Application of β,β-dimethylacryloyl alkannin in preparing a synergist for polymyxin E

Abstract

The present invention relates to the field of biomedical technologies, and particularly to the application of β,β-dimethylacryloylalkanin in the preparation of a synergist for polymyxin E. The present invention provides the application of β,β-dimethylacryloylalkanin in the preparation of a synergist for polymyxin E. The present invention discovers for the first time that β,β-dimethylacryloylalkanin can restore the inhibitory effect of polymyxin E on polymyxin E-resistant bacteria, reverse the resistance to polymyxin E while reducing the dosage of polymyxin E, and can also improve the antibacterial activity of polymyxin E against polymyxin E-sensitive bacteria, having important application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the use of β,β-dimethylacryloyl alkannin in the preparation of a synergist for polymyxin E. Background Art

[0002] Polymyxin E is a polypeptide antibiotic produced by Bacillus polymyxa, and has an inhibitory effect on most Gram-negative bacteria. The mechanism by which polymyxin E exerts its antibacterial effect is that it can interact with the lipid A part in the outer membrane LPS of bacteria, resulting in changes in bacterial membrane permeability and the outflow of contents, thereby inhibiting bacterial growth and causing bacterial death. In recent years, with the increasingly severe form of bacterial drug resistance, polymyxin E has become one of the important drugs for the clinical treatment of multi-drug resistant bacterial infections. At the same time, the continuous increase in the drug resistance rate of multi-drug resistant pathogens to polymyxin E has increased the mortality rate of sepsis patients, which has further increased the clinical application burden of polymyxin E.

[0003] Research has shown that mutations in the chromosomal-mediated two-component systems PhoPQ and PmrAB can regulate the expression of eptA or arnT, insert pEtN or L-Ara4N into lipid A, thereby reducing the affinity of polymyxin for LPS. In addition, the emergence of the plasmid-mediated polymyxin resistance gene mcr-1 in 2015 and the widespread prevalence of the mcr family have seriously threatened the clinical application of polymyxin E. Therefore, it is urgent to find a synergist that can increase the sensitivity of bacteria to polymyxin E to restore the sensitivity of bacteria to polymyxin E.

[0004] β,β-Dimethylacryloyl alkannin is a hydroxy naphthoquinone compound and is one of the active ingredients of the traditional Chinese herbal medicine Lithospermum erythrorhizon commonly used in clinics. It has various pharmacological effects such as antibacterial, anti-inflammatory, antioxidant, anti-tumor, immunomodulatory, and promoting wound healing. Research has shown that β,β-dimethylacryloyl alkannin inhibits the proliferation of colorectal cancer cells and skin cancer cells by directly targeting fibroblast growth factor receptor 1 (FGFR1) and inhibiting its activity. In addition, β,β-dimethylacryloyl alkannin can also be used as an antagonist of TLR7 / 8, providing ideas for the treatment of autoimmune diseases similar to the pathogenesis of psoriasis. However, up to now, there have been no reports on the combination of β,β-dimethylacryloyl alkannin and polymyxin E to inhibit polymyxin E-resistant bacteria. Summary of the Invention

[0005] The purpose of the present invention is to provide the use of β,β-dimethylacryloyl alkannin in the preparation of a synergist for polymyxin E.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the use of β,β-dimethylacryloyl alkannin in the preparation of a synergist for polymyxin E.

[0008] The present invention also provides an antibacterial composition comprising β,β-dimethylacryloyl alkannin and polymyxin E.

[0009] Preferably, the mass ratio of β,β-dimethylacryloyl alkannin to polymyxin E is 0.5-512:1.

[0010] Preferably, the final concentration of β,β-dimethylacryloyl alkannin in the composition is 1-8 μg / mL;

[0011] Preferably, the final concentration of polymyxin E in the composition is 0.0078-2 μg / mL.

[0012] The present invention also provides the use of the antibacterial composition in inhibiting polymyxin E-resistant bacteria or polymyxin E-sensitive bacteria.

[0013] Preferably, the polymyxin E-resistant bacteria are plasmid-mediated mcr-positive bacteria or bacteria resistant due to mutations in the two-component regulatory system; the polymyxin E-sensitive bacteria are Gram-negative bacteria sensitive to polymyxin E.

[0014] Preferably, the mcr-positive bacteria and / or bacteria resistant due to mutations in the two-component regulatory system are: Klebsiella pneumoniae 20 positive for mcr-1, Klebsiella pneumoniae 126 positive for mcr-8, Klebsiella pneumoniae 57 positive for mcr-9, Klebsiella pneumoniae 251 with mgrB mutation, Escherichia coli ZJ71 positive for mcr-1, Escherichia coli pHSG299-mcr-3 positive for mcr-3, two-component regulatory system mutant Escherichia coli BW25113-pmrBWT, or Salmonella 5 positive for mcr-9;

[0015] Preferably, the Gram-negative bacteria sensitive to polymyxin E are Klebsiella pneumoniae, Escherichia coli, Salmonella, Acinetobacter baumannii, or Pseudomonas aeruginosa.

[0016] Preferably, when the antibacterial composition acts on Klebsiella pneumoniae, the mass ratio of β,β-dimethylacryloyl alkannin to polymyxin E is 0.5-16:1; at this time, the final concentration of β,β-dimethylacryloyl alkannin in the antibacterial composition is 1-4 μg / mL, and the final concentration of polymyxin E is 0.125-2 μg / mL;

[0017] Preferably, when the antibacterial composition acts on Escherichia coli, the mass ratio of β,β-dimethylacryloyl alkannin to polymyxin E is 8-64:1; at this time, the final concentration of β,β-dimethylacryloyl alkannin in the antibacterial composition is 4-8 μg / mL, and the final concentration of polymyxin E is 0.125-0.5 μg / mL;

[0018] Preferably, when the antibacterial composition acts on Salmonella resistant to polymyxin E, the mass ratio of β,β-dimethylacryloyl alkannin to polymyxin E is 12-20:1; at this time, the final concentration of β,β-dimethylacryloyl alkannin in the antibacterial composition is 6-10 μg / mL, and the final concentration of polymyxin E is 0.4-0.6 μg / mL;

[0019] Preferably, when the antibacterial composition acts on polymyxin E-sensitive bacteria, the mass ratio of β,β-dimethylacryloyl alkannin to polymyxin E is 16-512:1; at this time, the final concentration of β,β-dimethylacryloyl alkannin in the antibacterial composition is 2-4 μg / mL, and the final concentration of polymyxin E is 0.0078-0.125 μg / mL (except for Salmonella and Pseudomonas aeruginosa).

[0020] The present invention also provides an antibacterial drug, comprising the antibacterial composition described above.

[0021] Preferably, the dosage form of the antibacterial drug is tablet, cream, capsule, sustained-release tablet, controlled-release tablet, oral liquid, syrup, dripping pill, injection dosage form or freeze-dried powder injection dosage form.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Through the broth microdilution checkerboard method and time-kill curve experiment, the present invention finds that β,β-dimethylacryloyl alkannin can synergistically inhibit plasmid-mediated mcr-positive bacteria and drug-resistant bacteria caused by mutations in the two-component regulatory system (such as mcr-1 positive Klebsiella pneumoniae 20, mcr-8 positive Klebsiella pneumoniae 126, mcr-9 positive Klebsiella pneumoniae 57, mgrB mutant Klebsiella pneumoniae 251, mcr-1 positive Escherichia coli ZJ71, mcr-3 positive Escherichia coli pHSG299-mcr-3, two-component regulatory system mutant Escherichia coli BW25113-pmrBWT, mcr-9 positive Salmonella 5). While reducing the dosage of polymyxin E, it restores the sensitivity of drug-resistant bacteria to polymyxin E. In addition, β,β-dimethylacryloyl alkannin can also improve the antibacterial activity of polymyxin E against polymyxin-sensitive bacteria (such as Klebsiella pneumoniae ATCC 13883, Escherichia coli ATCC 25922, Acinetobacter baumannii ATCC 19606).

[0024] The present invention for the first time discovers that β,β-dimethylacryloylalkanin can restore the sensitivity of polymyxin E to polymyxin E-resistant bacteria, improve the antibacterial activity of polymyxin E against polymyxin E-resistant bacteria, and at the same time improve the antibacterial activity of polymyxin E against polymyxin-sensitive bacteria, which has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0026] Figure 1 It is a heat map of the broth microdilution checkerboard method for detecting the minimum inhibitory concentration of β,β-dimethylacryloylalkanin and polymyxin E against polymyxin E-resistant Klebsiella pneumoniae 20;

[0027] Figure 2 It is a heat map of the broth microdilution checkerboard method for detecting the minimum inhibitory concentration of β,β-dimethylacryloylalkanin and polymyxin E against polymyxin E-resistant Klebsiella pneumoniae 126;

[0028] Figure 3 It is a heat map of the broth microdilution checkerboard method for detecting the minimum inhibitory concentration of β,β-dimethylacryloylalkanin and polymyxin E against polymyxin E-resistant Klebsiella pneumoniae 57;

[0029] Figure 4 It is a heat map of the broth microdilution checkerboard method for detecting the minimum inhibitory concentration of β,β-dimethylacryloylalkanin and polymyxin E against polymyxin E-resistant Klebsiella pneumoniae 251;

[0030] Figure 5 It is a heat map of the broth microdilution checkerboard method for detecting the minimum inhibitory concentration of β,β-dimethylacryloylalkanin and polymyxin E against polymyxin E-resistant Escherichia coli ZJ71;

[0031] Figure 6 It is a heat map of the broth microdilution checkerboard method for detecting the minimum inhibitory concentration of β,β-dimethylacryloylalkanin and polymyxin E against polymyxin E-resistant Escherichia coli pHSG299-mcr-3;

[0032] Figure 7 It is a heat map of the broth microdilution checkerboard method for detecting the minimum inhibitory concentration of β,β-dimethylacryloylalkanin and polymyxin E against polymyxin E-resistant Escherichia coli BW25113-pmrBWT;

[0033] Figure 8 It is a heat map of the minimum inhibitory concentration of β,β-dimethylacryloylakannin and polymyxin E against polymyxin E-resistant Salmonella 5 detected by the broth microdilution checkerboard method;

[0034] Figure 9 It is a heat map of the minimum inhibitory concentration of β,β-dimethylacryloylakannin and polymyxin E against polymyxin E-sensitive Klebsiella pneumoniae ATCC 13883 detected by the broth microdilution checkerboard method;

[0035] Figure 10 It is a heat map of the minimum inhibitory concentration of β,β-dimethylacryloylakannin and polymyxin E against polymyxin E-sensitive Escherichia coli ATCC 25922 detected by the broth microdilution checkerboard method;

[0036] Figure 11 It is a heat map of the minimum inhibitory concentration of β,β-dimethylacryloylakannin and polymyxin E against polymyxin E-sensitive Salmonella ATCC 13076 detected by the broth microdilution checkerboard method;

[0037] Figure 12 It is a heat map of the minimum inhibitory concentration of β,β-dimethylacryloylakannin and polymyxin E against polymyxin E-sensitive Acinetobacter baumannii ATCC 19606 detected by the broth microdilution checkerboard method;

[0038] Figure 13 It is a heat map of the minimum inhibitory concentration of β,β-dimethylacryloylakannin and polymyxin E against polymyxin E-sensitive Pseudomonas aeruginosa PAO1 detected by the broth microdilution checkerboard method;

[0039] Figure 14 It is the time-kill curve of different treatment groups against polymyxin E-resistant Klebsiella pneumoniae 126. Specific implementation manners

[0040] The technical solutions provided by the present invention will be described in detail below in conjunction with embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0041] Materials and reagents used in the present invention:

[0042] Polymyxin E is a product of Shanghai Aladdin Biochemical Technology Co., Ltd., with a potency of ≥19,000 U / mg.

[0043] β,β-Dimethylacryloylakannin is a product of Shanghai TargetMol Co., Ltd., with an effective content of 98%.

[0044] Polymyxin E-resistant bacteria (20 Klebsiella pneumoniae positive for mcr-1, 126 Klebsiella pneumoniae positive for mcr-8, 57 Klebsiella pneumoniae positive for mcr-9, 251 Klebsiella pneumoniae with mgrB mutation, Escherichia coli ZJ71 positive for mcr-1, Escherichia coli pHSG299-mcr-3 positive for mcr-3, Escherichia coli BW25113-pmrBWT with two-component regulatory system mutation, 5 Salmonella positive for mcr-9) and Polymyxin E-sensitive bacteria (Klebsiella pneumoniae ATCC 13883, Escherichia coli ATCC 25922, Salmonella ATCC13076, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa PAO1) were provided by the National Veterinary Drug Safety Evaluation Center (Beijing).

[0045] M-H II (cation-adjusted) liquid medium: Weigh 11 g of M-H II (cation-adjusted) liquid medium powder (BD) into 500 mL of distilled water, sterilize it by autoclaving at 121 °C for 20 min, and cool for later use.

[0046] MH solid medium: Weigh 19 g of MH agar medium powder (Beijing Land Bridge Technology Co., Ltd.) and add it to 500 mL of distilled water, sterilize it by autoclaving at 121 °C for 20 min, and cool for later use.

[0047] Example 1

[0048] According to the CLSI standard, the broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of β,β-dimethylacryloylakannin and polymyxin E against polymyxin E-resistant bacteria and sensitive bacteria. The specific steps are as follows:

[0049] 1. Adjust the bacterial suspensions of polymyxin E-resistant bacteria and sensitive bacteria in the logarithmic growth phase to 0.5 McFarland turbidity using a McFarland turbidimeter, and then dilute them 100-fold with M-H II (cation-adjusted) broth medium to obtain diluted solutions of polymyxin E-resistant bacteria and sensitive bacteria with a concentration of approximately 1×10 6 CFU / mL. The specific types of bacteria are shown in Table 1.

[0050] 2. Weigh an appropriate amount of polymyxin E powder, dissolve it in deionized water to obtain a polymyxin E solution with a concentration of 5120 μg / mL, and then filter it through a sterile filter membrane (pore size 0.22 μm) to obtain a polymyxin E stock solution.

[0051] Weigh an appropriate amount of β,β-dimethylacryloylakannin powder, dissolve it in dimethyl sulfoxide (DMSO) to obtain a β,β-dimethylacryloylakannin solution with a concentration of 5120 μg / mL, and then filter it through a sterile filter membrane (pore size 0.22 μm) to obtain a β,β-dimethylacryloylakannin stock solution.

[0052] 3. Take two 96-well microplates. Add polymyxin E stock solution and β,β-dimethylacryloylakannin stock solution to the first wells respectively, and perform 2-fold serial dilution with M-H II (cation-adjusted) broth medium. Then inoculate 100 μL of the polymyxin E-resistant bacteria dilution into each well, so that the concentration ranges of β,β-dimethylacryloylakannin and polymyxin E applied are 0 - 128 μg / mL. Among them, the positive control is the diluted bacterial solution (without drugs), and the blank control is M-H II (cation-adjusted) broth.

[0053] 4. Incubate the 96-well microplates after step 3 at 37 °C for 16 - 18 h.

[0054] Experimental results: As shown in Table 1, the MIC of β,β-dimethylacryloylakannin against polymyxin E-resistant bacteria is >128 μg / mL, and the MIC of polymyxin E against polymyxin E-resistant bacteria is ≥2 μg / mL, indicating that these strains are all resistant to polymyxin E. The MIC of β,β-dimethylacryloylakannin against polymyxin E-sensitive bacteria is >128 μg / mL, and the MIC of polymyxin E against polymyxin E-sensitive bacteria is 0.125 - 1 μg / mL.

[0055] Table 1 MICs (μg / mL) of β,β-dimethylacryloylakannin and polymyxin E

[0056]

[0057]

[0058] Example 2

[0059] The broth microdilution checkerboard method was used to detect the fractional inhibitory concentration index (FICI) of β,β-dimethylacryloylakannin and polymyxin E against the polymyxin E-resistant bacteria and sensitive bacteria in Example 1. The specific steps are as follows:

[0060] 1. Adjust the bacterial solutions of the polymyxin E-resistant bacteria and sensitive bacteria in the logarithmic growth phase to 0.5 McFarland turbidity with a McFarland turbidimeter, and then dilute them 100-fold with M-H II (cation-adjusted) broth medium to obtain polymyxin E-resistant bacteria and sensitive bacteria dilution solutions with a concentration of about 1×10 6 CFU / mL.

[0061] 2. Weigh an appropriate amount of polymyxin E powder, dissolve it in deionized water to obtain a polymyxin E solution with a concentration of 5120 μg / mL, and then filter it through a sterile filter membrane (pore size 0.22 μm) to obtain a polymyxin E stock solution. Weigh an appropriate amount of β,β-dimethylacryloyl alkannin powder, dissolve it in dimethyl sulfoxide (DMSO) to obtain a β,β-dimethylacryloyl alkannin solution with a concentration of 5120 μg / mL, and then filter it through a sterile filter membrane (pore size 0.22 μm) to obtain a β,β-dimethylacryloyl alkannin stock solution.

[0062] 3. Take a 96-well microplate, and inoculate 100 μL of drug solution (prepared by diluting and mixing β,β-dimethylacryloyl alkannin and polymyxin E in proportion) and 100 μL of diluted polymyxin E-resistant bacteria and sensitive bacteria solution into each well.

[0063] For polymyxin E-resistant Klebsiella pneumoniae 20, in each row from top to bottom, the concentration of β,β-dimethylacryloyl alkannin in each well is 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in turn; in each column from left to right, the concentration of polymyxin E in each well is 0, 1, 2, 4, 8, 16, 32, 64 μg / mL in turn.

[0064] For polymyxin E-resistant Klebsiella pneumoniae 126, in each row from top to bottom, the concentration of β,β-dimethylacryloyl alkannin in each well is 0, 0.5, 1, 2, 4, 8, 16, 32 μg / mL in turn; in each column from left to right, the concentration of polymyxin E in each well is 0, 2, 4, 8, 16, 32, 64, 128 μg / mL in turn.

[0065] For polymyxin E-resistant Klebsiella pneumoniae 57, in each row from top to bottom, the concentration of β,β-dimethylacryloyl alkannin in each well is 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in turn; in each column from left to right, the concentration of polymyxin E in each well is 0, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1, 2 μg / mL in turn.

[0066] For polymyxin E-resistant Klebsiella pneumoniae 251, in each row from top to bottom, the concentration of β,β-dimethylacryloyl alkannin in each well is 0, 0.5, 1, 2, 4, 8, 16, 32 μg / mL in turn; in each column from left to right, the concentration of polymyxin E in each well is 0, 2, 4, 8, 16, 32, 64, 128 μg / mL in turn.

[0067] For the polymyxin E-resistant Escherichia coli ZJ71, in each row from top to bottom, the concentrations of β,β-dimethylacryloylakannin in each well are 0, 0.5, 1, 2, 4, 8, 16, 32 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.125, 0.25, 0.5, 1, 2, 4, 8 μg / mL in sequence.

[0068] For the polymyxin E-resistant Escherichia coli pHSG299-mcr-3, in each row from top to bottom, the concentrations of β,β-dimethylacryloylakannin in each well are 0, 0.5, 1, 2, 4, 8, 16, 32 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1, 2 μg / mL in sequence.

[0069] For the polymyxin E-resistant Escherichia coli BW25113-pmrBWT, in each row from top to bottom, the concentrations of β,β-dimethylacryloylakannin in each well are 0, 0.5, 1, 2, 4, 8, 16, 32 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in sequence.

[0070] For the polymyxin E-resistant Salmonella 5, in each row from top to bottom, the concentrations of β,β-dimethylacryloylakannin in each well are 0, 0.5, 1, 2, 4, 8, 16, 32 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in sequence.

[0071] For the polymyxin E-sensitive Klebsiella pneumoniae ATCC 13883, in each row from top to bottom, the concentrations of β,β-dimethylacryloylakannin in each well are 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5 μg / mL in sequence.

[0072] For the polymyxin E-sensitive Escherichia coli ATCC 25922, in each row from top to bottom, the concentrations of β,β-dimethylacryloylakannin in each well are 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.0039, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25 μg / mL in sequence.

[0073] For the polymyxin E-sensitive bacterium Salmonella ATCC 13076, in each row from top to bottom, the concentrations of β,β-dimethylacryloyl alkannin in each well are 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5 μg / mL in sequence.

[0074] For the polymyxin E-sensitive bacterium Acinetobacter baumannii ATCC 19606, in each row from top to bottom, the concentrations of β,β-dimethylacryloyl alkannin in each well are 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5 μg / mL in sequence.

[0075] For the polymyxin E-sensitive bacterium Pseudomonas aeruginosa PAO1, in each row from top to bottom, the concentrations of β,β-dimethylacryloyl alkannin in each well are 0, 0.25, 0.5, 1, 2, 4, 8, 16 μg / mL in sequence; in each column from left to right, the concentrations of polymyxin E in each well are 0, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.5, 1 μg / mL in sequence.

[0076] 4. After completing step 3, take the 96-well microplate and incubate it at 37 °C for 16 - 18 h.

[0077] 5. After completing step 4, calculate the fractional inhibitory concentration index (FICI) value according to the minimum inhibitory concentration (MIC) of each drug to determine whether β,β-dimethylacryloyl alkannin and polymyxin E have a synergistic effect.

[0078] FICI = (MIC 多黏菌素E联合 / MIC 多黏菌素E单用 ) + (MIC β,β-二甲基丙烯酰阿卡宁联合 / MIC β,β-二甲基丙烯酰阿卡宁单用 )

[0079] The judgment criterion is: when FICI ≤ 0.5, it is determined that the two have a synergistic effect.

[0080] Test results: As Figures 1 to 13 shown. The optimal drug combination converted by the FICI method is marked with a triangle. The experiment was repeated three times, and the results were averaged. The statistical results are shown in Table 2.

[0081] Table 2 The combined antibacterial effect of β,β-dimethylacryloyl alkannin combined with polymyxin E on polymyxin E-resistant bacteria and sensitive bacteria

[0082]

[0083]

[0084] The results showed that β,β-dimethylacryloylalkanin combined with polymyxin E could significantly reduce the MIC values of polymyxin E against polymyxin E-resistant Klebsiella pneumoniae, Escherichia coli and Salmonella, as well as the MIC values of polymyxin E against polymyxin E-sensitive Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumannii, and there was no synergistic effect on polymyxin E-sensitive Salmonella and Pseudomonas aeruginosa.

[0085] For polymyxin E-resistant Klebsiella pneumoniae, the mass ratio of β,β-dimethylacryloylalkanin to polymyxin E was 0.5 - 16:1; at this time, the minimum inhibitory concentration of β,β-dimethylacryloylalkanin was 1 - 4 μg / mL, and the minimum inhibitory concentration of polymyxin E was 0.125 - 2 μg / mL.

[0086] For polymyxin E-resistant Escherichia coli, the mass ratio of β,β-dimethylacryloylalkanin to polymyxin E was 8 - 64:1; at this time, the minimum inhibitory concentration of β,β-dimethylacryloylalkanin was 4 - 8 μg / mL, and the minimum inhibitory concentration of polymyxin E was 0.125 - 0.5 μg / mL.

[0087] For polymyxin E-resistant Salmonella, the mass ratio of β,β-dimethylacryloylalkanin to polymyxin E was 16:1; at this time, the minimum inhibitory concentration of β,β-dimethylacryloylalkanin was 8 μg / mL, and the minimum inhibitory concentration of polymyxin E was 0.5 μg / mL.

[0088] For polymyxin E-sensitive bacteria, the mass ratio of β,β-dimethylacryloylalkanin to polymyxin E was 16 - 512:1; at this time, the minimum inhibitory concentration of β,β-dimethylacryloylalkanin was 2 - 4 μg / mL, and the minimum inhibitory concentration of polymyxin E was 0.0078 - 0.125 μg / mL (except for Salmonella and Pseudomonas aeruginosa).

[0089] The above results showed that β,β-dimethylacryloylalkanin could synergistically exert antibacterial effects with polymyxin E, thereby treating infections caused by polymyxin E-resistant bacteria and simultaneously increasing the sensitivity of polymyxin E-sensitive bacteria to polymyxin E.

[0090] Example 3

[0091] Time-kill curve of β,β-dimethylacryloylalkanin combined with polymyxin E against polymyxin E-resistant Klebsiella pneumoniae 126:

[0092] 1. Adjust the bacterial suspension of polymyxin E-resistant Klebsiella pneumoniae 126 in the logarithmic growth phase to 0.5 McFarland turbidity using a McFarland turbidimeter, and then dilute it 100-fold with MH broth medium to obtain a diluted solution of polymyxin E-resistant bacteria with a concentration of approximately 1×10 6 CFU / mL.

[0093] 2. Take 12 test tubes and randomly divide them into a blank group, a β,β-dimethylacryloylakannin group, a polymyxin E group, and a combination group, with 3 test tubes in each group. Perform the following treatments:

[0094] Blank group (Control): Do not add any drugs to the test tubes.

[0095] β,β-Dimethylacryloylakannin group: According to the MIC value of β,β-dimethylacryloylakannin against Klebsiella pneumoniae 126 in the broth microdilution checkerboard method experiment, add a β,β-dimethylacryloylakannin stock solution of 5120 μg / mL to make the final concentration of β,β-dimethylacryloylakannin in the system 1 / 8×MIC value (16 μg / mL) of β,β-dimethylacryloylakannin against Klebsiella pneumoniae 126;

[0096] Polymyxin E group: According to the MIC value of polymyxin E against Klebsiella pneumoniae 126 in the broth microdilution checkerboard method experiment, add a polymyxin E stock solution of 5120 μg / mL to make the final concentration of polymyxin E in the system 1 / 32×MIC value (16 μg / mL) of polymyxin E against Klebsiella pneumoniae 126;

[0097] Combination group (Combination): Add β,β-dimethylacryloylakannin and polymyxin E to the system so that the final concentrations are 1 / 8×MIC and 1 / 32×MIC values of β,β-dimethylacryloylakannin and polymyxin E against Klebsiella pneumoniae 126, respectively.

[0098] 3. Then add an equal volume (1 mL) of the diluted resistant bacteria solution to each test tube. Subsequently, place the above 12 test tubes in a shaker at 37 °C and incubate with shaking at 200 rpm. This is 0 h. During the incubation, 100 μL of the culture solution is taken at 0, 2, 4, 8, 12, and 24 h respectively for 10-fold serial dilution, and then spread on MH solid medium and cultured at 37 °C for 24 h for colony counting.

[0099] 4. After completing step 3, plot a time-kill curve with the culture time as the abscissa and the log10 value of the colony count as the ordinate.

[0100] The time-kill curve of polymyxin E-resistant Klebsiella pneumoniae 126 is shown in Figure 14 (CFU is colony-forming unit).

[0101] The results showed that Figure 14 ), compared with the blank group, β,β-dimethylacryloyl alkannin group and polymyxin E group, the combined group could completely inhibit bacterial growth at 2 h, and the colony-forming units decreased by about 9 log10 values at 24 h, significantly enhancing the antibacterial activity of polymyxin E against polymyxin E-resistant bacteria and showing a synergistic bactericidal effect.

[0102] As can be seen from the above examples, β,β-dimethylacryloyl alkannin combined with polymyxin E can enhance the antibacterial activity of polymyxin E against polymyxin E-resistant Klebsiella pneumoniae 126, that is, β,β-dimethylacryloyl alkannin combined with polymyxin E can inhibit polymyxin E-resistant Klebsiella pneumoniae 126.

[0103] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of an antibacterial composition in the preparation of a therapeutic drug for inhibiting polymyxin E-resistant bacteria or polymyxin E-sensitive bacteria, characterized in that: The antibacterial composition comprises β,β-dimethylacryloyl akanin and polymyxin E, with a mass ratio of 0.5-512:1; the final concentration of β,β-dimethylacryloyl akanin in the composition is 1-8 μg / mL; the final concentration of polymyxin E in the composition is 0.0078-2 μg / mL; The polymyxin E-resistant bacteria are plasmid-mediated mcr-positive bacteria or resistant bacteria caused by mutations in the binary regulatory system; the polymyxin E-sensitive bacteria are gram-negative bacteria that are sensitive to polymyxin E; Said mcr Positive bacteria and / or resistance caused by mutations in the binary regulatory system are: mcr-1 Positive Klebsiella pneumoniae 20, mcr-8 Positive Klebsiella pneumoniae 126, mcr-9 Positive Klebsiella pneumoniae 57, mgB Mutant Klebsiella pneumoniae 251, mcr-1 Positive Escherichia coli ZJ71, mcr-3 Positive E. coli pHSG299- mcr-3 , dual regulatory system mutant Escherichia coli BW25113-pmrBWT or mcr-9 positive Salmonella 5; The Gram-negative bacteria that are sensitive to polymyxin E are Klebsiella pneumoniae, Escherichia coli, Salmonella, Acinetobacter baumannii or Pseudomonas aeruginosa.

2. The use according to claim 1, characterized in that: When the antibacterial composition acts on Klebsiella pneumoniae, the mass ratio of β,β-dimethylacryloyl akanin to polymyxin E is 0.5-16:1; at this time, the final concentration of β,β-dimethylacryloyl akanin in the antibacterial composition is 1-4 μg / mL, and the final concentration of polymyxin E is 0.125-2 μg / mL; When the antibacterial composition acts on Escherichia coli, the mass ratio of β,β-dimethylacryloyl akanin to polymyxin E is 8-64:1; at this time, the final concentration of β,β-dimethylacryloyl akanin in the antibacterial composition is 4-8 μg / mL, and the final concentration of polymyxin E is 0.125-0.5 μg / mL; When the antibacterial composition acts on Salmonella resistant to polymyxin E, the mass ratio of β,β-dimethylacryloyl akanin to polymyxin E is 12-20:1; at this time, the final concentration of β,β-dimethylacryloyl akanin in the antibacterial composition is 6-10 μg / mL, and the final concentration of polymyxin E is 0.4-0.6 μg / mL; When the antibacterial composition acts on polymyxin E-sensitive bacteria, the mass ratio of β,β-dimethylacryloyl akanin to polymyxin E is 16-512:1; at this time, the final concentration of β,β-dimethylacryloyl akanin in the antibacterial composition is 2-4 μg / mL, and the final concentration of polymyxin E is 0.0078-0.125 μg / mL.

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