Application of polymyxin E combined with 5-fluorocytidine in the preparation of drugs for inhibiting Gram-negative bacteria

Abstract

The present invention relates to the field of medical technology, and provides the use of polymyxin E in combination with 5-fluorocytosine nucleoside in the preparation of a drug for inhibiting Gram-negative bacteria, and limits the concentration of 5-fluorocytosine nucleoside to 4-64 μg / mL; the concentration of the polymyxin E is 0.25-2 μg / mL. The present invention provides the use of 5-fluorocytosine nucleoside combined with polymyxin E in the preparation of a drug for inhibiting mcr-positive bacteria, and 5-fluorocytosine nucleoside can cooperate with polymyxin to exert an antibacterial effect, and reverse the drug resistance of resistant bacteria to polymyxin, while reducing the amount of antibacterial drugs used, achieving the effect of killing bacteria.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of polymyxin E combined with 5-fluorocytidine in the preparation of a drug for antibacterial use. Background Art

[0002] Polymyxin (colistin) is a polycationic antimicrobial peptide that has a strong and rapid bactericidal effect on most Gram-negative bacteria. The antibacterial mechanism of polymyxin is that it can bind to the negatively charged LPS in the cell membrane of Gram-negative bacteria, causing changes in the permeability of the bacterial cell membrane, leading to the leakage of important substances such as bacterial nucleic acids and amino acids, thereby inhibiting bacterial growth and causing bacterial death. In recent years, with the increasingly severe form of bacterial resistance, polymyxin has become one of the "last lines of defense" in the clinical treatment of multidrug-resistant Gram-negative bacteria infections.

[0003] At the end of 2015, Chinese scholars first reported the isolation of the plasmid-mediated polymyxin resistance gene mcr-1 from Escherichia coli isolated from humans and animals. Subsequently, the mcr gene was detected in more and more CRE strains. It is currently found that the mcr-1 gene is mainly present on IncI2, IncX4 and IncHI2 plasmids of Escherichia coli, and has been widely prevalent in more than 60 countries on six continents around the world. The emergence and widespread prevalence of mcr seriously threatens the clinical application of polymyxin. Therefore, it is urgent to find a polymyxin enhancer to restore the sensitivity of bacteria to polymyxin.

[0004] 5-Fluorocytidine is a nucleic acid analogue, but up to now, there has been no application of 5-Fluorocytidine combined with polymyxin in the preparation of drugs to inhibit mcr-positive bacteria at home and abroad. Summary of the invention

[0005] The object of the present invention is to provide an application of polymyxin E combined with 5-fluorocytidine in the preparation of a drug for inhibiting Gram-negative bacteria, increase the sensitivity of Gram-negative bacteria to polymyxin E, and reduce the usage of polymyxin E and 5-fluorocytidine by combined use.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of polymyxin E combined with 5-fluorocytidine in the preparation of a drug for inhibiting Gram-negative bacteria.

[0008] Preferably, the Gram-negative bacteria are polymyxin E-resistant Gram-negative bacteria.

[0009] Preferably, the polymyxin E-resistant Gram-negative bacteria is mcr-1 positive Escherichia coli.

[0010] The present invention also provides a drug for inhibiting Gram-negative bacteria, wherein the drug comprises 5-fluorocytidine and polymyxin E.

[0011] Preferably, the concentration of the 5-fluorocytidine is 4 to 64 μg / mL; and the minimum concentration of the polymyxin E is 0.25 to 2 μg / mL.

[0012] The present invention provides an application of 5-fluorocytosine nucleoside combined with polymyxin E in the preparation of a drug for inhibiting mcr-1 positive bacteria. The present invention verifies through a chessboard method that the combined use of 5-fluorocytosine nucleoside and polymyxin E can restore the effect of polymyxin E on mcr-1 positive Escherichia coli and reverse the drug resistance of drug-resistant bacteria to polymyxin, thereby achieving the effect of killing bacteria while reducing the amount of antibacterial drugs used. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0014] Figure 1 It is the minimum inhibitory concentration of 5-fluorocytosine combined with polymyxin E against mcr-1 positive Escherichia coli by the chessboard method in Example 1. DETAILED DESCRIPTION

[0015] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0016] 1. Experimental drugs

[0017] Polymyxin E: effective content 98%, purchased from Beijing Coupling Technology Co., Ltd.;

[0018] 5-Fluorocytidine: effective content 99.19%, purchased from Shanghai Taoshu Biotechnology Co., Ltd.;

[0019] Polymyxin E was prepared into a stock solution with an initial concentration of 2560 μg / mL using deionized water. The diluted drug was filtered using a sterile syringe and a 0.22 μm filter membrane after autoclaving and stored at -80°C for later use;

[0020] 5-Fluorocytidine was prepared into a stock solution with an initial concentration of 1280 μg / mL using deionized water and stored at -80°C until use.

[0021] 2. Culture medium

[0022] The culture media used in the experiment include: MH broth medium, MH agar medium, LB broth medium, and LB agar medium; all purchased from Beijing Luqiao Technology Co., Ltd.

[0023] Preparation of solid culture medium (MH agar medium and LB agar medium) (500 mL system): weigh 19 g MH agar medium or 20 g LB agar medium, add about 500 mL of distilled water, and mix well; sterilize with high pressure steam at 121°C for 20 min, let it cool to about 50°C, add drugs as needed, pour it into a plate, cool and dry;

[0024] Preparation of liquid culture medium (MH broth and LB broth) (500 mL system): weigh 25 g MH broth or 25 g LB broth, add about 500 mL of distilled water, and mix well; sterilize with high pressure steam at 121°C for 20 min, and let it cool for later use.

[0025] 3. Instruments

[0026] Electronic analytical balance (Satrorius, Germany);

[0027] Automatic vertical pressure steam sterilizer (Sanyo Company, Japan);

[0028] Constant temperature shaking incubator (Shanghai Huayan Instrument Equipment Co., Ltd.);

[0029] Single-channel micro-adjustable pipette (Eppendorf, Germany);

[0030] Multichannel adjustable micropipette (Eppendorf, Germany);

[0031] Ultra-low temperature refrigerator (Thermo Scientific Inc, Germany);

[0032] 0.22 μm filter (Pall Corporation, USA);

[0033] Clean bench (Suzhou Purification Equipment Co., Ltd.).

[0034] 4. Experimental strains

[0035] The clinically isolated polymyxin E-resistant strain of Escherichia coli ZJ807 was mcr-1 positive (for details, see the inventor’s previous patent, patent publication number is CN 114869999 A).

[0036] Example 1

[0037] The minimum inhibitory concentration of 5-fluorocytosine combined with polymyxin E against mcr-1 positive Escherichia coli ZJ807 was detected by chessboard method

[0038] According to the CLSIM27-A3 drug sensitivity test standard, the checkerboard method was used to detect the antibacterial activity of 5-fluorocytosine nucleoside alone, polymyxin E alone, and "5-fluorocytosine and polymyxin E combined" against polymyxin E-resistant bacteria (mcr-1 positive Escherichia coli ZJ807), and then the minimum inhibitory concentration against polymyxin E-resistant bacteria was obtained. The specific steps are as follows:

[0039] 1. Take the mcr-1 positive E. coli ZJ807 bacterial solution that has grown to the logarithmic growth phase, adjust it to McFarland 0.5 with a McFarland turbidimeter, and then dilute it 100 times with MH broth medium to obtain the mcr-1 positive E. coli ZJ807 dilution. The concentration of mcr-1 positive E. coli ZJ807 in the mcr-1 positive E. coli ZJ807 dilution is about 1×10 6 CFU / mL.

[0040] 2. Take a 96-well microplate and inoculate each well with 100 μL of mcr-1-positive Escherichia coli ZJ807 dilution and 100 μL of drug solution (a mixture of polymyxin E stock solution and 5-fluorocytidine stock solution, and 5-fluorocytidine stock solution alone and polymyxin E stock solution alone).

[0041] 3. In a 96-well sterile microplate, use the chessboard method (such as Figure 1 The antibacterial activity of 5-fluorocytidine stock solution alone, polymyxin E stock solution alone, and the combination of the two against mcr-1 positive Escherichia coli was tested;

[0042] 4. Take the 96-well microplate in step 3 and incubate at 37°C for 17 hours;

[0043] 5. Calculate the MIC value of each well and further calculate the combined inhibition fraction FIC.

[0044] Evaluation methods and result interpretation

[0045] FIC = MIC (colistin combined) / MIC (colistin alone) + MIC (5-fluorocytosine combined) / MIC (5-fluorocytosine alone)

[0046] The results are shown in Table 1:

[0047] Table 1 Minimum inhibitory concentration and combined action index of polymyxin E and 5-fluorocytidine alone and in combination

[0048]

[0049] It can be seen that the combination of 5-fluorocytidine and polymyxin E can reduce the MIC value of polymyxin E to mcr-1 positive Escherichia coli by 8 times. Therefore, different concentrations of 5-fluorocytidine significantly reduced the MIC value of mcr-1 positive Escherichia coli ZJ807 to polymyxin E, making the MIC value of polymyxin E of most strains lower than the sensitive value (2μg / mL);

[0050] The judgment criteria of the FIC index are: FIC index ≤ 0.5 is synergistic; 0.5-1 is additive; 1-2 is irrelevant; ≥ 2 is antagonistic. It can be seen from the records in Table 1 that the FIC index is 0.156, which is less than 0.5. Therefore, the present invention has a good synergistic effect on 5-fluorocytidine and polymyxin E. Therefore, it can treat infections caused by mcr-1 positive Escherichia coli ZJ807.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of polymyxin E combined with 5-fluorocytidine in the preparation of a drug for inhibiting Gram-negative bacteria, characterized in that: The Gram-negative bacteria are mcr-1 positive Escherichia coli resistant to polymyxin E.

2. A drug for use according to claim 1, characterized in that: The drug contains 5-fluorocytidine and polymyxin E.

3. The drug according to claim 2, characterized in that The concentration of the 5-fluorocytidine is 4 to 64 μg / mL; the concentration of the polymyxin E is 0.25 to 2 μg / mL.

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