Application of Chelerythrine Combined with Polymyxin E in Inhibiting Polymyxin E-resistant Bacteria

Abstract

The application discloses application of chelerythrine combined with polymyxin E in inhibition of polymyxin E drug-resistant bacteria, and the polymyxin E drug-resistant bacteria are mcr positive bacteria. Experiments prove that chelerythrine can inhibit (such as mcr-1 positive escherichia coli ZJ807, mcr-8 positive klebsiella pneumoniae KP91 and mcr-9 positive salmonella SLM183) in cooperation with polymyxin E, and can reduce the use amount of polymyxin E and reverse the drug resistance to polymyxin E. Chelerythrine can restore the inhibitory effect of polymyxin E on the polymyxin E drug-resistant bacteria. The application has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of celandine combined with polymyxin E in inhibiting polymyxin E-resistant bacteria, which are mcr-positive bacteria. Background Technology

[0002] Polymyxins are polycationic antimicrobial peptides that exhibit strong and rapid bactericidal activity against most Gram-negative bacteria. The antibacterial mechanism of polymyxins involves their binding to negatively charged LPS molecules in the cell membrane of Gram-negative bacteria, altering the cell membrane permeability and causing leakage of essential substances such as nucleic acids and amino acids. This inhibits bacterial growth and ultimately leads to bacterial death. In recent years, with the increasing severity of bacterial resistance, polymyxins have become one of the "last lines of defense" in the clinical treatment of multidrug-resistant Gram-negative bacillus infections.

[0003] In late 2015, the plasmid-mediated polymyxin resistance gene mcr-1 was first reported to be isolated from *Escherichia coli* strains isolated from humans and animals. Subsequently, the mcr gene has been detected in an increasing number of CRE strains. Currently, multiple mcr genotypes have been reported, including mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7, mcr-8, mcr-9, and mcr-10. The emergence and widespread prevalence of the mcr gene seriously threaten the clinical application of polymyxins. Therefore, there is an urgent need to find synergists to enhance bacterial sensitivity to polymyxins, thereby restoring bacterial susceptibility to polymyxins.

[0004] Chelidonine is a benzophenanthridine isoquinoline alkaloid found in the plant *Cephalotaxus fortunei*. It possesses heat-clearing and detoxifying, antibacterial and anti-inflammatory, antihypertensive and anticancer effects. It is a widely used in vitro protein kinase C inhibitor and an effective antagonist of the G protein-coupled CB1 receptor. Studies have shown that chelidonine has anticancer effects against various types of human cancer cells and has become the basis for many potential new anticancer drugs. To date, there are no reports of chelidonine inhibiting *C. cristatus*-positive bacteria. Summary of the Invention

[0005] The purpose of this invention is to find a way to inhibit polymyxin E-resistant bacteria.

[0006] This invention first protects a drug combination for inhibiting polymyxin E-resistant bacteria, comprising chelidonine and polymyxin E.

[0007] The above-mentioned drug combination specifically consists of chelidonine and polymyxin E.

[0008] In the drug combination, the mass ratio of celandine to polymyxin E can be 32-128:1 (e.g., 32-64:1, 64-128:1, 32:1, 64:1 or 128:1).

[0009] In the drug combination, the minimum inhibitory concentration (MIC) of chelidonine can be 8-32 μg / mL (e.g., 8-16 μg / mL, 16-32 μg / mL, 8 μg / mL, 16 μg / mL, or 32 μg / mL). The MIC of polymyxin can be 0.25-1 μg / mL (e.g., 0.25-0.50 μg / mL, 0.50-0.75 μg / mL, 0.75-1 μg / mL, 0.25 μg / mL, 0.50 μg / mL, 0.75 μg / mL, or 1 μg / mL).

[0010] This invention also protects the application of celandine combined with polymyxin E, which can be a1) or a2):

[0011] a1) Inhibits polymyxin E-resistant bacteria;

[0012] a2) Prepare drugs for inhibiting polymyxin E-resistant bacteria.

[0013] This invention also protects the use of celandine in improving the susceptibility of polymyxin E-resistant bacteria to polymyxin E.

[0014] This invention also protects the application of celandine in restoring the inhibitory effect of polymyxin E on polymyxin E-resistant bacteria.

[0015] In any of the applications described above, the mass ratio of chelidonine to polymyxin E can be 32-128:1 (e.g., 32-64:1, 64-128:1, 32:1, 64:1, or 128:1). The minimum inhibitory concentration (MIC) of chelidonine can be 8-32 μg / mL (e.g., 8-16 μg / mL, 16-32 μg / mL, 8 μg / mL, 16 μg / mL, or 32 μg / mL). The MIC of polymyxin can be 0.25-1 μg / mL (e.g., 0.25-0.50 μg / mL, 0.50-0.75 μg / mL, 0.75-1 μg / mL, 0.25 μg / mL, 0.50 μg / mL, 0.75 μg / mL, or 1 μg / mL).

[0016] The polymyxin E resistant bacteria mentioned above can be mcr-positive bacteria.

[0017] The mcr-positive bacteria mentioned above may be mcr-1 positive Escherichia coli ZJ807, mcr-8 positive Klebsiella pneumoniae KP91, or mcr-9 positive Salmonella SLM183.

[0018] Through extensive experiments, the inventors of this invention discovered that chelidonine can synergistically inhibit polymyxin E-resistant bacteria (such as mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, or mgr-9 positive Salmonella SLM183), reversing the resistance of polymyxin E-resistant bacteria to polymyxin E. This achieves the effect of inhibiting polymyxin E-resistant bacteria while reducing the dosage of polymyxin E used. This invention is the first to discover the inhibitory effect of chelidonine on polymyxin E-resistant bacteria. This invention has significant application value. Attached Figure Description

[0019] Figure 1 To determine the minimum inhibitory concentration of celandine combined with polymyxin E against mcr-1 positive Escherichia coli ZJ807 using the checkerboard method.

[0020] Figure 2 To determine the minimum inhibitory concentration of celandine combined with polymyxin E against mcr-8 positive Klebsiella pneumoniae KP91 using the checkerboard method.

[0021] Figure 3 To determine the minimum inhibitory concentration of celandine combined with polymyxin E against mcr-9 positive Salmonella SLM183 using the checkerboard method.

[0022] Figure 4 The time-bactericidal curve of celandine combined with polymyxin E against mcr-1 positive Escherichia coli ZJ807 is shown. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] Polymyxin is a product of Beijing Coupling Technology Co., Ltd., with an effective content of 98%.

[0026] Chelidonine is a product of Shanghai Taoshu Biotechnology Co., Ltd., with an effective content of 99.19%.

[0027] The preparation method of the culture medium involved in the following examples is as follows:

[0028] MH liquid medium: Add 25g of MH broth medium (Beijing Luqiao Technology Co., Ltd.) to an appropriate amount of distilled water, then bring the volume to 500mL with distilled water, autoclave at 121℃ for 20min, and cool.

[0029] The mcr-1-positive *Escherichia coli* ZJ807 is described in the following literature: Y Wang, GB Tian, ​​R Zhang, Y Shen, JM Tyrrell, X Huang, H Zhou, L Lei, HY Li, Y Doi. Supplementary material: Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive *Enterobacteriaceae* in patients and healthy adults from China: anepidemiological and clinical study. *The Lancet Infectious Diseases*. 2017. The genome sequence of mcr-1-positive *Escherichia coli* ZJ807 has been uploaded to NCBI, BioSample Accession Number: SAMN05437814. *Escherichia coli* ZJ807 is a polymyxin E-resistant bacterium.

[0030] The mcr-8 positive Klebsiella pneumoniae KP91 is described in the following literature: X Wang, Y Wang, Z Ying, J Li, Y Wang. Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae. Emerging Microbes & Infections. 2018. The genome sequence of mcr-8 positive Klebsiella pneumoniae KP91 has been uploaded to NCBI, BioSample Accession Number: SAMN14228398. mcr-8 positive Klebsiella pneumoniae KP91 is a polymyxin E resistant bacterium.

[0031] The mcr-9 positive Salmonella SLM183 is described in the following literature: Na Lyu, et al. Genomic Characterization of Salmonella Enterica Isolates From Retail Meat in Beijing, China. Front. Microbiol. 2021. The genome sequence of mcr-9 positive Salmonella SLM183 has been uploaded to NCBI, BioSample Accession Number: SAMN16708049. mcr-9 positive Salmonella SLM183 is a polymyxin E resistant bacterium.

[0032] Example 1: Determination of the minimum inhibitory concentration (MIC) of celandine combined with polymyxin E against polymyxin E-resistant bacteria using the checkerboard method.

[0033] According to the CLSI M27-A3 antimicrobial susceptibility testing standard, the checkerboard method was used to detect the antimicrobial activity of chelidonine alone, polymyxin E alone, and the combination of chelidonine and polymyxin E against polymyxin E-resistant bacteria (mcr-1 positive Escherichia coli ZJ807, mcr-8 positive Klebsiella pneumoniae KP91, or mcr-9 positive Salmonella SLM183), thereby obtaining the minimum inhibitory concentration against polymyxin E-resistant bacteria. The specific steps are as follows:

[0034] 1. Take a bacterial suspension of polymyxin E-resistant bacteria grown to the logarithmic growth phase, adjust it to McFarland 0.5 using a McFarland turbidimeter, and then dilute it 100-fold with MH broth medium to obtain a polymyxin E-resistant bacterial dilution. The concentration of polymyxin E-resistant bacteria in the dilution is approximately 1 × 10⁻⁶. 6 CFU / mL.

[0035] 2. Take polymyxin E and dilute it with deionized water to obtain a polymyxin E solution with a concentration of 2560 μg / mL; then filter the polymyxin E solution through a sterile filter membrane (pore size 0.22 μm) to obtain a polymyxin E stock solution. Take chelidonine and dilute it with deionized water to obtain a chelidonine solution with a concentration of 1280 μg / mL; then filter the chelidonine solution through a sterile filter membrane (pore size 0.22 μm) to obtain a chelidonine stock solution.

[0036] 3. Take a 96-well microplate and inoculate each well with 100 μL of polymyxin E-resistant bacterial dilution and 100 μL of drug solution (a mixture of polymyxin E stock solution and chelidonine stock solution).

[0037] For the mcr-1 positive Escherichia coli ZJ807, in a 96-well microplate, the concentrations of chelidonine in each well of each column from left to right were 0 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL, and 64.0 μg / mL, respectively; and the concentrations of polymyxin E in each well of each row from top to bottom were 0 μg / mL, 0.125 μg / mL, 0.250 μg / mL, 0.500 μg / mL, 1.000 μg / mL, 2.000 μg / mL, and 4.000 μg / mL, respectively.

[0038] For mcr-8 positive Klebsiella pneumoniae KP91, in a 96-well microplate, the concentrations of chelidonine in each well of each column from left to right were 0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL, 64.0 μg / mL, and 128.0 μg / mL, respectively; and the concentrations of polymyxin E in each well of each row from top to bottom were 0 μg / mL, 0.250 μg / mL, 0.500 μg / mL, 1.000 μg / mL, 2.000 μg / mL, 4.000 μg / mL, 8.000 μg / mL, and 16.000 μg / mL, respectively.

[0039] For mcr-9 positive Salmonella SLM183, in a 96-well microplate, the concentrations of chelidonine in each well of each column from left to right are 0 μg / mL, 4.0 μg / mL, 8.0 μg / mL, 16.0 μg / mL, 32.0 μg / mL, 64.0 μg / mL, 128.0 μg / mL, and 256.0 μg / mL, respectively; and the concentrations of polymyxin E in each well of each row from top to bottom are 0 μg / mL, 0.0625 μg / mL, 0.125 μg / mL, 0.250 μg / mL, 0.500 μg / mL, 1.000 μg / mL, 2.000 μg / mL, and 4.000 μg / mL, respectively.

[0040] 4. After completing step 3, take the 96-well microplate and incubate it at 37°C for 16-18 hours.

[0041] 5. After completing step 4, calculate the MIC value for each well and further calculate the combined antimicrobial fraction (FIC).

[0042] In column 1, the concentration of celandine is 0 μg / mL. The concentration of polymyxin E is serially diluted. This column can be used to measure the MIC of polymyxin E, denoted as MIC. 多黏菌素E单用In the first row, the concentration of polymyxin E is 0 μg / mL. The concentration of chelidonine is serially diluted. This row can be used to measure the MIC of chelidonine, denoted as MIC. 白屈菜红碱单用 All others involved a combination of celandine and polymyxin E, with the MIC of polymyxin E denoted as MIC. 多黏菌素E联合 The MIC of celandine is denoted as MIC. 白屈菜红碱联合 Calculate the combined effect index FIC.

[0043] Combined effect index FIC = MIC 多黏菌素E联合 / MIC 多黏菌素E单用 +MIC 白屈菜红碱联合 / MIC 白屈菜红碱单用

[0044] Some test results can be found Figures 1-3 The optimal drug combination calculated using the FICI method is marked with a triangle.

[0045] The experiment was repeated three times, and the results were averaged. The statistical results are shown in Table 1.

[0046] Table 1

[0047]

[0048] The results showed that the combined use of chelidonine and polymyxin E significantly reduced the MIC values ​​of polymyxin E against mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, and mgr-9 positive Salmonella SLM183. For mgr-1 positive Escherichia coli ZJ807, the optimal mass ratio of chelidonine to polymyxin E was 32:1; at this ratio, the minimum inhibitory concentration (MIC) of chelidonine was 8 μg / mL, and the MIC of polymyxin was 0.25 μg / mL. For mgr-8 positive Klebsiella pneumoniae KP91, the optimal mass ratio of chelidonine to polymyxin E was also 32:1; at this ratio, the MIC of chelidonine was 32 μg / mL, and the MIC of polymyxin was 1 μg / mL. For the mcr-9 positive Salmonella SLM183, the optimal mass ratio of chelidonine to polymyxin E is 128:1; at this ratio, the minimum inhibitory concentration of chelidonine is 32 μg / mL, and the minimum inhibitory concentration of polymyxin E is 0.25 μg / mL.

[0049] The above results indicate that celandine can synergistically exert antibacterial effects with polymyxin E, thereby treating infections caused by polymyxin E-resistant bacteria.

[0050] Example 2: Time-bactericidal curve of celandine combined with polymyxin E against polymyxin E-resistant bacteria.

[0051] Polymyxin E resistant bacteria include mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, or mgr-9 positive Salmonella SLM183.

[0052] 1. Take a bacterial suspension of polymyxin E-resistant bacteria grown to the logarithmic growth phase and adjust it to McFarland 0.5 using a McFarland turbidimeter to obtain a diluted polymyxin E-resistant bacterial solution. The concentration of polymyxin E-resistant bacteria in the diluted polymyxin E-resistant bacterial solution is approximately 1 × 10⁻⁶. 6 CFU / mL.

[0053] 2. Take 12 test tubes, add 2970 μL of MH liquid culture medium and 30 μL of polymyxin E resistant bacteria dilution to each tube, and mix well; then randomly divide the 12 test tubes into 4 groups: chelidonine group, polymyxin E group, combined group, and blank group, with 3 test tubes in each group, and perform the following treatments:

[0054] Chelidonium alkaloid group: 37.5 μL of 1280 μg / mL chelidonium alkaloid solution was added to each test tube to make the concentration of chelidonium alkaloid in the system 16 μg / mL, which is the 0h point; then it was incubated at 37℃ and 200 rpm for 24h.

[0055] Polymyxin E group: Add 0.29 μL of polymyxin E solution with a concentration of 2560 μg / mL to each test tube to make the concentration of polymyxin E in the system 0.25 μg / mL, which is the 0h point; then incubate at 37℃ and 200 rpm for 24h.

[0056] Combined group: Add 37.5 μL of 1280 μg / mL chelidonine solution and 0.29 μL of 2560 μg / mL polymyxin E solution to each test tube, so that the concentration of chelidonine in the system is 16 μg / mL and the concentration of polymyxin E in the system is 0.25 μg / mL, which is the 0h point; then incubate at 37℃ and 200 rpm for 24h.

[0057] Blank group: No drugs were added to the test tube, which is the 0h point; then it was incubated at 37℃ and 200rpm for 24h.

[0058] During the incubation period, 100 μL of culture medium was taken at 0h, 2h, 4h, 8h, 12h and 24h and spread onto MH solid medium (19g of MH agar medium (Beijing Luqiao Technology Co., Ltd.) was added to an appropriate amount of distilled water, then the volume was adjusted to 500mL with distilled water, autoclaved at 121℃ for 20min, poured into plates and cooled), and incubated at 37℃ and 200rpm for 24h, and colony counting was performed.

[0059] 3. After completing step 2, plot the time-sterilization curve with the incubation time as the x-axis and the log10 value of the colony count as the y-axis.

[0060] The time-bacterial curve of mcr-1 positive Escherichia coli ZJ807 is shown below. Figure 4 (CFU stands for colony-forming unit).

[0061] The results showed that the chelidonine group, polymyxin E group, and blank group could not inhibit polymyxin E-resistant bacteria (such as mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, and mgr-9 positive Salmonella SLM183), and there was no significant difference among the three groups. The combined group significantly inhibited polymyxin E-resistant bacteria (such as mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, and mgr-9 positive Salmonella SLM183). Compared with the chelidonine group, polymyxin E group, and blank group, the combined group showed significantly improved and stable inhibition of polymyxin E-resistant bacteria (such as mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, and mgr-9 positive Salmonella SLM183).

[0062] Therefore, it can be seen that the combination of celandine and polymyxin E can restore the inhibitory effect of polymyxin E on polymyxin E-resistant bacteria (such as mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, and mgr-9 positive Salmonella SLM183), that is, the combination of celandine and polymyxin E can inhibit polymyxin E-resistant bacteria (such as mgr-1 positive Escherichia coli ZJ807, mgr-8 positive Klebsiella pneumoniae KP91, and mgr-9 positive Salmonella SLM183).

[0063] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A pharmaceutical composition for inhibiting polymyxin E-resistant bacteria, comprising chelerythrine and polymyxin E. In the pharmaceutical composition, the mass ratio of chelerythrine to polymyxin E is 32-128:

1.

2. The pharmaceutical composition of claim 1, wherein: In the pharmaceutical composition, the minimum inhibitory concentration of chelerythrine is 8-32 μg / mL, and the minimum inhibitory concentration of polymyxin is 0.25-1 μg / mL. 3.Use of chelerythrine in combination with polymyxin E in the preparation of a drug for inhibiting polymyxin E-resistant bacteria. The polymyxin E-resistant bacteria are mcr positive bacteria; The mcr The positive bacteria are mcr-1 The positive Escherichia coli ZJ807, mcr-8 The positive Klebsiella pneumoniae KP91 or mcr-9 The positive Salmonella SLM183.

4. Use according to claim 3, characterized in that: In the use, the mass ratio of chelerythrine to polymyxin E is 32-128: 1; the minimum inhibitory concentration of chelerythrine is 8-32 μg / mL, and the minimum inhibitory concentration of polymyxin is 0.25-1 μg / mL. 5.Use of chelerythrine in the preparation of a drug for improving the sensitivity of polymyxin E-resistant bacteria to polymyxin E. The polymyxin E-resistant bacteria are mcr positive bacteria; The mcr The positive bacteria are mcr-1 The positive Escherichia coli ZJ807, mcr-8 The positive Klebsiella pneumoniae KP91 or mcr-9 The positive Salmonella SLM183. 6.Use of chelerythrine in the preparation of a drug for restoring the inhibitory effect of polymyxin E on polymyxin E-resistant bacteria. The polymyxin E-resistant bacteria are mcr positive bacteria; The mcr The positive bacteria are mcr-1 The positive Escherichia coli ZJ807, mcr-8 The positive Klebsiella pneumoniae KP91 or mcr-9 The positive Salmonella SLM183.

Images