Antimicrobial properties of amphibians

Abstract

Methods of inhibiting growth of microorganisms, or killing microorganisms using samples of organs, cells, blastemas or biological fluids obtained from amphibians are disclosed. The microorganisms may include bacteria, viruses, or fungi. Compositions comprising the samples and cosmetically or pharmaceutically acceptable carriers are further disclosed.

Description

Attorney Docket No.: R217-0014PCTANTIMICROBIAL PROPERTIES OF AMPHIBIANSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 778,959, filed March 27, 2025, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The disclosure describes a novel method for using the extracellular matrix of axolotl as a source of antimicrobial agent.BACKGROUND

[0003] Antimicrobials are essential for the treating and preventing various infectious disease in humans, animals, and plants. Antimicrobials are used to eradicate the microorganisms at the site of infection. They can target cell walls, cell membranes, nucleic acids, and cellular processes to interfere with the growth of or destroy the microorganisms at the site of infection.

[0004] Examples of antimicrobials include small molecules, proteins, peptides, and nucleic acids. They can be isolated from plants and bacteria by extraction, culturing, and screening, and they can be obtained by chemical synthesis. Production of antimicrobials is time consuming and carries a significant cost burden. Accordingly, there is a need in developing new sources of antimicrobials.SUMMARY

[0005] This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0006] The present disclosure provides amphibians, including Urodeles, as a new source of antimicrobials. As described herein, biological samples obtained from amphibians have antimicrobial activity. They can inhibit the growth of various microorganisms including bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa,

[0007] Biological samples from axolotls can be used to treat various skin conditions including infections, such as infected wounds.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 shows the experimental design for wounding and treatment of the wounds. The treatment groups include the following which were applied for the entire study: (A) collagen sheet 3X3 cm comprising decellularized ECM (dECM), applied every 3 days; (B) gelatinized collagen solution comprising 3% gelatinized dECM, applied once daily; (C) supernatant of sonicated tissue, applied once daily; (D) collagen sheet comprising dECM, applied every 3 days; (E) vehicle controlAttorney Docket No.: R217-0014PCTfor gelatinized collagen solution comprising gelatinized solution without dECM, applied once daily; (F) vehicle control for supernatant of sonicated tissue comprising phosphate buffered saline (PBS), applied once daily; (G) positive control silver sulfadiazine, applied once daily; and (H) untreated control. All wounds were covered with polyurethane film dressings (Tegaderm®, 3M, St. Paul MN), after treatment was applied. 24-hour biofilms were established prior to applying the first treatment, and baseline wounds were recovered after 24 hours. Assessments were performed on Day 3 and Day 7 after treatment.

[0009] FIG. 2 shows observations of Methicillin Resistant Staphylococcus aureus MRSA USA300 and Pseudomonas aeruginosa ATCC 27312 infected wounds on assessment days in normal light and under ultraviolet light (Ultraviolet Woods Lamp).

[0010] FIG. 3 shows bacterial counts of Methicillin Resistant Staphylococcus aureus MRSA USA300 and Pseudomonas aeruginosa ATCC 27312 after treatment application on Day 3.

[0011] FIG. 4 shows bacterial counts of Methicillin Resistant Staphylococcus aureus MRSA USA300 and Pseudomonas aeruginosa ATCC 27312 after treatment application on Day 7.

[0012] FIG. 5 shows Methicillin Resistant Staphylococcus aureus MRSA USA300 bacterial counts after treatment application.

[0013] FIG. 6 shows Pseudomonas aeruginosa ATCC 27312 bacterial counts after treatment application.DETAILED DESCRIPTION

[0014] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0015] The term “antigen” refers to a molecule that is a toxin or is foreign to the subject and induces an immune response in the form of the production of antibodies against the molecule. The isolated ECM described herein can have reduced antigenicity as compared to a native ECM, such that it can be used in a subject.

[0016] The term “antimicrobial” refers to an agent that kills or stops the growth of microorganisms such as bacteria, viruses, fungi, and parasites. The agent can be a bioactive molecule such as a small molecule, protein, peptide, polypeptide, and peptoid, or the agent can be a biological sample from an animal, such as an amphibian. In embodiments, the biological sample can comprise one or more bioactive molecules that are antimicrobials. Accordingly, antimicrobials or antimicrobial agents are used interchangeably to include agents that have one or more antimicrobial activities including having the function of an antibiotic, antiviral, antifungal, antiparasitic, disinfectant, and / or antiseptics.

[0017] The term “bioactivity” refers to biological effects. A substance is bioactive or has bioactivity includes a substance having a biological function. In embodiments, the bioactivity is an antimicrobial activity,Attorney Docket No.: R217-0014PCT

[0018] The term "biocompatible" refers to a product and its normal degradation products in vitro, ex vivo, or in vivo that are substantially non-toxic and non-carcinogenic to a cell, tissue, organ, organism, or subject within useful, practical, and / or acceptable tolerances. The term "cytocompatible" refers to a product that can sustain the viability and growth of a population of cells.

[0019] The term “biological sample obtained from amphibians” includes any sample from an amphibian including: their body parts, such as tissues and organs; their extracellular matrix (ECM) or the decellularized ECM (dECM); their blastemas; their cells including in vivo cells and cells that are grown under in vitro conditions; the cultured media and spent media of in vitro cultured cells; amphibian skin activated by light exposure; and their biological fluids, including blood, urine, and secretions such as cutaneous surface liquid. In embodiments, the biological sample comprises one or more bioactive agents that have one or more antimicrobial activities.

[0020] The term “biomaterial” refers to a material suitable for in vitro, ex vivo, or in vivo use. As an example of in vivo use, the biomaterial is suitable for administering to a subject in need thereof. The material can be synthetic or natural. The decellularized ECM (isolated ECM) described herein is an example of a biomaterial.

[0021] The term "carrier" or “excipient” refers to a substance added to a composition that does not affect the active compound in the composition. The carrier can be a diluent. The excipient can be a substance added to the composition to facilitate the administration of the composition.

[0022] The term “supernatant of one or more sonicated amphibian tissues” refers to the supernatant obtained from one or more amphibian tissues that have been sonicated. The one or more amphibian tissues are harvested, sonicated in a sonicator, and centrifuged to separate the supernatant from the sonicated tissue.

[0023] The term “cosmetics” refers to products (excluding pure soap) intended to be applied to the human body for cleansing, beautifying, promoting attractiveness, or altering the appearance. Examples of cosmetic benefits can include improving the appearance of skin such as improving the appearance of wrinkling and fine lines, removing oil and excess sebum, reducing the appearance of skin blemishes, cleansing, and conditioning the skin, toning, and tightening the skin, soothing irritation, and refreshing and cooling the skin.

[0024] The term “drugs,” “pharmaceuticals,” or “therapeutics” refers to articles intended for use in the diagnosis, cure, mitigation, treatment, or prevention of conditions or disease and articles (other than food) intended to affect the structure or any function of the body of man or other animals.

[0025] The term "derive", "derived," or "derives" refers to a product obtained from any stated source by any useful method. For example, an extracellular matrix (ECM) derived from an amphibian refers to an ECM obtained from a member of the amphibian family.Attorney Docket No.: R217-0014PCT

[0026] The term “exogenous” refers to a product that originated outside of the organism, tissue, cell, organ, or subject. In contrast, the term “endogenous” refers to a product that originated from the organism, cell, tissue, organ, or subject.

[0027] The term “extracellular matrix” or “ECM” refers to a natural scaffolding having a three-dimensional structure including biomolecules and minerals that provide biochemical support to surrounding cells. The ECM includes structural and non-structural biomolecules, such as collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and / or growth factors. The ECM can be obtained from various sources of tissues including the skin and non-cutaneous tissues.

[0028] The term "decellularized extracellular matrix" or "decellularized ECM" refers to ECM prepared by removing and / or devitalizing cells from ECM found in multicellular organisms, for example, amphibians or mammals. Decellularized ECM is substantially free of intact cells, lysed cells, and cellular components including cellular and nuclear debris such that the decellularized ECM exhibits reduced immunogenicity so that it can be administered to a subject, for example, a mammalian subject, as a non-toxic xenograft or biomaterial. A decellularized ECM that is “substantially free of immunogenic components” refers to an ECM in which immunogenic components are at a level that is not sufficient to induce an adverse immune response in a subject.

[0029] The term "isolated" refers to being separated or removed from its native surroundings, such that it is substantially free from components that accompany it in its naturally-occurring state. For example, a cell or a protein can be isolated from its naturally-occurring state. The term “isolated ECM” refers to decellularized ECM.

[0030] The term “immunogenic” refers to relating to or producing an immune response. The term “immunogenicity” refers to the ability of a foreign substance, such as an antigen, to provoke an immune response in a subject. The isolated ECM described herein can have reduced immunogenicity as compared to the native ECM, such that it can be used as a biomaterial in a subject.

[0031] The term “native ECM” or “naive ECM” refers to a naturally occurring amphibian ECM (or corresponding ECM sample) that has not been decellularized.

[0032] The term "non-toxic" refers to a product that causes little or no adverse reaction or substantial harm to cells and tissues in vitro or ex vivo, and / or does not cause a substantial adverse or undesirable reaction or substantial harm to cells and tissues in the body (in vivo).

[0033] The terms “prevent” or “prevention” refers to the prevention of the onset, recurrence, or spread of a condition or one or more symptoms of the condition. As an example, the condition could be a skin condition. The term includes the administration of a product described herein before the onset of symptoms in particular to subjects at risk of developing a condition, such as a skin condition. The term includes the inhibition or reduction of one or more symptoms associatedAttorney Docket No.: R217-0014PCTwith the skin condition. The term “prevention” can be used interchangeably with the term “prophylactic treatment”.

[0034] The term “retain structural and functional integrity" used with reference to the ECM refers to retaining sufficient structure and function to permit and support the use of the matrix as a substrate for the growth of cells in vivo, ex vivo, or in vitro. For example, the isolated ECM retains the structure and functional properties of a naturally occurring ECM enabling its use as a biomaterial.

[0035] The terms “scaffold” and “bioscaffold” are used interchangeably to refer to a substrate on which cells can grow in vitro, ex vivo, and / or in vivo. A scaffold or bioscaffold is an example of a biomaterial.

[0036] The term “skin conditions” includes skin conditions that require therapeutic “drug” treatment including diseases, defects, and injuries including wounds. The term “cosmetic skin conditions” includes skin conditions that are related to tone, clarity, radiance, brightness, and / or hydration of the skin.

[0037] The term "subject" refers to an animal, for example, a mammal. Examples of mammals include a human, a dog, a cat, a horse, a cow, a goat, a sheep, a pig, or a non-human primate. A subject in need of treatment or a subject in need thereof includes a subject having a disease or condition that needs to be treated. A subject in need thereof also includes a subject that needs treatment and / or prevention of a skin condition.

[0038] The term “substrate” refers to a living organism or inanimate object. It includes the surface and the inside of a living organism or an inanimate object. A living organism can be a plant, an animal, or a human being. An inanimate object can be medical device.

[0039] The term "therapeutically effective amount" refers to an amount of a product or composition that provides a therapeutic benefit in the treatment, prevention, or management of a condition, such as a skin disease, an injury to the skin, or a wound, for example, a drug product. The term “therapeutically effective amount” also includes that amount of a compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the signs or symptoms of the condition or disease being treated.

[0040] The term "treatment" or "treating" in the context of administering a product, such as a biomaterial, to a subject refers to administering the product to achieve a desirable clinical / medical end-point, including alleviating symptoms of a disease or condition. Examples of such desirable end-points associated with skin disease or condition include wound healing, tissue closure, bulking tissue, preventing tissue adhesion, providing structural support to tissue, providing a protective barrier, and / or correcting a defect. Administering the product also includes applying the product on a subject.

[0041] The term “xenogenic” refers to a product derived or originated from a member of another species.Attorney Docket No.: R217-0014PCT

[0042] The term “amphibians” refers to cold-blooded vertebrate animals that include frogs, toads, newts, salamanders, and caecilians. They have an aquatic gill-breathing larval stage followed by a terrestrial lung-breathing adult stage. Amphibians include the class of amphibians and the orders of Anura (frogs ortoads), Urodela (newts or salamanders), and Apoda (caecilians). In embodiments, the amphibians described herein are young or neotenic amphibians. A young amphibian includes a young frog, such as a froglet, tadpole, or larval stage young Apoda. The amphibian can be a young amphibian and the young amphibian can include a larval stage of any order of amphibian. The amphibian can be neotenic.

[0043] The term “salamanders” refers to a group of amphibians characterized by a lizard-like appearance and having a tail throughout life. The families of salamanders include the Ambystomatidae (mole salamanders), Amphiumidae (Congo eels), Cryptobranchidae (giant salamanders), Dicamptodontidae (Pacific giant salamanders), Hynobiidae (Asiatic salamanders), Plethodontidae (lungless salamanders), Proteidae (mudpuppies and olms), Rhyacotritonidae (torrent salamanders), Salamandridae (newts and true salamanders), and Sirenidae (sirens). The Ambystomatidae family includes Ambystoma altamirani, Ambystoma amblycephalum, Ambystoma andersoni, Ambystoma annulatum, Ambystoma barbourin, Ambystoma bishop, Ambystoma bombypellum, Ambystoma californiense, Ambystoma cingulated, Ambystoma dumerilii, Ambystoma flavipiperatum, Ambystoma gracile, Ambystoma granulosum, Ambystoma jeffersonianum, Ambystoma laterale, Ambystoma leorae, Ambystoma lermaense, Ambystoma mabeei, Ambystoma macrodactylum, Ambystoma maculatum, Ambystoma mavortium, Ambystoma mexicanum, Ambystoma opacum, Ambystoma ordinarium, Ambystoma rivulare, Ambystoma rosaceum, Ambystoma silvense, Ambystoma subsalsum, Ambystoma talpoideum, Ambystoma taylori, Ambystoma texanum, Ambystoma tigrinum, and Ambystoma velasci.

[0044] The families of salamanders are grouped under the order Urodela (or Caudata). The term “Urodele” refers to a salamander of the order Urodela, in the class Amphibia. The amphibians can be from the orders Urodela, Anura, and Apoda. Urodeles begin life as aquatic animals in a larval state, and some undergo metamorphosis from a juvenile form with gills to an adult, terrestrial, air-breathing form with lungs. During metamorphosis, a Urodele's physical features are altered in preparation for life on land. These alterations include caudal fin resorption, thickening of the skin, the development of dermal glands, and resorption of gills. Sexual maturity also occurs during this time in most Urodeles. However, some families of Urodeles are "neotenic," which means that individuals of such families, even after reaching sexual maturity, retain their juvenile aquatic form throughout their lives. The axolotl (Mexican walking fish), Ambystoma mexicanum, and / or hybrids of A. mexicana and A. tigrinum are examples of neotenic salamanders. Instead of becoming a terrestrial amphibian, an adult axolotl remains aquatic and gilled. However, under certain circumstances, an axolotl will undergo metamorphosis and transform into a terrestrial form.Attorney Docket No.: R217-0014PCT

[0045] Axolotls possess pigment cells, called chromatophores, that are responsible for their colors. The chromatophores of axolotls include melanophores containing eumelanin, xanthophores containing pteridines, and iridophores containing crystallized purines. Eumelanin is a black-brown pigment; pteridine is a yellow and reddish pigment; and crystallized purine is an iridescent white pigment. These pigments which are encoded by their respective genes provide the different phenotypes of axolotls, such as wild-type, golden albino, leucistic, and melanistic.

[0046] Although the skin structure of amphibians is similar to mammals, Urodeles and anuran amphibians (frogs and toads) can regenerate their skin structures including the dermis and secretion glands without forming any scar after a deep skin injury. Moreover, axolotls have the ability to fully regenerate lost or damaged body parts including organs, limbs, and parts of the central nervous system, throughout their entire life. Axolotls undergo rapid re-epithelialization during wound healing and limb regeneration, both of which are scar-less processes. The axolotl wound healing process resembles the scar-free healing process of mammalian fetal and embryonic wounds. Such wounds exhibit re-epithelialization and basement membrane reformation that occur at a faster rate than do the corresponding events in postnatal mammals.

[0047] Further, the skin of amphibians contains ECM, which is rich in growth factors, which are favorable for wound healing. The ECM is a three-dimensional network of extracellular macromolecules and minerals including collagen, enzymes, glycoproteins, and hydroxyapatite which provide structural and biochemical support to surrounding cells. The ECM can include a combination of fibrous and network-type collagens. Examples of various types of collagens, such as one or more of type I, II, III, IV, V, and VI collagens. The ECM can also include elastin and / or elastic fibers. The ECM can also include laminin, fibronectin, hyaluronan, chondroitin sulfate, or both, and / or one or more proteoglycan, glycoprotein, glycosaminoglycan, or any combination thereof. The components and structure of the ECM play an important role in the healing process because the ECM components create scaffolding which provides the structural architecture of the matrix required for the healing process. Additionally, the ECM components are involved in stimulating the adhesion and migration of cells during the healing process as well as mediating the interactions among the cells and between the cells and the matrix, or between ECM proteins during the healing process. Further, the ECM components also serve as a reservoir and modulator of the action of the cytokines and growth factors to regulate wound repair activities. However, the ECM contains cells and genetic materials that need to be removed before it can be used as a biomaterial since the cells and genetic material can induce an adverse immune response.

[0048] Decellularization of the ECM is the removal of cells and cellular components from the ECM of a biological sample, such as a tissue, while retaining the ECM proteins and the native ECM architecture or structure for effective use as a biomaterial. The present disclosure describes a novel method of decellularizing amphibian ECM, for example, Urodele ECM, for use inAttorney Docket No.: R217-0014PCTpreventing and / or treating and healing various conditions including skin conditions. In contrast to known methods of decellularizing ECM which can involve the use of cross-linking agents and other harsh agents, the method described herein uses milder agents yet provides a decellularized amphibian ECM containing fewer cells and cellular debris, thus enabling the decellularized ECM to be used in vitro, ex vivo, and / or in vivo without causing undesirable adverse effects. Methods of decellularizing amphibian ECM, such as Urodele ECM are described in detail in in WO 2023 / 154873, WO 2023 / 154876, US 20250134801 , and US 20250134934, which are incorporated by reference in their entirety.

[0049] Some amphibians start their life in water and transform into air-breathing adults with lungs and appendages while others remain aquatic their entire lives. They can secrete various bioactive molecules having therapeutic functions from their skin. Such molecules include peptides, amino acids, fatty acids, steroids, mucopolysaccharides, glycoproteins, monosaccharides, oligosaccharides, biogenic amines, alkaloids, ions, electrolytes.

[0050] Amphibians have antimicrobials including antimicrobial peptides that protect them from pathogens. Accordingly, a biological sample from an amphibian can be used as a source of antimicrobials. An amphibian sample includes any biological sample from an amphibian, including a young amphibian, such as a young frog or neotenic Urodele. The biological sample can include any amphibian tissue such as connective tissues, adipose tissue, bone, skin, cartilage, tendon, dura mater, and fascia. Other tissues include the dermis, basement membrane, and epithelial tissue, for example, basement membrane or epithelial tissues that line the body cavities such as the parietal mesothelial tissues of the thoracic cavity, the abdominal cavity, and the pericardium. In embodiments, the tissue has an ECM. The biological sample can be a tissue from a young amphibian, such as a young frog, or neotenic Urodele. The biological sample can be obtained from any Urodele species including axolotl. The sample can be obtained from the skin of an axolotl. The sample can also can be decellularized ECM of an axolotl skin sample.

[0051] Other biological samples of an amphibian, such as an axolotl, including its biological fluids, blastemas, in vitro cultured cells, culture media and spent media of in vitro cultured cells, and skin activated by light exposure, can also be obtained from amphibians such as Urodeles. WO 2025 / 111477 describes obtaining blastemas from amphibians. PCT Application PCT / US2026 / 011279 describes culturing in vitro amphibian cells, and PCT Application PCT / US2026 / 011283 describes obtaining culture media and spent media from in vitro cultured cells. WO 2026 / 020149 descries methods of obtaining activated amphibian skin. These patent applications are incorporated by reference in their entirety.

[0052] The biological samples of an amphibian can also include the supernatant of one or more sonicated amphibian tissues. The process of obtaining the supernatant includes harvesting the one or more tissues from the amphibian, placing the one or more harvested tissues in solution, such as PBS, sonicating the one or more tissues in a sonicator to obtain a mixture, centrifugingAttorney Docket No.: R217-0014PCTthe mixture, removing the sonicated tissue to obtain the supernatant, and filtering the supernatant to obtain sterilized supernatant.

[0053] The biological samples from an amphibian also can be obtained and prepared by any known method. The biological sample can be a fresh sample or a frozen sample that has been in storage and thawed for use.

[0054] The biological samples described herein can be prepared into various forms including a powder containing the micronized particulates, which can be reconstituted with water, a buffered solution, or any suitable liquid for use as a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, gel, gelatin, a hydrogel, a dispersion, or an emulsion. The biological sample, such as the ECM, can also be gelatinized to form gelatin. The method of gelatinizing amphibian ECM and dECM is described in detail in WO 2023 / 154876 and US 2025134934, which are incorporated by reference in its entirety.

[0055] The present disclosure describes compositions comprising a biological sample from an amphibian. The compositions can include one or more carriers or excipients. Examples of carriers and excipients include saline, emulsion, a mixture of organic solvents with water, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, petrolatum, lanolin, mineral oil, dimethicone, humectant, and polyethylene glycols. Examples of humectants include glycerin, lecithin, and propylene glycol. In embodiments, the compositions described herein include cosmetic, or pharmaceutical compositions containing one or more cosmetically, or pharmaceutically acceptable carriers or excipients, respectively.

[0056] The compositions described herein can also include carriers for immediate or sustained release preparations include polymers. The polymers can be biodegradable, and / or bioabsorbable. As an example, for controlled release, the biomaterial can be coated with polymers such as acrylic polymer, acrylic / methacrylic copolymer, cellulose acetate phthalate (CAP), Opadry®, and Ethocel™. For immediate release, the biomaterial can be coated with cellulosic polymers, such as hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethylcellulose (HEC), methyl cellulose (MC), and sodium carboxymethyl cellulose (NaCMC); vinyl derivatives, such as polyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone-polyvinyl acetate copolymer, polyvinyl alcohol (PVA), and polyvinyl alcohol-polyethylene glycol copolymer; acrylic polymers, such as Eudragit®; or glycols such as polyethylene glycols.

[0057] The biological samples and the compositions described herein can be used as antibiotics, antivirals, antifungals, antiparasitics, disinfectants, or antiseptics for treating and / or preventing condition or disease caused by microorganiss and to clean surfaces including medical devices and clinical surfaces. They can also be applied to various surfaces including medical devices and implants. They can be used as a therapeutic or pharmaceutical composition or as a cosmeceutical composition. They can include one or more further agents that are xenogenic to the biological sample.Attorney Docket No.: R217-0014PCT

[0058] The compositions described herein can further include one or more other agents such as one or more therapeutic agents. Examples of therapeutic agents include known drugs such as retinoic acid, corticosteroids, antifungals, antivirals, antibiotics, antiseptics, local anesthetics, and antineoplastics.

[0059] The compositions described herein can further include one or more other agents such as one or more cosmetic agents. Examples of cosmetic agents include antioxidants, peptides, alpha or beta hydroxy acids, retinol, vitamins, plant extracts, skin clarifying agents such as arbutin, moisturizing agents such as hyaluronic acid, emollients, carbohydrates, glycoproteins, and / or polymers. The one or more agents can include a combination of agents. The agent can be exogenous or xenogenic to the isolated biological sample of an amphibian.

[0060] Examples of one or more peptides and proteins include growth factors, cytokines, and chemokines. Examples of growth factors include fibroblast growth factors (FGFs) including acidic FGF, basic FGF, FGF8, and FGF10; ciliary neurotrophic factor (CNTF); epidermal growth factor (EGF); granulocyte-macrophage colony stimulating factor (GM-CSF); hepatocyte growth factor (HGF); insulin-like growth factors 1 and 2 (IGF-1 and IGF-2); keratinocyte growth factor (KGF); nerve growth factor (NGF); neurotrophins such as neurotrophin-3, neurotrophin-4, neurotrophin-5; platelet derived growth factor (PDGF); vascular endothelial growth factor (VEGF); stromal derived factor 1 alpha (SDF-1 alpha); and transforming growth factor-alpha and -beta (TGF-a and TGF-p). Examples of cytokines and chemokines include tumor necrosis factor-alpha (TNF-a), interleukin-1 alpha and beta (IL-1 a and IL-1 P), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-18 (IL-18), CCL2, CCL3, CCL5, CXCL1 , CXCL4, CXCL5, CXCL7, CXCL8, and CXCL12. Examples of cosmetic peptides include acetyl hexapeptide, acetyl tetrapeptide, palmitoyl pentapeptide, and palmitoyl oligopeptide.

[0061] Examples of one or more therapeutic agents include antimicrobials and antiinflammatory agents. Examples of antimicrobials include antibiotics such as penicillin, streptomycin, amoxicillin, cephalexin, clindamycin, dicloxacillin, and doxycycline. Other antimicrobials include anti-microbial peptides, silver salts, clotrimazole, miconazole, and ketoconazole. Examples of anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs) such as salicylic acid, ibuprofen, naproxen, colchicine, fenoprofen, sulindac, diflunisal, diclofenac, indoprofen, and sodium salicylamide.

[0062] Examples of cosmetic agents include one or more glycoproteins include proteoglycans which are proteins covalently attached to glycosaminoglycans (GAGs), antioxidants, ascorbic acid, vitamin C, alpha hydroxy acids (AHAs), beta hydroxy acids (BHAs), exfoliants, skin whitening agents, light diffusers, UV absorbing agents, sunscreens, moisturizers, anti-wrinkle ingredients, and oil absorbing agents. Examples of AHAs include glycolic acid, lactic acid, malic acid, tartaric acid, and citric acid. Examples of BHAs include salicylic acid.Attorney Docket No.: R217-0014PCT

[0063] The compositions described herein can also include one or more natural and / or synthetic polymers. Natural polymers can be from an animal source or a non-animal source such as a plant source. Examples of natural polymers include natural polymers such as collagen, chitosan, alginate, glycosaminoglycans, fibrin, and hyaluronic acid. Examples of synthetic polymers include polyethylene, polyethylene glycol (PEG), polyethylene terephthalate (PET, or PETE), polytetrafluoroethylene (PTFE), polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol (PEG), polyethylene glycol) diacrylate (PEG diacrylate), poly(hydroxy acids), polydioxanone, polycaprolactone, poly(ortho esters), poly(anhydrides), polyphosphazene, poly(amino acids), pseudo-poly(amino acids), conductive polymers (such as polyacetylene, polypyrrole, polyaniline), polyurethane, polystyrene, and nitinol.

[0064] The polymer included in the compositions described herein can be biocompatible, biodegradable, and / or bioabsorbable, and can be a random copolymer, block copolymer, or blend of monomers, homopolymers, copolymers, and / or heteropolymers that contain these monomers. Exemplary biodegradable or bioabsorbable polymers include polylactides, poly-glycolides, polycaprolactones, polydioxanes, and their random and block copolymers. A biodegradable and / or bioabsorbable polymer can contain a monomer selected from the group consisting of glycolide, lactide, dioxanone, caprolactone, trimethylene carbonate, ethylene glycol, and lysine. The biodegradable and / or bioabsorbable polymers can contain bioabsorbable and biodegradable linear aliphatic polyesters such as polyglycolide (PGA) and its random copolymer poly(glycolide-co-lactide-) (PGA-co-PLA). Other examples of suitable biocompatible polymers include polyhydroxyalkyl methacrylate, ethylmethacrylate, polyvinylpyrrolidone, and polyacrylamides. Other suitable bioabsorbable materials are biopolymers which include collagen, gelatin, alginic acid, chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acid, polylysine, and copolymers of these materials. Any combination of polymers and copolymers or blend thereof of the above examples can also be included in the composition.

[0065] The compositions described herein can also include protectives, adsorbents, demulcents, emollients, preservatives, antioxidants, moisturizers, buffering agents, solubilizing agents, skin-penetration enhancers, and surfactants.

[0066] Any skin penetration enhancer can be added to the compositions, provided the skin penetration enhancer is safe and can effectively facilitate the passage of the desired substances in the isolated ECM across the skin membrane. Examples of skin penetration enhancers include dimethyl sulphoxide (DMSO), monoglycerides, C10-C20 fatty acid esters including ethyl palmitate and isopropyl myristate; acyl lactylates such as caproyl lactylic acid and lauroyl lactylic acid; dimethyl lauramide; dodecyl (lauryl) acetate; lactate esters such as lauryl lactate, and myristyl lactate; monoalkyl ethers of polyethyleneglycol and their alkyl or aryl carboxylic acid esters and carboxymethyl ethers such as polyethylene glycol-4 lauryl ether (Laureth-4) and polyethylene glycol-2 lauryl ether (Laureth-2); Myreth-3, myristyl sarcosine, and methyl laurate; polypropyleneAttorney Docket No.: R217-0014PCTglycol, polyethylene glycol, lecithin, urea, amino acids, 1-dodecylhexahydro-2H-azepine 2-one (Azone), oleic acid, linoleic acid, isopropyl linoleate, oleyl alcohol, 1-dodecyl-azacycloheptan-2-one, butanediol, and 2-(2-Ethoxyethoxy)ethanol (Transcutol).

[0067] The compositions comprising the biological samples described herein can be prepared as a dry powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, a dispersion, gel, hydrogel, gelatinized composition, or an emulsion. The compositions can be prepared into a variety of suitable shapes and sizes as they can be formed, laminated, homogenized, and reconstituted. They can be formed into two-dimensional or three-dimensional shapes. They can be formed into a sheet, mesh, graft, plug, or any shape or form for use. The sheets can include a backing with or without an adhesive. The backing can be biodegradable or non-biodegradable. Two or more sheets can be laminated together or somehow attached. The sheets can be oriented in the same direction, different direction, or at an angle. There can be two to fifteen layers of sheets.

[0068] The biological samples and compositions described herein can be used to kill or inhibit the growth of one or more microorganisms including bacteria, viruses, yeasts, fungi, parasite, or a combination thereof. The bacteria can be a gram-positive bacteria including Staphylococcus aureus or Enterococcus. Staphylococcus aureus can include methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-sensitive Staphylococcus aureus (MSSA), and Enterococcus can include Enterococcus faecalis. The bacteria can be a gram-negative bacteria such as Klebsiella, Escherichia coli, Pseudomonas, or Pantoea. Klebsiella can include Klebsiella pneumoniae. Pseudomonas can include Pseudomonas aeruginosa. Pantoea can include Pantoea agglomerans. Examples of viruses can include herpes simplex virus, varicella-zoster virus, measles morbillivirus, rubella virus, monkeypox virus, small pox virus, parvovirus, ebola virus, and coxsackievirus. Examples of yeasts and / or fungi include Candida, Trichophyton, Microsporum, Epidermophyton. Examples of parasites include protozoa, helminths, and ectoparasites. Examples of parasites include scabies mite, pediculosis capitis, pediculosis corporis, cutaneous lava migrans, bed bugs, and mosquitos. The biological samples and compositions described herein can inhibit the growth or kill a combination of different bacteria, different viruses, different yeasts and fungi, and different parasites.

[0069] The biological samples and compositions described herein are able to reduce the population of one or more microorganisms by inhibiting their growth and / or killing them. The biological samples and compositions described herein can reduce the population of microorganisms by 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 99%, or 80% to 90% in about 1 to 10 days, 2 to 10 days, 3 to 10 days, 4 to 10 days, 5 to 10 days, 2 to 8 days, 3 to 7 days, 4 to 7 days, or 5 to 7 days.

[0070] In embodiments, the biological samples and compositions described herein can reduce the population of one or more viruses by 40% to 100%, 50% to 100%, 60% to 100%, 70% toAttorney Docket No.: R217-0014PCT100%, 80% to 100%, 90% to 100%, 95% to 99%, or 80% to 90% in 5 mins to 60 mins, 10 mins to 50 mins, 15 mins to 45 mins, 20 mins to 40 mins, 25 mins to 35 mins, or 30 mins. The biological samples and compositions described herein are able to inhibit the growth of viruses with a TCID50 per 0.1 of 5.50 log10which is about 82.22% reduction in the population of viruses.

[0071] The biological samples and compositions described herein can be prepared as a formulation for treating and / or preventing various skin conditions. The formulation can be in the form of a dry powder, a solution, a paste, a liquid, an extract, a cream, a lotion, a serum, an emollient, an ointment, a dispersion, a gel, a hydrogel, a gelatin, or an emulsion. The amount of the biological sample in the formulation is 0.001% to 5.0% w / v, 0.01% 4%, or 0.05% to 1.0% w / v. The formulation can be placed on or impregnated into a bandage, such as a non-stick adhesive bandage, for applying to the subject.

[0072] For the treatment and / or prevention of skin conditions, the biological samples and compositions described herein can be administered to the target site topically, or by injection, implantation, microneedling, or radiofrequency microneedling, or using laser including an ablative fractional laser or orally as a pill, tablet, capsule, liquid or suspension. The compositions described herein also can be administered to the subject prior to, during, or after a dermatological or cosmetic procedure, such as dermabrasion, microdermabrasion, and ablative laser resurfacing. The dermatological or cosmetic procedure includes procedures wherein at least one cell of the stratum corneum is removed. The compositions described herein can also be delivered as an injectable or with a dermal or subdermal implant such as a volume filler, hyaluronic acid, or other dermal matrix protein including collagen or elastin, either naturally occurring, bioengineered, or recombinantly produced. The compositions described herein can be administered alone or in combination with one or more agents described herein, such as growth factors, peptides, and proteins. The compositions can also be administered with toxins, such as botulinum toxin.

[0073] Examples of skin conditions include acne, actinic keratosis, blister, cellulitis, cold blister, hives, impetigo, Keratosis pilaris, melasma, moles, ringworm, uticaria, vitiligo, and wart. Examples of inflammatory skin conditions include psoriasis; dermatitis, such as contact dermatitis, atopic dermatitis (eczema), seborrheic dermatitis, nummular dermatitis, generalized exfoliative dermatitis, statis dermatitis, lichen simplex chronicus; disorders of hair follicles and sebaceous glands, such as acne, rosacea and rhinophyma, perioral dermatitis, and pseudofolliculitis barbae; and inflammatory reactions, such as drug eruptions, erythema multiforme, erythema nodosum, and granuloma annulare. Examples of cancerous skin conditions include basal cell carcinoma, melanoma, and squamous cell carcinoma. Other skin conditions needing treatment include fine lines and / or wrinkles, aging, redness, abrasion, burn, cut, infection, razor bumps, scars, uneven skin tone, pain, stretch marks, skin elasticity and / or firmness, skin hydration, and hyperpigmentation. The burns include acute thermal burns including first, second, or third-degree burns. The burns can be treated to prevent infections.Attorney Docket No.: R217-0014PCT

[0074] Examples of skin conditions caused by viruses include warts, shingles, chicken pox, cold sores, genital herpes, vesicular stomatitis, measles, rubella, erythema infectiosum, roseola, monkeypox, ebola, small pox, and hand, foot, and mouth disease.

[0075] Examples of parasitic skin infections include scabies, lice, bedbugs, and hookworm,

[0076] The compositions described herein can also be used in a skincare regimen for protecting the skin from damage including UV rays and environmental pollution and as an aesthetic agent for improving the appearance of the skin.

[0077] The biological samples and compositions described herein can reduce or diminish infection, inflammation, scarring, reduce keloid formation, and / or healing time for various dermatological and cosmetic procedures. The compositions described herein can also be used to restore lost dermal matrix or subdermal volume.

[0078] The biological samples and compositions described herein are also useful in surgical applications, treating burns and open wounds including infected wounds, and / or expediting healing of injuries and surgical procedures. In embodiments, the biological samples and compositions described herein promote regeneration of bone, tissue, skin, nerves, blood vessels and organs.

[0079] The present disclosure also describes a medical device comprising the biological samples or compositions described herein for the various uses described herein. A medical device can be a material or an object used directly or indirectly to apply the compositions described herein. As an example, a medical device can be used to apply the compositions described herein on the skin of a subject. A medical device can contain the composition described herein or include the composition on its surface for implanting into a subject. An implant can be formulated to contain the compositions described herein for implantation into a subject.

[0080] The present disclosure describes methods of using the biological samples and compositions for treating skin conditions comprising applying the biological samples or compositions described herein to a target site, such as the skin or wound. The biological samples or compositions can be applied every 4, 6, 8 or 12 hours, daily, every other day (every two days), every three days, every four days, every five days, every six days, or once a week.

[0081] The present disclosure describes kits including the biological samples and compositions described herein for the various uses described herein. The kits can include sterilized biological samples or compositions thereof in any shape and form. The kits can include a solution for reconstituting the composition for use. The kits can include a device for administering the composition to a subject. The kits can include an implant to be coated with or containing the composition prior to being implanted in a subject. The kits can include components for the various uses described herein.

[0082] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, orAttorney Docket No.: R217-0014PCTexemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0083] Numbers expressing ranges or quantities of ingredients, constituents, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about." When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ± 20% of the stated value; ± 15% of the stated value; ± 10% of the stated value; ± 5% of the stated value; ± 4% of the stated value; ± 3% of the stated value; ± 2% of the stated value; ± 1% of the stated value; or ± any percentage between 1% and 20% of the stated value.

[0084] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of, or consist of its particular stated element, step, ingredient, or component. Thus, the terms “include” or “including" should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of’ excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. In embodiments, the lack of a material effect of a step is evidenced by the lack of a statistically-significant reduction in the process step in removing cellular debris such as DNA from the sample. Lack of a material effect of an embodiment can include a lack of a statistically-significant improvement in using the biological samples or compositions described herein as an antimicrobial in inhibiting the growth of one or more microorganisms.

[0085] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.5, 2.7, 3, 4, 5, 5.1 , 5.3, 5.8 and 6. Moreover, any ranges cited herein are inclusive.Attorney Docket No.: R217-0014PCT

[0086] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein.

[0087] The following exemplary embodiments and examples illustrate exemplary methods provided herein. These exemplary embodiments and examples are not intended, nor are they to be construed, as limiting the scope of the disclosure. It will be clear that the methods can be practiced otherwise than as particularly described herein. Numerous modifications and variations are possible in view of the teachings herein and, therefore, are within the scope of the disclosure.EXEMPLARY EMBODIMENTS

[0088] The following are exemplary embodiments:1. A method of inhibiting growth of and / or killing one or more microorganisms, wherein the method includes administering a composition comprising a biological sample of an amphibian to a substrate in need thereof.2. The method of embodiment 1 , wherein the composition further includes one or more other cosmetically or pharmaceutically acceptable carriers.3. The method of embodiment 1 or 2, wherein the amphibian is a baby amphibian, and optionally, wherein the baby amphibian is one day old (newborn) to 12-month old.4. The method of any one of embodiments 1-3, wherein the amphibian is a froglet, tadpole, Urodele, or larval stage young Apoda.5. The method of any one of embodiments 1-4, wherein the amphibian is a baby Urodele, and optionally the amphibian is a baby axolotl.6. The method of any one of embodiments 1 -5, wherein the biological sample includes organ, cell, blastema, biological fluid, or a combination thereof.7. The method of any one of embodiments 1-6, wherein the biological sample includes tissue, in vitro cells, in vivo cells, blood, urine, secretion, or a combination thereof; and optionally, wherein the secretion includes cutaneous surface liquid.8. The method of any one of embodiments 1-7, wherein the composition includes decellularized extracellular matrix (dECM) or gelatinized dECM.9. The method of any one of embodiments 1-8, wherein the composition includes supernatant of one or more sonicated amphibian tissues.10. The method of any one of embodiments 1 -9, wherein the composition further includes one or more agents that are xenogenic to the biological sample.11. The method of any one of embodiments 1-10, wherein the composition further includes one or more mammalian peptides, mammalian proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof.12. The method of any one of embodiments 1-11 , wherein the substrate includes the surface of an inanimate object or a living organism.Attorney Docket No.: R217-0014PCT13. The method of any one of embodiments 1-12, wherein the substrate includes the skin of an living organism, and optionally the living organism is a human; or wherein the substrate includes the surface of a medical device.14. The method of any one of embodiments 1-13, wherein the one or more microorganisms includegram-positive bacteria, gram-negative bacteria, viruses, yeasts, fungi, or a combination thereof.15. The method of any one of embodiments 1-14, wherein the one or more microorganisms include gram-positive bacteria including Staphylococcus aureus, Enterococcus, ora combination thereof, and optionally,wherein the Staphylococcus aureus includes methicillin-resistant Staphylococcus aureus (MRSA) and / or methicillin-sensitive Staphylococcus aureus (MSSA); and / orwherein the Enterococcus includes Enterococcus faecalis.16. The method of any one of embodiments 1-14, wherein the one or more microorganisms include gram-negative bacteria comprising Klebsiella, Escherichia coli, Pseudomonas, Pantoea, or a combination thereof, and optionally,wherein the Klebsiella includes Klebsiella pneumoniae;wherein the Pseudomonas includes Pseudomonas aeruginosa; and / orwherein the Pantoea includes Pantoea agglomerans.17. The method of any one of embodiments 1-14, wherein the one or more microorganisms include viruses including herpes simplex virus, varicella-zoster virus, measles morbillivirus, rubella virus, monkeypox virus, small pox virus, parvovirus, ebola virus, coxsackievirus or a combination thereof.18. The method of any one of embodiments 1-9, wherein the one or more microorganisms includes yeasts and / or fungi comprising Candida, Trichophyton, Microsporum, Epidermophyton, or a combination thereof.19. The method of any one of embodiments 1-18, wherein the method further treats and / or prevents a disease or condition.20. The method of any one of embodiments 1-19, wherein the method further treats and / or prevents a skin condition.21. The method of any one of embodiments 1-20, wherein the method further treats a wound, an infected wound, or the infection of a wound.22. The method of any one of embodiments 1-21 , wherein the method reduces a population of microorganisms on or in the substrate by 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 99%, or 80% to 90%.23. The method of any one of embodiments 1-22, wherein the method reduces a population of viruses on or in the substrate by 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 99%, or 80% to 90%.Attorney Docket No.: R217-0014PCTEXAMPLES

[0089] Example 1. Gelatinized Axolotl ECM Inhibits the Growth of Herpes Simplex Virus (HSV)

[0090] In this Example, the test virus is HSV 1 (HF Strain, ATCC VR -260) and the test substance is gelatinized axolotl decellularized ECM (dECM 3%). Virus control substance is a molecule that has no viral effect and will allow the virus to replicate. The method steps are summarized below and the testing conditions are summarized in Table 5.• Stock virus was thawed and was not supplemented with an organic soil load.• Test and virus control substances were dispensed in 9-part equivalent volumes into sterile vessels.• Test and virus control substances were each inoculated with 1 -part equivalent volumes of the test virus.• The test suspensions were held for 30 minutes and then neutralized by ten-fold serial dilutions with 0.90 mL of 100% fetal bovine serum (FBS).• The virus control suspension was neutralized with in the same manner as the test suspensions.• Following neutralization, the viral suspensions were quantified to determine the levels of infectious virus using standard cell culture (e.g. TCID50) assay techniques.• The inoculated cell culture plates were incubated for 7 days.• Following the incubation period, the assay was microscopically scored for the presence / absence of test virus and cytotoxic effects.o The appropriate calculations were performed (e.g. Spearman-Karber) to determine viral titers and levels of test substance cytotoxicity, where applicable. o Logic and percent reductions were computed for test suspensions relative to the control suspensions and reported to the Study Sponsor.

[0091] Success criteria are described below.• The following measures are met to ensure the acceptability of virucidal efficacy data: o A minimum of 104 infectious viruses (TCID50) are recovered from the virus control, o The virus titer control demonstrates obvious and / or typical cytopathic effects on the monolayers unless a detection method other than cytopathic effect is used. o Neutralization of the test substance with a low titer (e.g. 1000-5000 infective units) of the test virus is demonstrated.o Quantification of the test and control parameters is conducted at a minimum of four determinations per dilution.• The product performance criteria are described below.Attorney Docket No.: R217-0014PCTo The log and percent reduction of the test virus following exposure to the test substance are calculated; however, there is no minimum reduction level to qualify as “passing” or an “efficacious” product.

[0092] Calculations and Statistical Analysis• The TCID50 (Tissue Culture Infectivity Dose) represents the endpoint dilution where 50% of the cell cultures exhibit cytopathic effects due to infection by the test virus. The endpoint dilution at which 50% of the host cell monolayers exhibit cytotoxicity is termed the Tissue Culture Dose (TCD50). The TCID50 and TCD50 were determined using the Spearman- Karber method andcalculated as follows:Negative logarithm of endpoint Titer = [- Log of first dilution inoculated] - [((sum of % mortality at each dilution / 100) - 0.5) x Logarithm of dilution]The result of this calculation is expressed as TCID50 / 0.I mL (or volume of dilution inoculated) for the test, virus control, and neutralization control and TCD5o / O.1 mL (or volume of dilutioninoculated) for the cytotoxicity control.• Calculation of the Log-10 Reductiono The log 10 reduction in viral titer was calculated as follows:o Recovery Control Log10 TCID50 - Virus-Test Substance Log10TCID50• Calculation of the Percent Reductiono The percent reduction in viral titer was calculated as follows:o Percent Reduction = 1- (C / B) x 100, where:B = Average TCID50of virus in control suspensions.C = Average TCID50 of virus in virus-test suspensions.• The presence of any test substance cytotoxicity was taken into account when calculating the log and percent reductions in viral titer.• If multiple virus control and test replicates were performed, the average TCID50 of each parameter was calculated and the average result used to calculate the log reductions in viral titer.

[0093] ResultsTable 1: Virus Inoculum Titer Control and Time Zero ResultsVirus Inoculum Titer Virus Controlpell Control 0 0 0 0 N / A|1O-2+ + + ++ + + +|1O-3+ + + +|1O-4+ + + + + + + +Attorney Docket No.: R217-0014PCTQ-5 00+ +_ i+ 0 + 0 _ IR000 + 0 + 00 Ho-610’zjTCID50 per 0.1 mL 6.25 log- 6.25 log-Table 2: Test Results -Gelatinized dECM- Lot: SG051024-1Test Results IO'2 + + + +_ J i Q-oO : : _+ + + + i 4 :+ + + +]10i+ + 00 1 O’50 + 0 + I'j Q“6 :0000 |10“zI [TCID50 per 0.1 mL 5.50 log10Logw Reduction 0.75 log10Percent Reduction 82.22%Table 3: Test Substance Cytotoxicity Control ResultsJ Cytotoxicity ControlLot: SG051024-1 ji n-2 0 000 | I30000 K b o o o . iw4;TCD50 perO.1 mL<1.50 log10Table 4: Test Substance Neutralization Control Results— Neutralization Control Lot: SG 051024-1 Test Media 10'2+ + + + !+ + + ++ + + + !+ + + + 10'310~4TCID50 per 6.1 <1.50 logl0 <1.50 log10mLTable 5: Testing Conditions> Temperature (°C) Relative Humidity (%)Attorney Docket No.: R217-0014PCTParameter DurationStart / End Start / End!T reatment{Test sample 30 minutes 21.9 / 19.1 29.7 / 33.0 j - Cytotoxicity Control 30 minutes 22.2 / 21.3 29.6 / 30.2 I Incubation of Assay~7 days 36.0 / 36.0 6.0* / 6.0*Plates

[0094] Conclusion

[0095] The purpose of the study was to determine the virucidal efficacy of the test substance against Herpes Simplex Virus 1 (HF Strain, ATCC VR-260) propagated in 2% FBS with no additional organic soil load supplementation, at a contact time of 30 minutes with an exposure temperature of room temperature.

[0096] The Recovery Control demonstrated a viral titer of 6.25 log-io TCID50 per 0.1 ml_.

[0097] Taking the cytotoxicity and neutralization control results into consideration, the evaluated test substance demonstrated a 0.75 log-10 reduction (82.22%) in viral titer as compared to the titer of the corresponding Recovery Control.

[0098] No test substance cytotoxic effects to the host monolayer were observed.

[0099] The Test Substance Neutralization Control demonstrated that the test substance was neutralized at <1.50 log-10 TCID50 per 0.1 mL.

[0100] Example 2: Treatment of Infected Wounds

[0101] The objective of this study was to determine the antimicrobial efficacy of the prepared test substance on infected wounds with a polymicrobial suspension of Methicillin Resistant Staphylococcus aureus USA300 (MRSA USA300) and Pseudomonas aeruginosa ATCC 27312 (PA 27312) using a deep dermal porcine wound model.

[0102] Materials and Methods

[0103] Experimental Animals. A porcine model was used for our experimental research due to the morphological similarities between swine skin and humans. One animal was used forthis pilot study. The young female specific pathogen free (SPF: Looper Farms, North Carolina) pigs weighing 25-35 kg was kept in house for at least 4 days prior to initiating the experiment. The animal was fed a basal diet ad libitum and housed individually in our animal facilities (meeting American Association for Accreditation of Laboratory Animal Care [AAALAC] accredited) with controlled temperature (19-21°C) and lighting (12h / 12h LD).

[0104] Animal Preparation, Wounding and Treatment. The animal was sedated with Telazol HCI, Xylazine, Atropine (I.M.) followed by endotracheal tube inhalation of an Isofluorane (Isothesia; Abbott Laboratories, Chicago, IL) and oxygen combination during preparation, wounding, and treatment. The flank and back of experimental animal was clipped with standard animal clippers on the first day of experimentation. The skin on both sides of the animal wasAttorney Docket No.: R217-0014PCTprepared for wounding by washing with non-antibiotic soap (Neutrogena Soap Bar; Johnson and Johnson, Los Angeles, CA) and sterile water. The animal was blotted dry with sterile gauze.

[0105] Wounding: Deep Partial Thickness. Fifty-one (51) deep reticular dermal wounds measuring (10 mm x 10 mm x2 mm deep) were made in the paravertebral and thoracic area with a specialized electrokeratome fitted with a 10 mm blade. The wounds were separated from one another by 5-7 cm of unwounded skin. All wounds were inoculated within 20 minutes after wounding and individually dressed. Wounds were randomly divided into eight (8) groups of six (6) wounds in each group and three wounds were assigned as a baseline (FIG. 1). Untreated wounds are also included as negative controls.

[0106] Wound Inoculation

[0107] A fresh culture of mixed MRSA USA300 and PA 27312 was used for this study. The challenge inoculum suspension was prepared by scraping the overnight growth from a culture plate into 5 milliter (mL) of Tryptic Soy Broth (TSB). This resulted in a suspension concentration of approximately 1010colony forming units / mL (CFU / mL) for the bacteria. Serial dilutions were made until a concentration of 106CFU / mL (critically colonized level of bacteria associated in wound infection) was achieved. The inoculum was vortexed and 25 microliter (pL) of the suspension was inoculated into each wound. In addition, serial dilutions of the suspension were plated onto selective media (see below) and plates were incubated aerobically overnight (16 - 24 hours) at 37°C, in order to quantify the exact concentration of viable organisms used for this experiment. All sites were secured with surgical tape and the entire animal wrapped with Coban self-adhesive elastic wrap (3M, St. Paul MN).

[0108] Immediately after wounding and infection, wounds were covered with polyurethane film dressing (Tegaderm; 3M, St. Paul, MN) for 24 hours to allow for biofilm formation. After 24 hours, six wounds from each group received treatments as described below.

[0109] The test substances included compositions comprising collagen sheet, gelatinized collagen solution, supernatant, their respective controls. Also, treatment with silver sulfadiazine is included as a positive control. The collagen sheets comprising the dECM (Treatments A and D) were carefully removed from their packaging and applied to their assigned wounds with sterile forceps and then covered with Tegaderm dressing. These dressings were only applied on Day 0 and Day 3 after biofilm formation.

[0110] Gelatinized collagen solution comprising 3% dECM and supernatant comprising sonicated axolotl tissues(Treatments B and C) along with their respective vehicle control comprising gelatinized solution (without dECM) or PBS (T reatments E and F) were applied daily.

[0111] After collecting the treatment from each vial, each wound received 200 pL of their formulation via pipette and then were covered with Tegaderm dressings.

[0112] Wounds that were treated with positive control silver sulfadiazine (SSD) received 200 mg of treatment and then were covered with Tegaderm dressings. These wounds were alsoAttorney Docket No.: R217-0014PCTtreated daily. Untreated Control wounds were just covered with polyurethane film dressings. The dressings were secured along the edges using surgical tape. All areas were covered and secured by wrapping the animal with self-adherent elastic bandages (Coban®, 3M, St. Paul, MN).

[0113] Recovery Method

[0114] On Day 0 (24 hours after inoculation), three wounds inoculated with MRSA USA300 and PA27312 were biopsied (6 mm) as a baseline. Then the remaining wounds were biopsied (3 from each group) on days 3 and 7 after treatment application.

[0115] The biopsies (6mm) were weighed and immediately placed in 1mL of All Purpose Neutralizing Solution. The sample was combined with an additional 4ml_ of Neutralizing Solution and homogenized in a sterile homogenization tube. Serial dilutions were made from all culture samples and the extent of microbiological contamination assessed using the Spiral Plater System (Spiral Biotech, Norwood, MA). This system deposits a 50mL aliquot of the scrub bacterial suspension over the surface of a rotating agar plate. Oxacillin Resistance Screening Agar (ORSAB) was used to isolate MRSA from the wounds and Pseudomonas Agar-base with CN supplement was used to isolate PA. All plates were incubated aerobically overnight (24 hours) at 37°C, after which the number of viable colonies were counted, and the CFU / g calculated. This method has been used for over 38 years to evaluate the antimicrobial efficacy of various topical agents and / or dressings.

[0116] Clinical Observations

[0117] Representative photos were taken of wounds during treatments (before and after) as well as during the assessment days. Wounds were recorded for any signs erythema (redness) and edema. On Day 0 after 24-hour biofilm formation and before treatment application, all wounds had erythema observed. All wounds exhibited no erythema for the duration of the experiment (FIG. 2).

[0118] Results

[0119] After counting the colonies, the data was tabulated and the Log of colony forming units / mL (Log CFU / g) determined. The mean of the Log CFU / g and standard deviation was calculateed for each time and treatment. The Tables below show the raw data for the bacterial count.The Antimicrobial Effect of Various Topical Formulations on Methicillin Resistant Staphylococcus aureus and Pseudomonas aeruginosa Using a Porcine Model InoculumStrain Dilution Count CFU / ml LogCFU / ml Methicillin ResistantStaphylococcus aureus (MRSA -4 47 9.39E+06 6.97USA300)Attorney Docket No.: R217-0014PCTInoculumStrain Dilution Count CFU / ml LogCFU / ml Pseudomonas aeruginosa -4 45 8.99E+06 6.95ATCC27312ORSAB Bacterial Count- Baseline and Day 3BASELINE ORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlBaseline 3 -3 146 584E+06 6.775 -3 150 600E+06 6.78 6 -4 39 1 56E+07 7.19 STDVMean 9 15E+06 6.91 0.24 Number of organisms per gT reatment Biopsy Colonies Factor (D) Biopsy(g) CFU / g Log(N) Purpose X CFU / g Baseline 3 146 2 1000 0.195 1.50E+06 6.185 150 2 1000 0.159 1.89E+06 6.28 6 39 2 10000 0.117 6.67E+06 6.82 STDVMean 3.35E+06 6.42 0.34DAY 3ORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlA - Collagensheet 3x3cm 3 -3 23 9 19E+05 5.965 -2 163 652E+05 5.81 6 -4 22 879E+06 6.94 STDVMean 345E+06 6.24 0.61 Number of organisms per gTreatment Biopsy Colonies Factor Biopsy(g) Log(N) Purpose CFU / g(D) X CFU / gA - Collagensheet 3x3cm 3 23 3 1000 0.184 3.75E+05 5.575 163 3 100 0.212 2.31E+05 5.36 6 22 3 10000 0.242 2.73E+06 6.44 STDVMean 1.11E+06 5.79 0.57 ORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlB - Gelatinizedcollagen 4 -2 58 232E+05 5.37solution5 -3 32 1 28E+06 6.11 6 -3 22 879E+05 5.94 STDVMean 797E+05 5.81 0.39 Number of organisms per gTreatment Biopsy Colonies Purpose Factor Biopsy(g) CFU / g Log_ (N) _ (D) X CFU / gAttorney Docket No.: R217-0014PCTB - Gelatinizedcollagen 4 58 3 100 0.157 1.11E+05 504 solution5 32 3 1000 0.182 5.27E+05 5726 22 3 1000 0205 322E+05 551 STDVMean 3.20E+05 542 0.35ORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlC - Supernatant 3 -2 86 344E+05 5.545 -3 30 1 20E+06 6.08 6 -3 26 1 04E+06 6.02 STDVMean 861E+05 5.88 0.30 Number of organisms per gTreatment Biopsy Number Purpose Dilution Weight CFU / g Logof CFU / gC - Supernatant 3 86 3 100 0.128 2.02E+05 5.35 30 3 1000 0 151 596E+05 5786 26 3 1000 0.193 4.04E+05 561 STDVMean 4.01E+05 556 0.24 ORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / ml D - Collagen sheet 3X3cm 4 -2 145 5.80E+05 5.765 -2 43 1.72E+05 5.246 -3 38 1.52E+06 6.18 STDVMean 7.57E+05 5.73 047 Number of organisms per gVolume ofTreatment Biopsy Number Loof all Purpose Dilution Weight CFU / g g Neutralizer CFU / gD - CollagenSheet 3X3 4 145 3 100 0.156 2.79E+05 545 cm5 43 3 100 0.125 1.03E+05 5016 38 3 1000 0.191 5.97E+05 578 STDVMean 3.26E+05 541 0.38 ORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlE - Vehicle control forGelatinized collagen 2 -3 32 1.28E+06 6.11solution3 -2 59 2.36E+05 5.376 -3 29 1.16E+06 6.06 STDVMean 8.92E+05 5.85 0.41Attorney Docket No.: R217-0014PCTNumber of organisms per gVolume ofTreatment Biopsy Numberof all Purpose Dilution Weight CFU / g Log Neutralizer CFU / gE - Vehiclecontrol forGelatinized 2 32 2 1000 0.154 4.16E+05 562 collagensolution3 59 2 100 0.152 7.76E+04 489 6 29 2 1000 0.158 3.67E+05 556 STDVMean 2.87E+05 536 0.41 ORSAB Bacterial count in wounds recovered Day 3Treatment Biopsy Dilution Count CFU / ml Log CFU / mlF - Vehicle controlSupernatant 4 -3 48 1.92E+06 7.095 -3 76 7.40E+06 6.876 -3 41 1.68E+07 7.23 STDVMean 8.71E+06 7.06 0.18 Number of organisms per gVolume ofTreatment Biopsy Number all Purpose Dilution Weight CFU / g Logof Neutralizer CFU / gF - Vehiclecontrol 4 48 2 1000 0.165 5.82E+05 576 Supernatant5 76 2 1000 0.194 7.84E+05 589 6 41 2 1000 0.213 3.85E+05 559 STDVMean 5.84E+05 575 0.15 ORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlG - Positive Control SilverSulfadiazine 2 -3 89 356E+06 6553 -3 73 2.92E+06 6.476 -3 51 2.04E+06 6.31 STDVMean 2.84E+06 6.44 0.12 Number of organisms per gVolume ofTreatment Biopsy Number all Purpose Dilution Weight CFU / g Logof Neutralizer CFU / gG - PositiveControlSilver 2 89 2 1000 0.141 1.26E+06 6 10 Sulfadiazine3 73 2 1000 0.168 8.69E+05 594 6 51 2 1000 0223 457E+05 566 STDVMean 8.62E+05 590 0.22Attorney Docket No.: R217-0014PCTORSAB Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml LogCFU / mlH Untreated Control 2 -3 102 4.08E+06 6.613 -3 30 1.20E+06 6.086 -3 67 2.68E+06 6.43 STDV Mean 2.65E+06 6.37 0.27Number of organisms per gVolume ofT reatment Biopsy Number Logof all Purpose Dilution Weight CFU / gNeutralizer CFU / gH- Untreated 2 102 3 1000 0.111 2.76E+06 644 Control3 30 3 1000 0.200 4.50E+05 5656 67 3 1000 0.198 1.02E+06 601 STDV Mean 1.41E+06 603 0.39PA Agar Bacterial Count - Baseline & Day 3BASELINE PA Agar Bacterial count in wounds recovered Day 3Log T reatment Biopsy Dilution Count CFU / ml CFU / mlBaseline 1 -4 94 3.76E+07 7.572 -4 90 3.60E+07 7.564 -4 87 3.48E+07 7.54 STDV Mean 3.61E+07 7.56 002Number of organisms per gColonies Biopsy(g) Log T reatment Biopsy Purpose Factor (D) (N) X CFU / g CFU / g Baseline 1 94 2 10000 0.195 9.64E+06 6.982 90 2 10000 0.159 1.13E+07 7.05 4 87 2 10000 0.117 1.49E+07 7.17 STDV Mean 1.19E+07 7.07 0.10DAY 3PA Agar Bacterial count in wounds recovered Day 3Log T reatment Biopsy Dilution Count CFU / ml CFU / mlA - Collagen 1 -4 26 1 04E+07 702 sheet 3x3cm2 -3 131 5.24E+06 6.724 -4 37 1.48E+07 7.17 STDV Mean 1.01E+07 6.97 0.23Number of organisms per gColonies Factor Biopsy(g) Log T reatment Biopsy Purpose CFU / g (N) X CFU / g (D)A - Collagen 1 26 2 10000 0.184 2.83E+06 6.45 sheet 3x3cm2 133 2 1000 0.212 1.25E+06 6.1Attorney Docket No.: R217-0014PCT| | 4 | 37 | 2 10000 0.242 3.06E+06 6.49Mean 2.38E+06 6.35 0.21 PA Agar Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlB - Gelatinizedcollagen solution 1 -3 53 2.12E+06 6332 -3 87 3.48E+06 6543 -3 41 1 64E+06 621 STDVMean 2.41E+06 636 0.17 Number of organisms per gT reatment Biopsy Colonies Purpose Factor Biopsy(g) CFU / g Log (N) (D) X CFU / g B - Gelatinizedcollagen 1 53 2 1000 0.157 6.75E+05 5.83 solution2 87 2 1000 0.182 9.56E+05 5.98 3 41 2 1000 0.205 4.00E+05 5.6Mean 6.77E+05 5.8 0.19 PA Agar Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml LogCFU / mlC - Supernatant 1 -3 25 9.99E+05 62 -2 194 7.76E+05 5.894 -3 86 3.44E+06 6.54 STDVMean 1.74E+06 6.14 0.35 Number of organisms per gT reatment Biopsy Number Purpose Dilution Weight CFU / g Log of CFU / g C - Supernatant t 1 25 2 1000 0.128 3.91E+05 5.592 194 2 100 0.151 2.57E+05 5.41 4 86 2 1000 0.193 8.91E+05 5.95Mean 5.13E+05 5.65 0.27 PA Agar Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml LogCFU / mlD -Collagen sheet3X3cm 1 -4 92 368E+07 7572 -4 37 1.48E+07 7.173 -4 71 2.84E+07 7.45 STDVMean 2.67E+07 7.4 0.21 Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutr CFU / g (N) alizer (D) X_ IY) _Attorney Docket No.: R217-0014PCTD -Collagensheet 3X3cm 1 92 2 10000 0.156 1.18E+07 7.072 37 2 10000 0.125 5.92E+06 6.77 3 71 2 10000 0.191 7.43E+06 6.87 STDVMean 8.38E+06 6.91 0.15 PA Agar Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml LogCFU / mlE - Vehicle controlfor Gelatinized 1 -3 196 784E+06 628collagen solution4 -4 27 1.08E+07 6.485 -4 39 1.56E+07 6.21 STDVMean 1.14E+07 632 0 14 Number of organisms per gT reatment Biopsy Number Purpose Dilution Weight CFU / g Log of CFU / g E - Vehiclecontrol forGelatinized 1 196 2 1000 0 154 255E+06 641 collagensolution4 27 2 10000 0 152 355E+06 655 5 39 2 10000 0.158 4.94E+06 6.69 STDVMean 3.68E+06 6.55 0.14 PA Agar Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml LogCFU / mlF - Vehicle controlfor Supernatant 1 -4 31 1.24E+07 7.092 -3 185 7.40E+06 6.873 -4 42 1.68E+07 7.23 STDVMean 1.22E+07 7.06 0.18 Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutralizer CFU / (N) (D) X g (V)F - Vehiclecontrol for 1 31 2 10000 0.165 3.76E+06 6.57 Supernatant2 185 2 1000 0.194 1.91E+06 6.28 3 42 2 10000 0.213 3.94E+06 6.6 STDVMean 3.20E+06 6.48 0.18 PA Agar Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml LogCFU / mlG - Positive Control 1 -2 91 3.64E+05 5.56Silver SulfadiazineAttorney Docket No.: R217-0014PCT4 -3 47 1.88E+06 6.275 -2 110 440E+05 564 STDVMean 8.95E+05 5.82 0.39Number Volume ofreatment Biopsy of ALL Dilution WeightT purpose Factor Biopsy(g) CFU / g Log Colonies Neutralizer CFU / (N) (D) X g (V)G - PositiveControl Silver 1 91 2 100 0.141 1.29E+05 5.11 Sulfadiazine4 47 2 1000 0.168 5.60E+05 5.75 5 110 2 100 0.223 9.87E+04 4.99 STDVMean 2.63E+05 5.28 0.41 PA Agar Bacterial count in wounds recovered Day 3T reatment Biopsy Dilution Count CFU / ml Log CFU / mlH - UntreatedControl 1 -4 67 268E+07 7434 -4 66 3.28E+07 7.525 -4 73 2.92E+07 7.47 STDVMean 2.96E+07 7.47 0.05 Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutra CFU / g (N) lizer (D) X(V)H - UntreatedControl 1 67 2 10000 0.111 1.21E+07 7.084 66 2 10000 0.2 6.60E+06 6.82 5 73 2 10000 0.198 7.37E+06 6.87 STDVMean 8.69E+06 6.92 0.14ORSAB Bacterial Count Day 7ORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml Log CFU / mlA - Collagen sheet3x3 cm 1 -2 27 1.08E+05 5.032 -2 47 1.88E+05 5.273 -3 23 9.19E+05 5.96 STDVMean 4.05E+05 5.42 0.48 Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Log Colonies purpose Factor Biopsy(g) CFU / gNeutra CFU / g (N) lizer (D) X(V)A - Collagensheet 3x3cm 1 27 2 100 0.172 3.14E+04 4.52 47 2 100 0.174 5.40E+04 4.73Attorney Docket No.: R217-0014PCT| | 3 | 23 | 2 | 1000 0.217 2.12E+05 5.33 STDVMean 9.91E+04 4.85 0.43 ORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml Log CFU / mlB - Gelatinizedcollagen solution 1 -2 50 2.00E+05 5.32 -2 45 1.80E+05 5.263 -2 83 3.32E+05 5.52 STDVMean 2.37E+05 5.36 0.14 Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of purpose Factor Biopsy(g) CFU / g Log Colonies Neutr CFU / g (N) alizer (D) X(V)B - Gelatinizedcollagen 1 50 2 100 0.151 6.62E+04 4.82 solution2 45 2 100 0.205 4.39E+04 4.64 3 83 2 100 0.216 7.69E+04 4.89 STDVMean 6.23E+04 4.78 0.13 ORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml Log CFU / mlC - Supernatant 1 -2 89 3.56E+05 5.552 -2 94 376E+05 5573 -2 180 7.20E+05 5.86 STDVMean 4.84E+05 5.66 0.17 Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutr CFU / g (N) alizer (D) X(V)C - Supernatant 1 89 2 100 0.181 9.83E+04 4.992 94 2 100 0.165 1.14E+05 5.06 3 180 2 100 0.21 1.71E+05 5.23 STDVMean 1.28E+05 5.09 0.13 ORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml Log CFU / mlD -Collagen sheet3X3cm 1 -2 132 5.28E+05 5.722 -2 195 7.80E+05 5.893 -3 23 9.19E+05 5.96 STDVMean 7.42E+05 5.86 0.12 Number of organisms per gAttorney Docket No.: R217-0014PCTNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutra CFU / g (N) lizer (D) X(V)D -Collagensheet 3X3cm 1 132 2 100 0 146 1 81E+05 5262 195 2 100 0.138 2.83E+05 5.45 3 23 2 1000 0.148 3.11E+05 5.49 STDVMean 2.58E+05 5.4 0.13 ORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml Log CFU / mlE - Vehicle controlfor Gelatinized 1 -2 82 3.28E+05 5.52collagen solution2 -2 74 2.96E+05 5.473 -2 55 2.20E+05 5.34 STDVMean 281E+05 544 009 Number of organisms per gNumber Volume ofof ALL Dilution WeightT reatment Biopsy Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutra CFU / g (N) lizer (D) X(V)E - Vehiclecontrol forGelatinized 1 82 2 100 0.175 9.37E+04 4.97 collagensolution2 74 2 100 0.223 6.64E+04 4.82 3 55 2 100 0.23 4.78E+04 4.68 STDVMean 6.93E+04 4.82 0.15 ORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml Log CFU / mlF - Vehicle controlfor Supernatant 1 -2 195 780E+05 5892 -3 33 1.32E+06 6.123 -3 38 1.52E+06 6.18 STDVMean 1.21E+06 6.06 0.15 Number of organism per gNumber Volume ofof ALL Dilution WeightT reatment Biopsy Colonies purpose Factor Biopsy(g) CFU / g Log CFU / g (N) Neutralizer (D) X(V)F - Vehiclecontrol for 1 195 2 100 0.193 2.02E+05 5.31 Supernatant2 33 2 1000 0.208 3.17E+05 5.5 3 38 2 1000 0227 335E+05 552 STDVMean 2.85E+05 5.44 0.12Attorney Docket No.: R217-0014PCTORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml Log CFU / mlG - Positive ControlSilver Sulfadiazine 1 -3 24 9.59E+05 5.982 -3 30 1.20E+06 6.083 -3 21 8.39E+05 5.92 STDVMean 9.99E+05 5.99 0.08 Number of organism per gNumber Volume ofALL Dilution WeightT reatment Biopsy of purpose Factor Biopsy(g) CFU / g Log Colonies Neutralizer CFU / g (N) (D) X(V)G - PositiveControl Silver 1 24 2 1000 0.167 2.87E+05 5.46 Sulfadiazine2 30 2 1000 0 174 345E+05 554 3 21 2 1000 0.231 1.82E+05 5.26 STDVMean 2.71E+05 5.42 0.14 ORSAB Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlH - UntreatedControl 1 -3 90 3.60E+06 6.562 -3 70 2.80E+06 6.453 -3 34 1 36E+06 6 13 STDVMean 2.59E+06 6.38 0.22 Number of organism per gNumber Volume ofof ALL Dilution WeightT reatment Biopsy Colonies purpose Factor Biopsy(g) CFU / g Log CFU / g (N) Neutralizer (D) X(V)H - UntreatedControl 1 90 2 1000 0.204 882E+05 5.952 70 2 1000 0.159 881E+05 5.94 3 34 2 1000 0.184 370E+05 5.57 STDVMean 7 11E+05 5.82 0.22PA Bacterial Count Day 7PA Agar Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlA - Collagen sheet3x3cm 1 -3 88 3.52E+06 6.552 -2 101 4.04E+05 5.613 -3 49 1.96E+06 6.29 STDVMean 1.96E+06 6.15 0.49 Number of organisms per gAttorney Docket No.: R217-0014PCTNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutralizer CFU / (N) (D) X g (V)A - Collagensheet 3x3cm 1 88 2 1000 0 172 1.02E+06 6012 101 2 100 0.174 1 16E+05 5.06 3 49 2 1000 0.217 452E+05 5.65 STDVMean 529E+05 5.58 0.48 PA Agar Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlB - Gelatinizedcollagen solution 1 -2 164 6.56E+05 5.822 -3 30 1.20E+06 6.083 -3 42 1.68E+06 6.23 STDVMean 1 18E+06 604 021 Number of organisms per gNumber Volume ofof ALL Dilution WeightT reatment Biopsy Colonies purpose Factor Biopsy(g) CFU / g Log Neutralizer CFU / (N) (D) X g (V)B - Gelatinizedcollagen 1 164 2 100 0.151 2.17E+05 5.34 solution2 30 2 1000 0.205 2.93E+05 5.47 3 42 2 1000 0.216 3.89E+05 5.59 STDVMean 3.00E+05 5.46 0.13 PA Agar Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlC - Supernatant 1 -2 26 1.04E+06 6.022 -2 40 1 60E+06 6.23 -3 20 8.00E+05 59 STDVMean 1.15E+06 6.04 0.15 Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutra CFU / g (N) lizer (D) X(V)C - Supernatant 1 26 2 100 0.181 2.87E+04 4.462 40 2 100 0.165 4.85E+04 4.69 3 20 2 1000 0.21 1.90E+05 5.28 STDVMean 8.91E+04 4.81 0.42 PA Agar Bacterial count in wounds recovered Day 7Attorney Docket No.: R217-0014PCTT reatment Biopsy Dilution Count CFU / ml LogCFU / mlD - Collagen sheet3X3cm 1 -4 30 1.20E+07 7.082 -3 196 7.84E+06 6.893 -4 21 8.39E+06 6.92 STDVMean 9.41E+06 6.96 0.10Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutr CFU / g (N) alizer (D) X(V)D -Collagensheet 3X3cm 1 30 2 10000 0.146 4.11E+06 6.612 196 2 1000 0.138 2.84E+06 6.45 3 21 2 10000 0.148 2.84E+06 6.45 STDVMean 326E+06 6.51 009PA Agar Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlE - Vehicle controlfor Gelatinized 1 -4 21 8.39E+06 6.92collagen solution2 -3 177 7.08E+06 6.853 -3 192 7.68E+06 6.89 STDVMean 7.72E+06 6.89 0.04Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutralizer CFU / g (N) (D) X(V)E - Vehiclecontrol forGelatinized 1 21 2 10000 0.175 2.40E+06 6.38 collagensolution2 177 2 1000 0.223 1.59E+06 6.2 3 192 2 1000 0.23 1.67E+06 6.22 STDVMean 1.89E+06 6.27 0.10 PA Agar Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlF - Vehicle controlfor Supernatant 1 -3 197 7.88E+06 692 -4 20 8.00E+06 693 -3 179 7 16E+06 685 STDVMean 7.68E+06 6.88 0.03Attorney Docket No.: R217-0014PCTNumber of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of purpose Factor Biopsy(g) CFU / g Log Colonies Neutra CFU / g (N) lizer (D) X(V)F - Vehiclecontrol for 1 197 2 1000 0.193 2.04E+06 6.31 Supernatant2 20 2 10000 0.208 1.92E+06 6.28 3 179 2 1000 0.227 1.58E+06 6.2 STDVMean 1.85E+06 6.26 0.06 PA Agar Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlG - Positive ControlSilver Sulfadiazine 1 -1 170 6.80E+04 4.832 -1 99 3.96E+04 4.63 -1 102 4.08E+04 4.61 STDVMean 4.95E+04 4.68 0.13Number of organisms per gNumber Volume ofof ALL Dilution WeightT reatment Biopsy purpose Factor Biopsy(g) CFU / g Log Colonies Neutra CFU / g (N) lizer (D) X(V)G - PositiveControl Silver 1 170 2 10 0 167 204E+04 431 Sulfadiazine2 99 2 10 0.174 1.14E+04 4.06 3 102 2 10 0.231 8.83E+03 3.95 STDVMean 1.35E+04 4.1 0.19 PA Agar Bacterial count in wounds recovered Day 7T reatment Biopsy Dilution Count CFU / ml LogCFU / mlH - UntreatedControl 1 -4 42 1.68E+07 7.232 -4 67 2.68E+07 7.433 -4 54 2 16E+07 733 STDVMean 2.17E+07 7.33 0.10Number of organisms per gNumber Volume ofALL Dilution WeightT reatment Biopsy of Lo Colonies purpose Factor Biopsy(g) CFU / g g Neutra CFU / g (N) lizer (D) X(V)H - UntreatedControl 1 42 2 10000 0.204 412E+06 6.612 67 2 10000 0.159 843E+06 6.933 54 2 10000 0.184 587E+06 6.77 STDVMean 614E+06 6.77 016Attorney Docket No.: R217-0014PCT

[0120] Comparing the baseline counts, PA had a higher bacterial count with 7.07±10 Log CFU / g, while MRSA had a baseline count of 6.42±0.35 Log CFU / g (FIG. 3). This indicates that at the baseline, PA 27312 was present in higher numbers than MRSA USA300. After treatment application on Day 3, all treatments resulted in a reduction in bacterial counts for both MRSA and PA compared to their respective baselines. The untreated control group (H) showed a slight decrease in MRSA by 0.39±0.05 Log CFU / g and in PA to 0.15±0.04 Log CFU / g, indicating some natural reduction in the bacterial population over time without intervention, which is expected of the mammalian immune response system but the reduction is much lower as compared to the treatments with the test substances. The positive control, silver sulfadiazine (G), was the most effective treatment for PA, reducing PA counts to 5.28±0.41 Log CFU / g, which was the lowest count observed among all treatment groups for PA on Day 3, indicating that the experiments worked. Treatments with collagen sheet (D), gelatinized collagen solution (B) and its Vehicle Control (E) had the lowest bacterial loads for MRSA USA300 on Day 3 with 5.41 ±0.39 Log CFU / g, 5.42±0.35 Log CFU / g and 5.36±0.41, respectively, as compared to the positive control, silver sulfadiazine (G) with MRSA counts of 5.90±0.22 Log CFU / g.

[0121] The efficacy of the treatments varied between the two bacterial species. For MRSA, treatments with collagen sheets (A) and (D), gelatinized collagen solution (B), and supernatant (C) resulted in bacterial counts of 5.79±0.57, 5.41 ±0.38, 5.42±0.35, and 5.56±0.24 Log CFU / g, respectively. These treatments showed a bacterial reduction compared to baseline of 76.8%, 90.2%, 90.0% and 86.3%, respectively. The vehicle controls for gelatinized collagen solution and supernatant (E and F) showed similar or slightly higher counts compared to each respective treatment (5.36±0.41 and 5.75±0.15 Log CFU / g, respectively). This suggests that the vehicle itself might have some effect on MRSA, or that the treatments' efficacy is comparable to the controls. Both treatments, gelatinized collagen solution (B) and vehicle control for gelatinized collagen solution (E), showed a higher percentage of MRSA USA300 reduction compared to baseline wounds (90.0% and 91.43%, respectively) and compared to untreated control wounds (75.4 and 78.9%, respectively).

[0122] For PA, treatments with the collagen sheet (A) and (D), gelatinized collagen solution (B), and supernatant (C) resulted in bacterial counts of 6.35±0.21 , 6.91±0.15, 5.80±0.19, and 5.65±0.27 Log CFU / g, respectively. Treatment with collagen sheets (A) and (D) had the least effect, with a slight decrease from the baseline counts (7.07±.10 Log CFU / g) at Day 3 with just one application of the sheet. In contrast, treatments with gelatinized collagen solution (B) and supernatant (C) showed a higher reduction in PA counts (1.27±0.10 and 1.42±0.18 Log CFU / g, respectively). These values represent 94.6 and 96.2% of bacteria reduction compared to baseline wounds. Treatments with vehicle controls (E, and F) for gelatinized collagen solution (B), and supernatant (C), respectively, showed an increase in the bacterial count (0.75 and 0.83 LogAttorney Docket No.: R217-0014PCTCFU / g, respectively). Overall, treatments with the positive control (G) and supernatant (C) resulted in the lowest bacterial counts for PA, demonstrating the most significant efficacy in reducing this bacterial species on Day 3.

[0123] On Day 7, the results showed similar efficacy among the different treatments for both MRSA USA300 and PA, as shown in (FIG. 4). The untreated control group (H) showed an expected reduction in MRSA count (5.82±0.22) and an expected decrease in PA count (6.77±0.16). The positive control, Silver Sulfadiazine (G), resulted in the lowest bacterial counts for PA27312 (4.10±0.19) Log CFU / g, demonstrating the greatest overall efficacy over this Gram (-) bacteria, but the amount of MRSA was also decreased (5.42±0.14). (FIG. 4), indicating that the experiments worked.

[0124] The efficacy of the treatments for MRSA USA300 varied significantly. Treatments with collagen sheet (A) and (D) and gelatinized collagen solution (B) resulted in counts of 4.85±0.43 Log CFU / g, 5.40±0.13 Log CFU / g and 4.78±0.13 Log CFU / g, respectively, and exhibited more than 97.8% of reduction in bacterial load compared to baseline. The vehicle control (F) for active treatments had higher bacterial counts than their respective treatments. Treatments with gelatinized collagen solution (B) and its vehicle control (E) were similar in effectiveness, while treatments with supernatant (C) were more effective than their vehicle control (F).

[0125] For PA, treatments with the collagen sheet (A) and (D), gelatinized collagen solution (B), and supernatant (C) resulted in bacterial counts of 5.58±0.48, 6.51±0.09, 5.46±0.13, and 4.81 ±0.42 Log CFU / g, respectively. The gelatinized collagen solution and supernatant treatments showed a reduction in the bacterial population compared to their respective vehicle controls (E and F) with counts of 6.27±0.10 and 6.26±0.06 Log CFU / g, respectively. The higher reductions of PA were observed with the collagen sheet gelatinized collagen solution B and Supernatant C as compared to their respective vehicle controls E and F, highlighting their specific efficacy against this particular bacterial species.

[0126] Further differences can be observed when directly comparing the effects of the treatments across the two bacteria. Although treatments with collagen sheet A reduced the bacterial counts of both MRSA and PA, the collagen sheet (A) treatment was more effective at reducing MRSA (4.85±0.43 Log CFU / g) than PA27312 (5.58±0.48 Log CFU / g). Similarly, treatments with supernatant (C) reduced the bacterial counts of both MRSA and PA, while having greater efficacy against PA (4.81±0.42 Log CFU / g) than against MRSA (5.09±0.13 Log CFU / g). Same results were observed with the treatments of gelatinized collagen solution (B), as they also reduced the bacterial counts of both MRSA and PA, while having greater efficacy in reducing MRSA (4.78±0.13 Log CFU / g) than PA (5.46±0.13 Log CFU / g). These findings suggest a treatment-specific effectiveness, with some treatments showing a more significant impact on one bacterial species over the other. The positive control (G) also demonstrated a more substantialAttorney Docket No.: R217-0014PCTreduction for PA (4.10±0.19 Log CFU / g) than for MRSA (5.42±0.14 Log CFU / g) confirming some treatments have more significant impact on one bacterial species over the other.

[0127] Bacterial counts for Methicillin-Resistant Staphylococcus aureus (MRSA USA300) showed a continued reduction from Day 3 to Day 7 across most treatment and control groups (FIG. 5). Treatment with collagen sheet (A) demonstrated a reduction in bacterial count from Day 3 to Day 7, a decrease of 0.94±0.14 Log CFU / g. Treatment with gelatinized collagen solution (B) also showed increased efficacy from Day 3 to Day 7, with counts falling by 0.64±0.22 Log CFU / g. Similarly, treatment with supernatant (C) showed reduced bacterial load from Day 3 to Day 7, with a reduction of 0.47±0.11 Log CFU / g. This trend of continued decrease in bacterial numbers from Day 3 to day 7 suggests that the treatments have a cumulative effect on the bacterial population overtime.

[0128] A closer look at the controls also reveals interesting trends between Day 3 and Day 7. Treatments with vehicle control for Gelatinized Collagen (E) and supernatant (F) both showed a reduction in bacterial counts from Day 3 to Day 7. The positive control, Silver Sulfadiazine (G), remained highly effective, showing a further decrease of 0.48±0.08 Log CFU / g. The untreated control also showed a reduction, but is less than the positive control. These results indicate a general trend of decreasing MRSA bacterial load over the 7-day period, regardless of the treatment or control group.

[0129] Consistent reduction in bacterial counts was observed across all groups from Day 3 to Day 7 when wounds were assessed for Pseudomonas aeruginosa ATCC 27312 (PA27312) as shown in FIG. 6. The treatments with the most significant impact on reducing the bacterial load were wounds treated with positive control silver sulfadiazine (G), which showed a substantial decrease of 1 ,18±0.22 Log CFU / g (93.4% of reduction), and with supernatant (C), which reduced the count by 0.84±0.15 Log CFU / g (85.6% of reduction). Treatment with the gelatinized collagen sheet (A) also contributed to a reduction in bacterial numbers, decreasing by 0.77±0.26 Log CFU / g (83.0% of reduction). These outcomes highlight the sustained effectiveness of the treatments in actively reducing the PA27312 population over the course of the seven-day period.

[0130] Further examination of the control groups also reveals a trend of reduced bacterial counts as the study progressed. The vehicle controls for gelatinized collagen (E), and supernatant (F) exhibited some degree of reduction, with decreases of 0.28±0.05, and 0.22±0.12 Log CFU / g, respectively. The untreated control (H) also saw a reduction of 0.15±0.02 Log CFU / g, but is less than the positive control. This reduction in bacterial counts across all tested conditions supports the observation that the overall mixed environment and time itself played a role in the diminishing PA27312 population.

[0131] Conclusion

[0132] Based on the results, this study demonstrated that the competitive dynamics between Methicillin-Resistant Staphylococcus aureus (MRSA USA300) and Pseudomonas aeruginosaAttorney Docket No.: R217-0014PCT(PA) were influenced by the applied treatments. Results showed that the test substances have efficacy in reducing the bacterial load of both organisms over a seven-day period. The positive control, Silver Sulfadiazine (G), consistently proved to be the most effective treatment for PA confirming that the experiments worked. Treatments with collagen sheet (A) and gelatinized collagen solution (B) were the most effective at reducing bacterial load of MRSA USA300, while the vehicle control for gelatinized collagen solution (E) provided similar efficacy as gelatinized collagen solution (B). The study also found that treatment efficacy often increased with extended application, as a further reduction in bacterial counts was observed between Day 3 and Day 7 for the test substances, highlighting a cumulative effect overtime.

[0133] The results revealed treatment-specific effectiveness, with certain treatments having a greater impact on one bacterial species than the other, which may have altered the competitive balance within the mixed infection. For example, treatments with collagen sheet (A) and supernatant (C) were more effective at reducing the PA population, potentially allowing MRSA to persist more readily. Conversely, the gelatinized collagen solution (B) showed superior efficacy against MRSA, which could, in turn, provide PA with a competitive advantage. These findings underscore that the choice of treatment for mixed infections should be tailored to the specific bacterial species present. Furthermore, the study highlighted the significant role of vehicle controls, which often contributed to the reduction in bacterial load, particularly for PA. This suggests that the delivery method itself may possess antibacterial properties that also impact on the competitive dynamics between the two organisms.

[0134] Overall, the best reduction against PA was the treatment with supernatant on day 3. On this day the activity of the supernatant was comparable to that of silver sulfadiazine (SSD). By day 7 all treatments were better than vehicle and untreated controls. The SSD was the best overall treatment against PA on day 7. The active treatments only demonstrated a slight reduction in MRSA counts as compared to their vehicle and untreated controls. Interestingly, on day 7 all treatments were better at reducing MRSA than SSD control. It appears that the vehicle controls also had an influence on the bacterial loads.

[0135] Applying the Collagen Sheet daily can produce a more pronounced antimicrobial effect.

[0136] All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. While the foregoing has been described in terms of various embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof.Attorney Docket No.: R217-0014PCTREFERENCESDavis SC, Cazzaniga AL, Eaglstein WH, and Mertz PM. Over-The-Counter Antimicrobial Bandages and Proliferation of a Common Wound Pathogen. Arch Dermatol Res. 2005 Nov; 297(5): 190-5.Davis SC and Bouzari N. Development of Antimicrobials for Wound Care: In-Vitro and In-Vivo Assessments. Wounds 2004; 16(11):344-347.Mertz, PM., Davis, SC, Cazzaniga, A., Drosou, A., Eaglstein, W. Barrier and Antibacterial Properties of 2-Octyl Cyanoacrylate Derived Wound Treatment Films. J. Cutaneous Medicine and Surgery 2003; 7:1-12.Martineau L and Davis SC. Controlling Methicillin Resistant Staphyloccocus aureus and Pseudomonas aeruginosa wound infections with a novel biomaterial. J Invest Surgery 2007; 20:217-227.Pastar I, Nusbaum AG, Gil J, Patel SB, Chen J, Valdes J, Stojadinovic O, Plano LR, Tomic-Canic M, Davis SC. Interactions of Methicillin-Resistant Staphylococcus aureus USA300 and Pseduomonas aeruginosa in Polymicrobial Wound Infection. PLOS One, February 2013.Cazzaniga, A., Seralta, V., Davis SC, Orr R, Eaglstein, W., Mertz, P. The Effect of an Antimicrobial Gauze Dressing Impregnated with 0.2-Percent Polyhexamethylene Biguanide as a Barrier to Prevent Pseudomonas aeruginosa Wound Invasion. Wounds 2002; 14(5):169-176.Oliveria MF, Davis SC and Mertz PM: Can occlusive dressing composition influence proliferation of bacterial wound pathogens? Wounds. 1998; 10(1):4-11.Mertz PM, Oliverira-Gandia MF and Davis SC: The evaluation of a cadexomer iodine wound dressing on methicllin resistant staphylococcus aureus (MRSA) in acute wounds. Derm Surg 1999; 25:89-93.Davis SC and Mertz PM: Treatment of Wounds with an Oak Bark Formulation: Antimicrobial and Wound Healing Assessments. Ostomy Wound Management. 2008 Oct; 54(10): 16-25.

Claims

Attorney Docket No.: R217-0014PCTCLAIMS1. A method of inhibiting growth of and / or killing one or more microorganisms, wherein the method comprises administering a composition comprising a biological sample of an amphibian to a substrate in need thereof.

2. The method of claim 1, wherein the composition further comprises one or more other cosmetically or pharmaceutically acceptable carriers.

3. The method of claim 1 or 2, wherein the amphibian is a baby amphibian, and optionally, wherein the baby amphibian is one day old (newborn) to 12-month old.

4. The method of claim 1 , wherein the amphibian is a froglet, tadpole, Urodele, or larval stage young Apoda.

5. The method of claim 1, wherein the amphibian is a baby Urodele, and optionally the amphibian is a baby axolotl.

6. The method of claim 1, wherein the biological sample comprises organ, cell, blastema, biological fluid, or a combination thereof.

7. The method of claim 1 , wherein the biological sample comprises tissue, vitro cells, in vivo cells, blood, urine, secretion, or a combination thereof; and optionally, wherein the secretion comprises cutaneous surface liquid.

8. The method of claim 1, wherein the composition comprises decellularized extracellular matrix (dECM) or gelatinized dECM.

9. The method of claim 1, wherein the composition comprises supernatant of one or more sonicated amphibian tissues.

10. The method of claim 1, wherein the composition further comprises one or more agents that are xenogenic to the biological sample.

11. The method of claim 1, wherein the composition further comprises one or more mammalian peptides, mammalian proteins, drugs, nutrients, retinoids, emollients, steroids, carbohydrates, glycoproteins, polymers, or a combination thereof.

12. The method of claim 1 , wherein the substrate comprises the surface of an inanimate object or a living organism.

13. The method of claim 1, wherein the substrate comprises the skin of an living organism, and optionally the living organism is a human; or wherein the substrate comprises the surface of a medical device.

14. The method of claim 1 , wherein the one or more microorganisms comprise gram-positive bacteria, gram-negative bacteria, viruses, yeasts, fungi, or a combination thereof.

15. The method of claim 1 , wherein the one or more microorganisms comprise gram-positive bacteria comprising Staphylococcus aureus, Enterococcus, or a combination thereof, and optionally,Attorney Docket No.: R217-0014PCTwherein the Staphylococcus aureus comprises methicillin-resistant Staphylococcus aureus (MRSA) and / or methicillin-sensitive Staphylococcus aureus (MSSA); and / orwherein the Enterococcus comprises Enterococcus faecalis.

16. The method of claim 1 , wherein the one or more microorganisms comprise gram-negative bacteria comprising Klebsiella, Escherichia coli, Pseudomonas, Pantoea, or acombination thereof, and optionally,wherein the Klebsiella comprises Klebsiella pneumoniae;wherein the Pseudomonas comprises Pseudomonas aeruginosa; and / orwherein the Pantoea comprises Pantoea agglomerans.

17. The method of claim 1, wherein the one or more microorganisms comprise viruses comprising herpes simplex virus, varicella-zoster virus, measles morbillivirus, rubella virus, monkeypox virus, small pox virus, parvovirus, ebola virus, coxsackievirus or a combination thereof.

18. The method of claim 1 , wherein the one or more microorganisms comprises yeasts and / or fungi comprising Candida, Trichophyton, Microsporum, Epidermophyton, or a combination thereof.

19. The method of claim 1, wherein the method further treats and / or prevents a disease or condition.

20. The method of claim 1 , wherein the method further treats and / or prevents a skin condition.

21. The method of claim 1, wherein the method further treats a wound, infected wound, or prevents an infection of a wound.

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