Application of loriverde in preparation of bacteriostatic agent for improving bacteriostatic effect of polymyxin

Abstract

The invention provides application of lorivid in preparation of a bacteriostatic agent for improving the bacteriostatic effect of polymyxin, and belongs to the technical field of biology. Specifically, the invention provides a bacteriostatic agent composed of loriverder and polymyxin B or polymyxin E. The bacteriostatic agent can effectively improve the effect of polymyxin in inhibition of the activity of mcr-positive bacteria (such as mcr-1 positive escherichia coli, mcr-1 positive klebsiella pneumoniae, mcr-8 positive klebsiella pneumoniae and mcr-3 positive escherichia coli), and the effect of polymyxin in inhibition of the activity of mcr-positive bacteria (such as mcr-1 positive escherichia coli, mcr-1 positive klebsiella pneumoniae, mcr-8 positive klebsiella pneumoniae and mcr-3 positive escherichia coli). Experimental results show that when the lorivid and the polymyxin BE are combined for use, the minimum inhibitory concentration (MIC) can be remarkably reduced, the FIC value is less than 0.5, and the lorivid and the polymyxin BE show a synergistic bacteriostatic effect. In addition, due to the low toxicity of the loriverder to cells, the loriverder becomes a safe treatment choice and has high clinical application potential.

Description

Technical Field

[0001] The invention belongs to the field of biological technology, and in particular relates to the application of lorivid in preparing an antibacterial agent for improving the antibacterial effect of polymyxin. Background Art

[0002] Bacterial resistance is regarded as one of the most serious challenges facing human health in the 21st century and has become an important focus of global public health issues. With the abuse and over-reliance on antibiotics, bacterial resistance continues to increase, especially in the fields of food safety, clinical medicine, and the prevention and treatment of veterinary diseases, and the threat to human health is gradually increasing. In particular, the rise of multidrug-resistant strains has greatly reduced the efficacy of commonly used antibiotics in clinical applications, bringing unprecedented difficulties and challenges to disease treatment.

[0003] Among them, carbapenemase-producing Gram-negative enterobacteriaceae (such as Escherichia coli and Klebsiella) are one of the most serious pathogens. These strains can secrete carbapenemase, destroying the mechanism of action of broad-spectrum antibiotics, making antibiotic treatment almost ineffective. The widespread spread of these multidrug-resistant strains has narrowed the choice of clinical antibiotics and made the treatment of many infectious diseases more complicated.

[0004] As a last resort, polymyxin is often used to prevent and treat infections caused by multidrug-resistant strains and carbapenem-producing super-resistant bacteria (CRE), and plays a vital role in the face of serious and life-threatening infections caused by resistant bacteria. However, with the widespread spread of the mcr gene, the emergence of this resistance gene has made the effectiveness of polymyxins face greater challenges. The spread of the mcr gene not only makes bacteria resistant to polymyxin, but also may lead to the gradual loss of the therapeutic value of polymyxin in clinical practice, thereby further exacerbating the severity of the antibiotic resistance crisis. Faced with this increasingly severe situation, how to effectively respond to the challenges of resistant bacteria and find alternative treatments has become an important issue that needs to be urgently addressed in the fields of global medical research and public health.

[0005] Nonivamide, also known as synthetic capsaicin, CAS: 2444-46-4, molecular formula: C 17 H 27 NO3, molecular weight: 293.4g / mol, chemical structure is as follows:

[0006]

[0007] It is one of the synthetic analogues of capsaicin. The difference in its chemical structure lies in the long alkyl chain. Compared with capsaicin extracted from natural products, lorividone is cheaper and has only weaker irritation. It is mainly used as neuropeptide P-substance and has ideal analgesic, anti-inflammatory, blood circulation, blood circulation and blood stasis removing effects. However, there are few reports on its application in bacterial resistance. Summary of the invention

[0008] The purpose of the present invention is to use Lorivid in the preparation of an antibacterial agent for improving the antibacterial effect of polymyxin, thereby effectively solving the problem of bacterial resistance.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] First, the present invention provides the use of Lorivid in the preparation of an antibacterial agent for improving the antibacterial effect of polymyxin, characterized in that the antibacterial effect of polymyxin is the effect of inhibiting MCR-positive bacteria.

[0011] Preferably, the polymyxin is polymyxin B or polymyxin E;

[0012] The mcr-positive 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.

[0013] Secondly, the present invention provides an antibacterial agent for inhibiting the activity of mcr-positive bacteria, wherein the antibacterial agent consists of polymyxin B or polymyxin E and lorivid.

[0014] Preferably, among the antibacterial agents, the MIC of polymyxin B is 0.125-1 μg / ml, the MIC of polymyxin E is 0.25-2 μg / ml, and the MIC of lorivid is 32-64 μg / ml.

[0015] Preferably, the mcr-positive bacteria are mcr-1-positive Escherichia coli, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

[0016] Secondly, the present invention provides an application of a composition in the preparation of an antibacterial agent for inhibiting the activity of mcr-positive bacteria, wherein the composition consists of polymyxin B or polymyxin E and lorivid.

[0017] Preferably, in the composition, the MIC of polymyxin B is 0.125-1 μg / ml, the MIC of polymyxin E is 0.25-2 μg / ml, and the MIC of lorividin is 32-64 μg / ml.

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

[0019] The beneficial effects of the present invention are:

[0020] The present invention systematically evaluates the combined use of Lorivid and polymyxin B or polymyxin E, and finds that it exhibits significant inhibitory effects on a variety of drug-resistant strains carrying mcr genes (such as mcr-1 positive Escherichia coli, mcr-1 positive Klebsiella pneumoniae, mcr-3 positive Escherichia coli and mcr-8 positive Klebsiella pneumoniae). In particular, when used in combination, the minimum inhibitory concentration (MIC) of the drug is significantly reduced, and the FIC value is less than 0.5, which meets the synergistic effect standard, indicating that Lorivid and polymyxin B\E have significant synergistic effects. This synergistic effect effectively enhances the antibacterial activity of the two antibiotics, especially when dealing with drug-resistant bacterial infections, providing a new and potential treatment strategy for clinical use.

[0021] By using CCK-8 cytotoxicity assay, the present invention proves the biosafety of Lorivid on HepG2 cells. The experimental results show that Lorivid has no significant inhibitory effect on cell activity at different concentrations, indicating that the drug will not cause toxic damage to normal cells when used. This low cytotoxicity property makes it highly safe in clinical applications and can provide patients with effective and safe treatment options.

[0022] In summary, the present invention has significant antibacterial synergistic effect, good biosafety and high clinical application potential, and provides a new and effective treatment strategy for dealing with drug-resistant bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of different concentrations of Lorivid on the growth of mcr-positive strains;

[0024] Figure 2 The effect of Lorivid combined with polymyxin B on the growth of mcr-positive strains;

[0025] Figure 3 The effect of different concentrations of Lorivid on cell activity. DETAILED DESCRIPTION

[0026] In order to clearly illustrate the technical features of this solution, this solution is described below through a specific implementation method.

[0027] Example 1

[0028] Using the checkerboard method for drug susceptibility testing to detect the inhibitory effect of the combination of lorivudine and polymyxin on mcr-positive bacteria

[0029] The drugs selected in this example are colistin B and polymyxin E;

[0030] The strains selected 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;

[0031] The specific detection steps are as follows:

[0032] 1) Checkerboard microbroth dilution method for drug susceptibility testing: The strain was shaken and cultured in MHB broth at 37 °C for 18 h.

[0033] Add 180 μL of broth to the first row of the 96-well plate, and add 100 μL of broth to each of the second row to the last row. Add 20 μL of the prepared drug to the first row, pipette and mix well, then aspirate 100 μL and add it to the second row, and so on until the last row, then discard 100 μL. After diluting the prepared bacterial solution 1:100, add 100 μL of the diluted bacterial solution to each well, and perform 3 parallel experiments for each strain.

[0034] 2) Result determination: After culturing in a 37 °C incubator for 16 - 20 h, observe the bacterial growth from low concentration to high concentration in sequence, and read the MIC value of the first clear and transparent well.

[0035] At the same time, according to the drug combination judgment standard FIC calculation formula (FIC = (MIC of drug A in combination) / (MIC of drug A alone) + (MIC of drug B in combination) / (MIC of drug B alone)), the FIC value is obtained.

[0036] 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; use this as the standard to judge the interaction mode of lorivudine and each drug.

[0037] The results obtained are shown in Table 1 and Table 2:

[0038] Table 1 MIC and FIC values of different strains when lorivudine is combined with polymyxin B

[0039]

[0040] Table 2 MIC and FIC values ​​of different strains when Lorividin was used in combination with polymyxin E

[0041]

[0042]

[0043] From the experimental results in Tables 1 and 2, it can be seen that when Lorivid is used in combination with polymyxin B\E, it shows a significant inhibitory effect on resistant strains carrying mcr-1, mcr-3 and mcr-8 genes, and can effectively reduce the minimum inhibitory concentration (MIC) of these resistant strains. In addition, the FIC values ​​of the combined treatment are all less than 0.5, which meets the standard of FIC value for determining synergistic effects, further indicating that the combined use of Lorivid and polymyxin B\E has a synergistic effect. This result means that Lorivid can enhance the antibacterial effect of polymyxin B\E, providing a potential effective combination therapy for clinical use. The discovery of such synergistic effects provides a new treatment strategy for addressing the problem of drug resistance caused by the mcr gene, and also provides an important basis for the optimization of clinical antibiotic treatment plans.

[0044] Example 2

[0045] In order to detect whether Lorivid can be used in combination with other antibiotics to produce a synergistic effect, the present invention combines Lorivid with tigecycline or meropenem to detect the antibacterial effect.

[0046] The detection steps are the same as those in Example 1, and the detection results are shown in Table 3:

[0047] Table 3 MIC and FIC of Lorivid combined with Tigecycline or Meropenem

[0048]

[0049] From the results in Table 3, it can be seen that when Lorivid is used in combination with tigecycline, there is no synergistic effect on tet(X)-positive Escherichia coli LJ23. At the same time, it can be seen that when Lorivid is used in combination with meropenem, there is no synergistic effect on bla ndm-1 There was also no synergistic effect on positive Klebsiella pneumoniae 1-15, indicating that Lorivid can only produce the current synergistic effect when used in combination with polymyxin B\E and targeting mcr-positive bacteria. This result is unexpected.

[0050] Example 3

[0051] Growth curves were used to further test the synergistic effect of Lorivid combined with polymyxin

[0052] (1) Detection of the effect of different concentrations of Lorivid on the growth of mcr-1 positive Escherichia coli LJ22

[0053] The specific detection steps are as follows:

[0054] 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.

[0055] 8 μg / ml, 16 μg / ml, 32 μg / ml and 64 μg / ml of Lorividin 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 1 shown.

[0056] from Figure 1 It can be seen from the results that within the concentration range detected by the present invention, the use of Lorivid alone does not inhibit the growth of mcr-1 positive Escherichia coli LJ22.

[0057] (2) Effect of combining Lorividin with polymyxin B on the growth of mcr-1-positive Escherichia coli LJ22

[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] 32 μg / ml Lorivid, 0.5 μg / ml Polymyxin B, 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.

[0060] from Figure 2 The experimental results show that when 32μg / ml lorivid or 0.5μg / ml polymyxin B were used alone, the inhibitory effect on mcr-1-positive Escherichia coli LJ22 was not significant, indicating that the antibacterial activity of these two drugs against this strain was relatively weak at this concentration. However, when lorivid was used in combination with polymyxin B, it showed a significant inhibitory effect at the beginning of treatment and was able to effectively inhibit the growth of mcr-1-positive Escherichia coli LJ22. This observation further confirmed that the combination of lorivid and polymyxin can significantly enhance the inhibitory effect on drug-resistant Escherichia coli carrying the mcr-1 gene, indicating the synergistic effect of the two in combined treatment.

[0061] Example 4

[0062] Using CCK8 cytotoxicity to detect the effect of Lorivid on cell activity

[0063] HepG2 cells were seeded into 96-well cell culture plates and cultured in a cell culture incubator until the cells were completely adhered.

[0064] The culture medium was removed, and DMEM medium containing 8 μg / ml, 16 μg / ml, or 32 μg / ml of Lorividin was added, and the cells were placed in a cell culture incubator and cultured for 24 h.

[0065] 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.

[0066] from Figure 3 The results show that the addition of Lorivid has no effect on cell viability, indicating that the use of this drug does not cause cytotoxicity.

Claims

1. The use of Lorivid in the preparation of an antibacterial agent for improving the antibacterial effect of polymyxin, characterized in that: The polymyxin antibacterial effect is the effect of inhibiting mcr-positive bacteria.

2. The use according to claim 1, characterized in that: The polymyxin is polymyxin B or polymyxin E; The mcr-positive 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.

3. A bacteriostatic agent for inhibiting the activity of mcr-positive bacteria, characterized in that: The antibacterial agent consists of polymyxin B or polymyxin E and lorivid.

4. The antibacterial agent according to claim 3, characterized in that Among the antibacterial agents, the MIC of polymyxin B is 0.125-1 μg / ml, the MIC of polymyxin E is 0.25-2 μg / ml, and the MIC of lorivid is 32-64 μg / ml.

5. The antibacterial agent according to claim 4, characterized in that The mcr-positive bacteria are mcr-1-positive Escherichia coli, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

6. Use of a composition in the preparation of an antibacterial agent for inhibiting the activity of mcr-positive bacteria, characterized in that: The composition consists of polymyxin B or polymyxin E and lorivid.

7. The use according to claim 6, characterized in that: In the composition, the MIC of polymyxin B is 0.125-1 μg / ml, the MIC of polymyxin E is 0.25-2 μg / ml, and the MIC of lorivid is 32-64 μg / ml.

8. The use according to claim 7, characterized in that: The mcr-positive bacteria are mcr-1-positive Escherichia coli, mcr-1-positive Klebsiella pneumoniae 1-11-1CD, mcr-8-positive Klebsiella pneumoniae mcr-8 and mcr-3-positive Escherichia coli FY20.

Images