Anti-acinetobacter baumannii composition and application thereof

Abstract

The invention relates to the technical field of anti-infection drugs, in particular to an anti-acinetobacter baumannii composition and application thereof. The anti-acinetobacter baumannii composition comprises a compound D44 and an antibiotic, the mass ratio of the compound D44 to the antibiotic is 1: (0.01-16); the chemical structure of the compound D44 is shown as a formula I. The composition with an anti-infection effect is obtained through research and screening, the growth of the acinetobacter baumannii can be effectively inhibited by combining the compound D44 and antibiotics, and particularly, after the compound D44 and daptomycin are combined for use, structural disintegration of membrane lipid bilayers is triggered through a double-membrane targeting mechanism, so that the acinetobacter baumannii is cracked to die. The invention provides a new treatment strategy for serious infection caused by drug-resistant acinetobacter baumannii, and has remarkable and better application value.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-infection drugs, and in particular to an anti-Acinetobacter baumannii composition and application thereof. Background Art

[0002] Antibiotics are an important pillar of modern medicine. In many parts of the world, the emergence of multidrug-resistant or even pan-drug-resistant bacterial pathogens is becoming more and more frequent, posing a serious challenge to public health and medical safety. To address this threat, it is urgent to develop new antibiotics for highly prevalent pathogens. Enterobacteriaceae (such as Escherichia coli and Klebsiella pneumoniae) have become representatives of widespread extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Gram-negative bacteria. At the same time, non-fermenting bacteria Pseudomonas aeruginosa and Acinetobacter baumannii also show similar resistance trends. These multidrug-resistant and pan-drug-resistant Gram-negative bacteria have developed into a major threat to health.

[0003] Currently, single antibiotic therapy is still the dominant treatment for infections caused by Gram-positive or Gram-negative bacteria, and combination therapy is only the ultimate treatment option for severe infections caused by multidrug-resistant bacteria. However, bacteria will eventually develop varying degrees of resistance to new or approved antibiotics, so there is an urgent need to explore new therapies through more basic research, translational medicine and clinical practice. In this context, the application value of drug combination strategies is becoming increasingly prominent. It can shorten treatment, improve efficacy and reduce toxicity through synergistic effects, reduce the probability of bacteria developing resistance to new antibiotics, and effectively treat infections caused by resistant bacteria. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an anti-Acinetobacter baumannii composition and application thereof.

[0005] In a first aspect, the present invention provides an anti-Acinetobacter baumannii composition, comprising: Compound D44 and antibiotics; the mass ratio of compound D44 to antibiotics is 1:(0.01-16); The chemical structure of the compound D44 is shown in Formula I: .

[0006] Furthermore, the antibiotic is a cyclic lipopeptide antibiotic or a glycopeptide antibiotic, and the mass ratio of the compound D44 to the antibiotic is 1:(0.1~4).

[0007] Furthermore, the antibiotic is a cyclolipopeptide antibiotic; the mass ratio of the compound D44 to the cyclolipopeptide antibiotic is 1:(0.25~4).

[0008] Furthermore, the cyclolipopeptide antibiotic is daptomycin, and the mass ratio of the compound D44 to daptomycin is 1:(0.5~2).

[0009] Furthermore, the concentration of the compound D44 is 4~32µg / mL; the concentration of daptomycin is 4~32µg / mL.

[0010] During the research process, the present invention discovered that compound D44 and daptomycin themselves do not have the effect of inhibiting Acinetobacter baumannii, but after combining the two, the combined use of compound D44 and daptomycin can produce significantly superior synergistic antibacterial activity against Acinetobacter baumannii. Compound D44 destroys the permeability barrier of the bacterial outer membrane, promotes the penetration of daptomycin and targets the inner membrane, triggering the structural collapse of the membrane lipid bilayer, and ultimately triggering cell dehydration contraction, leading to bacterial lysis and death.

[0011] In addition, the present invention compares various combinations of compound D44 and antibiotics. Although the FICI index of compound D44 and daptomycin is not optimal, they show the best antibacterial activity and the best therapeutic effect on mouse infection.

[0012] In a second aspect, the present invention provides a product, which comprises the aforementioned composition; the product is a medicine, an antibacterial agent, a disinfectant or a preservative.

[0013] Furthermore, the product is one or more of a solid dosage form, a semisolid dosage form, a liquid dosage form or a gaseous dosage form.

[0014] The solid dosage form of the present invention includes: one or more of tablets, capsules, granules, powders, pills or suppositories. The semisolid dosage form of the present invention includes: one or more of ointments, creams or gels. The liquid dosage form of the present invention includes: one or more of solutions, syrups, injections, suspensions or emulsions. The gas dosage form of the present invention includes: aerosols.

[0015] In a third aspect, the present invention provides use of the aforementioned composition in the preparation of anti-infective drugs.

[0016] Furthermore, the anti-infective drug is a drug for resisting Acinetobacter baumannii infection.

[0017] The present invention has the following beneficial effects: The present invention obtains an anti-infection composition by combining compound D44 and daptomycin, a cyclic lipopeptide antibiotic. Each component in the anti-infection composition provided by the present invention cannot inhibit Acinetobacter baumannii by itself, but there is a synergistic effect after the two are combined, and the anti-infection composition has a significant inhibitory effect on Acinetobacter baumannii. The anti-infection composition provided by the present invention can be prepared in the form of a medicine or an antibacterial agent, and can be used to prevent and treat a variety of infectious diseases caused by Acinetobacter baumannii, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Picture 1 This is the bactericidal curve of D44 provided in Example 3 of the present invention in combination with daptomycin against Acinetobacter baumannii.

[0020] Picture 2 This is the bactericidal curve of D44 provided in Example 3 of the present invention in combination with vancomycin against Acinetobacter baumannii.

[0021] Picture 3 This is the bactericidal curve of D44 provided in Example 3 of the present invention in combination with meropenem against Acinetobacter baumannii.

[0022] Picture 4 This is the sterilization curve of D44 provided in Example 3 of the present invention in combination with ceftazidime against Acinetobacter baumannii.

[0023] Picture 5 This is the sterilization curve of D44 provided in Example 3 of the present invention in combination with aztreonam against Acinetobacter baumannii.

[0024] Picture 6 This is the bactericidal curve of D44 provided in Example 3 of the present invention in combination with linezolid against Acinetobacter baumannii.

[0025] Picture 7 This is the sterilization curve of D44 provided in Example 4 of the present invention in combination with daptomycin in different ratios against Acinetobacter baumannii.

[0026] Picture 8 The NPN method provided in Example 4 of the present invention is used to determine the effect of the combined use of D44 and daptomycin on the permeability of the bacterial outer membrane; wherein, p <0.01; p<0.001; p <0.0001.

[0027] Picture 9 The PI method provided in Example 4 of the present invention was used to determine the effect of the combined use of D44 and daptomycin on cell membrane integrity; wherein, the D44+daptomycin group was compared with the D44, daptomycin and control groups. p All <0.01.

[0028] Picture 10 is the OD provided in Example 4 of the present invention 600nm The effect of combined use of D44 and daptomycin on the growth curve of Acinetobacter baumannii was dynamically monitored by the method; among them, the D44+daptomycin group was compared with the D44, daptomycin and control groups at the same time point. p All <0.0001, D44 compared with the control group at the same time point p <0.0001.

[0029] Picture 11 This is the effect of the combined use of D44 and daptomycin provided in Example 5 of the present invention on the bacterial morphology of Acinetobacter baumannii. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The experimental methods involved in the following examples, unless otherwise mentioned, are all conventional methods in the art, for example, reference may be made to experimental manuals in the art, or the conditions recommended in the manufacturer's instructions.

[0032] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0033] The experimental instruments involved in the following embodiments are: Shaking incubator (ZWY-100H, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), round-bottom 96-well cell culture plate (3799, Corning), McFarland turbidimeter (DENSICHEK, Bio-Merieux), vortexer (MS 3digital, IKA), high-pressure sterilizer (SQL510C, YAMATO), biological safety cabinet (Class Ⅱ, Nuaire), and high-precision electronic balance (XS105, Mettler, USA).

[0034] Example 1 The present invention verifies the inhibitory effects of compound D44 in combination with daptomycin, vancomycin, meropenem, ceftazidime, and aztreonam on Acinetobacter baumannii, specifically including the following: 1. The determination methods include: The strains tested included A. baumannii ATCC 19606, A. baumannii ATCC 17978, A. baumannii 24-13, A. baumannii 24-14, A. baumannii 24-18, A. baumannii 24-22, A. baumannii 24-24, A. baumannii 24-25, A. baumannii 24-32, A. baumannii 24-33.

[0035] Two days before the test, take out the strains to be tested from the -80℃ freezer, inoculate them on MHA plates by ice streak method, and culture them overnight at 37℃. Pick 3 to 5 well-separated and uniformly morphological colonies from the agar plate and inoculate them in 3mL CAMH medium, and culture them at 37℃ overnight.

[0036] The minimum inhibitory concentration (MIC) of each drug combination against Acinetobacter baumannii was determined by the chessboard method and the fractional inhibitory concentration index (FICI) was calculated. The specific method is as follows: the McFarland concentration of the bacterial solution was measured by McFarland turbidimeter, and the bacterial solution was adjusted to 0.5 McFarland (about 1×10 8CFU / mL), and diluted 100-fold with CAMH medium for standby. The bacterial solution was used within 15 min after preparation. Referring to the CLSI standard, the mother solutions of D44 and each antibiotic drug were serially diluted with CAMH broth culture medium for standby. Add 100 μL of each antibiotic drug solution to the 12th column of the round-bottom 96-well plate, and add 50 μL of CAMH broth culture medium to the remaining wells. Perform serial dilution to the 2nd column by the two-fold dilution method, so that the concentration of each antibacterial drug in the 1st column is 0. Add 50 μL of D44 with different concentrations after two-fold dilution to each row from the 1st row to the 7th row, and add 50 μL of CAMH broth culture medium to the 8th row. The diluted drug concentrations are all 4 times the final experimental concentration. At this time, the solution in each well is 100 μL. Take 100 μL of the prepared bacterial solution and add it to the round-bottom 96-well plate, and the inoculation concentration is about 5×10 5 CFU / mL; Wrap the 96-well plate with plastic wrap and place it in a static culture at 37 °C for 16 - 20 h; Take out the 96-well plate and observe with the naked eye whether there is colony deposition at the bottom of the wells. The minimum concentration of the drug contained in the wells without bacterial growth is the MIC value (refer to the CLSI standard). Calculate the fractional inhibitory concentration index FICI, FICI = MIC 甲药联用 / MIC 甲药单用 +MIC 乙药联用 / MIC 乙药单用 . The judgment basis for synergistic effect is: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, irrelevant effect; FICI > 2, antagonistic effect. The test strains all include ATCC standard strains and clinical isolates.

[0037] The test results are shown in Table 1 - Table 6: The combination of D44 with daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, and linezolid has a broad synergistic antibacterial effect against Acinetobacter baumannii. After the combination of D44 with each of the above antibiotics, the MIC values are all significantly reduced. Among them, the MIC of daptomycin is significantly reduced by 8 - 64 times, the MIC of vancomycin is significantly reduced by 64 - 256 times, the MIC of meropenem is significantly reduced by 4 - 64 times, the MIC of ceftazidime is significantly reduced by 4 - 64 times, the MIC of aztreonam is significantly reduced by 4 - 32 times, and the MIC of linezolid is significantly reduced by 4 - 8 times; Through the calculation of the fractional inhibitory concentration index (FICI), the synergistic effect is obvious, and the FICI values are all less than 0.5. The average FICI values are <0.108; <0.076; <0.133; <0.155; <0.147; <0.413 respectively, indicating that the combination of the two produces a strong synergistic antibacterial effect. The antibacterial activities of the combination of D44 with daptomycin and vancomycin against Acinetobacter baumannii are better than those of the combination of D44 with meropenem, ceftazidime, aztreonam, and linezolid against Acinetobacter baumannii.

[0038] Table 1 FICI values ​​of D44 combined with daptomycin

[0039] Table 2 FICI values ​​of D44 combined with vancomycin

[0040] Table 3 FICI values ​​of D44 combined with meropenem

[0041] Table 4 FICI values ​​of D44 combined with ceftazidime

[0042] Table 5 FICI values ​​of D44 combined with aztreonam

[0043] Table 6 FICI values ​​of D44 combined with linezolid

[0044] Example 2 The present invention uses D44 in combination with daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, and linezolid to determine the time-kill curve of Acinetobacter baumannii, including the following: The measurement methods include: Store in -80℃ freezer A. baumannii ATCC19606 strain was taken out, quickly streaked on MHA plate, and cultured in 37℃ incubator overnight. 3~5 single colonies were picked from the plate, inoculated into 3mL CAMH medium, and cultured at 37℃ and 220rpm for 2~3h. The bacterial solution cultured to logarithmic growth phase was inoculated into 5mL fresh CAMH medium at 1:100, and cultured at 37℃ overnight. The next day, the bacterial solution was diluted to about 2×10 6 CFU / mL, divided into sterile small test tubes, 5 mL in each tube.

[0045] Referring to the MIC values ​​of D44 and antibiotics alone, the antibiotic alone group, D44 alone group, and antibiotic and D44 combination group were added to the tubes with a final concentration of 1 / 4×MIC, and a bacterial growth control group without antibiotics and D44 was set up, with 5 mL in each tube; in addition, in order to explore the optimal concentration ratio of D44 and daptomycin, the concentrations of the daptomycin and D44 combination group were set as: D44 (32µg / mL): daptomycin (32µg / mL) = 1:1, D44 (32µg / mL): daptomycin (16µg / mL) = 2:1, D44 (32µg / mL): daptomycin (8µg / mL) = 4:1, D44 (16µg / mL): daptomycin (32µg / mL) = 1:2, D44 (8µg / mL): daptomycin (32µg / mL) = 1:4. Samples were taken at 0, 2, 4, 6, 8 and 24 hours for colony counts using the drip method.

[0046] Drip colony counting method: Take 100µL of bacterial solution and add it to 900µL of 0.85% NaCl, and so on for ten-fold gradient dilution. Take 10µL of the appropriate dilution gradient of bacterial solution and drop it on the MHA plate. Tilt the plate so that it flows down in a linear manner, and set up 3 biological replicates for each. After standing and absorbing, place the plate in a 37℃ incubator and invert it for overnight culture. According to the test results, the test concentration can be adjusted later.

[0047] With Time (h) as the horizontal axis, Log 10 CFU / mL was used as the ordinate, and the bactericidal curve was drawn using GraphPad Prism 5. Drug synergy judgment criteria: During the observation period, the colony count of the combination group decreased by ≥2Log compared with the group with the strongest drug effect when the drug was used alone. 10 CFU / mL, and the colony count of the combination group decreased by ≥2Log compared with the initial inoculation volume 10 CFU / mL, it was judged to be a synergistic effect.

[0048] Test results such as Pictures 1~6 As shown: D44 combined with various antibiotics A. baumannii The bactericidal curve of ATCC19606 showed that the colony count of the group using D44 in combination with various antibiotics (daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, linezolid) decreased by ≥2Log compared with the group using the strongest drug alone. 10 CFU / mL, and the colony count of the combination group decreased by ≥2Log compared with the initial inoculation volume 10 CFU / mL, so the synergistic effect of D44 in combination with various antibiotics (daptomycin, vancomycin, meropenem, ceftazidime, aztreonam, linezolid) was verified; Picture 7As shown: Taking 8h as an example, the time point when the colony count of the combination group decreased the most compared with the strongest drug single-drug group, the antibacterial effects of D44 and daptomycin at different concentration ratios were compared, and the synergistic antibacterial effects at different concentration ratios were obtained: 1:1>1:2>2:1>1:4>4:1.

[0049] Example 3 The present invention uses D44 in combination with daptomycin, vancomycin and meropenem to determine its effects on A. baumannii The therapeutic effect of ATCC19606 in mice with systemic infection.

[0050] The measurement methods include: take out A. baumannii For the frozen strain of ATCC19606, pick ice chips and inoculate them in 5mL BHI medium, shake and culture at 37℃ and 220rpm for 6h, then transfer the above bacterial solution to fresh medium of the same culture medium at 1:50, and culture at 37℃ for 18h. Dilute the bacterial solution appropriately with 5% highly active yeast solution to prepare the infection bacterial solution for use; dilute it several times with 0.85% NaCl, and take 10µL and drip it on the MHA plate, wait for it to dry, and then invert it for colony counting. ICR female mice weighing 18-22g were randomly divided into groups according to their weight, with 5 mice in each group. After the abdomen was disinfected with iodine, 0.5mL of 100% minimum lethal dose (100%MLD) bacterial solution was injected into the peritoneal cavity of the mice. The D44 combined with daptomycin group and its single-use control group (D44 single-use group, daptomycin single-use group) were administered once 1h after infection; while the other combination groups and their corresponding single-use control groups were intravenously administered 1h and 6h after infection, respectively, for a total of 2 times. Each drug was prepared with normal saline, and the administration volume was 0.2mL / 20g. The infection control group was given an equal volume of normal saline. The number of animals surviving within 7d was observed, and the survival rate of each group was calculated.

[0051] The results are shown in Tables 7 to 9: Intraperitoneal infection of mice in each group A. baumanniiATCC19606 was administered intravenously and observed for 7 consecutive days. The results showed that the survival rate of the infected control group animals was 0%; the survival rate of the animals in the 40 mg / kg D44 single dose group was 0%; the survival rate of the animals in the daptomycin single dose groups of 40 mg / kg, 80 mg / kg, and 120 mg / kg was 0%. After the combined use of 40 mg / kg D44, the survival rate of the animals in each combination group increased to 100%, which was significantly higher than that of the single-dose group. This shows that after the combination of daptomycin and D44, the in vivo antibacterial activity is enhanced, and the D44 and daptomycin dose ratios of 1:1, 1:2, and 1:3 all have good synergistic antibacterial activity in vivo. In addition, after daptomycin 5mg / kg, 10mg / kg, 20mg / kg was combined with 40mg / kg of D44, the survival rates of animals in each combination group were 0%, 60%, and 100%, respectively, indicating that the in vivo synergistic antibacterial activity of D44 and daptomycin at a dose ratio of 2:1 was better than 4:1, and no in vivo synergistic antibacterial activity was shown at a dose ratio of 1:8. The survival rates of animals in the single-dose groups of meropenem 20mg / kg and 40mg / kg were 0% and 20%, respectively. After combined with 40mg / kg of D44, the survival rates of animals in the combined dose groups increased to 60% and 80%, respectively, which was significantly higher than the survival rates of animals in the single-dose groups, indicating that the in vivo antibacterial activity of meropenem and D44 was enhanced after combined use. The survival rate of animals in the single-dose groups of vancomycin 80mg / kg and 160mg / kg was 0%. After the combination of D44 at a dose of 40mg / kg, the survival rate of animals in each combined dose group increased to 100%, which was significantly higher than that of the single-dose group, indicating that the antibacterial activity in vivo was enhanced after the combination of vancomycin and D44. The above results show that the combination of D44 and meropenem, and the combination of 1901 and vancomycin can A. baumannii It has a protective effect against systemic infection and death of mice caused by ATCC19606. D44 combined with daptomycin, meropenem, and vancomycin all have good in vivo anti-Acinetobacter baumannii infection activity and have a good effect in treating Acinetobacter baumannii infection. The combination of D44 and daptomycin is more effective than the combination of D44 and meropenem or D44 and vancomycin.

[0052] Table 7 Daptomycin combined with D44 A. baumannii Death rate of mice in each group infected with ATCC19606

[0053] Infection dose 1×10 7 CFU / mouse.

[0054] Table 8 Meropenem combined with D44 A. baumannii Death rate of mice in each group infected with ATCC19606

[0055] Infection dose 1×10 7 CFU / mouse.

[0056] Table 9 Vancomycin combined with D44 antibiotic A. baumannii Death rate of mice in each group infected with ATCC19606

[0057] Infection dose 1×10 7 CFU / mouse.

[0058] Example 4 The present invention explores the level of damage to the inner and outer membranes of Acinetobacter baumannii by using D44 in combination with daptomycin, specifically including the following: The measurement methods include: Store in -80℃ freezer A. baumannii ATCC19606 strain was taken out and quickly streaked on MHA plate, and then cultured in a 37°C incubator overnight. 3-5 single colonies were picked from the plate and inoculated into 3 mL CAMH medium, and cultured at 37°C and 220 rpm for 2-3 hours. The bacterial suspension was diluted to 0.5 McFarland concentration (about 1×10 8 CFU / mL), and dispensed into sterile glass test tubes. D44, DAP, and D44+DAP were added to each tube, and a control group of bacteria without drug was set up, and cultured with shaking at 180 rpm and 37°C.

[0059] After 2 hours of culture, 1 mL of bacterial solution was taken from each well, centrifuged at 1,2300 g for 3 minutes, washed once with 5 mM HEPES (pH = 7.2), and resuspended in the same volume of 5 mM HEPES, and N-Phenyl- 1-naphthylamine (NPN) was added at a final concentration of 10 µM. Each group of resuspended solutions was added to a black-bottom 96-well plate, 100 µL per well. The fluorescence value of each well was measured using an ELISA reader, with an excitation wavelength of 355 nm and an emission wavelength of 405 nm. Calculate the NPN uptake factor = (fluorescence value 样品 +NPN - Fluorescence value 样品不含NPN ) / (fluorescence value HEPES+NPN - Fluorescence value HEPES 不含 NPN ).

[0060] Take 1 mL of bacterial solution at 0h, 2h, and 4h, centrifuge at 1,2300g for 3min, wash once with sterile 1×PBS, add the same volume of 1×PBS to fully resuspend the bacteria, and add PI fluorescent reagent with a final concentration of 2.5µM. Add each group of resuspended liquid to a black-bottom 96-well plate, 100µL in each well. Use an enzyme reader to measure the fluorescence value of each well. The excitation wavelength is 535nm and the emission wavelength is 617nm; take samples synchronously at each time point to measure the OD of the bacterial solution 600 nm value.

[0061] Test results such as Picture 8-10 As shown: NPN is a hydrophobic fluorescent probe that is soluble in the lipid bilayer. When the cell outer membrane is damaged, NPN can pass through the outer membrane into the phospholipid layer and produce fluorescence. The amount of NPN entering the cell is monitored by detecting the increase in the fluorescence intensity of the solution, thereby reflecting the permeability of the bacterial outer membrane. In this experiment, samples were taken at each time point and centrifuged, the compounds in the sample were removed and resuspended with HEPES, and then NPN was added to measure the fluorescence value. This method eliminates the influence of the compound fluorescence value. The measurement was carried out 2 hours after drug treatment, and the results showed that ( Picture 8 ), compared with the control group, there was no significant change in the NPN fluorescence intensity in the daptomycin alone group ( p >0.05); the fluorescence intensity of NPN in the D44 alone group increased significantly ( p <0.0001); the NPN fluorescence intensity in the group treated with D44 combined with daptomycin increased significantly ( p <0.01).

[0062] The results showed that: daptomycin alone could not damage the cell outer membrane and increase the outer membrane permeability; D44 alone and D44 combined with daptomycin could damage the cell outer membrane and increase the cell outer membrane permeability. It is worth noting that the fluorescence intensity of the D44 alone group was significantly higher than that of the combined group ( p <0.0001). The possible reason is that daptomycin is a cyclic lipopeptide antibiotic, and its target is cell membrane phospholipids. It inserts into the bacterial cell membrane through its lipophilic tail and can bind to negatively charged phospholipids (such as phosphatidylglycerol); while D44 destroys the permeability of the bacterial outer membrane by inhibiting lipopolysaccharide synthesis. It is speculated that the combination of D44 and daptomycin allows daptomycin to penetrate the outer membrane and target the inner or outer membrane phospholipid bilayer. In this process, daptomycin may occupy the hydrophobic binding domain of the outer membrane phospholipids and form competitive inhibition with NPN; in addition, bacterial death induced by the combination may cause membrane structure collapse, further reducing the binding sites of NPN, and ultimately resulting in the NPN fluorescence intensity in the D44 combination group being higher than that in the control group, but the increase is significantly lower than that in the D44 single group.

[0063] PI is a nuclear staining reagent. The intact bacterial cell membrane (inner membrane) has a barrier function. PI cannot penetrate the intact lipid bilayer structure and therefore cannot enter the cell. When the bacterial cell inner membrane increases permeability or forms pores due to toxins, environmental pressure or drugs (such as antibiotics), PI can penetrate the damaged membrane and enter the cell. PI is embedded in double-stranded DNA and releases red fluorescence. The measurement results show ( Picture 9 ), 4 hours after drug treatment, compared with the control group, there was no significant change in PI fluorescence values ​​in the D44 alone group and the daptomycin alone group ( p >0.05, while the D44 combined with daptomycin group showed a significant increase in PI fluorescence value ( p <0.01). Mechanism analysis showed that although D44 can destroy the permeability of the bacterial outer membrane, it has no significant effect on the inner cell membrane when used alone, and PI cannot penetrate the intact lipid bilayer into the cell. Daptomycin is a membrane lipid-targeted antibiotic. When used alone, it is difficult for it to contact the inner membrane phospholipid target because it cannot penetrate the intact outer membrane. When the two drugs are used in combination, D44-mediated outer membrane destruction significantly improves the transmembrane penetration efficiency of daptomycin, allowing it to fully contact the inner membrane phospholipids and cause membrane structural damage. This dual targeting of the outer membrane and inner membrane ultimately leads to the loss of membrane integrity, allowing PI to enter the cell and bind to DNA. OD 600nm Dynamic detection results of values ​​( Picture 10 ) further confirmed that 4 hours after D44 was used in combination with daptomycin, the absorbance of the bacterial solution decreased significantly and the turbidity of the bacterial solution decreased, indicating that membrane damage caused a large number of bacteria to lyse and die.

[0064] This study showed that the combination of D44 and daptomycin has a synergistically enhanced double-membrane targeting effect on Acinetobacter baumannii. After D44 destroys the outer membrane of the cell, it promotes daptomycin to penetrate the outer membrane and act on the inner membrane, thereby destroying the integrity of the cell membrane and ultimately leading to bacterial lysis and death.

[0065] Example 5 The present invention explores the effect of the combined use of D44 and daptomycin on the bacterial morphology of Acinetobacter baumannii, specifically including the following: The measurement methods include: Store in -80℃ freezer A. baumannii ATCC19606 strain was taken out and quickly streaked on MHA plate, and then cultured in a 37°C incubator overnight. 3-5 single colonies were picked from the plate and inoculated into 3 mL CAMH medium, and cultured at 37°C and 220 rpm for 4-5 hours. The bacterial suspension was diluted to 0.5 McFarland's concentration (about 1×10 8CFU / mL), and dispensed into sterile glass test tubes. D44 (32µg / mL), DAP (32µg / mL), and D44 (32µg / mL) + DAP (32µg / mL) were added to each tube, and a control group of bacteria without drug was set up, and cultured with shaking at 180rpm and 37C for 6h.

[0066] Take 1.5mL of bacterial solution and add it to 1.5mL EP tubes, centrifuge at 3000×g for 10min (centrifuge 3mL of bacterial solution in total, which can be centrifuged multiple times), discard the supernatant, wash the bacteria with 1×PBS 1~2 times, and discard the supernatant. Fill each EP tube with 2.5% glutaraldehyde fixative along the wall of the centrifuge tube, be careful not to let the bacteria disperse, seal the tube with sealing film and fix it at 4℃ overnight. The next day, send the sample to Zhongke Baice Company for scanning electron microscope (SEM) detection.

[0067] Test results such as Picture 11 As shown: In the D44 group alone, the bacteria changed from long rods to oval shapes, and the outer layer of the bacteria became smooth. In the daptomycin group alone, no obvious changes were observed in the bacteria. In the D44 and daptomycin combination group, due to the changes in the permeability of the bacterial cell outer membrane and the destruction of the inner membrane, the double membrane damage caused the cell dehydration shrinkage, the bacterial surface collapsed and shrunk, and the morphology was distorted.

[0068] Analysis of the antibacterial mechanism of the combination of D44 and daptomycin: Studies have confirmed that daptomycin, as a cyclic lipopeptide antibiotic mainly targeting Gram-positive bacteria, has a lipophilic tail that inserts into the bacterial cell membrane and specifically binds to negatively charged phospholipids (such as phosphatidylglycerol), inhibiting bacterial protein synthesis and preventing bacterial cell wall synthesis, thereby inhibiting bacterial growth. It is worth noting that neither daptomycin nor D44 has any antibacterial activity against Acinetobacter baumannii alone. This study found that the combination of D44 and daptomycin can produce synergistic antibacterial activity against Acinetobacter baumannii, and its dual membrane targeting mechanism is as follows: D44 destroys the permeability barrier of the bacterial outer membrane, promotes daptomycin penetration and targets the inner membrane, triggering the structural collapse of the membrane lipid bilayer, and ultimately triggering cell dehydration contraction, leading to bacterial lysis and death.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-Acinetobacter baumannii composition, characterized in that: include: Compound D44 and antibiotics; the mass ratio of compound D44 to antibiotics is 1:(0.01-16); The chemical structure of the compound D44 is shown in Formula I: 。 2. The composition according to claim 1, characterized in that The antibiotic is a cyclic lipopeptide antibiotic or a glycopeptide antibiotic, and the mass ratio of the compound D44 to the antibiotic is 1:(0.1~4).

3. The composition according to claim 2, characterized in that The antibiotic is a cyclolipopeptide antibiotic; the mass ratio of the compound D44 to the cyclolipopeptide antibiotic is 1:(0.25~4).

4. The composition according to claim 3, characterized in that The cyclic lipopeptide antibiotic is daptomycin, and the mass ratio of the compound D44 to daptomycin is 1:(0.5~2).

5. The composition according to claim 4, characterized in that The concentration of the compound D44 is 4-32 µg / mL; the concentration of daptomycin is 4-32 µg / mL.

6. A product, characterized in that The product comprises the composition according to any one of claims 1 to 5; the product is a medicine, an antibacterial agent, a disinfectant or a preservative.

7. The product according to claim 6, characterized in that The product is one or more of a solid dosage form, a semisolid dosage form, a liquid dosage form or a gaseous dosage form.

8. Use of the composition according to any one of claims 1 to 5 in the preparation of anti-infective drugs.

9. The use according to claim 8, characterized in that: The anti-infective drug is a drug for resisting Acinetobacter baumannii infection.

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