Shelf-stable preparation of ammonia-oxidizing microorganisms

Abstract

A method for distributing a preparation, the method comprising providing a preparation comprising an ammonia-oxidizing microorganism (AOM), wherein during distribution, the preparation is exposed to a temperature greater than about 4°C. [Solution] Also disclosed is a method of treating a subject comprising administering a preparation, wherein less than 70% of the AOM is viable. Also disclosed is a method of treating a subject comprising administering a preparation at room temperature. Also disclosed is a method of treating autosomal dominant hyper-IgE syndrome (AD-HIES) with a preparation comprising AOM. Also disclosed is a method of treating X-linked immunodysregulation polyendocrinopathy enteropathy (IPEX) with a preparation comprising AOM. Also disclosed is a method of producing a shelf-stable product from the preparation. Also disclosed is a shelf-stable preparation of AOM.

Description

[Technical Field]

[0001] Technology field Aspects relate generally to the microbiome, and more specifically to restoring ammonia-oxidizing microorganisms associated with the microbiome. [Background technology]

[0002] background Bacteria and other microorganisms are ubiquitous in the environment. The discovery of pathogenic bacteria and the bacteriology of disease have had a profound impact on health and disease states. Microorganisms are a normal part of the environment of all living things and can even be beneficial. For example, in the intestine, bacteria are not pathogenic under normal conditions and may actually improve health by making normal intestinal contents inhospitable to disease-causing organisms. Summary of the Invention [Means for solving the problem]

[0003] overview According to one aspect, a method of distributing a preparation is provided. The method can include providing a preparation comprising live ammonia-oxidizing microorganisms (AOM). In some embodiments, during distribution, the packaged end-use container is exposed to an environment having a temperature greater than about 4°C.

[0004] According to another aspect, a method of treating a subject is provided. The method can include administering to the subject a therapeutically effective amount of a preparation comprising ammonia-oxidizing microorganisms (AOM). In some embodiments, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable.

[0005] According to another aspect, a method of treating a subject is provided. The method can include administering to the subject a therapeutically effective amount of a preparation comprising an ammonia-oxidizing microorganism (AOM). In some embodiments, the preparation is at a temperature above or about room temperature, e.g., between about 20°C and 25°C, at the time of administration.

[0006] According to another aspect, there is provided a method of treating autosomal dominant hyper-IgE syndrome (AD-HIES) in a subject, which may include administering to the subject a therapeutically effective amount of a preparation comprising ammonia-oxidizing microorganisms (AOM).

[0007] According to another aspect, there is provided a method of treating X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX) in a subject, which may include administering to the subject a therapeutically effective amount of a preparation comprising an ammonia-oxidizing microorganism (AOM).

[0008] According to another aspect, a method for producing a shelf-stable cosmetic, therapeutic, or consumer product is provided. The method may include formulating a preparation containing ammonia-oxidizing microorganisms (AOM) into a powder, cream, ointment, or lotion. The method may include packaging the preparation into the cosmetic, therapeutic, or consumer product. In some embodiments, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms in the cosmetic or therapeutic product are viable.

[0009] The method may include providing a preparation comprising live ammonia-oxidizing microorganisms.

[0010] In some embodiments, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia oxidizing microorganisms are viable.

[0011] In some embodiments, the preparation is exposed to an environment having a temperature greater than about 4°C.

[0012] In some embodiments, the preparation is packaged into a container.

[0013] In some embodiments, the preparation is packaged into multiple containers, for example, at least 2, 3, 6, 8, 10, or 20 containers.

[0014] In some embodiments, the preparation is packaged into an end-use container.

[0015] In some embodiments, the preparation is packaged into a plurality of separate end-use containers, for example, at least 2, 4, 6, 8, 10, 20, 50, or 100 end-use containers.

[0016] The method may include the step of providing (or having a designee provide) the formulation or a packaged end-use container to the recipient.

[0017] In some embodiments, during distribution, the preparation or packaged end-use container is exposed to an environment having a temperature greater than about 10°C.

[0018] In some embodiments, during distribution, the formulation or packaged end-use container is exposed to an environment having a temperature of room temperature, for example, between about 20°C and 25°C.

[0019] In some embodiments, during distribution, the preparation or packaged end-use container reaches a temperature greater than about 4°C.

[0020] In some embodiments, during distribution, the preparation or packaged end-use container reaches a temperature greater than about 10°C.

[0021] In some embodiments, during distribution, the formulation or packaged end-use container reaches a temperature above room temperature, for example, between about 20°C and 25°C.

[0022] In some embodiments, during distribution, the preparation or packaged end-use container is exposed to the environment for a period of at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years.

[0023] The environment may be a storage environment.

[0024] The environment may be a delivery environment, for example, a postal or commercial delivery environment.

[0025] The environment may be a delivery environment, for example a cargo or freight shipping delivery environment.

[0026] In some embodiments, during distribution, the preparation or packaged end-use container reaches a temperature greater than about 4°C for at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years.

[0027] In some embodiments, during distribution, the preparation or packaged end-use container reaches a temperature greater than about 10° C. for at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years.

[0028] In some embodiments, during distribution, the preparation or packaged end-use container reaches a temperature above about room temperature, e.g., a temperature between about 20°C and 25°C, for at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years.

[0029] In some embodiments, the step of supplying (or having a designee supply) the preparation or packaged end-use container to the recipient may include making the packaged end-use container available on an internet-based outlet.

[0030] In some embodiments, the step of supplying (or having a designee supply) the preparation or packaged end-use container to the recipient may include making the packaged end-use container available through a non-internet-based outlet, e.g., a store.

[0031] In some embodiments, for example, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are viable upon packaging into the end-use container.

[0032] In some embodiments, for example, upon packaging into the end-use container, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are active.

[0033] In some embodiments, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable after distribution.

[0034] In some embodiments, after distribution, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are inactive.

[0035] In some embodiments, for example, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable upon packaging into an end-use container.

[0036] In some embodiments, for example, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are inactive upon packaging into the end-use container.

[0037] In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9%, or substantially all of the ammonia-oxidizing microorganisms are heat-killed, e.g., prior to packaging.

[0038] In some embodiments, the container, eg, the end-use container, comprises a polymeric bottle, eg, a spray, aerosol, or mist bottle.

[0039] In some embodiments, the container, eg, the end-use container, comprises a squeezable container, eg, a squeeze bottle or a tube.

[0040] In some embodiments, the container, eg, the end-use container, is substantially free of a vacuum bag.

[0041] In some embodiments, the container, for example, the end use container, is not configured to impede or reduce retrograde flow.

[0042] In some embodiments, the container, e.g., the end-use container, is made of a polymer, e.g., polyethylene terephthalate (PET), high density polyethylene (HDPE), polypropylene, polycarbonate, polytetrafluoroethylene (Teflon®), polyvinylidene fluoride (PVDF), or cellulosic. , glass, aluminum, or cardboard.

[0043] In some embodiments, the container, for example, the end-use container, is configured to allow the passage of oxygen.

[0044] In some embodiments, the container, e.g., the end-use container, is configured to allow at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 percent of the transmission of ionizing radiation, e.g., from an isotope, e.g., cobalt-60, e.g., by gamma rays, e.g., by x-rays, or by ultraviolet light, e.g., ultraviolet C (UVC), to pass through the container.

[0045] The method can include treating a disease or disorder modulated by activated immune cells in a subject.

[0046] In some embodiments, the activated immune cells are T helper cells or regulatory T cells.

[0047] In some embodiments, the activated immune cells are T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17), or regulatory T cells (Treg).

[0048] In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia oxidizing microorganisms are inactive.

[0049] In some embodiments, administration provides upregulation, activation, downregulation, or suppression of cytokines associated with activated immune cells, such as IL-5, IL-13, IL-4, IFNγ, IL-12, IL-2, IL-18, IL-17, IL-21, IL-22, IL-10, and TFG-β.

[0050] In some embodiments, the preparation is at a temperature greater than about 4° C. at the time of administration.

[0051] In some embodiments, the preparation is at a temperature greater than about 10° C. at the time of administration.

[0052] In some embodiments, the preparation is at a temperature above about room temperature, eg, between about 20°C and 25°C, at the time of administration.

[0053] In some embodiments, a period of at least about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years elapses between packaging and administration.

[0054] In some embodiments, the subject has been identified as having a disease or disorder that is modulated by activated immune cells.

[0055] In some embodiments, treating includes providing a therapeutic effect against a disease or disorder modulated by activated immune cells.

[0056] In some embodiments, the subject has been identified as having a T helper type 1 (Th1) cell-mediated disease.

[0057] In some embodiments, the Th1-mediated disease is celiac disease, multiple sclerosis, or diabetes, eg, type 1 diabetes.

[0058] In some embodiments, treating comprises providing a therapeutic effect against a Th1-mediated disease or symptom thereof.

[0059] In some embodiments, the subject has been identified as having a T helper type 2 (Th2) cell-mediated disease or disorder.

[0060] In some embodiments, the Th2-mediated disease or disorder is associated with atopic dermatitis, allergic rhinitis, asthma, or itch.

[0061] In some embodiments, treating comprises providing a therapeutic effect against a Th2-mediated disease or symptom thereof.

[0062] In some embodiments, the subject has been identified as having a T helper type 17 (Th17) cell-mediated disease or disorder.

[0063] In some embodiments, the Th17-mediated disease or disorder is autosomal dominant hyper-IgE syndrome (AD-HIES), rheumatoid arthritis, or irritable bowel syndrome.

[0064] In some embodiments, treating comprises providing a therapeutic effect against a Th17-mediated disease or symptom thereof.

[0065] In some embodiments, the subject has been identified as having a regulatory T cell (Treg) cell-mediated disease or disorder.

[0066] In some embodiments, the Treg-mediated disease or disorder is X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX).

[0067] In some embodiments, treating comprises providing a therapeutic effect against a Treg-mediated disease or symptom thereof.

[0068] In some embodiments, the preparation is formulated as a spray, aerosol, or mist.

[0069] In some embodiments, the preparation is formulated as a powder, cream, ointment, or lotion.

[0070] In some embodiments, the preparation comprises a thickener and / or an emulsifier.

[0071] In some embodiments, the preparation has a viscosity of at least about 1 mPa·s, 10 mPa·s, 100 mPa·s, 1 Pa·s, 5 Pa·s, 10 Pa·s, or 20 Pa·s at room temperature, e.g., a temperature between about 20°C and 25°C.

[0072] In some embodiments, the preparation includes talcum powder or cornstarch.

[0073] In some embodiments, the preparation includes a component that is toxic to AOM, such as an antimicrobial agent or a preservative, such as a preservative listed in Appendix VI.

[0074] The method may include combining the preparation with at least one preservative listed in Appendix VI.

[0075] The method may include combining the preparation with at least 500 ppb of at least one preservative listed in Appendix VI.

[0076] In some embodiments, the preparation or formulation is administered topically.

[0077] In some embodiments, the preparation or formulation is administered to the subject's body, for example, to one or more of the subject's face, neck, scalp, limbs, hands, feet, back, buttocks, torso, genitals, and chest.

[0078] In some embodiments, the preparation or formulation is administered intranasally.

[0079] The method may include administering the preparation or formulation to the subject orally, enterally, intranasally, parenterally, subcutaneously, ophthalmically, otically, or respiratoryly.

[0080] In some embodiments, the preparation comprises the AOM in a buffer solution, for example, an aqueous buffer solution.

[0081] In some embodiments, the buffer solution, eg, an aqueous buffer solution, comprises disodium phosphate and magnesium chloride in water, eg, 50 mM Na2HPO4 and 2 mM MgCl2.

[0082] In some embodiments, the buffer solution, eg, an aqueous buffer solution, consists essentially of disodium phosphate and magnesium chloride, eg, 50 mM Na2HPO4 and 2 mM MgCl2, in water.

[0083] In some embodiments, the buffer solution, eg, an aqueous buffer solution, consists of disodium phosphate and magnesium chloride in water, eg, 50 mM Na2HPO4 and 2 mM MgCl2.

[0084] In some embodiments, the AOM comprises ammonia oxidizing bacteria (AOB).

[0085] In some embodiments, the AOM consists essentially of AOB.

[0086] In some embodiments, the AOM consists of an AOB.

[0087] In some embodiments, the AOM comprises Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof.

[0088] In some embodiments, the AOM is Nitrosomonas eutropha (N. eutropha).

[0089] In some embodiments, the AOM is N. eutropha D23, which has ATCC accession number PTA-121157.

[0090] In some embodiments, the AOM comprises ammonia-oxidizing archaea (AOA).

[0091] Administration may provide treatment for one or more of headaches, cardiovascular diseases, inflammation, immune responses, autoimmune disorders, liver diseases, infectious diseases, neurological diseases, psychiatric disorders, pulmonary diseases, nitric oxide disorders, urea cycle disorders, congestion, vasodilation disorders, skin diseases, ophthalmic disorders, intestinal disorders, hearing disorders, wound healing, reactions to insect bites, connective tissue disorders, and certain viral, bacterial, or fungal infections.

[0092] Administration may provide treatment or amelioration of local effects.

[0093] Administration may provide treatment or amelioration of systemic effects.

[0094] The method may include obtaining a preparation containing ammonia-oxidizing microorganisms (AOM). The method may include preparing a cosmetic, therapeutic, or consumer product from the preparation. The method may include measuring at least one of the AOM metabolic activity and the Th1, Th2, Th17, or Treg inhibitory activity of the AOM in the preparation or product to provide an activity value. The method may include comparing the activity value to a predetermined range of values ​​corresponding to a predetermined range of amounts of AOM metabolic activity and Th1, Th2, Th17, or Treg inhibitory activity. The method may include determining whether the activity value is within the predetermined range of values. In some embodiments, if the activity value is within the predetermined range of values, the method may include classifying the preparation or product as acceptable. In some embodiments, if the activity value is outside the predetermined range of values, the method may include classifying the preparation or product as unacceptable.

[0095] In some embodiments, the method may further comprise the step of heat killing a target percentage of ammonia-oxidizing microorganisms if the preparation is not acceptable.

[0096] According to another aspect, there is provided a method of treating a subject, the method comprising administering to the subject a therapeutically effective amount of a preparation comprising an ammonia-oxidizing microorganism (AOM). In some embodiments, the preparation is distributed by a method according to any of the preceding claims.

[0097] According to another aspect, a shelf-stable preparation is provided. The shelf-stable preparation has a shelf-stable concentration of at least about 10 3The preparation may contain 1000 cells / mL of ammonia-oxidizing microorganisms (AOM). In some embodiments, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable. In some embodiments, the preparation has a viscosity of at least about 1 mPa·s, 10 mPa·s, 100 mPa·s, 1 Pa·s, 5 Pa·s, 10 Pa·s, or 20 Pa·s at room temperature, e.g., between about 20°C and 25°C. In some embodiments, the preparation is formulated as a powder, cream, ointment, salve, or lotion. In some embodiments, the preparation includes a component that is toxic to AOM, e.g., an antimicrobial agent or preservative, e.g., a preservative listed in Appendix VI. In some embodiments, the preparation is sterile.

[0098] According to another embodiment, a shelf-stable preparation is provided. The shelf-stable preparation contains at least about 10 3 CFU / mL of ammonia-oxidizing microorganisms (AOM). The shelf-stable preparation may contain at least one preservative listed in Appendix VI. In particular, the shelf-stable preparation may contain at least 500 ppb of at least one preservative.

[0099] According to another embodiment, a shelf-stable preparation is provided. The shelf-stable preparation contains at least about 10 3 CFU / mL of ammonia-oxidizing microorganisms (AOM). The shelf-stable preparation may include at least one preservative listed in Appendix VI. In some embodiments, the preparation does not support the growth of pathogenic microorganisms when exposed to challenge with pathogenic microorganisms.

[0100] In some embodiments, the preparation comprises at least 500 ppb of at least one preservative listed in Appendix VI.

[0101] In some embodiments, the preparation includes a component that is toxic to AOM, such as an antimicrobial agent.

[0102] In some embodiments, the preparation comprises at least about 10 3 cells / mL, 10 4 cells / mL, 10 5 cells / mL, or 10 6 Contains cells / mL.

[0103] In some embodiments, the preparation comprises at least about 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, or 10 6 Contains CFU / mL.

[0104] In some embodiments, the preparation is sterile.

[0105] In some embodiments, the preparation is substantially free of polyphosphates.

[0106] In some embodiments, the preparation, when challenged with a population of pathogenic microorganisms, sterilizes at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of a population of microorganisms.

[0107] In some embodiments, the preparation is formulated as a liquid, for example, a spray, aerosol, or mist.

[0108] In some embodiments, the preparation is formulated as a powder, cream, ointment, salve, or lotion.

[0109] In some embodiments, the preparation comprises a thickener and / or an emulsifier.

[0110] In some embodiments, the preparation has a viscosity of at least about 1 mPa·s, 10 mPa·s, 100 mPa·s, 1 Pa·s, 5 Pa·s, 10 Pa·s, or 20 Pa·s at room temperature, e.g., a temperature between about 20°C and 25°C.

[0111] In some embodiments, the preparation includes talcum powder or cornstarch.

[0112] In some embodiments, the preparation is packaged in an end-use container.

[0113] The end-use container may indicate one or more of the following: storage and handling of the preparation, the dosage form of the preparation, a description of the contents in the preparation, the viability status of the AOM, and directions for use of the preparation.

[0114] In other embodiments, the end-use container does not indicate one or more of the following: storage and handling of the preparation, the dosage form of the preparation, a description of the contents in the preparation, the viability status of the AOM, and directions for use of the preparation.

[0115] In some embodiments, the end-use container provides information to the subject to apply the preparation topically.

[0116] In some embodiments, the end use container instructs the subject to apply the preparation intranasally.

[0117] In some embodiments, the end-use container provides instructions to a subject to apply the preparation at least one of orally, enterally, intranasally, parenterally, subcutaneously, ophthalmically, otically, or respiratoryly.

[0118] In some embodiments, the end-use container comprises a polymeric bottle, for example, a spray, aerosol, or mist bottle.

[0119] In some embodiments, the end-use container comprises a squeezable container, for example, a squeeze bottle or a tube.

[0120] In some embodiments, the end-use container is substantially free of a vacuum bag.

[0121] In some embodiments, the end-use container is not configured to impede or reduce retrograde flow.

[0122] In some embodiments, the end-use container comprises a polymer, such as polyethylene terephthalate (PET), high density polyethylene (HDPE), polypropylene, polycarbonate, polytetrafluoroethylene (Teflon®), polyvinylidene fluoride (PVDF), or cellulosic, glass, aluminum, or cardboard.

[0123] In some embodiments, the end use container is configured to allow the passage of oxygen.

[0124] In some embodiments, the end-use container is configured to allow at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 percent transmission of ionizing radiation, e.g., from an isotope, e.g., cobalt-60, e.g., by gamma rays, e.g., by x-rays, or by ultraviolet light, e.g., ultraviolet C (UVC), through the container.

[0125] Preparations may be formulated for oral, enteral (e.g., buccal, sublingual, sublabial, and rectal), parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (e.g., fine particle dusts or mists that may be generated by various types of metered-dose pressurized aerosols, nebulizers, or inhalers, including intranasally or via the lungs), intranasal, ophthalmic, otic, rectal, injectable, urogenital, or topical (e.g., dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration.

[0126] The preparations may be formulated for the treatment of one or more of headaches, cardiovascular diseases, inflammation, immune responses, autoimmune disorders, liver diseases, infectious diseases, neurological diseases, psychiatric disorders, nitric oxide disorders, urea cycle disorders, congestion, vasodilation disorders, skin diseases, ophthalmological disorders, wound healing, reactions to insect bites, connective tissue disorders, and certain viral, bacterial, or fungal infections.

[0127] The preparation may be formulated for the treatment of a disease or disorder modulated by activated immune cells.

[0128] In some embodiments, the activated immune cells are T helper cells or regulatory T cells, e.g., T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17), or regulatory T cells (Treg).

[0129] In some embodiments, the preparation is formulated for upregulation, activation, downregulation, or suppression of cytokines associated with activated immune cells, such as IL-5, IL-13, IL-4, IFNγ, IL-12, IL-2, IL-18, IL-17, IL-21, IL-22, IL-10, and TFG-β.

[0130] In some embodiments, the preparation is formulated for the treatment of a Th1-mediated disease or disorder, such as celiac disease, multiple sclerosis, or diabetes, eg, type 1 diabetes.

[0131] In some embodiments, the preparation is formulated for the treatment of a Th2-mediated disease or disorder, such as atopic dermatitis, allergic rhinitis, asthma, or itch.

[0132] In some embodiments, the preparation is formulated for the treatment of a Th17-mediated disease or disorder, for example, Job's syndrome, rheumatoid arthritis, irritable bowel syndrome.

[0133] In some embodiments, the preparation is formulated for the treatment of a Treg-mediated disease or disorder, for example, X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX).

[0134] In some embodiments, the preparation comprises the AOM in a buffer solution, for example, an aqueous buffer solution.

[0135] In some embodiments, the buffer solution, eg, an aqueous buffer solution, comprises disodium phosphate and magnesium chloride in water, eg, 50 mM Na2HPO4 and 2 mM MgCl2.

[0136] In some embodiments, the buffer solution, eg, an aqueous buffer solution, consists essentially of disodium phosphate and magnesium chloride, eg, 50 mM Na2HPO4 and 2 mM MgCl2, in water.

[0137] In some embodiments, the buffer solution, eg, an aqueous buffer solution, consists of disodium phosphate and magnesium chloride in water, eg, 50 mM Na2HPO4 and 2 mM MgCl2.

[0138] In some embodiments, the AOM comprises ammonia oxidizing bacteria (AOB).

[0139] In some embodiments, the AOM consists essentially of AOB.

[0140] In some embodiments, the AOM consists of an AOB.

[0141] In some embodiments, the AOM comprises Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof.

[0142] In some embodiments, the AOM is Nitrosomonas eutropha (N. eutropha).

[0143] In some embodiments, the AOM is N. eutropha D23, which has ATCC accession number PTA-121157.

[0144] In some embodiments, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia oxidizing microorganisms are viable.

[0145] In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia oxidizing microorganisms are inactive.

[0146] According to another aspect, there is provided a method of treating a subject, comprising administering to the subject a therapeutically effective amount of a preparation according to any of the preceding claims.

[0147] The present disclosure contemplates all combinations of any one or more of the above aspects and / or embodiments, as well as combinations with any one or more of the embodiments set forth in the detailed description and any examples. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method of distributing a preparation, comprising: providing a preparation comprising live ammonia-oxidizing microorganisms (AOM); During distribution, the preparation is exposed to an environment having a temperature greater than about 4°C; This method allows the preparation to be distributed. (Item 2) 1. A method of treating a subject, comprising: administering to the subject a therapeutically effective amount of a preparation comprising ammonia oxidizing microorganisms (AOM); The method, wherein less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable. (Item 3) 1. A method of treating a subject, comprising: administering to the subject a therapeutically effective amount of a preparation comprising ammonia oxidizing microorganisms (AOM); The preparation may be heated to a temperature above or about room temperature, e.g., about 2 The method is carried out at a temperature between 0°C and 25°C. (Item 4) 1. A method of treating autosomal dominant hyper-IgE syndrome (AD-HIES) in a subject, comprising: administering to the subject a therapeutically effective amount of a preparation comprising ammonia oxidizing microorganisms (AOM); This method treats autosomal dominant hyper IgE syndrome (AD-HIES). 1. A method of treating X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX) in a subject, comprising: administering to the subject a therapeutically effective amount of a preparation comprising ammonia oxidizing microorganisms (AOM); Thereby, the X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX) is treated. (Item 6) 1. A method for producing a shelf-stable cosmetic, therapeutic, or consumer product, comprising: formulating the preparation containing ammonia oxidizing microorganisms (AOM) into a powder, cream, ointment, or lotion; and packaging said preparation into said cosmetic, therapeutic or consumer product. Including, The method, wherein less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms in the cosmetic or treatment product are viable. (Item 7) 10. The method of any of the preceding items, comprising providing said preparation comprising live ammonia-oxidizing microorganisms. (Item 8) 10. The method of any of the preceding items, wherein less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable. (Item 9) 10. The method of any of the preceding items, wherein the preparation is exposed to an environment having a temperature greater than about 4° C. (Item 10) The method of any of the preceding items, wherein the preparation is packaged into a container. (Item 11) The method of any of the preceding items, wherein the preparation is packaged into multiple containers, for example, at least 2, 3, 6, 8, 10, or 20 containers. (Item 12) The method of any of the preceding items, wherein the preparation is packaged into an end-use container. (Item 13) The method of any of the preceding items, wherein the preparation is packaged into a plurality of separate end-use containers, for example, at least 2, 4, 6, 8, 10, 20, 50, or 100 end-use containers. (Item 14) The method of any of the preceding items, comprising the step of providing (or having a designee provide) the preparation or packaged end-use container to a recipient. (Item 15) During distribution, the preparation or packaged end-use container may be heated to a temperature above about 10°C. 3. The method of any of the preceding items, wherein the substrate is exposed to an environment having a temperature. (Item 16) The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container is exposed to an environment having room temperature, for example, a temperature between about 20°C and 25°C. (Item 17) 10. The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container reaches a temperature greater than about 4°C. (Item 18) 10. The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container reaches a temperature greater than about 10°C. (Item 19) The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container reaches a temperature above about room temperature, for example, between about 20°C and 25°C. (Item 20) The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container is exposed to the environment for a period of at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years. (Item 21) 10. The method of any of the preceding items, wherein the environment is a storage environment. (Item 22) 10. The method of any of the preceding items, wherein the environment is a delivery environment, such as a postal or commercial delivery environment. (Item 23) 10. The method of any of the preceding items, wherein the environment is a delivery environment, such as a cargo or freight transport delivery environment. (Item 24) The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container reaches a temperature greater than about 4°C for at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years. (Item 25) The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container reaches a temperature greater than about 10° C. for at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years. (Item 26) The method of any of the preceding items, wherein during distribution, the preparation or packaged end-use container reaches a temperature above about room temperature, e.g., a temperature between about 20°C and 25°C, for at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years. (Item 27) The method of any of the preceding items, wherein the step of providing (or having the designee provide) the preparation or packaged end-use container to the recipient includes making the preparation or packaged end-use container available on an internet-based outlet. (Item 28) The method of any of the preceding items, wherein the step of providing (or having the designee provide) the preparation or packaged end-use container to the recipient includes making the preparation or packaged end-use container available through a non-internet-based outlet, e.g., a store. (Item 29) For example, the method of any of the preceding items, wherein at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are viable upon packaging into an end-use container. (Item 30) For example, the method of any of the preceding items, wherein at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are active upon packaging into an end-use container. (Item 31) 10. The method of any of the preceding items, wherein less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable after distribution. (Item 32) 10. The method of any of the preceding items, wherein at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are inactive after distribution. (Item 33) For example, the method of any of the preceding items, wherein less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable upon packaging into an end-use container. (Item 34) For example, the method of any of the preceding items, wherein less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are inactive upon packaging into an end-use container. (Item 35) The method of any of the preceding items, wherein at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9%, or substantially all of the ammonia-oxidizing microorganisms are heat-killed, e.g., prior to packaging. (Item 36) The method of any of the preceding items, wherein the container, e.g., end-use container, comprises a polymeric bottle, e.g., a spray, aerosol, or mist bottle. (Item 37) The method of any of the preceding items, wherein the container, e.g., end-use container, comprises a squeezable container, e.g., a squeeze bottle or a tube. (Item 38) 10. The method of any of the preceding items, wherein the container, e.g., end-use container, is substantially free of vacuum bags. (Item 39) 10. The method of any of the preceding items, wherein the container, e.g., end-use container, is not configured to impede or reduce retrograde flow. (Item 40) The method of any of the preceding items, wherein the container, e.g., end-use container, comprises a polymer, such as polyethylene terephthalate (PET), high density polyethylene (HDPE), polypropylene, polycarbonate, polytetrafluoroethylene (Teflon®), polyvinylidene fluoride (PVDF), or cellulosic, glass, aluminum, or cardboard. (Item 41) 10. The method of any of the preceding items, wherein the container, e.g., end-use container, is configured to allow the passage of oxygen. (Item 42) The method of any preceding item, wherein the container, e.g., end-use container, is configured to allow at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 percent of the transmission of ionizing radiation, e.g., from an isotope, e.g., cobalt-60, e.g., by gamma rays, e.g., by x-rays, or by ultraviolet light, e.g., ultraviolet C (UVC), to pass through the container. (Item 43) The method of any of the preceding items, comprising treating a disease or disorder modulated by activated immune cells in a subject. (Item 44) The method of any of the preceding items, wherein the activated immune cells are T helper cells or regulatory T cells. (Item 45) 2. The method of any of the preceding items, wherein the activated immune cells are T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17), or regulatory T cells (Treg). (Item 46) 10. The method of any of the preceding items, wherein at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are inactive. (Item 47) The method of any of the preceding items, wherein administration provides upregulation, activation, downregulation, or suppression of cytokines associated with activated immune cells, e.g., IL-5, IL-13, IL-4, IFNγ, IL-12, IL-2, IL-18, IL-17, IL-21, IL-22, IL-10, and TFG-β. (Item 48) The method of any of the preceding items, wherein the preparation is at a temperature greater than about 4°C at the time of administration. (Item 49) 10°C。 The method of claim 10, wherein the preparation is at a temperature above about 10°C at the time of administration. Any of the methods described above. (Item 50) The method of any of the preceding items, wherein the preparation is at a temperature above about room temperature, for example, between about 20°C and 25°C, at the time of administration. (Item 51) The method of any of the preceding items, wherein a period of at least about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years elapses between packaging and administration. (Item 52) The method of any of the preceding items, wherein the subject is identified as having a disease or disorder modulated by activated immune cells. (Item 53) The method of any of the preceding items, wherein treatment includes providing a therapeutic effect against a disease or disorder modulated by activated immune cells. (Item 54) The method of any of the preceding items, wherein the subject is identified as having a T helper type 1 (Th1) cell-mediated disease. (Item 55) The method of any of the preceding items, wherein the Th1-mediated disease is celiac disease, multiple sclerosis, or diabetes, e.g., type 1 diabetes. (Item 56) The method of any of the preceding items, wherein treating comprises providing a therapeutic effect against a Th1-mediated disease or symptom thereof. (Item 57) The method of any of the preceding items, wherein the subject is identified as having a T helper type 2 (Th2) cell-mediated disease or disorder. (Item 58) The method of any of the preceding items, wherein the Th2-mediated disease or disorder is associated with atopic dermatitis, allergic rhinitis, asthma, or itch. (Item 59) The method of any of the preceding items, wherein treating comprises providing a therapeutic effect against a Th2-mediated disease or symptom thereof. (Item 60) The method of any of the preceding items, wherein the subject is identified as having a T helper type 17 (Th17) cell-mediated disease or disorder. (Item 61) The method of any of the preceding items, wherein the Th17-mediated disease or disorder is autosomal dominant hyper-IgE syndrome (AD-HIES), rheumatoid arthritis, or irritable bowel syndrome. (Item 62) The method of any of the preceding items, wherein treating comprises providing a therapeutic effect against a Th17-mediated disease or symptom thereof. (Item 63) The method of any of the preceding items, wherein the subject is identified as having a regulatory T cell (Treg) cell-mediated disease or disorder. (Item 64) The method of any of the preceding items, wherein the Treg-mediated disease or disorder is X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX). (Item 65) The method of any of the preceding items, wherein treating comprises providing a therapeutic effect against a Treg-mediated disease or symptom thereof. (Item 66) The method of any of the preceding items, wherein the preparation is formulated as a spray, aerosol, or mist. (Item 67) The method of any of the preceding items, wherein the preparation is formulated as a powder, cream, ointment, or lotion. (Item 68) 10. The method according to any of the preceding items, wherein the preparation comprises a thickener and / or an emulsifier. (Item 69) 10. The method of any of the preceding items, wherein the preparation has a viscosity of at least about 1 mPa·s, 10 mPa·s, 100 mPa·s, 1 Pa·s, 5 Pa·s, 10 Pa·s, or 20 Pa·s at room temperature, e.g., a temperature between about 20°C and 25°C. (Item 70) The method of any of the preceding items, wherein the preparation comprises talcum powder or cornstarch. (Item 71) The method of any of the preceding items, wherein the preparation includes a component that is toxic to AOM, such as an antimicrobial agent or a preservative, such as a preservative listed in Appendix VI. The method of any of the preceding items, further comprising combining said preparation with at least one preservative listed in Appendix VI. (Item 73) The method of any of the preceding items, comprising combining said preparation with at least 500 ppb of at least one preservative listed in Appendix VI. (Item 74) 75. The method of any of the preceding claims, wherein the preparation or formulation is administered topically. The method of any of the preceding items, wherein the preparation or formulation is administered to one or more of the subject's body, such as the face, neck, scalp, limbs, hands, feet, back, buttocks, torso, genitals, and chest of the subject. (Item 76) 77. The method of any of the preceding claims, wherein the preparation or formulation is administered intranasally. The method of any of the preceding items, comprising administering the preparation or formulation to the subject orally, enterally, intranasally, parenterally, subcutaneously, ophthalmically, otically, or respiratoryly. (Item 78) The method of any of the preceding items, wherein the preparation comprises AOM in a buffer solution, e.g., an aqueous buffer solution. (Item 79) The method of any of the preceding items, wherein the buffer solution, e.g., an aqueous buffer solution, comprises disodium phosphate and magnesium chloride in water, e.g., 50 mM NaHPO and 2 mM MgCl. (Item 80) The method of any of the preceding items, wherein the buffer solution, e.g., an aqueous buffer solution, consists essentially of disodium phosphate and magnesium chloride in water, e.g., 50 mM NaHPO and 2 mM MgCl. (Item 81) The method of any of the preceding items, wherein the buffer solution, e.g., an aqueous buffer solution, consists of disodium phosphate and magnesium chloride in water, e.g., 50 mM NaHPO and 2 mM MgCl. (Item 82) 10. The method of any of the preceding items, wherein the AOM comprises ammonia oxidizing bacteria (AOB). (Item 83) 10. The method of any of the preceding items, wherein the AOM consists essentially of AOB. (Item 84) 10. The method of any of the preceding items, wherein the AOM consists of an AOB. (Item 85) The method of any of the preceding items, wherein the AOM includes Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof. (Item 86) The AOM is Nitrosomonas eutropha (N. eutropha ) a method according to any of the preceding items. (Item 87) The AOM is N. eutropha having ATCC accession number PTA-121157. The method of any of the preceding items, wherein D23. (Item 88) 2. The method of any of the preceding items, wherein the AOM comprises ammonia-oxidizing archaea (AOA). (Item 89) The method of any of the preceding items, wherein administering provides treatment for one or more of headache, cardiovascular disease, inflammation, immune response, autoimmune disorder, liver disease, infectious disease, neurological disease, psychiatric disorder, pulmonary disease, nitric oxide disorder, urea cycle disorder, congestion, vasodilation disorder, skin disease, ophthalmic disorder, intestinal disorder, hearing disorder, wound healing, reaction to insect bites, connective tissue disorder, and certain viral, bacterial, or fungal infections. (Item 90) The method of any of the preceding items, wherein administration provides treatment or amelioration of a local effect. (Item 91) The method of any of the preceding items, wherein administration provides treatment or amelioration of a systemic effect. (Item 92) obtaining said preparation comprising ammonia oxidizing microorganisms (AOM); preparing a cosmetic, therapeutic, or consumer product from said preparation; measuring at least one of the AOM metabolic activity and the Th1, Th2, Th17 or Treg inhibitory activity of the AOM in the preparation or product to provide an activity value; comparing the activity value to a range of predetermined values ​​corresponding to a range of amounts of AOM metabolic activity and Th1, Th2, Th17 or Treg inhibitory activity; determining whether the activity value is within the range of predetermined values; classifying the preparation or product as acceptable if the activity value is within the range of predetermined values; or classifying the preparation or product as unacceptable if the activity value is outside the range of predetermined values. 10. The method of any of the preceding items, comprising: (Item 93) The method of any of the preceding items, further comprising, for example, if the preparation is unacceptable, heat-killing a target percentage of the ammonia-oxidizing microorganisms. (Item 94) 1. A method of treating a subject, comprising: administering to the subject a therapeutically effective amount of a preparation comprising ammonia oxidizing microorganisms (AOM); The preparation is distributed by the method described in any of the preceding items. (Item 95) At least about 10 3 cells / mL of ammonia-oxidizing microorganisms (AOM); and One or more of the following characteristics: less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable; the preparation has a viscosity of at least about 1 mPa·s, 10 mPa·s, 100 mPa·s, 1 Pa·s, 5 Pa·s, 10 Pa·s, or 20 Pa·s at room temperature, e.g., a temperature between about 20°C and 25°C; the preparation is formulated as a powder, cream, ointment, salve, or lotion; The preparation contains a component that is toxic to AOM, such as an antimicrobial agent or a preservative, such as a preservative listed in Appendix VI; and The preparation is sterile 1. A shelf-stable preparation comprising: (Item 96) At least about 10 in 750-1000 mg 3 CFU / mL of ammonia-oxidizing microorganisms (AOM); and At least one preservative listed in Appendix VI wherein the at least one preservative is at least 500 ppb. (Item 97) At least about 10 in 750-1000 mg 3 CFU / mL of ammonia-oxidizing microorganisms (AOM); and At least one preservative listed in Appendix VI wherein the shelf-stable preparation does not support growth of a pathogenic microorganism when exposed to a challenge by said pathogenic microorganism. (Item 98) 10. A preparation according to any of the preceding items, comprising at least 500 ppb of said at least one preservative listed in Appendix VI. (Item 99) A preparation according to any of the preceding items, comprising a component toxic to AOM, e.g., an antimicrobial agent. (Item 100) At least about 10 3 cells / mL, 10 4 cells / mL, 10 5 cells / mL, or 10 6 2. The preparation of any of the preceding items, comprising cells / mL. (Item 101) At least about 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, or 10 6 2. The preparation of any of the preceding items, comprising CFU / mL. (Item 102) 10. The preparation of any of the preceding items, which is sterile. (Item 103) 10. The preparation of any of the preceding items, which is substantially free of polyphosphates. (Item 104) 10. The preparation of any of the preceding items, which, when challenged with a population of pathogenic microorganisms, sterilizes at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of said population of microorganisms. (Item 105) The preparation of any of the preceding items, formulated as a liquid, e.g., a spray, aerosol, or mist. (Item 106) 10. The preparation of any of the preceding items, formulated as a powder, cream, ointment, salve, or lotion. (Item 107) 10. The preparation of any of the preceding items, comprising a thickener and / or an emulsifier. (Item 108) 10. The formulation of any of the preceding items, having a viscosity of at least about 1 mPa·s, 10 mPa·s, 100 mPa·s, 1 Pa·s, 5 Pa·s, 10 Pa·s, or 20 Pa·s at room temperature, e.g., a temperature between about 20°C and 25°C. (Item 109) A preparation according to any of the preceding items, comprising talcum powder or cornstarch. (Item 110) 10. The preparation of any of the preceding items packaged in an end-use container. (Item 111) The end-use container comprises: Storage and handling of said preparations; the dosage form of said preparation; a description of the contents of said preparation; the viability status of the AOM; and Use of the preparation The preparation according to any of the preceding items, wherein the preparation exhibits one or more of the following: (Item 112) The end-use container comprises: Storage and handling of said preparations; the dosage form of said preparation; a description of the contents of said preparation; the viability status of the AOM; and Use of the preparation The preparation of any of the preceding items, without one or more of: (Item 113) 10. The preparation of any of the preceding items, wherein the end-use container provides instructions to the subject to apply the preparation topically. (Item 114) 10. The preparation of any of the preceding items, wherein the end-use container instructs the subject to apply the preparation intranasally. (Item 115) The preparation of any of the preceding items, wherein the end-use container provides instructions to apply the preparation to the subject at least one of orally, enterally, intranasally, parenterally, subcutaneously, ophthalmically, otically, or respiratoryly. (Item 116) The formulation of any of the preceding items, wherein the end-use container comprises a polymeric bottle, such as a spray, aerosol, or mist bottle. (Item 117) The preparation according to any of the preceding items, wherein the end-use container comprises a squeezable container, such as a squeeze bottle or a tube. (Item 118) 10. The formulation of any of the preceding items, wherein the end-use container is substantially free of a vacuum bag. (Item 119) 10. The preparation of any of the preceding items, wherein the end-use container is not configured to inhibit or reduce retrograde flow. (Item 120) The formulation of any of the preceding items, wherein the end-use container comprises a polymer, such as polyethylene terephthalate (PET), high density polyethylene (HDPE), polypropylene, polycarbonate, polytetrafluoroethylene (Teflon®), polyvinylidene fluoride (PVDF), or cellulosic, glass, aluminum, or cardboard. (Item 121) 10. The formulation of any of the preceding items, wherein the end-use container is configured to allow the passage of oxygen. (Item 122) The preparation of any of the preceding items, wherein the end-use container is configured to allow at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 percent transmission of ionizing radiation, e.g., from an isotope, e.g., cobalt-60, e.g., by gamma rays, e.g., by x-rays, or by ultraviolet light, e.g., ultraviolet C (UVC), through the container. (Item 123) A preparation according to any of the preceding items that is formulated for oral, enteral (e.g., buccal, sublingual, sublabial, and rectal), parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (e.g., fine particle dusts or mists that may be generated by various types of metered-dose pressurized aerosols, nebulizers, or inhalers, including intranasally or via the lungs), intranasal, ophthalmic, otic, rectal, injectable, urogenital, or topical (e.g., dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration. (Item 124) The preparation of any of the preceding items formulated for the treatment of one or more of headache, cardiovascular disease, inflammation, immune response, autoimmune disorder, liver disease, infectious disease, neurological disease, psychiatric disorder, nitric oxide disorder, urea cycle disorder, congestion, vasodilation disorder, skin disease, ophthalmological disorder, wound healing, reaction to insect bites, connective tissue disorder, and certain viral, bacterial, or fungal infections. (Item 125) The preparation of any of the preceding items, formulated for the treatment of a disease or disorder modulated by activated immune cells. (Item 126) The preparation of any of the preceding items, wherein the activated immune cells are T helper cells or regulatory T cells, such as T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17), or regulatory T cells (Treg). (Item 127) The preparation of any of the preceding items, wherein the preparation is formulated for upregulation, activation, downregulation, or suppression of cytokines associated with activated immune cells, such as IL-5, IL-13, IL-4, IFNγ, IL-12, IL-2, IL-18, IL-17, IL-21, IL-22, IL-10, and TFG-β. (Item 128) The preparation of any of the preceding items, formulated for the treatment of a Th1-mediated disease or disorder, e.g., celiac disease, multiple sclerosis, or diabetes, e.g., type 1 diabetes. (Item 129) The preparation of any of the preceding items, formulated for the treatment of a Th2-mediated disease or disorder, e.g., atopic dermatitis, allergic rhinitis, asthma, or itch. (Item 130) The preparation of any of the preceding items, formulated for the treatment of a Th17-mediated disease or disorder, e.g., Job's syndrome, rheumatoid arthritis, irritable bowel syndrome. (Item 131) The preparation of any of the preceding items, formulated for the treatment of a Treg-mediated disease or disorder, such as X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX). (Item 132) 10. The method of claim 1, further comprising administering to said patient a therapeutically effective amount of AOM in a buffer solution, e.g., an aqueous buffer solution. Preparation. (Item 133) The preparation of any of the preceding items, wherein the buffer solution, e.g., an aqueous buffer solution, comprises disodium phosphate and magnesium chloride in water, e.g., 50 mM Na2HPO4 and 2 mM MgCl2. (Item 134) The preparation of any of the preceding items, wherein the buffer solution, e.g., an aqueous buffer solution, consists essentially of disodium phosphate and magnesium chloride, e.g., 50 mM NaHPO and 2 mM MgCl, in water. (Item 135) The preparation of any of the preceding items, wherein the buffer solution, e.g., an aqueous buffer solution, consists of disodium phosphate and magnesium chloride in water, e.g., 50 mM Na2HPO4 and 2 mM MgCl2. (Item 136) 10. The preparation of any of the preceding items, wherein the AOM comprises ammonia oxidizing bacteria (AOB). (Item 137) 10. The preparation of any of the preceding items, wherein the AOM consists essentially of AOB. (Item 138) 10. The preparation of any of the preceding items, wherein the AOM consists of AOB. (Item 139) The preparation of any of the preceding items, wherein the AOM comprises Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof. (Item 140) 10. The preparation of any of the preceding items, wherein the AOM is Nitrosomonas eutropha (N. eutropha). (Item 141) The AOM is N. eutropha having ATCC accession number PTA-121157. Item D23. The preparation of any of the preceding items. (Item 142) 10. The preparation of any of the preceding items, wherein less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable. (Item 143) 10. The preparation of any of the preceding items, wherein at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are inactive. (Item 144) 1. A method of treating a subject, comprising: administering to the subject a therapeutically effective amount of a preparation according to any of the preceding items; thereby treating the subject.

[0148] The accompanying drawings are not intended to be drawn to scale. In the drawings, each of the identical or nearly identical components illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component is labeled in every figure. Illustrated in the drawings are: [Brief explanation of the drawings]

[0149] [Figure 1] FIG. 1 is a graph of D23 metabolic activity for live and heat-killed D23. [Figure 2A] FIG. 2A is a graph of PBMC percent activity and viability for PBMCs co-cultured with D23 or heat-killed D23 in the presence or absence of Th2 stimulation. [Figure 2B] FIG. 2B is a graph of nitrite production by PBMC, D23, and PBMC co-cultured with D23. [Figure 3A] FIG. 3A is a graph of IL-5 expression for Th2-stimulated PBMCs co-cultured with D23 and heat-killed D23. [Figure 3B] FIG. 3B is a graph of IL-13 expression for Th2-stimulated PBMCs co-cultured with D23 and heat-killed D23. [Figure 3C] FIG. 3C is a graph of relative IL-4 expression for Th2-stimulated PBMCs co-cultured with D23 and heat-killed D23. [Figure 4] Figure 4A is a graph of IL-5 expression for Th2-stimulated PBMCs co-cultured with D23 and heat-killed D23, and Figure 4B is a graph of IL-13 expression for Th2-stimulated PBMCs co-cultured with D23 and heat-killed D23. [Figure 5] Figure 5A is a graph of TNFα expression for unstimulated cells co-cultured with D23 and heat-killed D23, Figure 5B is a graph of TNFα expression for LpS-stimulated cells co-cultured with D23 and heat-killed D23, and Figure 5C is a graph of TNFα expression for CD3 / 28-stimulated cells co-cultured with D23 and heat-killed D23. [Figure 6]Figure 6A is a graph of IL-6 expression for unstimulated cells co-cultured with D23 and heat-killed D23, Figure 6B is a graph of IL-6 expression for LpS-stimulated cells co-cultured with D23 and heat-killed D23, and Figure 6C is a graph of IL-6 expression for CD3 / 28-stimulated cells co-cultured with D23 and heat-killed D23. [Figure 7] Figure 7A is a graph of IL-10 expression for unstimulated cells co-cultured with D23 and heat-killed D23, Figure 7B is a graph of IL-10 expression for LpS-stimulated cells co-cultured with D23 and heat-killed D23, and Figure 7C is a graph of IL-10 expression for CD3 / 28-stimulated cells co-cultured with D23 and heat-killed D23. [Figure 8] Figure 8A is a graph of IFNγ expression for unstimulated cells co-cultured with D23 and heat-killed D23, Figure 8B is a graph of IFNγ expression for LpS-stimulated cells co-cultured with D23 and heat-killed D23, and Figure 8C is a graph of IFNγ expression for CD3 / 28-stimulated cells co-cultured with D23 and heat-killed D23. [Figure 9] Figure 9A is a graph of IL-2 expression for unstimulated cells co-cultured with D23 and heat-killed D23, Figure 9B is a graph of IL-2 expression for LpS stimulated cells co-cultured with D23 and heat-killed D23, and Figure 9C is a graph of IL-2 expression for CD3 / 28 stimulated cells co-cultured with D23 and heat-killed D23. [Figure 10A] FIG. 10A is a graph of AOB metabolic activity (expressed as nitrite production) for live and heat-killed AOB. [Figure 10B] FIG. 10B is a graph of AOB metabolic activity (expressed as nitric oxide production) for live and heat-killed AOB. [Figure 10C] FIG. 10C is a graph of the fold reduction in IL-5 expression mediated by AOB and heat-killed AOB. [Figure 10D]FIG. 10D is a graph of the fold reduction in IL-13 expression mediated by AOB and heat-killed AOB. [Figure 10E] FIG. 10E is a graph of the fold reduction in MHC II expression mediated by AOB and heat-killed AOB. [Figure 10F] FIG. 10F is a graph of the fold reduction in CD86 expression mediated by AOB and heat-killed AOB. [Figure 11] FIG. 11 is a graph of the fold reduction of D23-mediated IL-5 expression by TLR1, TLR4, TLR6, TLR5, TLR2, TLR9 and TLR8 inhibitors. [Figure 12] FIG. 12 is a graph of the fold reduction of D23-mediated IL-5 expression by TLR8 inhibitors. [Figure 13] Figure 13A is a graph of IFNγ expression for Th2 cells, Th2 cells co-cultured with AOB, and Th2 cells co-cultured with heat-killed AOB. Figure 13B is a graph of IL-12 expression for Th2 cells, Th2 cells co-cultured with AOB, and Th2 cells co-cultured with heat-killed AOB. [Figure 14] FIG. 14 is a graph of IL-5 expression for live D23 cells, heat-killed cells, senescent cells, cells treated by freeze / thaw cycles, and ethanol-treated cells, according to one embodiment. [Figure 15A] FIG. 15A is a graph of Eczema Area and Severity Index (EASI) scores after administration of a shelf-stable composition according to one embodiment. [Figure 15B] FIG. 15B is a graph of Investigator's Global Assessment (IGA) scores after administration of a shelf-stable composition according to one embodiment. [Figure 16] FIG. 16 is a graph showing the percentage of subjects with improved EASI and IGA scores from baseline after administration of a shelf-stable composition according to an embodiment. [Figure 17] FIG. 17 is a graph of visual analog scale (VAS) scores for itching after administration of a shelf-stable composition according to one embodiment. [Figure 18] FIG. 18 is a graph of the Itchman scores after administration of a shelf-stable composition according to one embodiment. [Figure 19A] FIG. 19A is a graph showing the percentage of subjects with improved VAS scores from baseline after administration of a shelf-stable composition according to one embodiment. [Figure 19B] FIG. 19B is a graph showing the percentage of subjects with an improved Itchman score from baseline after administration of a shelf-stable composition according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0150] Detailed Description According to one or more embodiments, the present disclosure provides various methods or modes of introducing ammonia-oxidizing microorganisms into a subject. These methods or modes include administering to the subject ammonia-oxidizing microorganisms, such as preparations, compositions, formulations, or products containing ammonia-oxidizing microorganisms. Thus, in at least some embodiments, the ammonia-oxidizing microorganisms can generally be returned to the subject's microbiome. In at least some embodiments, the ammonia-oxidizing microorganism preparations, compositions, and formulations described herein can be shelf-stable.

[0151] Disclosed are preparations, compositions, and / or formulations comprising, consisting essentially of, or consisting of ammonia-oxidizing microorganisms, including, for example, cosmetic products, therapeutic products, consumer products, non-natural products, natural products, and enhanced natural products. These preparations, compositions, and / or formulations are disclosed herein for use in various applications, for example, cosmetic and / or therapeutic applications. The preparations, compositions, and / or formulations can be administered in an amount effective for the intended use, for example, cosmetic or therapeutic applications. Provided are preparations, compositions, and / or formulations comprising ammonia-oxidizing microorganisms for various modes of administration to a subject. Provided are preparations, compositions, and / or formulations comprising ammonia-oxidizing microorganisms for use in treating various conditions and / or disorders in a subject. Disclosed are methods of treating a subject for various conditions and / or disorders via administration of ammonia-oxidizing microorganisms. Devices for use in administering ammonia-oxidizing microorganisms to a subject are also provided.

[0152] microbiology According to one or more embodiments, essentially any ammonia-oxidizing microorganism (AOM) can be used or realized. Ammonia-oxidizing microorganisms can generally be autotrophic. Ammonia-oxidizing microorganisms can produce nitrite and / or nitric oxide from ammonia.

[0153] The characteristics of autotrophic ammonia-oxidizing bacteria (AOB) have been fully described, for example, by Whitlock in U.S. Patent No. 7,820,420. Since that filing, the class of autotrophic microorganisms that oxidize ammonia to produce ATP has been expanded to include ammonia-oxidizing archaea (AOA), which have been removed from the class of bacteria and placed in their own separate class. For purposes of this disclosure, any and all autotrophic ammonia-oxidizing microorganisms that share the property of ammonia oxidation to generate ATP may be considered. AOM, including both AOB and AOA, share the necessary property of ammonia oxidation to NO and nitrite, and all known AOM lack virulence capabilities due to their inability to use organic substrates for ATP production. Bacteria can utilize higher concentrations of ammonia, while archaea can utilize lower concentrations. Physiological levels of ammonia are within the range that can be utilized by both bacteria (AOB) and archaea (AOA). Any specific reference to ammonia-oxidizing bacteria throughout this disclosure should be considered equally applicable to any ammonia-oxidizing microorganism, e.g., any ammonia-oxidizing archaea, and all of these terms may be used interchangeably herein.

[0154] Ammonia-oxidizing bacteria (AOB) are ubiquitous, gram-negative, obligate bacteria with the unique ability to generate energy exclusively from the conversion of ammonia to nitrite. In some embodiments, ammonia-oxidizing bacteria (AOB) of the genus Nitrosomonas are gram-negative, obligately autotrophic (chemolithoautotrophic) bacteria with the unique ability to generate nitrite and nitric oxide exclusively from ammonia as an energy source. They are widespread in both soil and aquatic environments and are important components of the environmental nitrification process. These bacteria possess beneficial properties, for example, in conjunction with various cosmetic and therapeutic uses, according to one or more embodiments described herein. While not wishing to be bound by any particular theory, due to the role of nitrite and nitric oxide as key components of several physiological functions, such as vasodilation, inflammation, and wound healing, these bacteria may possess various properties beneficial to both healthy and immunopathological conditions. These bacteria are safe for human use because they are slow-growing, cannot grow on organic carbon sources, can be sensitive to soaps and antibiotics, and have never been associated with any disease or infection in animals or humans.

[0155] Ammonia-oxidizing microorganisms produce coenzyme Q8 (CoQ8) as a by-product of the process in which they produce nitrite and nitric oxide. CoQ8 is a coenzyme Q with eight carbons in its isoprenoid side chain. Without wishing to be bound by any particular theory, due to the role of coenzyme Q as a key component of several cellular functions, such as mediating cell signaling and preventing cell death (anti-aging), the beneficial properties of these microorganisms may be further enhanced by their particular ability to produce CoQ8.

[0156] In some embodiments, the ammonia oxidizing bacteria may catalyze the following reaction:

[0157] At neutral pH levels, ammonia, generated from ammonium under neutral pH conditions, is the substrate for the initial reaction. The conversion of ammonia to nitrite occurs in two steps, catalyzed by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), respectively, as follows: NH3+2H + +2e-+O2→NH2OH+H2O (A) NH2OH+H2O→NO2 - +4e-+5H + (B)

[0158] In some instances, reaction B is reported to show nitrous acid (HNO) formation at low pH, as follows: NH2OH+H2O→HNO2+4e-+4H +

[0159] In certain embodiments, NH + and NH3 may be used interchangeably throughout this disclosure.

[0160] Examples of ammonia-oxidizing bacteria include Nitrosomonas eutropha strains, such as D23 and C91 discussed herein, as well as other bacteria in the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, and Nitrosovibrio. The D23 Nitrosomonas eutropha strain refers to the strain designated AOB D23-100, deposited on April 8, 2014, with the American Tissue Culture Collection (ATCC) (10801 University Blvd., Manassas, VA, USA), with accession number PTA-121157. The nucleic acid sequence, e.g., genome sequence, of accession number PTA-121157 is incorporated herein by reference in its entirety for all purposes. "AOB D23-100" may also be referred to as D23 or B244 throughout this disclosure.

[0161] Examples of ammonia-oxidizing archaea include Methanobrevibacter, Methanosphaera, Methanosarcina, Nitroscaldus, Nitrosopumilus, and archaea in the genus Nitrososphaera (e.g., Nitrososphaera viennensis, Nitrososphaera gargensis). Different phylogenetic types of archaea, such as methanogens and halophilic archaea, can be included in the preparations disclosed herein. Examples of archaea also include archaea in the lineages of Euryarchaea (e.g., Methanosarcina), Crenarchaea, Aiguarchaea, and Thaumarchaea (e.g., Giganthauma karukerense, Giganthauma insulaporcus, Caldiarchaeum subterraneum, Cenarchaeum symbiosum).

[0162] Each and every nucleic acid and amino acid sequence disclosed in International (PCT) Patent Application Publication No. WO2015 / 160911 (International (PCT) Patent Application No. PCT / US2015 / 025909, filed April 15, 2015) is incorporated herein by reference in its entirety for all purposes. Similarly, any ammonia-oxidizing bacteria disclosed in International (PCT) Patent Application Publication No. WO2015 / 160911 (International (PCT) Patent Application No. PCT / US2015 / 025909, filed April 15, 2015) are also incorporated herein by reference in their entirety for all purposes. In certain embodiments, the ammonia-oxidizing microorganism is a strain described therein.

[0163] According to one or more embodiments, the ammonia-oxidizing microorganisms may exist in several metabolic states, for example, growth states, storage states, and / or polyphosphate-loaded states.

[0164] According to one or more embodiments, the ammonia-oxidizing microorganisms may have desirable properties, e.g., optimized properties, such as the ability to inhibit the growth of pathogenic bacteria and an enhanced ability to produce nitric oxide and nitric oxide precursors.

[0165] Optimized Nitrosomonas eutropha (N. eutropha), as that term is used herein, refers to optimized growth rate; optimized NH4 + oxidation rate; and / or NH4 + In one embodiment, optimized N. eutropha is characterized by the expression of ammonia monooxygenase, hydroxylamine oxidoreductase, cytochrome c554, and cytochrome c M At least one nucleotide, e.g., one nucleotide, in a gene selected from 552 differs from naturally occurring N. eutropha. The difference can arise, for example, through the selection of spontaneously occurring mutations, induced mutations, or directed genetic engineering of N. eutropha. In one embodiment, the optimized N. eutropha differs from naturally occurring N. eutropha in that it has a collection of alleles that do not occur together in nature. These differences can provide one or more of the following: treatment or prevention of a disease or condition, for example, but not limited to, one associated with low nitrite levels.

[0166] Any ammonia-oxidizing bacterium, such as N. eutropha, for example, N. eutropha called "D23," also known as "B244" or "AOB D23-100," may have some of the above characteristics. Any ammonia-oxidizing archaea (AOA) may also have some of the above characteristics.

[0167] The AOB contemplated in the present disclosure may contain mutations compared to wild-type AOB. These mutations may, for example, arise spontaneously, be introduced by random mutagenesis, or be introduced by targeted mutagenesis. For example, the AOB may lack one or more genes or regulatory DNA sequences that wild-type AOB typically contains. The AOB may also contain point mutations, substitutions, insertions, deletions, and / or rearrangements compared to sequenced or wild-type strains. The AOB may be a purified preparation of optimized AOB.

[0168] In certain embodiments, the AOB is transgenic. For example, the AOB may contain one or more genes or regulatory DNA sequences that wild-type ammonia-oxidizing bacteria lack. More particularly, the ammonia-oxidizing bacteria may contain, for example, a reporter gene, a selectable marker, an enzyme-encoding gene, or a promoter (including an inducible promoter or a repressible promoter). In some embodiments, the additional gene or regulatory DNA sequence is integrated into the bacterial chromosome; in some embodiments, the additional gene or regulatory DNA sequence is located on a plasmid.

[0169] In some embodiments, AOB differs from naturally occurring bacteria by at least one nucleotide.For example, AOB may differ from naturally occurring bacteria in the gene or protein that is part of a related pathway, such as ammonia metabolic pathway, urea metabolic pathway, or pathway for producing nitric oxide or nitric oxide precursors.More particularly, AOB may include mutations that increase the activity of a pathway, for example, by increasing the level or activity of the components of that pathway.

[0170] The above-mentioned mutations can be introduced using any suitable technique. Numerous methods are known for introducing mutations at a given position. For example, site-directed mutagenesis, oligonucleotide-directed mutagenesis, or site-specific mutagenesis can be used. Non-limiting examples of specific mutagenesis protocols are described, for example, in Mutagenesis, pp. 13.1-13.105 (Sambrook and Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3rd ed. 2001). Furthermore, various mutagenesis protocols can be obtained from suppliers. Non-limiting examples of well-characterized mutagenesis protocols available from Altered Sites® II in vitro Mutagenesis Systems (Promega Corp., Madison, Wis.); Erase-a-Base® System (Promega, Madison, Wis.); GeneTailor™ Site-Directed Mutagenesis System (Invitrogen, Inc., Carlsbad, Calif.); QuikChange® II Site-Directed Mutagenesis Kits (Stratagene, La Jolla, Calif.); and Transformer™ Site-Directed Mutagenesis Kit (BD-Clontech, Mountain View, Calif.).

[0171] In certain embodiments of the present disclosure, the ammonia-oxidizing microorganisms can be axenic. A preparation (formulation or composition) of ammonia-oxidizing microorganisms can comprise, consist essentially of, or consist of pure ammonia-oxidizing microorganisms.

[0172] The ammonia oxidizing bacteria of the present disclosure may be from a genus selected from the group consisting of Nitrosomonas, Nitrosococcus, Nitrosospria, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof.

[0173] The present disclosure provides, inter alia, a unique, e.g., optimized, strain of ammonia-oxidizing bacteria, N. eutropha strain D23, that can increase the production of nitric oxide and nitric oxide precursors on the surface of a subject, e.g., a human subject. The present disclosure also provides methods of administering and using the bacteria, as well as preparations, compositions, formulations, and products comprising the bacteria.

[0174] In embodiments, the ammonia-oxidizing bacterium, e.g., N. eutropha, is not naturally occurring. For example, it may have desirable mutations that have accumulated over a period of selection. In other embodiments, desirable mutations may be introduced by the experimenter. In some embodiments, the N. eutropha may be a purified preparation or may be an optimized N. eutropha.

[0175] In a preferred embodiment, the N. eutropha strain is autotrophic and therefore unable to cause infection. Preferred strains utilize urea as well as ammonia, such that hydrolysis of urea in sweat is not required before absorption and utilization by the bacteria. Also, in order to grow at low pH, the bacteria require NH4 + The strain selected must also be able to live on the external skin of a subject, e.g., a human, and tolerate the conditions therein.

[0176] Although this disclosure makes specific reference to N. eutropha strain D23, the preparations, methods, compositions, treatments, formulations and products may be used with one or more other strains of N. eutropha, one or more other species of Nitrosomonas, and one or more other ammonia-oxidizing microorganisms, such as ammonia-oxidizing bacteria or other ammonia-oxidizing archaea.

[0177] In certain embodiments, the bacteria having the sequence characteristics described above have (1) an optimized growth rate as measured by doubling time, (2) an optimized growth rate as measured by OD600, and (3) an optimized NH4 + Oxidation rate, (4) NH4 + and (4) optimized resistance to NO2 - Specific non-limiting subcombinations of these properties are identified in the following paragraphs.

[0178] In some embodiments, the ammonia-oxidizing bacteria, e.g., N. eutropha described herein, or pure compositions thereof, have (1) an optimized growth rate as measured by doubling time, (2) an optimized growth rate as measured by OD600, and (3) an optimized NH4 + Oxidation rate, (4) NH4 + and (4) optimized resistance to NO2 -and optimized resistance to. For example, the bacterium may have characteristics (1) and (2); (2) and (3); (3) and (4); or (4) and (5) from the first list of this paragraph. As another example, the bacterium may have characteristics (1), (2), and (3); (1), (2), and (4); (1), (2), and (5); (1), (3), and (4); (1), (3), and (5); (1), (4), and (5); (2), (3), and (4); (2), (3), and (5), or (3), (4), and (5). As a further example, the bacteria may have characteristics (1), (2), (3), and (4) from the first list of this paragraph; (1), (2), (3), and (5); (1), (2), (4), and (5); (1), (3), (4), and (5); or (2), (3), (4), and (5). In some embodiments, the bacteria have characteristics (1), (2), (3), (4), and (5) from the first list of this paragraph.

[0179] In certain embodiments, the N. eutropha strain comprises a nucleic acid sequence, e.g., a genome, that hybridizes under low stringency, medium stringency, high stringency, or very high stringency, or other hybridization conditions, to SEQ ID NO: 1 of International (PCT) Patent Application Publication No. WO2015160911 (International (PCT) Patent Application No. PCT / US2015 / 025909, filed April 15, 2015), or the genome of strain D23, deposited in the form of 25 vials with the ATCC Patent Depository on April 8, 2014, designated AOBD23-100, and under accession number PTA-121157, or its complement.

[0180] The D23 strain is not considered a natural product, but rather has acquired certain mutations and characteristics during an extended period of laboratory cultivation and selection. For example, D23 has been found to grow at temperatures above about 200 or 250 mM NH4 for periods longer than 24 hours. + It has the ability to grow in the conditions.

[0181] In some embodiments, the N. eutropha disclosed herein differ from naturally occurring bacteria in the abundance of siderophores. For example, N. eutropha may have elevated or reduced levels of siderophores compared to N. eutropha C91. Generally, siderophores are secreted iron-chelating compounds that help bacteria capture iron from their environment. Some siderophores are peptides, while others are small organic molecules.

[0182] The practice of the present invention may employ, unless otherwise indicated, conventional methods of immunology, molecular biology, and recombinant DNA technology within the skill of those in the art. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (Current Edition); and Current Protocols in Molecular Biology (FM Ausubel, et al. eds., current edition).

[0183] Selected Definitions An ammonia-oxidizing microorganism, e.g., an ammonia-oxidizing bacterium, refers to a microorganism that is capable of oxidizing ammonia or ammonium to nitrite at a rate, e.g., a substantial rate, e.g., a predetermined rate. The rate, e.g., a predetermined rate, can be determined or measured, for example, in an in vitro assay or when administered to a subject, e.g., a human, based on the amount of ammonium ion (NH + ) (e.g., about 200 mM) of nitrite (NO - The rate may refer to the conversion of NO2 to ATP, for example, at least about 1 picomole per mg of protein per minute, 0.01, 0.1, 1, 10, 25, 50, 75, 125, or 150 nanomoles of NO2 per mg of protein per minute for a continuous culture with an OD of about 0.5. -, e.g., about 0.01-1, 0.1-50, 50-100, 100-150, 75-175, 75-125, 100-125, 125-150, or 125-175 nanomoles / min / mg protein, e.g., about 125 nanomoles of NO per mg of protein per minute. - The rate of conversion can be between about 1 picomole per mg of protein per minute and about 1 millimole per mg of protein per minute. The rate of conversion can be at most about 1 mole of NO2 per mg of protein per minute. - , e.g., at least about, about, or at most about 1 decimol, 1 centimole, 1 millimole, or 1 micromole of NO per mg of protein per minute. - It could be.

[0184] As used herein, "pure" refers to a composition comprising an organism that is substantially free of other organisms. For example, a pure culture of ammonia-oxidizing bacteria is a culture that is substantially free of organisms other than ammonia-oxidizing bacteria. For example, a pure culture of N. eutropha is a culture that is substantially free of organisms other than N. eutropha. In some embodiments, "substantially free" indicates that other organisms are not detectable by methods used to detect them, such as plating cultures and examining colony morphology, or PCR for conserved genes, such as 16S RNA. A pure composition may include non-living elements, such as nutrients or excipients. Any of the embodiments, preparations, compositions, or formulations of ammonia-oxidizing bacteria discussed herein may optionally comprise, consist essentially of, or consist of pure ammonia-oxidizing bacteria.

[0185] Throughout this disclosure, a formulation may refer to a composition or a preparation or a product.

[0186] As used herein, an "autotroph," e.g., an autotrophic bacterium, is any organism capable of self-nutrition by using inorganic materials as a source of nutrients and photosynthesis or chemosynthesis as a source of energy. Autotrophic bacteria can survive on carbon dioxide and other sources, such as oxidation of ammonia to nitrite, oxidation of hydrogen sulfide, and oxidation of Fe. 2+ From Fe 3+ and ATP derived from the oxidation of ATP to ATP. The autotrophic bacteria of the present disclosure are unable to cause infectious diseases.

[0187] As used herein, "administered in combination" means that two (or more) different treatments are delivered to a subject during the course of the subject's suffering from a disorder, for example, two or more treatments are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated. In some embodiments, the delivery of one treatment is still occurring when the delivery of a second treatment begins, and thus there is overlap. This is sometimes referred to herein as "simultaneous" or "concomitant" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. This is sometimes referred to herein as "continuous" or "sequential delivery." In either case, the treatment is more effective due to the combined administration. For example, the second treatment may be more effective, e.g., an equivalent effect may be seen with less of the second treatment, or the second treatment may reduce symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation may be seen with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other treatment. The effects of the two treatments may be partially additive, fully additive, or greater than additive (i.e., synergistic). The delivery may be such that the effect of the first treatment delivered is still detectable at the time the second treatment is delivered. In some embodiments, one or more treatments may be delivered prior to diagnosis of a patient with a disorder.

[0188] The term "isolated" as used herein refers to a material that has been removed from its original or native environment (e.g., the natural environment in which it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide that has been separated by human intervention from some or all of the coexisting materials in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition may still be said to be isolated in that it is not part of the environment in which it is found in nature.

[0189] As used herein, the term "optimized growth rate" refers to one or more of the following: a doubling time of less than about 4, 5, 6, 7, 8, 9, or 10 hours when cultured under batch conditions described herein in Example 2; a doubling time of less than about 16, 18, 20, 22, 24, or 26 hours when grown under chemostat conditions described herein in Example 2; or growth from about 0.15 to an OD of at least about 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 over about 1 or 2 days. In certain embodiments, an optimized growth rate is one that has a doubling time that is at least 10, 20, 30, 40, or 50% shorter than the doubling time of naturally occurring N. eutropha.

[0190] As used herein, "optimized NH4 + The oxidation rate is determined by the ratio of NH3 or NH4 + NO2 - For example, the rate may be at least about 50, 75, 125, or 150 micromoles per minute of NH4 + (e.g., about 200 mM) NO2 - In some embodiments, the optimized NH4 +The oxidation rates are at least 10, 20, 30, 40, or 50% faster than those seen in naturally occurring N. eutropha to NH3 or NH4 + is NO2 - is the speed converted into

[0191] As used herein, "NH4 + "Optimized resistance to NH3 or NH4" refers to resistance to NH3 or NH4 of greater than 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mM for at least about 24 or 48 hours. + In some embodiments, the ability to grow in conditions of NH4 + Optimized resistance to selected concentrations of NH3 or NH4 is greater than that possible for naturally occurring N. eutropha. + "Growth" refers to the ability to grow at least 10, 20, 30, 40, or 50% more rapidly, or at least 10, 20, 30, 40, or 50% longer in the presence of

[0192] As used herein, "transgenic" means containing one or more exogenous pieces of DNA. The exogenous DNA is derived from another organism, such as another bacterium, a bacteriophage, an animal, or a plant.

[0193] As used herein, treating a disease or condition refers to reducing the severity or frequency of at least one symptom of that disease or condition compared to a similar but untreated patient.Treatment can also refer to stopping, slowing down, or reversing the progression of a disease or condition compared to a similar but untreated patient.Treatment can include addressing the underlying cause of the disease and / or one or more symptoms.

[0194] As used herein, a therapeutically effective amount refers to a dose sufficient to prevent the progression of a disease or condition, or to cause regression of a disease or condition, or capable of alleviating the symptoms of a disease or condition, or capable of achieving a desired result. A therapeutically effective dose can be measured, for example, as the number of bacteria or viable bacteria (e.g., in CFU), or as the mass of bacteria (e.g., in milligrams, grams, or kilograms), or as the volume of bacteria (e.g., mm 3 can be measured.

[0195] As used herein, the term "viability" refers to the ability of an autotrophic microorganism, e.g., an ammonia-oxidizing microorganism, to oxidize ammonia, ammonium, or urea to nitrite at a predetermined rate. In some embodiments, the rate is at least about 1 picomole, 0.01, 0.1, 1, 10, 25, 50, 75, 125, or 150 nanomoles of NO per minute. - , e.g., about 0.01 to 1, 0.1 to 50, 50 to 100, 100 to 150, 75 to 175, 75 to 125, 100 to 125, 125 to 150, or 125 to 175 nanomoles per minute, e.g., about 125 nanomoles per minute of NO2 - At a rate of + ) (e.g., about 200 mM) of nitrite (NO - The conversion rate is at most about 1 mole of NO2 per minute. - , e.g., at least about, about, or at most about 1 decimol, 1 centimole, 1 millimole, or 1 micromole of NO per minute. - Viable ammonia-oxidizing microorganisms may generally include culturable AOM or AOM that are otherwise capable of producing NO, nitrate, or nitrite.

[0196] As used herein, the term "activity" refers to the ability of an autotrophic microorganism, e.g., an ammonia-oxidizing microorganism, to reproduce. Active cells are referred to as colony-forming units (CFUs). Inactive AOM may be sterile, dead, or otherwise non-viable. In some embodiments, AOM may be inactivated by time, temperature, or contact with a toxic substance, e.g., a preservative. In certain embodiments, inactive AOM may be heat-killed. Inactive AOM may be killed by freeze / thaw, ethanol treatment, or aging. Inactive AOM may contain one or more structural or intracellular components, e.g., structural or intracellular components that have a cosmetic or therapeutic effect. In some embodiments, the structural or intracellular components may play a role in a signaling cascade, e.g., have an effect on the response of activated immune cells, e.g., have an inhibitory effect on the response of activated immune cells.

[0197] As used herein, a "subject" may include animals, mammals, humans, non-human animals, livestock animals, or companion animals. The term "subject" is intended to include humans and non-human animals, such as vertebrates, large animals, and primates. In certain embodiments, the subject is a mammalian subject, and in certain embodiments, the subject is a human subject. While human applications are clearly envisioned, veterinary applications, for example, in non-human animals, are also contemplated herein. The term "non-human animal" of the present disclosure includes all vertebrates, such as non-mammals (e.g., birds, e.g., chickens; amphibians; reptiles) and mammals, such as non-human primates, domesticated animals, and agriculturally useful animals, such as sheep, dogs, cats, cows, pigs, and rats, among others.

[0198] "Microbiome" refers to a population, e.g., one or more microorganisms, living on a subject, e.g., in the gut, mouth, skin, and / or other locations of a subject. The population may have one or more beneficial functions and / or benefits associated with supporting the life of the subject.

[0199] "Biome-friendly" refers to an object, e.g., a product, e.g., a cosmetic product, e.g., a finished cosmetic product, that can minimize disruption of a subject's microbiome. For example, biome-friendly refers to a product that can be applied to a subject that can maintain, minimize disruption of, and / or restore the microbiome at the time of application after a period of time after application of the product. In embodiments, biome-friendly can refer to ammonia-oxidizing microorganism-friendly, e.g., ammonia-oxidizing bacteria-friendly, in that the product can minimize disruption of ammonia-oxidizing bacteria in the subject. In embodiments, "biome-friendly" can be referred to as "biome-compatible."

[0200] A "natural product" is or may include a product that can be at least partially derived from nature. A natural product can be or include something produced by a living organism, including the organism itself. A natural product can include or comprise whole organisms, parts of organisms (e.g., plant leaves), extracts from organisms, organic compounds from organisms, and purified organic compounds from organisms. A natural product can be or include organic substances found in cells, including primary metabolites (amino acids, carbohydrates, and nucleic acids) and secondary metabolites (organic compounds found in a limited range of species, e.g., polyketides, fatty acids, terpenoids, steroids, phenylpropanoids, alkaloids, specialized amino acids and peptides, specialized carbohydrates). A natural product can be or include polymeric organic materials, such as cellulose, lignin, and proteins.

[0201] As used herein, "presence" or "level" can refer to the qualitative or quantitative amount of any one or more of a component, e.g., ammonia-oxidizing microorganisms, ammonia, ammonium ions, urea, nitrite, or nitric oxide. Presence or level can include a zero value or the absence of a component.

[0202] As used herein, the term "surfactant" includes compounds that can reduce the surface or interfacial tension between two liquids or between a liquid and a solid. Surfactants can act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. Surfactants can include one or more of the following, alone or in combination with the listed or other surfactants or surfactant-like compounds: cocamidopropyl betaine (ColaTeric COAB), polyethylene sorbitol esters (e.g., Tween®), ... 80), Ethoxylated Lauryl Alcohol (RhodaSurf 6 NAT), Sodium Laureth Sulfate / Lauryl Glucoside / Cocamidopropyl Betaine (Plantapon 611 L UP), Sodium Laureth Sulfate (e.g., RhodaPex ESB 70 NAT), alkyl polyglucosides (e.g., Plantaren 2000 N UP), sodium laureth sulfate (Plantaren 200), Dr. Bronner's Castile soap, Dr. Bronner's baby soap, lauramine oxide (ColaLux Lo), sodium dodecyl sulfate (SDS), polysulfonic acid alkyl polyglucoside (PolySufanate 160 P), sodium lauryl sulfate (Stepanol-WA Extra K), and combinations thereof. Dr. Bronner's Castile soap and baby soap contain water, organic coconut oil, potassium hydroxide, organic olive oil, organic fair deal hemp oil, organic jojoba oil, citric acid, and tocopherol. Surfactants may include sodium hydroxypropylsulfonate laurylglucoside (Suga® nate 160NC), lauramidopropyl betaine (Cola® Teric LMB); cocamidopropyl hydroxysultaine (Cola® Teric CBS); disodium cocoamphodiacetate (Cola® Teric CDCX-LV); sodium hydroxypropyl phosphate laurylglucoside (Suga® Fax D12). Surfactants may include sodium lauroyl methyl isethionate (Iselux® LQ-CLR-SB); sodium cocoyl methyl taurate (Pureact WS Conc.); water (and) sodium lauroyl methyl isethionate (and) cocamidopropyl betaine (and) sodium cocoyl isethionate (and) sodium oleoyl methyl taurate (Iselux® SFS-SB). Other surfactants are contemplated by the present disclosure.

[0203] As used herein, the term "storable" may refer to a preparation, composition, or formulation that has adequate shelf life after a period of storage at room temperature. The storage period may be greater than about 4 weeks. The storage period at room temperature may be greater than about 6 months, greater than about 1 year, greater than about 2 years, or greater than about 5 years. Room temperature may include an average daily temperature between about 20°C and 25°C. In certain embodiments, a "storable" preparation, composition, or formulation may have adequate shelf life after a period of at least about 6 months of storage at refrigerated temperatures. The storage period at refrigerated temperatures may be greater than about 1 year, greater than about 3 years, greater than about 5 years, greater than about 7 years, greater than about 10 years, or greater than about 12 years. Refrigerated temperatures may include an average daily temperature between about 2°C and 6°C, or about 4°C. Other conditions for the duration of storage may include, for example, an average daily relative humidity of less than about 80% and / or an average daily atmospheric pressure, or a pressure between about 12-15 psi.

[0204] A "unit" of a finished product refers to a single entity of the finished preparation, which may form an individual or whole component for use or sale by an end user. In some embodiments, a unit may be a single entity, but may form an individual component of a larger or more complex whole. In certain embodiments, a unit may be an individual end-use container containing the preparation for sale or use by an end user.

[0205] "End user," as that term is used herein, refers to a person who uses the finished preparation, for example, by applying the finished preparation to themselves, or who applies or provides the finished preparation to a subject, e.g., another person, or an animal, e.g., a companion animal.

[0206] "End-use container," as the term is used herein, refers to a vessel that houses a formulation, e.g., a finished formulation. The end-use container may allow for delivery of the finished formulation from the container to the outside environment. In certain embodiments, the end-use container may prevent or reduce retrograde flow of the contents of the container. In other embodiments, the end-use container may not prevent or reduce retrograde flow of the contents of the container. The end-use container may be configured to provide unidirectional flow and / or zero dead volume. The end-use container may not be configured to provide unidirectional flow and / or zero dead volume.

[0207] The end-use container may be constructed of any suitable material that is compatible with the contents of the container and the external environment. For example, the end-use container may be made of glass, aluminum, or one or more polymers, such as high-density polyethylene polymers.

[0208] Preparations, compositions, formulations, and products comprising ammonia-oxidizing microorganisms The present disclosure provides, inter alia, compositions comprising ammonia-oxidizing microorganisms, preparations comprising AOM, e.g., purified and / or optimized preparations, formulations comprising AOM, and various products comprising AOM, e.g., natural products, non-natural products, enhanced natural products, consumer products, therapeutic products, or cosmetic products. The terms preparation, composition, formulation, and product may be used interchangeably herein.

[0209] Any embodiment, preparation, composition, formulation, or product of ammonia oxidizing microorganisms discussed herein may comprise, consist essentially of, or consist of (optionally pure) ammonia oxidizing microorganisms, e.g., live ammonia oxidizing microorganisms.

[0210] The preparation may contain or be supplemented with a product or by-product of ammonia-oxidizing microorganisms, such as nitrite, nitrate, nitric oxide, or CoQ8. In at least some embodiments, the preparation may contain or be supplemented with a composition that promotes the growth or metabolism of ammonia-oxidizing microorganisms, promotes the production of a product or by-product of ammonia-oxidizing microorganisms, promotes urease activity, or has a synergistic effect with ammonia-oxidizing microorganisms, such as ammonia, ammonium salts, urea, and urease. For example, the preparation may be supplemented with one or more of NO, nitrite, nitrate, CoQ8, ammonia, ammonium salts, urea, and urease. The supplement may be included in the same formulation as the ammonia-oxidizing microorganisms or in a separate formulation for simultaneous or combined administration. The supplement formulation may be prepared for delivery via any delivery mode, such as inhaled NO, nitrite, or nitrate. The preparation may include, among others, at least one of ammonia, ammonium salts, and urea. The preparation may include or be supplemented with an anti-inflammatory agent or composition that provides an anti-inflammatory effect.

[0211] The present disclosure provides preparations comprising ammonia-oxidizing microorganisms for cosmetic use.

[0212] The present disclosure provides preparations comprising ammonia-oxidizing microorganisms for therapeutic use.

[0213] In some embodiments, a preparation of ammonia-oxidizing microorganisms may include a concentration or amount sufficient to have a desired cosmetic effect, e.g., an effective amount of ammonia-oxidizing microorganisms. The preparation may be formulated and / or delivered to impart the desired cosmetic effect locally and / or systemically.

[0214] In some embodiments, a preparation of ammonia-oxidizing microorganisms may include a concentration or amount sufficient to have a desired therapeutic effect, e.g., an effective amount of ammonia-oxidizing microorganisms, to at least partially treat a condition or disease. The preparation may be formulated and / or delivered to impart the desired therapeutic effect locally and / or systemically.

[0215] In some embodiments, the preparation of ammonia-oxidizing microorganisms can include a sufficient concentration or amount, e.g., an effective amount, of ammonia-oxidizing microorganisms to alter, e.g., reduce or increase, the amount, concentration, or proportion of bacteria, or genera of bacteria, in a subject. The bacteria can be non-pathogenic or pathogenic, or potentially pathogenic.

[0216] In some embodiments, the preparation of ammonia-oxidizing microorganisms may include a sufficient concentration or amount, e.g., an effective amount, of ammonia-oxidizing microorganisms to modulate the microbiome associated with a subject.

[0217] In some embodiments, the preparation of ammonia-oxidizing microorganisms may contain a concentration or amount, e.g., an effective amount, of ammonia-oxidizing microorganisms sufficient to deliver NO to a subject. The preparation of ammonia-oxidizing microorganisms may contain a concentration or amount, e.g., an effective amount, of ammonia-oxidizing microorganisms such that, when administered, the preparation modulates, changes, or alters the level of nitrite or NO in a target tissue or in the circulation. For example, the preparation of ammonia-oxidizing microorganisms may contain a concentration or amount, e.g., an effective amount, of ammonia-oxidizing microorganisms such that, when administered, the preparation causes an increase in the level of nitrite or NO in a target tissue or in the circulation.

[0218] The present disclosure provides, inter alia, non-limiting compositions comprising an ammonia-oxidizing microorganism, e.g., N. eutropha, e.g., a purified preparation of optimized N. eutropha. In some embodiments, the N. eutropha in the composition is optimized for growth rate, optimized NH4 production, or the like. + Oxidation rate, and NH4 +and optimized resistance to

[0219] In some embodiments, the present disclosure provides compositions with a specified number of species. The composition may contain only one type of species, for example, one type of ammonia-oxidizing microorganism. The present disclosure also provides compositions with, for example, N. eutropha and one other type of organism, but no other types of organisms. In other examples, the composition may contain, for example, N. eutropha and 2, 3, 4, 5, 6, 7, 8, 9, or 10 other types of organisms, but no other types of organisms. The other types of organisms in the composition may be, for example, bacteria, such as ammonia-oxidizing bacteria. Suitable ammonia-oxidizing microorganisms for this purpose include those in the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, or Nitrosovibrio. Similarly, the composition may also contain AOA.

[0220] In some embodiments, for example, a composition comprising N. eutropha provides conditions that support the viability of N. eutropha. For example, the composition may promote the growth and metabolism of N. eutropha, or may promote a dormant state (e.g., frozen) from which viable N. eutropha can be recovered. If the composition promotes growth or metabolism, the composition may contain water and / or nutrients consumed by N. eutropha, such as ammonium, ammonia, urea, oxygen, carbon dioxide, or trace minerals. In some embodiments, a composition comprising ammonia-oxidizing microorganisms provides conditions that support the viability of ammonia-oxidizing microorganisms. For example, the composition may promote the growth and metabolism of ammonia-oxidizing microorganisms, or may promote a dormant state (e.g., frozen) or storage state described herein from which viable ammonia-oxidizing microorganisms can be recovered. If the composition promotes growth or metabolism, the composition may contain water and / or nutrients consumed by ammonia-oxidizing microorganisms, such as ammonium ion, ammonia, urea, oxygen, carbon dioxide, or trace minerals.

[0221] In some embodiments, one or more other organisms, for example, organisms other than ammonia-oxidizing microorganisms, can be included in the preparation of ammonia-oxidizing microorganisms.For example, a community of organisms or organisms of a genus selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacter, and combinations thereof can be provided in the preparation of ammonia-oxidizing microorganisms.In some embodiments, the preparation can be substantially free of other organisms.

[0222] The preparation of ammonia-oxidizing microorganisms was prepared at approximately 10 3 ~about 10 14 In some embodiments, the preparation of ammonia oxidizing microorganisms may comprise between about 10 CFU / ml. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 2 × 10 11 , 5×10 11 , 10 12 , 2 × 10 12 , 5×10 12 , 10 13 , 2 × 10 13 , 5×10 13 , or 10 14 ; or about 10 3 ~10 4 , 10 4 ~10 5 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , 10 9 ~10 10 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14CFU / ml, or approximately 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 2 × 10 11 , 5×10 11 , 10 12 , 2 × 10 12 , 5×10 12 , 10 13 , 2 × 10 13 , 5×10 13 , or 10 14 ; or about 10 3 ~10 4 , 10 4 ~10 5 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , 10 9 ~10 10 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 It may contain more than CFU / ml.

[0223] In some embodiments, the preparation of ammonia oxidizing microorganisms comprises about 1 x 10 9 ~About 10×10 9 In some embodiments, the administered dose of the preparation may comprise between about 3 x 10 CFU / ml per day. 10 CFU, e.g., 3 x 10 10 In some embodiments, the administered dose of the preparation may contain about 1 x 10 CFU per day. 9 ~About 10×10 9 CFU, e.g., about 1 x 10 per day 9 ~About 10×10 9In some embodiments, the administered dose of the preparation may contain about 10 CFU per administration or per day. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 2 × 10 11 , 5×10 11 , 10 12 , 2 × 10 12 , 5×10 12 , 10 13 , 2 × 10 13 , 5×10 13 , or 10 14 ; or about 10 3 ~10 4 , 10 4 ~10 5 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , 10 9 ~10 10 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 It may contain CFU.

[0224] In some embodiments, the administered dose of the preparation is at least about 7×10 per week 10 CFU, e.g., 21 x 10 10 In some embodiments, the administered dose of the preparation may comprise about 1 x 10 CFU per week. 9 ~About 10×10 9 CFU, e.g., approximately 1 x 10 per week 9 ~About 10×10 9 In some embodiments, the administered dose of the preparation may contain about 10 CFU per week. 3 , 10 4, 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 2 × 10 11 , 5×10 11 , 10 12 , 2 × 10 12 , 5×10 12 , 10 13 , 2 × 10 13 , 5×10 13 , or 10 14 ; or about 10 3 ~10 4 , 10 4 ~10 5 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , 10 9 ~10 10 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 CFU, or approximately 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 2 × 10 11 , 5×10 11 , 10 12 , 2 × 10 12 , 5×10 12 , 10 13 , 2 × 10 13 , 5×10 13 , or 10 14 ; or about 10 3 ~10 4 , 10 4 ~10 5 , 10 6 ~107 , 10 7 ~10 8 , 10 8 ~10 9 , 10 9 ~10 10 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 It may contain more than CFU.

[0225] In some embodiments, the administered dose of the preparation is at least about 30×10 per month 10 CFU, e.g., 90 x 10 10 In some embodiments, the administered dose of the preparation may comprise about 1 x 10 CFU per month. 9 ~About 10×10 9 CFU, e.g., approximately 1 x 10 per month 9 ~About 10×10 9 In some embodiments, the administered dose of the preparation may contain about 10 CFU per month. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 2 × 10 11 , 5×10 11 , 10 12 , 2 × 10 12 , 5×10 12 , 10 13 , 2 × 10 13 , 5×10 13 , or 10 14 ; or about 10 3 ~10 4 , 10 4 ~10 5 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~109 , 10 9 ~10 10 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 CFU, or approximately 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 2 × 10 11 , 5×10 11 , 10 12 , 2 × 10 12 , 5×10 12 , 10 13 , 2 × 10 13 , 5×10 13 , or 10 14 ; or about 10 3 ~10 4 , 10 4 ~10 5 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , 10 9 ~10 10 , 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 It may contain more than CFU.

[0226] In some embodiments, a preparation of ammonia-oxidizing microorganisms can contain between about 0.1 milligrams (mg) and about 1000 mg of ammonia-oxidizing microorganisms. In certain aspects, a preparation can contain between about 50 mg and about 1000 mg of ammonia-oxidizing microorganisms. Preparations can contain between about 0.1-0.5 mg, 0.2-0.7 mg, 0.5-1.0 mg, 0.5-2 mg, 0.5-5 mg, 2.5-5 mg, 2.5-7.0 mg, 5.0-10 mg, 7.5-15 mg, 10-15 mg, 15-20 mg, 15-25 mg, 20-30 mg, 25-50 mg, 25-75 mg, 50-75 mg, 50-100 mg, 75-100 mg, It may contain between 100-200mg, 200-300mg, 300-400mg, 400-500mg, 500-600mg, 600-700mg, 700-800mg, 800-900mg, 900-1000mg, 100-250mg, 250-500mg, 100-500mg, 500-750mg, 750-1000mg, or 500-1000mg.

[0227] The formulations disclosed herein may be shelf-stable. In certain embodiments, the formulations may have suitable efficacy after a period of storage at room temperature. Suitable efficacy may be cosmetic or therapeutic. The shelf-stable preparations may provide cosmetically effective results after a period of storage. The shelf-stable preparations may provide therapeutically effective results after a period of storage.

[0228] In certain embodiments, the shelf-stable formulation may contain CO2. The shelf-stable formulation may contain at least about 400 ppm CO2. For example, the shelf-stable formulation may contain at least about 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, or 650 ppm CO2. The shelf-stable formulation may be substantially free of polyphosphate. The shelf-stable formulation may contain less than about 1 μM polyphosphate. For example, the shelf-stable formulation may contain less than about 1 μM, 0.5 μM, 0.1 μM, 50 nM, or 10 nM polyphosphate.

[0229] Advantageously, the formulation can have a pH level that promotes the viability, e.g., metabolic activity, of AOM, e.g., N. eutropha. Urea hydrolyzes to ammonia, raising the pH to 7 to 8. AOB is very active in this pH range, lowering the pH to about 6 as NH3 is converted to ammonium and becomes unavailable. Lower pH levels, e.g., about pH 4, are also acceptable.

[0230] Ammonia-oxidizing microorganisms, such as N. eutropha, can be combined with one or more pharmaceutically or cosmetically acceptable excipients. In some embodiments, a "pharmaceutically acceptable excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In some embodiments, each excipient is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009;Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007;Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, Fla., Please refer to 2009.

[0231] In some embodiments, a cosmetically acceptable excipient refers to a cosmetically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In some embodiments, each excipient is cosmetically acceptable in the sense that it is compatible with the other ingredients of the cosmetic formulation and is suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0232] While it is possible for the active ingredient, e.g., an ammonia-oxidizing microorganism, e.g., N. eutropha, to be administered alone, in many embodiments, the active ingredient is present in a pharmaceutical formulation or composition. Thus, the present disclosure provides pharmaceutical formulations comprising an ammonia-oxidizing microorganism, e.g., N. eutropha, and a pharmaceutically acceptable excipient. The pharmaceutical composition may take the form of a pharmaceutical formulation described below.

[0233] According to one or more embodiments, preparations of ammonia-oxidizing microorganisms can be formulated to facilitate a desired delivery mechanism or mode of administration. Formulations described herein, e.g., pharmaceutical or cosmetic formulations, include those suitable for, for example, oral, enteral (including buccal, sublingual, sublabial, and rectal), parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (including various types of metered-dose pressurized aerosols, fine particle dusts or mists that can be generated by nebulizers or inhalers, including intranasally or via the lungs), intranasal, ocular, otic, rectal, injectable, urogenital, and topical (including dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration, although the most suitable route can depend, for example, on the condition or disorder of the recipient.

[0234] In some embodiments, the preparation of ammonia oxidizing microorganisms may comprise, consist essentially of, or consist of ammonia oxidizing microorganisms in a buffer solution comprising, consisting essentially of, or consisting of disodium phosphate and magnesium chloride, e.g., 50 mM NaHPO and 2 mM MgCl.

[0235] According to one or more non-limiting embodiments, a preparation comprising an ammonia-oxidizing microorganism may be administered to a subject, e.g., for cosmetic or therapeutic purposes, as a solution, suspension, powder, liquid, drops, spray, aerosol, mist, emulsion, foam, cream, ointment, gel, hydrogel, resin, tablet, capsule, film, suppository, enema, douche, vaginal suppository, insert, patch, e.g., a transdermal patch, or implantable device, e.g., a stent, catheter, vaginal ring, or intrauterine device.

[0236] Also disclosed are devices configured to deliver preparations containing live ammonia-oxidizing microorganisms via a desired mode of administration or otherwise targeted delivery.

[0237] According to one or more embodiments, the preparation can be formulated for targeted delivery to a subject, for example, to a target tissue, region, system, or organ of the subject. For example, the preparation can be formulated for delivery to the subject's eye, ear, nose, urogenital system, respiratory system, or gastrointestinal system. In some embodiments, targeted delivery can be based on the subject's condition or disorder. For example, the formulation for targeted delivery can be based on the desired local or systemic effect to be achieved, for example, a local or systemic therapeutic or cosmetic effect. In some embodiments, the target tissue, region, system, or organ of the subject can be selected for its association with the desired local or systemic effect.

[0238] The formulation can be conveniently presented in unit dosage form and can be prepared by any method known in the field of pharmacy.Typically, the method comprises the step of combining the active ingredient (for example, ammonia-oxidizing microorganism, for example, N. eutropha) with a pharmaceutical carrier that constitutes one or more accessory ingredients.Generally, the formulation is prepared by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.

[0239] The formulations may be presented as discrete units, such as capsules, cachets, or tablets, each containing a predetermined amount of N. eutropha, etc.; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil liquid emulsions. Formulations, such as solutions, aerosols, sprays, and mists, may be presented in multi-dosage forms, such as packaged units containing a predetermined number of doses, or in single-dosage forms, such as packaged units containing a single dose. The active ingredient may also be presented as a bolus, electuary, or paste. Various pharmaceutically acceptable carriers and their formulations are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences by EW Martin. Wang, YJ, and Hanson, M. See also A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2 S, 1988.

[0240] The composition of ammonia-oxidizing microorganisms, such as N. eutropha, can be administered in a form suitable for immediate release or extended release. Suitable examples of sustained-release systems include suitable polymeric materials, such as semipermeable polymer matrices in the form of shaped articles, such as films, or microcapsules; suitable hydrophobic materials, such as emulsions in acceptable oils; or ion exchange resins. The sustained-release system can be administered orally; rectally; parenterally; intracisternally; vaginally; intraperitoneally; externally, for example, as powders, ointments, gels, drops, or transdermal patches; bucally; or as a spray.

[0241] The preparation for administration can be suitably formulated to provide controlled release of ammonia-oxidizing microorganisms, such as N. eutropha. For example, the pharmaceutical composition can be in the form of particles containing one or more of biodegradable polymers, polysaccharide gelling and / or bioadhesive polymers, or amphiphilic polymers. These compositions exhibit certain biocompatible features that allow controlled release of the active substance. See U.S. Patent No. 5,700,486.

[0242] Exemplary compositions include suspensions that may contain, for example, microcrystalline cellulose to provide bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, dicalcium phosphate, starch, magnesium stearate, and / or lactose, and / or other excipients, binders, fillers, disintegrants, diluents, and lubricants, such as mannitol, lactose, sucrose, and / or cyclodextrin. High molecular weight excipients, such as cellulose (avicel) or polyethylene glycol (PEG), may also be included in such formulations. Such formulations may also include excipients to aid mucoadhesion, such as hydroxypropyl cellulose (HPC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose (SCMC), maleic anhydride copolymers (e.g., Gantrez), and release-controlling agents, such as polyacrylic copolymers (e.g., Carbopol 934). Lubricants, glidants, flavors, colorants, and stabilizers may also be added for ease of fabrication and use. The surfactant may be a zwitterionic surfactant, a nonionic surfactant, or an anionic surfactant.

[0243] Excipients, e.g., surfactants, that may be used with embodiments of the present disclosure include cocamidopropyl betaine (ColaTeric COAB), polyethylene sorbitol esters (e.g., Tween 80), ethoxylated lauryl alcohol (RhodaSurf 6 NAT), sodium laureth sulfate / lauryl glucoside / cocamidopropyl betaine (Plantapon 611 L UP), sodium laureth sulfate (e.g., RhodaPex ESB 70 NAT), alkyl polyglucosides (e.g., Plantaren 2000 N UP), sodium laureth sulfate (Plantaren 200), Dr. Bronner's Castile soap, Dr. Bronner's Castile baby soap, lauramine oxide (ColaLux Lo), sodium dodecyl sulfate (SDS), polysulfonic acid alkyl polyglucoside (PolySufanate 160 P), sodium lauryl sulfate (Stepanol-WA Extra K), and combinations thereof. Dr. Bronner's Castile Soap and Dr. Bronner's Baby Soap contain water, organic coconut oil, potassium hydroxide, organic olive oil, organic fair deal hemp oil, organic jojoba oil, citric acid, and tocopherol.

[0244] In some embodiments, a surfactant may be used with the ammonia-oxidizing microorganism in an amount that allows nitrite production to occur. In some embodiments, the preparation may have less than about 0.0001% to about 10% surfactant. In some embodiments, the preparation may have between about 0.1% and about 10% surfactant. In some embodiments, the concentration of surfactant used may be between about 0.0001% and about 10%. In some embodiments, the preparation may be substantially free of surfactant.

[0245] In some embodiments, the formulation, e.g., preparation, may include other components that may enhance the effectiveness of the ammonia oxidizing microorganism, its delivery, or may enhance the treatment or indication.

[0246] In some embodiments, a chelator may be included in the preparation. The chelator may be a compound that can bind to another compound, for example, a metal. The chelator may assist in removing unwanted compounds from the environment, or may act in a protective manner to reduce or eliminate contact between certain compounds and the environment, for example, ammonia-oxidizing microorganisms, for example, ammonia-oxidizing microorganism preparations, for example, excipients. In some embodiments, the preparation may be substantially free of chelators.

[0247] Formulations may also contain aqueous and non-aqueous sterile suspensions, which may contain antioxidants, buffers, bacteriostats to prevent the growth of undesired microorganisms, solutes, and suspending and thickening agents. Formulations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as saline or water for injection, immediately prior to use. Extemporaneous solutions and suspensions may be prepared from powders, granules, and tablets of the type previously described. Exemplary compositions include solutions or suspensions that may contain, for example, suitable non-toxic pharmaceutically acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremaphor. The aqueous carrier can be, for example, an isotonic buffer solution having a pH of about 3.0 to about 8.0, about 3.5 to about 7.4, for example, a pH of 3.5 to 6.0, for example, a pH of 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphate, and sodium acetate / acetic acid buffers. The composition, in some embodiments, does not contain an oxidizing agent.

[0248] The excipient that can be included is, for example, protein, for example, human serum albumin or plasma preparation.If necessary, pharmaceutical compositions can also contain minor amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.In some embodiments, excipients, for example, pharmaceutically acceptable excipients or cosmetically acceptable excipients, can include anti-adhesion agents, binders, coatings, disintegrants, fillers, flavorings, pigments, lubricants, glidants, adsorbents, preservatives, or sweeteners.In some embodiments, the preparation can be substantially free of excipients.

[0249] In some embodiments, the preparation may be substantially free of one or more of the compounds or substances listed in the present disclosure.

[0250] Exemplary compositions for spray, aerosol, or mist administration include solutions in saline, which may contain, for example, benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other solubilizing or dispersing agents. Conveniently, in compositions for aerosol administration, the ammonia-oxidizing microorganism, e.g., N. eutropha, is delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges can be formulated to contain a powder mix of N. eutropha and a suitable powder base, e.g., lactose or starch. In certain embodiments, N. eutropha is administered as an aerosol from a metered-dose valve via an aerosol adapter, also known as an actuator. Optionally, stabilizers are also included, and / or porous particles are included for deep lung delivery (see, eg, US Pat. No. 6,447,743).

[0251] The formulation may be presented using carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are typically solid at ambient temperatures but liquefy and / or melt at body temperature to release the ammonia-oxidizing bacteria, e.g., N. eutropha.

[0252] Exemplary compositions for topical administration include a topical carrier, such as Plastibase (mineral oil gelled with polyethylene). In some aspects, the composition and / or excipients may be in one or more of the following forms: liquid, solid, or gel. For example, liquid suspensions may include, but are not limited to, water, saline, phosphate-buffered saline, or an ammonia-oxidized storage buffer. Gel formulations may include, but are not limited to, agar, silica, polyacrylic acid (e.g., Carbopol®), carboxymethylcellulose, starch, guar gum, alginate, or chitosan. In some embodiments, the formulation may be supplemented with an ammonia source, including, but not limited to, ammonium chloride or ammonium sulfate.

[0253] In some embodiments, ammonia-oxidizing microorganisms, such as N. eutropha, are formulated to improve NO penetration into the skin or other target tissues. Gel-forming materials, such as KY jelly or various hair gels, provide a diffusion barrier to NO loss into the surrounding air, thus improving dermal absorption of NO. NO levels in the skin generally do not exceed 20 nM / L because this level activates GCs, causing local vasodilation and oxidative destruction of excess NO.

[0254] It should be understood that in addition to the ingredients particularly mentioned above, the formulations described herein may include other agents conventional in the art having regard to the type of formulation in question.

[0255] The formulation, e.g., preparation, e.g., composition, may be provided in a container, delivery system, or delivery device that has a weight that may be less than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 grams, with or without the contents of the container.

[0256] Suitable unit dosage formulations are those containing an effective dose of ammonia oxidizing microorganism, eg, N. eutropha, as hereinbefore recited, or an appropriate fraction thereof.

[0257] The therapeutically effective amount of ammonia-oxidizing microorganism, such as N. eutropha, can be administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time.Thus, in pulse doses, a bolus dose of ammonia-oxidizing microorganism, such as N. eutropha, is provided, followed by a period in which ammonia-oxidizing microorganism, such as N. eutropha, is administered to the subject, followed by a second bolus dose.In specific, non-limiting examples, pulse doses are administered over the course of a day, a week, or a month.

[0258] In some embodiments, the ammonia-oxidizing microorganism preparation, e.g., formulation, e.g., composition, may be applied for a predetermined number of days. This may be based, at least in part, on, for example, the severity of the condition or disease, the response to treatment, the dosage and frequency of the doses applied. For example, the preparation may be applied for about 1-3, 3-5, 5-7, 7-9, 5-10, 10-14, 12-18, 12-21, 21-28, 28-35, 35-42, 42-49, 49-56, 46-63, 63-70, 70-77, 77-84, or 84-91 days, about 1 month, about 2 months, or about 3 months. In some embodiments, the ammonia-oxidizing bacteria are administered for an indeterminate period of time, e.g., for more than 1 year, more than 5 years, more than 10 years, more than 15 years, more than 30 years, more than 50 years, or more than 75 years. In certain embodiments, the preparation may be applied for about 16 days.

[0259] In some embodiments, the preparation of ammonia-oxidizing microorganisms, for example, formulations, for example, compositions, can be applied a predetermined number of times per day.This can be based at least in part on, for example, the severity of the condition or disease, response to treatment, the dosage and frequency of application.For example, the preparation can be applied 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 times per day.

[0260] In some embodiments, the preparation can be applied once a day. In other embodiments, the preparation can be applied twice a day. In some embodiments, the preparation can be applied in a first predetermined amount for a certain number of days, and in a second predetermined amount for a certain number of subsequent days. In some embodiments, the preparation can be applied for about 16 days.

[0261] According to one or more embodiments, the preparation may generally be compatible with the physiological environment associated with a subject. In at least some embodiments, the composition is formulated to have a substantially neutral pH or physiological pH, for example, the pH that normally prevails at the target site for the intended delivery, administration, or desired effect. The composition may be formulated to have a pH between about 5.5 and about 8.5. The composition may be formulated to include conditions, such as pH and osmolality, that are compatible with the physiological environment of the target site associated with a subject.

[0262] The preparation can be formulated for, for example, local or systemic transmucosal delivery and / or circulation. In some embodiments, the preparation can be formulated so that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the ammonia-oxidizing microorganism, its product, or its by-product (e.g., nitrate, nitrite, NO, or CoQ8) penetrates the deposition or target tissue. The preparation can be formulated so that 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the ammonia-oxidizing microorganism, its product, or its by-product penetrates the deposition or target tissue or enters the circulation.

[0263] According to one or more embodiments, the preparation may be in the form of a solution, suspension, emulsion, cream, ointment, gel, hydrogel, or liquid, such as a drop, spray, aerosol, or mist, tablet, capsule, or device for administration to a subject.

[0264] According to one or more embodiments, a preparation, composition, formulation, or product containing ammonia-oxidizing microorganisms may undergo quality control and / or testing while it is being made and / or upon completion. International (PCT) Patent Application Publication No. WO2015 / 179669 (filed May 21, 2015, International (PCT) Patent Application No. PCT / US2015 / 032017), the entire contents of which are incorporated herein by reference for all purposes, describes various methods for preparing and testing materials containing ammonia-oxidizing microorganisms. For example, one or more parameters, such as OD level, pH level, waste level, nutrient level, contaminant level, oxidation rate, nitrite level, protein concentration, can be compared to a predetermined value to assess or evaluate a preparation containing ammonia-oxidizing microorganisms.

[0265] The present disclosure provides, inter alia, kits containing the preparations of ammonia-oxidizing microorganisms disclosed herein. The preparations may contain separate units of ammonia-oxidizing microorganisms, such as solid, liquid, or gas preparations. The preparations, such as solutions, aerosols, sprays, and mists, may be presented in multi-dose forms (multiple uses), such as packaged units containing a predetermined number of doses, or in single-dose forms (single uses), such as packaged units containing a single dose. The preparations of ammonia-oxidizing microorganisms may be packaged in a device or container configured to hold a volume of at least about 1 ml, 1 ml, 5 ml, 10 ml, 20 ml, 25 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, less than 100 ml, or greater than about 100 ml.

[0266] The kit may further include one or more devices for administration of the preparation, such as a syringe, needle, catheter, enema, bulb, pipette (eye or ear dropper), and other devices known in the art for drug administration. The kit may include instructions for use, such as instructions for administering an ammonia-oxidizing microorganism disclosed herein, or instructions for a combination therapy including administration of an ammonia-oxidizing microorganism. The kit may include a second or subsequent composition for administration in conjunction with the ammonia-oxidizing preparation disclosed herein. For example, the kit may include a supplement or composition including a product or by-product of an ammonia-oxidizing microorganism, a composition that promotes the growth or metabolism of an ammonia-oxidizing microorganism, a composition that promotes the production of a product or by-product of an ammonia-oxidizing microorganism, a composition that promotes urease activity, or a composition that has a synergistic effect with an ammonia-oxidizing microorganism, or a composition or pharmaceutical agent that treats, e.g., is approved for or commonly used to treat, an associated disease, disorder, or symptom of an associated disease or disorder, such as an anti-inflammatory composition. The kit can include a "biome-friendly" or "biome-compatible" product disclosed herein, such as one or more microbiome-compatible cosmetic products. Any of the products contained in the kit can be specifically formulated to treat a target indication and / or for a desired mode of delivery as described herein.

[0267] natural products; consumer products In some specific embodiments, the preparations comprising the ammonia-oxidizing microorganisms discussed herein can be natural or consumer products, hi other embodiments, the preparations of ammonia-oxidizing microorganisms can instead be used in conjunction with natural or consumer products.

[0268] Ammonia-oxidizing microorganisms, such as N. eutropha, can be associated with a variety of natural products, examples of which are provided below. These natural products can be comprised of formulations, compositions, or preparations disclosed throughout this disclosure.

[0269] Natural products may be or include products for commercial purposes, and may refer to cosmetics, health supplements, and foods produced from natural sources, such as foods, dietary supplements, medical foods, food additives, nutraceuticals, or beverages. Natural products may have pharmacological or biological activity that may be of therapeutic benefit, for example, in treating a disease or condition. Natural products may be included in traditional medicines, cosmetic treatments, and spa treatments. The natural products referred to herein may include any one or more of the components described as natural products incorporated into a preparation or formulation that includes one or more other components, such as excipients. A preparation or formulation referred to as a natural product may include a natural product as defined herein and one or more additional components or ingredients. Any composition, preparation, or formulation discussed throughout this disclosure may be or include one or more natural products.

[0270] In some embodiments, the natural product or enhanced natural product may include at least one of mud, water, food-derived products, plant-derived products, extracts, and oils. The natural product or enhanced natural product may be used in spa treatments. In some embodiments, the natural product or enhanced natural product may be incorporated into at least one of powder, cream, lotion, wrap, scrub, eye mask, facial mask, body mask, aerosol, such as mist, spray, ointment, wipe, stick, bandage, or soak.

[0271] In some embodiments, the natural product or enhanced natural product is a baby product, such as baby shampoo, baby lotion, baby oil, baby powder, baby cream; bath preparations, such as bath oil, tablets, salts, bubble bath, bath capsules; eye makeup preparations, such as eyebrow pencils, eyeliner, eye shadow, eye lotion, eye makeup remover, mascara; fragrance preparations, such as colognes, toners, perfumes, powders (dusting and talcum), sachets; hair preparations, such as hair conditioners, hair sprays, hair straighteners, permanent waves, rinses, shampoos, tonics, styling products, hair cosmetics, wave sets; hair coloring preparations, such as hair dyes and colors, hair dyes, colored hair rinses, colored hair shampoos, colored hair lighteners, hair bleach; makeup preparations, such as face powders, foundations, leg and body paints, lipsticks. , makeup bases, lipsticks, makeup fixatives; nail polish preparations such as base coats and undercoats, cuticle softeners, nail creams and lotions, nail extenders, nail polish and enamel, nail polish and enamel removers; oral hygiene products such as dentrifices, mouthwashes and breath refreshers; bath soaps and detergents, deodorants, douches, feminine hygiene deodorants; shaving preparations such as aftershave lotions, beard softeners, talcum, preshave lotions, shaving creams, shaving soaps; skin care preparations such as cleansers, depilatories, powders and sprays for the face and neck, body and hands, feet, moisturizers, night preparations, paste masks, skin fresheners; and tanning preparations such as gels, creams, and liquids, and indoor tanning preparations.

[0272] Ammonia-oxidizing microorganisms, e.g., N. eutropha, can be associated with various consumer products, examples of which are set forth below and comprise the formulations, compositions, or preparations disclosed throughout this disclosure. In some embodiments, the ammonia-oxidizing bacteria, e.g., N. eutropha, associated with the product is mixed with the product, e.g., evenly spread throughout the product; in some embodiments, the ammonia-oxidizing bacteria, e.g., N. eutropha, associated with the product is layered on the product.

[0273] In some embodiments, the preparation may be disposed in or provided as a powder, a cosmetic, a cream, a stick, an aerosol, such as a mist, an ointment, a wipe, or a bandage.

[0274] In some embodiments, the ammonia-oxidizing bacteria, such as N. eutropha, are associated with powder. Powders are typically small, granular solids that do not adhere to each other and can flow freely when tilted. Exemplary powders for consumer use include talcum powder and some cosmetics (e.g., powder foundation).

[0275] In some embodiments, ammonia-oxidizing bacteria are related to cosmetics.Cosmetics can be a substance for external application that is intended to change a person's appearance, such as liquid foundation, powder foundation, blush, or lipstick, and can be called preparations.Cosmetics can be any substance listed in the regulations of the Food and Drug Administration, for example, under 21C.FR §720.4.

[0276] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with cosmetics. Cosmetics can be topical applications intended to alter a person's appearance, such as liquid foundation, powder foundation, blush, or lipstick. Other components selected by those skilled in the art of cosmetic formulations, such as water, mineral oil, coloring agents, perfume, aloe vera, glycerin, sodium chloride, sodium bicarbonate, pH buffer, UV blocking agent, silicone oil, natural oil, vitamin E, herbal concentrate, lactic acid, citric acid, talc, clay, calcium carbonate, magnesium carbonate, zinc oxide, starch, urea, and erythorbic acid, or any other excipients known to those skilled in the art, including those disclosed herein, can be added to these cosmetic preparations.

[0277] Preparations, for example cosmetics, include baby products, for example baby shampoos, baby lotions, baby oils, baby powders, baby creams; bath preparations, for example bath oils, tablets, salts, bubble baths, bath capsules; eye make-up preparations, for example eyebrow pencils, eyeliners, eye shadows, eye lotions, eye make-up removers, mascara; fragrance preparations, for example colognes, toners, perfumes, powders (dustings and talcum), sachets; hair preparations, for example hair conditioners, hair sprays, hair straighteners, permanent waves, rinses, shampoos, tonics, hair styling products, hair cosmetics, wave sets; hair colouring preparations, for example hair dyes and colours, hair dyes, coloured hair rinses, coloured hair shampoos, coloured hair lighteners, hair bleach; make-up preparations, for example face powders, foundations, leg and body paints. , lipsticks, makeup bases, lipsticks, makeup fixatives; nail polish preparations such as base coats and undercoats, cuticle softeners, nail creams and lotions, nail extenders, nail polish and enamel, nail polish and enamel removers; oral hygiene products such as toothpastes, mouthwashes and breath refreshers; bath soaps and detergents, deodorants, douches, feminine hygiene deodorants; shaving preparations such as aftershave lotions, beard softeners, talcum, preshave lotions, shaving creams, shaving soaps; skin care preparations such as cleansers, depilatories, powders and sprays for the face and neck, body and hands, feet, moisturizers, night preparations, paste masks, skin fresheners; and tanning preparations such as gels, creams, and liquids, and indoor tanning preparations.

[0278] In some embodiments, the formulations, compositions, or preparations described herein may comprise, be provided as, or be disposed in at least one of: baby products, such as baby shampoo, baby lotion, baby oil, baby powder, baby cream; bath preparations, such as bath oil, tablets, salts, bubble bath, bath capsules; powders (dusting and talcum), sachets; hair preparations, such as hair conditioners, rinses, shampoos, tonics, face powders, cuticle softeners, nail creams and lotions, oral hygiene products, mouthwash, bath soaps, douches, feminine hygiene deodorants; shaving preparations, such as aftershave lotions, skin care preparations, such as cleansers, powders and sprays for the face and neck, body and hands, feet, moisturizers, night preparations, paste masks, skin fresheners; and tanning preparations, such as gels, creams, and liquids.

[0279] In some embodiments, ammonia-oxidizing microorganisms, such as N. eutropha, are associated with an aerosol, spray, or mist, and these terms can be used interchangeably. Aerosols are typically colloids of fine solid particles or liquid droplets in a gas, such as air. Aerosols can be created by placing N. eutropha (and optionally a carrier) in a container under pressure and then opening a valve to release the contents. The container can be designed to exert only a pressure level compatible with the viability of N. eutropha. For example, high pressure can be exerted for only a short period of time, and / or the pressure can be low enough so as not to impair viability. Examples of consumer uses of aerosols include sunscreen, deodorant, perfume, hairspray, and insect repellent. Aerosols can be referred to as sprays or mists.

[0280] Compositions comprising an ammonia-oxidizing microorganism, such as N. eutropha, may also include one or more of a moisturizer, a deodorizer, a fragrance, a colorant, an insect repellent, a cleaning agent, or a UV blocker.

[0281] In some embodiments, ammonia-oxidizing microorganisms, such as N. eutropha, are associated with fabrics, threads, or twine. Clothing, such as shoes, insoles, pajamas, sneakers, belts, bonnets, shirts, underwear, athletic wear, helmets, towels, gloves, socks, bandages, and the like, can also be treated with ammonia-oxidizing bacteria, such as N. eutropha. Bedding, including sheets, pillows, pillowcases, and blankets, can also be treated with ammonia-oxidizing bacteria, such as N. eutropha. In some embodiments, areas of skin that cannot be washed for a period of time can also be contacted with ammonia-oxidizing bacteria, such as N. eutropha. For example, skin wrapped in an orthopedic cast that immobilizes an injured limb during the healing process, and areas adjacent to an injury that must be kept dry for proper healing, such as a sutured wound, can benefit from contact with ammonia-oxidizing bacteria, such as N. eutropha.

[0282] In some embodiments, the present disclosure provides a wearable article comprising the ammonia-oxidizing microorganisms described herein. The wearable article may be a lightweight article that can be closely attached to the user's body in a manner that does not interfere with walking. Examples of wearable articles include watches, bracelets, headbands, hair ties, hair nets, shower caps, caps, wigs, and jewelry. A wearable article comprising the ammonia-oxidizing bacteria described herein, such as a N. eutropha strain, may provide a concentration that provides one or more of the following: treatment or prevention of skin disorders, treatment or prevention of diseases or conditions associated with low nitrite levels, treatment or prevention of body odor, treatment to provide nitric oxide to a subject, or treatment to inhibit microbial growth.

[0283] In some embodiments, ammonia-oxidizing microorganisms, such as N. eutropha, are associated with products intended to come into contact with hair, such as brushes, combs, shampoos, conditioners, headbands, hair ties, hair nets, shower caps, caps, and wigs. Nitric oxide formed on hair away from the skin surface can be trapped in the cap, scarf, or face mask and directed into inhaled air.

[0284] Articles that come into contact with the surface of human subjects, such as diapers, can be associated with ammonia-oxidizing microorganisms, such as N. eutropha. Because diapers are designed to retain and contain the urine and feces excreted by incontinent individuals, the urea in urine and feces can be hydrolyzed by skin and fecal bacteria to form free ammonia, which is irritating and can cause diaper rash. The incorporation of bacteria that metabolize urea into nitrite or nitrate, such as ammonia-oxidizing bacteria, such as N. eutropha, can prevent the release of free ammonia and release nitrite and ultimately NO, which can help maintain healthy skin for both children and incontinent adults. The release of nitric oxide in diapers can also have an antimicrobial effect against disease-causing organisms present in human feces. This effect can continue even after disposable diapers are disposed of as waste, reducing the incidence of disease transmission through contact with soiled disposable diapers.

[0285] In some embodiments, a product containing an ammonia-oxidizing microorganism, such as N. eutropha, is packaged. The packaging may function to contain the product or to protect the product from damage, contamination, or deterioration. The packaging may comprise, for example, plastic, paper, cardboard, or wood. In some embodiments, the packaging is impermeable to bacteria. In some embodiments, the packaging is permeable to oxygen and / or carbon dioxide.

[0286] Shelf-stable preparation of ammonia-oxidizing microorganisms The preparations disclosed herein may be shelf stable. A shelf stable preparation may exhibit appropriate metabolic activity of AOM and / or Th1, Th2, Th17, or Treg inhibitory activity of AOM, and may also have reduced viability of AOM, for example, less than 70% viability.

[0287] Shelf-stable preparations should be at least approximately 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, or 10 7 CFU / mL of AOM. Shelf-stable preparations contain approximately 10 8 CFU / mL, 10 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, or 10 3 For example, shelf-stable preparations may contain less than about 10 CFU / mL of AOM. 2 CFU / mL ~10 3 CFU / mL, 10 2 CFU / mL ~10 4 CFU / mL, or 10 3 CFU / mL ~10 5 In particular, shelf-stable preparations may contain at least about 500 mg, at least about 750 mg, or between 750-1000 mg of AOM per dose.

[0288] The preparations disclosed herein are 3 cells / mL, 10 4 cells / mL, 10 5 cells / mL, or 10 6 cells / mL, or more.

[0289] A shelf-stable preparation may have less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of AOM in a viable state. In certain embodiments, a shelf-stable preparation may have at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of AOM in an inactive state. Inactive AOM may be sterilized, dead, or otherwise non-viable. In certain embodiments, inactive AOM may be heat-killed. For example, a target percentage of AOM in a shelf-stable preparation may be heat-killed. In certain embodiments, AOM may be treated by freeze / thaw, ethanol, and / or aging. For example, a target percentage of AOM in a shelf-stable preparation can be killed by one or more of heat treatment, freezing treatment, ethanol treatment, and / or aging.

[0290] The shelf-stable preparation may contain at least one preservative listed in Appendix VI. In particular, the shelf-stable preparation may contain at least 500 ppb of at least one preservative. The shelf-stable preparation may contain more than 500 ppb, more than 550 ppb, more than 600 ppb, more than 650 ppb, more than 700 ppb, more than 750 ppb, more than 800 ppb, more than 850 ppb, more than 900 ppb, more than 950 ppb, or more than 1000 ppb of preservative. The shelf-stable preparation may contain between about 500 ppb and 1000 ppb, between about 750 ppb and 1000 ppb, or between about 850 ppb and 1000 ppb of preservative.

[0291] In some embodiments, the preparation does not support the growth of pathogenic microorganisms when challenged by the pathogenic microorganisms. For example, in certain embodiments, the preparation sterilizes at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of a population of microorganisms when challenged by the pathogenic microorganisms. The pathogenic microorganisms can be, for example, disease-causing microorganisms.

[0292] In some embodiments, the shelf-stable preparation may be sterilized, for example, the shelf-stable preparation may be sterilized by heat or ultraviolet light.

[0293] The shelf-stable preparation (e.g., preparation or composition) can be conveniently presented in a unit dosage form and can be prepared by any method known in the fields of pharmacy or cosmetology. Typically, the method includes combining an active ingredient (e.g., an ammonia-oxidizing microorganism) with a pharmaceutical carrier that constitutes one or more accessory ingredients. Generally, pharmaceutical or cosmetic preparations are prepared by uniformly and intimately combining the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation.

[0294] Shelf-stable formulations can be presented as discrete units, each containing a predetermined amount of the active ingredient, as a solution or suspension in an aqueous or non-aqueous liquid, as a powder or granules, or as an oil-in-water or water-in-oil liquid emulsion.Various pharmaceutically acceptable carriers and their formulations are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences by EW Martin.Wang, YJ and Hanson, MA, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2 S, 1988;Aulton, M. and Taylor, K., Aulton's Pharmaceutics: The Design and Manufacture of Medicines, 5 th Edition, 2017;Antoine, See also A., Gupta MR, and Stagner, WC, Integrated Pharmaceutics: Applied Preformulation, Product Design, and Regulatory Science, 2013; Dodou K. Exploring the Unconventional Routes - Rectal and Vaginal Dosage Formulations, The Pharmaceutical Journal, 29 Aug. 2012.

[0295] The shelf-stable preparation can be in aqueous form.For example, the preparation can be formulated as a liquid, such as a spray, aerosol, or mist.The shelf-stable aqueous preparation can comprise, essentially consist of, or consist of ammonia-oxidizing microorganisms in a buffer solution.The aqueous buffer solution can comprise, essentially consist of, or consist of disodium phosphate and magnesium chloride, for example, 50 mM Na2HPO4 and 2 mM MgCl2.

[0296] The shelf-stable preparation may be substantially solid or gel-like. For example, the shelf-stable preparation may be formulated as a powder or gel. The preparation may be in cream form. For example, the shelf-stable preparation may be formulated as a cream, ointment, or lotion. The preparation may be formulated as an ointment. The shelf-stable powder preparation may contain lyophilized ammonia-oxidizing microorganisms. The powder preparation may contain talcum powder or cornstarch. The ointment may contain, for example, an anhydrous dispersion of an active agent in a mineral oil-white petrolatum base. The gel may contain polymers such as poloxamer, xanthan gum, gellan gum, locust bean gum, and carrageenan. Gel, lotion, ointment, and ointment preparations may contain thickeners and / or emulsifiers. Exemplary thickeners and emulsifiers include emulsifying wax, polysorbate 20, ceteareth 20, cellulose derivatives, guar gum, locust bean gum, xanthan gum, and gelatin. Such preparations may have a viscosity of at least about 1 mPa·s, 10 mPa·s, 100 mPa·s, 1 Pa·s, 5 Pa·s, 10 Pa·s, or 20 Pa·s at room temperature, e.g., a temperature between about 20°C and 25°C.

[0297] Ointments, lotions, salves, and gels with higher viscosities can provide longer residence times than, for example, aqueous solutions. Longer residence times can further enable shorter dosing intervals. Emulsions can include microspheres, microcapsules, nanoparticles, nanocapsules, micelles, liposomes, niosomes, dendrimers, or cyclodextrin complexes. Films can include water-soluble polymeric films or polyvinyl alcohol polymeric films that dissolve and release active agents when in contact with body fluids.

[0298] Certain viscous liquid formulations may contain a gel or gelling agent. For example, the gelling agent may be a thermoreversible gel. A thermoreversible gel may be liquid at lower temperatures or room temperature and may return to a gel when inserted into a body cavity, such as the nasal cavity, rectum, or vaginal cavity. The gel or gelling agent may allow for easier administration and positioning of the dosage form. For example, the gel or gelling agent may prevent the dosage form from leaking out of the body cavity. Thermoreversible polymers include poloxamers. Mucoadhesive polymers include sodium alginate. The gel or gelling agent may further contain a solubility enhancer, such as hydroxypropyl-betacyclodextrin.

[0299] The onset time of action for the formulations disclosed herein may depend on the dosage form and may range from a few seconds to a few minutes to a few hours. The ammonia-oxidizing microorganism composition may be administered, for example, in a form suitable for immediate release or extended release. Preparations for administration may be appropriately formulated to provide controlled or extended release of the ammonia-oxidizing microorganism. In some embodiments, the controlled-release formulation may be formulated as an ointment, gel, foam, or emulsion. Extended-release pharmaceutical compositions may be formulated with one or more mucoadhesive agents, for example, a mucoadhesive gel or a dry mucoadhesive tablet. The mucoadhesive agent may aid in adhesion to the mucosa of a body cavity, for example, the rectum or nasal cavity. Similarly, solid dosage forms, such as films, may be formulated with one or more mucoadhesive agents that can enhance the positioning of the dosage form within a body cavity or that can remain present when a portion of the solid dosage form melts or disintegrates. For example, the solid dosage form may be formulated to rapidly dissolve upon contact with body fluids, becoming a mucoadhesive viscous solution that adheres to the wall of the body cavity and gradually washes away without requiring removal.

[0300] In certain embodiments, the shelf-stable preparation may include CO. The shelf-stable preparation may include at least about 400 ppm CO. For example, the shelf-stable preparation may include at least about 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, or 650 ppm CO.

[0301] The shelf-stable preparation may be substantially free of polyphosphate.The shelf-stable preparation may contain less than about 1 μM polyphosphate.For example, the shelf-stable preparation may contain less than about 1 μM, 0.5 μM, 0.1 μM, 50 nM, or 10 nM polyphosphate.

[0302] Shelf-stable preparations can be formulated for oral, enteral (e.g., buccal, sublingual, sublabial, and rectal), parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, and intraarticular), inhalation (e.g., fine particle dusts or mists that may be generated by various types of metered-dose pressurized aerosols, nebulizers, or inhalers, including intranasally or via the lungs), intranasal, ophthalmic, otic, rectal, injectable, urogenital, or topical (e.g., dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration.

[0303] The shelf-stable preparations may be formulated for the treatment of one or more of headaches, cardiovascular diseases, inflammation, immune responses, autoimmune disorders, liver diseases, infectious diseases, neurological diseases, psychiatric disorders, nitric oxide disorders, urea cycle disorders, congestion, vasodilation disorders, skin diseases, ophthalmological disorders, wound healing, reactions to insect bites, connective tissue disorders, and certain viral, bacterial, or fungal infections.

[0304] Methods for distributing preparations of ammonia-oxidizing microorganisms According to one or more embodiments, the methods disclosed herein can include distributing the preparation or formulation, for example, to a recipient. The recipient can be a distributor, wholesaler, retailer, or consumer, e.g., an end-use consumer. The aspects disclosed herein can be implemented at any one or more steps during manufacturing or distribution, e.g., during manufacturing, storage, or transportation.

[0305] The shelf-stable preparations disclosed herein can be maintained under non-refrigerated conditions.In particular, the preparations disclosed herein can have suitable cosmetic and therapeutic efficacy at temperatures higher than about 4°C.Therefore, the preparations disclosed herein can be stored at approximately room temperature for an extended period of time.Storage at room temperature is beneficial during production and distribution.For example, shelf-stable preparations can require fewer resources during production, storage, and transportation than non-shelf-stable preparations.In addition, the storage and transportation of packaged containers, such as end-use containers, containing shelf-stable preparations can have environmental benefits, for example, by reducing the use and dependence on insulating delivery materials and / or refrigerants and coolants.

[0306] The method of distribution can include packaging the preparation containing live ammonia-oxidizing microorganisms into one or more containers, e.g., one or more end-use containers. In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are viable during manufacturing and / or packaging.

[0307] The end-use container may include a reservoir in which the formulation is placed and a dispenser through which the formulation from the reservoir can be dispensed. The reservoir may be a bottle, jar, or tube. The reservoir may be formed of plastic, paperboard, glass, aluminum, or any other suitable metal. The dispenser may be squeeze-activated or pump-activated. In some embodiments, the dispenser may be an orifice in the container. In some embodiments, the dispenser may be a pump. In other embodiments, the dispenser may be a valve, e.g., a pressure-activated valve. In certain embodiments, the dispenser may inhibit the reverse flow of the dispensed formulation and / or atmospheric aerosol into the reservoir.

[0308] The total volume of the preparation packaged in the container can be between about 0.1 and about 100 fluid ounces, between about 0.2 and about 50 fluid ounces, between about 0.5 and about 25 fluid ounces, between about 1.0 and about 10 fluid ounces, between about 2.0 and about 7 fluid ounces, or between about 3 and about 5 fluid ounces. In some embodiments, the volume can be about 3.4 fluid ounces.

[0309] The container can be constructed to contain between about 0.1 and about 100 fluid ounces, between about 0.2 and about 50 fluid ounces, between about 0.5 and about 25 fluid ounces, between about 1.0 and about 10 fluid ounces, between about 2.0 and about 7 fluid ounces, or between about 3 and about 5 fluid ounces. In some embodiments, the container can be constructed to contain about 3.4 fluid ounces.

[0310] The end-use container may indicate one or more of the following: storage and handling of the preparation, the dosage form of the preparation, a description of the contents in the preparation, the viability status of the AOM, and instructions for use of the preparation. For example, the end-use container may instruct a subject to store the packaged preparation at a temperature above about 4°C. In certain embodiments, the end-use container may instruct a subject to store the packaged preparation at room temperature, for example, for a period of more than about 4 weeks. The end-use container may instruct a subject to store the packaged preparation at a temperature above about 4°C, for example, at about room temperature, for a period of more than about 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years. In certain embodiments, the end-use container may indicate the expiration date of the packaged preparation. The expiration date may be more than 6 months, more than 1 year, more than 5 years, or more than 10 years from the date of packaging or manufacture.

[0311] The end-use container can inform the subject that less than about 70% of AOM is viable.For example, the end-use container can inform the subject that less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5% or 0.1% of AOM is viable.The end-use container can inform the subject that at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5% or 99.9% of AOM is in an inactive state.The end-use container can inform the subject that the packaged preparation is sterile.

[0312] The end-use container may provide information to apply the preparation to a subject topically. The end-use container may provide information to apply the preparation to a subject intranasally. The end-use container may provide information to apply the preparation to a subject at least one of orally, enterally, intranasally, parenterally, subcutaneously, ophthalmically, otically, or respiratoryly.

[0313] In other embodiments, the end-use container does not indicate one or more of the following: storage and handling of the preparation, the dosage form of the preparation, a description of the contents in the preparation, the viability status of the AOM, and directions for use of the preparation.

[0314] A method of distribution can include, for example, providing (or having a designee provide) a recipient with a formulation packaged in a container, e.g., packaged in an end-use container. The recipient can be an intermediary or a consumer, e.g., an end-use consumer. The designee can be an entity designated by a consumer, e.g., a second end-user. In some embodiments, providing (or having a designee provide) the packaged end-use container to the recipient can include making the packaged end-use container available on an internet-based outlet. In other embodiments, providing (or having a designee provide) the packaged end-use container to the recipient can include making the packaged end-use container available in a non-internet-based outlet, e.g., a store.

[0315] During distribution, the formulation or packaged end-use container may be exposed to an environment having a temperature greater than about 4° C. For example, the formulation or packaged end-use container may be exposed to an environment having a temperature greater than about 10° C., or to an environment having a temperature between about 20° C. and 25° C.

[0316] The formulation or packaged end-use container may be exposed to an environment having a temperature greater than about 4°C for more than a short period of time. For example, the formulation or packaged end-use container may be exposed to this environment for a period of more than a few minutes or a few hours. In certain embodiments, the formulation or packaged end-use container may be exposed to this environment for a period of at least about 1 hour, 6 hours, 12 hours, 18 hours, 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years. The formulation or packaged end-use container may be exposed to an environment, for example, a temperature greater than about 4°C, a temperature greater than about 10°C, a temperature greater than about room temperature (20°C-25°C), or for a period of time sufficient for the formulation to reach temperature equilibrium with the environment such that the formulation has substantially the same temperature as the environment.

[0317] Thus, during distribution, the formulation or packaged end-use container may reach a temperature greater than about 4° C. For example, the formulation or packaged end-use container may reach a temperature greater than about 10° C. or room temperature, e.g., between about 20° C. and 25° C.

[0318] An environment having a temperature greater than about 4°C can be a storage environment. The environment can be a distribution environment, e.g., a mail or commercial delivery environment. The mail or commercial delivery environment can be a delivery environment to a consumer, e.g., an end-use consumer and / or a location designated by the consumer. The environment can be a distribution environment, e.g., a cargo or freight delivery environment. The cargo or freight delivery environment can be a delivery environment to a distributor, wholesaler, or retailer. Thus, in some embodiments, distribution of the preparation or formulation can include storing or transporting the packaged preparation in a non-refrigerated environment. In some embodiments, distribution of the preparation or formulation can include storing or transporting the packaged preparation in non-temperature-controlled packaging, e.g., non-insulated packaging and / or without a refrigerant or coolant.

[0319] In some embodiments, less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms are viable after distribution. For example, in at least some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms are inactive after distribution.

[0320] The distribution method may further include quality control of the preparation before packaging or quality control of the packaged preparation before providing the packaged preparation to a recipient. Thus, in certain embodiments, the methods may include measuring at least one parameter of the preparation or packaged preparation to determine a value. The methods may include comparing the value of the measured parameter with a predetermined range of values ​​corresponding to the parameter. The methods may include determining whether the value is within the predetermined range of the parameter. If the value is within the predetermined range, the preparation or packaged preparation may be classified as acceptable. If the value is outside the predetermined range, the preparation or packaged preparation may be classified as unacceptable.

[0321] These methods may include distributing acceptable preparations or packaged preparations. These methods may include modifying unacceptable preparations or packaged preparations to make them acceptable. Parameters may include, for example, metabolic activity of the AOM, viability of the AOM, or activated immune cell inhibitory activity of the AOM, such as cellular Th1, Th2, Th17, and / or Treg inhibitory activity. In some embodiments, the method may include heat-treating or heat-killing a target percentage of AOM to make the preparation or packaged preparation acceptable, for example, so that the metabolic activity of the AOM, viability of the AOM, or activated immune cell inhibitory activity of the AOM falls within a predetermined range. For example, the method may include heat-treating or heat-killing a target percentage of AOM to make the Th1, Th2, Th17, and / or Treg inhibitory activity of the AOM fall within a predetermined range. The predetermined range may be sufficient to treat a disease or disorder modulated by activated immune cells, or a symptom thereof.

[0322] Treatment method using ammonia-oxidizing microorganisms According to one or more embodiments, a subject can be treated by administering an ammonia-oxidizing microorganism, e.g., a preparation containing an ammonia-oxidizing microorganism. As used herein, treating a subject can include administering an ammonia-oxidizing microorganism composition for a cosmetic or therapeutic result. For example, treatment can include treating or alleviating a condition, symptoms, or side effects associated with a condition, or achieving a desired cosmetic effect.

[0323] The subject can include animals, mammals, humans, non-human animals, livestock animals, or companion animals. The subject can be female or male. The subject can have various skin types. The subject can have various health-related profiles, including medical history and / or genetic predisposition. The subject can generally have a normal microbiome, e.g., a physiological microbiome, or a disrupted microbiome. The subject can be characterized as one of the following ethnicities / races: Asian, Black or African American, Hispanic or Latino, Caucasian, or multiracial. The subject can have a Fitzpatrick Scale skin typing score of I-IV. The subject can be less than 1 year old, or between 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60 years old, or over 60 years old.

[0324] Ammonia-oxidizing microorganisms that can be used to treat a subject include any ammonia-oxidizing microorganism, such as the N. eutropha compositions described in this application, such as purified preparations of optimized ammonia-oxidizing microorganisms, such as strain D23.

[0325] Methods can be provided to administer or deliver therapeutic or cosmetic products. The methods can include administering or introducing to a subject a preparation comprising live ammonia-oxidizing microorganisms. The preparation can be formulated to treat a target indication and / or can be formulated for a desired mode of delivery.

[0326] According to one or more embodiments, a preparation containing live ammonia-oxidizing microorganisms can be administered to a first tissue of a subject. The first tissue can be a deposition tissue. The first tissue can be a target tissue or a tissue other than the target tissue. The live ammonia-oxidizing microorganisms, or their products, such as nitrite and / or nitric oxide, can then be transferred or transported to a second tissue, for example, by diffusion. The second tissue can be a target tissue. The target tissue can be associated with a desired local or systemic effect. The target tissue can be associated with a symptom, disorder, or condition to be treated.

[0327] Ammonia-oxidizing microorganism preparations can be administered to the skin, for example, for cosmetic or therapeutic effects. For example, administration can provide a cosmetic treatment, benefit, or effect. In some embodiments, administration can provide treatment or improvement of one or more of oily appearance, pore appearance, radiance, blemishes, even skin tone, visual smoothness, and tactile smoothness. In some embodiments, a subject's cosmetic appearance can be altered, such as as a result of improved skin health. Signs of aging can be reduced, delayed, or reversed. Administration can bring about qualitative improvements in the condition and / or quality of the skin and / or scalp. Skin smoothness, hydration, tightness, and / or softness can be improved in a subject. The present disclosure also provides a method for reducing body odor.

[0328] Administration can provide therapeutic treatment, benefit, or effect. The present disclosure provides a method for modulating nitrite and providing nitric oxide to a subject. The present disclosure provides various methods for suppressing, treating, or preventing diseases, disorders, infections, and conditions using ammonia-oxidizing microorganisms. Ammonia-oxidizing microorganisms can be used to treat, for example, various diseases associated with suboptimal nitrite levels, skin diseases, and diseases caused by pathogenic bacteria.

[0329] In some embodiments, administration can provide a reduction in inflammation. Indeed, local or systemic anti-inflammatory effects can be demonstrated. In at least some embodiments, microbial growth can be inhibited. Skin and overall health can be improved. Inappropriate circulation can be enhanced. Endothelial function can be promoted. Changes in nitrite or NO levels in target tissues or circulation can be demonstrated. In some embodiments, administration, for example, administration of an effective amount, can modulate, change, or alter the levels of nitrite or NO in target tissues or circulation. In some embodiments, administration, for example, administration of an effective amount, can result in changes in the levels of nitrite or NO in target tissues or circulation.

[0330] Administration of the compositions disclosed herein can provide, for example, local or systemic transmucosal delivery and / or circulation. In some embodiments, administration can provide at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% penetration of ammonia-oxidizing microorganisms, their products, or their by-products (e.g., nitrate, nitrite, NO, or CoQ8) into the deposition or target tissue. In at least some embodiments, after administration of the compositions disclosed herein, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the ammonia-oxidizing microorganisms, their products, or their by-products penetrate the deposition or target tissue or enter the circulation.

[0331] The preparations and methods of the present disclosure can provide a reduction in the amount of undesirable microorganisms from the environment related to the target.The ammonia-oxidizing microorganisms described herein can outcompete other organisms, for example, by consuming rare nutrients or producing by-products that are harmful to other organisms, for example, by changing the pH to a level that does not support the growth of undesirable organisms.

[0332] The present disclosure also provides methods for promoting wound healing, including chronic wounds in patients with impaired healing capacity, e.g., diabetic patients, etc. A dressing containing ammonia-oxidizing microorganisms can be applied to the wound as needed.

[0333] It is recognized that many modern degenerative diseases can be caused by the lack of NO species, and AOM can be administered to supply this species to target tissue directly or by diffusion to target tissue.The application of AOM can eliminate long-term medical conditions.In certain embodiments, AOM is applied to subject to counteract the modern bathing practice of using anionic surfactants, which removes AOM from external skin.

[0334] According to one or more embodiments, AOM converts ammonia into nitrite, an antimicrobial compound, and nitric oxide, a well-documented signaling molecule in the inflammatory process.

[0335] The present disclosure provides, inter alia, methods for modulating the composition of the microbiome, e.g., modulating or changing the proportion of the microbiome in an environment, e.g., a surface, e.g., the surface of a subject. This, in turn, can exhibit health-related benefits. The method can include administering to a subject a preparation comprising ammonia-oxidizing microorganisms. In some embodiments, the amount and frequency of administration, e.g., application, can be sufficient to reduce the proportion of pathogenic microorganisms.

[0336] Application of ammonia-oxidizing microorganisms to a subject, e.g., a human subject, can produce unexpected changes in the microbiome, which can result in an increase in the proportion of normal commensal non-pathogenic species and a decrease in the proportion of potentially pathogenic, pathogenic, or disease-causing organisms.

[0337] The increase in the proportion of non-pathogenic bacteria can occur in less than a predetermined period of time, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, or in less than 1 to 3, 3 to 5, 5 to 7, 7 to 9, 5 to 10, 10 to 14, 12 to 18, 12 to 21, 21 to 28, 28 to 35, 35 to 42, 42 to 49, 49 to 56, 46 to 63, 63 to 70, 70 to 77, 77 to 84, or 84 to 91 days.

[0338] The reduction in the proportion of pathogenic bacteria can occur in less than a predetermined period of time, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, or in less than 1 to 3, 3 to 5, 5 to 7, 7 to 9, 5 to 10, 10 to 14, 12 to 18, 12 to 21, 21 to 28, 28 to 35, 35 to 42, 42 to 49, 49 to 56, 46 to 63, 63 to 70, 70 to 77, 77 to 84, or 84 to 91 days.

[0339] According to one or more embodiments, a subject can be evaluated for need for treatment. In some embodiments, a subject can be selected based on the subject's need for treatment. The present disclosure can further provide for obtaining a sample from the subject and analyzing the sample.

[0340] According to one or more embodiments, administration can occur before, during, or after the onset of the health-related condition, or in response to a warning sign, trigger, or symptom thereof. According to one or more embodiments, a second amount, e.g., a second dose, of the preparation can be administered to the subject.

[0341] In certain aspects, the present disclosure provides a combination therapy comprising an ammonia-oxidizing microorganism, e.g., N. eutropha, and a second treatment, e.g., a therapeutic agent. For example, the present disclosure provides a physical mixture of two (or more) therapies that are physically mixed. In other embodiments, the two (or more) therapies are administered in combination as separate formulations. The second treatment can be, for example, a pharmaceutical agent, surgery, diagnostic procedure, or any other medical approach approved for or commonly used to treat a related disease, disorder, or symptom of a related disease or disorder. The second treatment can be administered before or after administration. An effective amount can be administered concurrently with the second treatment. The second treatment can be administered via the same or a different mode of delivery. The subject can have a therapeutic level of the second treatment after administration of the preparation. In certain embodiments, the second treatment can provide an anti-inflammatory effect or can be administered to reduce inflammation at the target site. In at least some embodiments, the preparation can be administered simultaneously or in conjunction with a product or by-product of ammonia-oxidizing microorganisms, such as nitrite, nitrate, nitric oxide, CoQ8. In at least some embodiments, the preparation can be administered simultaneously or in conjunction with a composition that promotes the growth or metabolism of ammonia-oxidizing microorganisms, promotes production of a product or by-product of ammonia-oxidizing microorganisms, promotes urease activity, or has a synergistic effect with ammonia-oxidizing microorganisms, such as ammonia, ammonium salts, urea, and urease.

[0342] The preparation can be administered together with a microbiome cleansing preparation, for example, topical or systemic antibiotics.The preparation can be administered after the administration of a cleansing preparation or intestinal cleansing.The preparation can be administered before or after a surgical procedure, a diagnostic procedure, or a natural event, for example, childbirth.The preparation can be administered before, during, or after the installation of an implantable or invasive device.

[0343] According to one or more embodiments, the preparation can be administered as an analgesic or prophylactic. The preparation can be self-administered. Administration of the preparation can be device-assisted.

[0344] In some embodiments, the ammonia oxidizing microorganism, e.g., the preparation of ammonia oxidizing microorganism, is administered at a dose of about 10 per application, per day, per week, or per month. 3 ~10 4 CFU, 10 4 ~10 5 CFU, 10 5 ~10 6 CFU, 10 6 ~10 7 CFU, 10 7 ~10 8 CFU, 10 8 ~10 9 CFU, 10 9 ~10 10 CFU, 10 10 ~10 11 CFU, 10 11 ~10 12 CFU, 10 12 ~10 13 CFU or 10 13 ~10 14 In some embodiments, the ammonia-oxidizing microorganisms are administered at a dose of about or greater than about 10 CFU per application or per day. 9 ~10 10 CFU, e.g., approximately 1 x 10 9 ~5×10 9 , 1×10 9 ~3×10 9 or 1×10 9 ~10×10 9 It is administered in a dose of CFU.

[0345] In some embodiments, the ammonia-oxidizing microorganism is administered in a volume of less than about 0.2, less than 0.5, less than 1, 1-2, 2-5, 5-10, 10-15, 12-18, 15-20, 20-25, or 25-50 ml per dose. 3 ~104 , 10 4 ~10 5 , 10 5 ~10 6 , 10 6 ~10 7 , 10 7 ~10 8 , 10 8 ~10 9 , 10 9 ~10 10 or 10 10 ~10 11 In some embodiments, the ammonia-oxidizing microorganism is administered as two 15 ml doses per day, each dose containing 10 CFU / ml. 3 The concentration is CFU / ml. In some embodiments, the ammonia-oxidizing microorganisms are administered once, twice, three times, or four times per day. In some embodiments, the ammonia-oxidizing microorganisms are administered once, twice, three times, four times, five times, or six times per week. In some embodiments, the ammonia-oxidizing microorganisms are administered immediately after a bath. In some embodiments, the ammonia-oxidizing microorganisms are administered immediately before bedtime.

[0346] In some embodiments, the ammonia-oxidizing microorganism is administered for about 1-3, 3-5, 5-7, 7-9, 5-10, 10-14, 12-18, 12-21, 21-28, 28-35, 35-42, 42-49, 49-56, 46-63, 63-70, 70-77, 77-84, or 84-91 days, e.g., about 1 month, about 2 months, or about 3 months. In some embodiments, the ammonia-oxidizing microorganism is administered for an indefinite period, e.g., more than 1 year, more than 5 years, more than 10 years, more than 15 years, more than 30 years, more than 50 years, or more than 75 years. Methods for treating diseases modulated by activated immune cells are disclosed herein. For example, the ammonia-oxidizing microbial composition can be administered in a form suitable for providing treatment for, e.g., diseases modulated by activated immune cells. Disclosed herein are methods of treating diseases modulated by activated T helper cells or regulatory T cells, e.g., T helper type 1 (Th1), T helper type 2 (Th2), T helper type 17 (Th17), or regulatory T cells (Treg).

[0347] In certain embodiments, the methods can include controlling a response characterized by upregulation, activation, downregulation, or suppression of immune-related cytokines. Exemplary cytokines that can be upregulated, activated, downregulated, or suppressed by administration of the ammonia-oxidizing microbial compositions disclosed herein include IL-5, IL-13, IL-4, IFNγ, IL-12, IL-2, IL-18, IL-17, IL-21, IL-22, IL-10, and TFG-β.

[0348] Disclosed herein are methods for treating autosomal dominant hyper-IgE syndrome (AD-HIES). For example, ammonia-oxidizing microbial compositions can be administered in a form suitable for providing treatment for autosomal dominant hyper-IgE syndrome (AD-HIES).

[0349] Disclosed herein are methods for treating X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX). For example, an ammonia-oxidizing microbial composition can be administered in a form suitable for providing treatment for, for example, X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX).

[0350] Exemplary diseases or disorders modulated by Th2 cells that can be treated by the methods disclosed herein include allergic diseases, such as atopic dermatitis, asthma, allergic rhinitis, and itching. In certain embodiments, treating a Th2-mediated disease or disorder can include upregulating or activating one or more Th2-associated cytokines, such as IL-5, IL-13, and IL-4. For example, the method can include administering a therapeutically effective amount of a preparation of ammonia-oxidizing microorganisms to modulate Th2-associated cytokines.

[0351] Exemplary diseases or disorders modulated by Th1 cells that can be treated by the methods disclosed herein include tissue damage associated with autoimmune diseases and chronic infections, e.g., celiac disease, multiple sclerosis, and diabetes, e.g., type 1 diabetes. In certain embodiments, treating a Th1-mediated disease or disorder can include upregulating or activating one or more Th1-associated cytokines, e.g., IFNγ, IL-12, IL-2, and IL-18. For example, a method can include administering a therapeutically effective amount of a preparation of ammonia-oxidizing microorganisms to modulate Th1-associated cytokines.

[0352] Exemplary diseases or disorders modulated by Th17 cells that can be treated by the methods disclosed herein include autoimmune diseases, e.g., autoimmune inflammatory diseases, e.g., autosomal dominant hyper-IgE syndrome (AD-HIES), rheumatoid arthritis, and irritable bowel syndrome. In certain embodiments, treatment of a Th17-mediated disease or disorder can include upregulating or activating one or more Th17-associated cytokines, e.g., IL-17, IL-21, and IL-22. For example, the method can include administering a therapeutically effective amount of a preparation of ammonia-oxidizing microorganisms to modulate Th17-associated cytokines.

[0353] Exemplary diseases or disorders modulated by Treg cells that can be treated by the methods disclosed herein include allergic and autoimmune diseases, e.g., diseases or disorders associated with the regulation of X-linked immunodysregulatory polyendocrinopathy enteropathy (IPEX). In certain embodiments, treating a Treg-mediated disease or disorder can include upregulating or activating one or more Treg-associated cytokines, e.g., IL-10 and TFG-β. For example, a method can include administering a therapeutically effective amount of a preparation of ammonia-oxidizing microorganisms to modulate Treg-associated cytokines.

[0354] The method can also include administering a therapeutically effective amount of a preparation of ammonia-oxidizing microorganisms to modulate a disease or disorder associated with MHC II expression. Exemplary diseases or disorders associated with MHC II expression include, for example, autoimmune diseases and allergic inflammation.

[0355] The method can also include administering a therapeutically effective amount of a preparation of ammonia-oxidizing microorganisms to modulate a disease or disorder associated with expression of CD86. Exemplary diseases or disorders associated with expression of CD86 include, for example, allergic inflammation.

[0356] The ammonia-oxidizing microbial composition can be administered in a form suitable for providing, for example, various local or systemic therapeutic treatments. For example, administration can provide treatment or amelioration of local effects. Suitable examples of local conditions that can be treated with the compositions disclosed herein include local infections, inflammation, and their associated symptoms. Localized conditions can vary widely depending on the intended deposition or target tissue. Administration can provide treatment or amelioration of systemic effects. Examples of systemic conditions that can be treated with the compositions disclosed herein include headaches, cardiovascular diseases, inflammation, immune response and autoimmune disorders, liver diseases, infectious diseases, neurological diseases, psychiatric disorders, pulmonary diseases, nitric oxide disorders, urea cycle disorders, congestion, vasodilation disorders, skin diseases, ophthalmic disorders, intestinal disorders, hearing disorders, wound healing, reactions to insect bites, connective tissue disorders, and certain viral, bacterial, and fungal infections.

[0357] For example, systemic conditions that may be treated with the compositions disclosed herein include cardiovascular diseases such as cardioprotection, heart failure, hypertension, pulmonary hypertension, pulmonary arterial hypertension; immune response and autoimmune disorders such as alopecia and vitiligo; liver diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH); neurological and psychological disorders such as depression, insomnia, and diabetic neuropathy; nitric oxide disorders such as erectile dysfunction; wound healing, e.g., from pressure ulcers and nursing home care, burns, diabetic ulcers, e.g., foot ulcers, venous leg ulcers, biofilms, and mouth ulcers. sore); skin diseases and disorders such as hyperhydrosis, pruritus, corns and subtypes of corns; ophthalmologic disorders such as blepharitis, dry eye, macular degeneration and glaucoma; intestinal disorders such as gluten sensitivity, irritable / inflammatory bowel disease, Crohn's disease, colitis and necrotizing enterocolitis; hearing disorders such as tinnitus, hearing loss, vertigo, pruritus, otitis externa and congenital anomalies; and vasodilation such as Renaud's disease, thermoregulation and migraines These include disorders. Various connective tissue disorders can also be treated. Certain viral, bacterial, and fungal infections can be treated with the formulations disclosed herein, including infections caused by human papillomavirus (HPV), yeast infections, tinea versicolor, tinea unguium, tinea pedis / mycosis, tinea cruris, jock itch, onychomycosis, dandruff, athlete's foot, sinusitis, methicillin-resistant Staphylococcus aureus (MRSA), staph, otitis media, otitis externa, and bacterial vaginosis. Additional systemic conditions that can be treated with the compositions disclosed herein include eczema, e.g., adult and pediatric eczema, urticaria, idiopathic urticaria (uriticaria), lichen planus, insect bites, e.g., mosquito bites and Demodex hives. These include insect bites, including allergic reactions to P. folliculorum mites, reactions to poison ivy, itching, keratosis pilaris, laryngitis, pemphigus, psoriasis, rosacea, folliculitis and subtypes of folliculitis, hidradenitis supportiva, perioral dermatitis, lupus rash, seborrheic dermatitis, e.g., adult and infantile seborrheic dermatitis, acne, e.g., adolescent acne, adult acne and cystic acne, diaper rash, occupational hand dermatitis, sunburn, and systemic inflammation such as dermatomyositis. Additionally, the compositions disclosed herein can be delivered or applied to treat certain cosmetic indications, including, but not limited to, contact dermatitis, diaper odor (e.g., adults and children), body odor, feminine odor, peeling, nail stiffness, body odor, oily skin, razor burn, skin appearance, skin blemishes, skin moisture, and sun spots. The compositions disclosed herein can be applied as insect repellents or antimicrobial agents.

[0358] The method can include administering a preparation having a temperature greater than about 4° C. For example, treatment can include administering a preparation having a temperature between about 4° C. and about 10° C., greater than about 10° C., between about 10° C. and about 20° C., greater than about 20° C., between about 20° C. and about 25° C., greater than about 25° C., between about 25° C. and about 37° C., greater than about 37° C., between about 37° C. and about 40° C., or greater than about 40° C.

[0359] The method can include administering a preparation exposed to temperatures described herein for a period of at least 1 day, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or 5 years.

[0360] The preparation can be shelf stable. Thus, in some embodiments, the method can include administering a preparation having less than about 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% of the ammonia-oxidizing microorganisms in a viable state. In certain embodiments, the method can include administering a preparation having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or 99.9% of the ammonia-oxidizing microorganisms in an inactive state.

[0361] The method can include administering the preparation topically. For example, the method can include administering the preparation to one or more of the subject's body, such as the face, neck, scalp, limbs, hands, feet, back, buttocks, torso, pubic area, and chest.

[0362] The method can include administering the preparation intranasally. For example, the method can include administering the preparation to one or more nasal tissues selected from the subject's nasal cavity, septum, nasal valve, nares, nasopharynx, vestibular area, turbinates (e.g., inferior, middle, superior), nasal meatus (e.g., inferior, middle, superior), turbinates (e.g., inferior, middle, superior), maxillary sinus, sphenoid sinus, sphenoethmoid recess, ethmoid bulla, semilunar hiatus, nasolacrimal duct, nasofrontal duct, or olfactory region.

[0363] The method can include administering the preparation orally, enterally, intranasally, parenterally, subcutaneously, ophthalmically, otically, or respiratoryly. For example, the method can include administering the preparation orally, enterally, topically, ophthalmically, via the auditory system, via the genitourinary system, via the respiratory system, or by injection.

[0364] Administration of ammonia-oxidizing microorganisms for the treatment of skin According to one or more embodiments, the preparations and methods disclosed herein can be used to treat the skin in a subject. According to one or more embodiments, the preparations and methods disclosed herein can be used to reduce the appearance or effects of aging in a subject. According to one or more embodiments, the preparations and methods disclosed herein can be used to improve the integrity or condition of the skin in a subject. According to one or more embodiments, the preparations and methods disclosed herein can be used to modulate the integrity or condition of the skin in a subject. According to one or more embodiments, the preparations and methods disclosed herein can be used to improve the skin surface topology in a subject.

[0365] An effective amount of a preparation comprising ammonia-oxidizing microorganisms can be administered to a subject to treat the skin, reduce the appearance or effects of aging, improve the integrity or condition of the skin, modulate the integrity or condition of the skin, or improve the skin surface topology. The preparation can be administered according to various modes disclosed herein, for example, topically.

[0366] The subject may have skin that shows signs of aging. The preparations and methods disclosed herein can be used to condition the skin of a subject that shows signs of aging. The preparations and methods disclosed herein can be used to prevent, limit, or inhibit the appearance or progression of aging effects in a subject that shows signs of aging. An effective amount of a preparation comprising ammonia-oxidizing microorganisms can be administered to a subject, thereby conditioning the skin of a subject that shows signs of aging. An effective amount of a preparation comprising ammonia-oxidizing microorganisms can be administered to a subject, thereby preventing, limit, or inhibit the appearance or progression of aging effects in a subject that shows signs of aging.

[0367] As disclosed herein, modulating can include altering, e.g., affecting or affecting, the integrity or condition of the skin. Conditioning can refer to a change in the condition of a subject, e.g., the subject's skin. Preventing can include slowing, e.g., substantially slowing or inhibiting progression. Limiting can include, e.g., reducing, diminishing, or restricting progression. Inhibiting can include, e.g., limiting or arresting progression.

[0368] In some embodiments, a subject can be determined to be in need of treatment for a skin condition. The subject may be predisposed to a skin condition based on, for example, age, race, skin type, eye color, habits, or genetics.

[0369] According to one or more embodiments, an effective amount of the preparation can be administered to the face of a subject. According to one or more embodiments, the preparation can be administered to the body of a subject. For example, the preparation can be applied to one or more of the subject's forehead, eye area, neck, scalp, head, shoulders, arms, hands, legs, underarms, torso, chest, feet, knees, ankles, back, buttocks, or pubic area.

[0370] The preparation can be administered before the onset of a skin condition in a subject. The preparation can be administered during the development of a skin condition in a subject. The preparation can be administered after at least partial reduction of a skin condition in a subject. The preparation can be administered in response to an insult to the skin of a subject. The preparation can be administered in response to a trigger or warning sign of a skin condition, such as aging, habitual sleeping posture, habitual facial expression, weight loss, ultraviolet (UV) light exposure, smoking, dehydration, or immersion.

[0371] In some embodiments, the preparation can be administered after washing the subject's skin. For example, the preparation can be administered 30, 60, 90, 120, 150, or 180 minutes before or after the subject's cleansing or shower. The preparation can be administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day. The preparation can be administered for about 1 to 3, 3 to 5, 5 to 7, 7 to 9, 5 to 10, 10 to 14, 12 to 18, 12 to 21, 21 to 28, 28 to 35, 35 to 42, 42 to 49, 49 to 56, 46 to 63, 63 to 70, 70 to 77, 77 to 84, or 84 to 91 days. The preparation can be administered within 30, 60, 90, 120, 150, or 180 minutes of the subject awakening from sleep. The preparation can be administered within 30, 60, 90, 120, 150, or 180 minutes prior to the subject sleeping. The preparation can be administered within 30, 60, 90, 120, 150, or 180 minutes of the subject eating. In at least some embodiments, administration can be device-assisted.

[0372] In some embodiments, a second amount of the preparation can be administered to the subject. In at least some embodiments, the second treatment can be administered in combination with the preparation. According to one or more embodiments, various combination therapies can be used for skin treatment. For example, the preparations disclosed herein can be administered in combination with moisturizers, sunscreens, wrinkle creams, retinoids, alpha-hydroxy acids, antioxidants, tretinoin, glycosaminoglycans (GAGs), lactic acid, malic acid, citric acid, tartaric acid, hydroquinone, kojic acid, L-ascorbic acid, licorice extract, N-acetylglucosamine, niacinamide, soy, dermal fillers or injections, such as hyaluronic acid or calcium hydroxylapatite, botulinum toxin, laser resurfacing procedures, ultrasound therapy, chemical peels, such as glycolic acid peels, trichloroacetic acid or salicylic acid, or dermabrasion procedures. In at least some embodiments, the preparations can be administered in combination with nitrites, nitrates, and / or NO. The preparation can be administered in conjunction with an anti-inflammatory agent.In at least some embodiments, the subject can have a therapeutic level of a second treatment.The second treatment can be performed before, simultaneously with, or after the treatment method disclosed herein.

[0373] According to one or more embodiments, the preparation can be administered in conjunction with the medical approach that is approved for treating or commonly used for treating skin condition or the symptoms of skin condition, for example.The preparation can be administered before or after surgical or diagnostic procedure.The second treatment can involve surgical procedure, for example, cosmetic surgical procedure, for example, lifting procedure or plastic surgery procedure.

[0374] According to one or more embodiments, the amount and / or frequency of administration may be sufficient to promote wound healing in a subject. The amount and / or frequency of administration may be sufficient to improve the barrier function associated with the subject's skin. The amount and / or frequency of administration may be sufficient to treat at least one of scarring (e.g., scarring associated with sunburn, pressure ulcers, wounds, inflammatory lesions, or burns), skin thickening (e.g., keloid scars), cracks, fissures, corns, sebum secretion, skin thickening, wrinkles, sun spots, skin tags, dark patches, stretch marks, spider veins, varicose veins, age spots, cellulitis, or the appearance of pores in a subject. The amount and / or frequency of administration may be sufficient to reduce blemishes or discoloration associated with the subject's skin (e.g., vitiligo or post-inflammatory hyperpigmentation). The amount and / or frequency of administration may be sufficient to reduce freckling associated with the subject's skin. The amount and / or frequency of administration may be sufficient to reduce hives, allergic reactions, dermatitis (e.g., seborrheic dermatitis), warts, cold sores, candidiasis, or carbuncles associated with the subject's skin. The amount and / or frequency of administration may be sufficient to promote firmness, moisture, elasticity, radiance, evenness of tone, visual smoothness, or tactile smoothness associated with the subject's skin.

[0375] According to one or more embodiments, administration of an effective amount of the preparation can alter or modify nitrite or NO levels in a subject. Administration of an effective amount of the preparation can modulate the microbiome associated with the skin of a subject.

[0376] According to one or more embodiments, the amount and / or frequency of administration may be sufficient to reduce the appearance, for example, severity, of wrinkles in a subject. The amount and / or frequency of administration may be sufficient to reduce the width of wrinkles in a subject. The amount and / or frequency of administration may be sufficient to reduce the depth of wrinkles in a subject. The amount and / or frequency of administration may be sufficient to reduce the length of wrinkles in a subject. The at least one wrinkle to be treated in a subject may be a fine line, a surface line, or a deep groove.

[0377] The subject can exhibit an improved condition following treatment, as determined, for example, by visual assessment or culture.

[0378] According to one or more embodiments, any treatment of the skin can be associated with, assist in, or result in the treatment, suppression, or prevention of a variety of local or systemic indications, both cosmetic and therapeutic.

[0379] According to one or more embodiments, the preparations, devices, and / or kits disclosed herein can be provided for treating the skin in a subject. Such preparations, devices, and / or kits can be used in conjunction with the methods of treating the skin disclosed herein.

[0380] Use of microbiome-compatible products with administration of ammonia-oxidizing microorganisms Microbiome-compatible products can be used in conjunction with the preparations and methods disclosed herein. Various products can be considered "biome-friendly" or "biome-compatible." Examples of biome-friendly products are disclosed in International (PCT) Patent Application Publication No. WO2017 / 004534 (International (PCT) Patent Application Serial No. PCT / US / 2016 / 040723, filed July 1, 2016), which is hereby incorporated by reference in its entirety for all purposes. Some biome-friendly products can be cosmetic or therapeutic in nature. According to one or more embodiments, a biome-friendly product can be used in combination with a microorganism, e.g., a non-pathogenic microorganism, e.g., an ammonia-oxidizing microorganism, and the combination can then be used in the form of a preparation or composition to be applied to a subject. The ammonia-oxidizing compositions disclosed herein can be administered for cosmetic or therapeutic indications in conjunction with a biome-friendly or biome-compatible product.

[0381] According to one or more embodiments, a preparation, composition, formulation, or product comprising an ammonia-oxidizing microorganism, for example for cosmetic or therapeutic use, may itself be considered biome-friendly. In other embodiments, a preparation comprising an ammonia-oxidizing microorganism may be used in conjunction with a biome-friendly product. In some embodiments, a preparation comprising an ammonia-oxidizing microorganism may be mixed with or otherwise co-administered with a biome-friendly product. In other embodiments, a preparation comprising an ammonia-oxidizing microorganism may be separate or distinct from, but potentially used in conjunction with, a biome-friendly product. In some embodiments, a biome-friendly product is used alone. An ammonia-oxidizing microorganism composition preparation for use in conjunction with a biome-friendly product may be formulated for cosmetic or therapeutic use.

[0382] Biome-friendly or biome-compatible products can be used in conjunction with ammonia-oxidizing microbial preparations formulated for any mode of delivery, for example, for targeted delivery to a subject, for example, to a target tissue, region, system, or organ of a subject. For example, ammonia-oxidizing microbial preparations to be used in conjunction with biome-friendly products can be formulated for delivery to the subject's eyes, ears, nose, urogenital system, respiratory system, or gastrointestinal system. In some embodiments, ammonia-oxidizing microbial compositions for use with biome-friendly products can be formulated for targeted delivery based on the subject's condition or disorder. For example, formulations for targeted delivery can be based on the desired local or systemic effect to be achieved, for example, a local or systemic therapeutic or cosmetic effect.

[0383] Biome-friendly cosmetic products that can be used with the present disclosure include baby products, such as baby shampoo, baby lotion, baby oil, baby powder, baby cream; bath preparations, such as bath oil, tablets, salts, bubble bath, bath capsules; eye makeup preparations, such as eyebrow pencils, eyeliner, eye shadow, eye lotion, eye makeup remover, mascara; fragrance preparations, such as colognes, toilet water, perfumes, powders (dusting and talcum), sachets; hair preparations, such as hair conditioners, hair sprays, hair straighteners, permanent waves, rinses, shampoos, tonics, dressings, hair grooming aids, wave sets, and the like. set); hair coloring preparations, such as hair dyes and colors, hair dyes, coloring hair rinses, coloring hair shampoos, colored hair lighteners, hair bleach; makeup preparations, such as face powders, foundations, leg and body paints, lipsticks, makeup bases, lipsticks, makeup fixatives; manicure preparations, such as base coats and undercoats, cuticle softeners, nail creams and lotions, false nails extenders), nail polishes and enamels, nail polish and enamel removers; oral hygiene products, such as toothpastes (dentrifices), mouthwashes and breath fresheners; bath soaps, such as foaming body cleansers and surfactants, deodorants, douches, feminine hygiene deodorants; shaving preparations, such as aftershave lotions, beard softeners, talcum, preshave lotions, shaving creams, shaving soaps; skin care preparations, such as cleansers, depilatories, powders and sprays for the face and neck, body and hands, feet, moisturizers, night preparations, paste masks, skin fresheners;and may be, contain, or be disposed within any one or more of tanning preparations, such as gels, creams, and liquids, and indoor tanning preparations;

[0384] The products described herein, such as microbiome-compatible cosmetic products, such as shampoos, conditioners, and cleansers, can be used in conjunction with the treatment of conditions, diseases, or disorders. Such cosmetic products can be used in conjunction with the administration of ammonia-oxidizing microorganisms for therapeutic or cosmetic purposes. For example, the microbiome-compatible cosmetic product can be used throughout the entire treatment or cosmetic period of administering ammonia-oxidizing bacteria to a subject. The microbiome-compatible cosmetic product can be used for a period before the start of therapeutic or cosmetic treatment of a condition by administering ammonia-oxidizing bacteria to a subject. The microbiome-compatible cosmetic product can be used for a period after the start of therapeutic or cosmetic treatment of a condition by administering ammonia-oxidizing bacteria to a subject. The microbiome-compatible cosmetic product can be used for a period after the discontinuation of therapeutic or cosmetic treatment of a condition by administering ammonia-oxidizing bacteria to a subject.

[0385] In some embodiments, a subject can apply one or more cosmetic products and wait a period of time before administering an ammonia-oxidizing microorganism, hi other embodiments, a subject can administer an ammonia-oxidizing microorganism and wait a period of time before applying one or more cosmetic products.

[0386] The period of time that the subject can wait can be about 1 minute, 5 minutes, 10, 15, 20, 25, 30, 45, 60, 90, 120 minutes, or 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 18 hours, 24 hours, or 30, 45, 60, 90, 120 minutes, or 30, 45, 60, 90, 120 minutes, or 30, 45, 60, 90, 120, 45, 60, 90, 120 minutes, or 30, 45, 60, 70, 80, 12 ...

[0387] The period of time that the subject can wait can be about 1 minute, 5 minutes, 10, 15, 20, 25, 30, 45, 60, 90, 120 minutes, or 3 hours, 4, 5, 6, 7, 8, 12, 18, 24 hours after administration of the ammonia-oxidizing microorganism and before application of the one or more cosmetic products.

[0388] While specific embodiments of the subject invention have been described, the foregoing specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art after review of this specification.

[0389] Containers, e.g., end-use containers, delivery devices The container and / or delivery device, e.g., the container, e.g., the delivery device, e.g., the end-use container, is provided as a housing for the preparation, e.g., the finished preparation. In some embodiments, the container or delivery device can also serve the purpose of delivering the preparation, e.g., the finished preparation, to, for example, a surface or a subject.

[0390] The container and / or delivery device can be configured to store and / or deliver any of the preparations or products disclosed herein. The preparation can be delivered, for example, to a site, environment, or surface of a subject, with or without additional components. In certain embodiments, other components can be delivered simultaneously or sequentially, e.g., at least partially before or at least partially after, delivery of the preparation begins. In certain embodiments, the container or delivery device can include or be referred to as a delivery system. In some embodiments, delivery of one component is still occurring when delivery of a second component begins, thus, there is an overlap. This is sometimes referred to herein as "simultaneous," "concomitant," or "concurrent delivery." In other embodiments, delivery of one component ends before delivery of the other treatment begins. This is sometimes referred to herein as "continuous," "sequential delivery," or "continuous delivery."

[0391] A barrier can be provided as part of or within the container to prevent fluid communication between the interior and exterior environments of the container. The barrier can be in the form of a valve, e.g., a check valve, a filtering material, a film, a wax, a lipid, a polymer, a controlled release material, e.g., a gel, and other materials that can provide either a permanent or temporary barrier between the interior and exterior environments of the container.

[0392] After actuation of the container, the barrier can be disrupted to allow placement of the cosmetic product from the container into the external environment or, for example, into a site, environment, or surface of interest, allowing the cosmetic product to come into contact with, for example, a site, environment, or surface of interest.

[0393] The container may include a delivery system, which may be an applicator or may be configured to deliver the contents of the formulation.

[0394] The delivery system can be configured to deliver the preparation to the surface of a subject, for example, the skin surface.The preparation can be in the form of a particle or particles with a particle size to enhance delivery or to enhance positioning or contact with the desired target site.The preparation can be in the form of a liquid, solid, suspension, or solution.The preparation can be in the form of a powder, cream, ointment, or lotion.

[0395] In certain embodiments, the delivery system can include a pump for delivering the contents of the interior of the container to a target site, eg, an environment, eg, a surface of a subject, eg, the skin of a subject.

[0396] In some embodiments, the container can be a single-use container. The container may or may not be pre-filled (e.g., pre-filled by the manufacturer or the user) with a content, e.g., a preparation, and can be used once by a user, e.g., a consumer or a medical professional, to deliver the content of the container to a target site, e.g., the environment, e.g., the surface of a subject, e.g., the skin of a subject.

[0397] In other embodiments, the container may be a multi-use container, in which case the container may or may not be pre-filled (e.g., filled by the manufacturer or user) with contents, e.g., a formulation, and can be used one or more times by a user, e.g., a consumer or medical professional, to deliver the contents of the container to a target site, e.g., an environment, e.g., a surface of a subject, e.g., the skin of a subject. The container may be refilled (e.g., filled by the manufacturer or user) with contents, e.g., a formulation including ammonia-oxidizing microorganisms, and can be used again by the same or a different user, e.g., a consumer or medical professional, to deliver the contents of the container to a target site, e.g., an environment, e.g., a surface of a subject, e.g., the skin of a subject.

[0398] The contents, eg, pre-filling or refilling of the formulation, may include a sterilization process to ensure that the contents of the container are sterile.

[0399] In some embodiments, the container, e.g., an end-use container, may be in the form of a syringe, bottle, ampoule, applicator, pouch, e.g., a spout pouch, e.g., with a screw cap. A pump may be attached to the bottle to dispense the contents from the container. The container may provide an aerosol spray or mist. The container may be a squeezable container, e.g., a squeeze bottle or a tube, to allow dispensing of the contents through an opening covered with a closure. The container may have a screw-type closure, a non-spill closure, a snap cap closure, or a snap flap closure. The container may have an overcap that resides over the dispensing area where dispensing of the contents occurs. The closure may be fully or partially removable, e.g., fully or partially removable from the body of the container and attached by a hinge. The container may be, for example, a single-use package, such as a laminated packet, that can be torn open to dispense the contents and disposed of after use.

[0400] The container, e.g., end-use container, may be configured to inhibit retrograde flow, e.g., backflow, e.g., reflux, e.g., backward movement, of a material, e.g., a formulation, into the end-use container. The container, e.g., end-use container, may be configured to inhibit retrograde flow, e.g., backflow, e.g., reflux, e.g., backward movement, of a material, e.g., a contaminant, into the end-use container. The contaminant may be atmospheric, e.g., an aerosol, or a liquid, e.g., water, or a solid, or a gas, e.g., oxygen.

[0401] In other embodiments, the container, e.g., end-use container, may not be configured to inhibit retrograde flow, e.g., backflow, e.g., reflux, e.g., backward movement, of a material, e.g., a cosmetic product, into the end-use container. The container, e.g., end-use container, may not be configured to inhibit retrograde flow, e.g., backflow, e.g., reflux, e.g., backward movement, of a material, e.g., a contaminant, into the end-use container.

[0402] The end-use container can include a reservoir into which the formulation is disposed and a dispenser capable of dispensing the formulation from the reservoir. In some embodiments, the dispenser inhibits retrograde flow of material into the reservoir. In other embodiments, the dispenser does not inhibit retrograde flow of material into the reservoir.

[0403] The end-use container can include a reservoir into which the formulation is placed and a dispenser capable of dispensing the formulation from the reservoir. In some embodiments, the dispenser inhibits retrograde flow of the dispensed formulation or atmospheric aerosol into the reservoir. In other embodiments, the dispenser does not inhibit retrograde flow of the dispensed formulation or atmospheric aerosol into the reservoir.

[0404] In certain embodiments, the end-use container may be an anti-backflow dispenser including a first pressure-activated valve disposed in the dispenser and proximal to the reservoir, and a second pressure-activated valve disposed in the dispenser and distal to the reservoir, wherein the actuation pressure of the first valve is higher than the actuation pressure of the second valve.

[0405] In certain embodiments, the end-use container may include an anti-backup mechanism configured to prevent movement of the formulation in a direction opposite to the operational direction associated with dispensing the product.

[0406] In some embodiments, the container may be substantially free of organisms, e.g., microorganisms. In embodiments, the container may be free of other organisms. The container may be sterilized to provide a container that is substantially free or free of organisms, e.g., microorganisms.

[0407] The container or preparation can be disposed in a powder, cosmetic, cream, stick, aerosol, ointment, wipe or bandage.The container or preparation can be provided as a powder, cosmetic, cream, stick, aerosol, ointment, wipe or bandage.

[0408] In some embodiments, the container may include an indicator component, which may be a color marker that can develop a color upon contact of the interior of the container with microorganisms.

[0409] The container can be constructed of any material suitable for containing the contents, for example, a cosmetic product, for example, the finished cosmetic product disclosed herein. For example, the container can be constructed and arranged to be at least partially resistant to at least one of gas exchange, water, and / or light. For example, the container can be constructed of glass or a polymer material. In other embodiments, the container can allow gas exchange, water, and / or light to pass through.

[0410] The end-use container may be composed of or include a polymer, such as polyethylene terephthalate (PET), high density polyethylene (HDPE), polypropylene, polycarbonate, polytetrafluoroethylene (Teflon®), polyvinylidene fluoride (PVDF), or cellulosic. The end-use container may be composed of or include glass.

[0411] A sensor, e.g., an oxygen sensor, that can indicate the presence of viable bacteria can be included in the end-use container. The end-use container can allow at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 percent transmission of ionizing radiation, e.g., by gamma rays, e.g., from an isotope such as cobalt-60, by x-rays, or by ultraviolet light, e.g., ultraviolet C (UVC), through the end-use container.

[0412] The containers described herein may be adapted to deliver one or more cosmetic products. The containers described herein may be adapted to deliver one or more therapeutic products.

[0413] The weight of the container, delivery system or delivery device, with or without the contents of the container, can be less than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500 or 2000 grams. [Example]

[0414] The function and advantages of the above and other embodiments can be better understood from the following examples, which are intended to be illustrative in nature and are not to be considered as limiting the scope of the invention.

[0415] Example 1 Production of heat-killed ammonia-oxidizing microorganisms to simulate inactivated ammonia-oxidizing microorganisms N. eutropha (D23) was heat-killed to simulate inactivated D23. Briefly, live D23 was kept in storage buffer (storage state). Secreted metabolites were washed out. D23 in storage buffer was heat-killed by exposure to 60°C for 2 hours. The metabolic activity of heat-killed D23 was measured by the Griess reaction after incubation in buffer with 50 mM ammonia at 37°C for 1 hour. As a control, live D23 was incubated for 1 hour.

[0416] The results are shown graphically in Figure 1. Heat-killed D23 produced no nitrite and had no measured metabolic activity.

[0417] Example 2 Peripheral blood mononuclear cells (PBMCs) treated with live and heat-killed ammonia-oxidizing microorganisms Heat-killed D23 was prepared as described in Example 1. PBMCs were treated with live and heat-killed D23 to determine the treatment effect of the D23 microbial component compared to live microorganisms. PBMCs were thawed and cultured at 7 x 10 cells in RPMI 1640 cell culture medium (Gibco™, distributed by Thermo Fisher Scientific, Waltham, MA) with 10% heat-inactivated fetal bovine serum (Hi-FBS) (distributed by Thermo Fisher Scientific, Waltham, MA) and 2 mM Glutamax™ (distributed by Thermo Fisher Scientific, Waltham, MA). 6 Cells / mL were seeded by culturing.

[0418] After 16 h of seeding, 3 × 10 7Live or heat-killed D23 cells were added to PBMC cultures at a concentration of CFU / mL (MOI = 4.3). After 1 hour, Th2 cells were stimulated with anti-CD3 antibody and a cytokine cocktail (Human Th2 Cell Differentiation Kit, CellxVivo™, distributed by R&D Systems, Minneapolis, MN). The cocktail did not contain anti-IFNg or anti-IL-12 antibodies.

[0419] After 3 days of Th2 stimulation, samples were collected and designated for WST-1, ELISA and RT-qPCR / flow cytometry experiments.

[0420] No toxicity was observed in co-cultures of PBMC with D23 or heat-killed D23. The results are shown in the graphs in Figures 2A-B. Figure 2A is a graph of percent PBMC activity and viability measured by WST-1 after 72 hours of stimulation. Figure 2B is a graph of nitrite levels measured by Griess reagent after 72 hours of stimulation.

[0421] Example 3 Th2 inhibition by ammonia-oxidizing microorganisms and heat-killed ammonia-oxidizing microorganisms Th2-stimulated cell cultures with D23 or heat-killed D23 were prepared as described in Example 2. IL-5 and IL-13 expression in treated cells from a single donor was measured by ELISA 72 hours after stimulation. The results are shown in the graphs of Figures 3A-3B. IL-4 expression in treated cells from a single donor was measured by RTqPCR 72 hours after stimulation. Relative IL-4 expression is shown in the graph of Figure 3C. IL-5 and IL-13 expression in treated cells from five donors was measured by ELISA 72 hours after stimulation. Mean expression is shown in the graphs of Figures 4A-4B.

[0422] As shown by the data presented in Figures 3A-3C and 4A-4C, Th2-stimulated cell cultures with D23 and heat-killed D23 show similar IL-5, IL-13, and IL-4 expression, suggesting that heat-killed D23 exhibits similar activity as live D23 through Th2 inhibition.

[0423] Example 4 Ammonia-oxidizing microorganisms, heat-killed ammonia-oxidizing microorganisms, and ammonia-bearing LpS and CD3 / 28 stimulated cells LpS and CD3 / 28 stimulated cells were co-cultured with D23 or heat-killed D23 as previously described. Cells were also co-cultured with 6 mM NH3 to determine microbial metabolic activation by ammonia.

[0424] TNFα and IL-6 expression were measured by CBA 24 hours after stimulation. The results are shown in Figures 5A-5C and 6A-6C, respectively. As shown in the graphs, heat-killed D23 induced innate immune responses comparable to those of live D23. However, the D23 ammonia oxidation phenotype was not detected in co-cultures with 6 mM NH3.

[0425] IL-10 and IFNγ expression were measured 24 hours after stimulation by CBA. The results are shown in the graphs in Figures 7A-7C and 8A-8C, respectively. As shown in the graphs, heat-killed D23 appear to drive adaptive immune responses independently of D23 ammonia oxidation metabolism. Live D23 induce slightly more IL-10 in the presence of LpS compared to heat-killed D23 (Figure 7B). Again, the D23 ammonia oxidation metabolism phenotype was not detected in co-cultures with 6 mM NH3.

[0426] IL-2 expression was measured 24 hours after stimulation by CBA. The results are shown in the graphs in Figures 9A-9C. As shown in the graphs, heat-killed and live D23 cells do not modulate IL-2 in the presence or absence of ammonia.

[0427] IL-4 and IL-17A expression was measured 24 hours after stimulation by CBA. Neither IL-4 nor IL-17A expression was detected by the assay, so data are not included.

[0428] As the data show, heat-killed D23 exhibits similar metabolic activity as live D23, as measured by cytokines.

[0429] Ammonia oxidizing microorganism preparations can have suitable efficacy (immune response) due to the population of inactive ammonia oxidizing microorganisms.

[0430] Example 5 Peripheral blood mononuclear cell (PBMC) stimulation and treatment PBMCs were thawed and washed in Hanks' medium (ATCC) with 10% heat-inactivated fetal bovine serum (FBS) (Gibco™, Thermo Fisher Scientific, Waltham, MA), then resuspended in PBMC culture medium (RPMI 1640 medium, phenol red- and glutamine-free (Gibco™, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated FBS (Gibco™, Thermo Fisher Scientific, Waltham, MA) and 2 mM Glutamax (Gibco™, Thermo Fisher Scientific, Waltham, MA)). PBMCs were seeded into 6- or 24-well plates (Corning, Inc., Corning, NY) at 7–8 × 10 6 cells / mL and incubated at 37°C with 5% CO. Live or heat-killed AOB, prepared as described in Example 1, were added at a multiplicity of infection (MOI) of 4 1 hour before Th2 stimulation.

[0431] Cells were then stimulated using either the CellxVivo™ Human Th2 Cell Differentiation Kit (R&D Systems, Minneapolis, MN) according to the manufacturer's recommendations or 1 μg / mL Staphylococcal Enterotoxin B (SEB) (Sigma-Aldrich, St. Louis, MO). Neutralizing antibodies were used at 10 μg / mL and refreshed daily until sample collection. The following neutralizing antibodies were used: mouse anti-human IFNγ clone K3.53 (MAB2852), mouse anti-human IL-12p70 clone 24910 (MAB219), mouse anti-human IL-10 clone 948505 (MAB9184), and isotype controls mouse IgG2a (MAB003) and mouse IgG1 (MAB002) (all from R&D Systems, Minneapolis, MN).

[0432] TLR-blocking antibodies were added to PBMCs at a final concentration of 10 μg / mL 1 h before AOB treatment and refreshed daily until sample collection. The following TLR-blocking antibodies were used: TLR1, TLR2, TLR4, TLR5, and TLR6 (TLR2 was from R&D Systems, Minneapolis, MN; all others were from Invivogen, San Diego, CA). The TLR8 inhibitor compounds CU-CPT9a (Invivogen, San Diego, CA) or CU-CPT8m (MedChemExpress, Monmouth Junction, NJ) were added to PBMCs at final concentrations between 100 nM and 10 μM 1 h before AOB treatment. The TLR9 antagonist ODN TTAGGG (A151) or negative control (Invivogen, San Diego, CA) was added at a final concentration of 1 μM and refreshed daily until sample collection.

[0433] After all treatments or stimulations, plates were shaken at 600 rpm for 30 seconds and incubated at 37°C with 5% CO2. Samples were collected 3 days after stimulation (unless otherwise indicated) and spun at 500 x g for 10 minutes. Cell-free supernatants were frozen at -20°C for later cytokine quantification, and cell pellets were processed for RNA extraction or flow cytometry analysis.

[0434] The stimulated and treated cells were used to generate the data presented in Examples 7 and 8 below.

[0435] Example 6 Bacterial metabolite quantification Nitrite and nitric oxide production were measured to determine the metabolic activity of live or heat-killed AOB. Nitrite was quantified using the Griess assay. Briefly, samples were mixed 1:1 with equal volumes of Griess reagent A (1.5 N hydrochloric acid, 58 mM sulfanilamide) and Griess reagent B (0.77 mM NNEQ) solutions, incubated for 20 minutes at room temperature protected from light, and then quantified by measuring OD540nm using a 96-well plate spectrophotometer. Nitrite concentrations were then calculated based on a sodium nitrite calibration curve.

[0436] Nitric oxide was quantified using the fluorescent dye DAF-2 (Abcam, Cambridge, UK). Briefly, samples were mixed 1:1 with PBMC / AOB culture medium containing 5 μM DAF-2 and incubated for 1 hour at 37°C with 5% CO2. Nitric oxide production was quantified using a calibration curve of the fluorescent molecule DAF-2T (Abcam, Cambridge, UK). Measurements were taken either after 1 hour in AOB culture medium or after 72 hours in PBMC culture medium.

[0437] Example 7 Th2 inhibition by ammonia-oxidizing microorganisms involves the RNA sensor Toll-like receptor 8 (TLR8) signaling Multiple types of bacterial immunomodulatory components were identified, ranging from metabolites (e.g., SCFAs, indoles) to cell wall components (e.g., LPS, PSA) and nucleic acids (e.g., CpG DNA, RNA). The ability of AOB to block Th2 polarization was shown to be substantially independent of metabolites. Heat-killed AOB appeared to be as effective as live AOB in Th2-mediated inhibition. Therefore, nitric oxide, an immunomodulatory molecule produced by AOB, is unlikely to be involved in this phenotype.

[0438] Briefly, studies were performed to identify the AOB bacterial components responsible for Th2 inhibition. To distinguish between secreted metabolites and cell wall / intracellular components, live AOB were compared with heat-killed AOB that had been washed to remove any residual secreted molecules. Heat-killed AOB were prepared as described in Example 1. In contrast to live AOB, heat-killed AOB were unable to produce nitrite or nitric oxide, indicating a lack of metabolic activity (Figures 10A-B, p<0.001). Heat-killed AOB were as efficient as live AOB in inhibiting the Th2 markers IL-5 and IL-13 (Figures 10C-D). Heat-killed AOB were as efficient as live AOB in inhibiting the CD11c+ surface proteins MHC II and CD86 (Figures 10E-F). These data indicate that AOB metabolic activity and associated secreted molecules, such as the immunomodulatory molecule nitric oxide, are not required for Th2 inhibition. Rather, these findings point toward a role for structural or intracellular components.

[0439] The role of toll-like receptors (TLRs) in Th2 inhibition was examined. Major TLR classes were tested by applying individual TLR inhibitors or neutralizing antibodies to PBMCs prior to AOB treatment and Th2 stimulation. The effect of TLR inhibitors or neutralizing antibodies on AOB-mediated Th2 inhibition was measured. None of TLR1 / 2 / 4 / 6 / 9 inhibition significantly affected the ability of AOB to affect IL-5 production (Figure 11).

[0440] Using a collection of TLR inhibitors and blocking antibodies, we found that TLR8 signaling appears to be critical for AOB-mediated Th2 inhibition: Inhibition of the RNA sensor TLR8 resulted in a 67.6% reduction in AOB-mediated IL-5 inhibition compared to vehicle control (Figure 12, p<0.05).

[0441] TLR8 is known to detect single-stranded RNA and is located inside endosomes. Without wishing to be bound by theory, it is thought that AOB may be endocytosed, and the RNA released by cell lysis triggers TLR8 signaling. TLR8 stimulation has been reported to induce IL-10 production in other models, but the exact mechanism remains unclear.

[0442] These data suggest that AOB RNA may play an important role in the early signaling cascade that leads to the suppression of Th2 responses by AOB.

[0443] Example 8 Mechanisms of ammonia-oxidizing microorganism-mediated Th2 modulation We determined that Th1 is not substantially involved in AOB-mediated Th2 modulation. Neutralization of the Th1 effector IFNγ or the Th1 differentiation factor IL-12 did not significantly impair the ability of AOB to block Th2. Heat-killed AOB, which blocked Th2 polarization as efficiently as live AOB, did not induce as strong a Th1 response as live AOB (Figures 13A-B).

[0444] Example 9 Therapeutic safety of shelf-stable ammonia-oxidizing microbial preparations AOB is predicted to be safe, as supported by the Generally Recognized As Safe (GRAS) label issued by the FDA for D23. There are no known reports of live AOB being associated with infection or pathology. A Phase I human safety trial with live AOB reported no adverse events. There are no known reports of adverse events with live AOB when used as a cosmetic.

[0445] As shown above, heat-killed AOB inhibits Th2 polarization as efficiently as live AOB. The potential use of heat-killed AOB would be expected to further enhance its safety profile by preventing colonization and avoiding any unexpected potential infectivity or in situ toxin production, which are common concerns with the use of live bacterial / biopharmaceutical products.

[0446] As also shown above, heat-killed AOB does not induce as strong a Th1 response as live AOB (Figures 13A-B). Thus, heat-killed AOB may be beneficial in the context of atopic patients who also suffer from atopic diseases with a Th1 component (e.g., celiac disease) or Th1-mediated diseases (e.g., multiple sclerosis, type 1 diabetes). Thus, heat-killed AOB can be used to treat Th2-mediated diseases without exacerbating the diagnosed Th1-mediated disease.

[0447] The nonviable, inactive, and / or heat-killed ammonia-oxidizing bacterium N. eutropha D23 shows promising therapeutic potential in atopic diseases due to its ability to block Th2 polarization and key cytokines involved in IgE production, eosinophilia, and itch, as well as a favorable safety profile. The mechanism of action leading to Th2 pathway inhibition is thought to be IL-10-mediated inhibition of dendritic cell activation triggered by TLR8 sensing of AOB RNA.

[0448] Example 10 Fold reduction of IL-5 mediated by D23 cells treated under different conditions Additional tests were performed to determine the effectiveness of D23 cells treated under different conditions. Freeze / thaw-treated, ethanol-treated, senescent, heat-treated, and live cells were tested in the same IL-5 assay. Frozen / thawed (F / T) cells were treated according to the following protocol: Live D23 cells were centrifuged at 16,000 x G for 5 minutes in a 1.5 mL centrifuge tube. The supernatant was removed. 1 mL of PBS was dispensed into each tube. 1 mL of D23 stock was dispensed into a 1.5 mL cryotube. The cells underwent two freeze-thaw cycles at -80°C and room temperature. After thawing, the cells were maintained at 4°C. The cells were considered to be killed by the freeze / thaw treatment.

[0449] Ethanol (EtOH) cells were treated according to the following protocol: 5 mL of D23 stock was pipetted into two 50 mL tubes. The tubes were centrifuged at 3000 x G for 45 minutes. The supernatant was removed. 5 mL of 70% ethanol was pipetted into each tube. The tubes were placed in an open biosafety cabinet in operation and allowed to dry completely. Once dry, 5 mL of PBS was pipetted into each tube. Both tubes were combined into one stock. The cells were considered to be killed by the ethanol treatment.

[0450] The senescent cells were stored at 4°C for the duration of aging. The first set of cells (Aged 1) was aged for approximately 27 months. The second set of cells (Aged 2) was aged for approximately 17 months. The third set of cells (Aged 3) was aged for approximately 12 months.

[0451] Heat-killed (HK) cells were treated as previously described. Briefly, live D23 cells were retained in storage buffer (storage state). Secreted metabolites were washed out. D23 cells in storage buffer were heat-killed by exposure to 60°C for 2 hours.

[0452] A reduction in IL-5 was observed for all cell treatments. The data are presented graphically in Figure 14. Thus, freeze / thawed, ethanol-treated, and senescent cells show similar effects as live and heat-killed D23 cells.

[0453] Example 11 Treatment of eczema by topical administration of a shelf-stable composition containing ammonia-oxidizing microorganisms Water (Aqua), Glycerin, Glyceryl Tricaprylate / Glyceryl Tricaprate, Jimmondsia Chinensis (Jojoba) Seed Oil, Squalane, Prunus Amygdalus Dulcis (Sweet Almond) Oil, Butyrospermum Parkii (Shea Butter), Carbomer, Polyacrylic Acid, Ceteareth 20, Glyceryl Stearate (Gyceryl), Potassium Sorbate, Citric Acid, Acetic Acid A shelf-stable N. eutropha D23 cream composition was prepared containing tocopheryl acetate, N. eutropha, sodium bicarbonate, and sodium hyaluronate. 7 It contained D23 cells / mL.

[0454] Twenty-one subjects applied the composition topically to target tissues exhibiting eczema symptoms twice daily as needed for 14 days. Treatment was discontinued on day 21, and subjects were followed up. Subjects were scored for the mean change in eczema appearance from baseline. The results are presented in Figures 15A-15B and 16.

[0455] As shown in the graph in FIG. 15A, the mean Eczema Area and Severity Index (EASI) score decreased after 14 days. Specifically, the mean EASI score decreased from about 2 to about 1. EASI is a validated scoring system that grades the physical signs of atopic dermatitis and eczema. Thus, administration of a composition disclosed herein can provide a decrease in the EASI score. In some examples, administration can provide a decrease in the EASI score of at least about 5 points, e.g., at least about 4 points, about 3 points, about 2 points, or about 1 point. In some examples, administration can provide a decrease in the EASI score of at least about 100%, e.g., at least about 75%, at least about 50%, or at least about 25%.

[0456] As shown in the graph in FIG. 15B, the mean Investigator's Global Assessment (IGA) score also decreased after 14 days. Specifically, the mean IGA score decreased from about 1.5 to about 1. The IGA is a 5-point scale providing a global clinical assessment of atopic dermatitis severity ranging from 0 to 4, where 0 indicates clear, 2 indicates mild, 3 indicates moderate, and 4 indicates severe atopic dermatitis. A decrease in score correlates with an improvement in signs and symptoms. In some examples, administration can provide a decrease in IGA score of at least about 5 points, e.g., at least about 4 points, about 3 points, about 2 points, about 1 point, about 0.5 points, or about 0.25 points. In some examples, administration can provide a decrease in IGA score of at least about 100%, e.g., at least about 75%, at least about 50%, at least about 25%, at least about 20%, at least about 15%, or at least about 10%.

[0457] As shown in the graph in Figure 16, the percentage of subjects who improved from baseline was high for EASI scores (slightly over 80%) and IGA scores (slightly over 50%). The mean change from baseline for EASI scores was approximately -1.1 points, while the mean change from baseline for IGA scores was approximately -0.5 points.

[0458] Thus, the shelf-stable D23 compositions disclosed herein are effective in treating eczema.

[0459] Example 12 Treatment of pruritus by topical administration of a shelf-stable composition containing ammonia-oxidizing microorganisms Fourteen adult subjects applied the shelf-stable D23 cream composition of Example 11 to target tissues twice daily as needed for 14 days to treat pruritus. On day 21, treatment was discontinued and follow-up was conducted. Subjects scored itch severity on a visual analog scale (VAS) of 1 to 10, with 0 being no pruritus; <3 being mild pruritus; ≥3 to <7 being moderate pruritus; ≥7 to <9 being severe pruritus; and ≥9 being very severe pruritus. Results are presented graphically in Figure 17.

[0460] As shown in the graph in Figure 17, the VAS scores decreased over the study period. In particular, the VAS scores decreased from an average of about 7 (severe pruritus) at baseline to an average of just under 3 (mild pruritus) on days 14 and 21. In some examples, administration can provide a reduction in the VAS score for itch of at least about 5 points, e.g., at least about 4 points, about 3 points, about 2 points, or about 1 point. In some examples, administration can provide a reduction in the VAS score for itch of at least about 100%, e.g., at least about 75%, at least about 50%, or at least about 25%.

[0461] Seven pediatric subjects applied the shelf-stable D23 cream composition of Example 11 to target tissues twice daily as needed for 14 days to treat pruritus. Treatment was discontinued on day 21 and follow-up was conducted. Subjects scored itch severity on the Ischman scale, with 0 being pleasant; 1 being slightly itchy; 2 being more itchy; 3 being very itchy; and 4 being the most severe itchy. Results are presented graphically in Figure 18.

[0462] As shown in the graph in Figure 18, the Itchman scores decreased over the study period. In particular, the Itchman scores decreased from an average of about 2.75 (more itchy to very itchy) at baseline to an average of 0.5 to 0.25 (comfortable to slightly itchy) on days 14 and 21, respectively. In some examples, administration can provide a reduction in the Itchman score of at least about 5 points, e.g., at least about 4 points, about 3 points, about 2 points, or about 1 point. In some examples, administration can provide a reduction in the Itchman score of at least about 100%, e.g., at least about 75%, at least about 50%, or at least about 25%.

[0463] As shown in the graphs in Figures 19A-19B, the percentage of adult subjects who improved from baseline was high for VAS scores (85%-100%), and the percentage of pediatric subjects who improved from baseline was high for Itchman scores (60%-100%). The mean change from baseline in VAS scores for adult subjects was -3.5 to -4.5 points, while the mean change from baseline in Itchman scores for pediatric subjects was -0.5 to -2.5 points.

[0464] Thus, the shelf-stable D23 compositions disclosed herein are effective in treating pruritus. Annex VI - ASEAN Cosmetics Document [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Regarding Annex VI: 1. Preservatives are substances that can be added to cosmetic products for the primary purpose of inhibiting the development of microorganisms in such products. 2. Substances marked with the symbol (+) may also be added to cosmetic products in concentrations other than those specified in this Annex, for example as deodorants in soaps or antidandruff agents in shampoos, or for other purposes that are clear from the product's presentation. 3. Other substances used in the formulation of cosmetic products may also have antimicrobial properties and thus help to preserve the product, such as many essential oils and some alcohols. These substances are not included in this Annex. 4. For purposes of this list: "Salts" is understood to mean salts of the cations sodium, potassium, calcium, magnesium, ammonium and ethanolamine; salts of the anions chloride, bromide, sulfate, acetate. "ester" is understood to mean methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl esters; 5. Any finished product containing formaldehyde or a formaldehyde-releasing substance in this Annex must be labeled with the warning "Contains formaldehyde" if the concentration of formaldehyde in the finished product exceeds 0.05%.

Claims

[Claim 1] An object, method or system as described in this specification and drawings.

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