A polymyxin antibacterial potentiator and antibacterial compositions and uses

Abstract

The application discloses a polymyxin antibacterial synergist, an antibacterial composition and application, the polymyxin antibacterial synergist includes compound G-749, the antibacterial composition includes antibacterial synergist and colistin sulfate, the antibacterial composition is applied synergistically to carry out antibacterial, especially for treating gram-negative bacterial infection, aiming at polymyxin drug-resistant strain, has significant bacteriostatic effect.The application finds that G-749 improves the antibacterial activity of strong colistin sulfate by destroying cell membrane permeability and inducing drug-resistant bacteria oxidative stress, the MIC of standard sensitive strain ATCC25922 is reduced by 16-64 times, the MIC of mcr-1 polymyxin drug-resistant strain is reduced by 4-16 times, and bacteria can be rapidly killed within 1-4h.The application first discloses that G-749 is used as a broad-spectrum synergist of polymyxin antibiotics, and can effectively solve the clinical polymyxin drug resistance problem, and is especially suitable for the treatment of mcr gene producing escherichia coli infection.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to a polymyxin antibacterial synergist, an antibacterial composition, and its application. Background Technology

[0002] Colistin sulfate, also known as polymyxin E, is a polypeptide antibiotic primarily used to combat Gram-negative bacterial infections. Due to its severe nephrotoxicity and neurotoxicity, it was gradually phased out of clinical use in the 1980s. In recent years, the increasing multidrug resistance rates of Gram-negative bacterial pathogens, including carbapenem-resistant Enterobacteriaceae such as Klebsiella pneumoniae, have led to the re-emergence of colistin as a last line of defense against Gram-negative bacteria due to its broad Gram-negative activity. With the increased use of colistin, resistance to colistin has inevitably emerged in Gram-negative bacteria, leading to the emergence of pan-drug-resistant strains. Furthermore, the clinical efficacy of polymyxins is affected by a low therapeutic index, even when treating susceptible strains. Therefore, there is an urgent need to determine methods to restore the antimicrobial activity of colistin sulfate and its closely related drug, polymyxin B, against drug-resistant bacteria, while preserving antibacterial activity and reducing drug toxicity. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a polymyxin antibacterial synergist, an antibacterial composition, and its application. Preliminary research is conducted on the in vitro and in vivo antibacterial activity and mechanism of the FLT-3 inhibitor compound G-749 in combination with colistin sulfate, to explore its application as a novel polymyxin antibacterial synergist.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] In a first aspect, the present invention provides a polymyxin antibacterial synergist, comprising compound G-749. The synergist enhances the antibacterial activity of colistin sulfate against Gram-negative bacteria, particularly against polymyxin-resistant strains.

[0006] In a second aspect, the present invention provides an antibacterial composition comprising the polymyxin antibacterial synergist described above, and colistin sulfate.

[0007] As some specific embodiments of the present invention, in the antibacterial composition, the concentration of compound G-749 is 0.25-16 μg / mL, and the concentration of colistin sulfate is 0.004-1 μg / mL;

[0008] The concentration ratio of compound G-749 to colistin sulfate is 8:1 to 1000:1.

[0009] Thirdly, the present invention provides an application of the above-described antibacterial composition in synergistic antibacterial activity.

[0010] As some specific embodiments of the present invention, its application in the fight against Gram-negative bacteria is included.

[0011] As some specific embodiments of the present invention, the Gram-negative bacteria include Escherichia coli, and the Escherichia coli includes either Escherichia coli ATCC25922 or mcr-1 polymyxin-resistant Escherichia coli.

[0012] As some specific embodiments of the present invention, the antibacterial composition is used against Escherichia coli ATCC25922, and the concentration of the composition is: G-749 0.25~4 μg / mL + colistin sulfate 0.004~0.016 μg / mL;

[0013] The antibacterial composition is used against mcr-1 polymyxin-resistant Escherichia coli, and the concentration of the composition is: G-7494~16μg / mL + colistin sulfate 0.125~0.5μg / mL.

[0014] As some specific embodiments of the present invention, for standard sensitive Escherichia coli ATCC25922, G-749 reduces the MIC (minimum inhibitory concentration) of colistin sulfate by 16 to 64 times; for mcr-1 resistant Escherichia coli, G-749 reduces the MIC of colistin sulfate by 4 to 16 times.

[0015] As some specific embodiments of the present invention, the antibacterial composition can achieve rapid bactericidal effect within 1 to 4 hours at a sub-inhibitory concentration and maintain antibacterial activity for 8 to 24 hours.

[0016] As some specific embodiments of the present invention, the Gram-negative bacteria include Gram-negative bacteria carrying transferable colistin resistance genes, specifically including polymyxin-resistant Escherichia coli producing the mcr gene.

[0017] As some specific embodiments of the present invention, in the antibacterial composition, colistin sulfate and G-749 are administered separately;

[0018] Alternatively, the colistin sulfate and compound G-749 can be formulated into a compound preparation and administered simultaneously.

[0019] As some specific embodiments of the invention, the application includes use in the preparation of a medicament for treating infections caused by Gram-negative bacteria, the medicament being administered via intravenous injection, intramuscular injection, or local administration. An effective amount of the antibacterial composition is administered to a subject in need.

[0020] As some specific embodiments of the present invention, in the antibacterial composition, compound G-749 enhances the antibacterial activity of colistin sulfate through the following mechanism:

[0021] a) Disrupts the integrity of bacterial cell membranes, increasing cell membrane permeability;

[0022] b) Promotes the loss of intracellular nucleic acids from bacteria;

[0023] c) Inducing oxidative stress in MCR-resistant strains.

[0024] Mechanistic studies have shown that G-749 disrupts cell membrane permeability, and the combined use of the drug significantly increases the fluorescence intensity of PI staining, indicating enhanced cell membrane permeability; DAPI staining shows that the nucleic acid content of bacteria in the combined group is reduced by more than 50%; it induces oxidative stress in drug-resistant bacteria, increasing the ROS level of mcr-1 polymyxin-resistant Escherichia coli by 2.3 times.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) This invention reveals that compound G-749 and colistin sulfate exhibit significant synergistic antibacterial activity in both in vitro and in vivo models. Combined administration reduced the MIC of colistin sulfate against susceptible strains (ATCC25922) by 16–64 times and against strains carrying the mcr resistance gene (mcr-1 polymyxin-resistant Escherichia coli) by 4–16 times. This result is consistent with recent research trends on other synergists (such as thymol and naringenin), but the synergistic effect of G-749 is more significant, especially among resistant strains. Time-bacterial action curves further confirmed that the combined administration achieved rapid bactericidal activity within 4 hours, and the antibacterial effect lasted for more than 24 hours, suggesting that it may exert its effect by disrupting bacterial regeneration or metabolic homeostasis. This rapid and sustained bactericidal characteristic is of great significance for treating severe infections, as it can shorten the treatment course and reduce the risk of drug resistance.

[0027] (2) This invention reveals a synergistic mechanism through multi-dimensional analysis of cell membrane integrity (PI staining), nucleic acid integrity (DAPI staining), and oxidative stress (ROS detection). The PI staining fluorescence value was significantly increased in the combined drug group, indicating that the drugs synergistically disrupt cell membrane permeability, promoting the targeting of colistin sulfate to the lipopolysaccharide (LPS) outer membrane. This is consistent with the traditional mechanism of action of polymyxin drugs. However, DAPI staining showed a decrease in intrabacterial nucleic acid content, suggesting that G-749 may enhance the penetration ability of colistin to the inner membrane by interfering with DNA metabolism or membrane-binding protein function. Notably, the ROS level was increased in mcr-1 polymyxin-resistant Escherichia coli, while this phenomenon was not observed in the sensitive strain ATCC25922. This indicates that the synergistic effect of G-749 on drug-resistant bacteria may involve the activation of the oxidative stress pathway. This mechanism contrasts with the characteristics of citrate-induced antibiotic resistance (reduced reactive oxygen species production, thereby weakening the bactericidal effect of antibiotics through the oxidative stress pathway).

[0028] (3) This invention discloses G-749 as a novel polymyxin synergist, which significantly enhances the antibacterial activity and resistance reversal ability of colistin sulfate through multiple mechanisms, including enhanced membrane disruption, interference with nucleic acid metabolism, and activation of oxidative stress in drug-resistant bacteria. Its low hemolytic properties and in vivo efficacy provide important evidence for clinical development. It can be combined with drug delivery systems (such as nanoparticles) to further improve targeting and safety, providing an innovative solution for addressing infections caused by widespread drug-resistant Gram-negative bacteria. Attached Figure Description

[0029] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0030] Figure 1 The image shows the checkerboard synergistic assay results of G-749 combined with colistin sulfate for antibacterial activity in Example 2. The left image shows the checkerboard synergistic assay results of Escherichia coli ATCC25922, and the right image shows the checkerboard synergistic assay results of mcr-1 polymyxin-resistant Escherichia coli.

[0031] Figure 2 The graph shows the synergistic time-bactericidal curve results of G-749 and colistin sulfate against ATCC25922 at a concentration of 4 + 0.004 μg / mL in Example 2.

[0032] Figure 3 This is a graph showing the synergistic time-bactericidal curve results of G-749 and colistin sulfate at a concentration of 16 + 0.125 μg / mL against mcr-1 polymyxin-resistant Escherichia coli in Example 2.

[0033] Figure 4 The graph shows the results of the hemolytic activity assay for compound G-749 in Example 2;

[0034] Figure 5 This is a graph showing the bacterial load test results of mouse thigh muscles after the combined use of G-749 and colistin sulfate in Example 2;

[0035] Figure 6 The graph shows the effect of G-749 combined with colistin sulfate on bacterial ROS in Example 2. The left graph shows the effect on ROS of the sensitive strain Escherichia coli ATCC25922; the right graph shows the effect on ROS level of mcr-1 polymyxin-resistant Escherichia coli.

[0036] Figure 7 This is a fluorescence result image of PI staining to identify the cell membrane integrity of Escherichia coli ATCC25922 after treatment with G-749 and colistin sulfate in Example 2; where a is the PBS blank group, b is the group treated with only 0.004 μg / mL colistin sulfate, c is the group treated with only 4 μg / mL G-749, d is the group treated with a combination of 0.004 μg / mL colistin sulfate and 4 μg / mL G-749, e is the group treated with only 0.016 μg / mL colistin sulfate, f is the group treated with only 0.25 μg / mL G-749, and g is the group treated with a combination of 0.016 μg / mL colistin sulfate and 0.25 μg / mL G-749.

[0037] Figure 8 This is a fluorescence result image of the intracellular nucleic acid content of *E. coli* ATCC25922 after DAPI staining in Example 2, where a is the PBS blank group, b is the group treated with only 0.004 μg / mL colistin sulfate, c is the group treated with only 4 μg / mL G-749, d is the group treated with a combination of 0.004 μg / mL colistin sulfate and 4 μg / mL G-749, e is the group treated with only 0.016 μg / mL colistin sulfate, f is the group treated with only 0.25 μg / mL G-749, and g is the group treated with a combination of 0.016 μg / mL colistin sulfate and 0.25 μg / mL G-749. Detailed Implementation

[0038] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0039] Example 1: Materials and Methods

[0040] 1.1 Bacterial strains, reagents, and laboratory animals

[0041] Escherichia coli ATCC 25922 was purchased from Shanghai Hongxin Biotechnology Co., Ltd.; mcr-1 polymyxin-resistant Escherichia coli SMHCC (SHBCC) D24800 was purchased from Beijing Huayuan Xingke Biotechnology Co., Ltd.; G-749 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; colistin sulfate and DAPI were purchased from Beijing Solarbio Science & Technology Co., Ltd.; MHB and MHA culture media were purchased from Qingdao Haibo Biotechnology Co., Ltd.; phosphate-buffered saline (PBS) and crystal violet staining solution were purchased from Sangon Biotech (Shanghai) Co., Ltd.; dimethyl sulfoxide (DMSO), glacial acetic acid and methanol were all purchased from Sinopharm Chemical Reagent Co., Ltd.; propidium iodide (PI) was purchased from Sigma-Aldrich; ROS detection kit was purchased from Beyotime Biotechnology; ICR mice were purchased from Shanghai JessJ Laboratory Animal Co., Ltd.

[0042] 1.2 Determination of minimum inhibitory concentration

[0043] According to the Clinical and Laboratory Standards Institute (CLSI) standards, the minimum inhibitory concentration (MIC) of the drug was determined using the microbroth dilution method. 96-well plates were incubated at 37°C for 18–24 hours. Turbidity did not inhibit bacterial growth, while clear broth inhibited bacterial growth. The minimum concentration at which bacterial growth was inhibited was defined as the MIC. Each experiment was performed in triplicate.

[0044] 1.3 Chessboard Coordination Measurement

[0045] The checkerboard method was used to evaluate the synergistic antibacterial effect of colistin sulfate and the target compound G-749. The fractional inhibition concentration index (FICI) was calculated as follows: FICI = (MIC A + B / MIC A) + (MIC B + A / MIC B), and FICI ≤ 0.5 indicated a synergistic effect. The experiment was independently repeated three times.

[0046] 1.4 Time-sterilization curve

[0047] Log-phase bacterial cultures (Escherichia coli ATCC25922 and mcr-1 polymyxin-resistant Escherichia coli) were collected and added to prepared drug solutions of appropriate concentrations, including G-749 alone, colistin sulfate alone, and G-749 in combination with colistin sulfate. A control group (culture medium only) was set up as a reference. Samples were taken at 0 min, 30 min, 1 h, 4 h, 8 h, 12 h, and 24 h and serially diluted in sterile physiological saline. 10 μL of the diluted solution was suspended on MHA agar plates and incubated at 37℃ for 18–24 h. After incubation, colony counts were performed, with three replicates per group. The effective counts were selected to calculate the logarithmic colony count and statistically analyze the results. A time-kill curve was plotted.

[0048] 1.5 Determination of the hemolytic activity of compound G-749

[0049] Add 100 μL of compound G-749 at different concentrations to each well of a 96-well plate. Use 2% Triton X-100 as a positive control and 1×PBS as a negative control. Add 100 μL of 4% erythrocyte suspension to each well of the 96-well plate and incubate at 37°C and 70 rpm for 1 h on a shaker. Centrifuge the 96-well plate at 3500 rpm for 10 min, transfer the supernatant to another 96-well plate, and measure the absorbance at OD540 nm using a microplate reader. Each experiment was performed in triplicate. Hemolysis rate was calculated using the formula: (OD sample measurement - OD negative control) / (OD positive control - OD negative control) * 100%.

[0050] 1.6 Establishment and treatment of mouse thigh muscle infection model

[0051] The experimental animals were SPF-grade female ICR mice, 5–6 weeks old, weighing 18–22 g, purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. The mice were acclimatized for one week at 25±1℃ and 55±5% relative humidity before being used in the experiments. Four days before challenge, mice were injected intraperitoneally with cyclophosphamide 150 mg / kg bw, and one day before challenge, mice were injected intraperitoneally with cyclophosphamide 100 mg / kg bw. 0.1 mL of 1×10⁻⁶ cyclophosphamide was injected into the muscles of both hind thighs of the mice. 7 A cfu / mL mcr-1 polymyxin-resistant Escherichia coli (PMC) suspension was used to establish a thigh muscle infection model. Five experimental groups were set up: G74950 mg / kg bw, colistin sulfate 5 mg / kg bw, G74950 mg / kg bw + colistin sulfate 5 mg / kg bw, a solvent group (Tween 80: polyethylene glycol 300: anhydrous ethanol = 2:2:1), and a control group. Each group contained 2 mice, resulting in 4 thigh muscle infection models. The medication was administered intraperitoneally 2 hours after infection. Administered every 12 hours, and after 24 hours, mice were sacrificed, and thigh muscle homogenates were collected, serially diluted, and plate-stamped to determine bacterial load.

[0052] 1.7 Determination of reactive oxygen species (ROS) in bacteria

[0053] Reactive oxygen species (ROS) in bacteria were measured using a ROS detection kit. Log-phase *E. coli* ATCC25922 and mcr-1 polymyxin-resistant *E. coli* cultures were centrifuged, resuspended in PBS, and their OD600nm was adjusted to 0.5. The fluorescent probe DCFH-DA (10 μmol / L) was added, and the mixture was incubated at 37°C in the dark for 30 min. After incubation, the cells were washed three times with PBS, and different concentrations of drug solutions were added, followed by incubation for 1 h. Then, 200 μL of the solution was added to a black 96-well plate, and fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Each experiment was performed in triplicate.

[0054] (1) A total of 8 experiments were set up for Escherichia coli ATCC25922:

[0055] control group: bacterial culture only, no drug administered;

[0056] Colistin 0.004: Colistin sulfate 0.004 μg / mL;

[0057] G-7494: G-7494 μg / mL;

[0058] G-7494 + Colistin 0.004: G-7494 μg / mL + Colistin sulfate 0.004 μg / mL;

[0059] Colistin 0.016: Colistin sulfate 0.016 μg / mL;

[0060] G-7490.25: G-7490.25μg / mL;

[0061] G-7490.25 + Colistin 0.016: G-7490.25 μg / mL + Colistin sulfate 0.016 μg / mL;

[0062] rosup: Use only the positive inducer from the ROS detection kit.

[0063] (2) Eight experimental groups were set up to target mcr-1 polymyxin-resistant Escherichia coli:

[0064] control group: bacterial culture only, no drug administered;

[0065] Colistin 0.125: Colistin sulfate 0.125 μg / mL;

[0066] G-7498: G-7498 μg / mL;

[0067] G-7498 + Colistin 0.125: G-7498 μg / mL + Colistin sulfate 0.125 μg / mL;

[0068] Colistin 0.125: Colistin sulfate 0.125 μg / mL;

[0069] G-74916: G-74916 μg / mL;

[0070] G-74916 + Colistin 0.125: G-74916 μg / mL + Colistin sulfate 0.125 μg / mL;

[0071] rosup: Use only the positive inducer from the ROS detection kit.

[0072] 1.8 Propidium iodide (PI) staining

[0073] A suspension of *E. coli* ATCC25922 in the logarithmic growth phase was collected by centrifugation, and the precipitate was washed three times with PBS and resuspended. The bacterial concentration was adjusted to OD600nm = 1.0. Different concentrations of drug solutions were added to the bacterial suspension and incubated in the dark for 1 hour. PI was then added to a final concentration of 10 μM and incubated in the dark for 30 minutes. The bacteria were imaged using a confocal laser scanning microscope, and a mixture of bacteria and dye was used as a blank control. Each experiment was performed in triplicate.

[0074] 1.9DAPI staining

[0075] A suspension of *E. coli* ATCC25922 in the logarithmic growth phase was collected by centrifugation, and the precipitate was washed three times with PBS and resuspended. The bacterial concentration was adjusted to OD600nm = 0.4. Different concentrations of drug solutions were added to the bacterial suspension and incubated in the dark for 1 h. 20 μL of DAPI (4',6-diamidinyl-2-phenylindole) solution was added to each milliliter of bacterial suspension and incubated in the dark for 30 min. The bacteria were imaged using a confocal laser scanning microscope, and a mixture of bacteria and dye was used as a blank control. Each experiment was performed in triplicate.

[0076] Example 2

[0077] 2.1 Synergistic antibacterial effect of G-749 and colistin sulfate in vitro

[0078] The results of the chessboard coordination test are as follows: Figure 1As shown, the MIC of colistin sulfate against *Escherichia coli* ATCC25922 was 0.25 μg / mL, which meets the CLSI (Clinical and Laboratory Standards Institute) quality control standard of 0.25-2 μg / mL. The MIC of colistin sulfate against mcr-1 polymyxin-resistant *Escherichia coli* was 2 μg / mL.

[0079] G-749 exhibits good synergistic bactericidal activity with colistin sulfate. At synergistic concentrations, it can reduce the MIC of colistin sulfate against the sensitive strain ATCC25922 by 16-64 times and the MIC against mcr-1 polymyxin-resistant Escherichia coli by 4-16 times, demonstrating a strong synergistic effect.

[0080] The calculation results of FICI are shown in the table below:

[0081] Table 1. Chessboard synergistic effect of G-749 and colistin sulfate against Escherichia coli ATCC25922.

[0082]

[0083] Table 2. Checkerboard synergistic effect of G-749 and colistin sulfate against mcr-1 polymyxin-resistant Escherichia coli.

[0084]

[0085]

[0086] 2.2 Synergistic time-bactericidal curve results of G-749 and colistin sulfate

[0087] G-749 and colistin sulfate at 4 + 0.004 μg / mL (e.g.) Figure 2 (As shown) At the synergistic concentration, the combined action on ATCC25922 resulted in sterilization after 4 hours and 1 hour of action, respectively, and maintained the sterilization effect for 24 hours without recovery of growth.

[0088] G-749 and colistin sulfate at a concentration of 16 + 0.125 μg / mL (e.g.) Figure 3 (As shown) When acting on mcr-1 polymyxin-resistant Escherichia coli, it can produce stronger antibacterial activity than colistin sulfate alone, and can kill bacteria in 4 hours and maintain antibacterial activity for about 12 hours.

[0089] Further analysis using time-bacterial action curves confirmed that the combined medication achieved rapid bactericidal activity within 4 hours, with the antibacterial effect lasting for over 24 hours, suggesting that it may exert its effects by disrupting bacterial regeneration or metabolic homeostasis. This rapid and sustained bactericidal characteristic is significant for treating severe infections, potentially shortening treatment duration and reducing the risk of drug resistance.

[0090] 2.3G-749 hemolytic activity

[0091] The results of the hemolytic activity assay for compound G-749 are as follows: Figure 4 As shown, the hemolysis rate of rabbit erythrocytes after treatment with G-749 was less than 8%. This indicates that compound G-749 has high safety for mammals.

[0092] In vivo antibacterial activity of 2.4G-749 in combination with colistin sulfate

[0093] like Figure 5 As shown, based on the results of the mouse thigh muscle infection experiment, the bacterial load in the mouse thigh muscle after the combined use of G-749 and colistin sulfate was significantly lower than that of G-749 or colistin sulfate alone, as well as the solvent group. This further demonstrates that the combined use of G-749 and colistin sulfate can significantly enhance the antibacterial effect of colistin sulfate against mcr-1 polymyxin-resistant Escherichia coli.

[0094] Effects of 2.5G-749 combined with colistin sulfate on bacterial ROS

[0095] This experiment determined the effects of G-749 and colistin sulfate alone and in combination on the ROS levels of polymyxin-resistant Escherichia coli ATCC25922 and mcr-1. Higher luminescence intensity was associated with higher ROS levels and more significant oxidative stress responses. Results are as follows: Figure 6 As shown, the ROS level of ATCC25922 did not change significantly after using G-749 alone or in combination with colistin sulfate, while the ROS level of mcr-1 polymyxin-resistant Escherichia coli increased by about 2.3 times.

[0096] The ROS level of mcr-1 polymyxin-resistant Escherichia coli was elevated, while that of the susceptible strain ATCC25922 was not, suggesting that the synergistic effect of G-749 on drug-resistant bacteria may involve the activation of oxidative stress pathways, which is different from the characteristics of citric acid-induced antibiotic resistance.

[0097] Effects of 2.6G-749 combined with colistin sulfate on bacterial cell membranes

[0098] like Figure 7 As shown, this experiment used PI staining to explore the cell membrane integrity of *E. coli* ATCC25922 after treatment with G-749 and colistin sulfate. The results showed that the fluorescence value after combined treatment with G-749 and colistin sulfate was significantly higher than that after single-drug treatment and the PBS blank group, indicating that the combined use of G-749 and colistin sulfate disrupted bacterial cell membrane integrity, increased cell membrane permeability, and thus led to bacterial death.

[0099] To further investigate the effect of the synergistic effect of G-749 and colistin sulfate on the cell membrane permeability of *E. coli* ATCC25922, this experiment performed DAPI staining on the nucleic acids within bacterial cells after the administration of G-749 and colistin sulfate, both alone and in combination. The results are as follows: Figure 8 As shown, compared with the control group and the single-use group, the fluorescence of DAPI staining imaging was significantly reduced after G-749 was treated with colistin sulfate, further indicating that the nucleic acid content in bacterial cells was reduced by more than 50% after the combined treatment of G-749 and colistin sulfate.

[0100] The combined drug group showed a significant increase in PI staining fluorescence value, indicating that the drugs promote the action of colistin sulfate targeting the lipopolysaccharide (LPS) outer membrane by synergistically disrupting cell membrane permeability. This is consistent with the traditional mechanism of action of polymyxin drugs. However, DAPI staining showed a decrease in the nucleic acid content in the bacteria, suggesting that G-749 may enhance the ability of colistin to penetrate the inner membrane by interfering with DNA metabolism or membrane-binding protein function.

[0101] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An antibacterial composition, characterized in that, It is composed of a polymyxin antibacterial synergist and colistin sulfate, wherein the polymyxin antibacterial synergist is compound G-749; In the antibacterial composition, the concentration of compound G-749 is 0.25~16 µg / mL, and the concentration of colistin sulfate is 0.004~1 µg / mL; The concentration ratio of compound G-749 to colistin sulfate is 8:1 to 1000:

1.

2. The use of the antibacterial composition as described in claim 1 in the preparation of a synergistic anti-Escherichia coli drug.

3. The application according to claim 2, characterized in that, The Escherichia coli is either Escherichia coli ATCC25922 or mcr-1 polymyxin-resistant Escherichia coli.

4. The application according to claim 3, characterized in that, The antibacterial composition is used against Escherichia coli ATCC25922, and the concentration of the composition is: G-749 0.25~4 μg / mL + colistin sulfate 0.004~0.016 μg / mL.

5. The application according to claim 3, characterized in that, The antibacterial composition is used against mcr-1 polymyxin-resistant Escherichia coli, and the concentration of the composition is: G-749 4~16 μg / mL + colistin sulfate 0.125~0.5 μg / mL.

6. The application according to claim 2, characterized in that, In the antibacterial composition, colistin sulfate and G-749 are administered separately. Alternatively, the colistin sulfate and compound G-749 can be formulated into a compound preparation and administered simultaneously.

7. The application according to claim 2, characterized in that, The drug is administered via intravenous injection, intramuscular injection, or local administration.

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