Application of natural product nootkatone in synergizing polymyxins in the fight against Gram-negative bacterial infections

Abstract

The present invention discloses the use of nootkatone in preparing a drug that enhances the antibacterial efficacy of polymyxin, as well as the use of a combination of nootkatone and polymyxin in preparing a drug with enhanced antibacterial efficacy. The present invention not only demonstrates the synergistic enhancement of the antibacterial activity of polymyxin by using a checkerboard minimum inhibitory concentration test and an in vitro bacterial growth curve, but also uses a mouse wound infection model to confirm that nootkatone can effectively enhance the antibacterial activity of polymyxin and its in vivo effectiveness. The present invention finds that the natural active substance nootkatone can significantly enhance the antibacterial activity of polymyxin, which can provide an auxiliary drug for the clinical treatment of multidrug-resistant Gram-negative bacterial infections, while reducing the dosage of polymyxin and reducing the occurrence of drug resistance, providing a new treatment strategy for the clinical treatment of bacterial infectious diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a new use of the natural active substance nootkatone as an antibacterial adjuvant to enhance the antibacterial activity of antibacterial drugs, more specifically to a new use of nootkatone to enhance the antibacterial activity of polymyxin against Gram-negative bacteria. Background Art

[0002] Bacterial resistance has become a global public health concern. In recent years, the rapid spread of carbapenem-resistant Gram-negative bacteria in both human and animal medicine has led to a dwindling supply of clinically available antibiotics. Furthermore, the recent stagnation in the development of new antimicrobial agents has led to the resurgence of the once-abandoned polymyxin class of antibiotics in clinical practice. In clinical practice, polymyxins primarily include polymyxin B and polymyxin E. Polymyxin E, also known as colistin, is primarily used in its sulfate (colistin sulfate) or sulfonate (colistin sulfonate). Currently, colistin is considered one of the most important antimicrobial agents for the treatment of infections such as carbapenem-resistant Escherichia coli, Pseudomonas aeruginosa, and multidrug-resistant Klebsiella pneumoniae. However, non-standard clinical use has led to the emergence of resistance, particularly the recent emergence of colistin resistance genes (MCRs), which have exacerbated the spread of polymyxin resistance in clinical practice. Therefore, how to reduce the incidence of polymyxin resistance and extend the lifespan of this clinically important antimicrobial drug has become a key concern for scientists worldwide. Polymyxin potentiators can effectively reduce the dosage of polymyxins, reduce the incidence of resistance and toxicity, and improve the clinical therapeutic index. Therefore, the development of polymyxin potentiators has become an effective strategy to curb polymyxin resistance.

[0003] The CAS number of nootkatone is 4674-50-4; the molecular formula is C 15 H 22 O, also known as nootkatone, has a molecular weight of 218.34 and is a bicyclic sesquiterpenoid ketone with a structure shown in Formula I. Nootkatone is found in a variety of plants and is the most characteristic aroma compound in grapefruit peel. It is primarily extracted from grapefruit or synthesized artificially. Nootkatone exhibits multiple biological activities, including antioxidant, anti-inflammatory, and anticancer activities. It also exhibits significant insect repellent and insecticide effects and is the first new mosquito repellent active ingredient approved by the US Environmental Protection Agency since 2009. To date, nootkatone's use as an antimicrobial adjuvant to enhance the antimicrobial activity of antimicrobial drugs has not been reported.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a new pharmaceutical use of nootkatone.

[0006] The novel pharmaceutical use of nootkatone provided by the present invention is its use in preparing a product for enhancing the antibacterial activity and / or bactericidal activity of polymyxin antibiotics.

[0007] Furthermore, the product can be an antibacterial synergist of polymyxin antibiotics or a drug for treating bacterial infectious diseases.

[0008] Furthermore, the polymyxin is selected from at least one of the following: 1) colistin (i.e., polymyxin E) or a pharmaceutically acceptable salt thereof (such as polymyxin E sulfate or colistin sulfonate); 2) polymyxin B or a pharmaceutically acceptable salt thereof (such as polymyxin B sulfate).

[0009] Furthermore, the bacterial infectious disease includes infection by drug-resistant bacteria carrying the MCR-1 gene.

[0010] Furthermore, the antibacterial activity and / or bactericidal activity has an inhibitory and / or killing effect on at least one of the following pathogens: 1. Gram-negative bacteria; 2. Proteobacteria; 3. γ-Proteobacteria; 4. Enterobacteriales or Pseudomonas; 5. Enterobacteriaceae or Pseudomonas; 6. Escherichia or Klebsiella or Pseudomonas; 7. Escherichia coli or Klebsiella pneumoniae or Pseudomonas aeruginosa or Salmonella; 8. Escherichia coli carrying the MCR-1 gene or Klebsiella pneumoniae carrying the MCR-1 gene or Salmonella carrying the MCR-1 gene.

[0011] Another object of the present invention is to provide an antibacterial composition.

[0012] The antibacterial composition provided by the present invention comprises nootkatone and polymyxin antibiotics.

[0013] Furthermore, the mass ratio of the nootkatone to the polymyxin antibiotic is (50-6410):1; specifically (50-200):1 or (3200-6410):1, more specifically 50:1, 200:1, 3200:1 or 6410:1.

[0014] Furthermore, the polymyxin antibiotic is selected from at least one of the following: 1) colistin (i.e., polymyxin E) or a pharmaceutically acceptable salt thereof (such as polymyxin E sulfate or colistin sulfonate); 2) polymyxin B or a pharmaceutically acceptable salt thereof (such as polymyxin B sulfate).

[0015] In the above-mentioned antibacterial composition, the active ingredients of the antibacterial composition may be only nootkatone and the polymyxin antibacterial drug; or it may further include other active ingredients, which can be determined by those skilled in the art based on the antibacterial effect.

[0016] The above composition can have a killing and / or inhibitory effect on at least one of the following pathogens: 1. Gram-negative bacteria; 2. Proteobacteria; 3. γ-Proteobacteria; 4. Enterobacteriales or Pseudomonas; 5. Enterobacteriaceae or Pseudomonas; 6. Escherichia or Klebsiella or Pseudomonas or Salmonella; 7. Escherichia coli or Klebsiella pneumoniae or Pseudomonas aeruginosa or Salmonella; 8. Escherichia coli carrying the MCR-1 gene or Klebsiella pneumoniae carrying the MCR-1 gene or Salmonella carrying the MCR-1 gene.

[0017] In addition, antibacterial products containing the above antibacterial composition also fall within the protection scope of the present invention.

[0018] The dosage form of the above-mentioned antibacterial product can be any of the following: tablets, creams, capsules, sustained-release tablets, controlled-release tablets, oral liquids, syrups, pills, injection dosage forms, and freeze-dried powder injection dosage forms.

[0019] The present invention demonstrates the synergistic antibacterial activity of nootkatone with polymyxins through a checkerboard minimum inhibitory concentration test, in vitro bacterial growth curves, and animal experiments. The Enterobacteriaceae used in the test primarily included MCR-1-positive Escherichia coli, MCR-1-positive Salmonella, MCR-1-positive Klebsiella pneumoniae, and the standard E. coli strain ATCC25922.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The present invention first discovered that nootkatone can be used to enhance the antibacterial activity of polymyxin and its therapeutic effect in bacterial infectious diseases. At the same time, the use of nootkatone in combination with polymyxin can significantly reduce the inflammatory response induced by polymyxin.

[0022] 2) This invention demonstrates that nootkatone can restore the sensitivity of polymyxin-resistant bacteria and further evaluates the effectiveness of their combined use in vivo and in vitro, which will contribute to the development of a new class of antibiotic synergists to alleviate the increasingly serious problem of bacterial resistance in clinical practice.

[0023] The present invention discovered that the natural active substance nootkatone can significantly enhance the antibacterial activity of polymyxin, and can provide an auxiliary drug for the clinical treatment of multidrug-resistant Gram-negative bacterial infections, while reducing the dosage of polymyxin and reducing the occurrence of drug resistance. Therefore, the invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The in vitro killing curves of polymyxin E sulfate and nootkatone alone and in combination against Escherichia coli B2 (E.coli B2) are shown;

[0025] Figure 2 The in vitro killing curves of polymyxin E sulfate and nootkatone alone and in combination against Escherichia coli 25922 (E.coli ATCC 25922);

[0026] Figure 3 To evaluate the therapeutic effects of polymyxin E sulfate and nootkatone alone or in combination on Escherichia coli B2 (E. coli B2) infection in mouse skin wounds. DETAILED DESCRIPTION

[0027] The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified.

[0028] Nootkatone, CAS number, 4674-50-4; molecular formula is C 15 H 22 O, molecular weight 218.34, purchased from Aladdin Reagent Company, item number N159076, purity> 97%. Colistin sulfate (polymyxin E sulfate) was purchased from Aladdin Reagent Company, item number C114323, activity> 19000U / mg. Polymyxin B sulfate was purchased from Aladdin Reagent Company, with a potency greater than or equal to 6000U / mg. Weigh a certain amount of colistin sulfate or polymyxin B sulfate to make a mother liquor concentration of 16mg / mL. Nootkatone was configured to a mother liquor concentration of 100mg / mL using DMSO. After preparation, store in a -20 degree refrigerator.

[0029] Pathogens used in the following tests:

[0030] The standard strain of Escherichia coli was Escherichia coli ATCC 25922 (hereinafter referred to as E. coli 25922), which was purchased from the China Veterinary Supervision Institute’s Culture Collection Center (China Veterinary Microbial Culture Collection Administration Center).

[0031] Escherichia coli B2 (E. coli B2) carries a colistin resistance gene 1 (MCR-1) plasmid and a New Delhi metallo-β-lactamase 5 (NDM-5) plasmid. Detailed information has been described in the following references: Song M, Liu Y, Huang X, Ding S, Wang Y, Shen J, Zhu KA. Broad-spectrum antibiotic adjuvant reverses multidrug-resistant Gram-negative pathogens. Nat Microbiol. 2020 Aug; 5(8): 1040-1050.

[0032] Klebsiella pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1), K. pneumoniae 1202 (ST11, KPC-2-producer) (detailed information has been described in the following references: Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, Doi Y, Tian G, Dong B, Huang X, Yu LF, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu JH, Shen J. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016 Feb;16(2):161-8.). Among them, K. pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1) carries the MCR-1 plasmid and is resistant to polymyxins.

[0033] The standard strain of Pseudomonas aeruginosa ATCC15692 (ie, Pseudomonas aeruginosa PA01) was purchased from the American Type Culture Collection (ATCC).

[0034] Salmonella SH16SF0764 was isolated from clinical samples and carried the MCR-1 plasmid, which was preserved and provided by the National Center for Veterinary Drug Safety Evaluation, China Agricultural University.

[0035] Bacterial culture media were used in the following experiments:

[0036] MHB broth culture medium was purchased from Beijing Luqiao Technology Co., Ltd. and prepared as follows: weigh 25.0 g into 1 L of distilled water, heat and boil until completely dissolved, sterilize under high pressure at 121°C for 15 min, and set aside.

[0037] MHA medium was purchased from Beijing Luqiao Technology Co., Ltd. and prepared as follows: weigh 38.0 g and dissolve in 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121°C under high pressure for 15 min, cool to 55°C and pour into a plate for later use.

[0038] Brain heart infusion medium (BHI) was purchased from Beijing Luqiao Technology Co., Ltd. and was prepared as follows: 38.5 g of the product was weighed and dissolved in 1000 mL of distilled water with heating and stirring, the pH was adjusted to 7.3, and the medium was sterilized by autoclaving at 121°C for 15 minutes and set aside.

[0039] BHI solid culture medium was purchased from Beijing Road & Bridge Technology Co., Ltd. and was prepared as follows: 50.0 g of the product was weighed and dissolved in 1000 mL of distilled water. The mixture was heated and boiled until completely dissolved. The medium was sterilized by autoclaving at 121°C for 20 min and cooled to 55°C before being poured into a plate for later use.

[0040] Example 1. Evaluation of the synergistic antibacterial activity of nootkatone and polymyxin combination

[0041] (1) Determination of the minimum inhibitory concentration (MIC) of nootkatone, polymyxin B, and polymyxin E when used alone

[0042] The minimum inhibitory concentration (MIC) of nootkatone, polymyxin B, and polymyxin E against different strains was determined according to the standard method of the Clinical and Laboratory Standards Institute (CLSI). The specific procedure was as follows: a single bacterial colony was picked and placed in BHI broth. The bacteria were cultured in a shaking incubator at 37°C until the logarithmic growth phase. The concentration of the bacterial solution was adjusted to a McFarland turbidity of 0.5 using a McFarland turbidimeter and diluted 100-fold (1.0 x 10 6 CFUs / mL) and then set aside. Nootkatone, polymyxin B, and polymyxin E were diluted in multiple concentration gradients using MHB medium, and 100 μL was added to a 96-well U-shaped plate. Subsequently, 100 μL of the diluted test bacterial solution was added to each well. The final drug concentration of nootkatone in each well was 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, and 31.125 μg / mL, respectively; the final drug concentrations of polymyxin B and polymyxin E were 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, and 0.0625 μg / mL. The negative control contained only MHB medium; the positive control contained 100 μL of the test bacteria solution, with 1 μL of DMSO added to each well. After drug addition, the plates were incubated at 37°C for 18 hours and the results were observed. The drug concentration in the wells that were visually clear in the 96-well plate was the minimum inhibitory concentration (MIC). The results are shown in Table 1.

[0043] Table 1 MIC values ​​of nootkatone, polymyxin E, and polymyxin B against various strains (μg / mL)

[0044]

[0045] As shown in Table 1, nootkatone exhibited antibacterial activity greater than 1000 μg / mL against the tested strains E. coli 25922, E. coli B2, ATCC 15692, K. pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1), K. pneumoniae 1202 (ST11, KPC-2-producer), and Salmonella SH16SF0764. There was no difference in the MICs of polymyxin B and polymyxin E against the above bacteria, with corresponding MICs of 0.125 μg / mL, 8 μg / mL, 1 μg / mL, 8 μg / mL, 0.25 μg / mL, and 8 μg / mL, respectively.

[0046] (2) Antibacterial activity test of combined application of nootkatone and antibacterial drug polymyxin

[0047] Determination of the combined use index (FICI) of nootkatone and the antimicrobial drug polymyxin against pathogens: The FICI values ​​of nootkatone combined with antimicrobial drugs against Escherichia coli standard strain ATCC25922 and MCR-1-producing E. coli B2, Klebsiella pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1) and K. pneumoniae 1202 (ST11, KPC-2-producer), Pseudomonas aeruginosa ATCC15692, and Salmonella SH16SF0764 were determined using the checkerboard method. The specific procedures are as follows:

[0048] The antimicrobial drugs polymyxin B (agent A) and nootkatone (agent B) were diluted in MHB broth at 2 MICs (maximum concentration) to 8-11 concentrations. 50 μL of MHB broth containing these drugs was added along the horizontal and vertical axes of a 96-well microplate. Subsequently, 50 μL of various pathogenic bacteria suspensions were added to achieve a final pathogen count of 1 × 10⁶ CFU / well. The plates were incubated at 37°C for 18-24 hours, and the results were observed. The MICs of the drugs, used alone and in combination, were recorded, and the FICI (fractional inhibitory concentration index) was calculated using the following formula.

[0049] FICI = MIC of drug A combined / MIC of drug A alone + MIC of drug B combined / MIC of drug B alone

[0050] Judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, no effect; FICI > 2, antagonistic effect. That is, the FICI of nootkatone and colistin = MIC (colistin combination) / MIC (colistin alone) + MIC (nootkatone combination) / MIC (nootkatone alone).

[0051] Table 2 FICI values ​​of nootkatone combined with antimicrobial drugs against Escherichia coli standard strain ATCC25922, E. coli B2, K. pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1), K. pneumoniae 1202 (ST11, KPC-2-producer), Pseudomonas aeruginosa ATCC15692, and Salmonella SH16SF0764

[0052]

[0053]

[0054] For all bacteria tested in this experiment, the results are as follows:

[0055] In vitro MIC assay results showed that the MIC of nootkatone alone was >1000 μg / mL. The combination of nootkatone and polymyxin B can enhance the antibacterial activity of polymyxin B:

[0056] For the standard strain of Escherichia coli ATCC25922, when colistin and nootkatone were used in combination, the two exerted the greatest synergistic effect when the colistin concentration was 0.0156 μg / mL and the nootkatone concentration was 100 μg / mL, with an FICI of 0.22.

[0057] Against K. pneumoniae 1202 (ST11, KPC-2-producer) + pHNSHP45 (mcr-1) strains, colistin and nootkatone exerted maximum synergistic antibacterial effects at 1 μg / mL and 50 μg / mL, respectively, with an FICI of 0.175.

[0058] Against the clinical Klebsiella pneumoniae 1202 (ST11, KPC-2-producer) strain, colistin and nootkatone exerted maximum synergistic antibacterial effects at 0.0625 μg / mL and 200 μg / mL, respectively, with an FICI of 0.45.

[0059] Against the clinical Pseudomonas aeruginosa ATCC15692 strain, colistin and nootkatone exerted maximum synergistic antibacterial effects at 0.25 μg / mL and 50 μg / mL, respectively, with an FICI of 0.3.

[0060] Against clinical E. coli B2 strain, colistin and nootkatone exerted maximum synergistic antibacterial effects at 1 μg / mL and 50 μg / mL, respectively, with an FICI of 0.175.

[0061] Against the clinical Salmonella SH16SF0764 strain, colistin and nootkatone exerted maximum synergistic antibacterial effects at 0.5 μg / mL and 100 μg / mL, respectively, with a FICI of 0.112.

[0062] In summary, the synergistic effect of nootkatone on the antibacterial activity of polymyxin against Gram-negative bacteria (including Escherichia coli, Salmonella and Klebsiella pneumoniae) was optimal when added at 50-200 μg / mL.

[0063] Example 2: Synergistic bactericidal curve of polymyxin combined with nootkatone

[0064] 1. Test materials

[0065] Nootkatone and colistin sulfate were the same as those in Experimental Example 1. CMHA medium and BHI broth were purchased from Solebro Reagent Company.

[0066] 2. In vitro sterilization curve test

[0067] After Escherichia coli B2 and Escherichia coli ATCC25922 were cultured in BHI broth for 6 hours, equal volumes of DMSO (final concentration 0.1%), colistin (i.e., polymyxin E), nootkatone, and a mixture of colistin (i.e., polymyxin E) and nootkatone were added.

[0068] (1) For Escherichia coli B2, the groups and dosages are as follows:

[0069] Control group (control): 0.1% DMSO;

[0070] Colistin group: colistin sulfate 4 μg / mL;

[0071] Nootkatone group: nootkatone 200 μg / mL;

[0072] Nootkatone and colistin combination group: Nootkatone concentration was 200 μg / mL, and colistin concentration was 4 μg / mL.

[0073] Then, 100 μL of the bacterial solution was spread on MHA agar plates at 1 h, 3 h, 6 h, 12 h, and 24 h, and the colonies were counted after overnight culture.

[0074] (2) For Escherichia coli ATCC25922, the groups and dosages are as follows:

[0075] Control group (control): 0.1% DMSO;

[0076] Colistin group: colistin sulfate 0.125 μg / mL;

[0077] Nootkatone group: nootkatone 200 μg / mL;

[0078] Nootkatone and colistin combination group: Nootkatone concentration was 200 μg / mL, and colistin concentration was 0.125 μg / mL.

[0079] After drug treatment, 100 μL of bacterial solution was spread on MHA agar medium at 1 hour, 3 hours, 6 hours, 12 hours and 24 hours respectively, and the colonies were counted after overnight culture.

[0080] Here are the results:

[0081] like Figure 1 and Figure 2 As shown, the combination of polymyxin and nootkatone significantly reduced the number of bacteria compared to polymyxin or nootkatone alone. For Escherichia coli B2, at 3 hours, the bacterial colony count in the nootkatone and colistin combination group was reduced to 0, and the colony count was reduced to 0 at the end of the experiment at 24 hours. For Escherichia coli ATCC25922, compared to the nootkatone or colistin alone treatment group, the bacterial colony count in the nootkatone and colistin combination group was significantly reduced at 1 hour, 3 hours, 6 hours, 12 hours and 24 hours, respectively, to 2.38Log 10 CFU / mL, 2.15Log 10 CFU / mL, 3.73Log 10 CFU / mL, 5.15Log 10 CFU / mL.

[0082] Example 3: Therapeutic effect of polymyxin E combined with nootkatone on bacterial clearance rate in wound infection in mice

[0083] Female BALB / c mice, weighing 18–20 g, were purchased from Beijing Weitonglihua Co., Ltd. Mice were housed in the experimental animal room of the National Center for Veterinary Drug Safety, College of Veterinary Medicine, China Agricultural University, at room temperature (25 ± 2°C, relative humidity 50 ± 10%), with a standard light schedule of 12 h day:12 h night, and fed a standard mouse maintenance pellet diet. Mice were acclimated to the animal room for 1 week before formal experiments. All animal experiments were approved by the Animal Ethics Committee of China Agricultural University.

[0084] After one week of acclimatization in the laboratory, all mice were subjected to a skin wound model. The main steps were as follows: the back hair was removed with an electric depilator, and the mice were anesthetized with an intraperitoneal injection of 4% chloral hydrate, each dosed at 10 mg / kg. The hair removal area on the back was disinfected with alcohol, a circular mark was made in advance with a marker pen, and the back skin was cut with autoclaved ophthalmic scissors to create a circular wound with a diameter of 1 cm. The skin was then excised. Subsequently, the mice were inoculated with E. coli B2, and 10 μL (1×10 9 CFU / mL) of E. coli B2 bacterial solution, and the amount of bacteria infected in each mouse was 1×10 7 In the control group, PBS was dripped onto the wound surface, and the skin was allowed to absorb the liquid. The mice were then observed until they regained consciousness. Mice infected with E. coli B2 were further divided into four groups: a vehicle-treated control group, a polymyxin E-treated group, a nootkatone-treated group, and a polymyxin E combined with nootkatone-treated group, with six mice in each group. At 1 and 24 hours after E. coli infection, a mixture of 10 μL of normal saline, 10 μL of polymyxin E (1 mg / mL, equivalent to a polymyxin E dose of 0.5 mg / kg body weight per mouse), 10 μL of nootkatone (40 mg / mL, equivalent to a nootkatone dose of 20 mg / kg body weight per mouse), or 10 μL of a mixture of polymyxin E (1 mg / mL) and nootkatone (40 mg / mL) (equivalent to a polymyxin E dose of 0.5 mg / kg body weight per mouse and a nootkatone dose of 20 mg / kg body weight per mouse) was dripped directly onto the skin infection site. 48 hours after infection, mice were anesthetized and killed. The wound and surrounding skin tissue were cut using autoclaved surgical instruments, and the subcutaneous adhesion tissue was peeled off. The skin tissue was placed in a 2mL grinding centrifuge tube, 500μL PBS and grinding beads were added, and the tissue was ground in a high-temperature, low-speed tissue grinder. The skin tissue grinding program was selected and the tissue was thoroughly ground at 4°C. The tissue homogenate was diluted to an appropriate multiple (500 times): 20μL of the stock solution was placed in a 2mL centrifuge tube and diluted in 1.98mL of sterile PBS. 100μL of the diluted bacterial solution was added dropwise to BHI solid culture medium containing polymyxin E (final concentration of 2μg / mL, the main purpose of which is to prevent contamination by other bacteria during the operation). The bacterial solution was evenly spread using a spreader and incubated in a 37°C incubator for 14-16 hours. The colony growth was observed and counted.

[0085] The results are as follows (see Figure 3 ):The bacterial load of the skin wound in the solvent treatment control group was 7.65Log 10 The bacterial loads in the polymyxin E and nootkatone treatment groups were 6.5Log 10 CFU and 7.5Log 10CFU; the colony count in the combined treatment group of polymyxin E and nootkatone decreased to 1.4Log 10 The CFU of the skin wounds was significantly different from that of the solvent control group and the groups treated with either polymyxin E or nootkatone alone (***p<0.001). This indicates that the combined treatment of polymyxin E and nootkatone significantly improved the bacterial clearance rate in skin wounds compared with either colistin E or nootkatone alone.

[0086] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of nootkatone in the preparation of a product for enhancing the antibacterial and / or bactericidal activity of a polymyxin antibiotic; the polymyxin antibiotic is selected from polymyxin E or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that: The product is a polymyxin antibiotic synergist or a drug for treating bacterial infectious diseases.

3. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on Gram-negative bacteria.

4. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on Proteobacteria bacteria.

5. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on γ-Proteobacteria bacteria.

6. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on Enterobacteriales bacteria or Pseudomonasales bacteria.

7. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on Enterobacteriaceae or Pseudomonadaceae bacteria.

8. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on Escherichia bacteria, Klebsiella bacteria or Pseudomonas bacteria.

9. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Salmonella.

10. The use according to claim 1 or 2, characterized in that: The antibacterial activity and / or bactericidal activity is an inhibitory and / or bactericidal effect on Escherichia coli carrying the MCR-1 gene, Klebsiella pneumoniae carrying the MCR-1 gene, or Salmonella carrying the MCR-1 gene.

11. An antibacterial composition, the active ingredients of which are only nootkatone and polymyxin antibiotics; The mass ratio of the nootkatone to the polymyxin antibiotic is (50-6410):1; The polymyxin antibiotic is selected from polymyxin E or a pharmaceutically acceptable salt thereof.

12. The antibacterial composition according to claim 11, characterized in that: The mass ratio of the nootkatone to the polymyxin antibiotic is (50-200):

1.

13. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on Gram-negative bacteria.

14. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on Proteobacteria bacteria.

15. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on gamma-proteobacteria bacteria.

16. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on Enterobacteriales bacteria or Pseudomonasales bacteria.

17. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on Enterobacteriaceae bacteria or Pseudomonadaceae bacteria.

18. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on Escherichia bacteria, Klebsiella bacteria or Pseudomonas bacteria.

19. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa or Salmonella.

20. The antibacterial composition according to claim 11 or 12, characterized in that: The antibacterial composition has an inhibitory and / or killing effect on Escherichia coli carrying the MCR-1 gene, Klebsiella pneumoniae carrying the MCR-1 gene, or Salmonella carrying the MCR-1 gene.

21. An antibacterial product comprising the antibacterial composition according to any one of claims 11 to 20.

22. The antibacterial product according to claim 21, characterized in that: The product is a pharmaceutical preparation, and the dosage form of the pharmaceutical preparation is selected from any one of the following: tablets, creams, capsules, oral liquids, syrups, pills, injection dosage forms, and freeze-dried powder injection dosage forms.

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