Cutibacterium acnes strain and its medical use

Abstract

The present invention relates to selected Cutibacterium acnes strains and / or their cell walls or postbiotics, as well as medical or nutritional uses thereof. The invention also relates to pharmaceutical or nutritional compositions containing the strains, their cell walls or postbiotics for the prevention or treatment of inflammatory diseases such as dermatitis or psoriasis or infectious diseases, particularly fungal or bacterial infections of the skin or mucous membranes.

Description

[Technical Field]

[0001] This invention relates to selected strains of bacteria belonging to the genus Cutibacterium, species acnes, and their medicinal uses, as well as pharmaceutical and nutritional compositions containing the same strains.

[0002] This invention originates in the field of microbiology and finds applications in the fields of pharmaceuticals, cosmetics, and nutrition.

[0003] Specifically, the present invention relates to selected Cutibacterium acnes strains, as well as postbiotics and cell wall fragments of strains that promote fibroblast proliferation via structural microbial components and / or by producing substances. The strains, wall fragments, and postbiotics of the present invention have anti-inflammatory and antibacterial activity. Preferably, the selected strains or wall fragments are for topical application to treat skin diseases, infections, or skin conditions. [Background technology]

[0004] Inflammation is essentially a biological response to harmful irritation or damage to an organism's tissues, which can be caused by external factors such as contact with irritants or microorganisms. Inflammation is considered the body's defense response aimed at eliminating the cause of tissue damage, removing damaged necrotic tissue from the original injury and inflammatory process, and initiating tissue repair. This response in organisms involves immune cells and molecular mediators.

[0005] Currently, inflammation is treated with systemic or topical administration of non-steroidal or steroidal anti-inflammatory drugs. Despite the widespread use of anti-inflammatory drugs, some risk of complications remains, such as the possibility of inflammation persisting after anti-inflammatory treatment. Furthermore, anti-inflammatory treatment, whether applied topically or systemically, is not without side effects.

[0006] In recent years, the incidence of side effects from topical administration has increased due to the misuse or abuse of steroid creams intended to treat skin inflammation.

[0007] Therefore, there is currently a need for novel products with anti-inflammatory activity that do not cause serious side effects even with long-term use.

[0008] In cases of skin or mucous membrane infections, similar problems arise in the fields of dermatology and gynecology.

[0009] The misuse of topical antibiotic products to treat skin infections is increasing the number of cases of resistance to topical antibiotic therapy, forcing physicians to prescribe second-generation antibiotics.

[0010] Therefore, there is currently a need for novel products that possess anti-inflammatory activity and / or even potentially antibacterial activity as alternatives to commercially available pharmaceuticals.

[0011] One of the objectives of the present invention is to provide a product that has anti-inflammatory activity and potentially antibacterial or antifungal activity, with substantially no side effects from use. It is also desirable to provide a product that is effective in controlling the human microbiome.

[0012] Another object of the present invention is to provide a non-steroidal product with anti-inflammatory activity, particularly intended for topical application to mucous membranes such as the skin or vaginal mucosa.

[0013] The latter generally targets pathogenic bacteria and / or fungi, acting as bactericides or bacteriostatic agents, or as fungicides or fungiostatic agents, depending on the target.

[0014] On the other hand, in contrast to pathogens, normalizing the commensal microbiome leads to a more rapid recovery of homeostasis. [Overview of the project]

[0015] The present invention stems from the finding that selected strains of the genus Cutibacterium, namely the acnes species, have the ability to promote fibroblast proliferation and inhibit the growth of most common bacteria and fungi, particularly those that affect human skin.

[0016] In particular, compositions containing selected Cutibacterium acnes strains as defined in claim 1, fragments of their cell walls or postbiotics, or as defined herein, exhibit improved inhibitory effects against skin pathogenic microorganisms combined with modulation of the immune system. These effects are partly related to the strain's interference with pathogen adhesion to host cells.

[0017] Furthermore, the inventors have found that living or dead strains or parts thereof, such as fragments of their walls according to the present invention, produce substances or by-products that promote the proliferation and migration of fibroblasts. This property further supports the use of selected strains or substances produced thereby as immunomodulatory agents, making them suitable candidates for use in the fields of dermatology or gynecology, particularly in the treatment of bacterial or fungal skin infections, especially Candida albicans or bacterial infections.

[0018] Accordingly, in a first embodiment, the present invention provides a strain of the species Cutibacterium acnes, or a variant essentially derived therefrom, deposited with the international depositary authority Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number (or application number) DSM 28251.

[0019] In one aspect, the present invention provides for the medical use of the above-mentioned strain Cutibacterium acnes deposited under accession number DSM 28251. According to this aspect, the strain of the present invention is for topical or systemic administration.

[0020] Systemic administration means a route of administering a pharmaceutical product or nutrient containing the strain of the present invention to the circulatory system so that the whole body is affected. Administration can be carried out by enteral, oral administration or parenteral administration, such as injection, infusion or implantation.

[0021] Topical administration or application is the preferred route of administration of Cutibacterium acnes DSM 28251 according to the present invention. Cutibacterium acnes DSM 28251, postbiotics, cell wall fragments and compositions containing them can be applied to the skin in any form suitable for topical application.

[0022] According to another aspect, the present invention provides a strain or variant of the above-identified strain DSM 28251 that is essentially derived by natural mutation, induced mutation and selection, hybridization and selection or other methods of genetic manipulation and can be traced back. In another aspect, the present invention relates to the postbiotics of the above strain and their medical or nutritional use.

[0023] The strain according to the present invention can be isolated and selected from healthy skin among a number of strains that make up the skin microbiota.

[0024] In yet another aspect, the present invention relates to the strain Cutibacterium acnes, deposited with the international depositary authority Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM 28251, or variants essentially derived from the said strain, particularly for use in the prevention or treatment of inflammatory diseases or infections of the skin or mucous membranes.

[0025] Preferably, the bacterial strains of the present invention find applications in the fields of dermatology and gynecology, for example, in the treatment of inflammation of the skin or mucous membranes or infections caused by bacteria, fungi, or protozoa. In particular, the above strains are especially effective in the prevention and / or treatment of skin infections.

[0026] The selected strains of the present invention are effective in treating fungi, particularly yeasts of the Candida genus, especially Candida albicans, or dermatophytes such as Malassezia species, both of which are the most common causative agents of opportunistic infections in humans. Furthermore, the selected strains of the present invention can be used to treat fungal infections resistant to commercially available antifungal products.

[0027] The present invention also provides for the rectal pathological use of Cutibacterium acnes DSM 28251, particularly the heat-sterilized, postbiotic, or wall fragments of this strain, or topical compositions thereof, in the treatment of hemorrhoids, anal canal lacerations, or skin scars.

[0028] In another aspect, the present invention relates to a hyaluronic acid composition containing fragments of the bacterial wall of C. acnes (DSM 28251) for use in the treatment of skin wounds, abrasions, ulcer formation, such as pressure ulcers.

[0029] A further aspect of the present invention relates to the cosmetic use of the strains identified above for improving aesthetic aspects of the skin, such as redness or cooper rose.

[0030] In another aspect, the present invention relates to the nutritional use of the above-mentioned strain or postbiotics obtained thereby.

[0031] The present invention will now be described in detail with reference to the attached drawings. [Brief explanation of the drawing]

[0032] [Figure 1] This bar graph shows the septum values ​​for Example 1, calculated using the ImageJ software. [Figure 2] This bar graph shows the area of ​​the partition wall in Example 1, calculated using the ImageJ software. [Figure 3] The bar graphs show the in vitro effects on the growth curves of Staphylococcus aureus, Escherichia coli, and C. albicans when heat-sterilized C. acnes DSM 28251 is added to culture media at different concentrations. [Figure 4] The preparation of the supernatant and Staphylococcus aureus used in the experiment in Example 3 is shown. [Figure 5A] The graph shows the survival rate of wax moth (Galleria mellonella) larvae after injection of the preparation tested according to Example 4 and the supernatant from a Staphylococcus aureus (S. aureus) ATCC BAA1680 culture pre-incubated for 1 hour. [Figure 5B]The graph shows the survival rate of *Galleria mellonella* larvae after injection of supernatant from *S. aureus* ATCC 29213 cultures pre-incubated for 1 hour with formulations tested according to Example 4. "Supernatant + Formulation A" represents supernatant incubated with LimpiAD A, "Supernatant + Formulation D" represents supernatant incubated with Formulation LimpiAD D, "Supernatant + Wall" represents supernatant incubated with the active ingredient of LimpiAD, "Supernatant" represents the positive control, "Culture medium" represents culture medium alone (no bacterial culture), and "Saline solution" represents a placebo treatment. [Figure 6A] The graph shows the survival rate of wax moth (Galleria mellonella) larvae after injection of the formulation tested according to Example 4 and the supernatant from a Staphylococcus aureus (S. aureus) ATCC BAA1680 culture pre-incubated for 4 hours. [Figure 6B] The graph shows the survival rates of Galleria mellonella larvae after injection of supernatant from Staphylococcus aureus (S. aureus) ATCC 29213 cultures that were pre-incubated for 4 hours with formulations tested according to Example 4. "Supernatant + Formulation A" represents supernatant incubated with formulation LimpiAD A (defined in Example 4), "Supernatant + Formulation D" represents supernatant incubated with LimpiAD D, "Supernatant + Wall" represents supernatant incubated with the active ingredient of LimpiAD, "Supernatant" represents the positive control, "Culture medium" represents culture medium alone (no bacterial culture), and "Saline" represents a placebo treatment. [Figure 7] The expression of the target gene, "normalized" in the control group, is shown and expressed as an increase or decrease ratio, as reported in Example 4. [Figure 8]Five graphs are shown, each illustrating the comparative growth curves of Staphylococcus aureus strains (three different strains), Staphylococcus epidermidis strains, and Candida albicans strains inoculated with the supernatants of seven known Cutibacterium acnes strains and (comparative) Cutibacterium acnes DSM 28251 strain (with a control without strains). [Figure 9] The following five graphs show comparative growth curves for three strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis, and one strain of Candida albicans in culture media inoculated with seven different heat-sterilized known strains of Cutibacterium acnes and Cutibacterium acnes DSM 28251 (comparative heat-sterilized strain). [Figure 10] The following five graphs show comparative growth curves for three strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis, and one strain of Candida albicans in culture media inoculated with bacterial wall fragments obtained by homogenizing and then gradient separating seven different known strains of Cutibacterium acnes and the Cutibacterium acnes DSM 28251 strain (comparative bacterial wall fragment). [Modes for carrying out the invention]

[0033] In a first aspect, the present invention relates to a strain of the species Cutibacterium acnes, or a variant essentially derived therefrom, filed with the international depositary authority Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM 28251 (ID 19-401).

[0034] The present invention also relates to postbiotics as defined in claims 2 and 3 and cell walls as defined in claims 4 and 5.

[0035] The above-mentioned strains possess both in vivo anti-inflammatory activity and at least in vitro antibacterial activity. Cell walls or postbiotics derived from these strains also possess these activities.

[0036] The above-mentioned activities have been demonstrated by experimental tests conducted by the inventors and are reported in the following examples. These tests provide a scientific basis for the use of the identified strains, particularly as anti-inflammatory agents, immunomodulators, and antibacterial agents for topical application.

[0037] In certain aspects of the present invention, compositions containing strains / walls or postbiotics as defined herein, particularly pharmaceutical or nutritional compositions, are provided herein.

[0038] In another aspect, the present invention relates to the use of the strains / walls / postbiotics identified above as pharmaceuticals, and in particular to their medical uses as defined in claims 9 to 13.

[0039] The unique properties of the selected strains provide anti-inflammatory effects, enabling physicians to treat a wide range of diseases, particularly those localized to the skin or mucous membranes of the human body.

[0040] Furthermore, the antibacterial and / or bacteriostatic properties of the selected strain make it useful for treating infections, particularly skin and mucous membrane infections.

[0041] The selected strains have been proven effective against common bacteria, particularly Gram-positive bacteria, especially cocci such as Staphylococcus aureus, as well as against Escherichia coli and fungi of the Candida genus, for example.

[0042] The strain according to the present invention is genotypic and identifiable in a clearly defined manner by specific traits identified within the genome. This strain has evolved spontaneously without direct intervention or genetic manipulation and possesses characteristics suitable for industrial applications.

[0043] The characteristics of the DSM 28251 strain were verified and confirmed, and genotypic characterization was performed using DSMZ to eliminate the possibility of overlap with strains described in prior art.

[0044] In one embodiment, the present invention is provided for the cosmetic use of topical compositions as defined in claims 7, 8, particularly for the cosmetic treatment or prevention of particularly sensitive skin, redness, cooper rose, and dry skin on the human face.

[0045] In certain embodiments, the present invention also relates to a strain or wall or postbiotic of the DSM 28251 C. acnes strain for medical use in the following treatments: - Gynecological diseases such as vaginitis, vaginal infection or inflammation, - Hemorrhoids, anal canal fissures, or other rectal pathological disorders such as skin scarring in the perianal area, - Skin wounds, injuries, abrasions, ulcers, such as pressure ulcers or sores To heal.

[0046] Definitions in this invention: - "DSM 28251 strain" is intended to contain a strain of the genus Cutibacterium, species acnes, filed on December 18, 2013 (identification number ULTIMO) and deposited with the international depositary authority Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under accession number DSM 28251 pursuant to the Budapest Convention of December 22, 2019. Typically, Cutibacterium acnes is a Gram-positive bacterium. - "Genetic manipulation" is intended to include any technical intervention aimed at directing the acquisition of a particular genetic feature expressed as a corresponding phenotypic trait, and such technical interventions include (Sturley & Young, 1986): (i) crossing and subsequently selecting natural strains; (ii) creating and subsequently selecting hybrids; (iii) transformation, i.e., insertion of exogenous DNA or other genetic factors such as plasmids into chromosomes or mitochondrial genomes; and (iv) induced random mutagenesis, followed by selection and / or production of hybrids, which may be added as a further mode of manipulation (Nevoigt 2008). - "Original variants" are intended to include variants of the strain of Cutibacterium acnes deposited with DSMZ under accession number DSM 28251 that can be traced back for specific genetic traits detectable by microsatellite DNA profiling or by genome sequencing and comparative analysis. - "Growth medium" (synonyms: culture medium, growth medium / culture broth / growth broth) refers to a substrate containing all the compounds (factors) that microorganisms, especially bacteria such as Gram+ bacteria, require for cell replication, which leads to an increase in the number of single cells and the growth of a population. The factors that microorganisms require for growth in the medium mainly belong to the following categories: carbon sources, similar nitrogen sources (both composed of ammonia and free amino acids, the latter also known as FAN), vitamins, and salts (trace elements). Typical carbon sources are sugarcane molasses, beet molasses, barley malt extract, and wheat malt extract.

[0047] The composition of non-living / non-viable cells, cell extracts, and cell lysates is closely related to the unique metabolic and physiological characteristics of the DSM 28251 strain. - "Postbiotics" refers to non-viable bacterial products, including metabolic by-products, secreted by microorganisms such as strains deposited with DSM-Z under accession number DSM 28251, which possess biological activity when applied to a host or to human tissues such as skin.

[0048] Postbiotics also include any substance of the Cutibacterium DSM 28251 strain, which may be advantageously released or acquired after bacterial lysis, including its wall, cytoplasm, cytoplasmic membrane, genetic material (nucleoid), and ribosomes. Postbiotics provide physiological benefits to the host and may be used to formulate compositions for the medical or cosmetic uses and for oral and / or topical administration as described herein.

[0049] Postbiotics of Cutibacterium DSM 28251 strain can be obtained by following a general procedure / method that includes the following steps: To obtain a suspension, the bacterial strain is grown in a culture medium or broth.

[0050] The suspension is centrifuged in a test tube using conventional techniques.

[0051] At the end of centrifugation, stratification of the suspension is achieved. The pellet containing cells, along with the upper layer containing the liquid / culture medium supernatant, is deposited at the bottom of the test tube.

[0052] Next, the supernatant is removed while the pellet remains settled at the bottom of the test tube, the pellet is collected, and washed with water. Then, the pellet is resuspended in physiological (saline) solution to obtain cells, and further centrifugation is performed to remove the remaining culture medium. The cells suspended in water are mechanically processed with a mechanical agitator such as an Ultratturrax (mechanical lysis).

[0053] Mechanical lysis of the cell wall releases the cell contents, yielding postbiotics (supernatant).

[0054] Specific embodiments of this procedure are described in the following detailed description and examples. - "Carrier" refers to an excipient, vehicle, diluent, or adjuvant, which may or may not be present in the composition of the present invention. - "Nutritional product" means a product that can be used to improve nutritional status, or to support or improve the functional activity of one or more organs within physiological boundaries or the functionality of the human body. -As used herein, “selected strain” or “strain” means the strain of the present invention deposited with DSMZ under accession number DSM 28251. - "Cell wall" or "(the strain wall of the present invention)" means the cell wall of the strain Cutibacterium acnes deposited under accession number DSM 28251.

[0055] The wall of the strain of this invention can be broken into parts or fragments.

[0056] The term "fragment" refers to a portion of the wall of the DSM 28251 strain of the present invention.

[0057] The fragment may also indicate cell lysates from the wall of the DSM 28251 strain.

[0058] Cell wall fragments or lysates of the DSM 28251 strain can be obtained, for example, by conventional or common methods of cell disruption as disclosed below. Bacteria in broth or culture medium are washed and degreased, typically using a Soxhlet extractor. The degreased bacteria are then suspended in water and subjected to mechanical dissolution, for example, using a mechanical stirrer, such as an Ultratturrax, and the contents are treated with ammonium sulfate to precipitate fragments of the bacterial wall.

[0059] Next, the fragments can be cleaned, for example, by washing them with water, to obtain wall fragments.

[0060] A specific method for obtaining wall fragments of the C. acnes DSM 28251 strain is described in Example 6.

[0061] Proper crushing / destruction of the stock or its wall can be achieved by subjecting the stock of the present invention to either a mechanical / dissolution method or a non-mechanical / dissolution method.

[0062] Destruction of strains by mechanical methods / devices Suitable mechanical methods for disrupting (lysing) the parietal cells of this strain and obtaining parietal fragments include either solid shearing or fluid shearing.

[0063] Solid shearing includes the use of bead mills, X-presses, or Hughes presses.

[0064] Liquid shearing includes sonication and high-pressure methods, including Hughes presses or French presses, and / or homogenization using a homogenizer or microfluider homogenizer.

[0065] Techniques using bead mills (or grinding) typically involve stirring a suspension of the strain with glass beads.

[0066] Typically, cell wall disruption by the bead mill method is performed in a bead mill containing a jacketed grinding chamber with a rotating shaft passing through its center. The shaft is fitted with a stirrer that imparts kinetic energy to the beads in the chamber, causing them to collide with one another (Chisti & Moo-Young, 1986; Middelberg, 1995). Suitable beads may be 0.10–0.15 mm in diameter to effectively disrupt bacteria. Larger industrial equipment can use beads 0.4–0.6 mm in diameter for the mechanism that separates the beads from the suspension (Kula & Shutter, 1987). A suitable tip velocity is at least 10 m for bacterial disruption. -1 (Kula & Shutter, 1987). Cell concentration can vary from 40–50% wet weight in the broth introduced into the chamber.

[0067] Appropriate solid shearing also includes sonication and high-pressure methods, including Hughes press or French press, in which the frozen suspension of cells is pushed through a small opening by high pressure (Engler, 1985).

[0068] Sonication involves the use of ultrasound, typically sound waves with frequencies above 15–20 kHz, which can disrupt cell walls in a suspension. An appropriate acoustic power is, for example, 35–95 W when sonicating 5–30 mL of a 20% bacterial suspension in a conventional liquid medium.

[0069] Alternatively, mechanical disruption can be obtained in a high-pressure valve homogenizer by passing a cell suspension of the strain through an adjustable, restricted orifice discharge valve under high pressure, as reported by Engler, 1985. Typically, a basic homogenizer design includes a positive displacement pump that pushes the cell suspension across the seat surface through the center of the valve seat. The pressure is controlled by adjusting the force applied to the valve. The fluid flows radially across the valve and collides with an impact ring (Middelberg, 1995). Disruption results from nonspecific tearing of the cell wall.

[0070] An exemplary homogenizer design is the Manton-Gaulin APV design (Middelberg, 1995). For example, the temperature is increased by approximately 21°C per 10 MPa in the homogenizer. The method of disruption in a homogenizer is strongly influenced by the operating pressure. By operating the homogenizer at higher pressures, it is possible to reduce the number of times the cell slurry passes through the homogenizer for a given degree of disruption (Chisti & Moo-Young, 1986; Bury et al., 2001).

[0071] A microfluider homogenizer can also be used as an instrument to obtain fragmented cell walls. In this apparatus, two flows of cell suspension are rapidly impacted against a stationary surface, and the energy input dissipates almost instantaneously at the point of impact, resulting in cell destruction (Middelberg, 1995; Agerkvist & Enfors, 1990). The residence time of the strain suspension in the microfluidizer destruction chamber, the hottest part of the device, is 25–40 ms. On-site cooling can be achieved by immersing the destruction chamber in an ice bath (Sauer et al., 1989; Geciova, personal experience). The percentage of destroyed cells increases with increasing pressure and number of passes.

[0072] Non-mechanical methods of destroying strains Non-mechanical methods are based on depressurization achieved by introducing pressurized subcritical or supercritical gas into cells, causing destruction after the pressure applied by expansion is released.

[0073] Another non-mechanical disruption of the cell wall can be achieved by osmotic shock, where the cell line suspension is equilibrated at high osmotic pressure under conventional conditions and then diluted with liquid medium / broth.

[0074] An alternative method of cell lysis is pyrolysis, which involves the heat treatment of cells under conventional conditions. Another non-mechanical method of cell lysis can be obtained by chemical permeation using substances selected from, in particular, antibiotics such as beta-lactam antibiotics such as penicillin, chelating agents such as EDTA, chaotropes such as urea, guanidine, ethanol, detergents such as Triton X series, sodium dodecyl sulfate, sodium lauryl sarcosinate, solvents such as toluene, acetone, chloroform, hydroxides such as sodium hydroxide, hypochlorites such as sodium hypochlorite, and mixtures thereof.

[0075] Cell lysis of strains can also be achieved by enzymatic lysis, for example, by using proteases and glucanases to first attack the mannoprotein complex of the cell wall, and then the glucan backbone (Kitamura, 1982). A suitable product for cell wall lysis is the commercially available Zymolase-20T (Seikagaku America, Inc., Rockville, Maryland). Lysozyme can also be used to lyse the peptidoglycan layer because it catalyzes the hydrolysis of β-1,4-glycosidic bonds.

[0076] According to a preferred embodiment, wall fragments of the C. acnes (C. acnes) DSM 28251 strain can be obtained by treating the strain with ammonium sulfate at a temperature preferably lower than room temperature, for example in the range of 10 to 2°C, and advantageously, the suspension is centrifuged after treatment to collect the precipitated fragments.

[0077] Advantageously, before treatment with ammonium sulfate, the C. acnes DSM 28251 strain is dried and optionally centrifuged with water. Optionally, after centrifugation, the supernatant obtained from centrifugation is heated to a temperature of, for example, 40–95°C, preferably 75–85°C, and then cooled to, for example, cold water, preferably 3–15°C. The precipitation step is then carried out by incubation with an ammonium sulfate solution at a concentration of 20–60% v / v, for example, at 2–10°C. Advantageously, after incubation, the resulting suspension can be centrifuged and the precipitated fragments collected.

[0078] For example, bacterial pellets are degreased by Soxhlet treatment using an organic solvent selected from ether-ethanol, chloroform, methanol-chloroform, and mixtures thereof, and then dried, for example, under laminar flow in a hood. After drying, the pellets are homogenized by Ultratturrax treatment, preferably in 1 / 2 step increments, with distilled water added in a ratio of 1:2 p / V. After centrifugation, the supernatant is heated to 80°C, then cooled to cold water, preferably 3-15°C, and finally cooled on ice. Subsequently, a fragment precipitation step is carried out by incubation with 40% v / v cold ammonium sulfate at 4°C for 24 hours. After incubation, the suspension is centrifuged, the precipitated fragments are collected, and lyophilized.

[0079] In some embodiments, cell wall fragments of Cutibacterium acnes deposited under accession number DSM 28251 are degreased, i.e., treated by chemical / biotechnology techniques to remove or significantly reduce the lipid components of the bacterial cell wall. For example, Cutibacterium acnes deposited under accession number DSM 28251 are degreased before crushing to produce cell wall fragments.

[0080] Typically, defatted fragments of the cell wall of the strains of the present invention contain sugars and peptide chains that bind together to form a densely woven mesh of glycopeptides. Typical sugars of the cell wall include N-acetylmuramic acid and N-acetylglucosamine.

[0081] Pharmaceutical composition The bacterial strain DSM 28251 and its essentially derived strains, or the fragments or postbiotics obtained therefrom, have proven to be extremely advantageous for industrial applications in the preparation of pharmaceutical compositions, particularly for topical applications.

[0082] According to one embodiment, the present invention relates to a pharmaceutical composition comprising the above-defined bacterial strain or a fragment or postbiotic obtained thereby, and a pharmaceutically or physiologically acceptable excipient.

[0083] A physiologically or pharmaceutically appropriate carrier, diluent, or excipient may be selected based on the intended route of administration of the resulting pharmaceutical composition.

[0084] The pharmaceutical compositions of the present invention encompass any composition prepared by mixing a strain, a fragment thereof, or its postbiotics as defined herein with a pharmaceutically acceptable carrier. Such compositions are suitable for pharmaceutical use in animals or humans.

[0085] The pharmaceutical composition of the present invention comprises a strain or fragment / postbiotic defined in therapeutically effective amounts, and a pharmaceutically acceptable carrier.

[0086] Pharmaceutical compositions may optionally contain other active ingredients. The term "carrier" refers to a vehicle, excipient, diluent, or adjuvant by which the therapeutic or active ingredient is administered. Any carrier and / or excipient suitable for the desired form of the preparation for administration is intended to be used in conjunction with the strains / walls / postbiotics disclosed herein.

[0087] The carrier can take on a wide variety of forms depending on the desired form of the preparation for administration, such as oral, parenteral, or intravenous. When preparing a composition as an oral dosage form, any of the usual pharmaceutical media can be used. For example, in the case of oral liquid preparations such as suspensions, elixirs, and solutions, these may be water, glycol, oil, alcohol, flavoring agents, preservatives, coloring agents, etc. Or, in the case of oral solid preparations such as powders, hard capsules, soft capsules, and tablets, these may be carriers such as starch, sugar, microcrystalline cellulose, diluents, granulators, lubricants, binders, disintegrants, etc. Solid oral preparations are preferred over liquid preparations.

[0088] In certain embodiments, the strain / wall / postbiotics of the present invention can be combined as an active ingredient closely mixed with a suitable pharmaceutical carrier and / or excipient according to conventional pharmaceutical formulation techniques.

[0089] The compositions include those suitable for parenteral administration, such as subcutaneous, intramuscular, and intravenous administration, as well as administration via the lung, nose, rectum, or topical injection. The appropriate route of administration in any given case depends in part on the nature and severity of the condition being treated and the nature of the active ingredient. An exemplary route of administration is the oral route. The compositions can be conveniently provided in unit dosage forms and can be prepared by any method well known in the field of pharmacy. Preferred compositions include those suitable for administration in oral, parenteral, topical, subcutaneous, or pulmonary, nasal, or buccal inhalation forms. The compositions can be prepared by any method well known in the field of pharmacy.

[0090] The pharmaceutical composition may be in the form of tablets, pills, capsules, liquids, suspensions, emulsions, powders, or suppositories, and may also be a sustained-release formulation.

[0091] If desired, the tablets may be coated by standard aqueous or non-aqueous techniques. In certain embodiments, such compositions and preparations may contain at least 0.1 percent of the strain. The proportion of the active strain / wall / postbiotic in these compositions may, of course, vary, and may conveniently be about 0.1 percent to about 60 percent, or 0.5 to 20 percent, of the weight of the unit. The amount of the active strain / wall / postbiotic in such therapeutically useful compositions is such that a therapeutically active dose is obtained. The strain / wall / postbiotic may also be administered intranasally, for example, as a droplet or spray.

[0092] Tablets, pills, capsules, etc., may also contain binders, such as tragacanth gum, acacia, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, or alginic acid; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose, or saccharin. If the unit dosage form is a capsule, the capsule may contain a liquid carrier, such as fatty oil, in addition to the above types of materials. Various other materials may be present as a coating or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar, or both. Syrups or elixirs may contain, in addition to the active ingredient, sucrose as a sweetener, methyl and propylparaben as preservatives, colorants, and flavorings such as cherry or orange flavor. To prevent disintegration while passing through the upper gastrointestinal tract, the composition is enteric-coated.

[0093] Within the framework of the present invention, topical application is preferred. Therefore, in certain preferred embodiments, the composition is for topical application. In this application, compositions containing strains / walls / postbiotics as defined herein may be applied to human skin.

[0094] Compositions for topical administration include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, sticks, liposomes, nanoparticles, patches, bandages, and wound dressings. In certain embodiments, the topical formulation includes a penetration enhancer.

[0095] Compositions for pulmonary administration include, but are not limited to, dry powder compositions comprising a strain / fragment / postbiotic powder and a suitable carrier and / or lubricant powder. Compositions for pulmonary administration can be inhaled from any suitable dry powder inhaler known to those skilled in the art.

[0096] Typically, compositions for topical use may contain the identified strain in amounts of 0.00001% to 10% by weight, 0.0001% to 3% by weight, or 0.1% to 2% by weight, based on the total weight of the composition.

[0097] Compositions for topical application may be in solid, semi-solid, or fluid form. Suitable solid formulations include creams, gels, ointments, pastes, unguents, and patches.

[0098] Compositions for topical application in fluid form may be in the form of lotions, gels, suspensions, or emulsions.

[0099] In the case of fluid or semi-fluid formulations, the strain can be diluted with a physiologically acceptable liquid carrier such as water, alcohol, water-alcohol solution, or glyceryl solution, or mixed with other liquids suitable for topical application.

[0100] As an example, compositions of the present invention in liquid form can be prepared by dissolving or dispersing a bacterial strain or its by-products in water and / or alcohol. The liquid composition may be buffered to a pH range conveniently selected from 5 to 7 to match the pH of the skin, then filtered and packaged in a suitable container such as a bottle or vial.

[0101] In one embodiment, the formulation for topical application is in the form of a cream or emulsion containing a bacterial strain supported on a suitable excipient.

[0102] According to other embodiments, the compositions of the present invention are in forms for systemic administration, particularly oral administration. In these cases, the compositions contain the previously defined bacterial strain and one or more vehicles or excipients suitable for systemic administration.

[0103] The composition is administered in a dose sufficient to reduce the target disease, according to the protocol.

[0104] In some embodiments, the active ingredient in the pharmaceutical composition of the present invention is generally formulated in dose units. Each dose unit may contain 0.00001 to 1000 mg of the strain / wall / postbiotic per daily dose.

[0105] In some embodiments, the effective amount of a topical formulation depends on the severity of the disease, disorder, or condition, previous treatments, the individual's health status, and their response to the drug. In some embodiments, the dose ranges from 0.001% to about 60% by weight of the formulation.

[0106] When used in combination with one or more other active ingredients, the strains / walls / postbiotics of the present invention and the other active ingredients may be used in lower doses than when each is used alone.

[0107] Regarding formulations for various routes of administration, methods for administering drugs and formulations are described in Remington's Pharmaceutical Sciences, 17. th Edition, Gennaro et al. Eds., Mack Publishing Co., 1985, and Remington's Pharmaceutical Sciences, Gennaro AR ed.20 thEdition, 2000, Williams & Wilkins PA, USA, and Remington: The Science and Practice of Pharmacy, 21 st Edition, Lippincott Williams & Wilkins Eds., 2005, and Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8 th These are disclosed in Edition, Lippincott Williams & Wilkins Eds., 2005, and are incorporated herein by reference.

[0108] In certain embodiments, the compositions of the present invention for oral administration are nutritional, dietary, or nutritional supplements.

[0109] Examples of embodiments and preferred procedures of the present invention are described below to illustrate the present invention.

[0110] Example 1 In vitro potential migration of human fibroblasts stimulated with supernatant derived from a culture of strain accession number DSM 28251.

[0111] Research objectives One of the mechanisms of action of the supernatant of the culture of strain accession number DSM 28251 is due to a postbiotic effect. The term postbiotics refers to metabolic byproducts of microorganisms, particularly bacteria and / or probiotics. Typically, postbiotics are produced by the metabolic activity or fermentation processes of bacteria, such as probiotics (live microorganisms that have beneficial effects on the host when administered in appropriate amounts).

[0112] Postbiotics play a crucial role in regulating health and maintaining a healthy microbiome.

[0113] One of the objectives of this study is to evaluate the postbiotic effects on the migration of immortalized human dermal fibroblasts and to assess whether the supernatant derived from cultures of strain accession number DSM 28251 promotes migration of this cell lineage.

[0114] Test sample The supernatant derived from a culture of a bacterial strain with accession number DSM 28251.

[0115] Materials and methods bacteria The bacterial strain having accession number DSM 28251 of this invention was grown overnight in Brain Heart Infusion Broth (BHI) medium at 37°C. Then, to evaluate the postbiotic effect, the supernatant from the bacterial growth was centrifuged and subsequently filtered (0.45 μm) to remove bacterial cells. Before use, aliquots were plated onto BHI agar and incubated at 37°C for 24 hours to verify that no microbial growth occurred.

[0116] cell lineage Non-tumor-forming human dermal fibroblasts were maintained in 25 cm² culture flasks in Dulbecco's modified Eagle medium (DMEM) supplemented with 1% L-glutamine 200 mM, penicillin (100 U / mL), streptomycin (100 μg / mL), and 10% FBS. After washing with DPBS, the cells were detached at room temperature using 0.25% trypsin-EDTA solution 1X, and observed under an inverted microscope until the cell layer dispersed (usually within 5 minutes). The cells were plated onto μ-dishes (35 mm in diameter) containing culture inserts (code 81176, Ibidi, Giemme, Italy). The culture inserts consisted of two wells, and when both wells were filled with adherent cells, a cell-free gap (channel) of approximately 500 μm was formed after removal of the culture insert. Cell suspensions were prepared at a density of 3–7 × 10⁵ cells / mL and applied to two wells of the culture insert (70 μL / well). The suspensions were maintained at 37°C in 5% CO₂ in DMEM supplemented with 1% L-glutamine 200 mg, penicillin (100 U / mL), streptomycin (100 μg / mL), and 10% FBS. After 24 hours of proper cell adhesion, the culture inserts were removed using sterile tweezers, and the μ-dishes were filled with culture medium (2 mL).

[0117] Cell experiments for migration analysis Cells were treated with pre-filtered supernatant derived from Aileens bacterial cultures diluted 1:10 and 1:100 with DPBS (Dulbeccio phosphate-buffered saline). Furthermore, fibroblasts treated with BHI diluted 1:10 and 1:100 with DPBS were used as controls.

[0118] The cells were maintained for an additional 24 hours. The degree of cell migration was captured using a Digital Microscope Eyepiece and measured using the image analysis software Image J (V 1.45s, provided by the National Institutes of Health). Subsequently, each image was negatively converted using the software's functions and then measured. The experiment was performed in three consecutive sets, and the results were expressed as mean ± SE. Student's t-test was used for statistical significance. A value of p > 0.05 is considered statistically significant.

[0119] Result The results obtained show that the supernatants of the strain deposited under accession number DSM 28251 diluted 1:10 and 1:100 with DPBS promote cell migration (Figures 1 and 2). Furthermore, BHI diluted 1:10 and 1:100 with DPBS does not interfere with fibroblast migration.

[0120] Example 2 Activity of the heat-killed strain deposited under accession number DSM 28251 by DSMZ against the growth of human pathogenic microorganisms.

[0121] Materials and methods The strain was grown overnight at 37 °C in brain heart infusion (BHI) medium and then heat-sterilized at 60 °C for 1 hour.

[0122] The cultures were washed with water to wash out the medium, and the cell-free cultures were diluted in Mueller-Hinton (MH) medium or Sabouraud medium at 10 7 、10 6 、and 10 5 CFU / ml. Inocula of 10 5 cells / ml of Staphylococcus aureus (S. aureus) ATCC 29213 and Escherichia coli (E. coli) ATCC 11775 were grown in triplicate at a final volume of 150 μl of each Mueller-Hinton dilution of the heat-sterilized culture. Similarly, an inoculum of 10 5 cells / ml of C. albicans was cultured in triplicate in 10 7 、10 6 、and 10 5 Sabouraud dilutions of the killed bacterial culture. As a control, all dilutions were made in triplicate in both media, and similarly, controls for strain growth were made with 10 5Cells / ml inoculum were used for preparation. Optical density (OD) at a wavelength of 600 nm was measured for each well before incubation at 37°C (T0) and after 3, 6, 20, and 24 hours (T3, T6, T20, T24). Growth trends, as observed from the OD values ​​of each strain in the medium containing heat-sterilized strains, were compared with those in Mueller-Hinton or Sabouraud medium alone.

[0123] The ability of strain accession number DSM 28251 to interfere with or inhibit the microbial growth of bacteria and fungi commonly found on human skin (see tested strain) was tested using the spectrophotometric method described by Hall et al.

[0124] Microbial growth inhibition was evaluated using 96-well polystyrene plates. Serial dilutions of the tindalized mixture (10 7 ~10 5 The test microorganisms were inoculated into CFU / ml (with the microorganism alone as a control) and with the culture medium (blank). All microbial strains were tested according to the CLSI (Clinical Standard Laboratory Institute) guidelines [3,4].

[0125] Spectrophotometric readings were taken at 600 nm at specified intervals (3, 6, 20, and 24 hours). Results are provided as growth curves (microorganism alone) and inhibition curves (microorganism and tindalized concentration). Each experiment was performed 8 times and repeated 3 times, and the results were expressed as mean ± SD.

[0126] Tested strains: - Staphylococcus aureus - Candida albicans -Escherichia coli

[0127] The results are reported in Figure 3.

[0128] As demonstrated in Figure 3, when the bacterial strain accession number DSM 28251 of the present invention is added to a Staphylococcus aureus (S aureus) culture, the logarithmic phase of growth is delayed in a dose-dependent manner.

[0129] Similar results can be seen with C. albicans.

[0130] These experimental data support the inhibitory effect of the bacterial strain with accession number DSM 28251 according to the present invention.

[0131] Comparative study using culture medium alone (i.e., without strain 28251).

[0132] Example 3 Tests on the proliferation of C. albicans using a formulation containing the DSM 28251 strain of the present invention. Materials and methods Tested formulations A) Basic prescription ( DSM 28251 shares (Does not include) B) 1% DSM 28251 shares prescriptions containing C) 2.5% DSM 28251 shares prescriptions containing D) 2.5% DSM 28251 shares Formula containing (2×C40)

[0133] The basic formulation contains water, caprylic / capric triglyceride, and glycerin butyrospermum parkii butter.

[0134] procedure Each formulation was inoculated into agar plates (20%) in four sectors. Different fungal dilutions (10⁻⁵ 10⁻⁸ CFU / ml) were plated using the four sectors, and the same dilutions were plated in a medium without the formulation as a control. After inoculation, plates containing the formulations were incubated at 37°C for 48 hours. Fungal growth in the medium containing the formulations was checked against the control. Furthermore, the pH of the medium alone and the formulation / medium was recorded to investigate the possibility of interference between the pathogenic state and the transition of C. albicans to a non-pathogenic state.

[0135] result In the control group, the formation of numerous colonies (2 × 10⁹ CFU / ml) with a typical white and cloudy morphology was clearly observed.

[0136] From microscopic findings, the inventors observed characteristic cell morphologies of pathogenic microorganisms that produce fungal hyphae.

[0137] For all formulations tested, the inventors observed the formation of an opaque patina, which is a failure of hyphae formation, a feature associated with the transition of the pathogen from a pathogenic state to a non-pathogenic state.

[0138] Furthermore, the pH of the culture medium alone (S) was similar to that of the formulation medium, and therefore, pH changes do not appear to be involved in phenotypic transformation.

[0139] conclusion In C. albicans, morphological variation of the colonies was observed. In the absence of the formulation, typical colonies with a milky white appearance were observed, while in the presence of the formulation, a uniform, translucent patina was obtained. This variation was also confirmed at the microscopic level; without the formulation, the microorganism was observed as both yeast and mold (in the presence of hyphae), but in the presence of the formulation, it was observed only as yeast.

[0140] This observation provides evidence of the inhibitory activity of the formulation containing the selected strain of the present invention against the proliferation of Candida albicans.

[0141] Example 4 Inhibitory effect of a formulation containing strain DSM 28251 (LimpiAD) against toxins / catabolic products produced by Staphylococcus aureus (S. aureus): In vivo study and gene expression assay in Galleria mellonella. Introduction The potential effects of formulations and active ingredients as drugs capable of reducing the effects of Staphylococcus aureus (S. aureus) toxin on specific biological targets were tested.

[0142] Staphylococcus aureus (S. aureus) is one of the most well-known microorganisms involved in the pathogenesis and development of atopic dermatitis. One of its main pathogenesis mechanisms is the production of a wide range of toxins.

[0143] The tested LimpiAD formulation contains water, caprylic / capric triglyceride, and glycerin butyrospermum parkii butter.

[0144] For this purpose, an in vivo assay was performed using the larvae of the wax moth (Galleria mellonella).

[0145] The greater wax moth Galleria mellonella (G. mellonella) is a model organism that has already been validated for bacterial infection experiments and pharmacological toxicity studies. It is an essential tool for preliminary screening of novel compounds and rapid and reliable assessment of potential inhibitory activity, and should therefore reduce the number of experiments required using mammalian models.[6]

[0146] The larvae of the wax moth (G. melonella) offer multiple options for the easy delivery of the pathogen, including topical application, oral delivery, and injection. The microorganism can be injected directly into the hemocalls of the larvae, thus the larvae receive a known amount of the pathogen.

[0147] Another advantage of the wax moth (G. melonella) model is the possibility of evaluating the expression of immune and stress-related genes. Therefore, to investigate the effects of the invention on wax moth (G. melonella) larvae inoculated with Staphylococcus aureus supernatant, the inventors performed qPCR assays to evaluate the effects of LimpiAD on immune and stress genes in wax moth (G. melonella). The inventors selected genes that play important roles in the insect immune response to infection: phagocytosis, cytokine regulation, cell adhesion, and metalloproteinase inhibition.

[0148] Materials and methods Tested microbial strains and culture conditions Staphylococcus aureus ATCC BAA1680 and Staphylococcus aureus ATCC 29213 were grown in Brain Heart Infusion Broth and incubated overnight under thermal conditions of 37°C.

[0149] Supernatant preparation: Overnight cultures of both Staphylococcus strains were centrifuged at 5000 rpm for 5 minutes. Residual bacterial cells were removed by filtration (0.22 μm) and diluted with physiological saline in a ratio of 1:2. The diluted supernatant was mixed with the following three different formulations (1:5 w / v) (Table 1). -LimpiAD A (basic formula with no active ingredients) -LimpiAD D (containing 2.5% active ingredient), - Active ingredient alone.

[0150] After incubating the formulation for 1 hour and 4 hours, the supernatant was collected by centrifugation and used for inoculation into Galleria mellonella larvae. Larvae treated with the supernatant (without formulation) and culture medium alone (Brainheart Infusion Broth) in a 1:2 ratio were used as controls (Figure 25).

[0151] To evaluate the larval survival rate of *Glaucus mellonella* larval infection, larvae were selected based on body weight and size and divided into experimental groups as listed in Table 1. Larvae were inoculated by injection through the last proleg, using an insulin syringe (BD, Wellington) and a repeating dispenser equipped with a 1 mL ultrafine needle. After inoculation, larvae under each condition were incubated in petri dishes at 35°C, and their survival was observed for 96 hours. [Table 1]

[0152] Gene expression assay: qPCR Three surviving larvae (control, physiological saline; infected, supernatant ATCC BAA1680; supernatant ATCC BAA1680 treated with Staphylococcus aureus) were collected from each group 96 hours after treatment. RNA was extracted in TRI reagent (Sigma Aldrich) according to the manufacturer's protocol, the RNA extract was quantified spectrophotometrically, and then reverse transcribed to cDNA using ReadyScript® cDNA compound mixture (Sigma Aldrich).

[0153] Table 2 lists the primer names, functions, and sequences used for gene expression. Gene expression ratios relative to the reference gene EF1 were determined in normalized samples using Rotor-Gene Q-QIAGEN with PowerSYBR® (AppliedBiosystems). Cycle conditions were 95°C for 5 minutes, followed by 42 cycles at 95°C for 5 seconds, annealing for 10 seconds, and 72°C for 20 seconds. An initial touchdown of 1°C per cycle from 65°C for the first 5 cycles resulted in optimal amplification for all loci. All experiments were performed in triplicate for three different measurements [7]. [Table 2]

[0154] The preparation of the supernatant and Staphylococcus aureus (S. aureus) used in the test is shown in Figure 4 (Fig. 5).

[0155] Larval survival rate Compared to the control group, the test formulation and the supernatant after 1 hour of pre-incubation were far less lethal.

[0156] Therefore, the increased larval survival rate in Staphylococcus aureus (S. aureus) ATCC BAA1680 is due to the action of its active ingredient.

[0157] Similarly, after 4 hours of incubation, the activity of the basic formula A was observed at a lower level, while formula D showed a higher larval survival rate.

[0158] Regarding Staphylococcus aureus (S. aureus) strain ATCC 29213, lower activity was observed in the basic formulation A compared to the other formulations tested. In this case, the active ingredient resulted in a greater increase in larval survival rate compared to formulation D, both with 1 hour and 4 hours of pretreatment.

[0159] Overall, these results suggest that the active ingredients may interfere with the pathogenesis associated with toxins / catabolites produced by Staphylococcus aureus (S. aureus) ATCC BAA1680. Furthermore, it can be seen that for both strains, as well as Staphylococcus aureus (S. aureus) ATCC 29213 strain, the intensity of interference increased with longer putative interactions with toxins / catabolites in the supernatant after 1 hour of incubation (Figures 27-28).

[0160] Conversely, the 18-wheeler gene was less expressed (99.98%) in the infected group compared to the treated group. Therefore, LimpiAD pre-incubation significantly increased the expression of this gene, which is involved in cell adhesion and migration.

[0161] Gene expression assay As demonstrated in Figure 7, the expression of two genes related to metabolic stress, IMPI and GLUT, did not change significantly in either group compared to the control. Therefore, LimpiAD does not alter the expression of these genes. The expression of two genes related to innate immunity differed in the infected group compared to the treated group. The CITOK gene (NF-Kappa B cascade), which modulates inflammatory cytokines, was overexpressed in the infected group (93.8%) compared to the treated group. Therefore, LimpiAD reduced the expression of pro-inflammatory cytokines. [Table 3]

[0162] conclusion Infection with pathogenic strains of Staphylococcus aureus is considered a harmful factor in atopic dermatitis because these catabolic microorganisms stimulate the production of inflammatory cytokines, contributing to damage to the epidermal barrier and the manifestation of characteristic disease symptoms.

[0163] The results suggest that the LimpiAD component reduces the impact of catabolisms produced by Staphylococcus aureus on the survival of injected wax moth (G. melonella) larvae.

[0164] Furthermore, treatment with LimpiAD reduced the expression of pro-inflammatory cytokines and increased the expression of the 18-wealer gene, which is involved in cell adhesion and migration. Data obtained from metabolic stress and immunogene expression provide further evidence regarding the mechanism of action of the LimpiAD invention.

[0165] Example 5 Comparative in vitro trial A comparative study of intermittently sterilized C. acnes DSM 28251 versus other Cutibacterium strains in terms of the proliferation of skin pathogenic microorganisms.

[0166] Purpose of the exam The inhibitory activity of C. acnes strain DSM 28251 against heat-killed bacteria colonizing the skin was compared with the activity of four different strains of the same species (Cutibacterium strains with the same peptidoglycan composition) and C. granulosum strains, which are very closely related from an evolutionary perspective (phylogenetic proximity).

[0167] The purpose of this study was to evaluate whether the C. acnes DSM 28251 strain possesses unique or improved characteristics compared to other phylogenetically related species.

[0168] For this purpose, four strains of Cutibacterium species different from the DSM 28251 strain (three Cutibacterium acnes and one Cutibacterium granulosum) were selected and tested based on either phylogenetic type or phylogenetic distance (test strains).

[0169] This study also includes two strains of C. acnes (DSM 30738 and 30753), whose characteristics are unknown, but they belong to the same species as the DSM 28251 strain of the present invention. [Table 4]

[0170] Pathogenic microorganisms of the skin The inhibitory activity of the six Cutibacterium strains mentioned above was tested against the following five well-known and widely represented skin pathogens. - Staphylococcus aureus (ATCC) 29213 - Staphylococcus aureus (ATCC) BAA-1680 - Staphylococcus aureus (S. aureus) DSM 20491 - Staphylococcus epidermidis (ATCC) 12228 - Candida albicans ATCC 90028

[0171] Materials and methods Preparation of heat-sterilized bacteria (Cutibacterium) The Cutibacterium tested were killed by tindalization (fractionation sterilization).

[0172] All Cutibacterium strains were grown in 20% Brain Heart Infusion (BHI) broth at 37°C until the maximum growth rate was confirmed by spectrophotometric reading (OD600nm). The medium was then removed by centrifugation and processed for supernatant testing. The bacterial pellet was washed with physiological saline (NaCl 0.9%) until all residual supernatant was removed, and then subjected to a 0.5 McFarland concentration (1.5 × 10⁻⁶). 8 Diluted to CFU / ml.

[0173] The titrated inoculum was subjected to tindalization.

[0174] The tindalization method involved heating at 80°C for 30 minutes to kill vegetative cells, followed by incubation at 37°C for 24 hours to promote germination of any remaining vegetative cells that were not killed by the heat treatment, and then returning the material to 80°C for 30 minutes. The total heat cycle was repeated three times. Aliquots of the tindalized material were seeded onto Colombian blood agar and incubated at 37°C for 24 hours under aerobic conditions to verify that there was no microbial growth and that the method had been performed correctly. Tindalized bacterial cells retain their cellular structure and cell wall while their replication and enzymatic activity is inactivated, making them physiologically intact and therefore immunologically active.

[0175] Evaluation of the activity of dead strains against the growth of pathogenic microorganisms The ability of heat-sterilized C. acnes strains to interfere with and / or inhibit the growth of tested Staphylococcus and C. albicans strains was evaluated using spectrophotometric method.

[0176] The heat-sterilized strains were tested using the same growth medium (BHI broth supplemented with 20% of the same concentration) following the procedure below. Bacteria that have been heat-sterilized, 10 5The solution was pre-diluted to the final concentration of CFU / ml, and then pathogenic microorganisms of the skin were inoculated as follows.

[0177] 100 µl of active cultures of the tested strains Staphylococcus aureus (S. aureus) ATCC® BAA-1680 (trademark), Staphylococcus aureus (S. aureus) DSM 20491, Staphylococcus aureus (S. aureus) ATCC® 29213 (trademark), Staphylococcus epidermidis (S. epidermidis) ATCC® 12228 (trademark), and C. albicans (C. albicans) ATCC® 90028 (trademark) were inoculated in three strips into the wells of each fragment. Each strain was pre-grown to the exponential growth stage in BHI medium at 37°C, and cells were collected by centrifugation at 3000 rpm for 5 minutes. The pellet was resuspended in fresh BHI medium and 1 × 10⁶ cells were collected. 5 A suspension with a concentration of CFU / ml was obtained.

[0178] These suspensions were used as inoculants in the wells of a 96-well plate.

[0179] For spectrophotometric calibration, three 96-well plates were prepared in a series, each containing an experimental control (i.e., the strain inoculation material alone without added fragments) and an experimental "blank" (BHI medium containing each fragment).

[0180] Optical density at 600 nm (OD600nm) was measured using a VICTOR multi-label plate reader (PerkinElmer) system and considered as the growth value for each strain and at treatment time 0 (T0). Subsequent measurements were performed at 2, 4, 6, 8, 18, 20, 22, and 24 hours during the incubation period. OD values ​​were normalized relative to the blank and control, and then analyzed to evaluate the growth trends of different pathogens with and without wall fragments (CTR). Results are reported as mean ± SD (standard deviation), and growth curves were obtained by nonlinear regression analysis using a sigmoid function suitable for bacterial growth. Analysis was performed using GraphPad Prism version 7.0a software.

[0181] result Figure 9 shows the growth curves of pathogenic microorganisms on the skin in the presence and absence (control) of heat-sterilized Cutibacterium strains. The initial AUC qualitative evaluation shows that heat-sterilized C. acnes DSM 28251 has an improved inhibitory effect on microbial growth for all pathogenic microorganisms tested on the skin.

[0182] Table 2a below reports the quantitative evaluation of the AUC of each derivative for all microorganisms tested. These values ​​support the fact shown in Figure 9 that heat-sterilized bacteria obtained from C. acnes (DSM 28251) exhibit significant effect / inhibitory activity against the growth of all skin microorganisms tested. [Table 5]

[0183] Table 2a shows the area under the curve (AUC) and relative standard error (light blue) of the growth curves, as shown in Figure 2, with and without heat-sterilized bacteria (control). The color scale for each strain indicates the range of AUC values: from small (dark red) to large (dark green).

[0184] The rate of decrease in proliferation compared to the control conditions is shown in Table 2b below. These data support the results discussed above and highlight the improved activity of heat-sterilized C. acnes DSM 28251 compared to common C. acnes strains. [Table 6]

[0185] Table 2b. Percentage reduction in growth compared to control conditions (no inoculation of heat-sterilized bacteria). The color scale for each strain indicates the range of reduction: from large (dark red) to small (dark green) reduction in growth (%).

[0186] Example 6 The comparative study in Example 5 was repeated using postbiotics / supernatant of all C. acnes strains (instead of the heat-sterilized strains in Example 5).

[0187] Purpose of the exam The inhibitory activity of postbiotics / supernatants obtained from the same six Cutibacterium strains in Example 5 against the same pathogenic microorganisms in the skin of Example 5 was compared.

[0188] The purpose of this study was to evaluate whether postbiotics derived from C. acnes DSM 28251 strain exhibit improved inhibitory activity against pathogenic microorganisms of the skin compared to postbiotics derived from the C. acnes species in Example 5.

[0189] Materials and methods Preparation of postbiotics for C. acnes DSM 28251. Bacterial supernatants previously obtained from Cutibacterium strains were filtered (through a 0.22 μm filter) to remove cellular residues. Sterility was confirmed by inoculating aliquots of each supernatant under investigation onto Colombian blood agar and incubating them under aerobic conditions at 37°C for 24 hours, after which no bacterial growth was observed.

[0190] To eliminate interference from the production of acidic substances typical of some strains of Cutibacterium species, the pH of each supernatant was carefully measured and neutralized with 1M sodium hydroxide solution as needed.

[0191] Spectrophotometric evaluation of the activity of postbiotics against the growth of pathogenic microorganisms.

[0192] The ability of postbiotics derived from the C. acnes strain mentioned in Example 5 to interfere with and / or inhibit the growth of the same Staphylococcus strain and C. albicans strain reported in Example 5 was evaluated using spectrophotometrics.

[0193] Postbiotics were tested using the same growth medium as in Example 5 (BHI broth supplemented with 20% concentration) following the procedure below. The supernatant was diluted in BHI broth at a ratio of 1:10 and then incubated with the tested skin microorganisms.

[0194] 100 µl of active cultures of the cutaneous pathogenic microorganisms Staphylococcus aureus (S. aureus) ATCC® BAA-1680 (trademark), Staphylococcus aureus (S. aureus) DSM 20491, Staphylococcus aureus (S. aureus) ATCC® 29213 (trademark), Staphylococcus epidermidis (S. epidermidis) ATCC® 12228 (trademark), and C. albicans (C. albicans) ATCC® 90028 (trademark) were inoculated into three wells in a series. Each strain was pre-grown to the exponential growth stage in BHI medium at 37°C, and cells were collected by centrifugation at 3000 rpm for 5 minutes. The pellet was resuspended in fresh BHI medium and 1 × 10⁶ cells were collected. 5 A suspension with a concentration of CFU / ml was obtained.

[0195] These suspensions were used as inoculants in the wells of a 96-well plate.

[0196] For spectrophotometric calibration, three 96-well plates were prepared in a series, each containing an experimental control (i.e., the strain inoculation material alone) and an experimental "blank" (BHI medium containing each fragment).

[0197] Optical density at 600 nm (OD600nm) was measured using a VICTOR multi-label plate reader (PerkinElmer) system and considered as the growth value for each strain and at treatment time 0 (T0). Subsequent measurements were performed at 2, 4, 6, 8, 18, 20, 22, and 24 hours during the incubation period. OD values ​​were normalized relative to the blank and control, and then analyzed to evaluate the growth trends of different pathogens with and without wall fragments (CTR). Results are reported as mean ± SD (standard deviation), and growth curves were obtained by nonlinear regression analysis using a sigmoid function suitable for bacterial growth. Analysis was performed using GraphPad Prism version 7.0a software.

[0198] result Figure 8 shows the growth curves of pathogenic microorganisms on the skin in the presence and absence (control) of C. acnes DSM 28251 strain.

[0199] The initial qualitative evaluation of AUC (Area Under the Curve) indicates that the probiotic (supernatant) obtained from C. acnes DSM 28251 strain exhibits the highest inhibitory effect on microbial growth against most of the pathogenic microorganisms tested.

[0200] Table 1a shows the quantitative AUC evaluation for each supernatant. These values ​​support what is shown in Figure 1. In fact, the postbiotic / supernatant C. acnes DSM 28251 inhibits the growth of all tested skin bacteria more than the other supernatants tested, with the exception of C. albicans ATCC 90028 (which is inhibited by a derivative of DSM 30738). [Table 7]

[0201] Table 1a. The area under the curve (AUC) and relative standard error (light blue) of the growth curves are shown in Figure 1, with and without bacterial supernatant (control). The color scale for each strain indicates the range of AUC values: from small (dark red) to large (dark green).

[0202] Table 1b shows the rate of growth reduction compared to control conditions obtained with the tested supernatants. These data further emphasize that the activity of supernatant DSM 30738 against C. albicans ATCC 90028 strain is only slightly higher and therefore comparable to the activity of supernatant C. acnes DSM 28251, supporting the results discussed so far. [Table 8]

[0203] Table 1b. Percentage reduction in growth compared to control conditions (no supernatant inoculum). The color scale for each strain indicates the range of reduction: from large (dark red) to small (dark green) growth reduction (%).

[0204] result The initial AUC qualitative assessment indicates that the postbiotic / supernatant of C. acnes DSM 28251 exhibits improved inhibitory effects against microbial growth for all pathogenic microorganisms tested on the skin.

[0205] Example 7 The comparative experiment in Example 5 was repeated using cell wall fragments from all C. acnes strains tested (instead of the heat-sterilized strains in Example 5).

[0206] Purpose of the exam The purpose of this study was to compare the inhibitory activity of wall fragments from the same six Cutibacterium strains in Example 5 against the same skin pathogenic microorganisms in Example 5.

[0207] The purpose of this study was to evaluate whether wall fragments derived from C. acnes DSM 28251 strain exhibit improved inhibitory activity against pathogenic microorganisms of the skin compared to bacterial wall fragments of the comparative C. acnes species in Example 5.

[0208] Materials and methods Preparation of wall fragments of C. acnes DSM 28251 and comparative C. acnes strains. C. acnes strain DSM 28251 and Cutibacterium strains ATCC® 11829, DSM 16379, DSM 30738, DSM 30753, and DSM 1897 were grown in BHI medium supplemented with 20% concentration at 37°C.

[0209] Culturing was performed using batch and scale-up systems with volumes of 5–1000 ml, and extended until a consistent cell aggregate was obtained (an average of 2 days with larger inoculum). The resulting bacterial pellets were then collected and subjected to standardized procedures to obtain the desired wall fragments, as described below. Hereafter, isolated fragments are referred to by their derived strain catalog codes.

[0210] Specifically, the isolation of the wall fragments was carried out as described below.

[0211] The bacterial pellets were first subjected to a degreasing procedure, followed by a Soxhlet treatment using an organic solvent (i.e., ether-ethanol, chloroform, methanol-chloroform, or a mixture thereof), and then dried under laminar flow in a hood. After drying, the pellets were homogenized by two-stage Ultratturrax treatments (20 seconds to 10 minutes each) with the addition of distilled water (at a ratio of 1:2 p / V). After centrifugation, the supernatant was heated to 80°C, then cooled to cold water, preferably 3–15°C, and finally cooled on ice. Subsequently, a fragment precipitation step was carried out by incubation with 15–40% v / v cold ammonium sulfate at 4°C for 24 hours. After incubation, the suspension was centrifuged, the precipitated fragments were collected, and lyophilized.

[0212] The freeze-dried specimens were finally sterilized using an ad-hoc multi-step procedure (ultrafreezing at -80°C, heating at 80°C, and UV sterilization for 1 hour).

[0213] Finally, we set up the experiment using them as described below.

[0214] Spectrophotometric evaluation of the activity of postbiotics against the growth of pathogenic microorganisms. The activity of wall fragments derived from the C. acnes strain (see above) in interfering with and / or inhibiting the growth of the same Staphylococcus strains and C. albicans strains reported in Example 5 was evaluated using spectrophotometric method.

[0215] The wall fragments were tested using the same growth medium as in Example 5 (BHI broth supplemented with 20% of the same concentration) according to the following procedure. The wall fragments were powdered and emulsified in BHI growth medium with a final concentration of 10 mg / ml. 100 μl aliquots were added to the wells of a 96-well flat-bottom plate.

[0216] 100 µl of active cultures of the cutaneous pathogenic microorganisms Staphylococcus aureus (S. aureus) ATCC® BAA-1680 (trademark), Staphylococcus aureus (S. aureus) DSM 20491, Staphylococcus aureus (S. aureus) ATCC® 29213 (trademark), Staphylococcus epidermidis (S. epidermidis) ATCC® 12228 (trademark), and C. albicans (C. albicans) ATCC® 90028 (trademark) were inoculated in three strips into the wells of each fragment. Each strain was pre-grown in BHI medium at 37°C to the exponential growth stage, and cells were collected by centrifugation at 3000 rpm for 5 minutes. The pellets were resuspended in fresh BHI medium to obtain a suspension with a concentration of 1 × 10⁵ CFU / ml.

[0217] These suspensions were used as inoculants in the wells of a 96-well plate.

[0218] For spectrophotometric calibration, three 96-well plates were prepared in a series, each containing an experimental control (i.e., the strain inoculation material alone without added fragments) and an experimental "blank" (BHI medium containing each fragment).

[0219] Optical density at 600 nm (OD600nm) was measured using a VICTOR multi-label plate reader (PerkinElmer) system and considered as the growth value for each strain and at treatment time 0 (T0). Subsequent measurements were performed at 2, 4, 6, 8, 18, 20, 22, and 24 hours during the incubation period. OD values ​​were normalized relative to the blank and control, and then analyzed to evaluate the growth trends of different pathogens with and without wall fragments (CTR). Results are reported as mean ± SD (standard deviation), and growth curves were obtained by nonlinear regression analysis using a sigmoid function suitable for bacterial growth. Analysis was performed using GraphPad Prism version 7.0a software.

[0220] result( C. acnes wall fragment ) Figure 10 shows the growth curves of skin bacteria cultured with and without various tested wall fragments (CTR). Based on a preliminary qualitative assessment of the "area under the curve" (AUC) parameter, it is highlighted how fragment DSM 28251 exerts the highest inhibitory effect on microbial growth for most of the tested skin pathogens.

[0221] Instead, Table 3a below reports the results of quantitative evaluation of the same AUC parameter for all tested skin pathogens. The calculated AUC values ​​support the hypothesis formulated by the qualitative evaluation. Bacterial cell wall fragment DSM 28251 showed a higher inhibitory effect on bacterial growth than the other fragments tested.

[0222] Looking more closely at the estimation of growth inhibition, the values ​​shown in Table 3b represent the percentage reduction in growth for each strain compared to the control condition, which is considered 100% of the growth rate under specific experimental conditions. The comparison of growth rate reductions was consistent with the conclusions mentioned above regarding the higher inhibitory effect of the DSM 28251 strain fragment.

[0223] The fragment obtained from C. acnes strain DSM 30738 shows similar inhibitory values ​​to the DSM 28251 fragment, but the latter consistently shows higher inhibition rates and also exhibits better performance against Staphylococcus aureus ATCC BAA-1680 strain (82.43% for DSM 30738 compared to 94.95% for DSM 28251).

[0224] Table 3a shows the area under the curve (AUC) and relative standard error (light blue) of the growth curves, as shown in Figure 3, with and without bacterial wall fragments (control). The color scale for each strain indicates the range of AUC values: from small (dark red) to large (dark green). [Table 9]

[0225] Table 3b. Percentage reduction in growth compared to control conditions (no fragment inoculation). The color scale for each strain indicates the range of reduction: from large (dark red) to small (dark green) growth reduction (%). [Table 10]

[0226] JPEG0007832676000011.jpg222166 [Sequence Listing Free Text]

[0227] Sequence Listing 1 <223> EF1 Forward Primer Sequence Listing 2 <223> EF1 Reverse Primer Sequence Listing 3 <223> Impai Forward Primer Sequence Listing 4 <223> Impi Reverse Primer Sequence Listing 5 <223> Glutaraldehyde forward primer Sequence Listing 6 <223> Glutaraldehyde Reverse Primer Sequence Listing 7 <223> Cytokine forward primers Sequence Listing 8 <223> Cytokine reverse primer Sequence Listing 9 <223> Phagoc Forward Primer Sequence Listing 10 <223> Phagoc Reverse Primer

Claims

1. A strain of Cutibacterium acnes deposited with the international depositary authority Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under depositary number DSM 28251.

2. A postbiotic product comprising cells of the bacterial strain described in claim 1 that have been killed by heat, or the supernatant of cells of the bacterial strain described in claim 1 that have been mechanically lysed.

3. A postbiotic product comprising a cell wall fragment of the bacterial strain described in claim 1.

4. The postbiotic product according to claim 2, wherein the lysed cells are obtained by mechanical lysis of the cell wall.

5. A fragment of the cell wall of the bacterial strain described in claim 1.

6. The bacterial strain according to claim 1, characterized in that it is inactivated by intermittent sterilization.

7. A bacterial strain according to claim 1, for use as a pharmaceutical product.

8. A postbiotic product according to claim 2 or 3 for use as a pharmaceutical product.

9. A cell wall fragment according to claim 5, for use as a pharmaceutical product.

10. A composition comprising an effective amount of Cutibacterium acnes deposited under deposit accession number DSMZ 28251 as described in claim 1, or its postbiotic product as described in claim 2, or its cell wall fragments and a physiologically acceptable carrier as described in claim 5.

11. The composition according to claim 10, which is a topical composition in the form of a cream, foam, ointment, paste, powder, gel, solution, formulation for topical application to the vaginal mucosa, irrigation solution, or emulsion.

12. The composition according to claim 10 for use as a pharmaceutical product.

13. The composition according to claim 10, for use in the treatment of inflammatory or allergic diseases or infections.

14. The composition according to claim 10, for use in the topical treatment of inflammatory or allergic diseases of the skin or mucous membranes.

15. A composition for use according to claim 13 or 14 in the prevention or treatment of bacterial or fungal infections of the skin or mucous membranes.

16. The composition for use according to claim 15, wherein the skin disease is eczema, atopic dermatitis, acne, seborrheic dermatitis, rosacea, psoriasis, erythema, or skin rash.

17. A composition for use according to claim 15, for treating fungal infections of the skin or mucous membranes.

18. The composition for use according to claim 17, wherein the fungal infection is a Candida infection.

19. The composition according to claim 10 or 11 for use in the treatment of vaginal infection or inflammation, which is a gynecological disease.

20. The composition for use according to claim 19, wherein the gynecological disorder is vaginitis.

21. The composition according to claim 10 or 11 for rectal pathological use in the treatment of hemorrhoids, anal canal lacerations, or skin scars.

22. The composition according to claim 10 or 11, for use in treating skin wounds, injuries, abrasions, ulcers, or pressure ulcers, or for healing wounds.

23. A method for producing a postbiotic product, comprising obtaining the entire supernatant after a fermentation process of the strain described in claim 1 as the postbiotic product.

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