Antimicrobial composition and method

Abstract

Provided are use of an antimicrobial peptide in inhibiting bacteria, and a composition comprising the peptide.

Description

Compositions and methods for antibacterial use

[0001] The present invention claims priority to Chinese patent application CN202410206761.6 filed on February 26, 2024, and the entire contents of the aforementioned patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of antibacterial technology, and more particularly to the use of antimicrobial peptides in inhibiting bacteria, and compositions and methods thereof. Background Art

[0003] Antimicrobial agents generally refer to drugs with bactericidal or bacteriostatic activity. The characteristic of antimicrobial drugs is to kill or inhibit foreign pathogens that invade the body and exert pharmacological effects. Their efficacy depends on the interaction between antimicrobial drugs, pathogens and the body.

[0004] Pathogenic bacteria are important factors in causing nosocomial and community-acquired infections, and can cause a variety of diseases or symptoms, including infections or conditions related to epithelial tissue, connective tissue, muscle tissue, and nervous tissue, or infections or conditions related to the musculoskeletal system, circulatory system (cardiocirculatory system, lymphatic system), respiratory system, digestive system, nervous system, endocrine system, urinary system, and reproductive system, such as skin and soft tissue infections, gastroenteritis, diarrhea-type diseases, abdominal pain-type diseases, vomiting-type diseases, mucus, pus, and blood in the stool, chills, high fever, rash, arthralgia, headache, myalgia, osteomyelitis, osteoarthritis, endocarditis, pneumonia, bacteremia, sepsis, tuberculosis, typhoid, convulsions, brain abscess, hydrocephalus, developmental delay, impaired consciousness, imbalance, and convulsions. Existing drugs for treating pathogenic bacteria infections mainly include β-lactams, macrolides, tetracyclines, and quinolones.

[0005] The discovery and application of antimicrobial drugs have made significant contributions to human and animal health. However, with the frequent and extensive use of antimicrobial drugs worldwide, pathogen resistance is becoming increasingly serious, and the emergence of "superbugs" has become a global problem, constantly threatening human and animal health.

[0006] Therefore, new mechanisms of action and novel structural types of antimicrobial drugs are urgently needed. Antimicrobial peptides have unique antibacterial mechanisms, are less likely to develop drug resistance, and are non-toxic to normal cells, making them a promising new generation of effective antimicrobial drugs. Their application prospects and value have attracted widespread attention. Summary of the Invention

[0007] In order to improve at least one of the above problems, in one aspect, the present disclosure provides an antimicrobial peptide comprising the amino acid sequence YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 1), or use of a functionally equivalent variant thereof in inhibiting bacteria.

[0008] In some embodiments, the amino acid sequence of the antimicrobial peptide disclosed herein is YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 1).

[0009] In some embodiments, the functionally equivalent variants of the antimicrobial peptides described herein have 1, 2, or 3 conservative amino acid substitutions, additions, or deletions relative to the antimicrobial peptides, such as hydrophobic amino acid substitutions or additions.

[0010] In some embodiments, the hydrophobic amino acids that can be selected in the present disclosure include tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline, and methionine (methionine), among others.

[0011] In some embodiments, the functionally equivalent variants of the antimicrobial peptides described herein have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the antimicrobial peptides.

[0012] In some embodiments, the bacteria described herein belong to the order Actinomycetales.

[0013] In some embodiments, the bacteria described in the present disclosure are one or more of Actinomycetaceae, Mycobacteriaceae, Bifidobacteriaceae, Dermatophilaceae, Frankiaceae, Rhodococcusaceae, Nephrobacteriaceae, Nocardiaceae, Streptomycetaceae, Thermoactinomycetaceae, and Enterobacteriaceae. In some specific embodiments, the bacteria described in the present disclosure are Mycobacteriaceae and / or Enterobacteriaceae.

[0014] In some embodiments, the bacteria described in the present disclosure are one or more of the genus Photorhabdus, Klebsiella, Proteobacterium, Hafnia, Escherichia, Shigella, Citrobacter, Erwinia, Salmonella, Serratia, Edwardsiella, Yersinia, Enterobacter, Xenorhabdus, and Mycobacterium. In some specific embodiments, the bacteria described in the present disclosure are Salmonella and / or Mycobacterium.

[0015] In some embodiments, the bacteria described in the present disclosure are one or more of Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium avium-intracellulare, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium ulcerans, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella dublin, Salmonella gallinarum, and Salmonella pullorum. In some specific embodiments, the bacteria described in the present disclosure are one or more of Mycobacterium smegmatis, Mycobacterium tuberculosis, or Salmonella typhimurium.

[0016] In some embodiments, the bacteria disclosed herein are one or more bacteria selected from the group consisting of Mycobacterium tuberculosis complex, Mycobacterium leprae, Mycobacterium avium complex, Mycobacterium gordonii subgroup, Mycobacterium kansasii subgroup, Mycobacterium terrestris subgroup, Mycobacterium simian subgroup, Mycobacterium chelonae subgroup, Mycobacterium fortuitum subgroup, Mycobacterium fortuitum group, and ungrouped mycobacteria.

[0017] In some aspects, the antimicrobial peptides disclosed herein or functionally equivalent variants thereof are used to prepare medicaments for preventing and / or treating diseases or conditions associated with bacterial invasion.

[0018] In some embodiments, the diseases or conditions associated with bacterial invasion described in the present disclosure are selected from infectious diseases or conditions related to epithelial tissue, connective tissue, muscle tissue and nervous tissue, or infectious diseases or conditions related to the musculoskeletal system, circulatory system, respiratory system, digestive system, nervous system, endocrine system, urinary system and reproductive system, such as skin and soft tissue infections, gastroenteritis, diarrhea-type conditions, abdominal pain-type conditions, vomiting-type conditions, mucus, pus and blood in the stool, chills, high fever, rash, joint pain, headache, myalgia, osteomyelitis, osteoarthritis, endocarditis, pneumonia, bacteremia, sepsis, tuberculosis, typhoid fever, convulsions, brain abscess, hydrocephalus and developmental delay, impaired consciousness, imbalance and convulsions.

[0019] In other aspects of the present disclosure, a pharmaceutical composition is provided, wherein the pharmaceutical composition comprises the antimicrobial peptide as described above and / or its functionally equivalent variant and a pharmaceutically acceptable excipient.

[0020] In some embodiments, the pharmaceutical compositions described herein comprise one, two, or more other antibacterial drugs.

[0021] In other aspects of the present disclosure, a method for inhibiting bacteria or preventing and / or treating diseases or conditions associated with bacterial invasion is provided, comprising administering to a subject the antimicrobial peptide and / or its functionally equivalent variant as described above, or administering the pharmaceutical composition as described above.

[0022] In other aspects of the present disclosure, the antimicrobial peptides and / or functionally equivalent variants thereof are used to inhibit bacteria or prevent and / or treat diseases or conditions associated with bacterial invasion, and the subject is a mammal, such as a human.

[0023] In other aspects of the present disclosure, the present disclosure provides the use of the antimicrobial peptide and / or its functionally equivalent variant in the preparation of an external-use product, wherein the external-use product is selected from a disinfectant, a cleaning agent or a care product; optionally, the disinfectant is selected from a medical disinfectant, an environmental disinfectant, a food disinfectant, such as a disinfectant wipe, a disinfectant cotton swab, a disinfectant gauze or a disinfectant solution; optionally, the detergent is selected from a medical detergent, a household detergent or an industrial detergent, such as a dishwashing liquid, a laundry detergent, a washing powder or a hand soap; optionally, the care product is selected from a skin care product and a lotion.

[0024] In some embodiments, the topical products described herein are prepared as solutions, lotions, creams, ointments, gels, or sprays.

[0025] In some embodiments, the present disclosure provides a method for inhibiting mycobacteria, comprising exposing the mycobacteria to an effective amount of S1 and / or S2 as defined in the present disclosure, such that the growth of the strain is inhibited.

[0026] In other embodiments, the present disclosure provides methods for inhibiting Salmonella, comprising exposing different strains of Salmonella typhimurium to an effective amount of S1 such that the growth of the strains is inhibited.

[0027] In some embodiments, the C-terminus or N-terminus of the antimicrobial peptides disclosed herein is covalently or non-covalently linked to a modification group. In some specific embodiments, the modification group includes an acetyl group, an amide group, an aldehyde group, a methyl group, an ester group, or an alkyl group. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1. Inhibition of Mycobacterium smegmatis growth by S1 and S2 at different doses.

[0029] Figure 2. Inhibition of Mycobacterium tuberculosis growth by S1 and S2 at different doses. DETAILED DESCRIPTION

[0030] For the purpose of promoting the understanding of the principle of the present disclosure, reference will now be made to the embodiments illustrated in the accompanying drawings and the embodiments will be described in detail. However, these descriptions are not intended to limit the scope of the present disclosure in any way.

[0031] Detailed description:

[0032] As used herein, the term "subject" refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like.

[0033] As used herein, the term "effective amount" refers to the concentration of the antimicrobial peptide molecule or its functionally equivalent variant molecule in the environment in which the bacteria are located. In one embodiment, the concentration of the antimicrobial peptide molecule or its functionally equivalent variant molecule includes, but is not limited to, about 0.01-2000 μg / mL, such as 0.05-1000 μg / mL, 0.1-500 μg / mL, 0.15-100 μg / mL, 0.2-50 μg / mL, 0.5-10 μg / mL, 1-9 μg / mL, 2-8 μg / mL, 3-7 μg / mL, 4-6 μg / mL or 5 μg / mL.

[0034] Antimicrobial peptides disclosed herein:

[0035] In one exemplary embodiment, antimicrobial peptides suitable for the present disclosure include a peptide (designated S1) having the amino acid sequence YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 1), or a sequence (or variant) thereof having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% identity thereto, for example, wherein the variant has 1, 2, or 3 conservative amino acid substitutions, additions, or deletions and exhibits substantially similar in vivo or in vitro activity as the peptide examples disclosed herein. In other exemplary embodiments, the antimicrobial peptides disclosed herein are peptides having the amino acid sequence YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 1). Variants of the peptides disclosed herein are also within the scope of the present disclosure. A peptide variant refers to an amino acid sequence in which one or more amino acids have been altered. A variant can have "conservative" alterations, wherein the substituted amino acids have similar structural or chemical properties, for example, replacing leucine with isoleucine. Alternatively, the variant may have "non-conservative" changes, such as replacing glycine with tryptophan. Similar minor changes may also include amino acid deletions or insertions, or both. A specific form of a "variant" peptide is a "functionally equivalent" peptide, i.e., a peptide that exhibits substantially similar in vivo or in vitro activity to the peptide examples disclosed herein. Guidance for determining which amino acid residues can be substituted, inserted, or deleted without losing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR software (DNASTAR, Inc., Madison, WI). In addition, specific guidance is provided below, including those provided within the cited references, which are incorporated herein by reference.

[0036] In some aspects, the peptides of the present invention comprise a membrane-penetrating peptide capable of guiding the peptides of the present invention across bacterial cell membranes at their N-terminus or C-terminus. Suitable membrane-penetrating peptides known in the art can be used in the present invention as long as they can guide the peptides of the present invention across cell membranes. Exemplary membrane-penetrating peptides can be selected from human HIV-1 Tat protein (TAT) sequences, such as "GRKKRRQRRR (SEQ ID NO: 2)", "YGRKKRRQRRR (SEQ ID NO: 3)", "YGRKKRRQRRRPPQ (SEQ ID NO: 4)" or "GRKKRRQRRRQ (SEQ ID NO: 5)", and can also be selected from model amphipathic peptide (modelamphipathic peptide, MAP) sequence "KLALKLALKALKAALKLA (SEQ ID NO: 6)", membrane translocation peptide (membrane translocating peptide, MTS) sequence "AAVALLPAVLLALLAP (SEQ ID NO: 7)" or R9 sequence "RRRRRRRRR (SEQ ID NO: 8)". YGRKKRRQRRR (SEQ ID NO: 3) of the present disclosure is designated as S2.

[0037] According to the present disclosure, the diseases or conditions associated with bacterial invasion include but are not limited to skin and soft tissue infections, gastroenteritis, diarrhea-type conditions, abdominal pain-type conditions, vomiting-type conditions, mucus, pus and blood in stool, chills, high fever, rash, joint pain, headache, myalgia, osteomyelitis, osteoarthritis, endocarditis, pneumonia, bacteremia, sepsis, tuberculosis, typhoid, convulsions, brain abscess, hydrocephalus and developmental delay, impaired consciousness, imbalance and convulsions, etc.

[0038] As used herein, the term "pharmaceutically acceptable excipient" includes an acceptable carrier, excipient, or stabilizer that is nontoxic to cells or mammals in contact at the dosages and concentrations employed. A physiologically acceptable carrier can be an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Polyethylene glycol (PEG) and

[0039] This disclosure also discloses the following technical solutions:

[0040] In some aspects, the present disclosure provides an active peptide comprising the amino acid sequence KKNRNKLRRQHSY (SEQ ID NO: 9) or consisting of the amino acid sequence shown in SEQ ID NO: 9, or its functionally equivalent variants for the preparation of drugs for antibacterial or prevention and / or treatment of diseases or conditions associated with invasion of foreign pathogens.

[0041] In other aspects, the present disclosure provides an active peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9, or its functionally equivalent variants for use in antibacterial or prevention and / or treatment of diseases or conditions associated with invasion of foreign pathogens.

[0042] In some embodiments, the functionally equivalent variants of the active peptides disclosed herein have 1, 2 or 3 conservative amino acid substitutions, additions or deletions relative to the active peptides, such as hydrophobic amino acid substitutions or additions, and the hydrophobic amino acids that can be selected include: tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline and methionine (methionine), etc.

[0043] In some embodiments, the functionally equivalent variant of the active peptide disclosed herein is an active peptide with a cell-penetrating peptide sequence added to the C-terminus or N-terminus.

[0044] In some embodiments, the cell-penetrating peptide sequence disclosed herein is selected from: a TAT sequence, a MAP sequence, an MTS sequence, or an R9 sequence.

[0045] In some embodiments, the functionally equivalent variant disclosed herein is an active peptide with a TAT sequence added to the N-terminus.

[0046] In some embodiments, the active peptide described in the present disclosure is an active peptide comprising the amino acid sequence YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 1).

[0047] In some specific embodiments, the active peptide disclosed herein is an active peptide having an amino acid sequence of YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 1).

[0048] In some embodiments, the active peptide disclosed herein is a functionally equivalent variant of the S1 active peptide, wherein the functionally equivalent variant has 1, 2 or 3 conservative amino acid substitutions, additions or deletions relative to the S1 active peptide.

[0049] In some embodiments, the functionally equivalent variants disclosed herein have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the active peptide.

[0050] In some embodiments, the antibacterial agent described in the present disclosure is anti-actinomycetales.

[0051] In some embodiments, the antibacterials disclosed herein are resistant to Actinomycetaceae, Mycobacteriaceae, Bifidobacteriaceae, Dermatophilaceae, Frankiaceae, Rhodococcusaceae, Nephrobacteriaceae, Nocardiaceae, Streptomycetaceae, and Thermoactinomycetaceae.

[0052] In some embodiments, the antibacterial agent disclosed herein is against Mycobacterium tuberculosis complex, Mycobacterium leprae, Mycobacterium avium complex, Mycobacterium gordonii subgroup, Mycobacterium kansasii subgroup, Mycobacterium terrestris subgroup, Mycobacterium simian subgroup, Mycobacterium chelonae subgroup, Mycobacterium fortuitum subgroup, Mycobacterium fortuitum group, and ungrouped mycobacteria.

[0053] In some embodiments, the antibacterial agent disclosed herein is against Mycobacterium tuberculosis, Mycobacterium smegmatis, Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium avium-intracellulare, Mycobacterium fortuitum, Mycobacterium chelonae, and Mycobacterium ulcerans.

[0054] In some embodiments, the antibacterial agent of the present disclosure is anti-Enterobacteriaceae.

[0055] In some embodiments, the antibacterial agent of the present disclosure is resistant to Photorhabdus, Klebsiella, Proteobacterium, Hafnia, Escherichia, Shigella, Citrobacter, Erwinia, Salmonella, Serratia, Edwardsiella, Yersinia, Enterobacter, and Xenorhabdus.

[0056] In some embodiments, the antibacterial agent of the present disclosure is anti-Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella dublin, Salmonella gallinarum, and Salmonella pullorum.

[0057] Another aspect of the present disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the active peptide as described above and a pharmaceutically acceptable excipient.

[0058] In some embodiments, the pharmaceutical compositions of the present disclosure comprise one, two, or more other antibacterial compounds.

[0059] Another aspect of the present disclosure provides a method for antibacterial or prevention and / or treatment of diseases or conditions associated with invasion of foreign pathogens, which comprises administering to a subject the active peptide and / or its functionally equivalent variant as described above, or administering the pharmaceutical composition as described above.

[0060] Another aspect of the present disclosure provides the active peptide and / or its functionally equivalent variant for use in antibacterial or prevention and / or treatment of diseases or conditions associated with invasion of foreign pathogens. The subject of antibacterial is a mammal, preferably a human.

[0061] In some embodiments, the diseases or conditions related to the invasion of foreign pathogens described in the present disclosure include infectious diseases or conditions related to epithelial tissue, connective tissue, muscle tissue and nervous tissue, or infectious diseases or conditions related to the musculoskeletal system, circulatory system (cardiocirculatory system, lymphatic system), respiratory system, digestive system, nervous system, endocrine system, urinary system and reproductive system, such as skin and soft tissue infections, gastroenteritis, diarrhea-type diseases, abdominal pain-type diseases, vomiting-type diseases, mucus, pus and blood in the stool, chills, high fever, rash, joint pain, headache, myalgia, osteomyelitis, osteoarthritis, endocarditis, pneumonia, bacteremia, sepsis, tuberculosis, typhoid, convulsions, brain abscess, hydrocephalus and developmental delay, impaired consciousness, imbalance and convulsions, etc.

[0062] Example

[0063] The following examples are only used to illustrate the present disclosure rather than to limit the present disclosure.

[0064] Example 1: Evaluation of the inhibitory effects of S1 and S2 on the growth of a test strain of Mycobacterium smegmatis.

[0065]

method

[0066] (1) Prepare stock solutions of compounds S1 and S2 of equal concentration, aliquot and store at -80°C. Dilute to the desired concentration before use.

[0067] (2) Add 100 μL of culture medium to each sterile culture tube. Add 100 μL of the test compound at the appropriate concentration to the first tube in each row, mix thoroughly, and add 100 μL to the second tube. Repeat this process, using a two-fold serial dilution method to obtain solutions of compound S1 and S2 at concentrations of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256.

[0068] (3) Take Mycobacterium smegmatis MC in the logarithmic growth phase 2 155. Determine the bacterial concentration by solid plate count method. Add 100 μL of bacterial solution diluted to the appropriate concentration to each tube so that the bacterial count in each tube is about 10 5 CFU / mL.

[0069] (4) Place the culture tubes in a 37°C constant temperature incubator for 2 days, add 30 μL of resazurin solution to each tube, and then place it in a 37°C constant temperature incubator for 20 minutes.

[0070] (5) After the incubation is completed, the culture tubes are removed and the color changes of each tube are observed and recorded (blue indicates no strain growth, and red indicates strain growth).

[0071] (6) For the test compound, eight concentration gradients were designed, and three parallel samples were tested at each concentration.

[0072] As shown in Figure 1, when the concentration of compound S1 is lower than a 1:64 dilution ratio, the growth of Mycobacterium smegmatis reduces resazurin to a pink solution; when the concentration of compound S1 is ≥ a 1:64 dilution ratio, the growth of Mycobacterium smegmatis is inhibited, and the color of resazurin remains blue. When the concentration of compound S2 is lower than a 1:8 dilution ratio, the growth of Mycobacterium smegmatis reduces resazurin to a pink solution; when the concentration of compound S2 is ≥ a 1:8 dilution ratio, the growth of Mycobacterium smegmatis is inhibited, and the color of resazurin remains blue.

[0073] Therefore, compounds S1 and S2 both have the effect of inhibiting the growth of Mycobacterium smegmatis and can be used for the purpose of combating Mycobacterium smegmatis.

[0074] Table 1 Minimum inhibitory concentration (MIC) of compounds S1 and S2 for inhibiting the growth of Mycobacterium smegmatis

[0075] As shown in Table 1, compound S2 can inhibit the growth of Mycobacterium smegmatis at a dilution ratio of 1:8; compound S1 can inhibit the growth of Mycobacterium smegmatis at a dilution ratio of 1:64.

[0076] Example 2: Compounds S1 and S2 inhibited the growth of a test strain of Mycobacterium tuberculosis at a certain dose.

[0077]

method

[0078] (1) Prepare two stock solutions of compounds S1 and S2 with the same mass concentration, freeze them at -80°C after aliquoting, and dilute them to the required concentration before use.

[0079] (2) Add 100 μL of culture medium to each sterile culture tube. Add 100 μL of the test compound at the appropriate concentration to the first tube in each row, mix thoroughly, and add 100 μL to the second tube. Repeat this process, using a two-fold serial dilution method to obtain solutions of compound S1 and S2 at concentrations of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256.

[0080] (3) Take Mycobacterium tuberculosis H37Ra in the logarithmic growth phase and determine the bacterial concentration by solid plate counting method. Add 100 μL of bacterial solution diluted to an appropriate concentration to each tube so that the number of bacteria in each tube is about 105 CFU / mL.

[0081] (4) Place the culture tubes in a 37°C constant temperature incubator for 2 days, add 30 μL of resazurin solution to each tube, and then place it in a 37°C constant temperature incubator for 20 minutes.

[0082] (5) After the incubation is completed, the culture tubes are removed and the color changes of each tube are observed and recorded (blue indicates no strain growth, and red indicates strain growth).

[0083] (6) For the test compound, eight concentration gradients were designed, and three parallel samples were tested at each concentration.

[0084] As shown in Figure 2, when the concentration of compound S1 is lower than a 1:8 dilution ratio, the growth of Mycobacterium tuberculosis reduces resazurin to a pink solution. When the concentration of compound S1 is ≥ a 1:8 dilution ratio, the growth of Mycobacterium tuberculosis is inhibited, and the color of resazurin remains blue. When the concentration of compound S2 is lower than a 1:4 dilution ratio, the growth of Mycobacterium tuberculosis reduces resazurin to a pink solution. When the concentration of compound S2 is ≥ a 1:4 dilution ratio, the growth of Mycobacterium tuberculosis is inhibited, and the color of resazurin remains blue.

[0085] Therefore, compounds S1 and S2 both have the effect of inhibiting the growth of Mycobacterium tuberculosis and can be used for the purpose of combating Mycobacterium tuberculosis.

[0086] Table 2 Minimum inhibitory concentration (MIC) of compounds S1 and S2 for inhibiting the growth of Mycobacterium tuberculosis

[0087] As shown in Table 2, compound S2 can inhibit the growth of Mycobacterium tuberculosis at a dilution ratio of 1:4; compound S1 can inhibit the growth of Mycobacterium tuberculosis at a dilution ratio of 1:8.

[0088] Example 3: Compound S1 inhibits the growth of various test strains of Salmonella typhimurium at a certain dosage.

[0089] Materials and Methods

[0090] The S1 compound stock solution was prepared using sterile water and diluted to an appropriate concentration. Serial dilution was then performed to obtain S1 compound solutions with concentrations of 1:5, 1:25, 1:125, 1:156, 1:390, 1:625, 1:976, and 1:3133.

[0091] In a clean bench or biosafety cabinet, pipette 0.1 mL of the dosing agent, 0.5 mL of 0.2M PBS, and 0.1 mL of the test strain solution into 2 mL of incubated surface culture medium. Repeat for each concentration in two replicate dishes.

[0092] Mix the surface culture medium and quickly pour the contents of the surface culture medium into the numbered bottom culture medium plates along the direction of sample addition. Rotate the plates to evenly distribute the surface culture medium on the bottom layer and solidify it in parallel. Incubate the plates upside down in a 37°C biochemical incubator for 48 to 72 hours and observe the results. Record the background bacterial moss and precipitation of each plate. The lowest concentration that causes the background bacterial moss to decrease is defined as the MIC.

[0093] Table 3 Minimum inhibitory concentration (MIC) of compound S1 for inhibiting the growth of various strains of Salmonella typhimurium

[0094] As shown in Table 3, compound S1 inhibited the growth of Salmonella typhimurium strains TA1535, TA97a, and TA100 at a dilution ratio of 1:125. Compound S1 inhibited the growth of Salmonella typhimurium strains TA98 and TA102 at a dilution ratio of 1:25.

[0095] Table 4 S1 inhibits the growth of various strains of Salmonella typhimurium at different doses

[0096] T0 indicates that the background moss is normal, T1 indicates that the background moss is slightly reduced, T2 indicates that the background moss is moderately reduced, T3 indicates that the background moss is severely reduced, and T4 indicates that the background moss disappears.

[0097] As shown in Table 4, compound S1, at a 1:125 dilution, initially caused a slight reduction in the background lawn of Salmonella typhimurium strains TA1535, TA97a, and TA100. As the dose increased, the reduction in background lawns for each of these strains increased until the background lawn disappeared. Compound S1, at a 1:25 dilution, initially caused a slight reduction in the background lawns of Salmonella typhimurium strains TA98 and TA102. As the dose increased, the reduction in background lawns for each of these strains increased.

[0098] Therefore, compound S1 exhibits an inhibitory effect on the growth of different strains of Salmonella typhimurium at different concentrations and can be used for anti-Salmonella purposes.

[0099] Although various embodiments of compositions and methods for inhibiting bacteria have been described in great detail herein, such embodiments are provided merely as non-limiting examples of the disclosure described herein. Therefore, those skilled in the art will appreciate that various changes and modifications may be made to the disclosure without departing from the scope of the disclosure. Indeed, the disclosure is not intended to be exhaustive or to limit the scope of the disclosure.

[0100] Furthermore, in the description of representative embodiments, the present disclosure has presented the methods and / or processes of the present disclosure in a specific order of steps. However, the methods or processes should not be limited to the specific order of steps described. Other order of steps are possible. Therefore, the specific order of steps disclosed herein should not be interpreted as limiting the present disclosure. In addition, the disclosure of the methods and / or processes should not be limited to performing their steps in the order described. Such an order can be varied and still be within the scope of the present disclosure.

Claims

1. Use of an antimicrobial peptide comprising the amino acid sequence YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 1) or a functionally equivalent variant thereof in inhibiting bacteria or in preparing a medicament for preventing and / or treating diseases or conditions associated with bacterial invasion; Optionally, the antimicrobial peptide amino acid sequence is the sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein: The functionally equivalent variant of the antimicrobial peptide has 1, 2 or 3 conservative amino acid substitutions, additions or deletions relative to the antimicrobial peptide.

3. The use according to any one of claims 1 to 2, wherein: The functionally equivalent variant has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the antimicrobial peptide.

4. The use according to any one of claims 1 to 3, wherein The bacteria are selected from one or more of Actinomycetaceae, Mycobacteriaceae, Bifidobacteriaceae, Dermatophilaceae, Frankiaceae, Rhodococcusaceae, Nephrobacteriaceae, Nocardiaceae, Streptomycetaceae, Thermoactinomycetaceae or Enterobacteriaceae.

5. The use according to any one of claims 1 to 4, wherein The bacteria are one or more of the genus Photorhabditis, Klebsiella, Proteobacterium, Hafnia, Escherichia, Shigella, Citrobacter, Erwinia, Salmonella, Serratia, Edwardsiella, Yersinia, Enterobacter, Xenorhabdus or Mycobacterium.

6. The use according to any one of claims 1 to 5, wherein The bacteria are one or more of Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium kansasii, Mycobacterium marineum, Mycobacterium avium-intracellulare, Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium ulcerans, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella dublin, Salmonella gallinarum, and Salmonella pullorum.

7. The use according to any one of claims 1 to 6, wherein: The disease or condition associated with bacterial invasion is selected from bacterial infection diseases or conditions associated with epithelial tissue, connective tissue, muscle tissue and nervous tissue, or bacterial infection diseases or conditions associated with the musculoskeletal system, circulatory system, respiratory system, digestive system, nervous system, endocrine system, urinary system and reproductive system, such as skin and soft tissue infection, gastroenteritis, diarrhea-type conditions, abdominal pain-type conditions, vomiting-type conditions, mucus, pus and blood in the stool, chills, high fever, rash, joint pain, headache, myalgia, osteomyelitis, osteoarthritis, endocarditis, pneumonia, bacteremia, sepsis, tuberculosis, typhoid fever, convulsions, brain abscess, hydrocephalus and developmental delay, impaired consciousness, imbalance and convulsions.

8. A pharmaceutical composition, wherein The pharmaceutical composition comprises (1) the antimicrobial peptide as defined in any one of claims 1 to 7 and / or its functionally equivalent variant, and (2) a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition comprises one, two or more additional bacteriostatic agents.

10. A method for inhibiting bacteria or preventing and / or treating diseases or conditions associated with bacterial invasion, comprising administering to a subject an antimicrobial peptide and / or a functionally equivalent variant thereof as defined in any one of claims 1 to 7, or administering the pharmaceutical composition according to claim 8 or 9.

11. The antimicrobial peptide as defined in any one of claims 1 to 7 and / or its functionally equivalent variant, for use in inhibiting bacteria or preventing and / or treating diseases or conditions associated with bacterial invasion.

12. Use of the antimicrobial peptide as defined in any one of claims 1 to 7 and / or its functionally equivalent variants in the preparation of a product for external use, wherein the product for external use is selected from a disinfectant, a cleaning agent or a care product; Optionally, the disinfectant is selected from medical disinfectants, environmental disinfectants, and food disinfectants, such as disinfectant wipes, disinfectant cotton swabs, disinfectant gauze, or disinfectant solution; Optionally, the detergent is selected from medical detergents, household detergents or industrial detergents, such as dishwashing liquid, laundry detergent, washing powder or hand soap; Optionally, the care products are selected from skin care products and lotions.

13. The use according to claim 12, wherein: The topical product is prepared as a solution, emulsion, cream, ointment, gel or spray.

14. The use according to any one of claims 1 to 7, wherein: A modification group is covalently or non-covalently connected to the C-terminus or N-terminus of the antimicrobial peptide, and the modification group includes an acetyl group, an amide group, an aldehyde group, a methyl group, an ester group, and an alkyl group.

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