Application of tea polyphenol combined with meropenem in preparation of carbapenem drug-resistant bacterium resisting medicine

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of tea polyphenol combined with meropenem to preparation of a medicine for resisting carbapenem drug-resistant bacteria, and the carbapenem drug-resistant bacteria are animal source blaNDM-5 positive escherichia coli and / or klebsiella pneumoniae. A trace bouillon chessboard method, a cefoxitin hydrolysis test, a time-sterilization curve, a greater wax moth infection model and the like prove that the tea polyphenol and carbapenem antibiotic meropenem generate a good synergistic antibacterial effect, and the tea polyphenol can also inhibit the activity of NDM-5 enzyme; meanwhile, the treatment and protection effects of meropenem on a greater wax moth infection model are improved. It is found for the first time that tea polyphenol can reverse the drug resistance of carbapenem drug-resistant bacteria to meropenem and enhance the antibacterial activity of meropenem to carbapenem drug-resistant bacteria, the synergistic effect is remarkable, use is easy, popularization is easy, and good application value is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically discloses the application of tea polyphenols combined with meropenem in preparing a drug for resisting carbapenem-resistant bacteria. Background Art

[0002] Carbapenem antibiotics are considered the last line of defense for treating multidrug-resistant Gram-negative bacterial infections. However, in recent years, carbapenem-resistant bacteria have been widely detected in livestock and poultry farming, food, water, and humans, demonstrating a trend of cross-border transmission. This phenomenon not only causes significant economic losses to the livestock industry but also poses a serious public health challenge through the food chain and the environment. Particularly concerning is that cross-border transmission severely undermines the therapeutic efficacy of carbapenems, leaving related infections with no effective treatment options. Given their potential threat, the World Health Organization has designated carbapenem-resistant bacteria as "key drug-resistant pathogens" requiring priority containment. While the development of new antimicrobial agents is considered a key strategy for combating drug resistance, the R&D cycle for traditional antibiotics is lengthy, costly, and often associated with the emergence of new resistance mechanisms. Therefore, the development of meropenem-potentiators to combat carbapenem-resistant bacteria is of paramount importance.

[0003] Tea active ingredients possess a variety of pharmacological and physiological functions. Tea polyphenols, among them, are used as natural products in the livestock industry due to their diverse functions. For example, adding tea extract to broiler chicken diets improves growth performance, optimizes blood lipids and antioxidant status, and enhances immune response. Tea polyphenols also have potential in the development of new natural medicines, such as lipid-lowering drugs, natural antibacterial agents, antidepressants, cancer prevention drugs, immune-enhancing drugs, and drugs for vascular diseases.

[0004] However, there are currently no reports on tea polyphenols as meropenem enhancers at home and abroad. Summary of the Invention

[0005] In order to overcome the above problems existing in the prior art, the present invention provides an application of tea polyphenols, a tea active substance, combined with meropenem in the preparation of a drug for resisting carbapenem-resistant bacteria.

[0006] In response to the widespread spread of carbapenem-resistant bacteria in clinical practice, the primary purpose of the present invention is to provide the use of tea polyphenols (CAS No.: 84650-60-2; English name: Tea polyphenols) to enhance the antibacterial activity of meropenem in the preparation of drugs against carbapenem-resistant bacteria. This provides broad prospects for the development of new antibiotic adjuvants and provides a scientific basis for preventing and controlling the spread of carbapenem-resistant bacteria.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides the use of tea polyphenols combined with meropenem in preparing a synergist against carbapenem-resistant bacteria.

[0009] The present invention also provides a composition for combating carbapenem-resistant bacteria, comprising tea polyphenols and meropenem.

[0010] Furthermore, carbapenem-resistant bacteria are bla NDM-5 Positive Escherichia coli and Klebsiella pneumoniae, both strains carry bla NDM-5 Gene.

[0011] Furthermore, the carbapenem-resistant bacteria are strains of animal origin.

[0012] Furthermore, for bla NDM-5 For positive Escherichia coli, the minimum inhibitory concentration of tea polyphenols was 1250 μg / mL, and the minimum inhibitory concentration of meropenem was 128 μg / mL.

[0013] Furthermore, for bla NDM-5 For positive Klebsiella pneumoniae, the minimum inhibitory concentration of tea polyphenols was 2500 μg / mL, and the minimum inhibitory concentration of meropenem was 256 μg / mL.

[0014] Furthermore, tea polyphenols can inhibit the activity of NDM-5 enzyme and restore the effect of meropenem on bla NDM-5 Positive antibacterial activity against Escherichia coli and Klebsiella pneumoniae.

[0015] Furthermore, the results of the chessboard method showed that tea polyphenols combined with meropenem had an effect on bla NDM-5 The FICI values ​​of positive Escherichia coli and Klebsiella pneumoniae were 0.25 and 0.375, respectively, showing significant synergistic effect.

[0016] Preferably, the mass ratio of the tea polyphenols to meropenem is 2.5-78:1.

[0017] More preferably, when the carbapenem-resistant bacteria are animal-derived bla NDM-5 When positive for Escherichia coli, the mass ratio of tea polyphenols to meropenem is 2.5~78:1.

[0018] More preferably, when the carbapenem-resistant bacteria are animal-derived bla NDM-5 When positive for Klebsiella pneumoniae, the mass ratio of tea polyphenols to meropenem is 5~20:1.

[0019] Furthermore, tea polyphenols can significantly inhibit the activity of NDM-5 enzyme, IC 50 It is 18.13μg / mL.

[0020] Furthermore, tea polyphenols combined with meropenem had an effect on bla NDM-5 The positive strains showed good synergistic bactericidal effect.

[0021] Furthermore, the greater wax moth infection model demonstrated that tea polyphenols could effectively enhance the therapeutic effect of meropenem in vivo.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0023] The present invention uses the checkerboard microdilution method, time-kill curve test and enzyme inhibition test to find a new application of tea polyphenols combined with meropenem in the preparation of drugs for inhibiting carbapenem-resistant bacteria, proving that tea polyphenols can restore the activity of meropenem against bla NDM-5 The antibacterial activity against Escherichia coli and Klebsiella pneumoniae was further established by NDM-5 A positive Escherichia coli (Galleria mellonella) infection model demonstrated that tea polyphenols combined with meropenem have a significant therapeutic effect against infections caused by carbapenemase-resistant bacteria. This study is the first to demonstrate that tea polyphenols can reverse meropenem resistance in carbapenem-resistant bacteria. The study is simple to use and readily available, demonstrating broad medical potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For tea polyphenols combined with meropenem on bla NDM-5 Checkerboard microdilution results for positive E. coli;

[0025] Figure 2 For tea polyphenols combined with meropenem on bla NDM-5 Checkerboard microdilution results for positive Klebsiella pneumoniae;

[0026] Figure 3 The inhibitory effect of tea polyphenols on NDM-5 enzyme activity;

[0027] Figure 4 The effect of tea polyphenols (TPP) combined with meropenem (MEM) on bla NDM-5 Time-kill curve of positive E. coli;

[0028] Figure 5 The effect of tea polyphenols (TPP) combined with meropenem (MEM) on bla NDM-5 Time-kill curve of positive Klebsiella pneumoniae;

[0029] Figure 6 This is the survival curve of greater wax moth treated with tea polyphenols (TPP) combined with meropenem (MEM). DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention below will provide a detailed and comprehensive explanation of the technical solutions of the present invention. It should be noted that the embodiments provided represent only a portion of the present invention, not all of it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the test methods used in the examples of the present invention are conventional methods; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified, and the commercially available raw materials are filtered to remove insoluble matter and other impurities before use.

[0032] Example 1: Minimum inhibitory concentration determination

[0033] The quality control strains 25922 and bla NDM-5 Positive strains were streaked onto the corresponding agar medium (conventional agar medium) and cultured at 37°C for 16–18 h. A single colony was picked and inoculated into 500 μL MH broth and cultured at 37°C with constant shaking at 180 rpm until the logarithmic growth phase.

[0034] Add 180 μL of MH broth to the first column of wells in a 96-well plate, and 100 μL of MH broth to the remaining wells. Next, add 20 μL of the appropriate concentrations of tea polyphenols and meropenem to the wells in the first column. Mix thoroughly with a pipette, then aspirate 100 μL to the next well. Repeat this process until 100 μL is aspirated and discarded from the last well. Dilute the logarithmically growing bacteria at a ratio of 1:100 with MH broth, then add 100 μL to each well. Additionally, add negative and positive controls to each 96-well plate. Incubate at 37°C for 16–18 hours before interpreting the results.

[0035] As shown in Table 1, meropenem has an inhibitory effect on bla NDM-5 The minimum inhibitory concentrations of positive Escherichia coli and Klebsiella pneumoniae were 128 μg / mL and 256 μg / mL respectively; tea polyphenols had an inhibitory effect on bla NDM-5 The minimum inhibitory concentrations of positive Escherichia coli and Klebsiella pneumoniae were 1250 μg / mL and 2500 μg / mL, respectively.

[0036] Table 1 Effects of meropenem and tea polyphenols on bla NDM-5 Minimum inhibitory concentration results of positive strains

[0037] Example 2: Microbroth Checkerboard Method

[0038] The present invention is based on bla NDM-5Positive Escherichia coli (DP01) and Klebsiella pneumoniae (AH2786K) (deposited in the Pharmacology Laboratory of the College of Veterinary Medicine of South China Agricultural University and available from commercial channels) were used as test strains. The microbroth checkerboard method was used for drug sensitivity testing to obtain the efficacy of tea polyphenols combined with meropenem against bla NDM-5 Combined inhibition index (FICI) of positive strains.

[0039] The strain to be tested was inoculated into MH broth and cultured at 37°C and 180 rpm until the logarithmic growth phase. The strain was then diluted 100-fold with MH broth to obtain a concentration of 1×10 6 CFU / mL bacterial solution concentration for future use.

[0040] For bla NDM-5 For positive Escherichia coli, add 50 μL of MH broth to each well from the 1st to the 8th column of a 96-well culture plate, add 50 μL of meropenem as drug A to each well in the 1st row, mix by pipetting, and then dilute the drug in a 2-fold gradient from high to low in the vertical direction, with 50 μL per well. The concentrations of meropenem in each well in each column from top to bottom are 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 0 μg / mL. Tea polyphenols were used as drug B alone in 7 dilution gradients with a 2-fold dilution ratio, and then 50 μL was added to each well in a 2-fold decreasing concentration. From right to left, the concentrations of meropenem in each well in each column were 1250 μg / mL, 625 μg / mL, 312 μg / mL, 156 μg / mL, 78 μg / mL, 30 μg / mL, 19 μg / mL, and 0 μg / mL. NDM-5 The mass ratio of positive Escherichia coli was in the range of 78:1 to 2.5:1.

[0041] For bla NDM-5For positive Klebsiella pneumoniae, add 50 μL of MH broth to each well from the 1st to the 8th column of a 96-well culture plate, add 50 μL of meropenem as drug A to each well in the 1st row, mix by pipetting, and then dilute the drug in a 2-fold gradient from high to low in the vertical direction, with 50 μL per well. The concentrations of meropenem in each well in each column from top to bottom are 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 0 μg / mL. Tea polyphenols were used as drug B alone and diluted 7 times in a 2-fold gradient. Then 50 μL was added to each well in a 2-fold decreasing concentration. From right to left, the concentration of meropenem in each well in each column was 2500 μg / mL, 1250 μg / mL, 625 μg / mL, 312 μg / mL, 156 μg / mL, 78 μg / mL, 30 μg / mL, and 0 μg / mL. NDM-5 The mass ratio of positive Klebsiella pneumoniae was in the range of 20:1 to 5:1.

[0042] Finally, 100 μL of diluted bacterial solution was pipetted from each well and added to columns 1 to 8. The cells were then incubated at 37°C for 18 hours. The results were determined and the FICI value was calculated. The combined drug sensitivity test results were based on: FICI = MIC 美罗培南联合 / MIC 美罗培南单用 +MIC 茶多酚联用 / MIC 茶多酚单用 .

[0043] Conclusion: Tea polyphenols can enhance the effect of meropenem on bla NDM-5 Among them, tea polyphenols and meropenem combined with bla NDM-5 The FICI value of positive Escherichia coli DP01 was 0.25 (≤0.5 means synergistic effect) ( Figure 1 and Table 2), tea polyphenols and meropenem combined with bla NDM-5 The FICI value of positive Klebsiella pneumoniae AH2786K was 0.375 ( Figure 2 ). This indicates that the combination of the two targets bla NDM-5 Positive E. coli and bla NDM-5 The positive Klebsiella pneumoniae produced a significant synergistic effect.

[0044] Table 2 Drug sensitivity results of meropenem and tea polyphenols combined

[0045] Example 3: Enzyme inhibition assay

[0046] The effect of tea polyphenols on NDM-5 enzyme activity was examined using a nitrocefin hydrolysis assay. NDM-5 is a metallo-β-lactamase that contributes to bacterial resistance to multiple β-lactam antibiotics. Inhibition of NDM-5 can restore the activity of these antibiotics.

[0047] The experimental treatment was as follows: NDM-5 protein was diluted to an appropriate concentration in HEPES buffer. Tea polyphenols (0-39 μg / mL) were then added to a total volume of 200 μL in a 96-well plate. After incubation at 37°C for 20 minutes, nitrocefin was added to a final concentration of 25 μM and incubated at 37°C for another 20 minutes. The absorbance of each well was measured at 492 nm using a microplate reader, and the residual enzyme activity was calculated.

[0048] Conclusion: Tea polyphenols can inhibit the activity of NDM-5 enzyme in a concentration-dependent manner ( Figure 3 ), IC 50 It is 18.13μg / mL.

[0049] Example 4: Time-kill curve

[0050] This experimental method uses the time-kill curve to evaluate the time-dependent changes in the growth and killing effects of antibiotics on bacteria.

[0051] The experimental treatment was as follows: the strain was cultured in LB liquid medium until the logarithmic growth phase, washed by centrifugation, and resuspended in PBS to adjust the bacterial solution concentration to about 1×10 7 CFU / mL. Bacteria were inoculated into test tubes containing a single drug (meropenem or tea polyphenols) and a combination of drugs, and a blank control group (no drug) was set up. Each group was set up with three replicates. The culture was shaken at 37°C, and samples were taken at 0, 2, 4, 6, 8, 10, and 24 hours. At each time point, 100 μL of bacterial solution was taken out, spread on LB agar plates after ten-fold gradient dilution, and the number of colonies (CFU / mL) was counted after incubation at 37°C for 18-24 hours. A bactericidal curve was drawn with time as the horizontal axis and the logarithm of the colony count as the vertical axis to evaluate the bactericidal effect of the combination drug at different time points.

[0052] Conclusion: The combined effect of tea polyphenols and meropenem on bla NDM-5 Positive Escherichia coli ( Figure 4 ) and bla NDM-5 Positive Klebsiella pneumoniae ( Figure 5 ) reflects a good synergistic bactericidal effect.

[0053] Example 5: Greater wax moth infection model

[0054] Healthy, uniform-sized larvae of the greater wax moth were selected and randomly divided into seven groups, each with 10 larvae: a blank control group, a model group (given sterile PBS), a 5 mg / kg meropenem treatment group, a 10 mg / kg meropenem treatment group, a 50 mg / kg tea polyphenols treatment group, a 5 mg / kg meropenem and 50 mg / kg tea polyphenols combined treatment group, and a 10 mg / kg meropenem and 50 mg / kg tea polyphenols combined treatment group. Both tea polyphenols and meropenem were prepared using sterile PBS, which was then filtered through a sterile filter membrane for sterilization. A microsyringe was used to inject 10 µL of bacterial suspension into the abdomen of the greater wax moth, with a bacterial concentration of approximately 1 × 10 5 CFU / mL. A blank control group received no bacterial solution or drug. One hour after infection, the corresponding drug was injected according to the group: the model group received PBS, the single-drug treatment group received a single drug, and the combination treatment group received a combination of drugs. The injection volume was 10 µL for each group. All G. mellonella were housed in a constant temperature chamber (28°C, dark conditions) and observed for 5 days. The number of surviving individuals in each group was recorded daily, and survival curves were plotted to analyze the survival differences between different treatment groups.

[0055] Conclusion: Tea polyphenols combined with meropenem treatment significantly improved menstrual blood NDM-5 The survival rate of G. mellonella infected with positive E. coli ( Figure 6 ).

[0056] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of tea polyphenols combined with meropenem in the preparation of drugs against carbapenem-resistant bacteria.

2. The use according to claim 1, characterized in that The carbapenem-resistant bacterial strain is an animal-derived strain.

3. The use according to claim 1, characterized in that The carbapenem-resistant bacterial strain carries bla NDM-5 Gene.

4. The use according to claim 3, characterized in that The carbapenem-resistant bacteria are Escherichia coli and / or Klebsiella pneumoniae.

5. The use according to claim 1, characterized in that The mass ratio of the tea polyphenols to meropenem is 2.5-78:

1.

6. The use according to claim 4, characterized in that When the carbapenem-resistant bacteria is bla NDM-5 When positive for Escherichia coli, the mass ratio of tea polyphenols to meropenem is 2.5~78:

1.

7. The use according to claim 4, characterized in that When the carbapenem-resistant bacteria is bla NDM-5 When positive for Klebsiella pneumoniae, the mass ratio of tea polyphenols to meropenem is 5~20:

1.

8. A composition, characterized in that It is composed of tea polyphenols and meropenem.

9. The composition according to claim 8, characterized in that The mass ratio of the tea polyphenols to meropenem is 2.5-78:

1.

10. A method for preparing the composition according to claim 9, characterized in that: Take tea polyphenols and meropenem in proportion, make a solution, and mix well.

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