A tritrpticin-based derivative antibacterial peptide, its synthesis method and application in antibacterial aspect

Abstract

The application belongs to the technical field of medicine, and particularly relates to design, preparation and application of an antibacterial peptide derivative. The application takes Tritrpticin antibacterial peptide as a structural template, and designs and prepares a series of terminal symmetrical polypeptide derivatives containing a beta-turn structure. The polypeptide derivative in the application has high antibacterial activity and safety, and has a good application prospect in antibacterial aspects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a derivative antibacterial peptide based on Tritrpticin and a synthetic method and application thereof in antibacterial aspects. BACKGROUND

[0002] At present, the abuse of antibiotics has become a global public health problem, which accelerates the generation of multi-drug resistant bacteria and poses a serious threat to human health. Therefore, it is particularly crucial to develop antibiotic alternative drugs which are not prone to cause bacterial resistance in terms of containing drug-resistant bacterial infections. As a kind of bioactive molecules widely existing in nature, antibacterial peptides universally exhibit broad-spectrum antibacterial activity. So far, more than 10,000 natural antibacterial peptides have been identified, which exhibit various conformations including alpha-helix, beta-sheet and random coil. These natural antibacterial peptides mainly exert antibacterial effect through a non-specific membrane disruption mechanism. Such antibacterial mechanism has a fast action speed and does not require receptor mediation, and the bacterial killing is lethal and not prone to cause bacterial resistance. Natural antibacterial peptides are usually composed of cationic residues and hydrophobic residues, and their positive charge and amphiphilicity are crucial for the exertion of membrane disruption effect.

[0003] Tritrpticin (VRRFPWWWPFLRR) is a natural antibacterial peptide with broad-spectrum antibacterial activity, which is composed of a middle beta-turn structure (FPWWWPF) and two terminal extension chains rich in cationic and hydrophobic residues (VRRF, FLRR) and mainly inhibits bacterial growth or kills bacteria through a membrane disruption mechanism. Among them, the three consecutive tryptophan residues (WWW) in the beta-turn structure can be anchored on the bacterial cell membrane through the indole ring side chain, thereby enhancing the interaction between the polypeptide and the bacterial cell membrane; the cationic and hydrophobic residues in the two terminal extension chains can promote the adsorption and insertion of the polypeptide to the bacterial cell membrane through electrostatic interaction and hydrophobic interaction, respectively. The unique structure of Tritrpticin provides an ideal template for antibacterial peptide design, and the antibacterial peptides based on the combination of the beta-turn structure fragment and the terminal symmetric amphiphilic structure fragment show good application prospects in antibiotic alternatives. SUMMARY

[0004] The present application provides a kind of derivative antibacterial peptide based on Tritrpticin, the antibacterial peptide has amino acid sequence as shown in general formula (1) or (2):

[0005] X (YX) n ZZ (XY) n X (1)

[0006] (YX) n ZZ (XY) n (2)

[0007] According to an embodiment of the present application, n is selected from 1, 2, 3;

[0008] According to an embodiment of the present application, ZZ is selected from a β-turn sequence FPWWWPF or PWWWP;

[0009] According to an embodiment of the present application, each X is the same or different, independently selected from a basic amino acid or a side chain with a positive charge of an amino acid derivative, for example, can be selected from one of lysine (Lys, K), arginine (Arg, R), and other side chains with a positive charge of an amino acid derivative, such as ornithine (Orn, O), homoarginine (Har);

[0010] According to an embodiment of the present application, each Y is the same or different, independently selected from a hydrophobic amino acid or a hydrophobic amino acid derivative, for example, can be selected from one of tryptophan (Trp, W), tyrosine (Tyr, Y), phenylalanine (Phe, F), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), alanine (Ala, A), and other hydrophobic amino acid derivatives, such as naphthylalanine (Nal).

[0011] According to an embodiment of the present application, the amino acid or amino acid derivative is L-type or D-type.

[0012] According to an embodiment of the present application, the N-terminal group of the antibacterial peptide is a free amino group or acetylated; for example, the N-terminal group is an acetylamino group.

[0013] According to an embodiment of the present application, the C-terminal group of the antibacterial peptide is a carboxyl group or amidated; for example, the C-terminal group is amidated to an amide group.

[0014] According to a preferred embodiment of the present application, the antibacterial peptide has an amino acid sequence as shown below:

[0015] SEQ ID NO. 1 (FP-11): KVKVKFPWWWPFKVKVK-NH2

[0016] SEQ ID NO. 2 (P-07): KIKIKPWWWPKIKIK-NH2

[0017] According to a preferred embodiment of the present application, the antibacterial peptide consists of L-type amino acids, the N-terminal group of the antibacterial peptide is acetylated, and / or the C-terminal is amidated.

[0018] According to a preferred embodiment of the present application, the key physicochemical property information of the antibacterial peptides FP-11 and P-07 is shown in Table 1:

[0019] Table 1 Key physico-chemical properties of FP-11 and P-07

[0020]

[0021] According to an embodiment of the present application, the antibacterial peptide is synthesized by solid phase synthesis.

[0022] The present application also provides an antibacterial medicament comprising a therapeutically effective amount of the antibacterial peptide.

[0023] The present application also provides use of the antibacterial peptide in the preparation of an antibacterial medicament.

[0024] According to an embodiment of the present application, the antibacterial medicament is used for treating an infection caused by a pathogenic bacterium (e.g. a skin wound infection). According to an embodiment of the present application, the pathogenic bacterium is selected from at least one of Gram-positive bacteria or Gram-negative bacteria, for example selected from at least one of Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella oxytoca, Streptococcus, Bacillus, or a multi-drug resistant strain thereof. Preferably, it is a drug-resistant Escherichia coli.

[0025] The present application also provides a combination antibacterial medicament, the active ingredients of which comprise a therapeutically effective amount of the antibacterial peptide and an antibacterial antibiotic.

[0026] According to an embodiment of the present application, the combination antibacterial medicament is used for treating an infection caused by a pathogenic bacterium (e.g. a skin wound infection). According to an embodiment of the present application, the antibacterial peptide is preferably antibacterial peptide P-07.

[0027] According to an embodiment of the present application, the antibiotic is selected from at least one of a polypeptide antibiotic, a penicillin antibiotic, a beta-lactam antibiotic, a quinolone antibiotic, a cephalosporin antibiotic, a semi-synthetic carbapenem antibiotic. Preferably, it is a quinolone antibiotic levofloxacin.

[0028] According to an embodiment of the present application, the pathogenic bacterium is selected from at least one of Gram-positive bacteria or Gram-negative bacteria, for example selected from at least one of Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Klebsiella oxytoca, Streptococcus, Bacillus, or a multi-drug resistant strain thereof. Preferably, it is a drug-resistant Escherichia coli.

[0029] Advantages

[0030] The Tritrpticin-based derivative antibacterial peptide according to the present application is composed of a beta-turn structure and a terminal symmetric hydrophilic-hydrophobic alternating amino acid sequence, has a good structural basis for exerting antibacterial effect, shows strong activity against drug-resistant bacteria and high safety, for example, shows broad-spectrum bacteriostatic activity against multi-drug resistant strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii, and has low hemolytic rate and HEK-293T cytotoxicity. As an antibiotic synergistic adjuvant, the antibacterial peptide in combination with the antibiotic levofloxacin can effectively treat skin wound infections caused by drug-resistant E. coli. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 High performance liquid chromatogram of antibacterial peptide FP-11;

[0032] Figure 2 High performance liquid chromatogram of antibacterial peptide P-07;

[0033] Figure 3 Mass spectrum of antibacterial peptide FP-11;

[0034] Figure 4 Mass spectrum of antibacterial peptide P-07;

[0035] Figure 5 Hemolysis of Tritrpticin and derivatives (FP-11, P-07);

[0036] Figure 6 Cytotoxicity of Tritrpticin and derivatives (FP-11, P-07) to HEK-293T;

[0037] Figure 7The effect of the combination of antibacterial peptide P-07 and antibiotic levofloxacin on promoting wound healing in mice infected with bacteria; (A) Schematic diagram of the development of wound infection and treatment procedure; bacterial load of E. coli ATCC 25922 (B) and drug-resistant E. coli (C) at the wound site of mice three days after administration; (D) Colony count chart of E. coli ATCC 25922 and drug-resistant E. coli at the wound site of mice three days after administration; (E) Body weight change of mice infected with drug-resistant E. coli during administration; (F) Representative photographs of the skin wounds of mice infected with drug-resistant E. coli within 11 days (scale bar: 5 mm); (G) Wound size change of mice infected with drug-resistant E. coli during administration; (H) H&E and interleukin-6 (IL-6) analysis of wound tissue of infected mice on the 11th day after administration (scale bar: 100 pm). The epidermal layer of different groups is in the red box. Black arrows indicate the absence and damage of the epidermis. Yellow arrows indicate inflammatory cell infiltration. Red arrows indicate vascular hyperemia and dilation. Green arrows indicate tissue hemorrhage. Un-inf is the negative control group; NS is the normal saline group; Normal is the blank control group; L is the levofloxacin group. One-way ANOVA and T-test were used to analyze whether there was a significant difference between the data (p < 0.05: *, p < 0.01: **, p < 0.001: ***). DETAILED DESCRIPTION

[0038] The technical solutions of the present disclosure will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present disclosure. Any technology realized based on the above description of the present disclosure is covered within the scope intended to be protected by the present disclosure.

[0039] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0040] Example 1

[0041] Synthesis, identification and purification of antibacterial peptides:

[0042] 1. Solid-phase synthesis of polypeptides (FP-11 and P-07):

[0043] ① The reaction was carried out at room temperature, and the reaction scale was 0.25 mmol. Rink Amide-AM resin (loading capacity of 0.53 mmol / g) was used as the starting point for synthesis, and the peptide chain was elongated from the C-terminal to the N-terminal.

[0044] ② Add the resin to the reactor, add 10 mL of dichloromethane (DCM) to swell for 30 min, remove the solvent, wash 3 times each with N,N-dimethylformamide (DMF) and DCM, stirring for 3 min each time, and remove the solvent.

[0045] ③ Add 5 mL of 20% piperidine / DMF (v / v), stir for 5 min, dry under vacuum, then add an equal amount of deprotection reagent, stir for 20 min, dry under vacuum, wash with DMF and DCM 3 times each, stirring for 3 min each time, and dry under vacuum.

[0046] ④ Take 4 equivalents of Fmoc-protected amino acids, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBT) and 8 equivalents of N,N-diisopropylethylamine (DIEA) and add them to 5 mL of DMF. Activate for 10 min, then add them to the reactor and stir. React for 1 h, then dry under vacuum. Wash DMF and DCM 3 times each, stirring for 3 min each time, and then dry under vacuum.

[0047] ⑤ Repeat steps ③ and ④, linking the amino acids sequentially to the peptide chain one by one. Wash with DMF and DCM three times each, stirring for 3 minutes each time, and then dry. Finally, remove the N-terminal Fmoc protecting group, wash with DMF and DCM three times each, stirring for 3 minutes each time, and then dry.

[0048] 2. Detection of free amino groups:

[0049] The Kaiser ninhydrin hydrate method is the most commonly used method for detecting free amino groups. It can qualitatively determine whether the amide condensation reaction has been completed through a color reaction. The specific operating steps are as follows:

[0050] After each condensation reaction, clean the resin thoroughly. Take about 20 resin grains, add 2-3 drops of ninhydrin indicator, and heat at 100°C for 5 minutes. If the resin is colorless and transparent, the reaction is complete and the next step can be carried out. If it is blue, the condensation needs to be repeated.

[0051] 3. Reagent preparation:

[0052] Ninhydrin indicator: Take 0.5g of ninhydrin powder and add it to 10mL of anhydrous ethanol. Mix well until completely dissolved.

[0053] Phenol solution: The phenol solution is obtained by mixing redistilled anhydrous phenol with anhydrous ethanol at a volume ratio of 4 / 1.

[0054] Pyridine solution: Pyridine solution after redistillation.

[0055] Pyrolysis reagent: Trifluoroacetic acid / triisopropylsilane / water = 90:5:5 (volume ratio)

[0056] 4. Pyrolysis and freeze-drying:

[0057] After the completion of the peptide resin preparation, 5 mL of anhydrous ether was added, and stirring was performed for 3 min, which was repeated 3 times. After the resin was fully dried, it was transferred to a 250 mL round-bottom flask, and a cleavage reagent (10 mL / mg) was added. Stirring was performed under ice bath conditions for 30 min, and after the ice bath was removed, the reaction was continued for 2.5 h. After the completion of the reaction, anhydrous ether was added, and stirring was performed for 30 min. Precipitation was performed by standing, and the crude peptide was obtained by filtration.

[0058] Freeze-drying: The crude peptide solution was collected and transferred to a centrifuge tube, and rapid freezing was performed under liquid nitrogen conditions. After freeze-drying, a crude peptide solid powder was obtained.

[0059] 5. Analysis and purification:

[0060] Analytical high-performance liquid chromatography (HPLC) chromatographic conditions:

[0061] Chromatographic column: C18 column (250 mm x 4.6 mm, 5 μm)

[0062] Mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile

[0063] Flow rate: 1 mL / min; detection wavelength: 210 nm; injection volume: 20 μL

[0064] The elution program is shown in Table 2

[0065] Analysis was performed by analytical HPLC, the main peak was collected, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to determine the molecular weight of the polypeptide. After the molecular weight was determined to be consistent with the theoretical value, the polypeptide was purified.

[0066] Table 2 HPLC detection of crude peptide purity elution program

[0067]

[0068] 6. Polypeptide purification:

[0069] 50 mg of crude peptide was weighed, dissolved in 50 mL of phase A, and filtered through a 0.45 μm water-based microporous filter for preparation liquid chromatography separation and purification.

[0070] Preparative HPLC chromatographic conditions:

[0071] Chromatographic column: C18 preparation column (250 mm x 20 mm, 5 μm)

[0072] Mobile phase A: 0.1% TFA / water, mobile phase B: 0.1% TFA / acetonitrile

[0073] Flow rate: 15 mL / min; detection wavelength: 210 nm; injection volume: 5000 μL

[0074] The elution procedure of purification was adjusted according to the physicochemical properties of the target polypeptide, gradient elution was performed at room temperature, and the purity of the purified polypeptide was detected using analytical HPLC. The polypeptide solution with a purity greater than 95% was collected, the acetonitrile was removed by a rotary evaporator, the polypeptide aqueous solution was collected and freeze-dried to obtain pure peptide solids, which were collected, weighed, sealed in a centrifuge tube, and stored at -40°C.

[0075] Example 2

[0076] Evaluation of the in vitro antibacterial activity of antibacterial peptides:

[0077] The minimal inhibitory concentration (MIC) of Tritrpticin and its derivatives in drug-resistant strains of E. faecalis, S. epidermidis, S. aureus, E. coli, K. pneumoniae, and A. baumannii was determined by the doubling dilution method.

[0078] The strains were incubated overnight at 37°C with shaking at 150 rpm. The bacterial suspension was transferred to fresh culture medium and incubated until the logarithmic growth phase. The MIC of the polypeptide was determined by the standard broth dilution method: the bacterial suspension was diluted to 1 × 10 6 CFU / mL, and the polypeptide solution was serially diluted 2-fold with culture medium to 1-256 μM. 50 μL of the bacterial suspension was mixed with an equal volume of the polypeptide solution and added to a 96-well plate, which was incubated in a 37°C constant-temperature shaking incubator at 180 r / min for 18 h. After incubation, the growth of bacteria in the 96-well transparent plate was observed, and the minimum polypeptide concentration corresponding to the liquid clear well was the MIC of the polypeptide. The experimental results are shown in Table 3. The modified antibacterial peptides FP-11 and P-07 had good antibacterial activity against both Gram-positive and Gram-negative bacteria.

[0079] Table 3 MIC values of Tritrpticin and its derivatives

[0080]

[0081] Example 3

[0082] Hemolytic experiment

[0083] Hemolysis of the antibacterial peptides was evaluated using human red blood cells. Fresh red blood cells were washed with PBS for 3 times and resuspended into a cell suspension (8%, v / v). The well-dispersed cell suspension was quickly added into a sterile 96-well transparent plate, 5 parallels for each concentration, 50 μL per well, and 50 μL of the polypeptide solution was added into each well, untreated red blood cells were used as negative control, and Triton-X100-treated red blood cells were used as positive control. After incubation, the plate was centrifuged at 1800 r / min for 5 min, and 50 μL of supernatant was taken from each well and added into a new 96-well transparent plate. The absorbance at 490 nm was measured by a microplate reader. The hemolytic rate was calculated according to the following formula:

[0084] Hemolytic rate = [OD 490 nm(treatment) - OD 490 nm(negative control) ] / [OD 490 nm(positive control) - OD 490 nm(negative control) ] x 100%.

[0085] OD 490 nm(treatment) : represents the absorbance of the cell wells

[0086] OD 490 nm(positive control) : represents the absorbance of the positive control wells

[0087] OD 490 nm(negative control) : represents the absorbance of the negative control wells

[0088] The experimental results are shown in Table 1. Figure 5 The antibacterial peptide FP-11 showed slight hemolysis in the detection range, and no hemolysis was found for P-07 at the highest detection concentration.

[0089] Example 4

[0090] In vitro cell proliferation toxicity experiment:

[0091] The in vitro cytotoxicity of the antibacterial peptides was evaluated using human kidney epithelial cells (HEK-293T). The cells were cultured to the logarithmic growth phase, and a uniformly dispersed cell suspension was quickly added to sterile 96-well plates, 5 replicates for each concentration, 100 μL per well, containing about 10,000 cells per well, and incubated at 37°C for 24 h. After 24 h, the original culture medium in the wells was aspirated, and the prepared polypeptide solutions of various concentrations were added to the culture plates. The negative control group was added with blank culture medium without cells, and the positive control group was added with blank culture medium with the same number of cells as the drug group. The plates were incubated at 37°C for 24 h. At the end of the incubation, the 96-well plates were removed, the original culture medium was aspirated, CCK-8 working solution was added, and the plates were incubated for another 1 h. The absorbance at 450 nm was measured by a microplate reader, and the cell survival rate was calculated according to the value. The cell survival rate calculation formula is as follows:

[0092] Cell survival rate = [OD 450 nm(treatment) - OD 450 nm(negative control) ] / [OD 450nm(positive control) - OD 450 nm(negative control) ] x 100%.

[0093] OD 450 nm(treatment) : represents the absorbance value of the drug cell well

[0094] OD 450 nm(positive control) : represents the absorbance value of the positive control well

[0095] OD 450 nm(negative control) : represents the absorbance value of the negative control well

[0096] The experimental results are shown in Figure 6 , and the antibacterial peptides FP-11 and P-07 have less cytotoxicity to HEK-293T cells, and no cytotoxicity was detected within the effective antibacterial concentration range.

[0097] Example 5

[0098] In vivo wound healing experiment

[0099] Male Kunming mice (6 weeks old, about 20 g) were acclimated for 1 week. The full-thickness wound model of bacterial infection in mice was established using E. coli ATCC25922 and drug-resistant E. coli. First, the mice were anesthetized with sodium pentobarbital, then the hair on the back of each mouse was shaved, and the exposed skin was disinfected with an alcohol cotton ball, after which a circular full-thickness wound with a diameter of about 8 mm was cut on the back of the mouse. The bacteria in the logarithmic growth phase were centrifuged, washed with PBS and resuspended in PBS solution (1 x 10 5 CFU / mL). The bacterial suspension (100 μL per wound) was dropped onto the wound site, and 24 hours after bacterial infection, a biofilm appeared on the wound site on the back of the mouse, indicating that the full-thickness wound model was established. The model mice were randomly divided into four groups, including a normal saline group, a P-07 group, a levofloxacin group, and a P-07 + levofloxacin group, with 5 mice in each group. In addition, 5 mice with only a wound but no bacterial infection and 5 normal mice were used as negative and blank controls, respectively. The drug concentration was set at 100 μM, and the antibacterial agent solution (50 μL) was dropped on the wound once a day for 11 days. In order to record the process of wound healing, the wound was photographed daily, and the wound size was measured with a caliper. On the 3rd day, the wound tissue of the mouse was taken to make a homogenate, which was diluted with sterile normal saline, and then bacterial counting was performed by plate coating method. On the 11th day, the skin tissue at the wound site was collected for hematoxylin and eosin (H&E) staining and immunohistochemical analysis.

[0100] The experimental results are shown in Figure 7 . As shown in Figure 7 B, P-07, levofloxacin, and P-07 combined with levofloxacin can significantly reduce the bacterial load of E. coli ATCC 25922 on the wound, and the bactericidal effect of levofloxacin and P-07 combined with levofloxacin is more significant than that of P-07. However, in the treatment of drug-resistant E. coli infection, the bactericidal effect of P-07 exceeds that of levofloxacin Figure 7 C and D. As shown in Figure 7 E, F, the wound size in each group gradually decreased within 11 days, indicating the healing process of the wound under natural and drug conditions. The body weight of each group of mice rapidly decreased at the beginning of modeling, and gradually returned to normal levels after the start of treatment (7G). In order to further verify the efficacy of different treatment drugs, hematoxylin and eosin (H&E) staining was used to examine the histopathological changes at the infection site at the end of treatment. The epidermal layer recovered well in the P-07 + levofloxacin group, and there was no vascular congestion, tissue hemorrhage, or inflammatory cell infiltration. Immunohistochemical detection of interleukin-6 (IL-6) expression levels in infected tissues Figure 7H) IL-6 expression in the saline group was stronger, suggesting that the inflammatory response at the infection site was more severe. The expression level of IL-6 in the levofloxacin group was comparable to that in the saline group. In contrast, the expression level of IL-6 in the P-07 and P-07 + levofloxacin groups was much lower, similar to that in the uninfected group of mice. In summary, P-07 significantly reduced the bacterial load and inflammatory response at the E. coli infection site, thereby promoting wound healing. Compared with levofloxacin or P-07 alone, the combination of the two drugs had a better effect on wound healing.

[0101] The above has exemplarily described the embodiments of the technical scheme of the present disclosure. It should be understood that the protection scope of the present disclosure is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present application.

Claims

1. The following are tritrpticin-derived antimicrobial peptides. SEQ ID NO. 2 (P-07): KIKIKPWWWPKIKIK-NH2.

2. The Tritrpticin-derived antimicrobial peptide as described in SEQ ID NO.2 (P-07), characterized in that, The antimicrobial peptide is composed of L-type amino acids, and the N-terminal group of the antimicrobial peptide is acetylated and / or the C-terminus is amidated.

3. The method for preparing the antimicrobial peptide according to claim 1 or 2, wherein the peptide is prepared by solid-phase synthesis.

4. An antimicrobial agent comprising a therapeutically effective amount of the antimicrobial peptide of claim 1 or 2.

5. The use of the antimicrobial peptide according to claim 1 or 2 in the preparation of antimicrobial drugs; The bacteria are selected from at least one of Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, or their multidrug-resistant strains.

6. The application according to claim 5, characterized in that, The bacteria in question is a drug-resistant Escherichia coli.

7. A combination antimicrobial drug, wherein the active ingredients comprise a therapeutically effective amount of the antimicrobial peptide and an antibacterial antibiotic as described in claim 1 or 2; And / or, the combined antimicrobial agents are used to treat infections caused by pathogens; And / or, the antibiotic is selected from polypeptide antibiotics, penicillin antibiotics, β - At least one of the following: lactam antibiotics, quinolone antibiotics, cephalosporin antibiotics, and semi-synthetic carbapenem antibiotics; And / or, the bacteria are selected from at least one of Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, or their multidrug-resistant strains.

8. The combined antibacterial drug according to claim 7, characterized in that, The antibiotic in question is levofloxacin; And / or, the bacteria are drug-resistant Escherichia coli.

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