria could mimic the warm exposure phenotype. Among the most consistently altered bacteria were Parabacteroides gordosteinii, Akkermansia muciniphila, Lactobacillus reuteri, and Lactobacillus gasseri, all of which increased during warm exposure. To assess their individual effects on the observed phenotype, these bacteria were freshly grown and administered orally every other day to ovariectomized mice.

[0070] Additional studies were conducted using heat-inactivated (HI) Parabacteroides gordosteinii. Supplementation with heat-inactivated Parabacteroides gordosteinii (OVA Gold) was able to prevent ovariectomy-induced hyperglycemia, and this effect was observed after 12 or 6 hours of fasting (Figure 3A). Furthermore, supplementation with HI Parabacteroides gordosteinii improved oral glucose tolerance (Figure 3B) and, in part, insulin sensitivity (Figure 3C) in ovariectomized mice. The effect of HI Parabacteroides gordosteinii was observed to be more pronounced than that mediated by the bacterium Akkermansia muciniphila, which has been described to improve metabolic status in obesity. HI P. gordosteinii was also observed to reduce lipid accumulation in the liver (Figure 4). These data support the idea that HI P. gordosteinii could be used to treat or prevent fatty liver disease.

[0071] Supplementation with heat-inactivated P. gordosteinii also increased the levels of P. gordosteinii detected 48 hours after the last administration. As shown in Figure 5, supplementation with heat-inactivated P. gordosteinii prevented the loss of endogenous P. gordosteinii induced by ovariectomy. While not wishing to be bound by any theory, because clearance of supplemented heat-inactivated P. gordosteinii should not normally take more than 24 hours, one possibility is that this treatment may suppress the loss of endogenous P. gordosteinii induced by ovariectomy or promote its proliferation.

[0072] To further explore the therapeutic potential of heat-inactivated Parabacteroides gordosteinii in improving metabolic outcomes, we can test the effects of supplementation with heat-inactivated Parabacteroides gordosteinii in a mouse model of diet-induced obesity. It is also possible to obtain secretory vesicles from Parabacteroides gordosteinii and use them to test whether they can replicate the effects of supplementation with heat-inactivated Parabacteroides gordosteinii. Because supplementation of ovariectomized mice with Lactobacillus reuteri and Lactobacillus gasseri also improved glucose tolerance and insulin sensitivity, secretory vesicles from these bacteria can be isolated and used to determine whether they exhibit similar therapeutic effects. Growth media from Parabacteroides gordosteinii, Lactobacillus reuteri, and / or Lactobacillus gasseri can be obtained and further tested, for example, in a mouse model of diet-induced obesity.

[0073] Example 2 Obesity treatment methods Browning of visceral adipose tissue: To address the importance of the microbiota during long-term cold exposure, we exposed mice treated with antibiotics (Abx) and depleted of the microbiota to cold (referred to as "combined treatment"). Surprisingly, our preliminary results not only showed that the mice exhibited a further improvement in glucose tolerance compared to untreated or single-stimulus-treated mice, but also showed that cold exposure of the microbiota-depleted mice led to a massive and preferential loss of fat from the VAT (Figures 6A-6B). Under non-stimulus conditions, all treatment groups showed a significant increase in the [ 3 H]-2-deoxyglucose ([ 3 Although cold-exposed microbiota-depleted mice showed a similar increase in glucose uptake ([H]-2-DG), VAT from cold-exposed microbiota-depleted mice showed significantly higher glucose uptake compared to all other groups (Figure 6C). Cold-exposed microbiota-depleted mice showed a dramatic decrease in VAT mass despite increased glucose uptake in this tissue, even under basal conditions, suggesting that the combined treatment induces remodeling and increased energy dissipation primarily restricted to visceral fat.

[0074] As shown in Figures 6A-6C, a significant increase in glucose uptake in epiVAT and rpVAT was observed in animals after the combination treatment, along with a decrease in total epiVAT and rpVAT volume. To gain further insight into the morphological changes in epiVAT, H&E staining of epiVAT and rpVAT sections from mice in all groups was performed. Notably, in contrast to control animals or single-treated cold-exposed or abx animals, the combination treatment resulted in approximately 40% multilocular cells within the VAT depot (Figure 7A). This multilocular appearance is a classic characteristic of beige adipocytes in vivo. Accordingly, visceral adipocytes from cold-exposed microbiota-depleted mice exhibited increased oxygen consumption rates compared to RT-treated or single-treated cold-exposed mice (Figure 7B). Together, these data (combined with the gene expression data shown in Figures 8A-8E) strongly suggest that the combination of cold exposure and microbiota depletion leads to extensive VAT remodeling and the appearance of multilocular cells typical of beige fat, a phenomenon unique to the visceral adipose tissue depot in dual-treated animals. These data support the idea that significant visceral fat browning is possible without genetic intervention and indicate that VAT browning can contribute to altered host physiology and metabolic state.

[0075] To gain insight into the requirement for UCP1 in visceral fat browning and remodeling, we compared groups of Ucp1-KO mice maintained at RT or cold, or after combined cold and abx treatment. Interestingly, Ucp1-KO mice responded similarly to WT mice; combined treatment significantly improved glucose tolerance and promoted the development of multilocular cells in the visceral fat depot of cold / abx combined-treatment mice (Figures 8A-8B). Gene expression profiling of epiVAT in WT mice showed that all thermogenic markers, except for Ucp1, were significantly increased after combined treatment compared to the remaining groups. Specifically, cold exposure increased Ucp1 expression in pgVAT of WT mice, but its levels did not differ after combined cold / abx treatment (Figure 8C). Consistent with these observations and the data from WT mice, Ucp1-KO animals showed a similar increase in thermogenic gene expression as WT controls after combined treatment; this further suggests that UCP1 is nonessential for visceral fat browning. Without wishing to be bound by any theory, these data support the idea that the emergence of visceral multilocular adipocytes and increased thermogenic gene expression can occur in a UCP1-independent manner and emphasize the importance of uncovering the molecular mechanisms orchestrating this process.

[0076] Identification of bacteria with beneficial metabolic effects Using a multi-omics approach, we identified Parabacteroides gordosteinii as consistently deregulated in various conditions, leading to improved glucose tolerance and weight loss. Because Parabacteroides gordosteinii is classified as a commensal bacterium but potentially pathogenic, we examined its metabolic effects using heat-inactivated (HI) bacteria. Oral supplementation with HI Parabacteroides gordosteinii abolished ovariectomy-induced hepatic lipid accumulation (e.g., Figure 9). These data support the idea that this bacterium may have a protective effect against fatty liver disease. Furthermore, it also improved overall glycemic control, reducing blood glucose levels and ovariectomy-induced hyperglycemia.

[0077] To investigate whether oral administration of Parabacteroides gordosteinii can treat diet-induced obesity in vivo, we performed the following experiment. C57Bl / 6 mice were fed a high-calorie diet (HCD) for 3 months. Subsequently, the mice were orally administered Parabacteroides gordosteinii. Body weight measurements showed that Parabacteroides gordosteinii suppressed weight gain in HCD-fed mice to a similar extent as chow-fed controls (Figure 10). These effects were consistent with reduced adipose tissue mass (Figure 11A) and lower liver weight (Figure 11B), which was consistent with reduced liver triglyceride content. These results support the idea that Parabacteroides gordosteinii can ameliorate diet-induced obesity, leading to weight loss and improved liver health. The full version of these data was provided to the Clayton Foundation.

[0078] Polyamines promote the growth of beneficial symbiotic bacteria Next, we investigated whether the presence of polyamines in the growth medium affected the growth of several metabolically beneficial bacteria. Supplementation of various concentrations of spermine and spermidine (Figure 12) enhanced the growth of Parabacteroides gordosteinii, Lactobacillus reuteri, and / or Lactobacillus gasseri, all of which were identified as metabolically beneficial bacteria. To further investigate whether the general growth of bacteria present in fresh fecal samples was affected, fresh mouse fecal contents were inoculated into growth medium supplemented with various concentrations of spermine or spermidine. Both polyamines increased the optical density during the exponential growth phase (Figure 13), indicating that polyamines promoted the growth of bacteria in the bacterial mix.

[0079] Aging is associated with a decline in polyamine levels, and polyamine supplementation may prevent several age-related diseases, such as memory impairment, cardiovascular disease, and cancer, and may extend the lifespan of some organisms. To investigate whether some of these beneficial effects are mediated in part by changes in the microbiota, we administered a mixture of spermine and spermidine to mice in vivo and transplanted the microbiota from the polyamine-treated animals into ovariectomized mice. Surprisingly, the polyamine-adapted microbiota reduced total fat mass due to reductions in subcutaneous, visceral, and brown adipose tissue. Transplantation of a control warm-adapted microbiota had no effect on these parameters.

[0080] Furthermore, the polyamine-adapted microbiota also reduced total liver weight (Figure 14). These data indicate that microbiota changes induced by the presence of polyamines in vivo can have beneficial metabolic effects, supporting such therapeutic strategies.

[0081] Example 3 material and method Animals: All C57BL / 6J mice were purchased from Janvier Labs and housed in individually ventilated cages in a specific pathogen-free (SPF) facility. All mice were housed under a 12-hour day / night cycle and fed a standard chow diet (total energy 16.2 MJ / kg; 9 kJ% fat, 33 kJ% protein, 58 kJ% carbohydrate; V1534-727, Ssniff, Germany). All mice were males, enrolled at 8 weeks of age, or females (for ovariectomy experiments) enrolled at 16 weeks of age. Acclimatized animals were assigned to groups based on body weight to ensure an equal starting point. Heat exposure was performed under SPF conditions in individually ventilated cages in a light- and humidity-controlled climate chamber (TSE, Germany) at 34°C. All mice were sacrificed after 5 hours of fasting. All animal experiments were approved by the Swiss Federal and Geneva Cantonal Authorities for Animal Experimentation (Office Veterinaire Federal and Commission Cantonale pour les Experiences sur les animaux de Geneve).

[0082] Ovariectomy: Mice were anesthetized with xylazine / ketamine (120 μl of a mixture of 120 mg / kg ketamine and 16 mg / kg xylazine was injected) and the hair was shaved under the dorsal ribs. Betadine was applied to the area for proper disinfection. After a 1-2 cm incision was made through the skin and muscle layer just below the ribs, the ovaries were located, the fallopian tubes were ligated with dissolvable sutures, and the ovaries were removed. The muscle layer was sutured with dissolvable sutures, and the wound was closed with staples and disinfected. The same procedure was performed on the contralateral side. Tamgesic was administered 4 hours after surgery, and the staples were removed under isoflurane anesthesia 7 days after surgery. Sham-operated animals underwent the same procedure without fallopian tube ligation and ovarian removal.

[0083] Microbiota transplantation: In microbiota transplant experiments in ovariectomized mice (with existing conventional microbiota), donor fecal pellets were freshly collected every two days and immediately homogenized in 1 ml of anaerobic PBS. After a brief centrifugation (300 g, 30 seconds), the supernatant was immediately administered orally to each recipient. In this condition, one cage of donors (one pellet per animal from both mice) was used to repopulate one cage of recipients. Each recipient received 200 μl of the donor mixture every two days.

[0084] Single-microorganism transplants: Lactobacillus gasseri (DSM 20604) and Parabacteroides gordosteinii (DSM 19948) were purchased from DSMZ. Lactobacillus reuteri (PTA-6475) and Akkermansia muciniphila (BAA835) were purchased from ATCC. Lactobacillus gasseri and L. reuteri were grown in MRS medium (deMan, Rogosa, and Sharpe, USbiological Life Sciences, L1021-01), P. gordosteinii was grown in anaerobe basal broth (Thermo Scientific Oxoid Microbiology Products, CM0957), and A. muciniphila was grown in Schaedlers broth plus vitamin K3 (Biomerieux ref. 42106) in an anaerobic incubator (Coy vinyl anaerobic chamber type C) set at 37°C using a gas mixture of 5% CO2, 5% H2, and 90% N2. Freshly prepared bacteria were diluted in anaerobic PBS to a final concentration equivalent to 1 OD at 600 nm. Ovariectomized mice were orally gavaged with 300 μl of this suspension every other day for 2 months, starting on postoperative day 3, until sacrifice. Oral gavage of the bacterial suspension to mice fed a high-calorie diet (HCD) was initiated 1 week after the start of the HCD to examine the protective effect against diet-induced obesity, and 3 months after the HCD to examine the healing effect. The P. gordosteinii preparation was heat-inactivated at 100°C for 15 minutes before administration to confirm its inactivation.

[0085] Metabolic experiments: Oral glucose tolerance tests (OGTTs) were performed by oral gavage of a glucose bolus (2 mg / kg body weight) after a 10-h overnight fast. Insulin tolerance tests were performed in the morning after a 5-h fast by intraperitoneal injection of 0.5 U / kg (I9278, Sigma-Aldrich). Insulin levels during the OGTT were measured using a Mouse Insulin ELISA kit (ref. 10-1247-01, Mercodia) according to the manufacturer's instructions.

[0086] Liver lipid measurement: Lipids were extracted from 50 mg of liver using 1 ml of hexane:isopropanol (3:2) in a bead-based TissueLyser device (Qiagen) by shaking at 30 Hz for 30 seconds in the presence of one bead per tube. The lysate was spun at full speed in a tabletop centrifuge for 3 minutes. The supernatant was removed. The pellet was re-extracted with 0.5 ml of hexane:isopropanol, spun again, and the supernatants were combined. 0.5 ml of a 1 g / 15 ml Na2SO4 solution was added, and the tube was mixed. The sample was spun at full speed for 3 minutes, and the upper organic phase was collected in a pre-weighed Eppendorf tube. After overnight evaporation, the tube was reweighed, and the lipid weight was recorded.

[0087] In vivo polyamine supplementation and inhibitor treatment: Six-week-old C57BL / 6J female mice were administered a freshly dissolved mixture of spermine (Sigma-Aldrich) and spermidine (Sigma-Aldrich) in drinking water at a concentration of 0.5 mM every other day at room temperature for an additional 45 days. Diaminazene acetureate (Sigma-Aldrich) was added to drinking water at a concentration of 50 μM and supplemented every other day for 45 days to 16-week-old C57BL / 6J female mice; the mice were maintained in a temperature-controlled chamber at 34°C in a conventional facility and provided with food and water ad libitum.

[0088] Micro-CT analysis: Mice were scanned with a micro-CT (VivaCT40 / Scanco system; Zurich, Switzerland). Before ovariectomy, limbs were scanned in vivo to assess basal conditions. After xylazine / ketamine anesthesia, mouse limbs were scanned for 18 minutes. Final scans were performed postmortem on isolated bones. For the trabecular bone area of ​​the femur and tibia, 100 slices were analyzed, starting from 50 slices below the distal growth plate. The cortical structure of the femur and tibia was assessed through 60 consecutive CT slides (600 μm) from the midshaft of the bone. Images were segmented using an adaptive-iterative thresholding approach rather than a fixed threshold. Morphometric variables were calculated from the binarized images using a direct 3D technique that does not rely on a priori assumptions about the underlying structure. For trabecular bone area, bone volume / total volume (BV / TV) is assessed. For cortical bone of the mid-shaft of the femur and tibia, cortical bone volume (mm3) and mean cortical thickness (μm), also called cortical width, are measured.

[0089] Bone biomechanical analysis: A three-point bending test was used to measure bone biomechanical parameters. The femur was mounted on two supports separated by a distance of 9.9 mm, and a load was applied to the midpoint of the shaft (creating three-point bending). Mechanical fracture resistance (displacement and applied load) was measured using a servo-controlled electromechanical system (Instron 1114, Instron, High Wycombe, UK) by displacing the actuator at 2 mm / min. Ultimate force (maximum load, measured in Newtons [N]), yield point (N), stiffness (elastic energy, N / mm), and fracture energy (subsurface of the curved plastic zone, N*mm) were calculated. Young's modulus (MPa) was determined by a previously described formula.

[0090] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods and in the steps or in the order of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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 invention as defined by the appended claims.

[0091] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007761556000001.tif190137TIFF0007761556000002.tif142128

Claims

1. 1. A composition for use in a method of treating a metabolic disease or disorder in a mammalian subject, comprising inactivated Parabacteroides goldsteinii, The method includes administering the composition to the gastrointestinal system of the subject; The metabolic disease or disorder comprises obesity, fatty liver disease, or insulin resistance. The composition.

2. 2. The composition of claim 1, wherein the inactivated Parabacteroides gordosteinii is heat-inactivated.

3. 2. The composition of claim 1, wherein the inactivated Parabacteroides gordosteinii is inactivated via exposure to peroxide.

4. 4. The composition of claim 3, wherein the peroxide is hydrogen peroxide.

5. 4. The composition of claim 3, wherein the peroxide is hydrogen peroxide vapor.

6. 2. The composition of claim 1, wherein the inactivated Parabacteroides gordosteinii is inactivated through exposure to radiation or ionizing radiation.

7. 7. The composition of claim 6, wherein the radiation comprises or consists of light having a wavelength of about 400 to 420 nm.

8. 2. The composition of claim 1, wherein the inactivated Parabacteroides gordosteinii is inactivated via exposure to air plasma, ultrasound under pressure, alcohol, high hydrostatic pressure (HHP), or pulsed electric fields (PEF).

9. 9. The composition of claim 8, wherein the alcohol is ethanol.

10. Approximately 1×10 8 ~Approx. 1×10 13 10. The composition according to any one of claims 1 to 9, comprising inactivated cfu of Parabacteroides gordosteinii.

11. 11. The composition of any one of claims 1 to 10, further comprising Lactobacillus gasseri, Lactobacillus reuteri, or Akkermansia muciniphila.

12. 12. The composition of any one of claims 1 to 11, further defined as a pharmaceutical composition.

13. 12. The composition of any one of claims 1 to 11, further defined as a probiotic composition.

14. 14. The composition of any one of claims 1 to 13, further comprising Lactobacillus gasseri or Lactobacillus reuteri.

15. The composition of any one of claims 1 to 13, further comprising extracellular vesicles derived from Lactobacillus gasseri or Lactobacillus reuteri.

16. 16. The composition of any one of claims 12 to 15, wherein the pharmaceutical or probiotic composition is administered orally, colonically, via enema, via an orogastric tube, or via a nasogastric tube.

17. 17. The composition of any one of claims 12 to 16, wherein the inactivated Parabacteroides gordosteinii is contained in a pharmaceutical or probiotic composition that is resistant to degradation in a subject's stomach but releases the bacteria in the small intestine and / or large intestine.

18. The composition of any one of claims 12 to 17, wherein the pharmaceutical or probiotic composition comprises an enteric coating, chitosan-alginate beads, or a hydrogel.

19. 19. The composition of claim 18, wherein the enteric coating is a fatty acid, a wax, a shellac, a plastic such as a phthalate, CAP, CAT, PVAP, HPMCP, or a vegetable fiber.

20. 18. The composition of any one of claims 12 to 17, wherein the pharmaceutical or probiotic composition does not include an enteric coating.

21. 21. The composition of any one of claims 12 to 20, wherein the pharmaceutical or probiotic composition is a tablet or capsule.

22. The composition of any one of claims 1 to 21, wherein the subject is a human.

23. 23. The composition of claim 22, wherein the human is a postmenopausal woman.

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

25. 25. The composition of claim 24, wherein the fatty liver disease is non-alcoholic fatty liver disease (NAFLD).

26. The composition according to any one of claims 1 to 25, wherein the Parabacteroides gordosteinii in the composition is purified or cultured.

27. 27. The composition of any one of claims 1-2 and 10-26, wherein the Parabacteroides gordosteinii has been inactivated by heating to about 95-105°C for about 10-20 minutes.

28. 28. The composition of claim 27, wherein the Parabacteroides gordosteinii is inactivated by heating to about 100°C for about 15 minutes.

29. 29. The composition of any one of claims 1 to 28, wherein the method further comprises enterally administering spermine and / or spermidine to the subject.

30. 30. The composition of claim 29, wherein the method comprises enterally administering both spermine and spermidine to the subject.

31. 30. The composition of claim 29, wherein the method comprises administering to the subject about 1 to 50 mg / kg body weight / day of spermine.

32. 30. The composition of claim 29, wherein the method comprises administering to the subject about 1 to 50 mg / kg body weight / day of spermidine.

33. 33. The composition of any one of claims 1 to 32, comprising spermine and / or spermidine.

34. 34. The composition of claim 33, wherein the composition comprises both spermine and spermidine.

35. 27. The composition of claim 26, wherein the inactivated Parabacteroides gordosteinii is cultured or grown in a medium containing spermidine or spermine.

36. 36. The composition of claim 35, wherein the medium comprises about 0.1 to 6 mM spermidine.

37. 36. The composition of claim 35, wherein the medium comprises about 0.1 to 6 mM spermine.

38. 1. A pharmaceutical or probiotic composition for treating a metabolic disease or disorder in a mammalian subject, comprising inactivated Parabacteroides gordosteinii, Formulated for delivery to the gastrointestinal system, The metabolic disease or disorder comprises obesity, fatty liver disease, or insulin resistance. The pharmaceutical or probiotic composition.

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