Antimicrobial composition and method for determining whether or not to be administered to subject

Abstract

The present invention provides: an antibacterial composition comprising a bacterium exhibiting antibacterial properties against a bacterium positively correlated with a SCORAD value indicating the severity of atopic dermatitis; and a method for determining whether or not to administrate the antibacterial composition to a subject. The present invention comprises an antibacterial composition for pro-inflammatory bacteria other than skin resident bacteria, wherein the antibacterial composition contains the skin resident bacteria as an active ingredient.

Description

Technical Field

[0001] The present invention relates to an antibacterial composition and a method for determining whether to administer the antibacterial composition to a subject. Background Art

[0002] Atopic dermatitis (atopic dermatitis, atopic eczema) is a disease characterized by repeated exacerbations and relief, with eczema with itching as the main lesion. The pathological process of atopic dermatitis is the combined effect of various pathogenic factors against the background of organ allergies including the skin caused by atopic factors and barrier function fragility.

[0003] The Japanese Dermatological Association defines three basic criteria for the diagnosis of atopic dermatitis: (1) pruritus, (2) characteristic rash, and (3) chronic relapsing course. As long as these basic criteria are met, the patient is diagnosed with atopic dermatitis regardless of the severity of the symptoms.

[0004] In order to select the appropriate treatment for atopic dermatitis, its severity needs to be accurately assessed. As one of the severity classification methods that has been verified for statistical reliability and appropriateness, SCORAD (Severity Scoring of Atopic Dermatitis: Atopic Dermatitis Severity Score) can be cited. SCORAD can be calculated, for example, by the method described in the Atopic Dermatitis Diagnosis and Treatment Guidelines 2021 of the Japanese Society of Allergy (https: / / www.jsaweb.jp / huge / atopic_gl2021.pdf).

[0005] As treatment methods for atopic dermatitis, depending on the pathology, basic methods include (a) drug therapy, (b) topical therapy / skin care for physiological abnormalities of the skin, and (c) detection and treatment of exacerbating factors.

[0006] As a drug therapy, steroid topical drugs are mostly used as drugs for relieving inflammation of atopic dermatitis. When using steroid topical drugs, it is necessary to consider the characteristics of the lesions and the dosage form corresponding to the site, and select the appropriate steroid topical drug according to the severity. However, although steroid topical drugs are the basic drugs for atopic dermatitis, some people point out that due to the long-term use of high-grade steroid topical drugs and the use of them in a state of damaged skin barrier, they are prone to systemic or local side effects.

[0007] Among them, research has focused on Staphylococcus aureus (S. aureus), which is considered to be involved in the onset of atopic dermatitis and the aggravation of inflammation.

[0008] For example, Patent Document 1 discloses an agent for improving the balance of resident bacteria on the skin, which contains lanolin fatty acid or a salt thereof as an active ingredient, which has a proliferative effect on Staphylococcus epidermidis and an antibacterial effect on Staphylococcus aureus.

[0009] On the other hand, Non-Patent Document 1 discloses a bacterial therapy against Staphylococcus aureus using Staphylococcus hominis (S. hominis) isolated from the skin of a healthy person.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent Application Publication No. 2021-161034

[0013] Non-patent literature

[0014] Non-patent document 1: Development of a human skin commensal microbe forbacteriotherapy of atopic dermatitis and use in a phase 1randomized clinical trial, Nakatsuji et.al., Nature Medicine 27, 700-709 (2021) Summary of the invention

[0015] Technical problem to be solved by the invention

[0016] Lanolin fatty acid, an active ingredient of the skin resident bacteria balance improving agent of Patent Document 1, may cause allergies depending on the subject, so there is still room for improvement.

[0017] Non-Patent Document 1 shows that human Staphylococcus has antibacterial activity against Staphylococcus aureus and is safe, but the skin flora of atopic dermatitis patients is diverse, and there is still room for improvement from the perspective of bacterial therapy that takes into account the severity of atopic dermatitis.

[0018] In view of the above circumstances, the present invention aims to provide an antibacterial composition comprising bacteria showing antibacterial activity against bacteria positively correlated with the SCORAD value indicating the severity of atopic dermatitis, and a method for determining whether to administer the antibacterial composition to a subject.

[0019] Technical solutions adopted to solve technical problems

[0020] The present invention includes the following inventions [1] to

[21] .

[0021] [1] An antibacterial composition targeting pro-inflammatory bacteria other than skin-resident bacteria, comprising the skin-resident bacteria as an active ingredient.

[0022] [2] The antibacterial composition described in [1], wherein the skin-resident bacteria include one or more bacteria selected from bacteria having 16S rDNA, and the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 1 to 6 or any of the base sequences having an identity of 95% or more to any of the base sequences.

[0023] [3] The antibacterial composition described in [1] or [2], wherein the skin-resident bacteria include two or more bacteria selected from bacteria having 16S rDNA, and the 16S rDNA includes any one of the base sequences described in sequence numbers 1 to 6 or base sequences having an identity of 95% or more to the base sequences.

[0024] [4] The antibacterial composition of any one of [1] to [3], wherein the skin-resident bacteria include one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in sequence numbers 1 to 3 or base sequences having an identity of 95% or more to the base sequences.

[0025] [5] The antibacterial composition of any one of [1] to [4], wherein the skin-resident bacteria include two or more selected from bacteria having 16S rDNA, and the 16S rDNA includes any of the base sequences described in sequence numbers 1 to 3 or base sequences having an identity of 95% or more to the base sequences.

[0026] [6] An antimicrobial composition according to any one of [1] to [5], wherein the skin-resident bacteria include bacteria having 16S rDNA comprising the base sequence described in sequence number 1 or a base sequence with an identity of 95% or more to the base sequence, bacteria having 16S rDNA comprising the base sequence described in sequence number 2 or a base sequence with an identity of 95% or more to the base sequence, and bacteria having 16S rDNA comprising the base sequence described in sequence number 3 or a base sequence with an identity of 95% or more to the base sequence.

[0027] [7] The antimicrobial composition according to any one of [2] to [6], further comprising one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA comprises the base sequence described in sequence numbers 7 to 8 or any base sequence having an identity of 95% or more to the base sequence.

[0028] [8] The antibacterial composition described in [1], wherein the skin-resident bacteria include one or more bacteria selected from bacteria having 16S rDNA, and the 16S rDNA includes any of the base sequences described in sequence numbers 1 to 13 or base sequences having an identity of 95% or more to the base sequences.

[0029] [9] The antibacterial composition of any one of [1] to [8], wherein the pro-inflammatory bacteria comprises one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA comprises the base sequence described in sequence numbers 14 to 15 or any base sequence having an identity of 95% or more to the base sequence.

[0030]

[10] The antibacterial composition of [1], wherein the skin-resident bacteria comprises one or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, and Corynebacterium tuberculostearicum.

[0031]

[11] The antibacterial composition of [1] or

[10] , wherein the skin-resident bacteria include two or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, and Corynebacterium tuberculostearicum.

[0032]

[12] The antibacterial composition of any one of [1] or

[10] to

[11] , wherein the skin-resident bacteria include one or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

[0033]

[13] The antibacterial composition of any one of [1] or

[10] to

[12] , wherein the skin-resident bacteria include two or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

[0034]

[14] The antibacterial composition of any one of [1] or

[10] to

[13] , wherein the skin-resident bacteria include Actinomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

[0035]

[15] The antimicrobial composition according to any one of

[10] to

[14] , wherein the skin-resident bacteria further comprises one or more bacteria selected from Staphylococcus capitis and Staphylococcus epidermidis.

[0036]

[16] The antibacterial composition of [1], wherein the skin-resident bacteria comprises one or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, Corynebacterium tuberculostearicum, Staphylococcus capitis, Staphylococcus epidermidis, Moraxella osloensis, Dermacoccus nishinomiyaensis, Micrococcus luteus, Enhydrobacterae erosaccus, and Finegoldia magna.

[0037]

[17] The antibacterial composition according to any one of [1] or

[10] to

[16] , wherein the pro-inflammatory bacteria include one or more bacteria selected from Staphylococcus aureus and Staphylococcus hominis.

[0038]

[18] The antibacterial composition according to any one of [1] to

[17] , which is used for the treatment or prevention of a disease or condition caused by the pro-inflammatory bacteria.

[0039]

[19] The antibacterial composition described in

[18] , wherein the disease or condition is one or more diseases or conditions selected from atopic dermatitis, impetigo contagiosum, staphylococcal scalded skin syndrome, suppurative mastitis, furunculosis, acne vulgaris, acne rosacea, contact dermatitis, seborrheic dermatitis, epidermolytic ichthyosis, superficial skin infection, harlequin ichthyosis, congenital ichthyosis-like erythroderma, lamellar ichthyosis, Netherton syndrome, Sjögren-Larson syndrome, KID (keratitis-ichthyosis-deafness) syndrome, Dorfman-Channerin syndrome, neutral lipidosis, multiple sulfatase deficiency and X-linked ichthyosis.

[0040]

[20] A method for determining whether to administer the antimicrobial composition described in any one of [1] to

[19] to a subject, wherein the composition ratio or bacterial quantity of a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 1 to 13 or base sequences with an identity of 95% or more thereto, and a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 14 to 15 or base sequences with an identity of 95% or more thereto relative to the subject's skin flora is calculated, and whether the antimicrobial composition is administered to the subject is determined based on the composition ratio or bacterial quantity.

[0041]

[21] The method of

[20] , wherein at least one of the following requirements (A) or (B) is satisfied:

[0042] (A) when the composition ratio (the group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in SEQ ID NOs. 1 to 13 or base sequences having an identity of 95% or more thereto: the group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in SEQ ID NOs. 14 to 15 or base sequences having an identity of 95% or more thereto) is 0:10 to 9:1, it is determined that the antimicrobial composition described in any one of [1] to

[19] is suitable for administration to the subject;

[0043] (B) the bacterial count of the group consisting of one or more bacteria having 16S rDNA comprising any of the base sequences described in SEQ ID NOs. 1 to 13 or base sequences having an identity of 95% or more to the base sequences is 1,000,000 CFU / cm 2 The bacterial count of the group consisting of one or more bacteria having 16S rDNA including any of the base sequences described in SEQ ID NOs. 14 to 15 or base sequences having an identity of 95% or more to the base sequences is 10 CFU / cm 2 If the above, it is determined that it is appropriate to administer the antibacterial composition described in any one of [1] to

[19] to the subject.

[0044] Effects of the Invention

[0045] According to the present invention, an antibacterial composition comprising bacteria showing antibacterial activity against bacteria positively correlated with a SCORAD value indicating the severity of atopic dermatitis and a method for determining whether to administer the antibacterial composition to a subject can be provided.

[0046] Furthermore, by allowing skin-resident bacteria to colonize the skin, the number of repeated administrations required as with existing antimicrobial drugs can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a graph showing the correlation between 13 negative bacteria in atopic dermatitis patients and objective SCORAD, and the correlation between 13 reference bacteria and objective SCORAD. The vertical axis represents the total number of reads of each bacterial group obtained by 16S rRNA analysis, and the horizontal axis represents the objective SCORAD value.

[0048] Figure 2-1 to Figure 2-3 The graph shows the growth curve of Staphylococcus aureus when Staphylococcus aureus was cultured with the culture supernatant of negative bacteria for different days. The vertical axis represents the optical density (OD) and the horizontal axis represents the culture time (hours).

[0049] Figure 3 is based on Figure 2-1 to Figure 2-3 The graph summarizes the growth inhibitory activity when Staphylococcus aureus was cultured using the culture supernatant of the negative bacteria, calculated from the results. The vertical axis represents the value obtained by normalizing the optical density (OD) value at the middle time of the logarithmic growth phase, and the horizontal axis represents the bacterial species.

[0050] Figure 4-1 to Figure 4-2 The graph shows the growth curve of Staphylococcus aureus when Staphylococcus aureus was cultured with the culture supernatant of reference bacteria for different days. The vertical axis represents the optical density (OD) and the horizontal axis represents the culture time (hours).

[0051] Figure 5 is based on Figure 4-1 to Figure 4-2 The graph summarizes the growth inhibitory activity when Staphylococcus aureus was cultured using the culture supernatant of the reference bacteria, calculated from the results. The vertical axis represents the value obtained by normalizing the optical density (OD) value at the center time of the logarithmic growth phase, and the horizontal axis represents the bacterial species.

[0052] Figure 6-1 to Figure 6-2 The graph shows the growth curve of human Staphylococcus aureus when human Staphylococcus aureus is cultured with the culture supernatant of negative bacteria for different days. The vertical axis represents the optical density (OD) and the horizontal axis represents the culture time (hours).

[0053] Figure 7 is based on Figure 6-1 to Figure 6-2 The graph summarizes the growth inhibitory activity when human Staphylococcus aureus was cultured using the culture supernatant of negative bacteria, calculated from the results. The vertical axis represents the value obtained by normalizing the optical density (OD) value at the middle time of the logarithmic growth phase, and the horizontal axis represents the bacterial species.

[0054] Figure 8 This is a graph showing the growth curve of Staphylococcus aureus when Staphylococcus aureus is cultured using the culture supernatant of a reference bacterium. The vertical axis represents the optical density (OD), and the horizontal axis represents the culture time (hours).

[0055] Fig. 9 It means according to Figure 8 The graph shows the growth inhibitory activity when Staphylococcus aureus was cultured using the culture supernatant of the reference bacteria calculated from the results. The vertical axis represents the value obtained by normalizing the optical density (OD) value at the middle time of the logarithmic growth phase, and the horizontal axis represents the bacterial species.

[0056] Fig.10 The graph shows the change over time in the thickness of the auricle of BALB / cAJcl mice in which inflammation was induced with MC903 and infected with Staphylococcus aureus, followed by treatment with culture supernatant of ozenoxacin (OZXN), negative bacteria (10mix) or reference bacteria (reference 6mix).

[0057] Fig.11 The photographs show the state of the mouse ears when the auricles of BALB / cAJcl mice, which were induced with inflammation by MC903, were infected with Staphylococcus aureus and then treated with culture supernatant of ozenoxacin (OZXN), negative bacteria (10mix) or reference bacteria (reference 6mix). A is a photograph taken on day 0 of treatment, and B is a photograph taken on day 6 after treatment.

[0058] Fig.12The graph shows the change over time in the thickness of the auricle when the auricle of BALB / cAJcl mice in which inflammation was induced with MC903 was infected with Staphylococcus aureus and then treated with vancomycin (VCM) or the culture supernatant of negative bacteria (10mix).

[0059] Fig.13 The photographs show the state of the mouse ears on day 6 after the auricles of BALB / cAJcl mice, in which inflammation was induced with MC903, were infected with Staphylococcus aureus and then treated with vancomycin (VCM) or the culture supernatant of negative bacteria (10mix).

[0060] Fig.14 The graph shows the change over time in the thickness of the auricle of BALB / cAJcl mice in which inflammation was induced with MC903 and infected with human Staphylococcus aureus (Sh), followed by treatment with the culture supernatant of negative bacteria (12mix) or reference bacteria (reference 6mix).

[0061] Fig.15 This is a graph showing the growth curve of each Staphylococcus aureus when each Staphylococcus aureus is cultured using the culture supernatant of oral Streptococcus. The vertical axis represents the optical density (OD) and the horizontal axis represents the culture time (hours).

[0062] Fig.16 is based on Fig.15 The results show a summary of the growth inhibitory activity of each Staphylococcus aureus cultured with the culture supernatant of oral Streptococcus. The vertical axis represents the value obtained by normalizing the optical density (OD) value at the middle time of the logarithmic growth phase, and the horizontal axis represents the bacterial species.

[0063] Fig.17 This is a graph showing the growth curve of Staphylococcus aureus (MRSA) when Staphylococcus aureus (MRSA) is cultured using the culture supernatant of negative bacteria. The vertical axis represents the optical density (OD), and the horizontal axis represents the culture time (hours).

[0064] Fig.18 The graph shows the change over time in the thickness of the auricle of BALB / cAJcl mice in which inflammation was induced with MC903 and then infected with Staphylococcus aureus and then treated with live bacteria of negative bacteria (12mix), killed bacteria of negative bacteria (12mix), live bacteria of oral Streptococcus, or killed bacteria of oral Streptococcus.

[0065] Fig.19 The graph shows the change over time in the thickness of the auricle when the auricle of C57BL / 6 mice, in which inflammation was induced with MC903, was infected with Staphylococcus aureus and then treated with the culture supernatant of negative bacteria (12mix) or oral Streptococcus.

[0066] Fig. 20 The graph shows the change over time in the thickness of the auricle when the auricle of BALB / cAJcl mice in which inflammation was induced with MC903 was infected with Staphylococcus aureus and then treated with a negative bacteria (12mix) obtained by redispersing freeze-dried negative bacteria in soybean oil. DETAILED DESCRIPTION

[0067] The preferred embodiment described below is an example of a representative embodiment of the present invention, and the scope of the present invention is not to be construed as being limited thereby.

[0068] <Antibacterial Composition>

[0069] One embodiment of the present invention is an antibacterial composition targeting pro-inflammatory bacteria other than skin-resident bacteria, wherein the composition comprises the skin-resident bacteria as an effective ingredient.

[0070] The skin resident bacteria contained in the antibacterial composition can be live bacteria or dead bacteria. The form of the composition containing skin resident bacteria is not particularly limited, and can be any one of liquid or solid. In the case of live bacteria, the form of the culture obtained after the culture (including the culture supernatant containing live bacteria and the culture supernatant not containing live bacteria), the form of diluting or concentrating it, or the form of drying the thalline, more preferably the form of the mixture of them with buffer and base, etc. is not particularly limited. As long as the culture medium corresponding to the strain is used, aerobic culture or anaerobic culture is appropriately selected according to the culture stage. For example, for the following granular epidermidis (Cutibacteriumgranulosum), oral streptococcus (Streptococcus oralis), acne epidermidis (Cutibacteriumacnes), Finegoldia magna (Finegoldia magna) in the embodiment, from the perspective of good inhibition of the proliferation of proinflammatory bacteria, it is preferred to perform aerobic culture in pre-culture, and then perform anaerobic culture in the stage of obtaining the culture supernatant. In the case of dead bacteria, the form of dried bacteria is preferred, and the form of a mixture with a culture medium, a buffer, a base, etc. is more preferred. The drying treatment can be performed, for example, by heat drying, freeze drying, spray drying, etc. of a suspension dispersed in a solvent such as water, among which freeze drying is preferred. In addition, it can also be a culture containing dead bacteria obtained by heating a culture of live bacteria.

[0071] The antibacterial composition may also contain: substances contained in skin resident bacteria such as proteins, nucleic acids, lipids, sugars, sugar chains, etc.; buffers such as phosphates, citrates, acetates, etc.; culture media for culturing skin resident bacteria such as sterile water, physiological saline, ACX medium, Clostridium medium, BHI medium, BL medium, LB medium, EG medium, etc.; vegetable oils such as soybean oil, rice bran oil, corn oil, olive oil, rapeseed oil, coconut oil, linseed oil, sesame oil, castor oil, peanut oil, etc.; bases such as beeswax, paraffin, liquid paraffin, stearic acid, white vaseline, etc.; sugars or sugar alcohols such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, mannoacetate, etc.; amino acids such as arginine and histidine; stabilizers such as polyols such as propylene glycol, glycerol, polyethylene glycol, and polypropylene glycol, etc. In addition, when freeze-drying the skin resident bacteria, it is particularly preferred to contain glycerol.

[0072] [Resident bacteria on the skin]

[0073] The skin-resident bacteria contained in the antibacterial composition of the present invention are bacteria that exhibit antibacterial activity against the following proinflammatory bacteria by combining one or more bacteria present on the skin of healthy persons or patients with skin diseases.

[0074] In this specification, the concept of antibacterial includes not only inhibition of bacterial proliferation, but also sterilization and sterilization. The presence or absence of antibacterial effect can be confirmed by known techniques, such as the method of evaluating based on the change of proliferation curve in the examples, the disc diffusion method and the micro-liquid dilution method.

[0075] As skin-resident bacteria, bacteria that show antibacterial activity against pro-inflammatory bacteria that are presumed to increase the value of SCORAD (including objective SCORAD obtained by omitting subjective symptoms from SCORAD), an index of severity of atopic dermatitis, are preferred.

[0076] For example, for patients with atopic dermatitis, the composition ratio of the skin flora before and after treatments such as bleaching bath treatment, anti-inflammatory topical drugs and antihistamines, and ultraviolet therapy is calculated through flora analysis such as 16S rRNA analysis or metagenomic analysis. Next, select bacterial species with a composition ratio of more than 10%, preferably more than 15%, and more preferably more than 20%. In the single regression of the linear mixed model with the bacterial species as the explanatory variable and SCORAD as the target variable, the Benjamini-Hochberg method is used for correction. For the case where the p-value of the F test of the corrected regression is less than 0.1, preferably less than 0.05, more preferably less than 0.01, further preferably less than 0.001, and particularly preferably less than 0.0001, bacteria that are positively correlated with SCORAD can be selected as pro-inflammatory bacteria, and bacteria that are negatively correlated with SCORAD can be selected as skin resident bacteria.

[0077] As skin resident bacteria, bacteria negatively correlated with SCORAD can be cited, such as bacteria having 16S rDNA including base sequences of sequence numbers 1 to 13 encoding the V1-V2 region of 16S rRNA or any one of base sequences with an identity of 95% or more, preferably 96% or more, more preferably 97% or more, further preferably 98% or more, and particularly preferably 99% or more. The bacterium may have more than one 16S rDNA, and the base sequences of the 16S rDNA have different identities although the sequence numbers are the same. For example, a 16S rDNA having a base sequence of sequence number 1 and a 16S rDNA having a base sequence with an identity of 95% or more with sequence number 1 may be included in one bacterium. In addition, the 16S rDNA possessed by the skin resident bacteria used in the following examples includes any one of the base sequences of sequence numbers 1 to 13 or a base sequence with an identity of 95% or more with the base sequence.

[0078] As a preferred form of the present invention, the antibacterial composition of the present invention comprises preferably one or more, more preferably two or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes the base sequence described in sequence numbers 1 to 6 or any one of the base sequences having an identity of more than 95%, preferably more than 96%, more preferably more than 97%, further preferably more than 98%, and particularly preferably more than 99% with the base sequence.

[0079] As a more preferred embodiment of the present invention, the antibacterial composition of the present invention comprises preferably one or more, more preferably two or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes the base sequence described in sequence numbers 1 to 3 or any one of the base sequences having an identity of more than 95%, preferably more than 96%, more preferably more than 97%, further preferably more than 98%, and particularly preferably more than 99% with the base sequence.

[0080] As a more preferred embodiment of the present invention, the antibacterial composition of the present invention comprises a bacterium having a 16S rDNA comprising the base sequence described in sequence number 1 or a base sequence having an identity of 95% or more, preferably 96% or more, more preferably 97% or more, further preferably 98% or more, and particularly preferably 99% or more, a bacterium having a 16S rDNA comprising the base sequence described in sequence number 2 or a base sequence having an identity of 95% or more, preferably 96% or more, more preferably 97% or more, further preferably 98% or more, and particularly preferably 99% or more, and a bacterium having a 16S rDNA comprising the base sequence described in sequence number 3 or a base sequence having an identity of 95% or more, preferably 96% or more, more preferably 97% or more, further preferably 98% or more, and particularly preferably 99% or more.

[0081] As a particularly preferred embodiment of the present invention, the antibacterial composition of the present invention comprises one or more, preferably two or more bacteria having 16S rDNA, wherein the 16S rDNA comprises the base sequence described in sequence numbers 1 to 6 or a base sequence having an identity of 95% or more, preferably 96% or more, more preferably 97% or more, further preferably 98% or more, and particularly preferably 99% or more to the base sequence, or comprises one or more, preferably two or more bacteria having 16S rDNA, wherein the 16S rDNA comprises the base sequence described in sequence numbers 1 to 3 or a base sequence having an identity of 95% or more, preferably 96% or more, more preferably 97% or more, further preferably 98% or more, and particularly preferably 99% or more to the base sequence, and in addition comprises one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S The rDNA includes the base sequences described in sequence numbers 7 to 8 or any one of the base sequences having an identity of more than 95%, preferably more than 96%, more preferably more than 97%, further preferably more than 98%, and particularly preferably more than 99% with the base sequences.

[0082] As one form of the antibacterial composition of the present invention, it contains one or more bacteria having 16S rDNA, and the 16S rDNA includes the base sequence described in the above-mentioned sequence numbers 1 to 13 or a base sequence having an identity of more than 95%, preferably more than 96%, more preferably more than 97%, further preferably more than 98%, and particularly preferably more than 99% with the base sequence.

[0083] When the antibacterial composition of the present invention contains two or more bacteria having 16S rDNA, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen bacteria, the combination of the bacteria can be arbitrarily selected, and the 16S rDNA includes the base sequence described in sequence numbers 1 to 13 or a base sequence having an identity of more than 95%, preferably more than 96%, more preferably more than 97%, further preferably more than 98%, and particularly preferably more than 99%.

[0084] As a combination of bacteria, when a bacterium having 16S rDNA including the base sequence described in sequence number n or any of the base sequences having an identity of 95% or more to the base sequence is simply referred to as "bacteria n", examples include: for example, a combination of bacteria 1 and bacteria 3, a combination of bacteria 2 and bacteria 3, a combination of bacteria 1 and bacteria 3, a combination of bacteria 1, bacteria 2, bacteria 3, bacteria 4, bacteria 5 and bacteria 6, a combination of bacteria 1, bacteria 2, bacteria 4, bacteria 5, bacteria 6, bacteria 7, bacteria 8, bacteria 9, bacteria 10 and bacteria 11, a combination of bacteria 1, bacteria 2, bacteria 4, bacteria 5, bacteria 6, bacteria 7, bacteria 8, bacteria 9, bacteria 10, bacteria 11 and bacteria 12, a combination of bacteria 1, bacteria 2, bacteria 3, bacteria 4, bacteria 5, bacteria 6, bacteria 7, bacteria 8, bacteria 9, bacteria 10, bacteria 11 and bacteria 13, a combination of bacteria 1, bacteria 2, bacteria 3, bacteria 4, bacteria 5, bacteria 6, bacteria 7, bacteria 8, bacteria 9, bacteria 10, bacteria 11 and bacteria 13, The combination of bacteria 10, bacteria 11, bacteria 12 and bacteria 13, the combination of bacteria 3 and bacteria 4, the combination of bacteria 3 and bacteria 5, the combination of bacteria 3 and bacteria 6, the combination of bacteria 3, bacteria 4 and bacteria 7, the combination of bacteria 3, bacteria 4 and bacteria 8, the combination of bacteria 3, bacteria 4 and bacteria 9, the combination of bacteria 3, bacteria 4 and bacteria 10, the combination of bacteria 3, bacteria 4 and bacteria 11, the combination of bacteria 3, bacteria 5 and bacteria 7, the combination of bacteria 3, bacteria 5 and bacteria 8, the combination of bacteria 3, bacteria 5 and bacteria 9, the combination of bacteria 3, bacteria 5 and bacteria 10, the combination of bacteria 3, bacteria 5 and bacteria 11, the combination of bacteria 3, bacteria 6 and bacteria 7, the combination of bacteria 3, bacteria 6 and bacteria 8, the combination of bacteria 3, bacteria 6 and bacteria 9, the combination of bacteria 3, bacteria 6 and bacteria 10, and the combination of bacteria 3, bacteria and bacteria 11.

[0085] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 1 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Actinomyces viscosus, and the strain number of the bacterium is preferably ATCC15987.

[0086] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 2 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Cutibacterium granulosum, and the strain number of the bacterium is preferably ATCC25564.

[0087] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 3 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Streptococcus oralis, and the strain number of the bacterium is preferably JCM12997.

[0088] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 4 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Staphylococcus warneri, and the strain number of the bacterium is preferably ATCC27836.

[0089] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 5 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Cutibacterium acnes, and the strain number of the bacterium is preferably ATCC6919.

[0090] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 6 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Corynebacterium tuberculostearicum, and the strain number of the bacterium is preferably ATCC35692.

[0091] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 7 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Staphylococcus capitis, and the strain number of the bacterium is preferably ATCC27840.

[0092] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 8 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Staphylococcus epidermidis, and the strain number of the bacterium is preferably ATCC14990.

[0093] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 9 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Moraxella osloensis, and the strain number of the bacterium is preferably ATCC27840.

[0094] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 10 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Dermacoccus nishinomiyaensis, and the strain number of the bacterium is preferably ATCC29093.

[0095] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 11 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Micrococcus luteus, and the strain number of the bacterium is preferably ATCC4698.

[0096] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 12 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Enhydrobacter aerosaccus, and the strain number of the bacterium is preferably ATCC27094.

[0097] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 13 or any of the base sequences having an identity of 95% or more, and preferably 97% or more to the base sequence is preferably Finegoldia magna, and the strain number of the bacterium is preferably JCM1766.

[0098] As a preferred embodiment of the present invention, the antibacterial composition of the present invention comprises preferably one or more, more preferably two or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, and Corynebacterium tuberculostearicum.

[0099] As a more preferred embodiment of the present invention, the antibacterial composition of the present invention contains preferably one or more, more preferably two or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

[0100] As a further preferred embodiment of the present invention, the antibacterial composition of the present invention contains Actinomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

[0101] As a particularly preferred embodiment of the present invention, the antibacterial composition of the present invention comprises one or more bacteria selected from the group consisting of Actionomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, and Corynebacterium tuberculostearicum, and in addition, comprises one or more bacteria selected from the group consisting of Staphylococcus capitis and Staphylococcus epidermidis.

[0102] One form of the antibacterial composition of the present invention comprises one or more bacteria selected from the group consisting of Actinomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, Corynebacterium tuberculostearicum, Staphylococcus capitis, Staphylococcus epidermidis, Moraxella osloensis, Dermacoccus nishinomiyaensis, Micrococcus luteus, Enhydrobacter aerosaccus, and Finegoldia magna.

[0103] [Pro-inflammatory bacteria]

[0104] Proinflammatory bacteria refer to bacteria that can cause skin inflammation other than the above-mentioned skin resident bacteria, including bacteria that not only cause inflammation but also worsen the inflammation that has already occurred. In addition, these bacteria can also be multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), penicillin-resistant Streptococcus pneumoniae (PRSP), quinolone-resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa and other multidrug-resistant bacteria. Among these multidrug-resistant bacteria, the antibacterial composition of the present invention has a better effect on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE).

[0105] The pro-inflammatory bacteria are not particularly limited as long as they are bacteria that cause inflammation, but the above-mentioned bacteria that are positively correlated with SCORAD are preferred, and more preferably bacteria having 16S rDNA comprising the base sequence of sequence numbers 14 to 15 encoding the V1-V2 region of 16S rRNA or any one of the base sequences having an identity of 95% or more, preferably 96% or more, more preferably 97% or more, further preferably 98% or more, and particularly preferably 99% or more to the base sequence.

[0106] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 14 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Staphylococcus aureus, and the strain number of the bacterium is preferably ATCC12600.

[0107] The bacterium having 16S rDNA including the base sequence described in SEQ ID NO: 15 or any base sequence having an identity of 95% or more, preferably 97% or more to the base sequence is preferably Staphylococcus hominis, and the strain number of the bacterium is preferably ATCC27844.

[0108] The pro-inflammatory bacteria may be Staphylococcus aureus with a strain number of ATCC29213 or ATCC25923, or Staphylococcus aureus with a strain number of ATCC33591 or ATCC43300 (MRSA: methicillin-resistant Staphylococcus aureus).

[0109] [use]

[0110] The antibacterial composition can be used for the treatment or prevention of diseases or conditions caused by the above-mentioned pro-inflammatory bacteria, and can be used as pharmaceutical compositions such as pharmaceuticals and quasi-pharmaceuticals, cosmetics having the effect of treating or preventing diseases or conditions caused by the above-mentioned pro-inflammatory bacteria, or for research purposes. "Treatment" includes not only complete recovery from the above-mentioned disease or condition, but also alleviation of the symptoms or condition of the above-mentioned disease or maintenance of alleviation, or inhibition of its progression. "Prevention" includes inhibition or delay of the onset of the above-mentioned disease or condition, or inhibition of its recurrence.

[0111] The disease or condition caused by proinflammatory bacteria may be a disease including dermatitis among its symptoms or a condition in which dermatitis can be observed even if the name of the disease cannot be determined, preferably a disease or condition dependent on Staphylococcus aureus or Staphylococcus hominis, more preferably a disease or condition selected from atopic dermatitis, impetigo contagiosum, staphylococcal scalded skin syndrome, suppurative mastitis, furunculosis, acne vulgaris, acne rosacea, contact dermatitis, seborrheic dermatitis, epidermolytic ichthyosis, superficial skin infection, harlequin ichthyosis, congenital ichthyosis-like erythroderma, lamellar ichthyosis, Netherton syndrome, Sjögren-Larsson syndrome, KID (keratitis-ichthyosis-deafness) syndrome, Dorfman-Channerin syndrome, neutral lipidosis, multiple sulfatase deficiency and X-linked ichthyosis, further preferably a disease or condition selected from atopic dermatitis, acne vulgaris and superficial skin infection.

[0112] The antibacterial composition is administered to the skin for use. The content of skin resident bacteria in the antibacterial composition can be appropriately set without particular limitation. The dosage of the antibacterial composition administered to the subject can be appropriately selected according to the subject's age, weight, disease symptoms, health status, etc.

[0113] The antibacterial composition can be formulated by known pharmaceutical methods, for example, it can be formulated into lotions, liniments, creams, ointments, plasters, poultices, sprays, or percutaneous absorption preparations, and preferably formulated into ointments, creams, lotions, or shower gels.

[0114] When formulating, the antibacterial composition can be appropriately combined with a pharmaceutically acceptable carrier, specifically sterile water, physiological saline, buffer, culture medium, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, fragrance, excipient, carrier, preservative, binder, diluent, isotonic agent, buffer, lubricant, colorant, thickener, other additives, etc.

[0115] The antibacterial composition can be used on animals including humans, but is not particularly limited to animals other than humans, and can be used on various livestock, poultry, pets, experimental animals, and the like.

[0116] <Method for determining administration of antibacterial composition>

[0117] One embodiment of the present invention is a method for determining whether to administer the antibacterial composition of the present invention to a subject, wherein the composition ratio or bacterial quantity of a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 1 to 13 or base sequences having an identity of 95% or more thereto (hereinafter also referred to as a "negative mix") and a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 14 to 15 or base sequences having an identity of 95% or more thereto (hereinafter also referred to as a "positive mix") relative to the subject's skin flora is calculated, and whether to administer the antibacterial composition to the subject is determined based on the composition ratio or bacterial quantity.

[0118] The above determination method preferably satisfies at least one of the following requirements (A) or (B):

[0119] (A) When the composition ratio (the group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 1 to 13 or the base sequences having an identity of 95% or more thereto: the group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 14 to 15 or the base sequences having an identity of 95% or more thereto) is 0:10 to 9:1, it is determined that the administration of the above-mentioned antibacterial composition to the subject is appropriate.

[0120] (B) the bacterial count of the group consisting of one or more bacteria having 16S rDNA comprising any of the base sequences described in SEQ ID NOs. 1 to 13 or base sequences having an identity of 95% or more to the base sequences is 1,000,000 CFU / cm 2 The bacterial count of the group consisting of one or more bacteria having 16S rDNA including any of the base sequences described in SEQ ID NOs. 14 to 15 or base sequences having an identity of 95% or more to the base sequences is 10 CFU / cm 2 If the above values ​​are obtained, it is determined that administration of the antibacterial composition to the subject is appropriate.

[0121] The above-mentioned composition ratio can be obtained by flora analysis such as 16S rRNA analysis or metagenomic analysis. When the ratio of each composition ratio (negative mixing: positive mixing) is, for example, 0:10-9:1, 0:10-8:2, 0:10-7:3, 0:10-6:4, 0:10-5:5, 0:10-4:5, 0:10-3:7, 0:10-2:9, 0:10-1:9, preferably 0:10-5:5, more preferably 0:10-3:7, it is determined that the antibacterial composition of the present invention is given to the object. When within the above-mentioned numerical range, by administering the antibacterial composition, the proliferation of pro-inflammatory bacteria is inhibited, which is effective for the treatment or prevention of diseases or symptoms caused by pro-inflammatory bacteria.

[0122] The bacterial counts of the negative mix and the positive mix can be determined, for example, by quantitative PCR using known 16S rRNA universal primers. 2 Below, preferably 100000 CFU / cm 2 Below, more preferably 10000 CFU / cm 2 Below, more preferably 1000 CFU / cm 2 Below, the amount of bacteria mixed is 10CFU / cm 2 Above, preferably 100 CFU / cm 2 More preferably, 1000 CFU / cm 2 Above, more preferably 10000 CFU / cm2 When the above value is above, it is determined that the antibacterial composition of the present invention is given to the object. When within the above numerical range, by administering the antibacterial composition, the proliferation of pro-inflammatory bacteria is suppressed, and the treatment or prevention of the disease or symptom caused by the pro-inflammatory bacteria is effective. In addition, although the above determination can be carried out using the value of each bacterial amount of only one of the negative mixing and the positive mixing, it can also be carried out using the value of each bacterial amount of both, but it is preferably determined by using the value of each bacterial amount of both.

[0123] <Methods for treating or preventing diseases or conditions caused by pro-inflammatory bacteria>

[0124] One embodiment of the present invention is a method for treating or preventing a disease or condition caused by pro-inflammatory bacteria, comprising: a step of calculating the composition ratio or bacterial quantity of a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 1 to 13 or a base sequence with an identity of 95% or more thereto, and a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 14 to 15 or a base sequence with an identity of 95% or more thereto, relative to the skin flora of a subject; a step of determining whether to administer the above-mentioned antibacterial composition to the above-mentioned subject based on the composition ratio or bacterial quantity obtained in the above-mentioned step; and a step of administering the above-mentioned antibacterial composition to the subject determined to be suitable for administration by the above-mentioned step.

[0125] <Antibacterial Composition for Treatment or Prevention>

[0126] One embodiment of the present invention is an antibacterial composition for use in the treatment or prevention of a disease or condition caused by pro-inflammatory bacteria.

[0127] <Application of antibacterial composition in the preparation of therapeutic or preventive drugs>

[0128] One embodiment of the present invention is the use of an antibacterial composition in the preparation of a therapeutic or preventive drug for a disease or condition caused by pro-inflammatory bacteria.

[0129] Example

[0130] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples and can be implemented with appropriate changes within the scope of the technical concept.

[0131] [Preparation Example]

[0132] [1] Network analysis

[0133] Bacteria that are positively correlated with the objective SCORAD and bacteria that are negatively correlated with the objective SCORAD are selected by the following method.

[0134] For 26 patients with atopic dermatitis, swab samples of skin flora were obtained from four locations (center of eyebrows, cubital fossa, inner forearm, and center of upper back) at a total of six time points, including one month before the start of a three-month bleaching bath treatment, once a month during the treatment, and one month after the end of the treatment. The composition ratio at the species level was determined by 16S rRNA analysis of each of the following samples. The readings of 34 species of bacteria present in more than 25% of the samples among all the species were logarithmically transformed with a constant of 2, and the obtained values ​​were subjected to multiple regression of linear mixed models. The linear mixed model is a type of statistical analysis model, which is represented by a formula defined by y=Xβ+Zb+ε. The Xβ term is called a fixed effect, the Zb term is called a variable effect, y is called a target variable, X is called an explanatory variable, and Z is called a random effect variable. The linear mixed model with objective SCORAD as the target variable was used as a correlation evaluation model for the linkage between the various species, and a linear mixed model with each single species as the target variable was performed. In the linear mixed model with objective SCORAD as the target variable, 34 bacterial species were used as explanatory variables, and patients and collection sites were used as random effects. In the linear mixed model with each bacteria as the target variable, 33 bacterial species other than the target bacterial species were used as explanatory variables, and patients and collection sites were used as random effects. The p-value of the F test of regression was corrected by the Benjamini-Hochberg method. For variables with a p-value of <0.0001, it was defined as having a correlation. Two bacteria that were positively correlated with objective SCORAD (hereinafter also referred to as "positive bacteria") were selected, 13 bacteria that were negatively correlated with these two bacteria (hereinafter also referred to as "negative bacteria") were selected, and 13 control bacteria for negative bacteria (skin resident bacteria other than positive bacteria and negative bacteria) were selected (hereinafter also referred to as "control bacteria" or "reference bacteria"). In addition, as the above-mentioned 13 reference bacteria, the 13 species with the highest average relative abundance ratio among the skin resident bacteria other than positive bacteria and negative bacteria in the sample were selected. The species names and strain numbers are shown in Table 1. In the case where the scientific name of the bacterial species corresponding to the strain number in Table 1 has been changed due to a change in classification resulting from the progress of taxonomic research, the bacterial species after the change in scientific name is referred to.

[0135] [Table 1]

[0136]

[0137]

[0138] The correlation between the 13 negative bacteria in atopic dermatitis patients and the objective SCORAD and the correlation between the 13 reference bacteria and the objective SCORAD based on the 16S rRNA analysis results before the bleaching bath treatment are shown in Figure 1 .

[0139] [2] 16S rRNA analysis

[0140] The above-mentioned 16S rRNA analysis was performed as described below. The bacteria in the swab samples obtained from the center of the eyebrows, elbows, inner forearms, and upper back of patients with atopic dermatitis were suspended in sterile water and then centrifuged (3,000×g, 4 minutes, room temperature) to recover the bacteria. The genomic DNA of the whole bacteria was extracted from the obtained bacteria using the Maxwell (registered trademark) RSC Blood DNA Kit [AS1400] (manufactured by Promega). PCR was performed using the following primers using the obtained genomic DNA as a template. The temperature and cycle conditions of PCR are as follows.

[0141] (a) 96°C, 2 minutes

[0142] (b) 96°C, 30 seconds

[0143] (c) 55°C, 45 seconds

[0144] (d) 72°C, 60 seconds

[0145] (b) to (d) for 30 cycles

[0146] (e) 72°C, 10 minutes

[0147] (f) Maintain at 4°C.

[0148] Fw primer: AGRGTTTGATYMTGGCTCAG (SEQ ID NO: 16)

[0149] Rv primer: TGCTGCCTCCCGTAGGAGT (SEQ ID NO: 17)

[0150] The amplified DNA fragment of about 300 bp encoding the V1-V2 region of 16S rRNA was purified using AMPure (Beckman Coulter). The obtained amplified fragment (amplicon) was provided for next generation sequencing (NGS) analysis. NGS was performed using Miseq and Miseq Ragent kit v2 (500-cycle, Illumina). For the two paired end reads, after removing the primer sequence with cutadapt, filterAndTrim of dada2 R package (version 1.14.1) was used to filter with the conditions of maxEE=c(2,2) and truncLen=c(200,180). The denoised paired end reads were merged with dada, and the chimeras were removed with removeBimeraDenovo to create an amplicon sequence variant (ASV) table. The ASV table was standardized to 3000 reads per sample by random sampling. Each ASV was taxonomically divided by searching the homology with respect to the Ribosomal Database Project (RDP) (rekaese 11) and the National Center for Biotechnology Information (NCBI) genome database using the GLSEARCH program. This search, division, etc. were performed on June 18, 2019. The relationship between the name of each strain and the sequence number representing the sequence of the amplified fragment of each strain is shown in Table 2 below.

[0151] [Table 2]

[0152]

[0153] [Example 1]

[0154] Each negative bacterial strain in Table 1 above, which had been pre-cultured in ACX liquid medium (39.5 g / L blood agar base medium No. 2 (manufactured by Lab MIDG), 3 g / L yeast extract (manufactured by Oxoid), 2 g / L glucose, 5 mL / L Tween 80, 50 mL / L defibrinated horse blood (manufactured by TSC biosciences)) at 37°C for 24 hours, was diluted with ACX liquid medium to prepare a single bacterial solution having an optical density (OD) of 0.05 at a wavelength of 600 nm. Each obtained single bacterial solution was cultured at 37°C for 48 hours. The culture solution was centrifuged (10,000×g, 5 minutes) and filtered with a 0.22 μm filter to obtain a culture supernatant.

[0155] Staphylococcus aureus pre-cultured in ACX liquid medium was diluted with ACX liquid medium to prepare a positive bacterial solution with an OD of 0.01. The positive bacterial solution was mixed with the culture supernatant obtained above in equal amounts, and then inoculated into a 96-well culture plate (product name "VIOLAMO 96-well plate flat bottom", manufactured by VIOLAMO) and cultured at 37°C. In addition, ACX liquid medium and physiological saline that were not used for culture were used as comparison controls for the mixed culture supernatant. The OD of the bacterial solution during the culture process was monitored using an ELISA reader (product name "ELISA reader Spark", manufactured by TECAN) to obtain a proliferation curve. This operation was performed three times on different days, and the results of the proliferation curves of each single bacterial solution are shown in Figure 2-1 to Figure 2-3 .

[0156] The central time of the logarithmic growth phase under the culture conditions of the ACX liquid medium as the comparison control group was defined as the evaluation time, and the OD value at the evaluation time was used as an indicator of the growth inhibitory activity of the culture supernatant of each negative bacteria against the positive bacteria. The results of the growth inhibitory activity of each single bacterial solution are shown in Figure 3 .

[0157] [Example 2]

[0158] The culture supernatant of each mixed bacterial solution was obtained in the same manner as in Example 1, except that each mixed bacterial solution of negative bacteria, which was obtained by mixing and combining the bacterial species shown in Table 3 below in equal amounts, was used instead of the single bacterial solution. The results of each growth curve of each mixed solution are shown in Figure 2-1 to Figure 2-3 , and in Figure 2-1 to Figure 2-3 In the growth curve of , the value obtained by normalizing the optical density (OD) value at the central time of the logarithmic growth phase was summarized as the growth inhibitory activity and the results are shown in FIG. Figure 3 .

[0159] [Table 3]

[0160]

[0161] [Reference Example 1]

[0162] The growth curve of each single bacterial solution was obtained in the same manner as in Example 1, except that the reference bacteria were used instead of the negative bacteria in Table 1. This operation was performed twice on different days, and the results of the growth curve of each single bacterial solution are shown in Figure 4-1 to Figure 4-2 , and the results of proliferation inhibitory activity are shown in Figure 5 .

[0163] [Reference Example 2]

[0164] The culture supernatant of the mixed bacterial solution was obtained in the same manner as in Example 2, except that a mixed bacterial solution of reference bacteria (reference 6mix) was used instead of the mixed bacterial solution of negative bacteria, in which single bacterial solutions of 6 reference bacteria (Anaerococcus octavius, Corynebacterium simulans, Corynebacterium striatum, Kocuria rhizophila, Staphylococcus saccharolyticus, and Staphylococcus saprophyticus) were mixed in equal amounts and combined, and the operation was performed twice on different days. The results of the proliferation curve of the mixed solution are shown in FIG. Figure 4-1 and Figure 4-2 , and the results of proliferation inhibitory activity are shown in Figure 5 .

[0165] [Example 3]

[0166] The growth curve of each single bacterial solution was obtained in the same manner as in Example 1, except that human Staphylococcus aureus was used instead of Staphylococcus aureus and the operation was performed twice on different days. The results of the growth curve of each single bacterial solution are shown in Figure 6-1 and Figure 6-2 , and the results of proliferation inhibitory activity are shown in Figure 7 .

[0167] [Example 4]

[0168] The culture supernatant of each mixed bacterial solution was obtained in the same manner as in Example 2, except that human Staphylococcus aureus was used instead of Staphylococcus aureus and the operation was performed twice on different days. The results of each growth curve of each mixed solution are shown in Figure 6-1 and Figure 6-2 , and the results of proliferation inhibitory activity are shown in Figure 7 .

[0169] [Reference Example 3]

[0170] The growth curve of each single bacterial solution was obtained in the same manner as in Example 1, except that the reference bacteria were used instead of the negative bacteria in Table 1 in Example 3 and the operation was performed once. The results of the growth curve of each single bacterial solution are shown in Figure 8 , and the results of proliferation inhibitory activity are shown in Fig. 9 .

[0171] [Reference Example 4]

[0172] The growth curves of the single bacterial solutions were obtained in the same manner as in Example 4, except that a mixed bacterial solution of reference bacteria (reference 6mix) was used instead of the mixed bacterial solution of negative bacteria, in which single bacterial solutions of 6 reference bacteria (Anaerococcus octavius, Corynebacterium simulans, Corynebacterium striatum, Kocuria rhizophila, Staphylococcus saccharolyticus, and Staphylococcus saprophyticus) were mixed and combined in equal amounts. The results of the growth curves of the single bacterial solutions are shown in FIG. Figure 8 , and the results of proliferation inhibitory activity are shown in Fig. 9 .

[0173] [Example 5]

[0174] [1] Preparation of mice

[0175] BALB / cAJcl mice (8 weeks old, male, manufactured by Japan Corea Co., Ltd.) were acclimated for 1 week in a temperature and humidity controlled (22-24°C, 40-60%) and dimming environment (200 lux / 12 h day and night cycle) and used in the following 5 comparative experiments (a) to (e).

[0176] (a) Untreated group

[0177] (b) MC903 treatment group only

[0178] (c) MC903+Staphylococcus aureus treatment group

[0179] (d) MC903+Staphylococcus aureus+antibiotic treatment group

[0180] (e) MC903+Staphylococcus aureus+bacteria mixed treatment group

[0181] [2] Preparation of MC903-induced inflammation model

[0182] Using a micropipette, 20 μL of 100 μM calcipotriol (product name "MC903", manufactured by Funakoshi Co., Ltd.) ethanol solution was applied to both ears of mice in groups (b) to (e) so that the entire auricle was covered. The MC903-induced inflammation model was established by applying once a day for one week.

[0183] [3] Staphylococcus aureus infection

[0184] Staphylococcus aureus (hereinafter also referred to as "Sa") was cultured in ACX liquid medium at 37°C to obtain a bacterial solution in a constant growth phase. The bacterial solution was diluted with ACX liquid medium and adjusted to an OD of 2.5. Using a micropipette, 50 μL of each diluted bacterial solution was applied to both ears of mice in groups (c) to (e) in which inflammation was induced with MC903, so that it covered the entire auricle. Staphylococcus aureus was applied only once, and the day of application was defined as Day 0 (DAY0), and each day was defined as Day 1 (DAY1), Day 2 (DAY2), etc.

[0185] [4] Antibiotic application

[0186] For the group (d) infected with Staphylococcus aureus, 50 μL of Zebiax (ABX) (product name "Zebiax Lotion", manufactured by Maruho Co., Ltd.) was applied to both ears of the mice using a micropipette so that it covered the entire auricle. The application was performed once a day from day 1 (DAY1) to day 4 (DAY4).

[0187] [5] Application of bacterial mixture

[0188] Each strain of 10mix in Table 3 pre-cultured in ACX liquid medium or each strain of 6 reference bacteria (Anaerococcus octavius, Corynebacterium simulans, Corynebacterium striatum, Kocuria rhizophila, Staphylococcus saccharolyticus and Staphylococcus saprophyticus) was diluted with ACX liquid medium, and each strain reaching an equal OD value was mixed in equal amounts to prepare 10mix and reference 6mix, and glycerol was further added to prepare a storage solution of 20 volume % glycerol. Each storage solution was frozen and stored at -80°C and thawed before coating. The thawed bacterial storage solution was centrifuged (5,000×g, 5 minutes), the supernatant was removed, and then dispersed in ACX liquid medium to prepare a bacterial mixture with an OD of 2.5. For the group (e) infected with Staphylococcus aureus, 50 μL of the bacterial mixture was applied to both ears of the mouse using a micropipette to cover the entire auricle. The application was performed once a day from day 1 (DAY1) to day 4 (DAY4). In addition, for the group (c) infected with Staphylococcus aureus, ACX medium without bacteria was applied in the same manner.

[0189] [6] Determination of ear thickness

[0190] Determine 3 measurement points in the auricle on each ear, and use a digital vernier caliper to measure a total of 6 points on both sides, and take the average value as the auricle thickness. The auricle thickness is used as an indicator of the degree of skin inflammation to evaluate the degree of inhibition of inflammation by the bacterial mixture treatment. In addition, take pictures of the mouse ears. The ear thickness was measured every day from the day before day 0 (DAY0) (day -1 (DAY-1)) to day 8 (DAY8). Photos were taken on day 0 (DAY0) and day 6 (DAY6). The results are shown in Fig.10 and Fig.11 .

[0191] [Example 6]

[0192] The degree of inhibition of inflammation by treatment with the bacterial mixture was evaluated in the same manner as in Example 5, except that the following "[4'] administration of antibiotics" was performed instead of "[4] application of antibiotics" in Example 5. The results are shown in Fig.12 and Fig.13 In addition, the state of the auricle after 0 days of treatment can be referred to Fig.11 The state shown in A.

[0193] [4'] Administration of antibiotics

[0194] The group (d) infected with Staphylococcus aureus was intraperitoneally administered with 200 μL of 18 mg / mL vancomycin (VCM) PBS solution (manufactured by Towa Pharmaceutical Co., Ltd.) The administration was performed twice a day for 4 days from Day 1 (DAY1) to Day 4 (DAY4).

[0195] [Example 7]

[0196] The degree of inhibition of inflammation by the bacterial mixture treatment was evaluated in the same manner as in Example 5, except that human Staphylococcus aureus (hereinafter also referred to as "Sh") was used instead of Sa in the item "[3] Staphylococcus aureus infection" in Example 5, and 12 mixes of each strain were used instead of 10 mixes of each strain in Table 3. The results are shown in Fig.14 .

[0197] [Example 8]

[0198] In Example 1, the growth curves were obtained in the same manner as in Example 1, except that the Staphylococcus aureus shown in Table 4 below was used instead of Staphylococcus aureus (ATCC12600) as the positive bacteria and Streptococcus oralis (JSM12997) was used as the negative bacteria, and the growth inhibition activity was evaluated. The growth curves of each single bacterial solution are shown in Fig.15 , and the results of proliferation inhibitory activity are shown in Fig.16 . Fig.15 In the figure, "300K" and "30K" mean that the supernatant was passed through filters with separation molecular weights of 300,000 and 30,000, respectively, and "×2sup" means that the supernatant was diluted 2 times.

[0199] [Table 4]

[0200] Strain number Species name ATCC29213 Staphylococcus aureus ATCC25923 Staphylococcus aureus ATCC33591 Staphylococcus aureus (MRSA) ATCC43300 Staphylococcus aureus (MRSA) ATCC12600 Staphylococcus aureus

[0201] [Example 9]

[0202] The growth curve was obtained in the same manner as in Example 1 except that Staphylococcus aureus (ATCC33591) which is MRSA was used as the positive bacteria and the bacteria shown in Table 1 were used as the negative bacteria. The growth curve results of each single bacterial solution are shown in Fig.17 In addition, the growth curve of Staphylococcus aureus was also obtained for the culture supernatant obtained by performing anaerobic culture (the same operation as in Example 1 was performed in an anaerobic chamber Bactron 300 (manufactured by Shellab)) instead of aerobic culture in Example 1 for the four types of negative bacteria. In the figure, the prefix "an" is added to the name of the strain for the strain subjected to the anaerobic culture.

[0203] [Example 10]

[0204] The degree of inhibition of inflammation by treatment with the bacterial mixture or oral streptococcus solution was evaluated in the same manner as in Example 5, except that the following "[5'] application of the bacterial mixture solution" was performed instead of "[5] application of the bacterial mixture solution". The results are shown in Fig.18 .

[0205] [5'] Application of bacterial mixture

[0206] Each strain pre-cultured in ACX liquid medium was diluted with ACX liquid medium, and each strain reaching the same OD value was mixed in equal amounts to prepare 12mixes, and glycerol was further added to prepare a preservation solution with 20 volume% glycerol. Each preservation solution was frozen and stored at -80°C, and thawed before coating. The thawed bacterial preservation solution was centrifuged (5,000×g, 5 minutes), and after removing the supernatant, it was heated at 121°C for 10 minutes, and then dispersed in ACX liquid medium to prepare HK (Heat Killing) 12mix solution. Similarly, oral streptococci were also heat-treated and prepared into HK oral streptococcal solution.

[0207] [Example 11]

[0208] The degree of inhibition of inflammation by treatment with the bacterial mixture was evaluated in the same manner as in Example 5, except that the following "[1'] Preparation of mice" was performed instead of "[1] Preparation of mice". The results are shown in Fig.19 .

[0209] [1'] Preparation of mice

[0210] C57BL / 6 mice (6-8 weeks old, male, manufactured by Japan Corea Co., Ltd.) were acclimated for 1 week in a temperature and humidity controlled (22-24°C, 40-60%) and dimming environment (200 lux / 12 h day and night cycle) and used in the following five comparative experiments (a') to (e').

[0211] (a') Untreated group

[0212] (b') MC903 treatment group only

[0213] (c') MC903+Staphylococcus aureus treatment group

[0214] (d') MC903+Staphylococcus aureus+bacteria mixed treatment group

[0215] (e') MC903+Staphylococcus aureus+Oral Streptococcus treatment group

[0216] [Example 12]

[0217] The degree of inhibition of inflammation by treatment with the bacterial mixture or oral streptococcus solution was evaluated in the same manner as in Example 5, except that the following "[5] application of the bacterial mixture solution" was performed instead of "[5] application of the bacterial mixture solution". The results are shown in Fig. 20 .

[0218] [5”] Spreading of bacterial mixture

[0219] (1) Bacteria culture method

[0220] The 12 strains of bacteria frozen and stored in the ACX medium of the composition in Table 5 below were revived, and bacterial inoculation and culture were started under the conditions in Table 6 below. After about 24 hours, the mixture was adjusted to OD = 0.01 / 50 mL and subcultured, and then subcultured in the same manner after about 24 hours. After subcultured twice from the start of revival, the mixture was transferred to freeze drying.

[0221] [Table 5]

[0222] [Table 6]

[0223]

[0224] (2) Preparation of bacterial mixture

[0225] Weigh 12 kinds of bacteria according to the amount in Table 7 below, add to 36mL soybean oil (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model 190-03776) and stir. After stirring, pass through a filter with a pore size of 100μm to prepare a stock solution for administration. On the day of coating, mix the stock solution for administration 3: the base material for the liquid for administration (soybean oil (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model 190-03776)) 7 in a volume ratio to prepare a bacterial mixed solution. The number of viable bacteria was adjusted to 4.0E+09 cells / mL.

[0226] [Table 7]

[0227]

[0228] From the above results, it can be seen that the composition of the present invention can inhibit the proliferation of pro-inflammatory bacteria, namely Staphylococcus aureus (including MRSA) and Staphylococcus aureus. In addition, it can be seen that the composition of the present invention can inhibit the progression of inflammation in animals caused by pro-inflammatory bacteria, namely Staphylococcus aureus and Staphylococcus aureus.

[0229] This application claims the priority based on the Japanese application (Japanese Patent Application No. 2022-151298) filed on September 22, 2022, and all the disclosed contents are incorporated herein.

Claims

1. An antibacterial composition targeting pro-inflammatory bacteria other than skin-resident bacteria, comprising the skin-resident bacteria as an active ingredient.

2. The antibacterial composition according to claim 1, wherein The skin-resident bacteria include one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 1 to 6 or any of the base sequences having an identity of 95% or more to any of the base sequences.

3. The antibacterial composition according to claim 1, wherein The skin-resident bacteria include two or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 1 to 6 or base sequences having an identity of 95% or more to the base sequences.

4. The antibacterial composition according to claim 1, wherein The skin-resident bacteria include one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 1 to 3 or base sequences having an identity of 95% or more to the base sequences.

5. The antibacterial composition according to claim 1, wherein The skin-resident bacteria include two or more species selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 1 to 3 or base sequences having an identity of 95% or more to the base sequences.

6. The antibacterial composition according to claim 1, wherein The skin-resident bacteria include bacteria having 16S rDNA including the base sequence described in sequence number 1 or a base sequence with an identity of more than 95% to the base sequence, bacteria having 16S rDNA including the base sequence described in sequence number 2 or a base sequence with an identity of more than 95% to the base sequence, and bacteria having 16S rDNA including the base sequence described in sequence number 3 or a base sequence with an identity of more than 95% to the base sequence.

7. The antibacterial composition according to any one of claims 2 to 6, wherein The present invention further includes one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 7 to 8 or base sequences having an identity of 95% or more to the base sequences.

8. The antibacterial composition according to claim 1, wherein The skin-resident bacteria include one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 1 to 13 or base sequences having an identity of 95% or more to the base sequences.

9. The antibacterial composition according to claim 1, wherein The pro-inflammatory bacteria include one or more bacteria selected from bacteria having 16S rDNA, wherein the 16S rDNA includes any of the base sequences described in SEQ ID NOs. 14 to 15 or base sequences having an identity of 95% or more to the base sequences.

10. The antimicrobial composition according to claim 1, wherein The skin resident bacteria include one or more bacteria selected from the group consisting of Actionomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, and Corynebacterium tuberculostearicum.

11. The antimicrobial composition of claim 1, wherein: The skin resident bacteria include two or more bacteria selected from the group consisting of Actionomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, and Corynebacterium tuberculostearicum.

12. The antibacterial composition according to claim 1, wherein The skin resident bacteria include one or more bacteria selected from the group consisting of Actionomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

13. The antibacterial composition according to claim 1, wherein The skin resident bacteria include two or more bacteria selected from the group consisting of Actionomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

14. The antibacterial composition according to claim 1, wherein The skin-resident bacteria include Actionomyces viscosus, Cutibacterium granulosum, and Streptococcus oralis.

15. The antibacterial composition according to any one of claims 10 to 14, wherein The skin-resident bacteria further include one or more bacteria selected from Staphylococcus capitis and Staphylococcus epidermidis.

16. The antimicrobial composition according to claim 1, wherein The skin-resident bacteria include one or more bacteria selected from the group consisting of Actionomyces viscosus, Cutibacterium granulosum, Streptococcus oralis, Staphylococcus warneri, Cutibacterium acnes, Corynebacterium tuberculostearicum, Staphylococcus capitis, Staphylococcus epidermidis, Moraxella osloensis, Dermacoccus nishinomiyaensis, Micrococcus luteus, Enhydrobacterae erosaccus, and Finegoldia magna.

17. The antimicrobial composition according to claim 1, wherein The pro-inflammatory bacteria include one or more bacteria selected from Staphylococcus aureus and Staphylococcus hominis.

18. The antimicrobial composition according to claim 1, wherein For use in the treatment or prevention of diseases or conditions caused by the pro-inflammatory bacteria.

19. The antimicrobial composition according to claim 18, wherein The disease or condition is one or more diseases or conditions selected from atopic dermatitis, impetigo contagiosum, staphylococcal scalded skin syndrome, suppurative mastitis, furunculosis, acne vulgaris, acne rosacea, contact dermatitis, seborrheic dermatitis, epidermolytic ichthyosis, superficial skin infection, harlequin ichthyosis, congenital ichthyosis-like erythroderma, lamellar ichthyosis, Netherton syndrome, Sjogren-Larson syndrome, KID (keratitis-ichthyosis-deafness) syndrome, Dorfman-Channerin syndrome, neutral lipidosis, multiple sulfatase deficiency and X-linked ichthyosis.

20. A method for determining whether to administer the antimicrobial composition of claim 1 to a subject, wherein: The composition ratio or bacterial quantity of a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 1 to 13 or the base sequences having an identity of 95% or more thereto, and a group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in sequence numbers 14 to 15 or the base sequences having an identity of 95% or more thereto, relative to the skin flora of the subject is calculated, and whether the antibacterial composition is administered to the subject is determined based on the composition ratio or the bacterial quantity.

21. The method of claim 20, wherein: Satisfy one or more of the following requirements (A) or (B): (A) when the composition ratio (the group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in SEQ ID NOs. 1 to 13 or base sequences having an identity of 95% or more thereto: the group consisting of one or more bacteria having 16S rDNA including any one of the base sequences described in SEQ ID NOs. 14 to 15 or base sequences having an identity of 95% or more thereto) is 0:10 to 9:1, it is determined that the antimicrobial composition according to claim 1 is suitable for administration to the subject; (B) the bacterial count of the group consisting of one or more bacteria having 16S rDNA comprising any of the base sequences described in SEQ ID NOs. 1 to 13 or base sequences having an identity of 95% or more to the base sequences is 1,000,000 CFU / cm 2 The bacterial count of the group consisting of one or more bacteria having 16S rDNA including any of the base sequences described in SEQ ID NOs. 14 to 15 or base sequences having an identity of 95% or more to the base sequences is 10 CFU / cm 2 If the above, it is determined that the antibacterial composition according to claim 1 is suitable for administration to the subject.

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