Pharmaceutical composition for inhibiting gram-negative bacteria and application thereof

Abstract

The invention discloses a pharmaceutical composition for inhibiting gram-negative bacteria and application of the pharmaceutical composition, and belongs to the technical field of biological medicines. The medicine composition is prepared from vitamin K3 and polymyxin. A chessboard method minimum inhibitory concentration test proves that the vitamin K3 can obviously enhance the antibacterial efficiency of the polymyxin, and especially has obvious effects on resisting mcr-1 positive polymyxin drug-resistant escherichia coli, high-level polymyxin drug-resistant escherichia coli and acinetobacter baumannii. In addition, an in-vitro bacterial growth curve experiment is also utilized to further verify the time-dependent bactericidal dynamic characteristics of the pharmaceutical composition. The vitamin K3 disclosed by the invention can recover the antibacterial effect of the polymyxin on drug-resistant strains, and the drug resistance of bacteria to the polymyxin is reversed while the use amount of the polymyxin is reduced. The invention is helpful to alleviate the increasingly serious bacterial drug resistance problem, provides a new treatment strategy for clinical treatment of bacterial infectious diseases, and has important application value.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for inhibiting Gram-negative bacteria and its application. More specifically, the present invention relates to a pharmaceutical composition composed of vitamin K3 and polymyxin and its application in inhibiting Gram-negative bacteria, especially polymyxin-resistant bacteria. The present invention belongs to the field of biomedicine technology. Background Art

[0002] With the widespread use of antibiotics, bacterial resistance has become one of the major challenges facing global public health. In particular, the emergence of multi-drug-resistant and extensively drug-resistant strains in Gram-negative bacteria has made traditional antibiotic treatment gradually ineffective. The results of The Lancet's analysis of data from 204 countries around the world show that in 2019, antimicrobial resistance infections directly caused about 1.27 million deaths worldwide and indirectly caused about 4.95 million deaths. It is estimated that by 2050, this number will exceed 10 million.

[0003] Polymyxins are cationic polypeptide antibiotics that have a strong and rapid bactericidal effect on most Gram-negative bacteria. The antibacterial mechanism of polymyxins is that they can bind to negatively charged LPS in the cell membrane of Gram-negative bacteria, causing changes in the permeability of bacterial cell membranes, leading to the leakage of important substances such as bacterial nucleic acids and amino acids, thereby inhibiting bacterial growth and causing bacterial death. In recent years, with the increasingly severe form of bacterial resistance, polymyxins have become one of the "last lines of defense" for the clinical treatment of multidrug-resistant Gram-negative bacteria infections. However, due to the emergence of polymyxin resistance genes (mcr), the therapeutic effect of clinical polymyxins has been significantly reduced, seriously threatening the service life of this important antibacterial drug, posing a great threat to human medicine, veterinary clinics and food safety, and has attracted global attention. At present, the development of new antibiotics is difficult, time-consuming and costly, and can no longer cope with the current severe problem of bacterial resistance. Therefore, screening antibiotic synergists to restore the antibacterial activity of existing antibacterial drugs is more economical and effective than developing new drugs.

[0004] Menadione (vitamin K3, VK3) is a fat-soluble vitamin belonging to the vitamin K class that is present in food and is non-toxic to humans. Fat-soluble vitamins are present in small amounts in food but are essential for body function and, like other vitamins, have recently been recognized to play further physiological roles. Previous studies on VK3 have focused on studies related to VK3 and its analogs as anticancer agents to induce apoptosis in cancer cells, and have been found to be effective in vitro against breast, bladder, liver, breast, oral, pharyngeal and blood cancers, as well as parental cancer and multidrug-resistant leukemia cell lines. However, there are no reports on VK3 enhancing the antibacterial activity of polymyxins. Summary of the invention

[0005] The object of the present invention is to provide a pharmaceutical composition for inhibiting Gram-negative bacteria, especially polymyxin-resistant bacteria, and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The present invention confirms that vitamin K3 can significantly enhance the antibacterial efficacy of polymyxin through the chessboard method minimum inhibitory concentration test, especially against mcr-1 positive polymyxin-resistant Escherichia coli, high-level polymyxin-resistant Escherichia coli and Acinetobacter baumannii. In addition, the present invention further verifies the time-dependent bactericidal kinetic characteristics of the pharmaceutical composition by using an in vitro bacterial growth curve experiment. The present invention reveals that vitamin K3 can restore the antibacterial effect of polymyxin on resistant strains, and reverse bacterial resistance to polymyxin while reducing the amount of polymyxin used.

[0008] Based on the above research, the present invention proposes a pharmaceutical composition for inhibiting Gram-negative bacteria, wherein the pharmaceutical composition consists of vitamin K3 and polymyxin.

[0009] Furthermore, the present invention also proposes the use of the pharmaceutical composition in the preparation of drugs for inhibiting Gram-negative bacteria.

[0010] Among them, preferably, the Gram-negative bacteria are polymyxin-resistant bacteria.

[0011] Among them, preferably, the polymyxin-resistant bacteria include standard Escherichia coli ATCC MG1655, mcr-1 positive Escherichia coli, high-level polymyxin-resistant Escherichia coli YPE3, standard Acinetobacter baumannii strain ATCC 19606, standard Klebsiella pneumoniae strain ATCC 700603, standard Pseudomonas aeruginosa strain ATCC 27853, standard Shigella ATCC12022 and standard Salmonella ATCC SL1344.

[0012] Wherein, preferably, when the pharmaceutical composition is used to inhibit standard Escherichia coli ATCC MG1655, the mass ratio of VK3 to polymyxin is 256.8:1; at this time, the minimum inhibitory concentration of VK3 is 0.391mM, and the minimum inhibitory concentration of polymyxin is 0.25μg / mL;

[0013] When the pharmaceutical composition is used to inhibit mcr-1 positive Escherichia coli, the optimal mass ratio of VK3 to polymyxin is 256.8:1; at this time, the minimum inhibitory concentration of VK3 is 0.391mM, and the minimum inhibitory concentration of polymyxin is 0.25μg / mL;

[0014] When the pharmaceutical composition is used to inhibit high-level polymyxin-resistant Escherichia coli YPE3, the optimal mass ratio of VK3 to polymyxin is 8.025:1; at this time, the minimum inhibitory concentration of VK3 is 0.391mM, and the minimum inhibitory concentration of polymyxin is 8μg / mL;

[0015] When the pharmaceutical composition is used to inhibit Acinetobacter baumannii strain ATCC 19606, the optimal mass ratio of VK3 to polymyxin is 16.02:1; at this time, the minimum inhibitory concentration of VK3 is 0.0488mM, and the minimum inhibitory concentration of polymyxin is 0.5μg / mL;

[0016] When the pharmaceutical composition is used to inhibit Klebsiella pneumoniae strain ATCC 700603, the optimal mass ratio of VK3 to polymyxin is 128:1; at this time, the minimum inhibitory concentration of VK3 is 0.781mM, and the minimum inhibitory concentration of polymyxin is 1μg / mL;

[0017] When the pharmaceutical composition is used to inhibit Pseudomonas aeruginosa strain ATCC 27853, the optimal mass ratio of VK3 to polymyxin is 3.94:1; at this time, the minimum inhibitory concentration of VK3 is 0.024mM, and the minimum inhibitory concentration of polymyxin is 1μg / mL;

[0018] When the pharmaceutical composition is used to inhibit Shigella ATCC 12022, the optimal mass ratio of VK3 to polymyxin is 7.88:1; at this time, the minimum inhibitory concentration of VK3 is 0.0244mM, and the minimum inhibitory concentration of polymyxin is 0.5μg / mL;

[0019] When the pharmaceutical composition is used to inhibit Salmonella ATCC SL1344, the optimal mass ratio of VK3 to polymyxin is 512:1; at this time, the minimum inhibitory concentration of VK3 is 0.781mM, and the minimum inhibitory concentration of polymyxin is 0.25μg / mL.

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

[0021] The present invention discloses for the first time the synergistic effect of vitamin K3 and polymyxin. The chessboard method minimum inhibitory concentration test confirms that vitamin K3 can significantly enhance the antibacterial efficacy of polymyxin, especially against mcr-1 positive polymyxin-resistant Escherichia coli, high-level polymyxin-resistant Escherichia coli and Acinetobacter baumannii. In addition, the present invention further verifies the time-dependent bactericidal kinetic characteristics of the drug combination using an in vitro bacterial growth curve experiment. The present invention reveals that vitamin K3 can restore the antibacterial effect of polymyxin on resistant strains, and reverse bacterial resistance to polymyxin while reducing the amount of polymyxin used. The present invention helps to alleviate the increasingly serious problem of bacterial resistance, provides a new treatment strategy for the clinical treatment of bacterial infectious diseases, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 To detect the minimum inhibitory concentration of VK3 combined with polymyxin against Escherichia coli MG1655 using the checkerboard method;

[0023] Figure 2 The checkerboard method was used to detect the minimum inhibitory concentration of VK3 combined with polymyxin against mcr-1 positive Escherichia coli B5;

[0024] Figure 3 To detect the minimum inhibitory concentration of VK3 combined with polymyxin against high-level polymyxin-resistant Escherichia coli YPE3 using the chessboard method;

[0025] Figure 4 The checkerboard method was used to detect the minimum inhibitory concentration of VK3 combined with polymyxin against Acinetobacter baumannii ATCC 19606.

[0026] Figure 5 To detect the minimum inhibitory concentration of VK3 combined with polymyxin against Klebsiella pneumoniae strain ATCC 700603 using the checkerboard method;

[0027] Figure 6 To detect the minimum inhibitory concentration of VK3 combined with polymyxin against Pseudomonas aeruginosa strain ATCC 27853 using the checkerboard method;

[0028] Figure 7 To detect the minimum inhibitory concentration of VK3 combined with polymyxin against Shigella ATCC 12022 using the checkerboard assay;

[0029] Figure 8 To detect the minimum inhibitory concentration of VK3 combined with polymyxin against Salmonella ATCC SL1344 using the checkerboard assay;

[0030] Fig. 9 This is the time-killing curve of VK3 combined with polymyxin against mcr-1 positive Escherichia coli B5. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] Polymyxin is a product of Shanghai Yuanye Biotechnology Co., Ltd.

[0034] Vitamin K3 (VK3) is a product of Merck KGaA.

[0035] The preparation method of the culture medium involved in the following examples is as follows:

[0036] MHB broth medium (dry powder) was purchased from Beijing Luqiao Technology Co., Ltd. The preparation method is as follows: weigh 25.0 g of MHB broth medium dry powder into 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121°C for 15 min, and set aside.

[0037] MHA culture medium (dry powder) was purchased from Beijing Luqiao Technology Co., Ltd. The preparation method is as follows: weigh 38.0 g of MHA culture medium dry powder into 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121°C for 15 min, cool to 55°C and pour into a plate for later use.

[0038] mcr-1 positive E. coli B5 and high-level polymyxin-resistant E. coli YPE3 were wild E. coli strains isolated in our laboratory. Other strains were purchased from the American Type Culture Collection (ATCC).

[0039] Example 1: Detection of the minimum inhibitory concentration of VK3 combined with polymyxin against polymyxin-resistant bacteria using the chessboard method

[0040] According to the CLSI drug sensitivity test standard, the checkerboard method was used to detect the antibacterial activity of VK3 alone, polymyxin alone, and "VK3 and polymyxin combined" against polymyxin-resistant bacteria (mcr-1 positive Escherichia coli B5, high-level polymyxin-resistant Escherichia coli YPE3, standard Escherichia coli ATCC MG1655, standard Acinetobacter baumannii strain ATCC 19606, standard Klebsiella pneumoniae strain ATCC700603, standard Pseudomonas aeruginosa strain ATCC 27853, standard Shigella ATCC 12022, standard Salmonella ATCCSL1344), and then the minimum inhibitory concentration against polymyxin-resistant bacteria was obtained. The specific steps are as follows:

[0041] 1. Take the polymyxin E-resistant bacteria solution that has grown to the logarithmic growth phase, first adjust it to McFarland 0.5 using a McFarland turbidimeter, and then dilute it 100 times with MH broth medium to obtain the polymyxin E-resistant bacteria dilution solution. The concentration of polymyxin E-resistant bacteria in the polymyxin E-resistant bacteria dilution solution is about 1×10 6 CFU / mL.

[0042] 2. Take polymyxin E and dilute it with deionized water to obtain a polymyxin solution with a concentration of 320 μg / mL; then filter the polymyxin solution with a sterile filter membrane (pore size of 0.22 μm) to obtain a polymyxin stock solution. Take VK3 and dilute it with DMSO to obtain a VK3 solution with a concentration of 31.25 mM; then filter the VK3 solution with a sterile filter membrane (pore size of 0.22 μm) to obtain a VK3 stock solution.

[0043] 3. Take a 96-well microplate and inoculate each well with 100 μL of polymyxin-resistant bacteria dilution and 100 μL of drug solution (mixed from polymyxin E stock solution and VK3 stock solution). For mcr-1-positive Escherichia coli B5 and Salmonella ATCCSL1344, the concentrations of VK3 in each well in each column from left to right in the 96-well microplate are 1.563 mM, 0.781 mM, 0.391 mM, 0.195 mM, 0.098 mM, 0.049 mM, 0.024 mM, and 0.012 mM; the concentrations of polymyxin in each well in each row from top to bottom are 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL. For high-level polymyxin-resistant Escherichia coli YPE3, in a 96-well microplate, the concentrations of VK3 in each well in each column from left to right were 1.563mM, 0.781mM, 0.391mM, 0.195mM, 0.098mM, 0.049mM, 0.024mM, and 0.012mM, respectively; the concentrations of polymyxin in each well in each row from top to bottom were 512μg / mL, 256μg / mL, 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, and 4μg / mL, respectively. For Escherichia coli ATCC MG1655 and Pseudomonas aeruginosa strain ATCC27853, in a 96-well microplate, the concentrations of VK3 in each well in each column from left to right were 1.563mM, 0.781mM, 0.391mM, 0.195mM, 0.098mM, 0.049mM, 0.024mM, and 0.012mM, respectively; the concentrations of polymyxin in each well in each row from top to bottom were 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, 0.125μg / mL, and 0.0625μg / mL, respectively. For Shigella ATCC 12022 and Acinetobacter baumannii strain ATCC 19606, in a 96-well microplate, the concentration of VK3 in each well in each column from left to right was 0.195mM, 0.098mM, 0.049mM, 0.024mM, 0.012mM, 0.006mM, 0.003mM, and 0.001mM, respectively; the concentration of polymyxin in each well in each row from top to bottom was 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, 0.25μg / mL, 0.125μg / mL, and 0.0625μg / mL, respectively.For Klebsiella pneumoniae strain ATCC700603, in a 96-well microplate, the concentrations of VK3 in each well in each column from left to right were 1.563mM, 0.781mM, 0.391mM, 0.195mM, 0.098mM, 0.049mM, 0.024mM, and 0.012mM, respectively; the concentrations of polymyxin in each well in each row from top to bottom were 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, 0.5μg / mL, and 0.25μg / mL, respectively.

[0044] 4. After completing step 3, take the 96-well microplate and incubate at 37°C for 16-18 hours.

[0045] 5. After completing step 4, calculate the MIC value of each well and further calculate the combined inhibition score FICI.

[0046] Combined effect index FICI = MIC 多黏菌素联合 / MIC 多黏菌素单用 +MIC VK3联合 / MIC VK3单用

[0047] The experiment was repeated three times and the results were averaged.

[0048] The results are as follows Figure 1-8 As shown, the results indicate that the combination of VK3 and polymyxin can reduce the MIC values ​​of polymyxin against standard Escherichia coli ATCC MG1655, mcr-1 positive Escherichia coli B5, high-level polymyxin-resistant Escherichia coli YPE3, standard Acinetobacter baumannii strain ATCC 19606, standard Klebsiella pneumoniae strain ATCC 700603, standard Pseudomonas aeruginosa strain ATCC 27853, standard Shigella ATCC 12022, and standard Salmonella ATCC SL1344.

[0049] For E. coli ATCC MG1655, the optimal mass ratio of VK3 to polymyxin was 256.8:1; at this time, the minimum inhibitory concentration of VK3 was 0.391mM, and the minimum inhibitory concentration of polymyxin was 0.25μg / mL ( Figure 1 ).

[0050] For mcr-1 positive E. coli B5, the optimal mass ratio of VK3 to polymyxin was 256.8:1; at this time, the minimum inhibitory concentration of VK3 was 0.391mM, and the minimum inhibitory concentration of polymyxin was 0.25μg / mL ( Figure 2 ).

[0051] For high-level polymyxin-resistant Escherichia coli YPE3, the optimal mass ratio of VK3 to polymyxin was 8.025:1; at this time, the minimum inhibitory concentration of VK3 was 0.391mM, and the minimum inhibitory concentration of polymyxin was 8μg / mL ( Figure 3 ).

[0052] For Acinetobacter baumannii strain ATCC 19606, the optimal mass ratio of VK3 to polymyxin was 16.02:1; at this time, the minimum inhibitory concentration of VK3 was 0.0488mM, and the minimum inhibitory concentration of polymyxin was 0.5μg / mL ( Figure 4 ).

[0053] For Klebsiella pneumoniae strain ATCC 700603, the optimal mass ratio of VK3 to polymyxin was 128:1; at this time, the minimum inhibitory concentration of VK3 was 0.781mM, and the minimum inhibitory concentration of polymyxin was 1μg / mL ( Figure 5 ).

[0054] For Pseudomonas aeruginosa strain ATCC 27853, the optimal mass ratio of VK3 to polymyxin was 3.94:1; at this time, the minimum inhibitory concentration of VK3 was 0.024mM, and the minimum inhibitory concentration of polymyxin was 1μg / mL ( Figure 6 ).

[0055] For Shigella ATCC 12022, the optimal mass ratio of VK3 to polymyxin was 7.88:1; at this time, the minimum inhibitory concentration of VK3 was 0.0244mM, and the minimum inhibitory concentration of polymyxin was 0.5μg / mL ( Figure 7 ).

[0056] For Salmonella ATCC SL1344, the optimal mass ratio of VK3 to polymyxin was 512:1; at this time, the minimum inhibitory concentration of VK3 was 0.781mM, and the minimum inhibitory concentration of polymyxin was 0.25μg / mL ( Figure 8 ).

[0057] The above results indicate that VK3 can synergize with polymyxin to exert antibacterial effects, thereby treating infections caused by polymyxin-resistant bacteria.

[0058] Example 2: Time-kill curve of VK3 combined with polymyxin against polymyxin-resistant bacteria

[0059] The polymyxin-resistant bacteria were mcr-1-positive Escherichia coli B5.

[0060] 1. Take the overnight culture solution and dilute it 100 times with MH broth medium to obtain a bacterial dilution solution with a bacterial concentration of about 1×10 8 CFU / mL.

[0061] 2. Take 12 test tubes, add 2970 μL of MH liquid culture medium and 30 μL of polymyxin-resistant bacteria dilution to each tube, and mix well; then randomly divide the 12 test tubes into 4 groups: VK3 group, polymyxin group, combination group and blank group, with 3 test tubes in each group, and perform the following treatments:

[0062] VK3 group: add 37.5 μL of 31.25 mM VK3 solution to each test tube, so that the concentration of VK3 in the system is 0.390625 mM, which is the 0th hour; then culture at 37°C and 200 rpm for 24 hours;

[0063] Polymyxin group: 37.5 μL of 320 μg / mL polymyxin solution was added to each test tube to make the concentration of polymyxin E in the system 4 μg / mL, which was the 0th hour; then the cells were cultured at 37°C and 200 rpm for 24 hours;

[0064] Combined group: 37.5 μL of 31.25 mM VK3 solution and 37.5 μL of 320 μg / mL polymyxin solution were added to each test tube, so that the concentration of VK3 in the system was 0.390625 mM and the concentration of polymyxin in the system was 4 μg / mL. This time was the 0th hour. Then, the cells were cultured at 37°C and 200 rpm for 24 hours.

[0065] Blank group: No drug was added to the test tube, which was the 0th hour; the cells were then cultured at 37°C and 200 rpm for 24 hours.

[0066] During the culture period, 100 μL of the culture solution was spread on MH solid medium at 0 h, 4 h, 8 h, 12 h and 24 h, respectively, and cultured at 37° C. and 200 rpm for 24 h, and the colonies were counted.

[0067] 3. After completing step 2, draw a time-sterilization curve with the culture time as the horizontal axis and the log10 value of the colony count as the vertical axis.

[0068] The time-kill curve of mcr-1 positive Escherichia coli B5 is shown in Fig. 9 (CFU is colony forming unit).

[0069] The results showed that the VK3 group, the polymyxin group and the blank group could not inhibit polymyxin-resistant bacteria, and there was no significant difference among the three groups; the combined group could significantly inhibit polymyxin-resistant bacteria. Compared with the VK3 group, the polymyxin group and the blank group, the combined group had a significantly improved and stable inhibitory effect on polymyxin-resistant bacteria.

[0070] It can be seen from this that VK3 combined with polymyxin can restore the inhibitory effect of polymyxin on polymyxin-resistant bacteria, that is, VK3 combined with polymyxin can inhibit polymyxin-resistant bacteria.

[0071] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A pharmaceutical composition for inhibiting Gram-negative bacteria, characterized in that: The pharmaceutical composition consists of vitamin K3 and polymyxin.

2. Use of the pharmaceutical composition according to claim 1 in the preparation of drugs for inhibiting Gram-negative bacteria.

3. The use according to claim 2, characterized in that The Gram-negative bacteria are polymyxin-resistant bacteria.

4. The use according to claim 3, characterized in that The polymyxin-resistant bacteria include standard Escherichia coli ATCC MG1655, mcr-1 positive Escherichia coli, high-level polymyxin-resistant Escherichia coli YPE3, standard Acinetobacter baumannii strain ATCC 19606, standard Klebsiella pneumoniae strain ATCC 700603, standard Pseudomonas aeruginosa strain ATCC27853, standard Shigella ATCC 12022 and standard Salmonella ATCC SL1344.

5. The use according to claim 4, characterized in that When the pharmaceutical composition is used to inhibit standard Escherichia coli ATCC MG1655, the mass ratio of VK3 to polymyxin is 256.8:1; at this time, the minimum inhibitory concentration of VK3 is 0.391mM, and the minimum inhibitory concentration of polymyxin is 0.25μg / mL; When the pharmaceutical composition is used to inhibit mcr-1 positive Escherichia coli, the optimal mass ratio of VK3 to polymyxin is 256.8:1; at this time, the minimum inhibitory concentration of VK3 is 0.391mM, and the minimum inhibitory concentration of polymyxin is 0.25μg / mL; When the pharmaceutical composition is used to inhibit high-level polymyxin-resistant Escherichia coli YPE3, the optimal mass ratio of VK3 to polymyxin is 8.025:1; at this time, the minimum inhibitory concentration of VK3 is 0.391mM, and the minimum inhibitory concentration of polymyxin is 8μg / mL; When the pharmaceutical composition is used to inhibit Acinetobacter baumannii strain ATCC 19606, the optimal mass ratio of VK3 to polymyxin is 16.02:1; at this time, the minimum inhibitory concentration of VK3 is 0.0488mM, and the minimum inhibitory concentration of polymyxin is 0.5μg / mL; When the pharmaceutical composition is used to inhibit Klebsiella pneumoniae strain ATCC 700603, the optimal mass ratio of VK3 to polymyxin is 128:1; at this time, the minimum inhibitory concentration of VK3 is 0.781mM, and the minimum inhibitory concentration of polymyxin is 1μg / mL; When the pharmaceutical composition is used to inhibit Pseudomonas aeruginosa strain ATCC 27853, the optimal mass ratio of VK3 to polymyxin is 3.94:1; at this time, the minimum inhibitory concentration of VK3 is 0.024mM, and the minimum inhibitory concentration of polymyxin is 1μg / mL; When the pharmaceutical composition is used to inhibit Shigella ATCC 12022, the optimal mass ratio of VK3 to polymyxin is 7.88:1; at this time, the minimum inhibitory concentration of VK3 is 0.0244mM, and the minimum inhibitory concentration of polymyxin is 0.5μg / mL; When the pharmaceutical composition is used to inhibit Salmonella ATCC SL1344, the optimal mass ratio of VK3 to polymyxin is 512:1; at this time, the minimum inhibitory concentration of VK3 is 0.781mM, and the minimum inhibitory concentration of polymyxin is 0.25μg / mL.

Images