Antibacterial composition for treating escherichia coli infection and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an antibacterial composition for treating escherichia coli infection and a preparation method and application thereof. The antibacterial composition is prepared from TBP-TA and polymyxin B, wherein the TBP-TA and the polymyxin B have aggregation-induced emission characteristics; wherein the mass ratio of the polymyxin B to the TBP-TA is 1 to (1 to 32). The antibacterial composition provided by the invention can be used for treating gram-negative bacterium infection, and the antibacterial activity of PMB on gram-negative bacteria can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibacterial composition for treating Escherichia coli infection, its preparation method, and its application. Background Technology

[0002] Polymyxins are often used clinically as a "last line of defense" in the treatment of Gram-negative drug-resistant bacterial infections. However, the discovery and rapid spread of polymyxin resistance in Gram-negative bacteria has hindered the effective prevention and control of animal diseases.

[0003] To reduce polymyxin dosage and alleviate polymyxin resistance, current research often employs combined use of traditional Chinese medicine (TCM) ingredients with polymyxins. Studies show that the combination of TCM ingredients and polymyxins can synergistically combat multidrug-resistant bacteria, reducing polymyxin dosage and thus alleviating resistance. However, the TCM ingredients used are also substances with antibacterial activity against Gram-negative bacteria, which may easily exacerbate the resistance of Gram-negative drug-resistant bacteria. Furthermore, there is still room for improvement in reducing polymyxin dosage even with synergistic effects. Therefore, there is an urgent need to develop new strategies for combining polymyxins with TCM ingredients. Summary of the Invention

[0004] This invention aims to develop a novel strategy for combining polymyxin with other antibacterial ingredients, addressing the problems in existing technologies where traditional Chinese medicine components, also active against Gram-negative bacteria, easily exacerbate drug resistance in Gram-negative bacteria, and there is still room for improvement in reducing the dosage of polymyxin during synergistic effects. This invention provides an antibacterial composition for treating Escherichia coli infections, its preparation method, and its application. To achieve the above objectives, this invention adopts the following technical solution.

[0005] Aggregation-induced emission (AIE) molecules have demonstrated excellent antibacterial activity as novel antimicrobial agents and have been extensively studied in the field of in vitro and in vivo antimicrobial applications. Their mechanism involves targeting bacteria and generating high concentrations of reactive oxygen species in an aggregated state, disrupting the microbial membrane structure, proteins, nucleic acids, and lipids to achieve antibacterial effects. This mechanism offers advantages such as precise control, minimal side effects, and negligible resistance. However, due to the structure of Gram-negative bacteria, most AIE antimicrobial agents cannot penetrate the bacterial outer membrane barrier to exert their antimicrobial activity. This invention can enhance the antimicrobial activity against Gram-negative bacteria by combining AIE antimicrobial agents with polymyxins without altering their molecular structure.

[0006] Currently, AIE (Alternating Infectious Microorganisms) is used for antibacterial purposes, but its antibacterial effects have limitations. The innovation of this invention lies in broadening the application of AIE in antibacterial treatment by employing a combination therapy strategy, combining the AIE material TBP-TA with polymyxin B for the treatment of Gram-negative bacterial infections. Based on this, the first objective of this invention is to provide an antibacterial composition for treating Escherichia coli infections, consisting of polymyxin B and TBP-TA with luminescent properties.

[0007] The mass ratio of polymyxin B to TBP-TA is 1:1 to 32.

[0008] The structure of the TBP-TA is shown below: .

[0009] A second object of the present invention is to provide a method for preparing the antibacterial composition, comprising the following steps: The antibacterial composition is obtained by mixing TBP-TA and polymyxin B.

[0010] A third object of the present invention is to provide the use of the said antimicrobial composition in the preparation of medicaments for treating and / or preventing diseases caused by Gram-negative bacteria.

[0011] Preferably, the drug is made from the antibacterial composition and supplemented with pharmaceutically acceptable excipients.

[0012] Preferably, the drug is any one of an injection, an aerosol, and an oral liquid.

[0013] Preferably, the antibacterial composition is used to inhibit or kill Gram-negative bacteria.

[0014] Preferably, the Gram-negative bacteria include Escherichia coli (Escherichia coli). Escherichia coli ).

[0015] Preferably, the diseases caused by the Gram-negative bacteria include peritonitis-septicemia.

[0016] The fourth objective of this invention is to provide a method for establishing a mouse peritonitis-septicemia model, comprising the following steps: a) Inject a lethal dose of Gram-negative bacteria into the peritoneum of mice.

[0017] b) Inject the antibacterial composition intraperitoneally 1 hour later.

[0018] c) Monitor survival rate, organ bacterial load, and inflammatory factor levels to verify the in vivo treatment effect.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides an antibacterial composition for treating Escherichia coli infection. The antibacterial composition comprises TBP-TA, which has aggregation-induced emission (AIE) properties, and polymyxin B. The mass ratio of polymyxin B to TBP-TA is 1:1 to 32. TBP-TA enters the cell via the disruptive effect of polymyxin B on the outer membrane of E. coli. The two work synergistically to exert their antibacterial effect, allowing the combined treatment regimen to achieve in vivo therapeutic effects comparable to high-concentration monotherapy while reducing the dosage of polymyxin B. This invention, by innovatively combining the aggregation-induced emission (AIE) material TBP-TA with polymyxin B, fundamentally solves the problems of increased drug resistance from traditional Chinese medicine ingredients and insufficient reduction in polymyxin dosage in existing technologies. First, as an AIEgens photosensitizer, TBP-TA has a completely different antibacterial mechanism from traditional Chinese herbal medicine ingredients: after being activated by light, TBP-TA enhances fluorescence emission and reactive oxygen species (ROS) production efficiency when it aggregates on the bacterial surface. It achieves sterilization by using multi-target action (including destroying the bacterial antioxidant system, interfering with DNA metabolism, and damaging the cell membrane). This non-single-target synergistic attack mode is less likely to induce drug resistance gene mutations, thus avoiding the problem of traditional Chinese herbal medicine ingredients aggravating drug resistance due to their single mechanism of action. Secondly, this combination utilizes the unique ability of polymyxin B to specifically disrupt the lipopolysaccharide (LPS) of the outer membrane of Gram-negative bacteria, exerting a "gateway opening" effect and significantly improving the penetration efficiency of TBP-TA into the bacterial outer membrane, allowing TBP-TA to fully exert its photodynamic antibacterial effect. In this synergistic mechanism, polymyxin B is no longer used as the main bactericidal component, but as a synergist, thus significantly reducing the dosage (the mass ratio is set at polymyxin B:TBP-TA = 1:1~32, with TBP-TA dominating). This overcomes the nephrotoxicity and neurotoxicity side effects caused by high-dose polymyxin B, and the high ROS generation capacity of TBP-TA compensates for the potential potency loss due to dosage reduction, achieving a dual optimization of synergistic effect and dosage reduction. By organically combining two materials with different mechanisms of action, this approach avoids the drug resistance risk of traditional antibacterial components and achieves a synergistic effect of using less but more, providing a novel antibacterial strategy with low drug resistance risk and high safety for the treatment of Escherichia coli infections.

[0020] 2. The antibacterial composition provided by the present invention also has the following effects: (1) Reduce the clinical dosage of polymyxin B and alleviate drug resistance.

[0021] (2) It broadened the application of AIE in antibacterial applications.

[0022] 3. Compared to similar products, the method for improving the antibacterial activity of PMB against Gram-negative bacteria in this invention is simple, easy to operate, has good repeatability, and is widely applicable. This invention can provide a new option for reducing the dosage of polymyxin and alleviating its resistance, and provides experimental evidence for the clinical treatment of Gram-negative bacterial infections. Attached Figure Description

[0023] Figure 1 This is a heatmap showing the FICI assay results of the combined drug (antibacterial composition) against different strains of Escherichia coli in this invention; wherein: A is the FICI heatmap of combined drug therapy on Escherichia coli standard strain ATCC 25922; B is a FICI heatmap of combined drug therapy against drug-resistant Escherichia coli NXGC9; C is the FICI heatmap of combined drug therapy against drug-resistant Escherichia coli SJ 10; D is the FICI heatmap of combined drug therapy against drug-resistant Escherichia coli SJ 19; E represents the FICI heatmap of combined drug therapy against drug-resistant Escherichia coli SJ 34; G is the FICI heatmap of combined drug therapy against drug-resistant Escherichia coli SJE 39; H represents the FICI heatmap of combined drug therapy against drug-resistant Escherichia coli HK 16; I is a FICI heatmap of combined drug therapy against drug-resistant Escherichia coli HK 20.

[0024] Figure 2 This invention uses plate counting to determine the bactericidal efficiencies of different combined drug concentrations against *Escherichia coli* standard strain ATCC25922 and drug-resistant *Escherichia coli* strain SJ10; wherein: A is a statistical chart of the sterilization rate of each group; B is a photograph of the Escherichia coli specimen ATCC 25922 smear. C is a plate photograph of drug-resistant Escherichia coli SJ 10.

[0025] Figure 3 These are the time-bactericidal curves of Escherichia coli under different combinations of drug concentrations in this invention; wherein: A represents the time-bactericidal curve of the combined drug against Escherichia coli standard strain ATCC 25922; B represents the time-bactericidal curve of the combined drug treatment against drug-resistant Escherichia coli NXGC 9; C represents the time-bactericidal curve of the combined drug against drug-resistant Escherichia coli SJ 10; D represents the time-bactericidal curve of the combined drug treatment against drug-resistant Escherichia coli SJ 19; E represents the time-bactericidal curve of the combined drug treatment against drug-resistant Escherichia coli SJ 34; F represents the time-bactericidal curve of the combined drug against drug-resistant Escherichia coli SJE 39; G represents the time-bactericidal curve of the combined drug treatment against drug-resistant Escherichia coli HK 16; H represents the time-bactericidal curve of the combined drug against drug-resistant Escherichia coli HK 20.

[0026] Figure 4 The hemolysis method in this invention is used to evaluate the biosafety of combined drug use.

[0027] Figure 5 This is an experimental flowchart for establishing the mouse peritonitis-septicemia model in this invention.

[0028] Figure 6 This is a statistical analysis of the survival rate of mice in the mouse peritonitis-septicemia model of this invention.

[0029] Figure 7 This invention uses a plate count method to observe the bacterial load in different organs of a mouse peritonitis-septicemia model.

[0030] Figure 8 This is a statistical analysis of the cardiac bacterial load in the mouse peritonitis-septicemia model of this invention; PC is the positive control group, which was injected intraperitoneally with 200 μL of physiological saline; T64 is TBP-TA at a concentration of 64 μg / mL; P8 is polymyxin B at a concentration of 8 μg / mL; P2T8 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 8 μg / mL; P2T4 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 4 μg / mL; P2T2 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 2 μg / mL.

[0031] Figure 9 This is a statistical analysis of the bacterial load in the liver of the mouse peritonitis-septicemia model of this invention; wherein, PC is the positive control group, which is injected intraperitoneally with 200 μL of physiological saline; T64 is TBP-TA at a concentration of 64 μg / mL; P8 is polymyxin B at a concentration of 8 μg / mL; P2T8 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 8 μg / mL; P2T4 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 4 μg / mL; P2T2 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 2 μg / mL.

[0032] Figure 10This is a statistical analysis of the bacterial load in the spleen of the mouse peritonitis-septicemia model of this invention; PC is the positive control group, which is injected intraperitoneally with 200 μL of physiological saline; T64 is TBP-TA at a concentration of 64 μg / mL; P8 is polymyxin B at a concentration of 8 μg / mL; P2T8 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 8 μg / mL; P2T4 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 4 μg / mL; P2T2 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 2 μg / mL.

[0033] Figure 11 This is a statistical analysis of the bacterial load in the lungs of the mouse peritonitis-septicemia model of this invention; PC is the positive control group, which was injected intraperitoneally with 200 μL of physiological saline; T64 is TBP-TA at a concentration of 64 μg / mL; P8 is polymyxin B at a concentration of 8 μg / mL; P2T8 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 8 μg / mL; P2T4 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 4 μg / mL; P2T2 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 2 μg / mL.

[0034] Figure 12 This is a statistical analysis of the bacterial load in the kidneys of the mouse peritonitis-septicemia model of this invention; PC is the positive control group, which is injected intraperitoneally with 200 μL of physiological saline; T64 is TBP-TA at a concentration of 64 μg / mL; P8 is polymyxin B at a concentration of 8 μg / mL; P2T8 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 8 μg / mL; P2T4 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 4 μg / mL; P2T2 is polymyxin B and TBP-TA at concentrations of 2 μg / mL and 2 μg / mL.

[0035] Figure 13 This is a histopathological analysis of different organs in the mouse peritonitis-septicemia model of this invention after different treatment regimens. Detailed Implementation

[0036] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0037] Example 1: Determination of the combined inhibitory index (FICI) of combined drug therapy against standard Escherichia coli and its polymyxin-resistant strains. The combined antimicrobial index of TBP-TA and polymyxin B (PMB) was determined using the micro-checkerboard dilution method. After incubation at 37℃ for 19 h ± 1 h, the MIC was recorded, and the synergistic effect was determined by calculating the FICI.

[0038] The structure of TBP-TA is shown below: .

[0039] TBP-TA is disclosed in the literature: "Dou L, Zhou P, Qi F, et al. When AI meets AIE: Machine learning in design function-oriented AIEgens for antibacterialability[J]. Chemical Engineering Journal, 2025: 169426.".

[0040] Polymyxin B, also known as polymyxin B sulfate or polymyxin B sulfate, was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with product number S17056.

[0041] The specific method is as follows: 1. Experimental Preparation 1) Preparation of basic culture medium: Prepare CAMHB broth (culture medium), autoclave and cool to 48℃ for later use.

[0042] The CAMHB broth was purchased from Qingdao Haibo Biotechnology Co., Ltd., product number HB6231-1. Its components (unit: g / L) are as follows: 3.0g beef extract powder, 17.5g acid-hydrolyzed casein, 1.5g soluble starch and 0.05g calcium chloride.

[0043] 2) Method for preparing bacterial suspensions of Escherichia coli standard strain ATCC 25922 and drug-resistant Escherichia coli NXGC 9, drug-resistant Escherichia coli SJ10, drug-resistant Escherichia coli SJ 19, drug-resistant Escherichia coli SJ 34, drug-resistant Escherichia coli SJE 39, drug-resistant Escherichia coli HK 16 and drug-resistant Escherichia coli HK 20 (in "Example 1", the same below): After thawing the cryopreserved bacterial solutions stored at -80℃, inoculate each bacterial solution onto LB solid plates and incubate at 37℃ for 15 hours. Single colonies of freshly cultured bacteria are then inoculated onto LB liquid medium and cultured at 37℃ with shaking until the logarithmic growth phase. The concentration is then adjusted to 1×10⁻⁶ using CAMHB liquid medium. 6 CFU / mL (calibrated by McFarland turbidimetric method) was used to obtain bacterial suspensions of each bacterium.

[0044] Among them, the bacteria include Escherichia coli standard strain ATCC 25922 and drug-resistant Escherichia coli NXGC 9, drug-resistant Escherichia coli SJ 10, drug-resistant Escherichia coli SJ 19, drug-resistant Escherichia coli SJ 34, drug-resistant Escherichia coli SJE 39, drug-resistant Escherichia coli HK 16 and drug-resistant Escherichia coli HK 20.

[0045] All of the above strains can be found in the literature: "Feng J, Xiang Q, Ma J, et al. Characterization of carbapenem-resistant Enterobacteriaceae cultured from retail meat products, patients, and porcine excrement in China[J]. Frontiers in Microbiology, 2021, 12: 743468."

[0046] 2. Serial dilution and cross-inoculation of drugs in 96-well plates (1) Layout design of 96-well plate: the horizontal direction is the gradient of drug A, and the vertical direction is the gradient of drug B. Among them, drug A is polymyxin B, and drug B is TBP-TA.

[0047] (2) Drug A and drug B were serially diluted twice using sterile CAMHB broth, resulting in 8 dilution concentrations (dilution ratios of 2, ... 2 2 3 2 4 2 5 2 6 2 7 and 2 8 ).

[0048] (3) Cross-drug concentration setting: the test concentration of drug A is 0.125μg / mL~16μg / mL, and the test concentration of drug B is 1μg / mL~128μg / mL, so that the two drugs are cross-combined in a checkerboard pattern.

[0049] (4) For both horizontal and vertical rows of drugs, add the drugs sequentially from low to high concentration (drug A and drug B), adding 50 μL of each different drug solution to each well, for a total of 100 μL. Then add 1×10 6 Add 100 μL of bacterial suspension of each bacterium (CFU / mL) to each well and gently shake the 96-well plate to mix thoroughly.

[0050] 3. Cultivation and Determination of Antibacterial Concentration Place the 96-well plate in a 37°C incubator and incubate for 18 hours.

[0051] Record the minimum inhibitory concentration of combined drugs: Record the MIC of drug A during combined use, the MIC of drug A when used alone, the MIC of drug B during combined use, and the MIC of drug B when used alone to calculate the FICI index.

[0052] 4. Calculation of FICI and result determination Calculate the inhibitory concentration index or fractional inhibitory concentration index (FICI) according to the following formula: FICI = (MIC a ' / MIC a ) + (MICᵦ' / MICᵦ); In the above formula, MIC a ' refers to the MIC of drug A during combined use, MIC a refers to the MIC of drug A when used alone, MICᵦ' refers to the MIC of drug B during combined use, and MICᵦ refers to the MIC of drug B when used alone.

[0053] The criteria for judging the interaction between two drugs (drug A and drug B) are as follows: FICI ≤ 0.5 indicates that the interaction type between drug A and drug B is synergistic, indicating that the combined use of drug A and drug B has a significantly better effect than the single drug effect, and the total drug effect is greater than the sum of each drug.

[0054] 0.5 < FICI ≤ 1 indicates that the interaction type between drug A and drug B is additive, indicating that the combined use of drug A and drug B has an effect equal to the sum of the effects of each drug.

[0055] 1 < FICI ≤ 2 indicates that the interaction type between drug A and drug B is irrelevant, indicating that the combined use of drug A and drug B has an effect similar to the single drug effect and there is no significant mutual influence.

[0056] FICI > 2 indicates that the interaction type between drug A and drug B is antagonistic, indicating that the combined use of drug A and drug B has a worse effect than the single drug effect and there is mutual interference.

[0057] The FICI results of the combined use of the measured TBP-TA and polymyxin B are shown in Figure 1 .

[0058] The FICI value is between 0.0625 and 0.5, indicating that the combined use of drug A and drug B has synergistic antibacterial activity against the standard strain of Escherichia coli and multiple polymyxin-resistant bacteria, and can reduce the dosage of polymyxin by about 16 times.

[0059] Example 2: Statistically analyze the bactericidal efficiency of combined drugs by plate counting (1) Take the bacterial suspensions of Escherichia coli ATCC 25922 and drug-resistant Escherichia coli SJ 10 cultured overnight in BHI broth, and adjust the bacterial suspension to McFarland turbidity of 0.5 with CAMHB broth. Take 100 μL of the 0.5 McFarland bacterial suspension and add it to 10 mL of MH broth.

[0060] Among them, BHI broth, CAMHB broth and MH broth are all commercially available culture media, purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0061] (2) Take a 96-well plate and add 50 μL of PMB and 50 μL of TBP-TA at a specific concentration combination (PC,T64,P0.25,P0.25T8,P0.25T4,P0.25T2,P1, where P is the abbreviation for polymyxin B, T is the abbreviation for TBP-TA, and the numbers correspond to the concentrations. For example, P2T8 refers to a dosage of 2 μg / mL polymyxin B and 8 μg / mL TBP-TA). Add 100 μL of the solution diluted in step (1) (concentration of 1×10⁻⁶) to the plate. 6 A culture of drug-resistant Escherichia coli SJ 10 (CFU / mL) was prepared in a 96-well plate and incubated at 37°C. A specific concentration combination (PC, T64, P2, P2T16, P2T8, P2T4, P4, P8, where P is an abbreviation for polymyxin B and T is an abbreviation for TBP-TA, and the numbers correspond to the concentrations. For example, P2T8 refers to a dosage of 2 μg / mL polymyxin B and 8 μg / mL TBP-TA) of 50 μL PMB and 50 μL TBP-TA. Then, 100 μL of a diluted solution (concentration 1×10⁻⁶ CFU / mL) was added. 6 The bacterial suspension of Escherichia coli standard strain ATCC 25922 (CFU / mL) was used to incubate 96-well plates at 37°C as a positive control group (hereinafter referred to as the control group). A positive control group was also established, consisting of either the standard strain or a drug-resistant strain without the drug solution.

[0062] (3) Take 100 μL from the 96-well plate every 0 min, 10 min and 30 min, and dilute it with CAMHB broth by a specific multiple (using the 10-fold serial dilution method, with a dilution gradient range of 10~10). 6 100 μL of diluted Escherichia coli standard culture ATCC 25922 was spread onto LB agar plates and incubated at 37°C for 17 h.

[0063] Every 0 min, 30 min, and 60 min, 100 μL of the sample was taken from the 96-well plate and diluted with CAMHB broth at specific folds (using a 10-fold serial dilution method, with a dilution gradient range of 10~10). 6100 μL of diluted Escherichia coli SJ 10 bacterial suspension was spread onto LB agar plates and incubated at 37°C for 17 h.

[0064] (4) Count the colonies formed on the plate. Calculate the bactericidal efficiency of the combined two-drug treatment at a specific concentration using the following formula:

[0065] Sterilization efficiency = (number of colonies in control group - number of colonies in test group) / number of colonies in control group × 100%; In the above formula, the unit of sterilization efficiency is %; the colony count in the control group refers to the colony count of Escherichia coli standard strain ATCC 25922; the colony count in the experimental group refers to the colony count of drug-resistant Escherichia coli SJ 10.

[0066] The results are as follows Figure 2 As shown, combined drug administration significantly improves the bactericidal rate of polymyxin B against Escherichia coli. Even with reduced antibiotic concentrations, the colony count in the combined drug group was comparable to that in the high-concentration antibiotic monotherapy group, indicating that combined drug administration enhances the bactericidal efficiency of the drugs.

[0067] In this context, combined drug use is equivalent to mixing TBP-TA and polymyxin B to form an antibacterial composition before use.

[0068] Example 3: Plotting the time-bacterial control curve of combined drug administration Pick a single colony and incubate it in BHI broth at 37°C and 200 rpm until the logarithmic growth phase. Adjust the bacterial turbidity to McFarland turbidity of 0.5 using a McFarland turbidimeter and dilute it 100-fold with CAMHB broth. Add 100 μL of the diluted bacterial solution to a 96-well plate.

[0069] A negative control containing only CAMHB broth and a positive control containing the test bacterial culture were also set up. OD was detected. 600nm The absorbance at the point was measured hourly for a total of 12 hours. Three biological replicates were set up for each treatment.

[0070] The results are as follows Figure 3 As shown, low-concentration antibiotics alone are insufficient to inhibit bacterial growth, but the addition of a certain concentration of TBP-QA significantly inhibits bacterial growth. This indicates that the combination of the two can better inhibit the growth of standard strains of Escherichia coli or polymyxin-resistant bacteria.

[0071] Example 4: Hemolysis test to evaluate the biosafety of combined drug therapy (1) Take 2 mL of defibrinated sheep blood and centrifuge at 3,000 rpm for 10 min at 4°C. Then, gently rinse twice with PBS. Repeat until the supernatant is colorless and clear to obtain a red blood cell suspension. The source of the defibrinated sheep blood is:

[0072] The PBS was purchased from Beijing Solarbio Science & Technology Co., Ltd., at a concentration of 1×, 0.01M, and a pH of 7.3. (2) Take 1.6 mL of red blood cell suspension from step (1) and add 18.4 mL of PBS to prepare an 8% (v / v) red blood cell suspension.

[0073] (3) Add 100 μL of 8% (v / v) PBS suspension of red blood cells to each well. Add 100 μL of different concentrations (P2T8, P2T4, P2T2, where P is the abbreviation for polymyxin B and T is the abbreviation for TBP-TA, and the numbers correspond to the concentrations. For example, P2T8 refers to a drug combination (antibacterial composition) of TBP-TA and PMB with a drug concentration of 2 μg / mL polymyxin B and 8 μg / mL TBP-TA. Add 100 μL of 0.2% (v / v) Triton X-100 to the positive control group. Add 100 μL of PBS buffer solution to the negative control group. PBS buffer solution is PBS.

[0074] (4) After placing in a 37°C water bath for 1 hour, take 120 μL of supernatant into a new 1.5 mL EP tube, centrifuge at 3,000 rpm for 10 min, take 100 μL of supernatant and measure its absorbance at 576 nm.

[0075] The hemolysis rate is calculated as follows: Hemolysis rate = [(OD)] 576样本 -OD 576阴性 ) / (OD 576 阳性 -100-OD 576阴性 ]×100%; In the above formula, the unit of hemolysis rate is %; OD 576样本 This refers to the absorbance of the experimental group at 576 nm; OD 576阴性 This refers to the absorbance of the negative control group at 576 nm; OD 576阳性 This refers to the absorbance of the positive control group at 576 nm.

[0076] The results are as follows Figure 4 As shown, under specific drug concentration combinations, the hemolysis rate of combined drug use meets the clinical use standard (less than 5%) and has biocompatibility.

[0077] Example 6: Establishing a mouse model of peritonitis-septicemia caused by polymyxin B-resistant Escherichia coli infection and evaluating the in vivo therapeutic effect of combined drug therapy. The therapeutic effect of combined drug therapy in a mouse model of drug-resistant Escherichia coli peritonitis-septicemia was evaluated by smear counting and pathological section staining of major mouse organs. The establishment process of the drug-resistant Escherichia coli peritonitis-septicemia mouse model is as follows: Figure 5 As shown.

[0078] The mice were 7-week-old SPF-grade BALB / c female mice, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

[0079] (1) Preparation of bacterial culture: Drug-resistant Escherichia coli SJ 10 was cultured overnight at 37℃ and then adjusted to McFarland 0.5. 15 mL of the McFarland 0.5 bacterial culture was taken and concentrated (after centrifugation to obtain bacterial precipitate, it was resuspended in physiological saline) to 5 mL, so that the final bacterial count was 3 × 10⁻⁶. 8 CFU / mL was used to obtain a bacterial suspension at 3 times the 0.5 McFarland concentration for subsequent intraperitoneal injection. The source of the drug-resistant *E. coli* SJ 10 was disclosed in the literature: “Feng J, Xiang Q, Ma J, et al. Characterization of carbapenem-resistant Enterobacteriaceae cultured from retail meat products, patients, and porcine excrement in China[J]. Frontiers in Microbiology, 2021,12: 743468.” Overnight refers to a time ≥12 hours.

[0080] (2) Injection of bacterial solution: Seven-week-old female BALB / C mice weighing 18g-22g were randomly divided into 6 groups, with 6 mice in each group. All mice were injected intraperitoneally with 3×10 8 200 μL of bacterial culture with CFU / mL.

[0081] (3) Injection of drugs: 1 hour after bacterial infection, the mice in the six groups were given different drugs via intraperitoneal injection.

[0082] The drug was dissolved and diluted with physiological saline, and the corresponding concentration of drug was prepared according to the dosage of 200 μL per mouse, which is 0.02 kg of body weight.

[0083] The treatment and dosage of the 6 groups of mice were as follows: Group 1 was injected with sterile saline, Group 2 was injected with TBP-TA, Group 3 was injected with PMB, and Groups 4 to 6 were injected with drug combinations of different concentrations of TBP-TA and PMB.

[0084] The drug combinations of different concentrations of TBP-TA and PMB are as follows: Group 1: 200 μL of normal saline was injected.

[0085] Group 2-T64: Inject 200 μL of 6.4 mg / mL PMB solution.

[0086] Group 3 - P8: Inject 200 μL of 0.8 mg / mL PMB solution.

[0087] Group 4 - P2T8: Inject 100 μL of 0.4 mg / mL PMB solution + 100 μL of 1.6 mg / mL TBP-TA solution.

[0088] Group 5 - P2T4: Inject 100 μL of 0.4 mg / mL PMB solution + 100 μL of 0.8 mg / mL TBP-TA solution.

[0089] Group 6-P2T2: Inject 100 μL of 0.4 mg / mL PMB solution + 100 μL of 0.4 mg / mL TBP-TA solution.

[0090] PMB solution is obtained by dissolving polymyxin B in sterile physiological water and mixing thoroughly. TBP-TA solution is obtained by dissolving TBP-TA in dimethyl sulfoxide cell cryopreservation solution (DMSO) and mixing thoroughly.

[0091] (4) Observation and Recording: The survival rate of mice in each group was continuously observed and recorded for 48 hours after injection. All surviving mice were sacrificed after 48 hours. The survival rate statistics are shown in the graph below. Figure 6 The survival rate of the combination therapy group was higher than that of the other groups.

[0092] Immediately after the mice died, their hearts, livers, spleens, lungs, and kidneys were harvested. The harvested organs were ground into tissue homogenates and serially diluted with sterile saline (using a 10-fold serial dilution method, with a dilution gradient of 10-10). 6 Add the diluted homogenate to LB medium and incubate overnight at 37°C for colony counting; or fix with 4% (v / v) paraformaldehyde tissue fixative and send for pathological HE staining. "Overnight" refers to a time ≥12 hours.

[0093] Bacterial load in mouse visceral organs is shown in Figure 7 and Figures 8-12 The bacterial load in the internal organs of mice in the combined drug group was significantly lower than that in the other groups.

[0094] Results of HE staining pathological sections of mouse internal organs as follows Figure 13 As shown, the tissue structure damage and inflammatory cell infiltration were more significant in the non-combination therapy group. This indicates that combination therapy has good in vivo efficacy in treating this infection model.

[0095] The experimental results above show that the combination of TBP-TA and polymyxin B has a significant synergistic antibacterial effect against Escherichia coli, good biosafety, and excellent in vivo therapeutic effect in a mouse peritonitis-septicemia infection model.

[0096] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0097] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. An antibacterial composition for treating Escherichia coli infection, characterized in that, It is composed of polymyxin B and TBP-TA, which has aggregation-induced emission properties; The mass ratio of polymyxin B to TBP-TA is 1:1~32; The structure of the TBP-TA is shown below: 。 2. The method for preparing the antibacterial composition according to claim 1, characterized in that, Includes the following steps: The antibacterial composition is obtained by mixing TBP-TA and polymyxin B.

3. The use of the antimicrobial composition of claim 1 in the preparation of a medicament for treating and / or preventing diseases caused by Gram-negative bacteria.

4. The application according to claim 3, characterized in that, The drug is made from the antibacterial composition and supplemented with pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The drug is any one of injection, aerosol, and oral liquid formulation.

6. The application according to claim 3, characterized in that, The antimicrobial composition is used to inhibit or kill Gram-negative bacteria.

7. The application according to claim 6, characterized in that, The Gram-negative bacteria include Escherichia coli (Escherichia coli) Escherichia coli ).

8. The application according to claim 3, characterized in that, Diseases caused by Gram-negative bacteria include peritonitis-septicemia.

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