Pharmaceutical composition aiming at mcr positive drug-resistant bacteria and application thereof

Abstract

The invention provides a pharmaceutical composition aiming at mcr positive drug-resistant bacteria and application of the pharmaceutical composition, and belongs to the technical field of biomedicine. The composition is composed of patchouli alcohol and polymyxin B or polymyxin E, and the antibacterial effect on mcr positive drug-resistant bacteria is remarkably enhanced. Experimental results show that when patchouli alcohol and polymyxin B or E are combined for use, the minimum inhibitory concentration (MIC) of polymyxin can be remarkably reduced, and the FIC is less than 0.5, so that the patchouli alcohol and the polymyxin B or E have a synergistic effect. In addition, a cytotoxicity experiment shows that patchouli alcohol has no obvious toxicity to normal cells in a certain concentration range and has relatively high safety. The composition can be used as a synergistic enhancer of polymyxin, can effectively cope with mcr positive drug-resistant bacterial infection, provides a safe and efficient new way for treatment of drug-resistant bacterial infection, and is helpful for delaying the progress of antibiotic drug resistance crisis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a pharmaceutical composition for MCR-positive drug-resistant bacteria and an application thereof. Background Art

[0002] Bacterial resistance has become one of the major challenges in the field of global public health in the 21st century, seriously threatening human health. The abuse and over-reliance on antibiotics have gradually increased bacterial resistance, especially in food safety, clinical medicine and veterinary disease prevention, and the risks it brings are becoming increasingly significant.

[0003] As the last treatment option, polymyxin is widely used to combat infections caused by multidrug-resistant strains, and plays a key role in combating serious drug-resistant bacterial infections. However, due to the rapid spread of the mcr gene, the effectiveness of polymyxin treatment faces great challenges. The spread of the mcr gene not only makes bacteria resistant to polymyxin, but also may lead to the gradual loss of its clinical application value, further exacerbating the severity of the antibiotic resistance crisis.

[0004] Patchouli alcohol, also known as patchouli alcohol, is a naturally occurring tricyclic sesquiterpene. Its CAS: 5986-55-0, molecular formula: C 15 H 26 O, molecular weight: 222.37g / mol, chemical structure:

[0005]

[0006] Patchouli alcohol is the main active ingredient extracted from patchouli, which has been widely studied and confirmed to have multiple pharmacological effects. These effects include immunomodulation, anti-inflammatory, antioxidant, anti-tumor, antibacterial, insecticidal, anti-atherosclerotic and antiemetic effects, making it have a wide range of application potential in the medical field. Patent application number CN201310083795.2 shows that Patchouli alcohol has the effect of resisting methicillin-resistant bacteria, but there is no research on the effect of Patchouli alcohol on MCR-positive resistant bacteria. Summary of the invention

[0007] The purpose of the present invention is to provide a pharmaceutical composition for MCR-positive drug-resistant bacteria and its application, so as to effectively alleviate the antibiotic resistance crisis.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides a pharmaceutical composition for mcr-positive drug-resistant bacteria, wherein the composition consists of tricholoma and polymyxin B or polymyxin E.

[0010] Preferably, in the composition, the MIC of the patchouli alcohol is 32-64 μg / ml, the MIC of the polymyxin B is 0.06-0.125 μg / ml, and the MIC of the polymyxin E is 0.06-0.125 μg / ml.

[0011] Preferably, the mcr-positive drug-resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

[0012] In a second aspect, the present invention provides the use of patchouli alcohol in the preparation of an enhancer for enhancing the antibacterial effect of polymyxin, characterized in that the antibacterial effect of polymyxin is the effect of polymyxin in inhibiting MCR-positive resistant bacteria.

[0013] Preferably, the polymyxin is polymyxin B or polymyxin E;

[0014] The mcr-positive resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

[0015] Preferably, in the enhancer, the MIC of patchouli alcohol is 32-64 μg / ml.

[0016] In a third aspect, the present invention provides an antibacterial agent for inhibiting mcr-positive drug-resistant bacteria, wherein the core active ingredients of the antibacterial agent are composed of patchouli alcohol and polymyxin B or polymyxin E.

[0017] Preferably, in the antibacterial agent, the MIC of the patchouli alcohol is 32-64 μg / ml, the MIC of the polymyxin B is 0.06-0.125 μg / ml, and the MIC of the polymyxin E is 0.06-0.125 μg / ml;

[0018] The mcr-positive resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

[0019] In a fourth aspect, the present invention provides a pharmaceutical composition for use in the preparation of an antibacterial agent for MCR-positive drug-resistant bacteria, characterized in that the pharmaceutical composition consists of patchouli alcohol and polymyxin B or polymyxin E.

[0020] Preferably, in the composition, the MIC of the patchouli alcohol is 32-64 μg / ml, the MIC of the polymyxin B is 0.06-0.125 μg / ml, and the MIC of the polymyxin E is 0.06-0.125 μg / ml;

[0021] The mcr-positive resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae Mcr-8 and mcr-3-positive Escherichia coli FY20.

[0022] The beneficial effects of the present invention are:

[0023] The pharmaceutical composition provided by the present invention, combined with Patchouli alcohol and polymyxin B or polymyxin E, exhibits a significant antibacterial effect on mcr-positive resistant bacteria (such as mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae Mcr-8, and mcr-3-positive Escherichia coli FY20). Compared with a single antibiotic, the combined use can effectively reduce the minimum inhibitory concentration (MIC) of polymyxin, significantly enhance the inhibitory effect on mcr-positive resistant bacteria, and reduce the risk of infection by resistant bacteria.

[0024] The experimental results show that the FIC values ​​of the combination of Patchouli alcohol and polymyxin B or E are all less than 0.5, which meets the standard of synergistic effect. This synergistic effect not only significantly improves the antibacterial effect of polymyxin, but also reduces the side effects of antibiotic use by reducing the drug dosage, providing a more efficient solution for the treatment of drug-resistant bacterial infections in clinical practice.

[0025] CCK-8 cytotoxicity experiments showed that within the concentration range of 8μg / ml to 32μg / ml, Patchouli alcohol had no significant toxic effect on HepG2 cells, and the cell survival rate remained stable. This result proves the safety of Patchouli alcohol at this concentration, laying the foundation for its further application in pharmaceutical compositions, and showing that it is suitable for safe treatment of MCR-resistant bacteria.

[0026] In summary, the present invention not only expands the medicinal value of Patchouli alcohol, but also provides a new and effective combined medication regimen for the treatment of MCR-positive drug-resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effect of different concentrations of patchouli alcohol on the growth of mcr-positive strains;

[0028] Figure 2 The effect of patchouli alcohol combined with polymyxin B on the growth of mcr-positive strains;

[0029] Figure 3 The figure shows the effect of different concentrations of patchouli alcohol on cell activity. DETAILED DESCRIPTION

[0030] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0031] Example 1

[0032] The inhibitory effect of Patchouli alcohol combined with polymyxin on mcr-positive bacteria was detected by chessboard method drug sensitivity test

[0033] The drugs used in this embodiment are colistin B and polymyxin E;

[0034] The strains used in the present invention are: mcr-1 positive Escherichia coli LJ22, mcr-1 positive Klebsiella pneumoniae 1-11-1CD, mcr-8 positive Klebsiella pneumoniae mcr-8 and mcr-3 positive Escherichia coli FY20;

[0035] The specific detection steps are as follows:

[0036] 1) Checkerboard microbroth dilution test: The strain was cultured in MHB broth at 37°C with shaking for 18 h.

[0037] Add 180uL broth to the first row of the 96-well plate, add 100μL broth to each of the second to last rows, add 20μL of prepared drugs to the first row, mix well with a spray gun, then pipette 100μL and add to the second row, and so on, until the last row. Then discard 100μL and dilute the prepared bacterial solution 1:100, add 100μL of the diluted bacterial solution to each well, and perform 3 parallel experiments for each strain.

[0038] 2) Result determination: After culturing in a 37°C incubator for 16-20 hours, observe the bacterial growth from low concentration to high concentration, and read the MIC value of the first clear and transparent well.

[0039] At the same time, the FIC value was obtained according to the FIC calculation formula (FIC = (MIC of drug A incombination) / (MIC of drug A alone)+(MIC of drug B in combination) / (MIC of drug B alone) of the drug combination judgment standard.

[0040] When FICI ≤ 0.5, the interaction mode of the two drugs is synergistic; when 0.5 < FICI ≤ 1, the interaction mode of the two drugs is additive; when 1 < FICI ≤ 2, the interaction mode of the two drugs is irrelevant; when FICI > 2, the interaction mode of the two drugs is antagonistic; based on this standard, the interaction mode of patchouli alcohol and each drug is judged.

[0041] The results obtained are shown in Table 1 and Table 2 as follows:

[0042] Table 1 MIC and FIC values of different strains when patchouli alcohol is combined with polymyxin B

[0043]

[0044]

[0045] Table 2 MIC and FIC values of different strains when patchouli alcohol is combined with polymyxin B

[0046]

[0047] It can be seen from the data in Table 1 and Table 2 that when patchouli alcohol is combined with polymyxin B or polymyxin E, the minimum inhibitory concentration (MIC) of mcr-1, mcr-3 and mcr-8 positive drug-resistant strains is significantly reduced. For example, in the mcr-1 positive Escherichia coli LJ22 strain, the MIC of polymyxin B or polymyxin E alone is 2 μg / ml, while after combining with patchouli alcohol, it is reduced to 0.25 μg / ml and 0.5 μg / ml. In addition, the MIC of patchouli alcohol alone for each strain exceeds 512 μg / ml, but after combining with polymyxin B or E, this value decreases significantly. For example, the MIC of mcr-1 positive Escherichia coli LJ22 drops to 32 μg / ml.

[0048] From the fractional inhibitory concentration (FIC) of the combined drugs, the FIC values of all strains are less than 0.5, meeting the judgment criteria for synergistic effects, further indicating that patchouli alcohol and polymyxin B and E produce a synergistic effect in inhibiting the growth of drug-resistant strains. This synergistic effect significantly enhances the antibacterial effect of polymyxin against mcr positive drug-resistant bacteria, especially showing potential in combating drug resistance caused by the mcr gene. This finding indicates that patchouli alcohol can be used as an effective enhancer of polymyxin, providing a possible treatment strategy for dealing with mcr drug-resistant bacterial infections and helping to delay the crisis brought about by antibiotic resistance.

[0049] Example 2

[0050] In order to detect whether patchouli alcohol can be combined with other antibiotics to produce a synergistic effect, the present invention combines patchouli alcohol with tigecycline and detects the antibacterial effect.

[0051] The detection steps are the same as those in Example 1, and the detection results are shown in Table 3:

[0052] Table 3 MIC and FIC of tigecycline combined with patchouli alcohol

[0053]

[0054] From the results in Table 3, it can be seen that when patula alcohol is used in combination with tigecycline, there is no obvious antibacterial effect on tet(X)-positive Escherichia coli LJ18, and although it has a certain effect on tmexCD1-toprj1-positive Klebsiella pneumoniae XN, it does not achieve a synergistic effect. Therefore, patula alcohol will only produce a significant synergistic effect when used in combination with polymyxin B or polymyxin E and targeting mcr-positive bacteria, which has certain specificity.

[0055] Example 3

[0056] Growth curves were used to further test the synergistic effect of the combination of patchouli alcohol and polymyxin

[0057] The specific detection steps are as follows:

[0058] After overnight culture of mcr-1-positive Escherichia coli LJ22, the bacterial solution after overnight culture was diluted 1:100 and 100 μL was added to a 96-well plate.

[0059] 8 μg / ml, 16 μg / ml, 32 μg / ml and 64 μg / ml of patchouli alcohol were added to a 96-well plate, and the absorbance was measured at OD=600nm every hour to draw a bacterial growth curve. The results are shown in the figure. Figure 1 shown.

[0060] from Figure 1 It can be seen from the results that within the concentration range detected by the present invention, the use of patchouli alcohol alone does not inhibit the growth of mcr-1 positive Escherichia coli LJ22.

[0061] (2) Effect of combining patchouli alcohol with polymyxin E on the growth of mcr-1 positive Escherichia coli LJ22

[0062] After overnight culture of mcr-1-positive Escherichia coli LJ22, the bacterial solution after overnight culture was diluted 1:100 and 100 μL was added to a 96-well plate.

[0063] 32 μg / ml of patchouli alcohol, 0.5 μg / ml of polymyxin E, and the combination of the two were added to a 96-well plate, and the absorbance was measured at OD = 600 nm every hour to draw a bacterial growth curve. The results are shown in the figure. Figure 2 shown.

[0064] from Figure 2 It can be observed that the untreated mcr-1 positive E. coli LJ22 showed a normal growth curve. The addition of 32 μg / ml of patchouli alcohol alone did not significantly inhibit the growth of mcr-1 positive E. coli LJ22, indicating that patchouli alcohol had a weak inhibitory effect on the strain when used alone. The use of polymyxin E alone can slow down the growth rate of mcr-1 positive E. coli LJ22 to a certain extent, but the inhibitory effect is limited and cannot completely inhibit its growth.

[0065] In contrast, after the combination of Patchouli alcohol and polymyxin E, the growth of mcr-1 positive Escherichia coli LJ22 was completely inhibited, and the growth curve showed a significant inhibitory effect. This result further verified the synergistic effect of Patchouli alcohol and polymyxin E in combination, providing new possibilities for the treatment of drug-resistant bacteria.

[0066] Example 4

[0067] Using CCK8 cytotoxicity to detect the effect of Patchouli alcohol on cell activity

[0068] HepG2 cells were seeded into 96-well cell culture plates and cultured in a cell culture incubator until the cells were completely adhered.

[0069] The culture medium was removed, and DMEM culture medium containing 8 μg / ml, 16 μg / ml, or 32 μg / ml trichol was added, and the cells were placed in a cell culture incubator and cultured for 24 h.

[0070] 10 μl CCK-8 reagent was added to each well. After incubation for 3 hours, the absorbance at 450 nm was detected and the cell viability was calculated. The results were as follows: Figure 3 shown.

[0071] from Figure 3 The results show that different concentrations of Patchouli alcohol (8μg / ml, 16μg / ml, 32μg / ml) have no significant effect on the activity of HepG2 cells, and the cell survival rate remains basically stable. This shows that Patchouli alcohol has no obvious cytotoxicity to HepG2 cells at these concentrations and has a high safety. This result supports the potential application of Patchouli alcohol within a safe range and lays the foundation for its application in further antibacterial or drug resistance research.

Claims

1. A pharmaceutical composition for mcr-positive drug-resistant bacteria, characterized in that: The composition consists of patchouli alcohol and polymyxin B or polymyxin E.

2. The pharmaceutical composition according to claim 1, characterized in that In the composition, the MIC of the patchouli alcohol is 32-64 μg / ml, the MIC of the polymyxin B is 0.06-0.125 μg / ml, and the MIC of the polymyxin E is 0.06-0.125 μg / ml.

3. The pharmaceutical composition according to claim 2, characterized in that The mcr-positive resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

4. The use of Patchouli alcohol in the preparation of an enhancer for enhancing the antibacterial effect of polymyxin, characterized in that: The polymyxin antibacterial effect is the effect of polymyxin inhibiting mcr-positive resistant bacteria.

5. The use according to claim 4, characterized in that: The polymyxin is polymyxin B or polymyxin E; The mcr-positive resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

6. The use according to claim 5, characterized in that: In the enhancer, the MIC of the patchouli alcohol is 32-64 μg / ml.

7. A bacteriostatic agent for inhibiting mcr-positive drug-resistant bacteria, characterized in that: The core active ingredients of the antibacterial agent are composed of patchouli alcohol and polymyxin B or polymyxin E.

8. The antibacterial agent according to claim 7, characterized in that Among the antibacterial agents, the MIC of the patchouli alcohol is 32-64 μg / ml, the MIC of the polymyxin B is 0.06-0.125 μg / ml, and the MIC of the polymyxin E is 0.06-0.125 μg / ml; The mcr-positive resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

9. A pharmaceutical composition is used in the preparation of an antibacterial agent for mcr-positive drug-resistant bacteria, characterized in that: The pharmaceutical composition consists of Patchouli alcohol and polymyxin B or polymyxin E.

10. The use according to claim 9, characterized in that: In the composition, the MIC of the patchouli alcohol is 32-64 μg / ml, the MIC of the polymyxin B is 0.06-0.125 μg / ml, and the MIC of the polymyxin E is 0.06-0.125 μg / ml; The mcr-positive resistant bacteria are mcr-1-positive Escherichia coli LJ22, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

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