Ammonia-oxidizing bacteria for treatment of acne

Abstract

To provide ammonia-oxidizing bacteria for treatment of acne.SOLUTION: A method for treating skin disorders, for example acne, e.g., acne vulgaris, in a subject is provided. This method comprises administering a preparation containing an ammonia oxidizing bacterium to the surface of the subject, for example applying, for example, topically administering. Also provided is a preparation containing an ammonia oxidizing bacteria for treating skin disorders, for example acne, e.g., acne vulgaris, in a subject. The administration, for example the amount and frequency of application may be sufficient to reduce the amount or concentration of pathogenic bacteria, for example Propionibacterium acnes, on the surface of the subject.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - References to Related Applications This application is a continuation - in - part of International Patent Application No. PCT / US2015 / 025909, filed on Apr. 15, 2015, which claims priority based on Greek Patent Application No. 20140100217, filed on Apr. 15, 2014; U.S. Provisional Application No. 62 / 002084, filed on May 22, 2014; U.S. Provisional Application No. 62 / 012811, filed on Jun. 16, 2014; U.S. Provisional Application No. 62 / 053588, filed on Sep. 22, 2014; and Greek Patent Application No. 20150100115, filed on Mar. 13, 2015. The contents of these applications are hereby incorporated by reference in their entirety. This application also claims priority based on U.S. Provisional Application No. 62 / 188343, filed on Jul. 2, 2015 and U.S. Provisional Application No. 62 / 189105, filed on Jul. 6, 2015.

Background Art

[0002] Background Beneficial bacteria can be used to inhibit the growth of pathogenic bacteria. Bacteria and other microorganisms are ubiquitous in the environment. The discovery of pathogenic bacteria and the germ theory of disease have had a profound impact on health and disease states. Bacteria are a normal part of the environment of all living things. In the intestine, these bacteria are not pathogenic under normal conditions and actually improve health by making the normal intestinal contents less suitable for organisms that cause disease. Disease prevention is carried out in many ways: consuming nutrients so that there is little left for pathogens; creating conditions such as pH and oxygen pressure that are not suitable for pathogens; producing compounds that are toxic to pathogens; these microorganisms consuming pathogens as food; reducing the physical space available to pathogens; and occupying specific binding sites to reduce the binding sites available to pathogens. The presence of these desirable bacteria is seen as useful in the prevention of disease states.

[0003] For example, with respect to the treatment of skin conditions such as acne, such as common acne, there is a need in the art for improved beneficial bacteria that can inhibit the growth of pathogenic bacteria.

[0004] Acne is the most common skin disease and the leading reported cause of dermatologist visits, accounting for approximately one - quarter of the patients seen by dermatologists in the United States. Traditionally considered a minor "normal" condition, acne has in recent years been re - defined medically as a chronic disease that can have a significant impact on an individual's quality of life due to social, psychological, and emotional dysfunctions comparable to those reported by patients with epilepsy, asthma, diabetes, or arthritis. In the United States alone, nearly 40 to 50 million people suffer from acne, and 20 - 25% have moderate to severe acne. The annual sales rate of the top 10 branded acne treatments in the United States is $3 billion, broken down as $1.5 billion for systemic retinoids, $700 million for oral antibiotics, $600 million for topical antibiotics, and $240 million for topical retinoids. Worldwide, the prevalence of acne is approximately 80% among adolescents and 50% among young adults. Despite significant expenditures on prescription and over - the - counter medications, acne remains a major unmet medical need due to treatments being only marginally effective (topical antibiotics, topical retinoids, astringents) and / or associated with serious risks (oral retinoids). In addition to the need for safe and effective products to prevent and treat inflammatory and non - inflammatory acne lesions, there is a substantial unmet need and commercial opportunity for topical therapies that target excessive sebum production. [Table 1]

[0005] The ideal target product has the following characteristics: being self-administered (e.g., once a day, e.g., twice a day); being odorless, colorless, and invisible to the eye; not being accompanied by an increased sensitivity to sunlight; being safe and non-toxic when ingested / inhaled, and having good tolerance in the skin, adjacent mucous membranes, and eyes; being suitable for both treatment and maintenance therapy without the risk of promoting antibiotic resistance; and acting locally to (in order of priority): reduce the frequency of lesions (antibiotics, anti-inflammatory drugs); reduce the duration of lesions (improved healing); reduce the inflammation of lesions (anti-inflammatory drugs); potentially prevent or reduce abnormal pigmentation and scarring (improved healing); accelerate the healing of damaged skin (improved healing); reduce the production of excessive sebum; and reduce the presence of pathogenic bacteria (antibiotics), and may have one or more of them.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0007] Abstract The present disclosure provides a method for treating a skin disorder of a subject, such as acne, such as acne vulgaris. The method includes administering, such as applying, such as topically administering, a preparation containing ammonia-oxidizing bacteria, such as ammonia-oxidizing bacteria, to the surface of the subject.

[0008] In some embodiments, the amount and frequency of administration, such as application, are sufficient to reduce the amount or concentration of pathogenic bacteria, such as Propionibacterium acnes, on the surface of the subject. In some cases, this amount is a therapeutically effective dose of ammonia-oxidizing bacteria. In some cases, the skin disorder is acne, such as acne vulgaris.

[0009] In some embodiments, administering provides treatment of inflammatory lesions such as papules, pustules, cysts / nodules. In some embodiments, administering provides treatment of non-inflammatory lesions such as open comedones, closed comedones. In some embodiments, administering provides treatment or improvement of post-inflammatory hyperpigmentation / post-inflammatory erythema (PIH / PIE) lesions. In some embodiments, administering provides treatment or improvement of one or more of erythema, edema, desquamation, stinging, burning, and pruritus. In some embodiments, administering provides treatment or improvement of one or more of a greasy appearance, appearance of pores, gloss, spots, skin tone uniformity, visual smoothness, and tactile smoothness. In some embodiments, administering provides treatment or improvement of sebumeter measurements.

[0010] In some embodiments, administering includes pre-treating the subject with ammonia-oxidizing bacteria. In some embodiments, topically administering includes topically administering prior to the onset of a skin disorder. In some embodiments, topically administering includes topically administering an effective dose of ammonia-oxidizing bacteria to the subject. In some cases, the effective dose is about 0.1×10 9 、0.2×10 9 、0.3×10 9 、0.4×10 9 、0.5×10 9 、0.6×10 9 、0.7×10 9 、0.8×10 9 、0.9×10 9 、1.0×10 9 、1.2×10 9 、1.4×10 9 、1.5×10 9 、1.6×10 9 、1.8×10 9 、2.0×10 9 、2.2×10 9 、2.4×10 9 、2.6×10 9 、2.8×10 9 、3.0×10 9 、3.2×109 , 3.4×10 9 , 3.6×10 9 , 3.8×10 9 , 4.0×10 9 , 4.2×10 9 , 4.4×10 9 , 4.6×10 9 , 4.8×10 9 , 5.0×10 9 , 5.5×10 9 , 6.0×10 9 , 6.5×10 9 , 7.0×10 9 , 7.5×10 9 , 8.0×10 9 , 8.5×10 9 , 9.0×10 9 , 9.5×10 9 , 10.0×10 9 , 12×10 9 , 14×10 9 , 16×10 9 , 18×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , 40×10 9 , 50×10 9 CFU.

[0011] In some embodiments, the skin disorder is at a target site of the subject and comprises one or more undesirable bacteria, such as pathogenic bacteria. In some cases, the target site comprises Propionibacterium acnes.

[0012] In some embodiments, the method further comprises determining whether the subject is in need of treatment for a skin disorder, e.g., determining whether the subject is in need of treatment for acne, such as acne vulgaris. In some embodiments, the method further comprises selecting a subject in need of treatment for a skin disorder.

[0013] In some embodiments, the ammonia-oxidizing bacteria, e.g., the preparation containing ammonia-oxidizing bacteria, can be in the form of an aerosol, spray or mist, i.e., it may be in the form of a mist. In some embodiments, the ammonia-oxidizing bacteria, e.g., the preparation, may be applied as an aerosol or mist in an aqueous medium, for example. In some embodiments, the ammonia-oxidizing bacteria, e.g., the preparation of ammonia-oxidizing bacteria, may be an ammonia-oxidizing bacteria in a buffer solution, e.g., an aqueous buffer solution. In some cases, the buffer solution, e.g., the aqueous buffer solution, is sodium dihydrogen phosphate and magnesium chloride in water, e.g., 50 mM Na 2 HPO 4 and 2 mM MgCl 2 In some cases, the buffer solution, e.g., the aqueous buffer solution, is sodium dihydrogen phosphate and magnesium chloride in water, e.g., 50 mM Na 2 HPO 4 and 2 mM MgCl 2 In some cases, the buffer solution, e.g., the aqueous buffer solution, consists essentially of sodium dihydrogen phosphate and magnesium chloride in water, e.g., 50 mM Na 2 HPO 4 and 2 mM MgCl 2 In some cases, the buffer solution, e.g., the aqueous buffer solution, consists of sodium dihydrogen phosphate and magnesium chloride in water, e.g., 50 mM Na

[0014] In some embodiments, the method further includes selecting a subject based on the subject having a need to reduce the amount or concentration of pathogenic bacteria, e.g., Propionibacterium acnes, on the surface of the subject. In some embodiments, the ammonia-oxidizing bacteria are self-administered.

[0015] In some embodiments, the ammonia-oxidizing bacteria are applied to any one or more of the subject's face, neck, and scalp.

[0016] In some embodiments, the preparation comprises at least one of ammonia, ammonium salts, and urea. In some cases, the preparation comprises a controlled-release material, such as a delayed-release material. In some cases, the preparation of ammonia-oxidizing bacteria further comprises an additive, such as one of a pharmaceutically acceptable additive or a cosmetically acceptable additive. In some cases, an additive, such as one of a pharmaceutically acceptable additive and a cosmetically acceptable additive, is a surfactant. In some cases, the preparation is substantially free of other organisms. In some cases, the preparation is disposed in a powder, cosmetic, cream, stick, aerosol, such as a mist, plaster, wipe, or dressing. In some cases, the preparation is provided as a powder, cosmetic, cream, stick, aerosol, such as a mist, plaster, wipe, or dressing. In some cases, the preparation comprises a humectant, deodorant, fragrance, colorant, insect repellent, cleansing agent, or UV blocker. In some cases, additives, such as pharmaceutically acceptable additives or cosmetically acceptable additives, include anti-adhesives, binders, coating agents, disintegrants, fillers, fragrances, colorants, lubricants, flow promoters, adsorbents, preservatives, or sweeteners.

[0017] In some cases, a preparation comprising ammonia-oxidizing bacteria comprises from about 10 8 to about 10 14 CFU / L. In some cases, a preparation comprising ammonia-oxidizing bacteria comprises from about 10 8 to about 10 14 CFU / mL. In some cases, the preparation comprises between about 1×10 9 CFU / L and about 10×10 9 CFU / L. In some cases, the preparation comprises between about 1×10 9 CFU / mL and about 10×10 9 CFU / mL.

[0018] In some embodiments, the preparation containing ammonia-oxidizing bacteria contains ammonia-oxidizing bacteria in an amount between about 50 milligrams (mg) and about 1000 mg. In some cases, the mass ratio of ammonia-oxidizing bacteria to an additive, such as a pharmaceutically acceptable additive or a cosmetically acceptable additive, ranges from about 0.1 gram to about 1 gram per liter.

[0019] In some embodiments, the ammonia-oxidizing bacteria are selected from the group consisting of Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof. In some cases, the ammonia-oxidizing bacteria are Nitrosomonas eutropha (N. eutropha). In some cases, the ammonia-oxidizing bacteria are N. eutropha D23 having ATCC accession number PTA-121157.

[0020] In some embodiments, the preparation contains organisms selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacter, and combinations thereof.

[0021] In some embodiments, methods are provided for delivering cosmetic products. In some embodiments, methods are provided for delivering therapeutic products.

[0022] In some embodiments, the preparation is provided in a container, and the preparation and the container have a weight of less than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 grams.

[0023] In some embodiments, the preparation is applied 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. In some cases, the preparation is applied once per day. In some cases, the preparation is applied twice per day.

[0024] In some embodiments, the preparation is 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. In some cases, the preparation is applied for about 7 days. In some cases, the preparation is applied for about 14 days. In some cases, the preparation is applied for about 21 days. In some cases, the preparation is applied for about 28 days.

[0025] In some embodiments, the method further includes obtaining a sample from the surface of the skin. In some cases, the method further includes isolating the DNA of bacteria in the sample. In some cases, the method further includes sequencing the DNA of bacteria in the sample. In some cases, the bacteria is Propionibacterium acnes.

[0026] In some embodiments, administering ammonia - oxidizing bacteria provides a reduction in Propionibacterium acnes. In some cases, after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, Propionibacterium acnes decreases, for example, the concentration of Propionibacterium acnes decreases.

[0027] In some embodiments, the method further comprises administering, e.g., self-administering, one or more of a shampoo, a conditioner, and a cleaning agent, such as a facial cleanser. In certain cases, any one or more of the shampoo, the conditioner, and the facial cleanser are applied once a day. In certain cases, the shampoo used was Lot 293178. In certain cases, the cleaning agent used was Lot 293162. In certain cases, at least one of the shampoo, the conditioner, and the soap was used following discontinuation of the administration of ammonia-oxidizing bacteria. In certain cases, at least one of the shampoo, the conditioner, and the soap was used following discontinuation of the preparation of ammonia-oxidizing bacteria.

[0028] In some embodiments, the subject was evaluated before beginning treatment, e.g., before administering ammonia-oxidizing bacteria. In some embodiments, the subject was evaluated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 days after beginning treatment, e.g., after administering ammonia-oxidizing bacteria; or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 weeks after beginning treatment, e.g., after administering ammonia-oxidizing bacteria; or 2, 3, 4, or 5 years after beginning treatment, e.g., after administering ammonia-oxidizing bacteria.

[0029] In some embodiments, administering a preparation of ammonia-oxidizing bacteria, such as ammonia-oxidizing bacteria, provides one or more of the following: reducing the inflammation of the lesions, reducing the frequency of the lesions, and reducing the presence of pathogenic bacteria, such as Propionibacterium acnes. In some cases, administering the preparation provides a reduction in the inflammation of the lesions. In some cases, administering the preparation provides a reduction in the inflammation of the lesions in adults. In some cases, administering the preparation provides a reduction in the frequency of the lesions. In some cases, administering the preparation provides a reduction in the presence of pathogenic bacteria, such as Propionibacterium acnes. In some cases, administering the preparation provides an improvement in the emotional assessment of the subject with respect to the disease of the subject as measured by the Skindex16 quality of life survey. In some cases, administering the preparation provides one or more improvements in the following: the pain of the skin condition in the subject, the persistence / recurrence of the skin condition in the subject, and the appearance of the skin condition in the subject. In some cases, administering the preparation provides an improvement (reduction) in one or more of the following assessment scores according to clinical assessment: visual and tactile smoothness, and scarring.

[0030] In some embodiments, the subject is female. In other embodiments, the subject is male. In some cases, the subject is one of the following ethnic / racial groups: Asian, Black or African American, Hispanic or Latino, White, or multiracial. In some cases, the subject is characterized as having at least one of the following skin types: normal skin, oily skin, and combination skin. In some cases, the subject is characterized as having one of the following Fitzpatrick skin types: I, II, III, IV, V. In some cases, acne is characterized as adolescent acne. In some cases, acne is characterized as adult acne. In some cases, the age of the subject is between about 12 - 15 years, 16 - 18 years, 19 - 28 years, or over 28 years.

[0031] In some embodiments, an acne treatment selected from the group consisting of: topical retinoids, azelaic acid, salicylic acid, topical antimicrobials, oral antibiotics, benzoyl peroxide, oral antiandrogens, oral isotretinoin, and combinations thereof is administered to a subject. In some embodiments, the acne treatment is administered for a period of time before initiating administration of the ammonia-oxidizing bacteria. In some embodiments, the acne treatment is continued throughout the period of administration of the ammonia-oxidizing bacteria. In some embodiments, the acne treatment is administered for a period of time before initiating administration of the ammonia-oxidizing bacteria and is stopped before initiation of administration of the ammonia-oxidizing bacteria. In some embodiments, the acne treatment is stopped during administration of the ammonia-oxidizing bacteria. In some embodiments, the acne treatment is continued throughout the period of administration of the ammonia-oxidizing bacteria. In some embodiments, the acne treatment is initiated subsequent to stopping administration of the ammonia-oxidizing bacteria.

[0032] In some embodiments, administration is performed 30, 60, 90, 120, 150, or 180 minutes before the subject washes or takes a shower.

[0033] In some embodiments, provided is a preparation comprising ammonia-oxidizing bacteria as described above or in any part of the present disclosure for treating a skin condition, such as acne, such as acne vulgaris.

[0034] The present disclosure contemplates any one or more combinations of any of the foregoing aspects and / or embodiments, as well as any one or more combinations with any of the embodiments described in the detailed description and examples. Examples of embodiments of the present invention include the following items. (Item 1) A method for treating a skin disorder of a subject, such as acne, such as acne vulgaris, comprising: administering, such as applying, such as topically administering, a preparation comprising ammonia-oxidizing bacteria, such as ammonia-oxidizing bacteria, to the surface of the subject The method comprising. (Item 2) The method according to item 1, wherein the administration, for example, the amount and frequency of application, is sufficient to reduce the amount or concentration of pathogenic bacteria, such as Propionibacterium acnes, on the surface of the subject. (Item 3) The method according to item 2, wherein the amount is a therapeutically effective dose of ammonia-oxidizing bacteria. (Item 4) The method according to item 1, wherein the skin disorder is acne, such as acne vulgaris. (Item 5) The method according to item 4, wherein the administration provides treatment of inflammatory lesions, such as papules, pustules, cysts / nodules. (Item 6) The method according to item 5, wherein the administration provides treatment of non-inflammatory lesions, such as open comedones, closed comedones. (Item 7) The method according to item 5, wherein the administration provides treatment or improvement of post-inflammatory hyperpigmentation / post-inflammatory erythema (PIH / PIE) lesions. (Item 8) The method according to item 5, wherein the administration provides treatment or improvement of one or more of erythema, edema, desquamation, stinging, burning, and pruritus. (Item 9) The method according to item 5, wherein the administration provides treatment or improvement of one or more of a greasy appearance, the appearance of pores, gloss, spots, skin color uniformity, visual smoothness, and tactile smoothness. (Item 10) The method according to item 5, wherein the administration provides treatment or improvement of sebumeter measurements. (Item 11) The method according to item 1, wherein the administration includes pre-treating the subject with ammonia-oxidizing bacteria. (Item 12) The method according to item 1, wherein the topical administration includes topical administration before the onset of the skin disorder. (Item 13) The method according to item 1, wherein the local administration comprises administering to the subject an effective dose of ammonia-oxidizing bacteria. (Item 14) The effective dose is about 0.1×10 9 , 0.2×10 9 , 0.3×10 9 , 0.4×10 9 , 0.5×10 9 , 0.6×10 9 , 0.7×10 9 , 0.8×10 9 , 0.9×10 9 , 1.0×10 9 , 1.2×10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.8×10 9 , 2.0×10 9 , 2.2×10 9 , 2.4×10 9 , 2.6×10 9 , 2.8×10 9 , 3.0×10 9 , 3.2×10 9 , 3.4×10 9 , 3.6×10 9 , 3.8×10 9 , 4.0×10 9 , 4.2×10 9 , 4.4×10 9 , 4.6×10 9 , 4.8×10 9 , 5.0×10 9 , 5.5×10 9 , 6.0×10 9 , 6.5×10 9 , 7.0×10 9 , 7.5×10 9 , 8.0×10 9 , 8.5×10 9 , 9.0×10 9 , 9.5×10 9 , 10.0×10 9 , 12×10 9 , 14×10 9 , 16×10 9 , 18×10 9 , 20×109 、 25 × 10 9 、 30 × 10 9 、 40 × 10 9 、 50 × 10 9 The method according to item 13, which is CFU. (Item 15) The method according to item 1, wherein the skin disorder is at the target site of the subject and contains one or more undesirable bacteria, such as pathogenic bacteria. (Item 16) The method according to item 15, wherein the target site contains Propionibacterium acnes. (Item 17) The method according to item 1, further comprising determining whether the subject needs treatment for the skin disorder, for example, determining whether the subject needs treatment for acne, such as acne vulgaris. (Item 18) The method according to item 17, further comprising selecting the subject in need of treatment for the skin disorder. (Item 19) The method according to item 1, wherein the ammonia-oxidizing bacteria, for example, the preparation containing ammonia-oxidizing bacteria, can be in the form of an aerosol, spray or mist, that is, can be in the form of a mist. (Item 20) The method according to item 19, wherein the ammonia-oxidizing bacteria, for example, the preparation, is applied as an aerosol or mist in an aqueous medium, for example. (Item 21) The method according to item 20, wherein the ammonia-oxidizing bacteria, for example, the preparation of ammonia-oxidizing bacteria, may be an ammonia-oxidizing bacteria in a buffer solution, for example, an aqueous buffer solution. (Item 22) The method according to item 21, wherein the buffer solution, for example, the aqueous buffer solution, contains disodium phosphate and magnesium chloride in water, for example, 50 mM Na 2 HPO 4 and 2 mM MgCl 2 and the method according to item 21. (Item 23) The buffer solution, for example, an aqueous buffer solution, is disodium phosphate and magnesium chloride in water, for example, 50 mM Na 2 HPO 4 and 2 mM MgCl 2 and consisting essentially of, the method according to item 21. (Item 24) The buffer solution, for example, an aqueous buffer solution, is disodium phosphate and magnesium chloride in water, for example, 50 mM Na 2 HPO 4 and 2 mM MgCl 2 and consisting of, the method according to item 21. (Item 25) The method according to item 1, further comprising selecting the subject based on the subject's need to reduce the amount or concentration of pathogenic bacteria, such as Propionibacterium acnes, on the surface of the subject. (Item 26) The method according to item 1, wherein the ammonia-oxidizing bacteria are self-administered. (Item 27) The method according to item 1, wherein the ammonia-oxidizing bacteria are applied to any one or more of the face, neck, and scalp of the subject. (Item 28) The method according to item 1, wherein the preparation contains at least one of ammonia, ammonium salts, and urea. (Item 29) The method according to item 1, wherein the preparation contains a controlled-release material, such as a delayed-release material. (Item 30) The method according to item 1, wherein the preparation of ammonia-oxidizing bacteria further contains an additive, such as one of a pharmaceutically acceptable additive or a cosmetically acceptable additive. (Item 31) The method according to item 30, wherein one of the additives, such as one of the pharmaceutically acceptable additives and the cosmetically acceptable additives, is a surfactant. (Item 32) The method according to item 1, wherein the preparation substantially does not contain other organisms. (Item 33) The method according to item 1, wherein the preparation is disposed in a powder, a cosmetic, a cream, a stick, an aerosol, for example, a mist, a plaster, a wipe, or a bandage. (Item 34) The method according to item 1, wherein the preparation is provided as a powder, a cosmetic, a cream, a stick, an aerosol, for example, a mist, a plaster, a wipe, or a bandage. (Item 35) The method according to item 1, wherein the preparation contains a humectant, a deodorant, a fragrance, a coloring agent, an insect repellent, a cleansing agent, or a UV blocker. (Item 36) The method according to item 30, wherein the additive, for example, the pharmaceutically acceptable additive or the cosmetically acceptable additive, contains an anti-adhesive, a binder, a coating agent, a disintegrant, a filler, a fragrance, a coloring agent, a lubricant, a flow promoter, an adsorbent, a preservative, or a sweetening agent. (Item 37) The preparation containing ammonia-oxidizing bacteria contains about 10 8 to about 10 14 CFU / L of ammonia-oxidizing bacteria. The method according to item 1. (Item 38) The preparation containing ammonia-oxidizing bacteria contains about 1×10 9 CFU / mL to about 10×10 9 CFU / mL of ammonia-oxidizing bacteria. The method according to item 1. (Item 39) The preparation containing ammonia-oxidizing bacteria contains about 50 milligrams (mg) to about 1000 mg of ammonia-oxidizing bacteria. The method according to item 1. (Item 40) The method according to item 30, wherein the mass ratio of ammonia-oxidizing bacteria to the additive, for example, the pharmaceutically acceptable additive or the cosmetically acceptable additive, ranges from about 0.1 gram to about 1 gram per liter. (Item 41) The method according to item 1, wherein the ammonia-oxidizing bacteria are selected from the group consisting of Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof. (Item 42) The method according to item 41, wherein the ammonia-oxidizing bacteria are Nitrosomonas eutropha (N. eutropha). (Item 43) The method according to item 42, wherein the ammonia-oxidizing bacteria are N. eutropha D23 having ATCC accession number PTA-121157. (Item 44) The method according to item 1, wherein the preparation contains organisms selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacter, and combinations thereof. (Item 45) The method according to item 1 for delivering a cosmetic product. (Item 46) The method according to item 1 for delivering a therapeutic product. (Item 47) The method according to item 1, wherein the preparation is provided in a container, and the preparation and the container have a weight of less than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 grams. (Item 48) The method according to item 1, wherein the preparation is applied 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. (Item 49) The method according to item 48, wherein the preparation is applied once per day. (Item 50) The method according to item 49, wherein the preparation is applied twice per day. (Item 51) The method according to item 1, wherein the preparation is applied 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. (Item 52) The method according to item 51, wherein the preparation is applied for about 7 days. (Item 53) The method according to item 51, wherein the preparation is applied for about 14 days. (Item 54) The method according to item 51, wherein the preparation is applied for about 21 days. (Item 55) The method according to item 51, wherein the preparation is applied for about 28 days. (Item 56) The method according to item 1, further comprising obtaining a sample from the surface of the skin. (Item 57) The method according to item 56, further comprising isolating the DNA of bacteria in the sample. (Item 58) The method according to item 57, further comprising sequencing the DNA of bacteria in the sample. (Item 59) The method according to item 58, wherein the bacteria is Propionibacterium acnes. (Item 60) The method according to item 1, wherein administering the ammonia-oxidizing bacteria provides a reduction in Propionibacterium acnes. (Item 61) The method according to item 60, wherein Propionibacterium acnes decreases, for example, the concentration of Propionibacterium acnes decreases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days later. (Item 62) The method according to item 1, further comprising administering one or more of shampoo, conditioner, and a cleaning agent, for example, a facial cleanser, for example, self-administering. (Item 63) The method according to item 62, wherein any one or more of shampoo, conditioner, and facial cleanser are applied once a day. (Item 64) The method according to item 62, wherein the shampoo used was Lot 293178. (Item 65) The method according to item 62, wherein the cleaning agent used was Lot 293162. (Item 66) The method according to item 62, wherein at least one of shampoo, conditioner, and soap was used following discontinuation of the administration of the ammonia-oxidizing bacteria. (Item 67) The method according to item 62, wherein at least one of the shampoo, conditioner, and soap was used following discontinuation of the preparation of the ammonia-oxidizing bacteria. (Item 68) The method according to item 1, wherein the subject was evaluated before starting the treatment, for example, before administering the ammonia-oxidizing bacteria. (Item 69) The method according to item 1, wherein the subject was evaluated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 days after starting the treatment, for example, after administering the ammonia-oxidizing bacteria; or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 weeks; or 2, 3, 4, or 5 years later. (Item 70) Administering the preparation provides one or more of the following: reducing the inflammation of the lesion, reducing the frequency of the lesion, and reducing the presence of pathogenic bacteria, such as Propionibacterium acnes, the method according to item 1. (Item 71) Administering the preparation provides reduction of the inflammation of the lesion, the method according to item 70. (Item 72) Administering the preparation provides reduction of the inflammation of the lesion in adults, the method according to item 71. (Item 73) Administering the preparation provides reduction of the frequency of the lesion, the method according to item 70. (Item 74) Administering the preparation provides reduction of the presence of pathogenic bacteria, such as Propionibacterium acnes, the method according to item 70. (Item 75) Administering the preparation provides improvement of the emotional assessment of the subject regarding the disease of the subject measured by the Skindex16 quality of life survey, the method according to item 1. (Item 76) Administering the preparation provides one or more of the following improvements: pain of the skin condition in the subject, persistence / recurrence of the skin condition in the subject, and appearance of the skin condition in the subject, the method according to item 1. (Item 77) Administering the preparation provides improvement (reduction) of one or more of the following according to clinical assessment: visual and tactile smoothness, and rating scores for pigmentation, the method according to item 1. (Item 78) The subject is female, the method according to item 1. (Item 79) The subject is male, the method according to item 1. (Item 80) The method according to item 1, wherein the subject is characterized as one of the following ethnic groups / races: Asian, Black or African American, Hispanic or Latino, White, or Multi-ethnic. (Item 81) The method according to item 1, wherein the subject is characterized as having at least one of the following skin types: normal skin, oily skin, and combination skin. (Item 82) The method according to item 1, wherein the subject is characterized as having one of the following Fitzpatrick skin types: I, II, III, IV, V. (Item 83) The method according to item 1, wherein the type of acne is adolescent acne. (Item 84) The method according to item 1, wherein the type of acne is adult acne. (Item 85) The method according to item 1, wherein the age of the subject is between about 12 - 15 years old, 16 - 18 years old, 19 - 28 years old, or over 28 years old. (Item 86) The following: Topical retinoids, azelaic acid, salicylic acid, topical antimicrobials, oral antibiotics, benzoyl peroxide, oral antiandrogens, oral isotretinoin, and combinations thereof The method according to item 1, wherein an acne treatment selected from the group consisting of is administered to the subject. (Item 87) The method according to item 86, wherein the acne treatment is administered for a period before starting the administration of the ammonia-oxidizing bacteria. (Item 88) The method according to item 86, wherein the acne treatment is continued throughout the administration period of the ammonia-oxidizing bacteria. (Item 89) The method according to item 86, wherein the acne treatment is administered for a period before starting the administration of the ammonia-oxidizing bacteria and stopped before the start of the administration of the ammonia-oxidizing bacteria. (Item 90) The method according to item 86, wherein the acne treatment is stopped during the administration of the ammonia-oxidizing bacteria. (Item 91) The method according to item 86, wherein the acne treatment is continued throughout the administration of the ammonia-oxidizing bacteria. (Item 92) The method according to item 86, wherein the acne treatment is started following the cessation of the administration of the ammonia-oxidizing bacteria. (Item 93) The method according to item 1, wherein the administration is carried out 30, 60, 90, 120, 150, or 180 minutes before the subject performs washing or takes a shower. (Item 94) A preparation comprising ammonia-oxidizing bacteria according to any one of items 1 to 93 for treating a skin condition, such as acne, such as acne vulgaris. (Item 95) A preparation comprising ammonia-oxidizing bacteria for treating a skin condition, such as acne, such as acne vulgaris. (Item 96) The preparation according to item 95, wherein the ammonia-oxidizing bacteria are in a buffer solution, such as an aqueous buffer solution. (Item 97) The preparation according to item 96, wherein the buffer solution, such as an aqueous buffer solution, contains disodium phosphate and magnesium chloride in water, such as 50 mM Na 2 HPO 4 and 2 mM MgCl 2 . (Item 98) The preparation according to item 96, wherein the buffer solution, such as an aqueous buffer solution, consists essentially of disodium phosphate and magnesium chloride in water, such as 50 mM Na 2 HPO 4 and 2 mM MgCl 2 . (Item 99) The preparation according to item 96, wherein the buffer solution, such as an aqueous buffer solution, contains disodium phosphate and magnesium chloride in water, such as 50 mM Na 2 HPO 4 and 2 mM MgCl 2The preparation according to item 96, comprising (Item 100) The preparation according to item 95, comprising at least one of ammonia, ammonium salts, and urea. (Item 101) The preparation according to item 95, comprising a controlled-release material, such as a sustained-release material. (Item 102) The preparation according to item 95, wherein the preparation of ammonia-oxidizing bacteria further comprises an additive, such as one of pharmaceutically acceptable additives or cosmetically acceptable additives. (Item 103) The preparation according to item 102, wherein the additive, such as one of the pharmaceutically acceptable additives and the cosmetically acceptable additives, is a surfactant. (Item 104) The preparation according to item 95, substantially free of other organisms. (Item 105) The preparation according to item 95, disposed in a powder, cosmetic, cream, stick, aerosol, such as a mist, plaster, wipe, or bandage. (Item 106) The preparation according to item 95, provided as a powder, cosmetic, cream, stick, aerosol, such as a mist, plaster, wipe, or bandage. (Item 107) The preparation according to item 95, comprising a humectant, deodorant, fragrance, coloring agent, insect repellent, cleansing agent, or UV blocker. (Item 108) The preparation according to item 102, wherein the additive, such as the pharmaceutically acceptable additive or the cosmetically acceptable additive, comprises an anti-adhesive, binder, coating agent, disintegrant, filler, fragrance, coloring agent, lubricant, flow promoter, adsorbent, preservative, or sweetener. (Item 109) The preparation containing ammonia-oxidizing bacteria is about 10 8 to about 10 14 CFU / L of ammonia-oxidizing bacteria. The preparation according to item 95. (Item 110) The preparation containing ammonia-oxidizing bacteria contains from about 1×10 9 CFU / mL to about 10×10 9 CFU / mL of ammonia-oxidizing bacteria, the preparation according to item 95. (Item 111) The preparation containing ammonia-oxidizing bacteria contains from about 50 milligrams (mg) to about 1000 mg of ammonia-oxidizing bacteria, the preparation according to item 95. (Item 112) The mass ratio of ammonia-oxidizing bacteria to the additive, for example, the pharmaceutically acceptable additive or the cosmetically acceptable additive, is in the range of about 0.1 gram to about 1 gram per liter, the preparation according to item 102. (Item 113) The ammonia-oxidizing bacteria are selected from the group consisting of Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof, the preparation according to item 95. (Item 114) The ammonia-oxidizing bacteria are Nitrosomonas eutropha (N. eutropha), the preparation according to item 113. (Item 115) The ammonia-oxidizing bacteria are N. eutropha D23 having the ATCC accession number PTA-121157, the preparation according to item 114. (Item 116) The preparation according to item 115, comprising an organism selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacter, and combinations thereof.

Brief Description of the Drawings

[0035]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0036] Detailed Description The present disclosure provides a method for treating a skin condition, such as acne, such as acne vulgaris. The method includes administering to a subject an ammonia-oxidizing bacterium, such as a preparation containing an ammonia-oxidizing bacterium.

[0037] Preparations, compositions, and formulations are contemplated for use in the treatment of skin conditions, such as acne, e.g., acne vulgaris, comprising, consisting essentially of, or consisting of ammonia-oxidizing bacteria, e.g., including non-natural products, natural products, and fortified natural products. Compositions for use in the treatment of skin conditions, such as acne vulgaris, e.g., acne, in a subject, comprising ammonia-oxidizing bacteria, e.g., preparations comprising ammonia-oxidizing bacteria, are contemplated.

[0038] Ammonia-oxidizing bacteria (AOB) of the genus Nitrosomonas are Gram-negative obligate chemolithoautotrophic bacteria that have the unique ability to produce nitrite and nitric oxide from ammonia only as an energy source. They are widely present in both soil and aquatic environments and are an essential component of the environmental nitrification process. Due to the role of nitrite and nitric oxide on human skin as important components of several physiological functions such as vasodilation, skin inflammation, and wound healing, these bacteria can have beneficial properties for both healthy and immunopathological skin conditions. These bacteria are safe for use in humans as they have slow growth, cannot grow on organic carbon sources, can be sensitive to soaps and antibiotics, and are not associated with any disease or infection in animals or humans.

[0039] Ammonia-oxidizing bacteria are ubiquitous Gram-negative obligate chemosynthetic inorganic autotrophic bacteria that have the unique ability to generate energy from the conversion of ammonia to nitrite only.

[0040] In some embodiments, the ammonia-oxidizing bacteria catalyze the following reaction.

[0041] At neutral pH, ammonia generated from ammonium near 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: NH 3+2H + +2e− + O 2 →NH 2 OH + H 2 O (A) NH 2 OH + H 2 O → NO 2 - +4e− + 5H + (B)

[0042] In some cases, reaction B is reported to show nitrous acid (HNO 2 ) formation at low pH as follows: NH 2 OH + H 2 O → HNO 2 +4e− + 4H +

[0043] In certain embodiments, NH 4 + and NH 3 can be used interchangeably throughout this disclosure.

[0044] This disclosure provides · reducing the inflammation of lesions (e.g., in adults), · reducing the frequency of lesions, and · reducing the presence of pathogenic bacteria in an ammonia-oxidizing bacterium, e.g., a preparation containing an ammonia-oxidizing bacterium, which is capable of doing so.

[0045] An ammonia-oxidizing bacterium, e.g., a preparation containing an ammonia-oxidizing bacterium, can also · reduce the duration of lesions, · prevent or reduce abnormal pigmentation and scarring, · shorten the healing period of damaged skin, as well as · reduce the production of excessive sebum (e.g., the fatty secretion of sebaceous glands) which is possible.

[0046] The present disclosure provides a preparation containing ammonia-oxidizing bacteria for use in the treatment of skin conditions, such as acne, such as acne vulgaris, which may have one or more of the following: being self-administered (e.g., once or twice daily), being odorless, being colorless, being invisible to the eye, not being accompanied by an increased sensitivity to sunlight, being safe, being non-toxic, having good tolerance in the skin (e.g., adjacent mucous membranes and eyes), being suitable for children under 12 years of age, and being suitable for both treatment and maintenance therapy without the risk of promoting antibiotic resistance.

[0047] Ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha referred to as "D23" and also known as "B244" or "AOB D23-100", may have some of the above characteristics. 1. Definitions

[0048] Ammonia-oxidizing bacteria refer to bacteria that can oxidize ammonia or ammonium to nitrite at a rate, such as a substantial rate, such as a predetermined rate. The rate, such as a predetermined rate, is at least 50, 75, 125, or 150 micromoles of NO per minute 2 - , such as about 100 - 150, 75 - 175, 75 - 125, 100 - 125, 125 - 150, or 125 - 175 micromoles / minute, such as about 125 micromoles of NO per minute 2 - at a rate of ammonium ions (NH 4 + )(e.g., of about 200 mM) to nitrite (NO 2 - ). In multiple embodiments, the rate, such as a predetermined rate, refers to, for example, at least 50, 75, 125, or 150 nanomoles of NO per minute per ml for a continuous culture having, for example, an OD of about 0.5 2 -, for example, about 100 - 150, 75 - 175, 75 - 125, 100 - 125, 125 - 150, or 125 - 175 nanomoles per minute per ml, for example, about 125 nanomoles of NO per minute per ml 2 - at a rate of ammonium ions (NH 4 + )(e.g., about 200 mM) to nitrite (NO 2 - ).

[0049] Examples of ammonia - oxidizing bacteria include Nitrosomonas eutropha strains, such as D23 and C91, and other bacteria of the genus Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, and Nitrosovibrio. The D23 Nitrosomonas eutropha strain is designated as AOB D23 - 100 and was deposited on April 8, 2014, with the American Tissue Culture Collection (ATCC) (10801 University Blvd., Manassas, VA, USA) under accession number PTA - 121157. The nucleic acid sequence of accession number PTA - 121157, such as the genomic sequence, is hereby incorporated by reference in its entirety. In certain embodiments, N. eutropha is the strain described in PCT application No. PCT / US2015 / 025909 filed on April 15, 2015, which is hereby incorporated by reference in its entirety. "AOB D23 - 100" may also be referred to as D23 or B244 throughout the present disclosure.

[0050] Any nucleic acid sequences and amino acid sequences disclosed in PCT application No. PCT / US2015 / 025909 filed on April 15, 2015, are hereby incorporated by reference in their entirety. Any ammonia - oxidizing bacteria disclosed in PCT application No. PCT / US2015 / 025909 filed on April 15, 2015, are hereby incorporated by reference in their entirety.

[0051] Optimized Nitrosomonas eutropha (N. eutropha), when the term is used herein, refers to optimized growth rate; optimized NH 4 + oxidation rate; or optimized NH 4 + -tolerant N. eutropha. In one embodiment, this is at least one nucleotide, for example, in genes selected from ammonia monooxygenase, hydroxylamine oxidoreductase, cytochrome c554, and cytochrome c M 552, which differs from naturally occurring N. eutropha. This difference can occur, for example, by spontaneously occurring mutations, induced mutations, or selected directed genetic engineering of N. eutropha. In one embodiment, this differs from naturally occurring N. eutropha in that it has a set of alleles that do not occur together in nature. These differences can provide one or more of the treatment or prevention of skin disorders, the treatment or prevention of diseases or conditions associated with low nitrite levels, the treatment or prevention of body odor, the treatment of supplying nitric oxide to a subject, and the treatment of inhibiting the growth of microorganisms.

[0052] As used herein, "axenic" refers to a composition containing organisms that are substantially free of other organisms. For example, an axenic culture of ammonia-oxidizing bacteria is a culture that is substantially free of organisms other than ammonia-oxidizing bacteria. For example, an axenic culture of N. eutropha is a culture that is substantially free of organisms other than N. eutropha. In some embodiments, "substantially free" indicates that the organisms are undetectable by methods used to detect other organisms, such as plating the culture and examining colony morphology, or by PCR for conserved genes such as 16S RNA. Axenic compositions may contain non-biological elements, such as nutrients or additives. Any embodiment, preparation, composition, or formulation of ammonia-oxidizing bacteria discussed herein may optionally comprise, consist essentially of, or consist of axenic ammonia-oxidizing bacteria.

[0053] Throughout the present disclosure, "formulation" may refer to a composition or preparation.

[0054] As used herein, "autotroph", e.g., autotrophic bacteria, refers to any organism capable of self-nourishment by using inorganic materials as a source of nutrients and photosynthesis or chemosynthesis as a source of energy. Autotrophic bacteria can synthesize organic compounds from ATP derived from the oxidation of ammonia to nitrite, the oxidation of hydrogen sulfide, and the oxidation of Fe 2+ of Fe 3+ to Fe. The autotrophic bacteria of the present disclosure are not capable of causing infection.

[0055] 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 disorder, e.g., after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated, two or more treatments are delivered. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second treatment begins, such that there is an overlap. This may be referred to herein as "simultaneous" or "co-administered" or "parallel delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. This may be referred to herein as "sequential" or "serial delivery". In multiple embodiments of either case, the treatments are more effective for combined administration. For example, the second treatment is more effective to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, e.g., an equivalent effect may be seen with a lesser amount of the second treatment or the second treatment reduces the symptoms or a similar situation is seen with the first treatment. In some embodiments, the delivery is such that the reduction of symptoms, or other parameters related to the disorder, exceeds what is 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 more than additive (i.e., synergistic). The delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered. In some embodiments, one or more treatments may be delivered before the patient is diagnosed with the disorder.

[0056] Complete N. europaea medium refers to the N. europaea growth medium described in Ensign et al., "In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper.", J Bacteriol., April 1993; 175(7):1971-80.

[0057] "Culturing" refers to the process of placing a quantity of the desired bacteria under conditions that promote its growth, i.e., promote cell division. The conditions can involve a specified culture medium, a set temperature range, and / or a stirring rate. The bacteria can be cultured in liquid culture or on a plate, e.g., an agar plate.

[0058] As used herein, the term "isolated" refers to a material that has been removed from its original or native environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting materials in the natural system by human intervention 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 is still isolated in that it is not part of the environment in which it is found in nature.

[0059] The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence", and "polynucleotide" are used interchangeably. They refer to polymers of nucleotides of any length, e.g., deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be either single-stranded or double-stranded, and if single-stranded, may be either the coding strand or the non-coding (antisense) strand. The polynucleotide may include modified nucleotides, e.g., methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, e.g., by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin that does not occur in nature or is non-naturally ligated to another polynucleotide.

[0060] 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 the batch conditions described in Example 2 herein; a doubling time of less than about 16, 18, 20, 22, 24, or 26 hours when grown under the chemostat conditions described in Example 2 herein; or growth from an OD600 of about 0.15 to at least about 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 over about 1 or 2 days. In one embodiment, the optimized growth rate is a rate having a doubling time that is at least 10, 20, 30, 40, or 50% shorter than the doubling time of naturally occurring N. eutropha.

[0061] As used herein, "optimized NH 4 + oxidation rate" refers to a rate of conversion of at least about 50, 75, 125, or 150 micromoles per minute of NH 3 or NH 4 + to NO 2 - For example, the rate may be a conversion of at least about 50, 75, 125, or 150 micromoles per minute of NH 4 + (e.g., of about 200 mM) to NO 2 - In one embodiment, the optimized NH 4 + oxidation rate is a rate at which NH 3 or NH 4 + is converted to NO 2 - at least 10, 20, 30, 40, or 50% more rapidly than is seen in naturally occurring N. eutropha.

[0062] The percent amino acid sequence identity (%) for an amino acid sequence (e.g., a protein expressed by N. eutropha D23) in this specification is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference sequence that can be a naturally occurring N. eutropha sequence or N. eutropha D23 sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in various ways within the means of those skilled in the art. By way of example, it can be achieved using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. By way of example, the WU-BLAST-2 software can be used to determine the amino acid sequence identity (Altschul et al., Methods in Enzymology, Vol. 266, pp. 460 - 480 (1996); http: / / blast.wustl / edu / blast / README.html). WU-BLAST-2 uses several search parameters, most of which are set to their initial values. The adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = I1. The HSP score (S) and HSP S2 parameters are dynamic values established by the program itself according to the composition of a particular sequence, but the minimum values can be adjusted as appropriate. Most are set to their initial values. The adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = I1. The HSP score (S) and HSP S2 parameters are dynamic values established by the program itself according to the composition of a particular sequence, but the minimum values can be adjusted as appropriate.

[0063] An amino acid substitution can be the replacement of one amino acid with another having similar structural and / or chemical properties, for example, the replacement of leucine with serine, which can be the result of a conservative amino acid substitution. Typical but non-limiting conservative substitutions are the mutual replacement among the aliphatic amino acids Ala, Val, Leu, and Ile; the exchange of Ser and Thr containing hydroxy residues, the exchange of the acidic residues Asp and Glu, the exchange between the amide-containing residues Asn and Gln, the exchange of the basic residues Lys and Arg, the exchange of the aromatic residues Phe and Tyr, and the exchange of the small amino acids Ala, Ser, Thr, Met, and Gly. Additional conservative substitutions include the replacement of an amino acid with another having a similar spatial or steric configuration, for example, the exchange of Asn with Asp, or Gln with Glu. An amino acid substitution can also be the replacement of one amino acid with another having dissimilar structural and / or chemical properties, i.e., it can be the result of a non-conservative amino acid substitution. Insertions or deletions can optionally be in the range of 1 to 5 amino acids. Permissible mutations can be determined by systematically making amino acid insertions, deletions, or substitutions in the sequence and testing the resulting variants for activity in in vivo or in vitro assays for, for example, the metabolism of urea or ammonia.

[0064] The percent sequence identity (%) for a nucleic acid sequence (e.g., the N. eutropha D23 genome and parts thereof) in this specification is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a reference sequence that can be a naturally occurring N. eutropha sequence or an N. eutropha D23 sequence, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity. Alignments for the purpose of determining nucleotide sequence identity percentages can be achieved in a variety of ways within the means of one of ordinary skill in the art, by way of example, using publicly available computer software, such as BLAST. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to achieve the maximum alignment over the full length of the sequences being compared.

[0065] The terms “polypeptide,” “peptide,” and “protein” (in the case of single chains) are used interchangeably herein to refer to amino acid polymers. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. These terms also include modified amino acid polymers; for example, those having disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. A polypeptide can be isolated from a natural source, produced from a eukaryotic or prokaryotic host by recombinant techniques, or be a product of synthetic procedures.

[0066] As used herein, “optimized NH 4 + tolerance” refers to the ability to grow for at least about 24 or 48 hours under conditions of greater than 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mM NH 3 or NH 4 + In one embodiment, optimized NH 4 +Tolerance means the ability to grow at least 10, 20, 30, 40, or 50% faster, or at least 10, 20, 30, 40, or 50% longer than the ability of naturally occurring N. eutropha to grow in the presence of the selected concentration of NH 3 or NH 4 + .

[0067] As used herein with respect to comparisons between nucleic acid or protein sequences, "similar" means having homology. Similar genes or proteins may, for example, include substitutions (such as conservative or non-conservative substitutions, etc.), insertions (such as at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 amino acids, and for example, up to 2, 3, 4, 5, 10, 15, 20, 25, 30, or 50 amino acids, or any positive combination thereof, or the number of nucleotides necessary to encode said amino acids), or deletions (such as at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 amino acids, and for example, up to 2, 3, 4, 5, 10, 15, 20, 25, 30, or 50 amino acids, or any positive combination thereof, or the number of nucleotides necessary to encode said amino acids), or any combination thereof. Substitutions, insertions, and deletions can each be located at the N-terminus, C-terminus, or central region of the protein or gene. In multiple embodiments, conservative substitutions are substitutions that do not change the charge and / or polarity and / or approximate size and / or shape at the position being substituted.

[0068] As used herein, "subject" may include an animal, mammal, human, non-human animal, livestock animal, or companion animal. 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. Although application in humans is clearly contemplated, veterinary applications in non-human animals, for example, are also envisioned herein. The term "non-human animal" as used in this disclosure includes all vertebrates, such as non-mammals (birds, such as chickens; amphibians; reptiles, etc.) as well as mammals, such as non-human primates, domesticated, and agriculturally useful animals, such as, among others, sheep, dogs, cats, cows, pigs, rats.

[0069] As used herein, "transgenic" means containing one or more exogenous DNA moieties. The exogenous DNA is derived from another organism, such as another bacterium, bacteriophage, animal, or plant.

[0070] As used herein, "treatment of a disease or condition" refers to reducing the severity or frequency of at least one symptom of that disease or condition as compared to an untreated patient. Treatment can also refer to stopping, slowing, or reversing the progression of a disease or condition as compared to an untreated patient. Treatment may include addressing the disease and / or the underlying cause of one or more symptoms.

[0071] As used herein, "therapeutically effective amount" refers to an amount sufficient to prevent the progression of, or cause regression of, a disease or condition, or to alleviate the symptoms of a disease or condition, or to achieve a desired result. A therapeutically effective dose can be measured, for example, as the number of bacteria or viable bacteria (e.g., in CFU units) or the mass of bacteria (e.g., in milligrams, grams, or kilograms), or the volume of bacteria (e.g., in mm 3 units).

[0072] As used herein, the term "viability" refers to the ability of autotrophic bacteria that oxidize ammonia, ammonium, or urea to nitrite at a predetermined rate, e.g., ammonia-oxidizing bacteria. In some embodiments, the rate is at least 50, 75, 125, or 150 micromoles of NO 2 - , e.g., about 100 - 150, 75 - 175, 75 - 125, 100 - 125, 125 - 150, or 125 - 175 micromoles per minute, e.g., about 125 micromoles of NO 2 - per minute, of the conversion of ammonium ions (NH 4 + )(e.g., of about 200 mM) to nitrite (NO 2 - ).

[0073] The "growth medium" or "AOB medium" referred to herein contains the components of Table 2 or Table 3 below:

Table 2

Table 3-1

Table 3-2

[0074] In some embodiments, the optimal state for the present disclosure is a state of growth, e.g., maximum growth, characterized by at least about 7.6 pH, ammonia, trace minerals, oxygen, and carbon dioxide. Another state may be characterized by a pH of about 7.4 or less and by the absence of carbon dioxide. Under low carbon dioxide conditions, ammonia-oxidizing bacteria, e.g., N. eutropha, continue to oxidize ammonia to nitrite and produce ATP, but in the absence of carbon dioxide, e.g., a sufficient amount of carbon dioxide for fixation and protein production, instead produce polyphosphate and use this as an energy storage medium. This may enable the ammonia-oxidizing bacteria to remain in a "stored state" for a period of time, e.g., a predetermined period, e.g., at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, or 5 years. In some embodiments, the ammonia-oxidizing bacteria may remain in a stored state for at least about 6 months to about 1 year.

[0075] As used herein, the term "growth state" refers to a state or environment that can have a pH of at least about 7.6, such as a medium, such as a culture medium, such as a growth medium, for autotrophic bacteria, such as ammonia-oxidizing bacteria. The level of at least one of ammonia, ammonium ions, and urea may be between about 1 micromolar concentration and 1000 millimolar concentration. The level of trace materials is between about 0.01 micromolar concentration of iron and 200 micromolar concentration of iron. The level of oxygen is an oxygen saturation between about 5% and 100% (e.g., of the medium). The level of carbon dioxide is a saturation between about 20 ppm and 10% (e.g., of the medium). In certain embodiments, the level of at least one of ammonia, ammonium ions, and urea may be between about 10 micromolar concentration and 100 millimolar concentration. The level of trace materials is between about 0.1 micromolar concentration of iron and 20 micromolar concentration of iron. The level of oxygen is an oxygen saturation between about 5% and 100%. The level of carbon dioxide is a saturation between about 200 ppm and 5% (e.g., of the medium).

[0076] As used herein, the term "polyphosphate-loading state" refers to a state or environment that can have a pH of about 7.4 or less, such as a medium, such as a culture medium, such as a growth medium, for autotrophic bacteria, such as ammonia-oxidizing bacteria. The level of at least one of ammonia, ammonium ions, and urea is between about 1 micromolar concentration and 2000 millimolar concentration. The level of trace materials is between 0.01 micromolar concentration of iron and 200 micromolar concentration of iron. The level of oxygen is an O 2 saturation between about 0% and 100% (e.g., of the medium). The level of carbon dioxide is between about 0 and 400 ppm / less than that, and the phosphate level is greater than about 1 micromolar concentration. In certain embodiments, the level of at least one of ammonia, ammonium ions, and urea is between about 10 micromolar concentration and 200 millimolar concentration. The level of trace materials is between 0.1 micromolar concentration of iron and 20 micromolar concentration of iron. The level of oxygen is an O2 It is saturation. The level of carbon dioxide is between / about 0 and 200 ppm or less, and the phosphate level is above / about 10 micromolar concentration.

[0077] The polyphosphate loading state can be induced for a period, for example, a predetermined period. The predetermined period may be a period that enables sufficient polyphosphate accumulation in ammonia-oxidizing bacteria. This predetermined period is a period suitable for providing a sufficient polyphosphate load so that the ammonia-oxidizing bacteria can be stored for a long period. The predetermined period may be at least partly based on a period of about 0.2 to 10 times, 0.3 to 5 times, 0.5 to 3 times, 0.5 to 1.5 times, or 0.5 to 1 times the doubling time of the ammonia-oxidizing bacteria. The predetermined period may be at least partly based on a period of about 1 doubling time of the ammonia-oxidizing bacteria. In some embodiments, the predetermined period is between about 8 hours and 12 hours. In some embodiments, the predetermined period is about 10 hours. In some embodiments, the predetermined period is about 24 hours.

[0078] The purpose of the polyphosphate loading state is to provide sufficient ammonia, ammonium ions, and / or urea, and O 2 to the AOB so that they can generate ATP, but they cannot use that ATP to fix CO 2 Instead, by not providing them with CO 2 and carbonate, it can cause them to produce polyphosphate that can be stored by the bacteria using that ATP.

[0079] As used herein, the term "storage state" refers to a state or environment having a pH of about 7.4 or less, such as in a medium, such as a culture medium, such as a growth medium, for autotrophic bacteria, such as ammonia-oxidizing bacteria (in some embodiments, the pH may be 7.6 or less). The level of at least one of ammonia, ammonium ions, and urea is between about 1 and 1000 micromolar concentration. The level of trace materials is between about 0.1 and 100 micromolar concentration. The level of oxygen is at a saturation of between about 0 and 100% (e.g., of the medium). The level of carbon dioxide is between about 0 and 800 ppm. In certain embodiments, the level of at least one of ammonia, ammonium ions, and urea is between about 10 and 100 micromolar concentration. The level of trace materials is between about 1 and 10 micromolar concentration. The level of oxygen is at a saturation of between about 0 and 100% (e.g., of the medium). The level of carbon dioxide is between about 0 and 400 ppm.

[0080] According to some embodiments of the present disclosure, AOB is produced by causing AOB biomass to be produced during a growth state, then exposing the AOB to a polyphosphate loading state, then removing the medium, and resuspending the AOB in a buffer, such as a storage buffer (i.e., the storage state).

[0081] The ammonia-oxidizing bacteria may remain in the "storage state" for a period of time, such as a predetermined period of time, such as at least 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, or 5 years. In some embodiments, the ammonia-oxidizing bacteria may remain in the storage state for at least about 6 months to about 1 year. Upon regeneration, the viability of the ammonia-oxidizing bacteria is at least about 50%, 60%, 70%, 80%, 90%, or 100% of the viability of the ammonia-oxidizing bacteria in the pre-storage, e.g., growth state. In some embodiments, the preparation of ammonia-oxidizing bacteria, when stored under selected conditions, NH 4 +It can be prepared such that the ability to oxidize is lost by 10%, 20%, 30%, 40%, 50%, 60%, or 70% or less.

[0082] The time required to regenerate ammonia-oxidizing bacteria from a storage state (or polyphosphate-loaded state) may be a predetermined period. For example, the predetermined period may be less than about 75 hours, or less than about 72 hours. The predetermined period may be at least partly based on a period of about 0.2 to 10 times, 0.3 to 5 times, 0.5 to 3 times, 0.5 to 1.5 times, or 0.5 to 1 times the doubling time of the ammonia-oxidizing bacteria. The predetermined period may be at least partly based on a period of about 1 doubling time of the ammonia-oxidizing bacteria. The predetermined period may be between about 8 hours and 12 hours. The predetermined period may be about 10 hours. The predetermined time may be less than about 75 hours, 72 hours, 70 hours, 68 hours, 65 hours, 60 hours, 55 hours, 50 hours, 45 hours, 40 hours, 35 hours, 30 hours, 25 hours, 20 hours, 15 hours, 10 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour. The predetermined period may be between about 5 minutes and 5 hours. The predetermined period may be about 5 - 10 minutes, 10 - 15 minutes, 15 - 20 minutes, 20 - 25 minutes, 25 - 30 minutes, 30 - 45 minutes, 45 - 60 minutes, 60 minutes - 1.5 hours, 1.5 hours - 2 hours, 2 hours - 2.5 hours, 2.5 hours - 3 hours, 3 hours - 3.5 hours, 3.5 hours - 4 hours, 4 hours - 4.5 hours, 4.5 hours - 5 hours. In some embodiments, the predetermined period may be about 2 hours. The predetermined period may be, for example, the time required to achieve the regeneration of ammonia-oxidizing bacteria, for example, the viability of the ammonia-oxidizing bacteria compared to the viability of the bacteria before storage (e.g., in a growing state), for example, to achieve at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% viability.

[0083] As used herein, "activation" is used with respect to autotrophic bacteria, such as ammonia-oxidizing bacteria. Activation refers to any action that can potentially place ammonia-oxidizing bacteria in a more active state, such as a growth state. Activation may be related to the stimulation of autotrophic bacteria, such as ammonia-oxidizing bacteria, that in some way assist the conversion of at least one of ammonia, ammonium ions, and urea to nitrite, nitric oxide, or a nitric oxide precursor. Activation may be related to, for example, assisting in the establishment of a bacterial colony such that autotrophic bacteria, such as ammonia-oxidizing bacteria, can compete with other existing bacteria. Activation may be related to providing an environment that can advantageously act on the persistence and / or growth of autotrophic bacteria, such as ammonia-oxidizing bacteria. Activation may be related to accelerating the availability of autotrophic bacteria, such as ammonia-oxidizing bacteria, to an environment or surface. "Activation" may provide that the ammonia-oxidizing bacteria are in an "activated" or "growing" state. "Activation" may occur by the use of an activator. The ammonia-oxidizing bacteria can come into contact with the activator and become ammonia-oxidizing bacteria in an "activated" or "growing" state. This may occur within or outside of a container, delivery device, or delivery system, for example, in a first chamber, a second chamber, a mixing chamber, a third or additional chamber, or a combination thereof. The activator may be at least one of ammonia, ammonium ions, or urea. The activator may be an ammonium salt, such as ammonium chloride or ammonium sulfate. The concentration of the activator, such as an ammonium salt, such as ammonium chloride or ammonium sulfate, may range from about 10 micromolar concentration to about 100 millimolar concentration. In certain embodiments, the concentration of the activator, such as an ammonium salt, such as ammonium chloride or ammonium sulfate, may range from about 0.5 mM to about 50 mM. The activator may be in a solution, suspension, powder, such as a crystalline form, medium, buffer, or may be disposed in or provided as a suitable carrier for maintaining the activator.The ammonia-oxidizing bacteria may be in any suitable form, such as an aqueous suspension, gel, or powder form, to maintain the AOB in a desired state, such as a storage state. At least one of ammonia, ammonium ions, or urea may be in a medium or buffer that promotes the growth of the ammonia-oxidizing bacteria, such as an AOB medium or growth medium. Slowly-releasing, or controlled-release, urea may be used as an activator.

[0084] As used herein, "operation" means that some action is taking place, such as a process being initiated or some thing being activated. In some embodiments, operation may mean breaking a barrier of a container or the beginning of the movement of one or more contents of the container, such as the delivery of one or more contents of the container outside of the container, such as to a surface or environment.

[0085] As used herein, "barrier" may mean any structure or configuration that can serve to prevent passage or maintain separation between a first chamber and a second chamber of a container. The barrier may be in the form of a valve, such as a check valve, a filtering material, a film, a wax, a lipid, a polymer, or a controlled-release material, such as a delayed-release material. The barrier may be a material that allows passage of contents from the first chamber to the second chamber, from the second chamber to the first chamber, or both, during operation of the container. The barrier may be broken during operation, for example, by piercing, puncturing, stabbing, perforating, penetrating, tearing, opening, or ripping through the barrier. The barrier may be a valve, such as a check valve, a flexible or non-flexible material that may not decompose upon contact with one or more contents of the container, or a flexible or non-flexible material that may decompose upon contact with one or more contents of the container, in the form of a filtering material. The barrier may be made of any material suitable for its purpose, such as a material that can serve to prevent passage or maintain separation, such as a polymeric material or a metallic material.

[0086] "Microbiome" refers to a population, e.g., one or more microorganisms, that live on the surface of a subject, e.g., in the gut, mouth, skin, and / or other locations in the subject. The population can have one or more beneficial functions and / or benefits with respect to supporting the life of the subject.

[0087] "Harmless to the biome" refers to any thing, e.g., a product, e.g., a cosmetic product, e.g., a finished cosmetic product, that can allow for minimal disruption of the subject's microbiome. For example, being harmless to the biome refers to a product that can be applied to a subject, where the microbiome at the time of application is maintained, minimally disrupted, and / or can return to that microbiome after a period of time after application of the product. In multiple embodiments, being harmless to the biome can refer to being harmless to ammonia-oxidizing bacteria in that the product can allow for minimal disruption of the subject's ammonia-oxidizing bacteria.

[0088] In multiple embodiments, "harmless to the biome" may be referred to as "compatible with the biome".

[0089] "Natural product" refers to a product that can be at least partially of natural origin or can include such. This can be any thing produced by a living organism or can include any thing, and can include the organism itself. Natural products can include whole organisms, and parts of organisms (e.g., leaves of a plant), extracts from organisms, organic compounds from organisms, purified organic compounds from organisms. Natural products can include primary metabolites (amino acids, carbohydrates, and nucleic acids) as well as secondary metabolites (organic compounds found in a limited range of species, e.g., polyketides, fatty acids, terpenoids, steroids, phenylpropanoids, alkaloids, special amino acids and peptides, special carbohydrates). Natural products can be or can include polymeric organic materials, e.g., cellulose, lignin, and proteins.

[0090] Natural products may be or include products for commercial purposes and can refer to cosmetics, dietary supplements, and foods produced from natural sources. Natural products can have pharmacological or biological activities that can be therapeutically beneficial, for example, in the treatment of diseases or conditions. Natural products can be included in traditional medicines, beauty 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 containing one or more other components, such as additives. Preparations or formulations referred to as natural products may include the natural products defined herein and one or more additional components or ingredients. Any of the compositions, preparations, or formulations discussed throughout this disclosure may be or include one or more natural products.

[0091] 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 a spa treatment.

[0092] In some embodiments, the natural product or enhanced natural product may be incorporated into at least one of a powder, cream, lotion, wrap, scrub, eye mask, facial mask, body mask, aerosol, such as a mist, spray, plaster, wipe, stick, bandage, or infusion.

[0093] In some embodiments, the natural product or enhanced natural product may be provided as or incorporated into 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; eye makeup preparations such as eye brow pencils, eye liners, eye shadows, eye lotions, eye makeup removers, mascaras; fragrance preparations such as colognes, eau de toilettes, perfumes, powders (dusting and talcum), sachets; hair preparations such as hair conditioners, hair sprays, hair straighteners, permanent wave agents, rinses, shampoos, tonics, hair dressings, hair styling aids, wave set agents; hair coloring preparations such as hair dyes and hair colors, hair bleaches, colored hair rinses, colored hair shampoos, colored hair lighteners, hair bleaches; makeup preparations such as face powders, foundations, leg and body paints, lipsticks, makeup bases, blushes, makeup setting agents; manicure preparations such as base coats and undercoats, cuticle softeners, nail creams and lotions, nail extenders, nail polishes and enamels, nail polish and enamel removers; oral hygiene products such as toothpastes, mouthwashes and breath fresheners; bath soaps and detergents, deodorants, bidets, feminine hygiene deodorants; shaving preparations such as aftershave lotions, beard softeners, talcum, pre-shave lotions, shaving creams, shaving soaps; skin care preparations such as cleansing, depilatory agents, face and neck, body and hand, foot powders and sprays, moisturizers, night preparations, paste masks, skin fresheners; and sunburn preparations such as gels, creams, and liquids, and indoor tanning preparations.

[0094] As used herein, "presence" or "level" can refer to the qualitative or quantitative amount of one or more of the components, such as ammonia-oxidizing bacteria, ammonia, ammonium ions, urea, nitrite, or nitric oxide. The presence or level may include a zero value or the absence of the component.

[0095] As used herein, the term "surfactant" includes compounds that can reduce the surface tension, 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 may include one or more of the following, alone or in combination with those listed, or other surfactants or surfactant-like compounds: cocoamidopropyl betaine (ColaTeric COAB), polyethylene sorbitol ester (e.g., Tween 80), ethoxylated lauryl alcohol (RhodaSurf 6 NAT), sodium lauryl sulfate / sodium lauryl glucoside / cocoamidopropyl betaine (Plantapon 611 L UP), sodium lauryl sulfate (e.g., RhodaPex ESB 70 NAT), alkyl polyglucoside (e.g., Plantaren 2000 N UP), sodium lauryl sulfate (Plantaren 200), Dr. Bronner's Castile Soap, Dr. Bronner's Baby Soap, lauramine oxide (ColaLux Lo), sodium dodecyl sulfate (SDS), polysulfonate 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 trade asafoetida oil, organic jojoba oil, citric acid, and tocopherol.

[0096] The surfactant may include sodium lauryl glucoside hydroxypropyl sulfonate (Suga® nate 160NC), lauramidopropyl betaine (Cola® Teric LMB); cocoamidopropyl hydroxysultaine (Cola® Teric CBS); disodium cocoamphodiacetate (Cola® Teric CDCX-LV); sodium lauryl glucoside hydroxypropyl phosphate (Suga® Fax D12).

[0097] The surfactant 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).

[0098] Other surfactants are contemplated by the present disclosure. 2. Ammonia-oxidizing bacteria (AOB), N. eutropha strain D23 and other ammonia-oxidizing bacteria

[0099] The autotrophic ammonia-oxidizing bacteria, which may be referred to herein as multiple AOBs or one AOB, are the obligate autotrophic bacteria described by Alan B. Hooper and A. Krummel et al. Alan B. Hooper, Biochemical Basis of Obligate Autotrophy in Nitrosomonas europaea, Journal of Bacteriology, February 1969, pp. 776 - 779. Antje Krummel et al., Effect of Organic Matter on Growth and Cell Yield page. Antje Krummel et al., Effect of Organic Matter on Growth and Cell Yield of Ammonia - Oxidizing Bacteria, Arch Microbiol (1982) 133:50 - 54. These bacteria obtain all their metabolic energy solely from the oxidation of ammonia to nitrite, but have nitric oxide (NO) as an intermediate product within their respiratory chain and obtain virtually all their carbon by fixing carbon dioxide. They cannot utilize carbon sources other than a few simple molecules.

[0100] Ammonia - oxidizing bacteria (AOB) are widely found in the environment and multiply by fixing carbon dioxide in the presence of ammonia, oxygen, and trace metals. AOB may grow slowly, and toxic levels of ammonia can kill fish and other organisms before the AOB can multiply, but reduce ammonia to non - toxic levels. The slow growth of AOB may also delay the health benefits of NO and nitrite produced by AOB when applied to the skin.

[0101] It is desirable to replenish an aquarium, skin, or process with sufficient live AOB that has grown and been stored for that purpose. Since AOB do not form spores, it is difficult to store them with high viability in a dry state, and storage in a wet state keeps the AOB metabolically active.

[0102] The decay of nitrification ability during storage of AOB for wastewater treatment has been studied, for example, as (Munz G, Lubello C, Oleszkiewicz JA. Modeling the decay of ammonium oxidizing bacteria. Water Res. January 2011;45(2):557 - 64, Oi:10.1016 / j.watres.2010.09.022).

[0103] The growth, long - term storage, and activity recovery of Nitrosomonas were studied by Cassidy et al. (US5 ,314,542), where they discuss growing Nitrosomonas, removing toxic waste, storing in sterile water with appropriate salinity for up to one year, and then restoring by adding buffer (CaCO 3 ) and 200 ppm ammonium, and disclose that the restoration takes 72 hours.

[0104] As absolute autotrophs, AOB use the energy and reducing equivalents generated by the oxidation of ammonia to nitrite to synthesize proteins by fixing CO 2 . Growth requires ammonia, oxygen, minerals, and carbon dioxide.

[0105] Nitrosomonas may exist in several metabolic states according to "Polyphosphate and Orthophosphate Content of Nitrosomonas europaea as a Function of Growth" by K.R. Terry and A.B. Hooper, Journal of Bacteriology, July 1970, pages 199 - 206, volume 103, number 1.

[0106] The AOB considered in this disclosure may contain mutations relative to wild - type AOB. These mutations may be introduced, for example, spontaneously, by random mutagenesis, or by targeted mutagenesis. As an example, the AOB may lack one or more genes or regulatory DNA sequences typically included in wild - type AOB. The AOB may also include point mutations, substitutions, insertions, deletions, and / or rearrangements relative to a sequenced strain or wild - type strain. The AOB may be a purified preparation of optimized AOB.

[0107] In certain embodiments, the AOB is transgenic. By way of example, it may contain one or more genes or regulatory DNA sequences lacking in wild-type ammonia-oxidizing bacteria. More specifically, the ammonia-oxidizing bacteria may contain, by way of example, a reporter gene, a selectable marker, a gene encoding an enzyme, or a promoter (including an inducible or repressible promoter). In some embodiments, the additional gene or regulatory DNA sequence is incorporated into the bacterial chromosome; in some embodiments, the additional gene or regulatory DNA sequence is located on a plasmid.

[0108] In some embodiments, the AOB differs by at least one nucleotide from a naturally occurring bacterium. By way of example, the AOB may differ from a naturally occurring bacterium in genes or proteins that are part of a related pathway, such as an ammonia metabolism pathway, a urea metabolism pathway, or a pathway for generating nitric oxide or a nitric oxide precursor. More specifically, the AOB may contain, for example, mutations that increase the activity of the pathway by increasing the level or activity of an element of the pathway.

[0109] The mutations described above can be introduced using any suitable technique. Numerous methods for introducing mutations at a given position are known. By way of example, site-directed mutagenesis, oligonucleotide-directed mutagenesis, or site-specific mutagenesis can be used. Non-limiting examples of specific mutagenesis protocols are, for example, Mutagenesis It is described on pages 13.1 to 13.105 (edited by Sambrook and Russell, Molecular Cloning A Laboratory Manual, 3 volumes, 3rd edition, 2001). In addition, non-limiting examples of well-characterized mutagenesis protocols available from commercial suppliers include 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.).

[0110] In certain embodiments of the present disclosure, the ammonia-oxidizing bacteria may be a pure culture. A preparation (formulation or composition) of ammonia-oxidizing bacteria may comprise, consist essentially of, or consist of a pure culture of ammonia-oxidizing bacteria.

[0111] The ammonia-oxidizing bacteria of the present disclosure may be derived from a genus selected from the group consisting of Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof.

[0112] The present disclosure provides, inter alia, a specific, e.g., optimized strain of ammonia-oxidizing bacteria, such as 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 using the bacteria and articles containing the bacteria.

[0113] In a plurality of embodiments, the ammonia-oxidizing bacteria, such as N. eutropha, do not occur naturally. By way of example, this may accumulate desirable mutations during a period of selection. In other embodiments, the desirable mutations may be introduced by an experimenter. In some embodiments, N. eutropha may be a purified preparation or an optimized N. eutropha.

[0114] In preferred embodiments, the N. eutropha strain is autotrophic and thus cannot cause infection. Preferred strains utilize urea and ammonia, such that hydrolysis of urea in sweat is not required prior to uptake and utilization by the bacteria. Also, in order to grow at low pH, the bacteria can absorb NH 4 + ions or urea. The selected strain should also be viable and withstand the conditions on the outer epidermis of a subject, e.g., a human.

[0115] Although the present disclosure refers specifically to N. eutropha strain D23, preparations, methods, compositions, treatments, wearable articles, and clothing items using one or more other strains of N. eutropha, one or more other species of Nitrosomonas, and one or more of one or more other ammonia-oxidizing bacteria may be used.

[0116] In certain embodiments, N. eutropha is the strain designated as AOB D23 - 100 (25 vials), deposited with the American Tissue Culture Collection (ATCC) under the accession number PTA - 121157 on April 8, 2014. The D23 Nitrosomonas eutropha strain refers to the strain designated as AOB D23 - 100 and deposited with the American Tissue Culture Collection (ATCC) (10801 University Blvd., Manassas, VA, USA) on April 8, 2014, having the accession number PTA - 121157. The nucleic acid sequence of accession number PTA - 121157, e.g., the genomic sequence, is hereby incorporated by reference in its entirety. In certain embodiments, N. eutropha is the strain described in PCT Application No. PCT / US2015 / 025909, filed on April 15, 2015, which is hereby incorporated by reference in its entirety.

[0117] In certain embodiments, the bacteria having the above-described sequence features have one or more of (1) an optimized growth rate as measured by doubling time, (2) an optimized growth rate as measured by OD600, (3) an optimized NH 4 + oxidation rate, (4) an optimized NH 4 + tolerance, and (4) an optimized NO 2 - tolerance. Specific sub-combinations of these properties are identified in the following paragraphs.

[0118] In some embodiments, the ammonia-oxidizing bacteria described herein, e.g., N. eutropha, have the following: (1) an optimized growth rate as measured by doubling time, (2) an optimized growth rate as measured by OD600, (3) an optimized NH 4 + oxidation rate, (4) an optimized NH 4 + tolerance, and (4) an optimized NO 2 -has one or more of the tolerances. As an example, the bacterium may have characteristics (1) and (2); (2) and (3); (3) and (4); or (4) and (5) from the list at the beginning 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) from the list at the beginning of this paragraph. As a further example, the bacterium may have characteristics (1), (2), (3), and (4); (1), (2), (3), and (5); (1), (2), (4), and (5); (1), (3), (4), and (5); or (2), (3), (4), and (5) from the list at the beginning of this paragraph. In some embodiments, the bacterium has characteristics (1), (2), (3), (4), and (5) from the list at the beginning of this paragraph.

[0119] The present disclosure also includes the following: (1) an optimized growth rate measured by doubling time, (2) an optimized growth rate measured by OD600, (3) an optimized NH 4 + oxidation rate, (4) an optimized NH 4 + tolerance, and (4) an optimized NO 2 -Provided is a pure culture composition of ammonia-oxidizing bacteria having one or more of the tolerances, e.g., N. eutropha. As an example, the bacterial composition may have characteristics (1) and (2); (2) and (3); (3) and (4); or (4) and (5) from the list at the beginning of this paragraph. As another example, the bacterial composition 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) from the list at the beginning of this paragraph. As a further example, the bacterial composition may have characteristics (1), (2), (3), and (4); (1), (2), (3), and (5); (1), (2), (4), and (5); (1), (3), (4), and (5); or (2), (3), (4), and (5) from the list at the beginning of this paragraph. In some embodiments, the bacterial composition has characteristics (1), (2), (3), (4), and (5) from the list at the beginning of this paragraph.

[0120] The N. eutropha strain D23, deposited with the ATCC Patent Depositary in the form of 25 vials on April 8, 2014, under accession number PTA-121157 and designated AOB D23-100, contains a circular genome having SEQ ID NO:1 or its complement. Accordingly, in some embodiments, the N. eutropha strain described herein contains a nucleic acid sequence similar to SEQ ID NO:1 of PCT Application No. PCT / US2015 / 025909, filed on April 15, 2015, or its complement, e.g., a genome.

[0121] In certain embodiments, the N. eutropha strain hybridizes under the low stringency, medium stringency, high stringency, or very high stringency, or other hybridization conditions described herein, to the nucleotide sequence of SEQ ID NO:1 of PCT Application No. PCT / US2015 / 025909, filed Apr. 15, 2015, or to the genome of the D23 strain deposited with the ATCC Patent Depository on Apr. 8, 2014, in the form of 25 vials and designated AOB D23-100 under accession number PTA-121157, or to the complement thereof, e.g., including the genome.

[0122] As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes the conditions of hybridization and washing. Guidelines for performing the hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), pages 6.3.1-6.3.6, which are incorporated by reference. Both aqueous and non-aqueous methods are described in that reference and either can be used. The specific hybridization conditions referred to herein are as follows: 1) Low stringency hybridization conditions are in about 6× sodium chloride / sodium citrate (SSC) at about 45° C. followed by washing at least twice in 0.2× SSC, 0.1% SDS at at least 50° C. (for low stringency conditions, the washing temperature can be increased up to 55° C.); 2) Medium stringency hybridization conditions are in about 6× SSC at about 45° C. followed by washing one or more times in 0.2× SSC, 0.1% SDS at 60° C.; 3) High stringency hybridization conditions are in about 6× SSC at about 45° C. followed by washing one or more times in 0.2× SSC, 0.1% SDS at 65° C.; 4) Very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65° C. followed by washing one or more times in 0.2× SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the appropriate conditions and are to be used unless otherwise specified.

[0123] The D23 strain does not appear to be a natural product, but rather has acquired certain mutations and characteristics during long-term culturing and selection in the laboratory. By way of example, D23 has the ability to grow for more than 24 hours under conditions of about 200 or greater than 250 mM NH 4 + .

[0124] In some embodiments, the N. eutropha disclosed herein differs from naturally occurring bacteria in the abundance of siderophores. By way of example, N. eutropha may have elevated or reduced siderophore levels compared to N. eutropha C91. Generally, siderophores are secreted iron chelate compounds that assist bacteria in capturing iron from their environment. Siderophores may be peptides or small organic molecules.

[0125] The AOB contemplated by the present disclosure, such as N. eutropha, may include mutations to wild-type N. eutropha and / or N. eutropha sequences disclosed herein. These mutations may occur, for example, spontaneously, be introduced by random mutagenesis, or be introduced by targeted mutagenesis. By way of example, ammonia-oxidizing bacteria, such as N. eutropha, may lack one or more genes or regulatory DNA sequences typically included in wild-type N. eutropha. Ammonia-oxidizing bacteria, such as N. eutropha, may also include point mutations, substitutions, insertions, deletions, and / or rearrangements relative to a sequenced or wild-type strain. Ammonia-oxidizing bacteria, such as N. eutropha, may be a purified preparation of an optimized ammonia-oxidizing bacteria, such as N. eutropha.

[0126] In certain embodiments, the ammonia-oxidizing bacteria, such as N. eutropha, are transgenic. By way of example, this may include one or more genes or regulatory DNA sequences that the wild-type N. eutropha D23 lacks. More specifically, the ammonia-oxidizing bacteria, such as N. eutropha, may, by way of example, include a reporter gene, a selectable marker, a gene encoding an enzyme, or a promoter (including an inducible or repressible promoter). In some embodiments, the additional gene or regulatory DNA sequence is incorporated into the bacterial chromosome, and in some embodiments, the additional gene or regulatory DNA sequence is on a plasmid, such as a plasmid associated with the plasmid found in N. eutropha N91.

[0127] In some preferred embodiments, the ammonia-oxidizing bacteria, such as N. eutropha, differ in that at least one nucleotide is different from the naturally occurring bacteria. By way of example, the ammonia-oxidizing bacteria, such as N. eutropha, may differ in that a gene or protein that is part of a related pathway, such as an ammonia metabolism pathway, a urea metabolism pathway, or a pathway for generating nitric oxide or a nitric oxide precursor, may be different from the naturally occurring bacteria. More specifically, the ammonia-oxidizing bacteria, such as N. eutropha, may include mutations that increase the activity of the pathway, such as by increasing the level or activity of an element of that pathway.

[0128] The above mutations can be introduced using any suitable technique. A number of methods for introducing mutations at a given position are known. By way of example, site-directed mutagenesis, oligonucleotide-directed mutagenesis, or site-specific mutagenesis can be used. Non-limiting examples of specific mutagenesis protocols are, for example, Mutagenesis, 1 It is described in pages 3.1 to 13.105 (edited by Sambrook and Russell, Molecular Cloning A Laboratory Manual, 3 volumes, 3rd supplementary edition, 2001). In addition, non-limiting examples of well-characterized mutagenesis protocols available from vendors include, but are not limited to, Altered Sites® II in vitro Mutagenesis System (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 Kit (Stratagene, La Jolla, Calif.); and Transformer™ Site-Directed Mutagenesis Kit (BD-Clontech, Mountain View, Calif.).

[0129] In some embodiments, the preparation of ammonia-oxidizing bacteria may contain the concentration or amount of ammonia-oxidizing bacteria for at least partially treating a condition or disease. The preparation of ammonia-oxidizing bacteria may contain the concentration or amount of ammonia-oxidizing bacteria for changing, e.g., reducing or increasing, the amount, concentration or ratio of bacteria or the genus of bacteria on a surface, e.g., the skin surface. The bacteria may be non-pathogenic or pathogenic, or potentially pathogenic.

[0130] In some embodiments, the preparation of ammonia-oxidizing bacteria may contain from about 10 8 to about 10 14 CFU / L. The preparation may be at least 10 8 、10 9 、10 10 、10 11 、2×10 11 、5×10 11 、10 12 、2×10 12 、5×10 12 、10 13 、2×1013 , 5×10 13 , or 10 14 , or about 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 may contain CFU / L.

[0131] In certain embodiments, the preparation may contain from about 1×10 9 CFU / L to about 10×10 9 CFU / L. In certain embodiments, the preparation may contain from about 1×10 9 CFU to about 10×10 9 CFU. In certain embodiments, the preparation may contain from about 1×10 9 CFU / mL to 10×10 9 CFU / mL. In certain embodiments, the preparation may contain about 1×10 9 CFU / mL.

[0132] In some embodiments, the preparation of ammonia-oxidizing bacteria may contain from about 0.1 milligram (mg) to about 1000 mg of ammonia-oxidizing bacteria. In certain embodiments, the preparation may contain from about 50 mg to about 1000 mg of ammonia-oxidizing bacteria. The preparation may 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, 100 - 200 mg, 200 - 300 mg, 300 - 400 mg, 400 - 500 mg, 500 - 600 mg, 600 - 700 mg, 700 - 800 mg, 800 - 900 mg, 900 - 1000 mg, 100 - 250 mg, 250 - 500 mg, 100 - 500 mg, 500 - 750 mg, 750 - 1000 mg, or between 500 - 1000 mg.

[0133] In some embodiments, the preparation of ammonia-oxidizing bacteria may contain the mass ratio of ammonia-oxidizing bacteria to an additive, such as a pharmaceutically acceptable additive or a cosmetically acceptable additive, in the range of from about 0.1 gram / liter to about 1 gram / liter. The preparation may contain the mass ratio of ammonia-oxidizing bacteria to the additive in the range of from about 0.1 - 0.2, 0.2 - 0.3, 0.1 - 0.5, 0.2 - 0.7, 0.5 - 1.0, or 0.7 - 1.0 gram / liter.

[0134] In some embodiments, the preparation of ammonia-oxidizing bacteria may comprise ammonia-oxidizing bacteria having the dosages described herein in combination with ammonia, for example, ammonia concentrations between about 0.01 mM and about 100 mM. For example, the ammonia concentration may be about 0.01, 0.05, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0 mM.

[0135] In some embodiments, the preparation of ammonia-oxidizing bacteria may comprise, consist essentially of, or consist of ammonia-oxidizing bacteria in an aqueous buffer solution containing sodium phosphate and magnesium chloride, for example, 50 mM Na 2 HPO 4 and 2 mM MgCl 2 In certain embodiments, the preparation may comprise, consist essentially of, or consist of about 1×10 9 CFU / mL in water, 50 mM Na 2 HPO 4 and 2 mM MgCl 2 .

[0136] In some embodiments, the preparation of ammonia-oxidizing bacteria may comprise components that provide pre-activation of the ammonia-oxidizing bacteria. For example, pre-activation may involve having a preparation with ammonia-oxidizing bacteria and ammonia.

[0137] In some embodiments, the preparation of ammonia-oxidizing bacteria may be in a form that provides activation upon delivery to a subject, such as co-activation. This provides delivery of the ammonia-oxidizing bacteria to ammonia present on or utilized by the subject either before or simultaneously with delivery of the ammonia-oxidizing bacteria. For example, the ammonia may be derived from the subject's own ammonia source or may be applied before or simultaneously with the ammonia-oxidizing bacteria. The ammonia may be provided in a separate container or may be naturally derived from the subject.

[0138] The preparation may contain a volume between about 0.1 and about 100 fluid ounces, about 0.2 and about 50 fluid ounces, about 0.5 and about 25 fluid ounces, about 1.0 and about 10 fluid ounces, about 2.0 and about 7 fluid ounces, about 3 and about 5 fluid ounces. In some embodiments, the preparation may contain a volume of about 3.4 fluid ounces.

[0139] The preparation may be provided in a container constructed to contain between about 0.1 and about 100 fluid ounces, about 0.2 and about 50 fluid ounces, about 0.5 and about 25 fluid ounces, about 1.0 and about 10 fluid ounces, about 2.0 and about 7 fluid ounces, about 3 and about 5 fluid ounces. In some embodiments, the container is constructed to contain about 3.4 fluid ounces. The container may be a one-chamber container or any other container disclosed herein.

[0140] In some embodiments, the preparation of ammonia-oxidizing bacteria may be in a growth state. The growth state can be provided by exposing the ammonia-oxidizing bacteria to an environment that can promote growth. The growth state is a state, for example, where ammonium ions (NH 4 + ) are converted to nitrite (NO 2 -) It may be ammonia-oxidizing bacteria in an environment that enables the immediate availability of ammonia-oxidizing bacteria for conversion to . The growth state may include providing ammonia-oxidizing bacteria in an environment having a pH greater than about 7.6. The growth state may also include providing ammonia-oxidizing bacteria in an environment having ammonia, ammonium salts, and / or urea, trace minerals, and sufficient oxygen and carbon dioxide as described in Section 1 .

[0141] In some embodiments, the preparation of ammonia-oxidizing bacteria may be in a polyphosphate-loaded state, and the state or environment, e.g., the medium, e.g., the culture medium, e.g., the growth medium, may have a pH of less than about 7.4. The level of at least one of ammonia, ammonium ions, and urea may be between about 10 micromolar concentration and 200 millimolar concentration. The level of trace materials may be between 0.1 micromolar concentration of iron and 20 micromolar concentration of iron. The level of oxygen may be between about 5% and 100% oxygen saturation. The level of carbon dioxide may be between / about 0 and 200 ppm / less than that, and the level of phosphate may exceed about 10 micromolar concentration. The purpose of the polyphosphate-loaded state is to provide ammonia and oxygen to AOB so that ATP can be generated, but instead of AOB being able to use that ATP to fix carbon dioxide, to cause AOB to use that ATP to generate polyphosphate that can be stored by not providing AOB with carbon dioxide and carbonate .

[0142] In some embodiments, the preparation of ammonia-oxidizing bacteria may be in a storage state. The storage state may be defined as ammonia-oxidizing bacteria in an environment that can be stored for later restoration. The storage state may be ammonia-oxidizing bacteria in an environment that enables the availability of ammonia-oxidizing bacteria, e.g., after being placed in an environment that promotes the growth state for a predetermined period after restoration .

[0143] The storage condition may include providing ammonia-oxidizing bacteria in an environment having a pH of less than about 7.4. The storage condition may also include providing ammonia-oxidizing bacteria in an environment having ammonia, ammonium salts, and / or urea, trace minerals, oxygen, and low concentrations of carbon dioxide as described in Section 1 above.

[0144] Storage can also be achieved by storing at 4°C for up to several months. The storage buffer may, in some embodiments, be 50 mM Na 2 HPO 4 ~2 mM MgCl 2 (pH 7.6).

[0145] In some embodiments, the ammonia-oxidizing bacteria may be stored at low temperature. 1.25 ml of a mid-logarithmic growth phase culture of ammonia-oxidizing bacteria can be added to a 2 ml cryotube and 0.75 ml of sterile 80% glycerol. The tube can be gently shaken and incubated at room temperature for 15 minutes to allow the cryoprotectant to be taken up by the cells. The tube can be stored directly in an -80°C freezer for freezing and storage.

[0146] For revival of the culture, the frozen stock can be thawed on ice for 10 - 20 minutes and then centrifuged at 8,000×g for 3 minutes at 4°C. The pellet can be washed by suspending it in 2 ml of AOB medium and then centrifuged again at 8,000×g for 3 minutes at 4°C to reduce the potential toxicity of the cryoprotectant. The pellet can be resuspended in 2 ml of AOB medium and seeded into 50 ml of AOB medium containing 50 mM NH 4 + and incubated at 30°C in the dark with shaking at 200 rpm.

[0147] In some embodiments, the preparation of ammonia-oxidizing bacteria may include ammonia-oxidizing bacteria in a storage state and / or ammonia-oxidizing bacteria in a polyphosphate-loaded state and / or ammonia-oxidizing bacteria in a growth state.

[0148] Although not desiring to be bound by theory, by maintaining ammonia-oxidizing bacteria in a low-carbon dioxide state or environment with sufficient oxygen and ammonia, the ammonia-oxidizing bacteria can accumulate polyphosphate for a predetermined period, such as a period of about double the doubling time, such as about 8 to 12 hours, such as about 10 hours. The ammonia-oxidizing bacteria can accumulate sufficient polyphosphate to extend its storage survival rate and storage time and accelerate its restoration. This can occur with or without the addition of buffer and ammonia.

[0149] The presence of sufficient stored polyphosphate can provide the ammonia-oxidizing bacteria with an ATP resource to maintain metabolic activity even in the absence of ammonia and oxygen and survive injuries that would otherwise be lethal.

[0150] The oxidation process in which ammonia generates ATP has two steps. The first step is the oxidation of ammonia to hydroxylamine by ammonia monooxygenase (Amo), followed by the conversion of hydroxylamine to nitrite by hydroxylamine oxidoreductase (Hao). The electrons from the second step (conversion of hydroxylamine to nitrite) are used to power the first step (oxidation of ammonia to hydroxylamine).

[0151] If ammonia-oxidizing bacteria do not have hydroxylamine for generating electrons for Amo, then hydroxylamine is not available to Hao. For example, acetylene irreversibly inhibits an enzyme that is important for the oxidation of ammonia to hydroxylamine, which is the first step in the oxidation of ammonia to nitrite. After AOB are exposed to acetylene, Amo is irreversibly inhibited, so new enzyme must be synthesized before hydroxylamine can be generated. In the habitats of normal community biofilms, AOB can share hydroxylamine with other AOB (still different strains with different sensitivities to inhibitors), and can receive it from other AOB, so the biofilm tends to be more resistant to inhibitors such as acetylene than individual organisms. AOB can synthesize new Amo using stored polyphosphate even in the absence of hydroxylamine.

[0152] Any embodiment, preparation, composition, or formulation of ammonia-oxidizing bacteria discussed herein may optionally include, consist essentially of, or consist of a pure culture of ammonia-oxidizing bacteria. 3. A composition comprising ammonia-oxidizing bacteria; a composition comprising N. eutropha, such as D23 N. eutropha

[0153] The present disclosure provides, inter alia, compositions comprising ammonia-oxidizing bacteria, such as preparations of ammonia-oxidizing bacteria, or purified preparations of ammonia-oxidizing bacteria, such as natural products, or enhanced natural products.

[0154] The compositions disclosed herein throughout the present disclosure may be provided for use in treating skin conditions, such as acne, such as acne vulgaris.

[0155] Compositions containing ammonia-oxidizing bacteria, such as preparations of ammonia-oxidizing bacteria, or purified preparations of ammonia-oxidizing bacteria, can be provided in cosmetic or therapeutic products. The preparation may contain, among other things, at least one of ammonia, ammonium salts, and urea. The ammonia-oxidizing bacteria may be any ammonia-oxidizing bacteria disclosed herein, or a combination of ammonia-oxidizing bacteria.

[0156] The present disclosure provides, among other things, compositions containing a purified preparation of N. eutropha, such as optimized N. eutropha. In some embodiments, the N. eutropha in the composition has at least one property selected from optimized growth rate, optimized NH 4 + oxidation rate, and optimized NH 4 + resistance.

[0157] In some aspects, the present disclosure provides compositions having a defined number of species. By way of example, the present disclosure provides a composition having N. eutropha and one other type of organism and no other types of organisms. In other examples, the composition has N. eutropha and 2, 3, 4, 5, 6, 7, 8, 9, or 10 other types of organisms and no other types of organisms. The other types of organisms in this composition may be, by way of example, bacteria such as ammonia-oxidizing bacteria. Suitable ammonia-oxidizing bacteria for this purpose include those of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, or Nitrosovibrio.

[0158] In some embodiments, a composition comprising ammonia-oxidizing bacteria, such as N. eutropha, provides conditions that support the viability of the ammonia-oxidizing bacteria, such as N. eutropha. By way of example, the composition may promote the growth and metabolism of the ammonia-oxidizing bacteria, such as N. eutropha, or may promote a dormant state (e.g., freezing) in which live ammonia-oxidizing bacteria, such as N. eutropha, can be recovered. When the composition promotes growth or metabolism, it may contain water and / or nutrients consumed by the ammonia-oxidizing bacteria, such as N. eutropha, such as ammonium, ammonia, urea, oxygen, carbon dioxide, or trace minerals.

[0159] In some embodiments, one or more other organisms other than ammonia-oxidizing bacteria may be included in a preparation of ammonia-oxidizing bacteria. For example, organisms of a genus selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacter, and combinations thereof may be provided in a preparation of ammonia-oxidizing bacteria. In some embodiments, the preparation may substantially contain no other organisms.

[0160] A preparation of ammonia-oxidizing bacteria may contain between about 10 8 and about 10 14 CFU / L. The preparation may contain at least about 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 8 to 10 9 , 10 9 to 10 10 , 10 10 to 10 11 , 10 11 to 1012 , 10 12 ~10 13 , or 10 13 ~10 14 CFU / L may be included. Other preparations may contain about 2×10 9 , 4×10 9 , or 8×10 9 CFU / L.

[0161] Preparations of ammonia-oxidizing bacteria may contain between about 10 8 and about 10 14 CFU / ml. The preparation may contain at least about 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 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 / ml may be included. Other preparations may contain about 2×10 9 , 4×10 9 , or 8×10 9 CFU / mL.

[0162] In some embodiments, the preparation may contain between about 1×10 9 and about 10×10 9 CFU / L. In some embodiments, the preparation may contain about 3×10 10 CFU, e.g., 3×10 10 CFU per day. In some embodiments, the preparation may contain about 1×109 to about 10×10 9 CFU, for example, about 1×10 per day 9 to about 10×10 9 CFU may be included.

[0163] In some embodiments, the preparation of ammonia-oxidizing bacteria may contain ammonia-oxidizing bacteria between about 0.1 milligrams (mg) and about 1000 mg. In certain embodiments, the preparation may contain ammonia-oxidizing bacteria between about 50 mg and about 1000 mg. The preparation may 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, 100 - 200 mg, 200 - 300 mg, 300 - 400 mg, 400 - 500 mg, 500 - 600 mg, 600 - 700 mg, 700 - 800 mg, 800 - 900 mg, 900 - 1000 mg, 100 - 250 mg, 250 - 500 mg, 100 - 500 mg, 500 - 750 mg, 750 - 1000 mg, or between 500 - 1000 mg.

[0164] In some embodiments, the preparation of ammonia-oxidizing bacteria may contain the mass ratio of ammonia-oxidizing bacteria to an additive, for example, a pharmaceutically acceptable additive or a cosmetically acceptable additive, in the range of about 0.1 gram / liter to about 1 gram / liter. The preparation may contain the mass ratio of ammonia-oxidizing bacteria to the additive in the range of about 0.1 - 0.2, 0.2 - 0.3, 0.1 - 0.5, 0.2 - 0.7, 0.5 - 1.0, or 0.7 - 1.0 gram / liter.

[0165] In some embodiments, the preparation of ammonia-oxidizing bacteria is sodium dihydrogen phosphate and magnesium chloride, for example, 50 mM Na 2 HPO 4 and 2 mM MgCl 2It may be an ammonia-oxidizing bacterium in an aqueous buffer solution that comprises, consists essentially of, or consists of. The ammonia-oxidizing bacterium may be at any concentration disclosed herein, for example, 1×10 9 CFU / ml. The preparation may be provided in a predetermined volume of buffer, for example, 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, between about 3 and about 5 fluid ounces. In some embodiments, the preparation may be provided in a container. The preparation may be provided in a container constructed to contain about 3.4 fluid ounces, or any other volume disclosed herein. The preparation may be in a form that can be made into an aerosol, spray or mist, i.e., in the form of a mist, for example, in the state of an AO+Mist product.

[0166] Advantageously, the formulation may have a pH that promotes the viability, for example, the metabolic activity, of AOB, such as N. eutropha. Urea is hydrolyzed to ammonia, raising the pH from 7 to 8. AOB is very active in this pH range and converts NH3 to ammonium, lowering the pH to about 6 where it becomes unavailable. Lower pH levels, for example, about pH 4 are also tolerated.

[0167] An ammonia-oxidizing bacterium, such as N. eutropha, can be combined with one or more pharmaceutically or cosmetically acceptable additives. In some embodiments, "pharmaceutically acceptable additive" 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 additive is compatible with the other components of the pharmaceutical formulation and is "pharmaceutically acceptable" in the sense that it has a reasonable benefit / risk ratio and is suitable for use in contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications. Remington: The Science and Practice of Pharmacy See, e.g., 21st Edition; Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of Pharmaceutical Excipients, 6th Edition; Edited by Rowe et al.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd Edition; Edited by Ash and Ash; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; Edited by Gibson; CRC Press LLC: Boca Raton, Fla., 2009.

[0168] In some embodiments, a cosmetically acceptable additive represents a cosmetically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In some embodiments, each additive is compatible with other components of the cosmetic formulation and is cosmetically acceptable in the sense that it is suitable, at a reasonable benefit / risk ratio, for use in contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications.

[0169] Although an active ingredient, such as ammonia-oxidizing bacteria, such as N. eutropha, can be administered alone, in many embodiments it is present in a pharmaceutical formulation or composition. Accordingly, the present disclosure provides a pharmaceutical formulation (preparation or composition) or a cosmetic formulation (preparation or composition) comprising ammonia-oxidizing bacteria and a pharmaceutically acceptable additive or a cosmetically acceptable additive. The pharmaceutical and cosmetic compositions can take the form of formulations as described below.

[0170] The pharmaceutical and cosmetic formulations (e.g., preparations or compositions) described herein may include those suitable for oral (e.g., by deposition within or for the alimentary tract), parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intra-articular), inhalation (including particulate dusts or mists that may be generated using various types of metered-dose, pressurized aerosols, nebulizers or inhalers, including intranasal (nasal) or transpulmonary (pulmonary)), renal, and topical (including dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration; however, the most appropriate route may depend, for example, on the condition and disorder of the recipient.

[0171] The formulations (e.g., preparations or compositions) can be conveniently presented as unit dosage forms and can be prepared by any of the methods well known in the pharmaceutical or cosmetic arts. Typically, the method includes the step of associating the active ingredient (e.g., ammonia-oxidizing bacteria, e.g., N. eutropha) with a pharmaceutical or cosmetic carrier comprising one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately associating the active ingredient with a liquid carrier or a finely divided solid carrier or both, and then shaping the product into the desired formulation, if necessary.

[0172] The formulations can be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of, for example, N. eutropha; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water type liquid emulsion or a water-in-oil type liquid emulsion. The active ingredient can also be presented as a bolus, a lozenge or a paste. Various pharmaceutically acceptable carriers and their formulations are described in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Suppl. 42:2S, 1988.

[0173] Ammonia-oxidizing bacteria, such as N. eutropha compositions, can be administered, for example, in a form suitable for immediate release or extended release. Suitable examples of sustained release systems include suitable polymeric materials, such as semipermeable polymeric matrices in the form of shaped articles, such as films, or microcapsules; suitable hydrophobic materials, such as in an acceptable emulsion in oil; or ion exchange resins. The sustained release system can be administered orally; rectally; parenterally; intracapsularly; intravaginally; intraperitoneally; topically, for example, as a powder, ointment, gel, drops or transdermal patch; buccally; or as a spray.

[0174] The dosage preparations can be appropriately formulated to provide controlled release of ammonia-oxidizing bacteria, such as N. eutropha. For example, the pharmaceutical composition may be in the form of particles containing one or more of a biodegradable polymer, a polysaccharide gelling and / or a bioadhesive polymer, or an amphiphilic polymer. These compositions exhibit certain biocompatible characteristics that allow for controlled release of the active substance. See U.S. Patent No. 5,700,486.

[0175] Exemplary compositions may include, for example, crystalline cellulose for bulking, 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 additives, binders, extenders, disintegrants, diluents and lubricants, mannitol, lactose, sucrose and / or cyclodextrin, and may include suspensions that may contain high molecular weight additives such as cellulose (Avicel) or polyethylene glycol (PEG). Such formulations may also include additives to assist in mucoadhesion such as hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), sodium carboxymethylcellulose (SCMC), maleic anhydride copolymers (e.g., Gantrez), and agents to control release such as polyacrylic acid copolymers (e.g., Carbopol 934). Lubricants, flow promoters, flavors, colorants and stabilizers may be added to facilitate fabrication and use. The surfactant may be an amphoteric surfactant, a nonionic surfactant, or an anionic surfactant.

[0176] Additives such as surfactants that may be used in embodiments of the present disclosure include cocamidopropyl betaine (ColaTeric COAB), polyethylene sorbitol ester (e.g., Tween 80), ethoxylated lauryl alcohol (RhodaSurf 6 NAT), sodium lauryl sulfate / sodium lauryl glucoside / cocamidopropyl betaine (Plantapon 611 L UP), sodium lauryl sulfate (e.g., RhodaPex ESB 70 NAT), alkyl polyglucoside (e.g., Plantaren One or more of 2000 N UP), sodium lauryl sulfate (Plantaren 200), Dr. Bronner's Castile soap, Dr. Bronner's baby Castile soap, lauramine oxide (ColaLux Lo), sodium dodecyl sulfate (SDS), alkyl polyglucoside polysulfonate (PolySufanate 160 P), sodium lauryl sulfate (Stepanol-WA Extra K) and combinations thereof may be included. Dr. Bronner's Castile soap and Dr. Bronner's baby soap contain water, organic coconut oil, potassium hydroxide, organic olive oil, fair trade organic hemp oil, organic jojoba oil, citric acid, and tocopherol.

[0177] In some embodiments, the surfactant can be used with ammonia-oxidizing bacteria in an amount that causes nitrite formation. In some embodiments, the preparation can have from less than about 0.0001% to about 10% surfactant. In some embodiments, the preparation can have between about 0.1% and about 10% surfactant. In some embodiments, the concentration of surfactant used can be between about 0.0001% and about 10%. In some embodiments, the preparation may substantially contain no surfactant.

[0178] In some embodiments, the formulation, e.g., the preparation, may include other components that can enhance the effectiveness of the ammonia-oxidizing bacteria or maintain or enhance the viability of the ammonia-oxidizing bacteria, or enhance treatment or adaptation.

[0179] In some embodiments, a chelating agent may be included in the preparation. The chelating agent may be another compound, e.g., a compound that can bind to a metal. The chelating agent may help remove unwanted compounds from the environment, or may act in a protective manner to reduce or eliminate contact between a particular compound and the environment, e.g., ammonia-oxidizing bacteria, e.g., a preparation of ammonia-oxidizing bacteria, e.g., an additive. In some embodiments, the preparation may substantially contain no chelating agent.

[0180] The formulation may also contain antioxidants, buffers, bacteriostatic agents to prevent the growth of undesirable bacteria, solutes, and may contain aqueous and non-aqueous sterile suspensions which may also contain suspending and thickening agents. The formulation may be presented in unit dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state which requires only the addition of a sterile liquid carrier, for example, saline or water for injection, immediately before use. Immediate solutions and suspensions may be prepared from powders, granules and tablets of the above types. Exemplary compositions may contain suitable non-toxic pharmaceutically acceptable diluents or solvents such as, for example, mannitol, 1,3-butanediol, water, Ringer's solution, and isotonic sodium chloride solution, or other suitable dispersing or wetting agents and suspending agents including synthetic monoglycerides or diglycerides, and fatty acids including oleic acid, or fatty acids containing Cremophor Contains a solution or suspension. The aqueous carrier may be, for example, an isotonic buffered solution having a pH of about 3.0 to about 8.0, a pH of about 3.5 to about 7.4, for example, from 3.5 to 6.0, for example, from 3.5 to about 5.0. Useful buffer solutions include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate / acetic acid buffer solutions. In some embodiments, the composition does not contain an oxidizing agent.

[0181] Additives that may be included are, by way of example, proteins such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition may also contain small amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate. In some embodiments, the additive, for example, a pharmaceutically acceptable additive or a cosmetically acceptable additive, may include an anti-adhesive, binder, coating agent, disintegrant, filler, fragrance, colorant, lubricant, flow promoter, adsorbent, preservative, or sweetening agent. In some embodiments, the preparation may substantially contain no additives.

[0182] In some embodiments, the preparation may substantially not contain one or more of the compounds or substances listed in this disclosure.

[0183] Exemplary compositions for aerosol administration may include, for example, solutions in saline that contain benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other solubilizing or dispersing agents. Advantageously, in compositions for aerosol administration, ammonia-oxidizing bacteria, such as N. eutropha, are delivered in the form of an aerosol spray presentation from a pump. In other embodiments, compositions for aerosol administration of ammonia-oxidizing bacteria, such as N. eutropha, are delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, nitrogen, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for delivering a fixed amount. For example, gelatin capsules and cartridges can be formulated to contain a powder mixture of N. eutropha and a suitable powder base, such as lactose or starch. In certain embodiments, N. eutropha is administered as an aerosol via an aerosol adapter, also known as an actuator, from a metering valve. Optionally, stabilizers are included and / or porous particles for deep lung delivery are included (see, for example, U.S. Patent No. 6,447,743). The composition or preparation may be in a form that can be aerosolized, sprayed, or misted, i.e., in the form of a mist. Preparations of ammonia-oxidizing bacteria can be ammonia-oxidizing bacteria in an aqueous buffer solution containing, consisting essentially of, or consisting of sodium phosphate dibasic and magnesium chloride, such as 50 mM Na 2 HPO 4 and 2 mM MgCl 2 and can be referred to as AO + Mist throughout this disclosure. The AO + Mist preparation is 50 mM Na 2 HPO4 and 2 mM MgCl 2 in an aqueous buffer solution containing ammonia-oxidizing bacteria at a concentration of 1×10 9 CFU / mL.

[0184] The formulation may be presented using a carrier such as cocoa butter, synthetic glyceride esters or polyethylene glycol. Such carriers are typically solid at room temperature but liquefy and / or dissolve at body temperature to release ammonia-oxidizing bacteria, such as N. eutropha.

[0185] Exemplary topical administration compositions include topical carriers such as plastibase (mineral oil gelled with polyethylene). In some embodiments, the composition and / or additive may be in one or more of the forms of a liquid, solid, or gel. For example, suspensions include, but are not limited to, water, saline, phosphate buffered saline, ammonia-oxidizing storage buffer, or an aqueous buffer solution containing, for example, Na 2 HPO 4 and MgCl 2 , for example, 50 mM Na 2 HPO 4 and 2 mM MgCl 2 .

[0186] Gel formulations may include, but are not limited to, agar, silica, polyacrylic acid (e.g., Carbopol®), carboxymethyl cellulose, starch, guar gum, alginate or chitosan.

[0187] In some embodiments, the formulation may be supplemented with an ammonia source including, but not limited to, ammonium chloride or ammonium sulfate.

[0188] In some embodiments, an ammonia-oxidizing bacterial composition, e.g., an N. eutropha composition, is formulated to improve the penetration of NO into the skin. Gel-forming materials such as KY jelly or various hair gels present a diffusion barrier to NO loss to the ambient air, thus improving the NO absorption of the skin. The NO level in the skin generally does not greatly exceed 20 nM / L. This is because that level activates GC and causes local vasodilation and oxidative destruction of excess NO.

[0189] It should be understood that in addition to the components specifically mentioned above, the formulations described herein may include other conventional agents in the art with respect to the type of formulation in question.

[0190] A formulation, e.g., a preparation, e.g., a composition, can be provided in a container, delivery system, or delivery device having a weight of less than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 grams, with or without including the contents of the container.

[0191] A suitable unit dosage formulation is a unit dosage formulation containing the effective dosage described herein of an ammonia-oxidizing bacterium, e.g., N. eutropha, or an appropriate fraction thereof.

[0192] A therapeutically effective amount of an ammonia-oxidizing bacterium, e.g., N. eutropha, can be administered as a single pulse dosage, as a bolus dosage, or as a pulse dosage administered over time. Thus, in a pulse dosage, a bolus administration of an ammonia-oxidizing bacterium, e.g., N. eutropha, is provided, the period during which the ammonia-oxidizing bacterium, e.g., N. eutropha, is administered to the subject continues, and a second bolus administration follows. In a specific non-limiting example, the pulse dosage is administered during a 1-day cool-down, a 1-week cool-down, or a 1-month cool-down.

[0193] A therapeutically effective amount of ammonia-oxidizing bacteria, such as N. eutropha, can be administered as a single spray or pump in the form of an aerosol or mist, or as multiple sprays or pumps. In a single administration, one or more sprays or pumps can be actuated. The therapeutically effective amount can be the result of a single administration or multiple administrations during one or more days of cooling, one or more weeks of cooling, one or more months of cooling, or a longer period.

[0194] A therapeutically effective amount can be an amount sufficient to improve any one or more of the number of acne lesions, PIH / PIE lesion assessment, SkinDex16 data, modified Griffiths 10-point scale, tolerance assessment, sebumeter measurement (value), and Investigator's Global Acne (IGA) scale by a physician in a clinical trial at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 weeks.

[0195] In some embodiments, a preparation of ammonia-oxidizing bacteria, such as a formulation, such as a composition, can be applied for a predetermined number of days. This can be based, for example, at least in part on the severity of the condition or disease, the response to treatment, the dosage and dosing frequency applied. For example, the preparation can 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, 84 - 91 days, about 1 month, about 2 months, about 3 months. In some embodiments, the ammonia-oxidizing bacteria are administered for an indefinite period, such as longer than 1 year, longer than 5 years, longer than 10 years, longer than 15 years, longer than 30 years, longer than 50 years, longer than 75 years. In certain aspects, the preparation can be applied for about 16 days. In certain aspects, the preparation can be applied for about 4 weeks.

[0196] In some embodiments, a preparation of ammonia-oxidizing bacteria, e.g., a formulation, e.g., a composition, may be applied a predetermined number of times per day. This may be based, for example, at least in part, on the condition or severity of the disease, the response to treatment, the dosage applied, and the dosing frequency. For example, the preparation may 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, or 24 times per day.

[0197] In some embodiments, the preparation may be applied once per day. In other embodiments, the preparation may be applied twice per day. In some embodiments, the preparation may be applied in a first predetermined amount for a certain number of days and in a second predetermined amount for a certain subsequent number of days. In some embodiments, the preparation may be applied for about 16 days. In certain aspects, the preparation may be applied for about 4 weeks. Consumer products

[0198] Ammonia-oxidizing bacteria, e.g., N. eutropha, may be associated with a variety of consumer products, examples of such products are described below, and may consist of the formulations, compositions, or preparations disclosed throughout the present disclosure. In some embodiments, the ammonia-oxidizing bacteria associated with the product, e.g., N. eutropha, are admixed with the product and, for example, spread uniformly throughout the product. In some embodiments, the ammonia-oxidizing bacteria associated with the product, e.g., N. eutropha, are laminated onto the product.

[0199] In some embodiments, ammonia-oxidizing bacteria, e.g., N. eutropha, are associated with a powder. The powder is typically a particulate solid that does 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).

[0200] In some embodiments, ammonia-oxidizing bacteria are associated with cosmetics. The cosmetics may be substances for topical application, such as liquid foundation, powder foundation, rouge, or lipstick, intended to change a human appearance, and may be referred to as preparations. The cosmetics may be any substance listed in Food and Drug Administration regulations, such as those listed in Title 21, Code of Federal Regulations, Section 720.4.

[0201] Preparations, for example, cosmetics, may be 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; eye makeup preparations, such as eye brow pencils, eye liners, eye shadows, eye lotions, eye makeup removers, mascaras; fragrance preparations, such as colognes, eau de toilettes, perfumes, powders (dusting and talcum), sachets; hair preparations, such as hair conditioners, hair sprays, hair straighteners, permanent wave agents, rinses, shampoos, tonics, hair dressings, hair styling aids, wave setting agents; hair coloring preparations, such as hair dyes and hair colors, hair bleaches, colored hair rinses, colored hair shampoos, colored hair lighteners, hair bleaches; makeup preparations, such as face powders, foundations, leg and body paints, lipsticks, makeup bases, blushes, makeup setting agents; manicure preparations, such as base coats and undercoats, cuticle softeners, nail creams and lotions, nail extenders, nail polishes and enamels, nail polish and enamel removers; oral hygiene products, such as toothpastes, mouthwashes and breath fresheners; bath soaps and detergents, deodorants, vides, feminine hygiene deodorants; shaving preparations, such as aftershave lotions, beard softeners, talcum, pre-shave lotions, shaving creams, shaving soaps; skin care preparations, such as cleansing, depilatory agents, face and neck, body and hand, foot powders and sprays, moisturizers, night preparations, paste masks, skin fresheners; and sunburn preparations, such as gels, creams, and liquids, and indoor tanning preparations.

[0202] In some embodiments, the formulations, compositions, or preparations described herein are provided as or may be disposed within 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 conditioner, rinse, shampoo, tonic, face powder, cuticle softener, nail cream and lotion, oral hygiene products, mouthwash, bath soap, bidet, feminine hygiene deodorant; shaving preparations such as aftershave lotion, skin care preparations such as cleansing, face and neck, body and hands, foot powders and sprays, moisturizers, night preparations, paste masks, skin fresheners; and sunburn preparations such as gels, creams, and liquids, including at least one of the foregoing.

[0203] In some embodiments, ammonia-oxidizing bacteria such as N. eutropha are associated with cosmetics. The cosmetics may be substances for topical application intended to change the appearance of a human, such as liquid foundation, powder foundation, rouge, or lipstick. Other components may be added to pharmaceutical formulations such as preparations, or cosmetic preparations such as water, mineral oil, colorants, perfumes, aloe, glycerin, sodium chloride, sodium bicarbonate, pH buffers, UV blockers, silicone oils, natural oils, vitamin E, herbal concentrates, lactic acid, citric acid, talc, clay, calcium carbonate, magnesium carbonate, zinc oxide, starch, urea, and erythorbic acid, or any other additives known to those of skill in the art, such as the additives disclosed herein.

[0204] In some embodiments, the preparation may be disposed within or provided as a powder, cosmetic, cream, stick, aerosol such as a mist, plaster, wipe, or bandage.

[0205] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with a cream. The cream may be a fluid containing a thickening agent and generally has a consistency that allows it to be spread evenly on the skin. Exemplary creams include moisturizing lotions, face creams, and body lotions.

[0206] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with a stick. The stick is typically a solid that, when placed in contact with a surface, transfers a portion of the stick's contents to the surface. Exemplary sticks include deodorant sticks, lipsticks, lip balms in stick form, and sunscreen applicator sticks.

[0207] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with an aerosol. An aerosol is typically a colloid of fine solid particles or fine liquid droplets in a gas such as air. An aerosol can be created by placing N. eutropha (and optionally a carrier) in a pressurized vessel and then opening a valve to release the contents. The vessel can be designed to apply only a pressure level compatible with the viability of N. eutropha. By way of example, a high pressure may be applied for only a short time and / or the pressure may be low enough so as not to compromise viability. Examples of consumer uses of aerosols include sunscreens, deodorants, perfumes, hairsprays, and insect repellents. An aerosol may also be referred to as a mist.

[0208] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with a plaster. The plaster can be a topical application agent having a liquid or cream-like consistency intended to protect the skin or promote healing. Examples of plasters include burn ointments and skin moisturizers.

[0209] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with a wipe. The wipe may be a flexible material suitable for topical application of a liquid or cream to the skin. The wipe may be, for example, paper-based or cloth-based. Exemplary wipes include tissues and wet wipes.

[0210] Compositions containing ammonia-oxidizing bacteria, such as N. eutropha, may also contain one or more of a humectant, a deodorant, a fragrance, a coloring agent, an insect repellent, a cleansing agent, or a UV blocker.

[0211] By way of example, a humectant can be an agent that reduces or prevents skin dryness. Exemplary humectants include water retention agents (e.g., urea, glycerin, alpha-hydroxy acids, and dimethicone) and emollients (e.g., lanolin, mineral oil, and petrolatum). For example, a humectant may be included in a cream, balm, lotion, or sunscreen containing ammonia-oxidizing bacteria, such as N. eutropha.

[0212] A deodorant can be an agent that reduces an unwanted odor. A deodorant can function by directly neutralizing an odor, preventing sweating, or preventing the growth of odor-producing bacteria. Exemplary deodorants include aluminum salts (e.g., aluminum chloride or aluminum chlorohydrate), cyclomethicone, talc, swelling powder, essential oils, inorganic salts, hops, and mintsuk. Deodorants are typically present in spray or stick deodorants and may also be found in some soaps and clothing.

[0213] An insect repellent can be an agent that is applicable to a surface (e.g., skin) to prevent insects and other arthropods from staying on the surface. Insect repellents include DEET (N,N-diethyl-m-toluamide), p-menthane-3,8-diol (PMD), icariside, nepetalactone, citronella oil, neem oil, Vitex trifolia, dimethylcarbamate, tricyclodecenyl allyl ether, and IR3535 (ethyl 3-[N-butyl-N-acetyl]-aminopropionate).

[0214] A cleansing agent can be an agent that removes dirt or unwanted bacteria from a surface such as the skin. Exemplary cleansing agents include bar soap, liquid soap, and shampoo.

[0215] A UV blocker can be an agent that is applicable to a surface to reduce the amount of ultraviolet light received by the surface. The UV blocker can block UV-A and / or UV-B light rays. The UV blocker can function by absorbing, reflecting, or scattering UV. Exemplary UV blockers include absorbers such as homosalate, octisalate (also called octyl salicylate), octinoxate (also called octyl methoxycinnamate or OMC), octocrylene, oxybenzone, and avobenzone, as well as reflectors (e.g., titanium dioxide and zinc oxide). UV blockers are typically present in sunscreens and can also be found in skin creams and some cosmetics.

[0216] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with the conditioner. The conditioner generally represents a substance having a cream-like consistency that can be applied to hair to improve its appearance, strength, or manageability.

[0217] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with a cloth. The cloth represents a flexible material that is generally suitable for making clothing and has, for example, a material strength sufficient to withstand daily movements by the wearer. The cloth can be fibrous, woven, or knitted and may be made from natural or synthetic materials. Exemplary cloth materials include cotton, linen, wool, ramie, silk, denim, leather, nylon, polyester, and spandex, as well as blends thereof.

[0218] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with a thread. A thread generally refers to a long, thin, spun, flexible material suitable for knitting or weaving. The thread may be made of, for example, wool, cotton, polyester, and blends thereof.

[0219] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are associated with a thread. A thread represents a long, thin, spun, flexible material generally suitable for sewing. A thread generally has a smaller diameter than a yarn. The thread may be made of, for example, cotton, polyester, nylon, silk, and blends thereof.

[0220] For example, clothing items such as shoes, shoe insoles, pajamas, sneakers, belts, hats, shirts, underwear, sportswear, helmets, towels, gloves, socks, bandages, and the like may also be treated with ammonia-oxidizing bacteria, such as N. eutropha. Bedding including sheets, pillows, pillowcases, and blankets may also be treated with ammonia-oxidizing bacteria, such as N. eutropha. In some embodiments, skin areas that cannot be washed for a period of time may also be contacted with ammonia-oxidizing bacteria, such as N. eutropha. For example, skin enclosed in an orthopedic cast that immobilizes an injured limb during the healing process and areas near an injury that must remain dry for proper healing, such as a sutured wound, may benefit from contact with ammonia-oxidizing bacteria, such as N. eutropha.

[0221] In some embodiments, the present disclosure provides an article of manufacture comprising the N. eutropha strains described herein. The article of manufacture may be a lightweight article that can closely follow the user's body in a manner that does not impede walking. Examples of articles of manufacture include watches, wristbands, headbands, hair rubber bands, hair nets, shower caps, hats, hair pieces, and jewelry. Articles of manufacture comprising the ammonia-oxidizing bacteria described herein, such as N. eutropha strains, may be provided at a concentration that provides, for example, 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 for supplying nitric oxide to a subject, or treatment for inhibiting the growth of microorganisms, one or more of which.

[0222] In some embodiments, the ammonia-oxidizing bacteria, such as N. eutropha, are associated with products intended to come into contact with hair, such as brushes, combs, shampoos, conditioners, headbands, hair rubber bands, hair nets, shower caps, hats, and hair pieces. Nitric oxide formed on the hair away from the skin surface may be trapped in a hat, scarf, or face mask and directed into the inhaled air.

[0223] Articles that come into contact with the surface of a human, such as diapers, may be accompanied by ammonia-oxidizing bacteria, such as N. eutropha. Since diapers are designed to hold and contain urine and feces produced by incontinent individuals, urea in urine and feces can be hydrolyzed by skin and fecal bacteria to form free ammonia, which may cause irritation and diaper rash. Incorporation of bacteria that metabolize urea, such as ammonia-oxidizing bacteria, e.g., N. eutropha, into nitrite or nitrate can avoid the release of free ammonia and may release nitrite and ultimately NO, which may help maintain healthy skin for both children and incontinent adults. Nitric oxide release within the diaper may also have an antimicrobial effect on disease-causing organisms present in human feces. This effect can persist even after disposable diapers are discarded as waste and can reduce the incidence of disease transmission through contact with soiled disposable diapers.

[0224] In some embodiments, products containing ammonia-oxidizing bacteria, such as N. eutropha, are packaged. The packaging can serve to compress the product or protect it from damage, soiling, or degradation. The packaging may comprise, for example, plastic, paper, cardboard, or wood. In some embodiments, the packaging is bacteria-impermeable. In some embodiments, the packaging is oxygen and / or carbon dioxide permeable. 4. Treatment methods using ammonia-oxidizing bacteria, such as N. eutropha

[0225] The present disclosure provides various methods of treating diseases and conditions using ammonia-oxidizing bacteria, such as N. eutropha. Ammonia-oxidizing bacteria that can be used to treat diseases and conditions, such as N. eutropha, include all ammonia-oxidizing bacteria, such as the N. eutropha compositions described in this application, such as optimized ammonia-oxidizing bacteria, such as N. eutropha, for example, a purified preparation of strain D23.

[0226] Ammonia-oxidizing bacteria administered for treating a skin condition, such as acne, such as acne vulgaris, can be selected from the group consisting of Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocystis, Nitrosolobus, Nitrosovibrio, and combinations thereof. In certain embodiments, the ammonia-oxidizing bacteria can be Nitrosomonas eutropha (N. eutropha). In certain embodiments, the ammonia-oxidizing bacteria is N. eutropha D23 having ATCC accession number PTA-121157.

[0227] The method can be provided for administering or delivering a therapeutic or cosmetic product.

[0228] Ammonia-oxidizing bacteria, such as N. eutropha, can be used for treating skin conditions such as acne, rosacea, eczema, or psoriasis.

[0229] In certain embodiments, the present disclosure provides ammonia-oxidizing bacteria for treating a skin condition or disease (e.g., inhibiting the growth of microorganisms on the skin of a subject), such as (1) optimized growth rate, (2) optimized NH 4 + oxidation rate, (3) optimized NH 3 tolerance, (4) optimized NH 4 + tolerance, and (5) optimized NO 2 -Provided is the use of a composition of a pure culture of N. eutropha having one or more of the tolerances. As an example, the pure culture N. eutropha composition may have the characteristics of (1) and (2); (2) and (3); (3) and (4); or (4) and (5) from the listing at the beginning of this section. As another example, the pure culture N. eutropha composition may have the characteristics of (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) from the listing at the beginning of this section. As a further example, the optionally pure culture N. eutropha composition may have the characteristics of (1), (2), (3), and (4); (1), (2), (3), and (5); (1), (2), (4), and (5); (1), (3), (4), and (5); or (2), (3), (4), and (5) from the listing at the beginning of this section. In some embodiments, the pure culture N. eutropha composition has the characteristics of (1), (2), (3), (4), and (5) from the listing at the beginning of this section.

[0230] Ammonia-oxidizing bacteria can be used to treat skin conditions such as acne, such as acne vulgaris.

[0231] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, such as optionally pure culture N. eutropha (e.g., strain D23) are used to treat a subject. The subject may include an animal, a mammal, a human, a non-human animal, a livestock animal, or a companion animal.

[0232] The subject may be female or male. The subject may be of one of the following ethnicities / races: Asian, Black or African American, Hispanic or Latino, White, or multi-ethnic. The subject can be characterized as having at least one of the following skin types: normal skin, oily skin, and combination skin. The subject can be characterized as having one of the following Fitzpatrick skin types: I, II, III, IV, V. The type of acne of the subject may be either adolescent acne or adult acne. The age of the subject may be between about 12 - 15, 16 - 18, 19 - 28, or higher than 28.

[0233] Adult acne represents acne in subjects 19 years of age or older. Adolescent acne represents acne in subjects under 19 years of age.

[0234] In some embodiments, a method of treating a skin disorder of a subject, such as acne, such as acne vulgaris, comprising administering, such as applying, such as topically administering, a preparation comprising ammonia-oxidizing bacteria, such as ammonia-oxidizing bacteria, to the surface of the subject is provided.

[0235] The amount and / or frequency of administration, such as application, may be sufficient to reduce the amount or concentration of pathogenic bacteria, such as Propionibacterium acnes, on the surface of the subject. The amount may be a therapeutically effective dose of the ammonia-oxidizing bacteria.

[0236] Administration, such as administering, may provide treatment of inflammatory lesions. The inflammatory lesions may be in any one or more forms of papules, pustules, and cysts / nodules.

[0237] Papules and pustules can be described as eruptions caused by inflamed pores. A papule is a small elevation that appears on the skin and typically has a rough texture and feels hard to the touch. Papules can occur when the hair follicle wall breaks and collapses. Pustules are similar to papules, except that they may contain a buildup of yellowish, liquid pus. White blood cells rise to the surface of the skin of the papule to form pustules.

[0238] Blocked pores can cause more significant inflammation, grow larger, and penetrate deeper into the skin, resulting in nodules and cysts. Cysts typically form beneath the skin surface, where the buildup of white blood cells, oil, and fluid begins to construct, causing the appearance of a cyst, or an area filled with pus. Nodules are hard and may form when the bottom of the hair follicle breaks, which may cause the hair follicle to collapse. This can result in the formation of a large, painful elevation on the skin surface called a nodule. Nodules can extend deep into the skin.

[0239] Administration, for example, administering, may provide treatment for non-inflammatory lesions. Non-inflammatory lesions may be acne, for example, open comedones and / or closed comedones.

[0240] A closed comedo or whitehead is a small, plugged hair follicle whose contents are not exposed to the skin. An open comedo or blackhead is a small hair follicle with an opening to the skin that oxidizes the debris within the follicle, resulting in a black coloring.

[0241] Administration, for example, administering, may provide treatment or improvement for post-inflammatory hyperpigmentation / post-inflammatory erythema (PIH / PIE) lesions. Hyperpigmentation is characterized by darkening of skin areas caused by the overproduction of pigments, such as melanin, in the skin. Post-inflammatory refers to local skin erythema following any type of dermatitis, such as acne-related dermatitis, including erythema that can lead to scarring.

[0242] Administration, for example, administering can provide one or more treatments or improvements of erythema, edema, scaling, prickling, burning, and itching. Erythema can represent superficial redness of the skin, typically patchy, as a result of injury or irritation that causes dilation of capillaries. Edema can represent a condition characterized by an excess of aqueous fluid collected within a cavity or tissue of a subject, for example, swelling.

[0243] Administration, for example, administering can provide one or more treatments or improvements of oily appearance, appearance of pores, gloss, spots, skin color tone uniformity, visual smoothness, and tactile smoothness.

[0244] Administration, for example, administering can provide a treatment or improvement in sebumeter measurements. Sebum is an oily secretion from sebaceous glands. Sebum may be found, for example, on any body area of a subject. Administration can provide one or more of the following: reducing inflammation of a lesion, reducing the frequency of a lesion, and reducing the presence of pathogenic bacteria, for example, Propionibacterium acnes.

[0245] Administration can provide a reduction in the presence of pathogenic bacteria, for example, Propionibacterium acnes. Administration can provide an improvement in the emotional assessment of the subject's disease by the subject, as measured by the Skindex16 quality of life survey. Administration can provide one or more improvements in the following: pain of the skin condition in the subject, persistence / recurrence of the skin condition in the subject, and appearance of the skin condition in the subject. Administration can provide an improvement (reduction) in one or more of the following assessment scores by clinical evaluation: visual and tactile smoothness, and spots.

[0246] Administration of ammonia-oxidizing bacteria, such as a preparation of ammonia-oxidizing bacteria, may include pre-treating the subject with ammonia-oxidizing bacteria. For example, pre-treatment may involve administration of ammonia-oxidizing bacteria before acne or acne-related outbreaks, such as before the appearance of acne-related symptoms or conditions. Administration may include topical administration before a skin disorder, such as acne, such as acne vulgaris, occurs.

[0247] Administration, such as topical administration, may include topically administering an effective dose of ammonia-oxidizing bacteria to the subject. The effective dose may be about 0.1×10 9 , 0.2×10 9 , 0.3×10 9 , 0.4×10 9 , 0.5×10 9 , 0.6×10 9 , 0.7×10 9 , 0.8×10 9 , 0.9×10 9 , 1.0×10 9 , 1.2×10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.8×10 9 , 2.0×10 9 , 2.2×10 9 , 2.4×10 9 , 2.6×10 9 , 2.8×10 9 , 3.0×10 9 , 3.2×10 9 , 3.4×10 9 , 3.6×10 9 , 3.8×10 9 , 4.0×10 9 , 4.2×10 9 , 4.4×10 9 , 4.6×10 9 , 4.8×10 9 , 5.0×10 9 , 5.5×10 9 , 6.0×10 9 , 6.5×10 9 , 7.0×10 9 , 7.5×10 9, 8.0×10 9 , 8.5×10 9 , 9.0×10 9 , 9.5×10 9 , 10.0×10 9 , 12×10 9 , 14×10 9 , 16×10 9 , 18×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , 40×10 9 , and 50×10 9 It may be any one or more of the CFU.

[0248] The effective dose may also contain ammonia-oxidizing bacteria having the doses described herein in combination with ammonia, for example, an ammonia concentration between about 0.01 mM and about 100 mM. For example, the ammonia concentration may be about 0.01, 0.05, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, or 100.0 mM.

[0249] Skin disorders, such as acne, such as acne vulgaris, may be present at the target site of a subject that may contain one or more undesirable bacteria, such as pathogenic bacteria. The target site may contain Propionibacterium acnes.

[0250] The treatment method may further include determining whether the subject needs treatment for skin disorders. This may involve determining whether the subject needs treatment for acne, such as acne vulgaris. The method may further include selecting a subject in need of treatment for skin disorders.

[0251] In certain embodiments, the method may include selecting a subject based on the need for the subject to reduce the amount or concentration of pathogenic bacteria, such as Propionibacterium acnes, on the surface of the subject.

[0252] Administration may include self - administration of ammonia - oxidizing bacteria by the subject. Administration may include administration of ammonia - oxidizing bacteria by a person other than the subject. The ammonia - oxidizing bacteria may be administered, for example, applied to any part of the subject's body, such as the head, shoulders, arms, legs, sides, torso, feet, knees, ankles, or buttocks. In certain aspects, the ammonia - oxidizing bacteria may be applied to any one or more of the subject's face, neck, and scalp.

[0253] In some embodiments, administration includes administering, for example, applying a preparation of ammonia - oxidizing bacteria an appropriate number of times to provide an effective treatment for acne, as explained by improvement of any one or more of the measurement techniques described herein. For example, administration may occur 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. Administration may include administering, for example, applying a preparation of ammonia - oxidizing bacteria once per day. Administration may include administering, for example, applying a preparation of ammonia - oxidizing bacteria twice per day.

[0254] In some embodiments, administration of the preparation may include administering, for example, an ammonia-oxidizing bacterium, such as a preparation of an ammonia-oxidizing bacterium, for a suitable period, for example, applying it, to provide an effective treatment for acne as explained by any one or more of the measurement techniques described herein. Administration of the preparation may include administering an ammonia-oxidizing bacterium, such as a preparation of an ammonia-oxidizing bacterium, 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, for example, applying it. For example, the preparation may be applied for about 7 days. For example, the preparation may be applied for about 14 days. For example, the preparation may be applied for about 21 days. For example, the preparation may be applied for about 28 days.

[0255] The ammonia-oxidizing bacterium, such as a preparation of an ammonia-oxidizing bacterium, may be in a form that can be aerosolized, sprayed, or misted, for example, in the form of a medium, i.e., a mist. The ammonia-oxidizing bacterium, such as a preparation of an ammonia-oxidizing bacterium, can be administered, for example, applied, for example, topically, as an aerosol or a mist. The aerosol or mist may contain an aqueous medium. The preparation of the ammonia-oxidizing bacterium may be in a preparation that maintains or promotes the viability of the ammonia-oxidizing bacterium. The preparation may be in a buffer solution, for example, an aqueous buffer solution. Buffer solution, for example, aqueous buffer solution. The aqueous buffer may contain disodium phosphate and magnesium chloride. The aqueous buffer may contain 50 mM Na 2 HPO 4 and 2 mM MgCl 2 in water. The concentration of the ammonia-oxidizing bacterium may be any concentration of AOB disclosed herein. In certain embodiments, the aqueous buffer contains 50 mM Na 2 HPO 4 and 2 mM MgCl 2 in water, and the ammonia-oxidizing bacterium, for example, 1×10 9It may further contain D23 N.eutropha at a concentration of CFU / mL.

[0256] The aqueous buffer solution before the addition of ammonia-oxidizing bacteria may consist essentially of or consist of sodium dihydrogen phosphate and magnesium chloride in water, for example, 50 mM Na 2 HPO 4 and 2 mM MgCl 2 and may contain, consist essentially of, or consist of the same.

[0257] In some embodiments, the preparation may contain at least one of ammonia, ammonium salts, and urea. The preparation may contain a controlled-release material. For example, the controlled-release material may be a sustained-release material. The preparation of ammonia-oxidizing bacteria may contain an additive. The additive may be a pharmaceutically acceptable additive or a cosmetically acceptable additive as disclosed throughout the present disclosure. The additive may be a surfactant, for example, a surfactant as disclosed throughout the present disclosure. In some embodiments, the additive, for example, a pharmaceutically acceptable additive or a cosmetically acceptable additive, includes an anti-adhesive, a binder, a coating agent, a disintegrant, a filler, a fragrance, a colorant, a lubricant, a flow promoter, an adsorbent, a preservative, or a sweetener.

[0258] The preparation may substantially contain no other organisms. The preparation may contain a second organism, for example, an organism selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacter, and combinations thereof.

[0259] In some embodiments, a preparation for treating a skin condition, for example, acne, for example, acne vulgaris, may be placed in or provided as a powder, a cosmetic, a cream, a stick, an aerosol, for example, a mist, an ointment, a wipe, or a dressing. In some embodiments, a preparation for treating a skin condition, for example, acne, for example, acne vulgaris, may contain a moisturizer, a deodorant, a fragrance, a colorant, an insect repellent, a cleansing agent, or a UV-blocking agent.

[0260] In some embodiments, a preparation for treating a skin condition, such as acne, such as acne vulgaris, may contain ammonia-oxidizing bacteria having an ammonia-oxidizing bacteria concentration of about 10 8 to about 10 14 CFU / mL. In certain embodiments, the preparation may contain ammonia-oxidizing bacteria between about 1×10 9 CFU / mL and about 10×10 9 CFU / mL. In some embodiments, the preparation may contain ammonia-oxidizing bacteria between about 50 milligrams (mg) and about 1000 mg of ammonia-oxidizing bacteria. In some embodiments, the mass ratio of ammonia-oxidizing bacteria to an additive, such as a pharmaceutically acceptable additive or a cosmetically acceptable additive, ranges from about 0.1 gram to about 1 gram / liter.

[0261] A preparation of ammonia-oxidizing bacteria that can be used to treat acne may be provided in a container, and the preparation and the container have a weight of less than about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 grams.

[0262] The method may further include obtaining a sample from the skin surface. The sample can be taken at any time before the administration of the ammonia-oxidizing bacteria or at any time after the administration of the ammonia-oxidizing bacteria. For example, the sample can be taken about 1 minute, 5 minutes, 10, 15, 20, 25, 30, 45, 60, 90, 120 minutes before administration, or 3 hours, 4, 5, 6, 7, 8, 12, 18, 24 hours before administration, or 1 week, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks before administration. Alternatively or additionally, the sample can be taken about 1 minute, 5 minutes, 10, 15, 20, 25, 30, 45, 60, 90, 120 minutes after administration, or 3 hours, 4, 5, 6, 7, 8, 12, 18, 24 hours after administration, or 1 week, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks after administration.

[0263] The method may further include isolating the DNA of bacteria in the sample. The bacteria may be Propionibacterium acnes. Administration of ammonia-oxidizing bacteria provides a reduction in Propionibacterium acnes. The reduction in Propionibacterium acnes may occur about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105 days later, or after a longer number of days.

[0264] The use of the cosmetic products described herein, such as cosmetic products adapted to the microbiome, such as shampoos, conditioners, and detergents, can be used in the treatment of skin conditions, such as acne, such as acne vulgaris. These cosmetic products can be used in conjunction with the administration of ammonia-oxidizing bacteria. For example, the cosmetic products can be used over a treatment period during which ammonia-oxidizing bacteria are administered to the subject. The cosmetic products can be used during a period prior to the start of the treatment of the skin condition by administering ammonia-oxidizing bacteria to the subject. The cosmetic products can be used during a period after the start of the treatment of the skin condition by administering ammonia-oxidizing bacteria to the subject. The cosmetic products can be used during a period after the cessation of the treatment of the skin condition by administering ammonia-oxidizing bacteria to the subject.

[0265] In some embodiments, the subject may apply one or more cosmetic products and wait for a period of time prior to the administration of ammonia-oxidizing bacteria. In other embodiments, the subject may be administered ammonia-oxidizing bacteria and wait for a period of time prior to applying one or more cosmetic products.

[0266] The period of time the subject may wait may 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 applying one or more cosmetic products and prior to the administration of ammonia-oxidizing bacteria.

[0267] The period during which the subject can wait may 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 bacteria and before application of one or more cosmetic products.

[0268] The subject can be evaluated before starting a treatment, such as administration of ammonia-oxidizing bacteria. The subject can be evaluated after starting the treatment, such as after the first administration, such as application of ammonia-oxidizing bacteria.

[0269] The subject can be evaluated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 days after starting a treatment, such as administration of ammonia-oxidizing bacteria; or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 weeks; or 2, 3 years, 4, or 5 years ago.

[0270] The subject can be evaluated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 days after starting a treatment, such as the first administration, such as application of ammonia-oxidizing bacteria; or 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 weeks; or 2, 3 years, 4, or 5 years later.

[0271] Administration can occur during a period before or after the subject washes, takes a bath, or showers. For example, administration can occur at a time 30, 60, 90, 120, 150, or 180 minutes before or after the subject washes, takes a bath, or showers.

[0272] In some embodiments, ammonia-oxidizing bacteria, such as, N. eutropha, such as, optionally, N. eutropha in pure culture described herein (e.g., N. eutropha described throughout the present disclosure, e.g., strain D23) are used to inhibit the growth of other organisms. As an example, N. eutropha D23 is well-suited for long-term colonization of human skin and, in some embodiments, it expels other undesirable bacteria on the skin. Undesirable skin bacteria include, for example, those that can cause wound infections, increase the risk or severity of disease, or produce odors. Certain undesirable skin bacteria include S. aureus, P. aeruginosa, S. pyogenes, and A. baumannii. The N. eutropha described herein can expel other organisms, for example, by consuming poor nutrients or generating by-products harmful to other organisms, such as, by changing the skin pH to a level that does not aid the growth of undesirable organisms.

[0273] Accordingly, the present disclosure provides, inter alia, a method of inhibiting the growth of microorganisms on the skin of a subject, the method comprising topically administering to a human in need thereof an effective dose of an ammonia-oxidizing bacterium as described herein, such as N. eutropha, such as N. eutropha (e.g., strain D23) in pure culture, optionally. Similarly, the present disclosure provides an ammonia-oxidizing bacterium, such as N. eutropha, such as N. eutropha as described herein in pure culture, optionally, for use in inhibiting the growth of microorganisms on the skin of a subject. Likewise, the present disclosure provides the use of an ammonia-oxidizing bacterium, such as N. eutropha, such as N. eutropha as described herein in pure culture, optionally, in the manufacture of a medicament for inhibiting the growth of microorganisms on the skin of a subject.

[0274] Accordingly, the present disclosure provides, inter alia, a method of treating a skin condition, such as acne, such as acne vulgaris, the method comprising topically administering to a human in need thereof an effective dose of an ammonia-oxidizing bacterium, such as N. eutropha, such as N. eutropha (e.g., strain D23) in pure culture, optionally. Similarly, the present disclosure provides an ammonia-oxidizing bacterium, such as N. eutropha, such as N. eutropha (e.g., strain D23) as described herein in pure culture, optionally, for use in treating a skin condition, such as acne, such as acne vulgaris, on the skin of a subject. Likewise, the present disclosure provides the use of an ammonia-oxidizing bacterium, such as N. eutropha, such as N. eutropha as described herein in pure culture, optionally, in the manufacture of a medicament for treating a skin condition, such as acne, such as acne vulgaris, on the skin of a subject.

[0275] The present disclosure also provides a method for supplying nitric oxide to a subject, which includes positioning an effective dose of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, which is optionally in pure culture, in the immediate vicinity of the subject. Similarly, the present disclosure provides ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, which is optionally in pure culture, for use in supplying nitric oxide to a subject. Likewise, the present disclosure provides the use of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, which is optionally in pure culture, in the manufacture of a suitable medicament or composition at a location in the immediate vicinity of a subject.

[0276] The present disclosure also provides a method for reducing body odor, which includes topically administering an effective dose of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, which is optionally in pure culture, to a subject in need thereof. Similarly, the present disclosure provides ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, which is optionally in pure culture, for use in reducing body odor in a subject. Likewise, the present disclosure provides the use of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, which is optionally in pure culture, in the manufacture of a medicament or composition for reducing body odor.

[0277] The present disclosure also provides a method for treating or preventing a disease associated with low nitrite levels, the method comprising topically administering a therapeutically effective dose of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, optionally in pure culture, to a subject in need thereof. Similarly, the present disclosure provides a topical formulation of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, optionally in pure culture, for use in treating a disease associated with low nitrite levels. Likewise, the present disclosure provides the use of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, optionally in pure culture, in the manufacture of a topical medicament for treating a disease associated with low nitrite levels.

[0278] The present disclosure also provides a method for treating or preventing a skin disorder or skin infection, the method comprising topically administering a therapeutically effective dose of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, optionally in pure culture, to a subject in need thereof. Similarly, the present disclosure provides ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, optionally in pure culture, for use in treating a skin disorder in a subject. Likewise, the present disclosure provides the use of ammonia-oxidizing bacteria, such as N. eutropha, such as N. eutropha (e.g., strain D23) described herein, optionally in pure culture, in the manufacture of a medicament for treating a skin disorder. In a plurality of embodiments, the skin disorder is acne, rosacea, eczema, psoriasis, or urticaria; and the skin infection is impetigo.

[0279] While not wishing to be bound by theory, it has been proposed that the treatment of acne with a therapeutically effective dose of the N. eutropha bacteria (e.g., strain D23) described herein in pure culture, optionally, may involve downregulation of inflammation by NO production; and / or limitation and / or inhibition of the expansion and propagation of Propionibacterium acnes associated with acne vulgaris by acidified nitrite and NO production.

[0280] As an example, the present disclosure provides for the use of a composition of ammonia-oxidizing bacteria for treating a condition or disease (e.g., inhibiting the growth of microorganisms on the skin of a subject). In a plurality of embodiments, the ammonia-oxidizing bacteria can be used, for example, to treat chronic wounds, acne, rosacea, eczema, psoriasis, urticaria, or skin infections.

[0281] The systems and methods of the present disclosure should provide or contain contents useful for treating or preventing skin disorders, treating or preventing diseases or conditions associated with low nitrite levels, treating or preventing body odor, treating to supply nitric oxide to a subject, or treating to inhibit the growth of microorganisms.

[0282] The systems and methods of the present disclosure can provide for reducing the amount of undesirable bacteria from an environment, e.g., the surface of a subject.

[0283] The systems and methods of the present disclosure should provide or can contain contents useful for treating at least one of acne, eczema, psoriasis, urticaria, rosacea, skin infections, and wounds, e.g., infected wounds.

[0284] In some embodiments, the ammonia-oxidizing bacteria can be used to treat a subject. The subject can include an animal, a mammal, a human, a non-human animal, a livestock animal, or a companion animal.

[0285] In some embodiments, the ammonia-oxidizing bacteria described herein are used to inhibit the growth of other organisms. By way of example, the ammonia-oxidizing bacteria may be well-suited for long-term colonization of human skin and, in some embodiments, it expels other unwanted bacteria on the skin. Unwanted skin bacteria include, for example, those that can cause wound infections, increase the risk or severity of disease, or produce odors. Unwanted bacteria may be referred to as pathogenic bacteria. Certain unwanted skin bacteria include Staphylococcus aureus (S. aureus), such as methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa (P. aeruginosa), Streptococcus pyogenes (S. pyogenes), Acinetobacter baumannii (A. baumannii), Propionibacterium, and Stenotrophomonas. The ammonia-oxidizing bacteria described herein can expel other organisms, for example, by consuming limited nutrients or generating by-products that are harmful to other organisms, such as changing the skin pH to a level that does not support the growth of unwanted organisms.

[0286] Accordingly, the present disclosure provides, inter alia, a method of inhibiting the growth of microorganisms on the skin of a subject, the method comprising topically administering to a human in need thereof an effective dose of the ammonia-oxidizing bacteria described herein. Similarly, the present disclosure provides the ammonia-oxidizing bacteria described herein for use in inhibiting the growth of microorganisms on the skin of a subject. Likewise, the present disclosure provides the use of the ammonia-oxidizing bacteria in the manufacture of a medicament for inhibiting the growth of microorganisms on the skin of a subject.

[0287] The present disclosure provides, inter alia, methods of changing, e.g., modulating, e.g., modulating or changing the ratio of, the composition of the skin microbiome in an environment, e.g., on a surface, e.g., on the surface of a subject. The method may include administering, e.g., applying, a preparation comprising ammonia-oxidizing bacteria to the environment, e.g., on the surface, e.g., on the surface of a subject. In some embodiments, the amount and frequency of the administration, e.g., application, may be sufficient to reduce the ratio of pathogenic bacteria on the skin surface. In some embodiments, the subject may be selected based on the need for the subject to reduce the ratio of pathogenic bacteria on the skin surface.

[0288] The present disclosure may further provide obtaining a sample from the skin surface and isolating the DNA of bacteria in the sample. Sequencing of the DNA of bacteria in the sample can also be performed to determine or monitor the amount or ratio of bacteria in the sample of the subject.

[0289] The present disclosure may also provide increasing the ratio of non-pathogenic bacteria on the surface. In some embodiments, the non-pathogenic bacteria may be commensal non-pathogenic bacteria. In some embodiments, the non-pathogenic bacteria may be of the genus Staphylococcus. In some embodiments, the non-pathogenic bacteria may be Staphylococcus epidermidis. In some embodiments, the non-pathogenic bacteria whose ratio is increased may be of the genus Staphylococcus comprising at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% Staphylococcus epidermidis.

[0290] An increase in the ratio of non-pathogenic bacteria can occur within a predetermined period, for example, less than 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, or less than 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, 84 - 91 days.

[0291] An increase in the ratio of Staphylococcus bacteria, for example, Staphylococcus epidermidis, can be observed within less than about 3 weeks, for example, about 16 days, for example, within about 2 weeks.

[0292] The present disclosure may provide for reducing the ratio of pathogenic bacteria, for example, potential pathogenic bacteria, for example, disease-related bacteria, on a surface. In some embodiments, the pathogenic bacteria may be Propionibacterium. In some embodiments, the pathogenic bacteria may be Stenotrophomonas.

[0293] A decrease in the ratio of pathogenic bacteria can occur within a predetermined period, for example, less than 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, or 4 weeks, or less than 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, 84 - 91 days.

[0294] A decrease in the ratio of Propionibacterium bacteria and / or Stenotrophomonas can be observed within less than about 3 weeks, for example, about 16 days, for example, within about 2 weeks.

[0295] The present disclosure also provides a method for supplying nitric oxide to a subject, the method comprising positioning an effective dose of the ammonia-oxidizing bacteria described herein in the immediate vicinity of the subject. Similarly, the present disclosure provides the ammonia-oxidizing bacteria described herein for use in supplying nitric oxide to a subject. Likewise, the present disclosure provides for use in the manufacture of a suitable medicament or composition at a location in the immediate vicinity of the subject.

[0296] The present disclosure also provides a method for reducing body odor, the method comprising topically administering an effective dose of the ammonia-oxidizing bacteria described herein to a subject in need thereof. Similarly, the present disclosure provides the ammonia-oxidizing bacteria described herein for use in reducing body odor in a subject. Likewise, the present disclosure provides for use of the ammonia-oxidizing bacteria described herein in the manufacture of a medicament or composition for reducing body odor.

[0297] The present disclosure also provides a method for treating or preventing a disease associated with low nitrite levels, the method comprising topically administering a therapeutically effective dose of the ammonia-oxidizing bacteria described herein to a subject in need thereof. Similarly, the present disclosure provides a topical formulation of the ammonia-oxidizing bacteria described herein for use in treating a disease associated with low nitrite levels. Likewise, the present disclosure provides for use of the ammonia-oxidizing bacteria described herein in the manufacture of a topical medicament for treating a disease associated with low nitrite levels.

[0298] The present disclosure also provides a method for treating or preventing a skin disorder or skin infection, the method comprising topically administering a therapeutically effective dose of the ammonia-oxidizing bacteria described herein to a subject in need thereof. Similarly, the present disclosure provides the ammonia-oxidizing bacteria described herein for use in treating a skin disorder in a subject. Likewise, the present disclosure provides for use of the ammonia-oxidizing bacteria described herein in the manufacture of a medicament for treating a skin disorder. In a plurality of embodiments, the skin disorder is acne, rosacea, eczema, psoriasis, or urticaria; and the skin infection is impetigo.

[0299] While not wishing to be bound by theory, it has been proposed that the treatment of rosacea with a therapeutically effective dose of ammonia-oxidizing bacteria, such as, optionally, the N. eutropha bacteria (e.g., strain D23) described herein in pure culture, may be accompanied by downregulation by NO generation. This is achieved by the expression of a Kazal-type KLK5 / KLK7 inhibitor that can reduce the formation of the human cathelicidin peptide LL-37 from its precursor propeptide hCAP18.

[0300] While not wishing to be bound by theory, it has been proposed that the treatment of eczema and / or atopic dermatitis with a therapeutically effective dose of ammonia-oxidizing bacteria, such as, optionally, the N. eutropha bacteria (e.g., strain D23) described herein in pure culture, downregulates inflammation by NO generation; and / or limits and / or inhibits the spread and growth of S. aureus and other skin pathogens, which are often associated with a very high colonization rate and skin burden in atopic dermatitis, by nitrite acidification and NO production.

[0301] While not wishing to be bound by theory, it has been proposed that the treatment of psoriasis with a therapeutically effective dose of ammonia-oxidizing bacteria, such as, optionally, the N. eutropha bacteria (e.g., strain D23) described herein in pure culture, may be accompanied by downregulation of inflammation by NO generation and reduced formation of the human cathelicidin peptide LL-37.

[0302] While not wishing to be bound by theory, it has been proposed that the treatment of psoriasis with a therapeutically effective dose of ammonia-oxidizing bacteria, such as, optionally, the N. eutropha bacteria (e.g., strain D23) described herein in pure culture, may be accompanied by downregulation of inflammation by NO generation.

[0303] While not wishing to be bound by theory, it has been proposed that the treatment of impetigo or other skin and soft tissue infections with a therapeutically effective dose of ammonia-oxidizing bacteria, such as the N. eutropha bacteria (e.g., strain D23) described herein in pure culture, if optionally so, may involve limiting and / or inhibiting the spread and growth of S. aureus and S. pyogenes.

[0304] The present disclosure also provides a method of promoting wound healing, the method comprising administering to the wound an effective dose of ammonia-oxidizing bacteria, such as the N. eutropha bacteria (e.g., strain D23) described herein in pure culture, if optionally so. Similarly, the present disclosure provides ammonia-oxidizing bacteria, such as N. eutropha (e.g., strain D23) described herein in pure culture, if optionally so, for use in treating a wound. Likewise, the present disclosure provides the use of ammonia-oxidizing bacteria, such as N. eutropha (e.g., strain D23) described herein in pure culture, if optionally so, in the manufacture of a medicament or composition for treating a wound.

[0305] Ammonia-oxidizing bacteria, such as N. eutropha (e.g., strain D23) described herein in pure culture, if optionally so, may be used to promote wound healing in patients with a reduced ability to heal, such as diabetic patients.

[0306] In some embodiments, the present disclosure provides a method of using ammonia-oxidizing bacteria, such as N. eutropha (e.g., strain D23) described herein in pure culture, if optionally so, to prevent a disease or disorder, such as a skin disorder. In certain embodiments, prevention means reducing the risk that a subject will develop the disease as compared to a similar untreated subject. It is not necessary to reduce the risk to zero.

[0307] In some embodiments, the present disclosure provides a method of treating a wound by applying a dressing comprising N. eutropha to the wound. Also provided is a method of making such a dressing. The dressing may include, for example, an adhesive portion for securing the dressing to the uninjured skin near the wound and a soft and flexible portion for covering or overlaying the wound. In some embodiments, the dressing does not contain organisms other than N. eutropha. The dressing may be made of a permeable material that allows gases such as oxygen and carbon dioxide to reach the N. eutropha when the dressing is applied to the wound. In certain embodiments, the dressing contains nutrients for N. eutropha, such as ammonium, ammonia, urea, or trace minerals. In certain embodiments, the dressing contains an antibiotic to which N. eutropha is resistant. The antibiotic resistance may be due to one or more endogenous resistance genes or one or more introduced genes.

[0308] In some embodiments, the ammonia-oxidizing bacterium, e.g., N. eutropha, is administered at a dose of about 10 8 ~10 9 CFU, 10 9 ~10 10 CFU, 10 10 ~10 11 CFU, or 10 11 ~10 12 CFU per application. In some embodiments, the ammonia-oxidizing bacterium, e.g., N. eutropha, is topically administered at a dose of about 10 10 ~10 11 CFU, e.g., about 1 × 10 10 ~5 × 10 10 , 1 × 10 10 ~3 × 10 10 , or 1 × 10 10 ~2 × 10 10 CFU. In some embodiments, the ammonia-oxidizing bacterium, e.g., N. eutropha, is topically administered at a dose of about 2 × 10 9 , 4 × 10 9 or 8 × 10 9 CFU.

[0309] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered in a volume of about 1-2, 2-5, 5-10, 10-15, 12-18, 15-20, 20-25, or 25-50 ml per dose. In some embodiments, the solution is about 10 8 ~10 9 、10 9 ~10 10 、or 10 10 ~10 11 CFU / ml in concentration. In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered twice daily at a dose of 15 ml, and each dose is 10 9 CFU / ml in concentration. In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered at a concentration of 2×10 9 、4×10 9 or 8×10 9 CFU / L. In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered at a concentration of 2×10 9 、4×10 9 or 8×10 9 CFU / mL.

[0310] In some embodiments, the preparation of ammonia-oxidizing bacteria may contain ammonia-oxidizing bacteria having the dosages described herein in combination with an ammonia concentration, for example, between about 0.01 mM and about 100 mM. For example, the ammonia concentration may be about 0.01, 0.05, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, or 100.0 mM.

[0311] In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered once, twice, three times, or four times per day. In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered once, twice, three times, four times, five times, or six times per week. In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered immediately after bathing. In some embodiments, ammonia-oxidizing bacteria, such as N. eutropha, are administered immediately before bedtime.

[0312] In certain aspects, the disclosure provides a combination therapy comprising ammonia-oxidizing bacteria, such as N. eutropha, and a second therapeutic agent. By way of example, the disclosure provides a physical mixture of two (or more) therapies. In other embodiments, two (or more) therapies are administered in combination as separate formulations. The second therapeutic agent may be, for example, a pharmaceutical agent, a surgical procedure, or any other medical approach for treating a related disease or disorder. The following sections describe possible combination therapies for chronic wounds, acne, rosacea, eczema, and psoriasis.

[0313] In a combination therapy capable of treating chronic wounds, the second therapy may be, for example, an antibiotic (e.g., topical or systemic, and bactericidal or bacteriostatic), such as penicillin, cephalosporin, polymyxin, rifamycin, lipiarmycin, quinolone, sulfonamide, macrolide, lincosamide, tetracycline, cyclic lipopeptide, glycylcycline, oxazolidinone, and lipiarmycin; an angiotensin, an angiotensin analog; debriding man; drainage; wound irrigation; negative pressure wound therapy; heat application; arterial revascularization; hyperbaric oxygen therapy; an antioxidant, such as ascorbic acid, glutathione, lipoic acid, carotene, α-tocopherol, or ubiquinol; low-level laser therapy; gastrocnemius recession; a growth factor, such as vascular endothelial growth factor, insulin-like growth factor 1-2, platelet-derived growth factor, transforming growth factor-β, or epidermal growth factor; the application of autologous platelets, such as those secreting one or more growth factors such as vascular endothelial growth factor, insulin-like growth factor 1-2, platelet-derived growth factor, transforming growth factor-β, or epidermal growth factor; the transplantation of cultured keratinocytes; allograft; a collagen, such as a coating material containing collagen; or a protease inhibitor such as SLPI. The combination therapy may include one or more of the above treatments.

[0314] In a combination therapy capable of treating acne, the second treatment method may include, for example, medication (e.g., systemic or topical), such as benzoyl peroxide, antibiotics (such as erythromycin, clindamycin, or tetracycline), salicylic acid, hormones (e.g., progestins, including desogestrel, norgestimate, or drospirenone), retinoids, such as tretinoin, adapalene, tazarotene, or isotretinoin. The second treatment method may be a procedure such as acne extraction, corticosteroid injection, or surgical incision. The combination therapy may include one or more of the above treatments. The combination therapy may include the treatment of acne selected from the group consisting of one or more of the treatment methods described in Table 1 above, such as topical retinoids, azelaic acid, salicylic acid, topical antimicrobials, oral antibiotics, benzoyl peroxide, oral antiandrogens, oral isotretinoin, and combinations thereof.

[0315] By way of example, the present disclosure provides a physical mixture of two (or more) treatment methods. In other embodiments, two (or more) treatment methods are administered in combination as separate formulations. These treatment methods may be administered as "simultaneous" or "combined" 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 "sequential" or "sequential delivery". The start of a treatment or treatment method may start before or after the subject is diagnosed with a skin condition or disorder. The start of treatment with ammonia-oxidizing bacteria can be started when there is an insufficient response to another treatment. The method may include starting the administration of ammonia-oxidizing bacteria and stopping and / or continuing the use of the other treatment.

[0316] Treatment methods, such as the treatment of acne, such as the treatments listed in Table 1 above, can be administered during the period before starting the administration of ammonia-oxidizing bacteria. Treatment methods, such as the treatment of acne, such as the treatments listed in Table 1 above, can be continued throughout the administration period of ammonia-oxidizing bacteria. In certain embodiments, treatment methods, such as the treatment of acne, such as the treatments listed in Table 1 above, can be discontinued during the administration of ammonia-oxidizing bacteria. Treatment methods, such as the treatment of acne, such as the treatments listed in Table 1 above, can be continued throughout the entire administration of ammonia-oxidizing bacteria. Treatment methods, such as the treatment of acne, such as the treatments listed in Table 1 above, can be started following the discontinuation of the administration of ammonia-oxidizing bacteria.

[0317] In combination therapies capable of treating rosacea, the second treatment method may be, for example, an antibiotic, such as an oral tetracycline antibiotic, such as tetracycline, doxycycline, or minocycline, or a topical antibiotic such as metronidazole; azelaic acid; alpha-hydroxy acid; isotretinoin may be prescribed; acne oil; clonidine; beta-blockers such as nadolol and propranolol; antihistamines (such as loratadine); mirtazapine; optionally methylsulfonylmethane or silymarin combined with each other; a dermatological vascular laser or a laser such as a CO 2 laser; or may include phototherapy such as intense pulsed light, low-level light therapy or photo rejuvenation. The combination therapy may include one or more of the above treatments.

[0318] In combination therapies capable of treating eczema, the second treatment method may include, for example, corticosteroid injections such as hydrocortisone or clobetasol propionate, immunosuppressants (topical or systemic), such as pimecrolimus, tacrolimus, cyclosporine, azathioprine or methotrexate, or phototherapy such as using ultraviolet light. The combination therapy may include one or more of the above treatments.

[0319] In a combination therapy capable of treating psoriasis, the second therapy may include, for example, corticosteroid injections such as desoxymethasone; retinoids; coal tar; vitamin D or its analogs, such as paricalcitol or calcipotriol; moisturizers and emollients, such as mineral oil, petrolatum, calcipotriol, decubal, or coconut oil; dithranol; or fluocinonide. The combination therapy may include one or more of the above treatments.

[0320] While not wishing to be bound by theory, it has been proposed that the treatment of psoriasis using a therapeutically effective dose of ammonia-oxidizing bacteria described herein may be accompanied by downregulation of inflammation by reduction in NO generation and formation of the human cathelicidin peptide LL-37.

[0321] While not wishing to be bound by theory, it has been proposed that the treatment of psoriasis using a therapeutically effective dose of ammonia-oxidizing bacteria described herein may be accompanied by downregulation of inflammation by NO generation.

[0322] While not wishing to be bound by theory, it has been proposed that the treatment of impetigo or other skin and soft tissue infections using a therapeutically effective dose of ammonia-oxidizing bacteria described herein may be accompanied by limiting and / or inhibiting the expansion and proliferation of Staphylococcus aureus (S. aureus), such as methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa (P. aeruginosa), Streptococcus pyogenes (S. pyogenes), Acinetobacter baumannii (A. baumannii), Propionibacterium, and Stenotrophomonas.

[0323] The present disclosure also provides a method for promoting wound healing, which includes administering an effective dose of the ammonia-oxidizing bacteria described herein to a wound. Similarly, the present disclosure provides the ammonia-oxidizing bacteria described herein for use in treating a wound. Likewise, the present disclosure provides the use of the ammonia-oxidizing bacteria described herein in the manufacture of a medicament or composition for treating a wound.

[0324] In some embodiments, the present disclosure provides a method of using the ammonia-oxidizing bacteria described herein to prevent a disease or disorder, such as a skin disorder. In certain embodiments, prevention means reducing the risk that a subject will develop the disease as compared to a similar untreated subject. The risk need not be reduced to zero.

[0325] Individuals with reduced bathing frequency, such as astronauts, submarine crew members, military personnel during military operations, civilian workers in remote areas, refugees, bedridden individuals, and many others, may maintain healthier skin by maintaining ammonia-oxidizing bacteria on the skin. With respect to bedridden individuals, in some embodiments, the ammonia-oxidizing bacteria reduce the frequency or severity of pressure ulcers by enhancing poor circulation.

[0326] It has been recognized that many modern degenerative diseases can be caused by a lack of NO species, that ammonia-oxidizing bacteria on the external skin can supply NO species by diffusion, and that application of ammonia-oxidizing bacteria to the skin can restore a long-term medical condition. In certain embodiments, in particular, ammonia-oxidizing bacteria are applied to a subject to counteract modern bathing habits using anionic detergents that remove ammonia-oxidizing bacteria from the external skin.

[0327] One suitable method of topical application is to apply sufficient ammonia-oxidizing bacteria and then wear sufficient clothing to induce sweating. However, many people would like to maintain their current bathing habits while also reaping the benefits of ammonia-oxidizing bacteria, in which case a culture of the bacteria can be applied along with a substrate sufficient for the bacteria to produce NO. A nutrient solution similar to the inorganic composition of human sweat can be used for this purpose. Using bacteria adapted to a medium similar to human sweat minimizes the time for adaptation when the bacteria are applied. Since sweat evaporates after being discharged onto the skin surface, it is desirable to use a culture medium with a higher ionic strength. A concentration about twice that of human sweat is appropriate, although other conditions are also considered. The nutritional requirements of ammonia-oxidizing bacteria typically include NH 3 or urea, O 2 , CO 2 , and inorganic substances. In some embodiments, the substrate includes trace inorganic substances including iron, copper, zinc, cobalt, molybdenum, manganese, sodium, potassium, calcium, magnesium, chloride, phosphate, sulfate, or any combination thereof.

[0328] In some embodiments, the present disclosure provides a method of treating a wound by applying a bandage containing ammonia-oxidizing bacteria to the wound. A method of making such a bandage is also provided. The bandage may include, for example, an adhesive portion for securing the bandage to the uninjured skin near the wound and a soft and flexible portion for covering or overlapping the wound. In some embodiments, the bandage does not contain organisms other than ammonia-oxidizing bacteria. The bandage may be made of a permeable material that allows gases such as oxygen and carbon dioxide to reach the ammonia-oxidizing bacteria when the bandage is applied to the wound. In certain embodiments, the bandage contains nutrients for ammonia-oxidizing bacteria, such as ammonium, ammonia, urea, or trace inorganic substances. In certain embodiments, the bandage contains an antibiotic to which the ammonia-oxidizing bacteria are resistant. Antibiotic resistance can be due to one or more endogenous resistance genes or one or more introduced genes.

[0329] In some embodiments, ammonia-oxidizing bacteria, for example, a preparation of ammonia-oxidizing bacteria, is administered at a dose of about 10 8 - 10 9 CFU, 10 9 - 10 10 CFU, 10 10 - 10 11 CFU, or 10 11 - 10 12 CFU per application or per day. In some embodiments, the ammonia-oxidizing bacteria are topically administered at a dose of about 10 9 - 10 10 CFU, for example, about 1×10 9 - 5×10 9 , 1×10 9 - 3×10 9 , or 1×10 9 - 10×10 9 CFU.

[0330] In some embodiments, the ammonia-oxidizing bacteria are administered in a volume of about 1 - 2, 2 - 5, 5 - 10, 10 - 15, 12 - 18, 15 - 20, 20 - 25, or 25 - 50 ml per dose. In some embodiments, the solution has a concentration of about 10 8 - 10 9 , 10 9 - 10 10 , or 10 10 - 10 11 CFU / ml. In some embodiments, the ammonia-oxidizing bacteria are administered twice daily at a dose of 15 ml per day, and each dose has a concentration of 10 9 CFU / ml.

[0331] In some embodiments, the ammonia-oxidizing bacteria are administered 1, 2, 3, or 4 times per day. In some embodiments, the ammonia-oxidizing bacteria are administered 1, 2, 3, 4, 5, or 6 times per week. In some embodiments, the ammonia-oxidizing bacteria are administered immediately after bathing. In some embodiments, the ammonia-oxidizing bacteria are administered immediately before bedtime.

[0332] In some embodiments, the ammonia-oxidizing bacteria are 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, 84 - 91 days, for example, about 1 month, about 2 months, about 3 months. In some embodiments, the ammonia-oxidizing bacteria are administered for an indefinite period, for example, longer than 1 year, longer than 5 years, longer than 10 years, longer than 15 years, longer than 30 years, longer than 50 years, longer than 75 years. 5. Use of microbiome-compatible cosmetics, such as detergents, shampoos, and / or conditioners, with administration of ammonia-oxidizing bacteria

[0333] The systems and methods of the present disclosure provide, among other things, finished cosmetic products that can be considered, for example, "biome-safe" or "biome-compatible." The systems and methods of the present disclosure may provide for the use of cosmetic products, for example, finished cosmetic products, that can be used in combination with bacteria, such as non-pathogenic bacteria, such as ammonia-oxidizing bacteria, which may be used in the form of a preparation or composition to be applied to a subject. Table 4 provides a listing of components that can be provided in one or more of a preparation or composition and that can provide a product that is biome-safe or biome-compatible.

Table 4-1

Table 4-2

Table 4-3

[0334] 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 eye brow pencils, eye liners, eye shadows, eye lotions, eye makeup removers, mascaras; fragrance preparations such as colognes, eau de toilettes, perfumes, powders (dusting and talcum), sachets; hair preparations such as hair conditioners, hair sprays, hair straighteners, permanent wave agents, rinses, shampoos, tonics, hair dressings, hair styling aids, wave set agents; hair coloring preparations such as hair dyes and hair colors, hair bleaches, colored hair rinses, colored hair shampoos, colored hair lighteners, hair bleaches; makeup preparations such as face powders, foundations, leg and body paints, lipsticks, makeup bases, blushes, makeup setting agents; manicure preparations such as base coats and undercoats, cuticle softeners, nail creams and lotions, nail extenders, nail polishes and enamels, nail polish and enamel removers; oral hygiene products such as toothpastes, mouthwashes and breath fresheners; bath soaps such as foaming body washes, and detergents, deodorants, bidets, feminine hygiene deodorants; shaving preparations such as aftershave lotions, beard softeners, talcum, pre-shave lotions, shaving creams, shaving soaps; skin care preparations such as cleansing, depilatory agents, face and neck, body and hand, foot powders and sprays, moisturizers, night preparations, paste masks, skin fresheners; and sunburn preparations such as gels, creams, liquids, and indoor tanning preparations, and may be any one or more of, or include, or be disposed within, the foregoing.

[0335] The cosmetic products may include any one or more of the components disclosed herein, such as those disclosed in Table 4.

[0336] The cosmetic product or finished cosmetic product may comprise, consist essentially of, or consist of the following as shown in Table 5 below: [Table 5] and may contain, consist essentially of, or consist of the composition of.

[0337] The product can be used as a cosmetic product, for example, for shampoo, or for body wash. The product contains water to make it 100%. In some embodiments, the cosmetic product, such as shampoo, may or may not contain citric acid, and citric acid may be required if pH stabilization is required or desired.

[0338] The cosmetic product or finished cosmetic product may have the following composition as shown in Table 6 below: [Table 6] and may contain, consist essentially of, or consist of the composition of.

[0339] The product can be used as a cosmetic product, for example, for a detergent, or for the body, hands, or face. The product contains water to make it 100%. In some embodiments, the cosmetic product, such as a detergent, may or may not contain citric acid, and citric acid may be required if pH stabilization is required or desired.

[0340] Other hydrolyzed proteins may be used, including, but not limited to, rice, soy, baobab, and amaranth. Other fragrance alternatives can be considered.

[0341] The finished cosmetic product may have one or more, or all, of the characteristics described herein.

[0342] The following as shown in Table 7 below: [Table 7] Other products containing, consisting essentially of, or consisting of a hair and / or skin conditioner are contemplated.

[0343] The product contains water for 100%. In some embodiments, the cosmetic product, e.g., the conditioner, may or may not contain citric acid, and citric acid may be required when pH stabilization is required or desired. 6. Mechanism of Therapeutic Benefit

[0344] While not wishing to be bound by theory, one or more of the following mechanisms are thought to underlie the beneficial effects of ammonia-oxidizing bacteria, e.g., N. eutropha, in the treatment of the diseases and conditions discussed herein. Additional mechanistic details are found in International Application WO / 2005 / 030147, which is hereby incorporated by reference in its entirety.

[0345] To understand the beneficial aspects of these bacteria, it helps to understand angiogenesis. All somatic cells except those in the outer atmosphere hundreds of microns away receive all their metabolic oxygen from the blood supply. Oxygen is absorbed by the blood in the lungs, carried by red blood cells as oxyhemoglobin to peripheral tissues, where it is exchanged for carbon dioxide, which is carried back and exhaled from the lungs. Oxygen must diffuse through the plasma from the red blood cells, through the endothelium, and through various tissues until it reaches the mitochondria within the cells that consume oxygen. Since the human body contains approximately 5 liters of blood, the volume of the circulatory system is small compared to the volume of the body. Oxygen is not actively transported. Oxygen diffuses passively down a concentration gradient from the air to the red blood cells, from the red blood cells to the cells, and from the cells to cytochrome oxidase where oxygen is consumed. The oxygen concentration at the site of consumption is the lowest in the body, O 2Flow is determined by diffusion resistance and concentration gradients. Achieving sufficient oxygen supply to all peripheral tissues requires exquisite control of capillary size and location. If the capillary spacing increases, achieving the same flow of oxygen will require a greater concentration difference and thus a lower O 2 concentration at cytochrome oxidase. If there are more cells between capillaries, the demand for O 2 will be greater. If the capillary spacing decreases, the space available to the cells performing the metabolic functions of the organ will be smaller.

[0346] In certain embodiments, it has been recognized that NO from ammonia-oxidizing bacteria is readily absorbed by the skin and converted to S-nitrosothiols since the skin does not contain hemoglobin. M. Stucker et al. showed in “The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis. (Journal of Physiology (2002), Volume 538.3, pages 985 - 994)” that the outer skin receives all of its oxygen from the outside air. This can be readily understood since it can be seen that the outer skin essentially does not contain red blood cells. These layers are alive and actually require other nutrients in the blood besides oxygen, so there is plasma circulation through these layers. The formed S-nitrosothiols are stable and can diffuse throughout the body, constituting a major source of authentic NO and a source of NO for transnitrosylation of protein thiols.

[0347] In some embodiments, capillary rarefaction may be one of the first signs that NO levels are insufficient. F. T. Tarek et al. showed that sparse capillaries, i.e., capillary rarefaction is commonly seen in people with essential hypertension (Structural Skin Capillary Rarefaction in Essential Hypertension. Hypertension. 1999; 33: 998 - 1001.

[0348] A very large number of conditions are associated with the density of capillaries that are becoming increasingly sparse. Hypertension is one of them, and researchers have reported that sparse capillaries are seen in children of people with essential hypertension as well as in people with diabetes. The major complications of diabetes are hypertension, diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy. R. Candido et al. found that the latter two conditions are characterized by a reduction in blood flow to the affected area before the symptoms are observed (Haemodynamics in microvascular complications in type 1 diabetes. Diabetes Metab Res Rev 2002; 18: 286 - 304). As shown by A Philip et al. in "Effect of Weight Loss on Muscle Fiber Type, Fiber Size, Capillarity, and Succinate Dehydrogenase Activity in Humans. The Journal of Clinical Endocrinology & Metabolism, 84(11): 4185 - 4190, 1999, reduced capillary density is associated with obesity, and simply losing weight increases capillary density.

[0349] The researchers showed that in primary Raynaud's phenomenon (PRP), the nailfold capillaries are sparser (slightly) than in normal controls and denser than in patients who progressed to systemic sclerosis (SSc). M. Bukhari, Increased Nailfold Capillary Dimensions In Primary Raynaud's Phenomenon And Systemic Sclerosis. British Journal of Rheumatology, 24(35): 1127 - 1131, 1996. They found that the capillary density is 35 loops / mm2 It was found to decrease from 33 (PRP) to 17 (SSc) from the (normal control). The average distance between capillary limbs was 18 μ, 18 μ, and 30 μ for the control, PRP, and SSc, respectively. respectively.

[0350] In certain embodiments, it is recognized that the mechanisms normally used by the body to sense "hypoxia" can affect the body systems that regulate capillary density. According to this embodiment of the invention, a key component of "hypoxia" is sensed not by a decrease in O2 levels, but rather by an increase in NO levels. A decrease in the basal level of NO interferes with this "hypoxia" sensing and thus affects numerous body functions regulated by "hypoxia". For example, anemia is typically defined as "insufficient hemoglobin", and one consequence of insufficient hemoglobin is "hypoxia", which is defined as "insufficient oxygen". According to some embodiments, these common definitions do not account for the manner in which both conditions are mediated by nitric oxide.

[0351] Acute isovolemic anemia is well tolerated at rest. A two-thirds reduction in hematocrit has a minimal effect on venous return PvO2 and shows no reduction in either systemic O 2 pressure or delivery. Weiskopf et al., Human cardiovascular and metabolic response to acute, severe isovolemic anemia. JAMA 1998, 279(3):217 - 221. At a 50% reduction (from 140 to 70 g Hb / L), the mean PvO2 (in over 32 subjects) decreased from approximately 77% (saturated) to approximately 74%. The O 2The reduction in volume is compensated for by vasodilation and tachycardia, and the heart rate increases from 63 to 85 bpm. It is easy to see that the compensation is effective, but the mechanism is unknown. A typical explanation is that a "hypoxia" sensor detects "hypoxia" and compensates with vasodilation and tachycardia. However, "hypoxia" could not be detected. There was a slight decrease in blood lactate (a marker of anaerobic respiration) from 0.77 to 0.62 mM / L, indicating less anaerobic respiration and less "hypoxia". The 3% reduction in venous return PvO2 is at the same level as the "hypoxia" that would be obtained by ascending to an altitude of 300 meters (which typically does not produce tachycardia). The O 2 concentration remains the same, and since the O 2 consumption remains the same, there is no location in the body where the O 2 concentration is reduced. The compensation during isovolemic anemia cannot be caused by O 2 sensing.

[0352] Therefore, the vasodilation observed in acute isovolemic anemia may be caused by an increase in NO concentration in the vessel wall. NO mediates the dilation of blood vessels in response to shear stress and other factors. Since the production rate of NO remains constant and continues to be equal to the decomposition rate, no change will be observed in the levels of NO metabolites. The lack of observed compensation for "hypoxia" with the replacement of metHb can be understood because metHb binds to NO in the same way as Hb, so the increase in NO concentration that occurs when Hb is removed does not occur with the replacement of metHb.

[0353] Nitric oxide plays a role in a number of metabolic pathways. It has been suggested that basal levels of NO exert an inhibitory response on tone and that a reduction in these basal levels leads to the disinhibition of those pathways. Zanzinger et al. showed that NO inhibits the basal tone of the sympathetic nervous system and weakens excitatory reflexes It has been reported that... (Inhibition of basal and reflex-mediated sympathetic activity in the RVLM by nitric oxide. Am. J. Physiol. 268, (Regulatory Integrative Comp. Physiol. 37): R958 - R962, 1995).

[0354] In some embodiments, a component of the major source of NO is recognized to be low molecular weight S - nitrosothiols produced in erythrocyte - free skin from NO produced on the outer skin by ammonia - oxidizing bacteria. These low molecular weight S - nitrosothiols are long - term stable and can freely diffuse and circulate in the plasma. Various enzymes can cleave NO from various S - nitrosothiols, releasing NO to the enzyme side. The loss of this major source of NO from AOB on the skin results in disruption of normal physiological functions. The advantage to the body of using S - nitrosothiols to generate NO far from capillaries is that 2 it does not require O. The generation of NO from nitric oxide synthase (NOS) actually 2 requires O. If background S - nitrosothiols are sufficient, NO may be generated even in anoxic regions. Since NO exerts its effects only when bound to another molecule such as the thiol of a cysteine residue or iron in heme, free NO is also unnecessary. Thus, if S - nitrosothiols and trans - nitrosation enzymes are present, the effects of NO can be mediated by the trans - nitrosation reaction even in the absence of free NO.

[0355] Frank et al. showed that angiogenesis associated with normal wound healing is partially generated by the increased VEGF induction by nitric oxide. (Nitric oxide triggers ​enhanced induction of vascular endothelial growth factor expression in (2002) and during cutaneous wound repair. FASEB J. Vol. 13, pp. 2002-2014 (1999).

[0356] NO has a role in carcinogenesis, indicating that the bacteria described herein may be used in methods of treating and preventing cancer. According to certain embodiments, it is recognized that the presence of NO during hypoxia can prevent cells from dividing while under hypoxic stress, when cells are at greater risk of miscopying DNA. One related cellular function is the regulation of the cell cycle, which is the regulatory program that controls how and when cells replicate their DNA, assemble into duplicated chromosomes, and divide. Cell cycle regulation is extremely complex and not fully understood. However, it is known that there are numerous points along the path of the cycle at which the cell cycle can be arrested and division ceased until conditions for cell division improve. The p53 tumor suppressor protein is a key protein in the regulation of the cell cycle, helping to initiate both cell arrest and apoptosis from a variety of cellular stress signals, including DNA damage, and is described by Ashcroft et al. in "Stress As reported in "Signals Utilize Multiple Pathways To Stabilize p53." (Molecular And Cellular Biology, May 2000, pp. 3224 - 3233), p53 is mutated in more than half of human cancers. Although hypoxia initiates p53 accumulation and hypoxia is important for regulating the cell cycle, hypoxia alone cannot induce the expression downstream of the p53 mRNA effector protein and thus cannot cause cell cycle arrest. Goda et al. reported that hypoxia-inducible factor-1 (HIF-1α) is required for hypoxia to induce cell arrest. (Hypoxia-Inducible Factor 1α Is Essential for Cell Cycle Arrest during Hypoxia. Molecular And Cellular Biology, January 2003, pp. 359 - 369). Britta et al. reported that NO is one of the major stimuli for HIF-1α. (Accumulation of HIF-1a under the influence of nitric oxide. Blood, February 15, 2001, Vol. 97, No. 4). In contrast, NO actually causes the accumulation of transcriptionally active p53, actually causes cell cycle arrest, and actually causes apoptosis. Wang et al., P53 Activation By Nitric Oxide Involves Down-Regulation Of Mdm2. THE JOURNAL OF BIOLOGICAL CHEMISTRY, 277 Vol. 18, Issued on May 3, pp. 15697 - 15702, 2002.

[0357] In certain aspects of the present invention, it is recognized that preventing cell necrosis by preventing capillary rarefaction leading to hypoxic cell death may prevent autoimmune disorders. When cells are chronically exposed to hypoxia, the production of reactive oxygen species (ROS) increases, leading to increased damage to the cell's metabolic machinery and ultimately to the cell's DNA. The decrease in metabolic capacity reduces the ability to repair damage caused by ROS and exposure to exogenous carcinogens. Over time, damage accumulates, increasing the likelihood of three events: the cell undergoes deletion of a cancer-preventing gene, the cell becomes cancerous, and the cell dies by necrosis or apoptosis. When cells die by either necrosis or apoptosis, the cell debris must be removed from the site. Dead cells are phagocytosed by immune cells, including dendritic cells and macrophages. When these cells phagocytose the body, the body is digested by various proteolytic enzymes into antigenic fragments, which then attach to the major histocompatibility complex (MHC1, MHC2), and the antigen-MHC complex migrates to the cell surface, where the complex can interact with T cells and activate the T cells in various ways. Any type of cell injury releases adjuvants that stimulate the immune system in various ways. Generally, cells undergoing necrosis stimulate a greater immune response than cells undergoing apoptosis. Thus, chronic exposure of immune cells to dead and dying cells can lead to autoimmune disorders.

[0358] In certain aspects, it is recognized that low basal NO leads to fibrotic hypertrophy. After dead cells are removed, sufficient O is required to support new cells 2Therefore, new cells cannot easily replace them. All such new cells will encounter the same fate. The gap may remain empty, in which case the organ will atrophy, the capillaries will come closer, and since the new cells now lack the VEGF previously produced by the currently missing cells, the capillaries will be severed and a hypoxic zone will reform. This may lead to general atrophy of the diseased tissue. In tissues that support fibrosis, relatively inactive collagen fibers may fill this gap. Since the metabolic requirements of the body for the specific organ in question are not reduced, the organ may attempt to grow larger but now has a significant fibrous content. This can lead to fibrotic hypertrophy in organs such as the heart and liver. Some organs such as the brain cannot grow larger or smaller because the three-dimensional connectivity of nerves and blood vessels is important, and it cannot be mapped continuously and simultaneously to an asymmetrically atrophied brain. Something must fill the gap, and β-amyloid could be a (not so inactive) gap-filling substance. The kidneys cannot grow larger due to the renal capsule, so the number of surviving cells becomes smaller and those cells are replaced by fibrous tissue. Dead cells are removed, the tissue atrophies, and the ratio of NO / O 2 decreases again, and the capillaries become more sparse again. This may create a vicious cycle in polyconnective tissue diseases starting from Raynaud's phenomenon and ending with systemic sclerosis and primary Sjögren's syndrome where capillary rarefaction is similarly observed in end-stage renal disease, congestive heart failure / cardiac hypertrophy, primary biliary cirrhosis, Alzheimer's disease, atherosclerosis, inflammatory bowel disease, hypertrophic scar formation, etc. Ferrini et al. showed that chronic inhibition of NOS with L-NAME leading to a reduction in basal NO levels is linked to systemic fibrosis in the heart and kidneys. (Antifibrotic Role of Inducible Nitric Oxide Synthase. Nitric Oxide: Biology and Chemistry, Volume 6, Number 3, pages 283 - 294 (2002)). Low basal NO may be linked to fibrotic hypertrophy.

[0359] In certain embodiments, capillary rarefaction is recognized to affect a subject's ability to control its appetite. Capillary rarefaction is observed in the brains of aged humans and animals. Capillary rarefaction is associated with a decline in circulating growth factors, including insulin-like growth factor-1. Neurogenesis in the adult brain is coordinated with angiogenesis. Since the brain regulates a number of homeostatic functions, an increase in the diffusion length between capillaries to control elements of the brain may be "interpreted" as inappropriate blood concentrations of these species. Glucose flux in the brain is very close to the requirements of normal metabolism, in which case glucose flux is 50 to 75% greater than glucose consumption, and the glucose transporters across the blood-brain barrier are saturable, stereospecific, and independent of energy or ion gradients. Most of the regulation of appetite is mediated by the brain, and capillary rarefaction may cause an insufficient blood concentration of "nutrients" (or marker compounds proportional to "nutrients") to be interpreted as such. This can be a cause of obesity.

[0360] According to certain embodiments, capillary rarefaction is recognized to be a cause of non-insulin-dependent diabetes. Non-insulin-dependent diabetes mellitus (NIDDM), also known as metabolic syndrome or type 2 diabetes, is characterized by insulin resistance. The body's sensitivity to insulin is reduced and insulin levels increase. People with NIDDM have high blood glucose, high blood triglycerides, are typically obese, hypertensive, and typically have significant visceral fat.

[0361] Other symptoms that can be pointed out as a cause of capillary rarefaction are associated with NIDDM. In a study of 40 male subjects, 10 obese (BMI 29) and 10 lean (BMI 24) with and without NIDDM, Konrad et al. reported that resting blood lactate levels were 1.78, 2.26, 2.42, and 2.76 (mM / L) for lean non-NIDDM, obese non-NIDDM, lean NIDDM, and obese NIDDM male subjects, respectively. (A-Lipoic acid treatment decreases serum lactate and pyruvate concentrations and improves glucose effectiveness in lean and obese patients with type 2 diabetes. Diabetes Care 22:280-287, 1999). Lactate is a measure of anaerobic glycolysis. O 2 When O is insufficient to generate ATP by oxidative phosphorylation, the cell can produce ATP by anaerobic glycolysis. One of the products of anaerobic glycolysis is lactate, which must be exported from the cell or else the pH will drop and function will be impaired. Blood lactate is usually measured during an exercise test, and an increase indicates the workload at which maximal oxidative work can be performed. Higher lactate levels at rest indicate increased anaerobic glycolysis at rest, which is consistent with capillary rarefaction.

[0362] Primary biliary cirrhosis is associated with Raynaud's phenomenon, pruritus, sicca syndrome, osteoporosis, portal hypertension, neuropathy, and pancreatic insufficiency, and liver abnormalities are associated with rheumatic diseases. Elevated liver enzymes are symptoms of hepatitis and are observed as an early symptom of "asymptomatic" primary biliary cirrhosis. Thus, the bacteria described herein can be used to treat hepatitis.

[0363] Torre et al. showed that Alzheimer's disease (AD) has microvascular It has been reported to be a vascular disorder and is partially due to insufficient nitric oxide. (Review: Evidence that Alzheimer's disease is a microvascular disorder: the role of constitutive nitric oxide, Brain Research Reviews 34 (2000) 119 - 136). Therefore, the bacteria described herein can be used to treat AD.

[0364] The health - harmful effects associated with hypertension can also be a result of low basal NO. The decreased response to vasodilation is also consistent with low basal NO. NO is a diffusible molecule that diffuses from the source to the sensor site, and NO has a signaling effect at the sensor site. In the case of low NO levels, each NO source has to produce more NO in order to generate an equivalent NO signal at a certain distance with a certain intensity. NO diffuses three - dimensionally, and the total volume within the diffusion range has to increase to a level that gives an appropriate signal at the location of the sensor. As a result, higher NO levels can occur at the source and between the source and the sensor. At that time, due to a too - low NO background, there is a risk of harmful local effects of the elevated NO near the source. There is some evidence that this scenario actually occurs. Henningsson et al. reported that in rat islets, inhibition of NOS with L - NAME increased total NO production by induction of iNOS. (Chronic blockade of NO synthase paradoxically increases islet NO production and modulates islet hormone release. Am J Physiol Endocrinol Metab 279 Volumes: E95 - E107, 2000). Increasing NO by increasing NOS activity works only up to a certain limit. When NOS is activated but not supplied with sufficient tetrahydrobiopterin (BH4) or L - arginine, NOS becomes "uncoupled" and generates superoxide (O2-) instead of NO. Next, this O 2 - may destroy NO. Attempts to generate NO at a rate exceeding the supply of BH4 or L - arginine may rather decrease the NO level. This is because low NO levels are worsened by NOS stimulation, and uncoupled NOS causes significant O 2 - generation, such as the local reactive oxygen species (ROS) damage observed in atherosclerosis, end - stage renal disease, Alzheimer's, and diabetes, which can lead to a positive feedback that generates more O

[0365] The bacteria described herein can also be used to delay the signs of aging. Calorie restriction extends lifespan. Holloszy reported that restricting food intake to 70% of the ad libitum control increased the lifespan of non - exercising rats from 858 days to 1,051 days, almost 25% (Mortality rate and longevity of food restricted exercising male rats: a reevaluation. J. Appl. Physiol. 82(2):399 - 403, 1997). The link between calorie restriction and long lifespan is well - established, but the underlying mechanism is not. Lopez - Torres et al. reported that examination of liver mitochondrial enzymes in rats showed a reduction in H 2 O 2 production due to reduced activity of complex I associated with calorie restriction. (Influence Of Aging And Long - Term Caloric Restriction On Oxygen Radical Generation And Oxidative DNA Damage In Rat Liver Mitochondria. Free Radical Biology & Medicine 32, no. 9, pp. 882 - 889 (2002). H 2 O 2 is the main ROS generated by mitochondria during respiration. O 2 - is generated by the disproportionation of O 2 - The main source of O and others is suggested to be Complex I, which catalyzes the NAD / NADH redox couple by the reverse flow of electrons from Complex III, the succinate reduction site. By Beckman The free radical theory of aging proposed by Beckman hypothesizes that damage to cellular DNA, antioxidant systems, and DNA repair systems by free radicals accumulates with age, and death occurs when critical systems are damaged beyond the scope of repair (The Free Radical Theory of Aging Matures. Physiol. Rev. 78:547 - 581, 1998).

[0366] As an additional mechanism, it has been demonstrated by Vasa et al. that NO activates telomerase and delays endothelial cell senescence. (Nitric Oxide Activates Telomerase and Delays Endothelial Cell Senescence. Circ Res. 2000; 87:54 0 - 542). Low basal NO increases the basal metabolic rate by derepressing cytochrome oxidase. The increased basal metabolism also increases the cell turnover and proliferation rate. Capillary rarefaction induced by chronic hypoxia may increase free radical damage and may also increase cell turnover, thereby accelerating aging by both mechanisms.

[0367] In some embodiments, autotrophic ammonia-oxidizing bacteria are recognized to be able to produce protective modalities against allergies and autoimmune disorders. The most well-known autoimmune disease is perhaps type 1 diabetes, which is caused by the destruction of insulin-producing cells in the pancreas by the immune system. Infertility is also associated with autoimmune disorders, and in infertility, the number of positive autoimmune antibodies was positively correlated with the number of infertility episodes. Systemic sclerosis, primary biliary cirrhosis, autoimmune hepatitis, and various rheumatic disorders are other examples of autoimmune disorders. The application of AOB has been observed to reduce allergies, hay fever, as described in WO / 2005 / 030147.

[0368] One mechanism by which AOB may exert its protective effect against allergies and autoimmune disorders is through the production of nitric oxide, mainly by inhibiting the regulation of NF-κB and preventing the activation of immune cells and the induction of inflammatory responses. NF-κB is a transcription factor that upregulates gene expression, and many of these genes, including those that cause the release of cytokines, chemokines, and various adhesion factors, are associated with inflammation and immune responses. These various immune factors cause the migration of immune cells to the site where they are released, leading to an inflammatory response. Constitutive NO production has been shown to inhibit NF-κB by stabilizing IκBα by preventing the degradation of IκBα (an NF-κB inhibitor).

[0369] The administration of NO donors has been shown by Xu et al. to prevent the onset of experimental allergic encephalomyelitis in rats. (SIN-1, a Nitric Oxide Donor, Ameliorates Experimental Allergic Encephalomyelitis in Lewis Rats in the Incipient Phase: The Importance of the Time Window. The Journal of Immunology, In 2001, 166 volumes: pages 5810 - 5816). In this study, it was demonstrated that administering an NO donor reduced macrophage infiltration into the central nervous system, reduced the proliferation of blood monocytes, and increased the apoptosis of blood monocytes. All of these results were expected to reduce the degree and severity of the induced autoimmune response.

[0370] Due to the mechanism that insufficient new connections in the brain are formed as a result of insufficient basal nitric oxide, low basal NO can lead to autism. Although not wishing to be bound by theory, in some embodiments, the formation of neural connections is modulated by NO. In these cases, any condition that reduces the range of NO diffusion may reduce the volume size of the brain elements capable of making connections. A brain developed in a state of low basal NO levels may be arranged as smaller volume elements due to the reduced effective range of NO.

[0371] Additional symptoms that appear in autistic individuals, including increased pitch discrimination, intestinal disorders, immune system dysfunction, reduced cerebral blood flow, increased cerebral glucose consumption, increased plasma lactate, attachment disorders, and hamming, can also be attributed to low NO as a cause. Each of these symptoms can be caused by low basal NO levels.

[0372] Takashi Ohnishi et al. reported that autistic individuals exhibit reduced blood flow. Takashi Ohnishi et al., Abnormal regional cerebral blood flow in childhood autism. Brain (2000), 123 volumes, pages 1838 - 1844. J. M. Rumsey et al., autism Individuals with the disorder have been reported to have increased glucose consumption. Rumsey JM, Duara R, Grady C, Rapoport JL, Margolin RA, Rapoport SI, Cutler NR. Brain metabolism in autism. Resting cerebral glucose utilization rates as measured with positron emission tomography. Arch Gen Psychiatry, May 1985; 42(5):448 - 455 (abstract). D. C. Chugani reported that individuals with autism have increased plasma lactate levels. Chugani DC, et al., Evidence of altered energy metabolism in autistic children. Prog Neuropsychopharmacol Biol Psychiatry. May 1999;23(4):635 - 641. The occurrence of these effects may be the result of capillary rarefaction in the brain, which may reduce blood flow and O 2 supply, and as a result, part of the brain's metabolic load may be generated by glycolysis instead of oxidative phosphorylation.

[0373] Nitric oxide has been demonstrated by B. A. Klyachko, et al. to increase neuronal excitability by increasing it after hyperpolarization through cGMP modification of ion channels. Vitaly A. Klyachko, et al., cGMP-mediated facilitation in nerve terminals by enhancement of the spike after hyperpolarization. Neuron, Vol. 31, 101 Pages 5 - 1025, September 27, 2001. C. Sandie et al. have shown that inhibition of NOS reduces startle. Carmen Sandi et al., Decreased spontaneous motor activity and startle response in nitric oxide synthase inhibitor - treated rats. European journal of pharmacology 277 (1995) 89 - 97. Attention - deficit hyperactivity disorder (ADHD) has been modeled using spontaneously hypertensive rats (SHR) and Naples high - excitability (NHE) rats. Both of these models have been shown by Raffaele Aspide et al. to exhibit increased attention deficits during the period of acute NOS inhibition. Raffaele Aspide et al., Non - selective attention and nitric oxide in putative animal models of attention - deficit hyperactivity disorder. Behavioral Brain Research 95 (1998) 123 - 133. Thus, the bacteria herein can be used for the treatment of ADHD.

[0374] Inhibition of NOS has also been shown by M. R. Dzoljic to inhibit sleep. M. R. Dzoljic, R. de Vries, R. van Leeuwen. Sleep and nitric oxide: effects of 7 - nitro indazole, inhibitor of brain nitric oxide synthase. Brain Research 718 (1996) 145 - 150. G. Zoccoli has shown that NOS is inhibited It has been reported that when this occurs, some of the physiological effects seen during sleep, including rapid eye movement and sleep-wake differences in cerebral circulation, change. G. Zoccoli et al., Nitric oxide inhibition abolishes sleep-wake differences in cerebral circulation. Am. J. Physiol. Heart Circ Physiol 280: H2598 - 2606, 2001 . NO donors have been shown by L. Kapas et al. to promote non-REM sleep, however, these increases persisted much longer than the duration of the NO donor, perhaps suggesting a rebound effect. Levente Kapas et al., Nitric oxide donors SIN-1 and SNAP promote nonrapid-eye-movement sleep in rats. Brain Research Bullitin, 41(5): 293 - 298, 1996. According to M. Rosaria et al., central NO facilitates both penile erection and yawning. Maria Rosaria Melis and Antonio Argiolas. Role of central nitric oxide in the control of penile erection and yawning. Prog Neuro-Psychopharmacol & Biol. Phychiat. 1997, 21: 899 - 922. P. Tani et al. reported that insomnia is a frequent finding in adults with Asperger's syndrome. Pekka Tani et al., Insomnia is a frequent finding in adults with Asperger's syndrome. BMC Psychiatry 2003, 3 Volume: 12 pages. Y. Hoshino has also observed sleep disturbances in autistic children. Hoshino Y, Watanabe H, Yashima Y, Kaneko M, Kumashiro H. An investigation on sleep disturbance of autistic children. Folia Psychiatr Neurol Jpn. 1984 Year; Volume 38(1): 45 - 51 pages (abstract). K. A. Schreck et al. have observed that the severity of sleep disturbances is correlated with the severity of autism symptoms. Schreck KA et al., Sleep problems as possible predictors of intensified symptoms of autism. Res Dev Disabil. January - February 2004; Volume 25(1): 57 - 66 pages (abstract). Therefore, the bacteria in this specification can be used for the treatment of insomnia.

[0375] W. D. Ratnasooriya et al. reported that inhibition of NOS in male rats reduces pre - copulatory activity, reduces libido, and reduces fertility. W. D. Ratnasooriya et al., Reduction in libido and fertility of male rats by administration of the nitric oxide (NO) synthase inhibitor N - nitro - L - arginine methyl ester. International journal of andrology, Volume 23: 187 - 191 pages (2000).

[0376] Several seemingly different disorders characterized by ATP depletion and eventual organ failure may actually be "caused" by nitropenia, which is caused by a global lack of basal nitric oxide. When this occurs in the heart, the result is dilated cardiomyopathy. When this occurs in the brain, the results are white matter hyperintense regions, Alzheimer's, vascular depression, vascular dementia, Parkinson's disease, and Lewy body dementia. When this occurs in the kidney, the result is end-stage renal disease, and when this occurs in the liver, the result is primary biliary cirrhosis. When this occurs in muscle, the progression is fibromyalgia, Gulf War syndrome, or chronic fatigue syndrome. When this occurs in the intestine, the progression is ischemic bowel disease. When this occurs in the pancreas, the progression is initially type 2 diabetes, followed by chronic pancreatitis, followed by autoimmune attack of the pancreas (or pancreatic cancer), followed by type 1 diabetes. When this occurs in connective tissue, the progression is systemic sclerosis.

[0377] In the remnant kidney model of end-stage renal disease, a portion of the kidney is removed (either surgically or using a toxin), thereby increasing the metabolic load on the remaining portion. Superoxide is generated, reducing NO and increasing O 2 diffusion into the renal mitochondria. Chronic overload leads to progressive renal capillary rarefaction and progressive renal failure. In acute renal failure, dialysis can be used to give the kidney "rest" and allow it to recover. In acute renal failure induced by rhabdomyolysis (muscle damage that releases myoglobin into the bloodstream), the kidney injury is characterized by ischemic injury. Myoglobin traps NO, just as hemoglobin does, causing vasoconstriction in the kidney and leading to ischemia. Myoglobin also induces a cascade of events leading to local nitropenia and further ATP depletion.

[0378] In some aspects, low NO levels lead to reduced mitochondrial biogenesis. Generating the same amount of ATP with a reduced mitochondrial density requires O 2It causes an increase in consumption or an accelerated basal metabolic rate. An accelerated basal metabolic rate is observed in several conditions including sickle cell anemia, congestive heart failure, diabetes, cirrhosis, Crohn's disease, amyotrophic lateral sclerosis, obesity, end-stage renal disease, Alzheimer's, and chronic obstructive pulmonary disease.

[0379] Some increased O 2 Although the increased O consumption can be productively utilized, uncoupling proteins are also upregulated in many of these conditions. This indicates that at least part of the increased metabolic rate is due to inefficiency. Conditions in which uncoupling proteins are known to be upregulated include obesity and diabetes.

[0380] O 2 When mitochondria that consume O to a lower O concentration are fewer, the diffusion of O driven by the O gradient becomes greater, and the path length of O diffusion may increase, leading to capillary rarefaction, which is observed in dilated cardiomyopathy, hypertension, type 2 diabetes, and renal hypertension. 2 concentration is less, the diffusion of O driven by the O gradient becomes greater, and the path length of O diffusion may increase, leading to capillary rarefaction, which is observed in dilated cardiomyopathy, hypertension, type 2 diabetes, and renal hypertension. 2 gradient becomes greater, and the path length of O diffusion may increase, leading to capillary rarefaction, which is observed in dilated cardiomyopathy, hypertension, type 2 diabetes, and renal hypertension. 2 diffusion becomes greater, and the path length of O diffusion may increase, leading to capillary rarefaction, which is observed in dilated cardiomyopathy, hypertension, type 2 diabetes, and renal hypertension. 2 The path length of O diffusion may increase, leading to capillary rarefaction, which is observed in dilated cardiomyopathy, hypertension, type 2 diabetes, and renal hypertension.

[0381] Copper, either as Cu2+ or as ceruloplasmin (CP) (present at 0.38 g / L in adult serum, 0.32% Cu, and the major Cu-containing serum protein containing 94% of the copper in serum), catalyzes the formation of S-NO-thiol from NO and thiol-containing groups (RSH). The Cu content of plasma is variable and increases in infectious states. Berger et al. reported that the Cu and Zn contents of burn wound exudates are substantial in patients with burns covering one-third of the skin, and they lose 20 to 40% of the normal body's Cu content and 5 to 10% of the Zn content in 7 days. (Cutaneous copper and zinc losses in burns. Burns. October 1992;18(5):373 - 80). If AOB had colonized the patient's skin, the wound exudate containing urea and Fe, Cu, and Zn required by AOB would be converted to NO and nitrite, and resources (O2 Without consuming, for example, L-arginine, etc., it greatly replenishes the local production of NO by iNOS. The high production of NO and nitrite by AOB on the wound surface is expected to inhibit infections caused by anaerobic bacteria such as Clostridium that cause tetanus, gas gangrene, and botulism, in particular.

[0382] The practice of the present invention may employ conventional methods of immunology, molecular biology, and recombinant DNA techniques within the skill of those in the art, unless otherwise indicated. Such techniques are well described in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (latest edition); and Current Protocols in Molecular Biology (edited by F. M. Ausubel et al., latest edition). 7. Modulation of the Skin Microbiome Using Ammonia-Oxidizing Bacteria

[0383] The present disclosure provides systems and methods for altering a skin microbiome, such as the human skin microbiome. The systems and methods may provide, for example, treatment of an infectious disease or condition related to the skin, such as a skin infection and / or a skin condition.

[0384] Ammonia-oxidizing bacteria (AOB) of the genus Nitrosomonas are Gram-negative obligate autotrophic bacteria with the unique ability to generate nitrite and nitric oxide solely from ammonia as an energy source. AOB are widely present in both soil and aquatic environments and are an essential component of the environmental nitrification process. Due to the role of nitrite and nitric oxide on human skin as important components of several physiological functions such as vasodilation, dermatitis, and wound healing, these bacteria can have beneficial properties regarding both healthy and immunopathological skin conditions. These bacteria are slow-growing, unable to grow on an organic carbon source, may be sensitive to soap and antibiotics, and have never been associated with any disease or infection in animals or humans, so they may be safe for use in humans.

[0385] Topical application of ammonia-oxidizing bacteria to a subject, such as a human subject, can result in unexpected changes in the skin microbiome, and more specifically, it can result in an increase in the proportion of normal commensal non-pathogenic species and a reduction in the proportion of potential pathogenic organisms, pathogens, or disease-causing organisms.

Example

[0386] (Example 1) Study on human skin using ammonia-oxidizing bacteria: Effects, safety, detection, and skin metagenomics of cosmetics A double-blind placebo-controlled study was conducted on 24 human volunteers, randomized with an AOB to placebo control ratio of 4:1. The subjects applied a Nitrosomonas suspension (10 9 CFU / ml, twice a day, total daily dose 3×10 10 CFU) to their face and scalp twice a day for 1 week and were followed up for an additional 2 weeks after application. The volunteers were instructed to refrain from using hair products during the 1 week of AOB application and the week following application and then return to their usual shampoo use at week 3. Scalp swabs were obtained on day 0 as a baseline control and on days 1, 3, 8, 14, and 21, and the presence or absence of AOB was assessed by PCR and 16S rRNA gene sequence analysis.

[0387] Severe adverse events were not associated with 1 week of AOB application at all, and the product was considered safe. As shown by the self-assessment reports filled out after the 7-day application period, clear improvements in skin condition and quality were reported by AOB users. Using AOB-specific PCR analysis of skin samples, it was demonstrated that bacteria were present in 83 - 100% of AOB users during the application period, while no AOB was detected in placebo control samples. None of the subjects showed AOB from the baseline swabs taken before the start of the study, which is consistent with the predicted susceptibility of these bacteria to soap and other commercial products. Amplifying the 16S rRNA gene and sequencing a portion of the sample suggested the potential tendency that the presence of AOB was confirmed in the corresponding samples and the skin microbiome was modulated by topical application of AOB. In summary, viable AOB-based products are safe and may be highly promising as a novel self-regulatory topical delivery agent of nitrite and nitric oxide to human skin.

[0388] As shown in Table 8 below, the proportion of Nitrosomonas (AOB) increased when comparing day 0 to day 8. The proportions of other bacteria, namely Propionibacterium, Enterobacter, and Citrobacter, decreased when comparing day 0 to day 8. The p-values shown in Table 8 demonstrate that the most significant changes between day 0 and day 8 were observed in Nitrosomonas (AOB), followed by Propionibacterium. Enterobacter and Citrobacter also showed changes between day 0 and day 8, but not as much.

Table 8

[0389] (Example 2) A placebo-controlled, double-blind, randomized, multi-site cosmetic trial to evaluate the performance and tolerability of a cosmetic product for improving the appearance of oily skin, facial pores, and other cosmetic parameters in subjects prone to acne Phase 2b study:

[0390] Treatment with AO + Mist (D23 N. eutropha preparation) demonstrated a strong efficacy signal in both the overall assessment of the Skindex treating physicians (adult cohort) and the inflammatory lesion count.

[0391] The AO + Mist preparation used in the assay of Example 2 contains ammonia-oxidizing bacteria at a concentration of 1 × 10 2 HPO 4 and 2 mM MgCl 2 in an aqueous buffer of 9 CFU / mL.

[0392] Over 4 weeks (2 weeks of treatment, 2 weeks of washout), 36 adults (aged 18+) with acne vulgaris were randomized (1:1:1:1) in a double-blind, placebo-controlled, dose-ranging study (2, 4, 8 × 10 9 CFU) with B244 (D23 N. eutropha). The study had 80% power to detect a net improvement rate of 30% in IGA (% improvement in subjects) at week 2 (pooled B244 vs placebo). The study also had 80% power to detect a 30% difference (p < 0.05) in the mean change in lesion count from baseline between the pooled treatment group and placebo.

[0393] The outline of the treatment protocol was as follows. Ammonia-oxidizing bacteria were administered to the subject's face, neck, and scalp for a total of 4 weeks. After 4 weeks, the application of ammonia-oxidizing bacteria was stopped for 1 week. A biome-compatible shampoo and conditioner were used for a total of 5 weeks. Clinical evaluations were performed at baseline (0 hours), week 4, and week 5. Objective

[0394] A four-week multi-site double-blind randomized placebo-controlled trial of cosmetics was conducted to assess the overall cosmetic efficacy and tolerability of AO+Mist mist on skin appearance when used by men and women aged 12 to 45 years with skin prone to mild to moderate acne. Summary

[0395] A total of 83 subjects completed the study, with 42 subjects using AO+Mist (Nitrosomonas eutropha preparation) and 41 subjects using placebo. During the study process, according to the pre-determined randomization, the subjects were assigned to use AO+Mist (Nitrosomonas eutropha preparation) or placebo. The test substance was applied to the subjects' hair, scalp, and face twice a day as instructed. The subjects used standard soap and shampoo as support materials ( "biomass compatibility"), namely shampoo (Lot 293178; shown as "preferred concentration" in Table 5) and detergent (Lot 293162; shown as "preferred concentration" in Table 6) once a day throughout the study process. After the four-week usage period, the subjects stopped using the test substance (while still using the support materials) and participated in a one-week washout period. Clinical evaluations were performed at the first visit (baseline), the second visit (4 weeks), and the third visit (5 weeks). The subjects participated in the following procedures at each time point (unless otherwise instructed). · Clinical assessment of acne

[0396] Trained clinical graders evaluated the subjects' VISIA and self-taken photos regarding the post-study acne condition respectively. · Acne counting

[0397] Trained clinical graders counted the inflammatory lesions (papules, pustules, and cysts / nodules) and non-inflammatory lesions (open and closed comedones) on the forehead, left cheek, jaw, and right cheek of each subject (individually). · Investigator's Global Improvement Assessment (IGIA)

[0398] The principal investigator or designee conducted a global facial acne assessment at week 4 and week 5. The description of IGA can be found in the U.S. Food and Drug Administration, e.g., http: / / www.fda.gov / downloads / Drugs / ... / Guidances / UCM071292.pdf: Guidance for Industry; Acne Vulgaris: Develping Drugs for Treatment (U.S. Department of Health and Human Services; Food and Drug Administration; Center for Drug Evaluation and Research (CDER); September 2005, available in Clinical / Medical). · PIH / PIE lesion assessment

[0399] Post-inflammatory hyperpigmentation / post-inflammatory erythema (PIH / PIE) lesions were selected for each subject, and trained clinical assessors evaluated the selected PIH / PIE lesions for color intensity and size. · Imaging procedures

[0400] Digital images of each subject's face (right side, left side, and central plane) were taken using a VISIA CR photo-station (Canfield Imaging Systems, Fairfield, NJ) equipped with a Canon Mark II 5D digital SLR camera (Canon Inc., Tokyo, Japan) using standard 1 and 2, cross-polarized, parallel-polarized, and UV-NF lighting conditions. Pores were analyzed in digital images taken using cross-polarized illumination. · Surveys and selfies

[0401] Subjects took standard selfies (''selfies'') once a week using their smartphones and filled out a questionnaire. · Tolerability assessment

[0402] Skilled clinical assessors evaluated the entire face of each subject with respect to objective stimulation parameters (erythema, edema, and desquamation), and the subjects reported the degree of subjective stimulation parameters (stinging, burning, and itching). · Clinical assessment of cosmetic efficacy parameters

[0403] Skilled clinical assessors evaluated the face of each subject with respect to oily appearance (lustre and feel), appearance of pores, gloss, spots, homogeneity of skin color (hue), visual smoothness, and tactile smoothness. · Sebumeter measurement

[0404] The forehead was measured three times with a Sebumeter SM 815 (Courage+Khazaka, Germany), and the sebum content of the skin was measured using photometry. · Collection of microbiome samples

[0405] Microbiome samples were collected from the back of the neck, behind the right ear, and from the right cheek (one sample from each location). Overall conclusion

[0406] The overall results from this 4-week multi-site double-blind randomized placebo-controlled cosmetics trial demonstrated that the use of the test substance, AO+Mist (Nitrosomonas eutropha preparation), was safe and effective for subjects with mild to moderate acne conditions. The severity of the disease and the inflammatory lesion count in adults were shown to be improved with AO+Mist treatment. The use of AO+Mist (Nitrosomonas eutropha preparation) had very good tolerance by the subjects and was not associated with a statistically significant increase (worsening) in the clinical assessment scores for erythema, desquamation, edema, burning, stinging, and itching at week 4 when compared to the baseline scores.

[0407] The study included the standard FDA investigator global acne (IGA) scale 1Using this, the acne condition was assessed using VISIA images under a completely blinded state. The subjects using AO + Mist (Nitrosomonas eutropha preparation) showed a statistically significant decrease (improvement) in the IGA score at the 4th and 5th weeks when compared to the baseline score. By comparing between test substances based on the changes from the baseline, a statistically significant difference in favor of AO + Mist (Nitrosomonas eutropha preparation) was shown for acne assessment at the 4th week.

[0408] The results of the acne count regarding the use of AO + Mist (Nitrosomonas eutropha preparation) showed a statistically significant decrease (improvement) in the count of papules and inflammatory lesions at the 4th and 5th weeks and in the overall lesion count at the 4th week when compared to the baseline count. No statistically significant difference was observed between AO + Mist (Nitrosomonas eutropha preparation) and placebo, but as shown in Figure 4, the data showed that the inflammatory lesion count was more greatly and persistently reduced in adult subjects using AO + Mist (Nitrosomonas eutropha preparation). The bars for AO + Mist shown in the graph are the 1st (from the left) and 3rd (from the left). The bars for placebo shown in the graph are the 2nd (from the left) and 4th (from the left).

[0409] The outcomes reported by the subjects were measured using the Skindex 16 quality of life survey. AO + Mist was associated with changes (improvement) in the group of questions that captured the emotional aspect of the subjects regarding their disease. Specifically, for pain in the skin condition (2nd week), the skin condition persisting / recurring (4th week), worry about the skin condition (5th week), and the appearance of the skin condition (2nd, 4th, and 5th weeks), a statistically significant improvement in favor of AO + Mist (Nitrosomonas eutropha preparation) was observed.

[0410] The results of the clinical assessment scores regarding the use of AO + Mist (a preparation of Nitrosomonas eutropha) showed a statistically significant decrease (improvement) in the clinical assessment scores regarding visual and tactile smoothness at week 4 and week 5 and regarding pigmentation at week 5 when compared to the baseline scores. Description of the test substance

[0411] Tables 9 and 10 show the descriptions of each test substance and the support material.

Table 9

Table 10

[0412] Breakdown and demographics of the subjects Table 11 summarizes the breakdown information of the subjects. Table 12 shows the demographic information regarding the per-protocol (PP) population. For applicable parameters, the number of subjects in each category is listed along with the percentage of all subjects in parentheses.

Table 11

Table 12

[0413] Before starting the study, potential subjects were screened by phone using an IRB-approved script regarding eligibility criteria. Adolescent and adult men and women between the ages of 12 and 45 who self-diagnosed as having normal, oily, and combination skin types were scheduled for eligibility screening at the clinic. Subject candidates were instructed to remove all makeup at least 3 hours before each scheduled visit.

[0414] At the first visit to the hospital (baseline), candidates aged 18 or older, or the parents / legal guardians of any minor candidate under 18 years old, received an explanation of the nature of the study, answered any questions related to the clinical trial, read the informed consent document, and signed it. Minor candidate subjects read and signed the assent document. Screening numbers were assigned to the candidate subjects who signed this initial document, and they were evaluated against the following eligibility criteria. · Fitzpatrick skin classification: Certified as types I - V

[0415] The Fitzpatrick skin classification is based on the reaction of unprotected skin during the first 30 - 45 minutes of sun exposure after sun exposure in winter when not exposed to sunlight. The categories of skin types are as follows in Table 13.

Table 13

[0416] Candidate subjects filled out questionnaires regarding eligibility and health. Screened subjects who met the eligibility requirements were enrolled in the study and assigned subject numbers. The subjects participated in the following procedures. · Clinical assessment of cosmetic efficacy parameters

[0417] The entire face of the subject was clinically evaluated for the following cosmetic efficacy parameters by a trained clinical assessor who was unaware of the treatment details, using the modified Griffiths 10 - point scale 3 as defined numerically below (assigning a half - point score if an exact description of the skin condition is required). 0 = None (best possible condition) 1 - 3 = Mild 4 - 6 = Moderate 7 - 9 = Severe (worst possible condition)

[0418] The following parameters (Table 14) were rated according to the scale anchors listed below. [Table 14] · PIH / PIE lesion assessment

[0419] Skilled clinical assessors who were not informed of the treatment content selected post-inflammatory hyperpigmentation / post-inflammatory erythema (PIH / PIE) lesions on the face of each subject and rated the lesions for color intensity and size using the following rating scale. Color intensity 1 Slightly lighter than the color of the surrounding skin 2 Equal to the color of the surrounding skin (PIH / PIE is not visually observable) 3 Slightly darker / redder than the color of the surrounding skin 4 Moderately darker / redder than the color of the surrounding skin 5 Significantly darker / redder than the color of the surrounding skin 6 Extremely darker / redder than the color of the surrounding skin Size 0 Not visually observable 1 Diameter 0.5 mm or less 2 Diameter greater than 0.5 mm to 1.5 mm 3 Diameter greater than 1.5 mm to 2.5 mm 4 Diameter greater than 2.5 mm · Acne count

[0420] Skilled clinical assessors who were not informed of the treatment content individually counted and recorded the number of inflammatory acne lesions (papules, pustules, and cysts / nodules) and non-inflammatory acne lesions (open comedones and closed comedones) on the forehead, left cheek, jaw, and right cheek (excluding lesions along the hairline including the nose, below the lower jaw contour, or eyebrows) of each subject. · Tolerance evaluation

[0421] The local skin tolerance was evaluated by assessing the signs and symptoms of objective and subjective irritation in the entire face (cosmetic treatment area) of each subject. The following irritation parameters were evaluated. - Objective irritation (clinical assessment): erythema, edema, and desquamation - Subjective irritation (assessed by the subject): stinging, burning, and itching

[0422] The results of the irritation assessment were recorded using the following scale (using half-point scores when a detailed description of the clinical condition was required). Erythema 0 = None There is no erythema in the cosmetic treatment area. 1 = Mild There is a slight but distinct redness in the cosmetic treatment area. 2 = Moderate There is a distinct redness in the cosmetic treatment area. 3 = Severe There is a prominent redness in the cosmetic treatment area. Edema 0 = None There is no edema / swelling in the cosmetic treatment area. 1 = Mild There is a slight but distinct edema in the cosmetic treatment area. 2 = Moderate There is a distinct edema in the cosmetic treatment area. 3 = Severe There is a prominent edema in the cosmetic treatment area. Desquamation 0 = None There is no desquamation in the cosmetic treatment area. 1 = Mild There is barely perceptible fine desquamation in limited areas of the cosmetic treatment area. 2 = Moderate There is fine desquamation throughout all areas of the cosmetic treatment area. 3 = Severe There is skin desquamation and peeling throughout all areas of the cosmetic treatment area. Stinging 0 = None There is no stinging in the cosmetic treatment area. 1 = Mild There is a slight stinging sensation in the cosmetic treatment area, but it is not really bothersome. 2 = Moderate There is a distinct stinging in the cosmetic treatment area and it is somewhat bothersome. 3 = There is a significant stinging sensation in the area of the cosmetic treatment, a distinct sense of discomfort occurs, and daily activities and / or sleep may be interrupted Sensation of heat 0 = None There is no sensation of heat in the area of the cosmetic treatment 1 = Mild There is a slight sensation of heat in the area of the cosmetic treatment, but it is actually not bothersome 2 = Moderate There is a distinct feeling of warmth and a sensation of heat in the area of the cosmetic treatment, and it is somewhat bothersome 3 = Severe There is a burning sensation of heat in the area of the cosmetic treatment, a distinct sense of discomfort occurs, and daily activities and / or sleep may be interrupted Itching sensation 0 = None There is no itching sensation in the area of the cosmetic treatment 1 = Mild There is a slight itching sensation in the area of the cosmetic treatment, but it is actually not bothersome 2 = Moderate There is a distinct itching sensation in the area of the cosmetic treatment, and it is somewhat bothersome 3 = Severe There is a significant itching sensation in the area of the cosmetic treatment, a distinct sense of discomfort occurs, and daily activities and / or sleep may be interrupted

[0423] The subjects were acclimated to the ambient temperature and humidity conditions for at least 15 minutes before participating in the biometric / imaging / microbiome sample collection procedures. During the study process, the applicable waiting room / measurement room was maintained at a temperature of 68°F - 75°F and a relative humidity range of 35% - 65%.

[0424] After acclimation, the subjects participated in the following procedures. · Sebumeter measurement

[0425] The forehead was measured three times with a sebumeter SM 815 (Courage + Khazaka, Germany), and the sebum content of the skin was measured independently of moisture using a photometric method. The light transmittance through sebutape, a synthetic material that becomes transparent when in contact with sebum, was measured. The amount of the skin surface (μg sebum / cm 2 ) based on the transparency of sebutape was calculated by the microprocessor in the sebumeter. · Imaging procedure

[0426] Prior to the imaging procedure, the healthcare staff confirmed that the subject's face was clean and free of makeup and that all ornaments had been removed from the area(s) where the subject was to be photographed. The subject was given a matte black or gray headband to keep hair off the face and the subject's clothing was covered with a matte black shirt or a matte black or gray cloth. The subject was instructed to assume an expression that was not an intermediate smile while gently closing their eyes and was carefully positioned for each photograph.

[0427] Images of the frontal views (frontal, right lateral, and left lateral images) of each subject were taken using a VISIA CR photo-station (Canfield Imaging Systems, Fairfield, New Jersey) equipped with a Canon Mark II 5D digital SLR camera (Canon Inc., Tokyo, Japan) under illumination conditions of Standard Illumination 1 (visible), Standard Illumination 2 (visible / fluorescent), cross-polarized light, parallel-polarized light, and UV-NF. · Microbiome procedure

[0428] Microbiome samples were collected from behind the neck, behind the right ear, and from the right cheek of each subject (one sample from each location). The swab was removed from the sterile packaging, held by the shaft away from the swab, rubbed strongly about 20 times, and rotated over a 3-inch portion of the area where sampling was being done. The swab was then placed into a (time-point labeled) test tube, the shaft was cut off, and the test tube was ensured to be closed. Skin swabs were collected, frozen at -80°C, and then DNA extraction and microbiome profiling were performed.

[0429] The subjects were instructed on how to take a standard selfie photograph (a "selfie") once a week using their smartphones and how to fill out a questionnaire.

[0430] The subjects were assigned to the support material units and were instructed to apply shampoo (Lot 293178) once a day to the hair and scalp and a cleanser (Lot 293162) once a day to the face during the study period. The subjects were assigned to a pre-weighed unit of test substance, namely AO + Mist (Nitrosomonas eutropha preparation) or placebo, according to a predetermined randomization.

[0431] The subjects were given the following oral and written instructions for use. Instructions for Use

[0432] The test substance is to be applied twice a day (morning and evening), at least 6 pumps each time, to the hair, scalp, and face after washing and before going to bed, and not immediately before taking a shower. Do not apply any makeup (foundation, eyeshadow, etc.) for 1 hour after applying the product and use the minimum amount.

[0433] The subjects were given written instructions for use, a study explanation, a calendar of clinic visit days, and a diary for recording the time of product application.

[0434] The subjects received a second (4th week) follow-up visit and participated in the following procedures. - The medical staff recorded the concomitant medications and asked the subjects about changes in their health status. Adverse events were recorded if applicable. - The diaries were collected, compliance was reviewed, and they were stored at the clinic. New diaries were distributed. - The test substance units were collected, visually inspected, weighed for compliance, and stored at the clinic. The support materials were visually inspected for compliance and returned to the subjects. - The subjects participated in the following procedures as described at baseline. · Clinical assessment of cosmetic efficacy parameters · PIH / PIE lesion assessment · Acne count · Tolerance evaluation - The subjects participated in the following procedures. · The overall improvement assessment (IGA) of the principal investigator was conducted using the following scale and numerical definitions. 1 = Worsening 2 = No improvement 3 = Slight improvement 4 = Moderate improvement 5 = Marked improvement - The subjects were acclimated for at least 15 minutes and then participated in the following procedures as described at baseline. · Sebumeter measurement · Imaging procedure · Microbiome sample collection

[0435] After the 4-week assessment was completed, the subjects participated in a 1-week washout period and discontinued the use of the test substance (the support material was still used). The subjects received a third (5th week) follow-up visit and participated in the following procedures. - The medical staff recorded the concomitant medications and asked the subjects about changes in their health status. Adverse events were recorded if applicable. - The diaries were collected, compliance was reviewed, and they were stored at the clinic. - The support material units were collected, visually inspected for compliance, and stored at the clinic. - The subjects participated in the following procedures as described at baseline and at week 4. · Clinical assessment of cosmetic efficacy parameters · PIH / PIE lesion assessment · Acne count · Overall improvement assessment by the principal investigator · Tolerability assessment - The subjects were acclimated for at least 15 minutes and then participated in the following procedures as described at baseline. · Sebumeter measurement · Imaging procedure · Microbiome sample collection After the 5-week study procedures were completed, the following procedures were performed by clinical assessors who were blinded to the treatment. · Acne assessment

[0436] The clinical assessment of acne was performed using the modified Griffiths 10 - point scale 3 (0 = no acne, 9 = severe acne) from VISIA images taken at the clinic at baseline, week 4, and week 5, and self - taken photos of the subjects taken at baseline and weeks 1, 2, 3, 4, and 5 to screen for any worsening of the subjects' acne itself.

[0437] Furthermore, the FDA IGA scale 1 (0 - 4) was used on the VISIA images to perform a clinical assessment regarding acne. 0 = Clear. No inflammatory or non - inflammatory lesions 1 = Almost clear. Very few non - inflammatory lesions and accompanied by one or fewer small inflammatory lesions 2 = Mild. Exceeding grade 1, with several non - inflammatory lesions and accompanied by two or three or fewer inflammatory lesions (papules / pustules only, no nodular lesions) 3 = Moderate. Exceeding grade 2, with up to a large number of non - inflammatory lesions and having several inflammatory lesions, but having one or fewer small nodular lesions 4 = Severe. Exceeding grade 3, with up to a large number of non - inflammatory and inflammatory lesions, but having two or three or fewer nodular lesions Biostatistics and data management

[0438] The per - protocol (PP) population was the primary population for all statistical analysis tests. The PP population included all subjects who were considered eligible to participate in the study and who completed the study according to the protocol. Only the data of the completed subjects were analyzed. Subjects might have been excluded from the analysis in case of AE, SAE, non - compliance, or at the discretion of the investigator in charge of the clinical trial.

[0439] For each subject and time point, the three Cebmeter measurements were averaged before performing the statistical analysis.

[0440] The digital images taken using cross-polarized illumination conditions were analyzed for pores using Macroversion "Pore_20121207" developed by Stephens&Associates and Image Pro Plus v7 software (MediaCybernetics, Inc., Rockville, Maryland). The target uneven-shaped area in the cheek region was selected for analysis. Pore analysis reported the values of the number of pores (count), the total area covered by pores, and the average depth of pores.

[0441] Acne count data were analyzed for the areas rated at a total of 4 locations (forehead, right cheek, left cheek, and chin) for each of the following rating positions. · Papules, pustules, cysts / nodules, open comedones, and closed comedones are separate · Inflammatory acne lesions (combinations of papules, pustules, cysts / nodules) and non-inflammatory acne lesions (combinations of open comedones and closed comedones) are separate · There are overall lesions (a total of 5 types of acne)

[0442] For the aesthetic efficacy assessment data, acne assessment data, PIH / PIE lesion assessment data, acne count, tolerance assessment data, sebumeter measurements, and pore image analysis parameters, descriptive statistical summaries including the number of observed patients (N), mean, median, standard deviation (SD), minimum value (MIN), and maximum value (MAX) are provided for all clinic visit days.

[0443] The average of the changes from the baseline (defined as the value after the baseline minus the value of the baseline) was estimated at each applicable time point after the baseline. The null hypothesis that the average change from the baseline is zero was estimated at the fourth week, and the Wilcoxon signed-rank test was used for the aesthetic effectiveness assessment parameters, acne assessment, acne count, image analysis of the number of pores (count), and tolerance assessment, and the paired t-test was used for the sebumeter measurement and image analysis of the pores (total area covered by the pores and average depth of the pores). For the regression period, a similar analysis including the test of the overall improvement assessment by the principal investigator using the Wilcoxon signed-rank test was performed at the fifth week for the change from the fourth week.

[0444] The percentage average change from the baseline / week 4 and the percentage of subjects showing improvement or deterioration were calculated using the following formulas. Percentage average change from the baseline = (Average score at visit - Average baseline score) × 100 / Average baseline score Percentage of subjects with improvement / deterioration = (Number of subjects with improvement / deterioration from the baseline) × 100 / Total number of subjects

[0445] Regarding the change from the baseline, a comparison between the treatment cells was made at the fourth week. The null hypothesis that the average change from the baseline is equal between the two treatment cells was tested at each applicable time point after the baseline for all parameters (excluding the overall improvement assessment by the principal investigator), using the Wilcoxon rank-sum test for the aesthetic effectiveness assessment parameters, acne assessment, acne count, image analysis of the number of pores (count), and tolerance assessment, and the two-sample t-test for the sebumeter measurement and image analysis of the pores (total area covered and average depth of the pores).

[0446] For the regression period, a similar analysis including the test of the overall improvement assessment by the principal investigator using the Wilcoxon rank-sum test was performed at the fifth week for the change from the fourth week.

[0447] Present the comprehensive statistical results as well as the statistical results stratified by age (12 - 15, 16 - 18, 19 - 28, 29 and over), gender (male / female), adult acne (19 years and over) and adolescent acne (under 19 years), and Fitzpatrick skin type (III, IV, and V [due to small sample size, analysis of I and II was not performed]) with respect to the clinical assessment of cosmetic efficacy parameters, PIH / PIE lesion assessment, acne assessment, acne count, overall improvement assessment by the treating investigator, Sebmeter measurement, and image analysis of pore size. Additionally, show the demographic aggregation by treatment with respect to age, gender, adult and adolescent acne, and Fitzpatrick skin type.

[0448] For acne assessment, the Spearman's rank correlation coefficient for scores using two scales (Griffiths scale 3 and FDA scale 1 ) was calculated for each applicable time point and treatment.

[0449] A statistical test regarding the null hypothesis that the correlation coefficient is equal to 0 was performed. Treatment comparisons were not made. Similar correlation coefficients were calculated from VISIA and selfie images for the modified Griffiths score.

[0450] Obtained an outcome assessing the quality of life reported by the subjects, i.e., SkinDex data including the responses of each subject to 16 questions at baseline and at weeks 2, 4, and 5. Frequency tables including counts and percentages were obtained for the SkinDex data. Similar analyses were performed for the clinical assessment data including the analysis of data by age, gender, adult and adolescent acne, and Fitzpatrick type.

[0451] Information on Skindex can be found at http: / / www.researchgate.net / publication / 51646907_Using_the_Skindex-16_and_Common_Terminology_Criteria_for_Adverse_Events_to_assess_rash_symptoms_results_of_a_pooled-analysis_(N0993).

[0452] A survey was obtained that included responses to 37 questions answered by the subjects at weeks 1, 2, 3, 4, and 5.

[0453] For questions 1 through 19, responses ranged from 0 (worse) to 6 (better). For the remaining questions, responses ranged from -1 (no / unchanged) to 1 (yes / favorable change).

[0454] A frequency table containing the count and percentage of subjects who selected each response option was obtained for the survey data. Additionally, a binomial (signed) test was performed to test whether the combined proportion of predefined favorable / positive responses matched the combined predefined unfavorable / negative responses for each question at each time point. Treatment comparisons were performed using Fisher's exact probability test. For questions 1 through 19, the null hypothesis to be tested was that the proportion of favorable / unfavorable / intermediate responses was equal between treatments. For the remaining questions, the null hypothesis to be tested was that the proportion of positive / negative responses was equal between treatments. The data analysis was also performed according to age, gender, adult and adolescent acne, and Fitzpatrick type.

[0455] All statistical tests were two-sided at a significance level of alpha = 0.05. p-values were reported to the third decimal place (0.000). Multiple testing correction was not considered in the study. Statistical analysis was performed using SAS software version 9.30 series (SAS Statistical Institute).

[0456] Clinical evaluations and biometric measurements were recorded using the Stephens Electronic Data Capture (EDC) system. Stephens EDC is a computerized system devised for collecting clinical data in electronic format. Three important aspects of the EDC consist of a graphical user interface for data entry, a validation component for checking user data, and a reporting tool for analyzing the collected data. Results Clinical evaluation of acne by image

[0457] For subjects using AO + Mist (Nitrosomonas eutropha preparation), when compared to the baseline score, on the 4th and 5th weeks, the FDA scale 1 was used, and on the 5th week, the Griffiths scale 3 was used. There was a statistically significant decrease (improvement) in the acne assessment score in the VISIA images. By comparing between test substances based on the change from the baseline regarding the acne assessment score, for the acne assessment by VISIA image using the FDA scale on the 4th week 1 a statistically significant difference in favor of AO + Mist (Nitrosomonas eutropha preparation) was shown.

[0458] By analyzing the correlation coefficients from the results of the image-based assessment, a statistically significant positive correlation (the correlation coefficients ranged from 0.495 to 0.847) was shown among the acne assessment scores in the VISIA images at baseline, the 4th week, and the 5th week, suggesting that self-taken photos could be a promising attempt for documenting the progress during clinical trials. Acne count All subjects

[0459] Subjects using AO+ Mist (a preparation of Nitrosomonas eutropha) had a statistically significant decrease (improvement) in the count of a total of 4 evaluated areas (forehead, left cheek, jaw, and right cheek) with respect to papules and inflammatory acne at weeks 4 and 5 and with respect to overall lesions at week 4, when compared to the baseline count. Subjects using placebo had a statistically significant decrease (improvement) in the count of a total of 4 evaluated areas with respect to open comedones, closed comedones, non-inflammatory acne, and overall lesions at weeks 4 and 5 and with respect to papules and inflammatory acne at week 4, when compared to the baseline count. By comparing between test substances based on the change from baseline in the acne count of a total of 4 evaluated areas, a statistically significant difference in favor of placebo was shown for closed comedones at week 4. Subjects with adult acne

[0460] Subjects using AO+ Mist (a preparation of Nitrosomonas eutropha) had a statistically significant decrease (improvement) in the acne count of a total of 4 evaluated areas (forehead, left cheek, jaw, and right cheek) with respect to papules, inflammatory acne, and overall lesions at weeks 4 and 5, when compared to the baseline count. Subjects using placebo had a statistically significant decrease (improvement) in the acne count of a total of 4 evaluated areas with respect to closed comedones, non-inflammatory acne, and overall lesions at weeks 4 and 5 and with respect to open comedones at week 5, when compared to the baseline count. By comparing between test substances based on the change from baseline in the acne count of a total of 4 areas, a statistically significant difference in favor of AO+ Mist (a preparation of Nitrosomonas eutropha) was shown for papules at week 4 and a statistically significant difference in favor of placebo was shown for non-inflammatory acne at week 5. PIH / PIE lesion assessment All subjects

[0461] Subjects using AO + Mist (a preparation of Nitrosomonas eutropha) or placebo had a statistically significant decrease (improvement) in the PIH / PIE lesion assessment score for the color intensity and size of lesions 1 and 2 at weeks 4 and 5 when compared to the baseline score. By comparing betwee...

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[Claim 1] A composition as described in the specification.

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