Application of tea polyphenols combined with meropenem in preparation of drug for resisting carbapenem-resistant bacteria
The combined use of tea polyphenols and meropenem, by inhibiting NDM-5 enzyme activity, solved the problem of carbapenem-resistant bacteria resistance, significantly enhanced the antibacterial effect of meropenem, and achieved effective treatment of blaNDM-5 positive strains.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2025-08-01
- Publication Date
- 2026-04-24
AI Technical Summary
There are no reports in the current technology of using tea polyphenols as meropenem synergists to combat carbapenem-resistant bacteria. The widespread spread of carbapenem-resistant bacteria has weakened the therapeutic effect of carbapenem drugs, resulting in the lack of effective drugs available for related infections.
The combined use of tea polyphenols and meropenem restored the antibacterial activity of meropenem against blaNDM-5 positive Escherichia coli and Klebsiella pneumoniae by inhibiting the activity of NDM-5 enzyme. The therapeutic effect was verified by checkerboard method, time-bactericidal curve test and large wax moth infection model.
Tea polyphenols significantly enhance the antibacterial activity of meropenem against carbapenem-resistant bacteria, restoring the therapeutic effect of meropenem. Its good therapeutic effect was verified through an in vivo infection model, demonstrating broad prospects for medical application.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and specifically discloses the application of tea polyphenols combined with meropenem in the preparation of drugs against carbapenem-resistant bacteria. Background Technology
[0002] Carbapenem antibiotics are considered the last line of defense against multidrug-resistant Gram-negative bacterial infections. However, in recent years, carbapenem-resistant bacteria have been widely detected in various sectors, including livestock farming, food, water, and humans, showing a trend of cross-species transmission. This phenomenon not only causes significant economic losses to the livestock industry but also poses a serious challenge to public health through the food chain and the environment. Particularly worrying is that their cross-species transmission severely weakens the therapeutic efficacy of carbapenems, leaving related infections in a "no effective drug available" predicament. Given this potential threat, the World Health Organization has listed carbapenem-resistant bacteria as a "key drug-resistant pathogen" requiring priority containment. Although the development of novel antimicrobial drugs is considered a key strategy for combating drug resistance, the development of traditional antibiotics is lengthy, costly, and often accompanied by the emergence of new resistance mechanisms. Therefore, developing meropenem potentiators to combat carbapenem-resistant bacteria is of great significance.
[0003] Tea's active substances possess a variety of pharmacological and physiological functions. Among them, tea polyphenols, due to their diverse functions, are used as natural products in animal husbandry. For example, adding tea extracts to broiler feed can improve broiler growth performance, optimize blood lipids and antioxidant status, and enhance immune responses. Furthermore, tea polyphenols also hold potential significance in the development of novel natural drugs, 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, either domestically or internationally, on the use of tea polyphenols as meropenem synergists. Summary of the Invention
[0005] To overcome the aforementioned problems in the prior art, this invention provides the application of tea polyphenols, an active substance in tea, in combination with meropenem in the preparation of drugs against carbapenem-resistant bacteria.
[0006] In response to the widespread problem of carbapenem-resistant bacteria in clinical practice, the primary objective of this invention is to provide tea polyphenols (CAS No.: 84650-60-2; English name: Tea polyphenols) to enhance the application of meropenem in the preparation of antibacterial drugs against carbapenem-resistant bacteria. This provides a broad prospect for the development of novel antibiotic adjuvants and offers a scientific basis for controlling the spread of carbapenem-resistant bacteria.
[0007] To achieve the above objectives, the present invention employs the following technical solution:
[0008] This invention provides the application of tea polyphenols combined with meropenem in the preparation of carbapenem-resistant synergists.
[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 strains, both carrying bacillus. NDM-5 Gene.
[0011] Furthermore, the carbapenem-resistant bacteria are animal-derived strains.
[0012] Furthermore, regarding bla NDM-5 For positive Escherichia coli, the minimum inhibitory concentration (MIC) of tea polyphenols was 1250 μg / mL, and the MIC of meropenem was 128 μg / mL.
[0013] Furthermore, regarding bla NDM-5 For Klebsiella pneumoniae positive, the minimum inhibitory concentration (MIC) of tea polyphenols is 2500 μg / mL, and the MIC of meropenem is 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 Antibacterial activity against positive Escherichia coli and Klebsiella pneumoniae.
[0015] Furthermore, the checkerboard method results 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 a significant synergistic effect.
[0016] Preferably, the mass ratio of tea polyphenols to meropenem is 2.5 to 78:1.
[0017] More preferably, when the carbapenem-resistant bacteria are of animal origin... NDM-5 When E. coli is positive, the mass ratio of tea polyphenols to meropenem is 2.5~78:1.
[0018] More preferably, when the carbapenem-resistant bacteria are of animal origin... NDM-5 When Klebsiella pneumoniae is positive, 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, IC50... 50 It was 18.13 μg / mL.
[0020] Furthermore, the combined use of tea polyphenols and meropenem has an effect on bla NDM-5 The positive strains demonstrated good synergistic bactericidal effects.
[0021] Furthermore, the in vivo therapeutic effect of tea polyphenols was verified using a large wax moth infection model.
[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are:
[0023] This invention, through checkerboard microdilution method, time-bacterial control curve test, and enzyme inhibition test, discovers a novel application of tea polyphenols combined with meropenem in the preparation of drugs to inhibit carbapenem-resistant bacteria, demonstrating that tea polyphenols can restore meropenem's resistance to carbapenem-resistant bacteria. NDM-5 Antibacterial activity against positive Escherichia coli and Klebsiella pneumoniae. Further research was conducted by establishing blavin... NDM-5 A positive Escherichia coli (E. coli) infection model was used to verify that tea polyphenols combined with meropenem have a good therapeutic effect on in vivo infections caused by carbapenemase-resistant bacteria. This invention is the first to discover that tea polyphenols can reverse the resistance of carbapenem-resistant bacteria to meropenem. It is simple to use, easy to promote, and has broad medical prospects. Attached Figure Description
[0024] Figure 1 For tea polyphenols combined with meropenem to treat blavin NDM-5 Results of checkerboard microdilution method for positive Escherichia coli;
[0025] Figure 2 For tea polyphenols combined with meropenem to treat blavin NDM-5 Results of checkerboard microdilution method for positive Klebsiella pneumoniae;
[0026] Figure 3 The inhibitory effect of tea polyphenols on NDM-5 enzyme activity;
[0027] Figure 4 Tea polyphenols (TPP) combined with meropenem (MEM) for the treatment of blavin NDM-5 Time-bacterialization curve of positive E. coli;
[0028] Figure 5 Tea polyphenols (TPP) combined with meropenem (MEM) for the treatment of blavin NDM-5 Time-bactericidal curve of positive Klebsiella pneumoniae;
[0029] Figure 6 Survival curves for large wax moth treated with tea polyphenols (TPP) combined with meropenem (MEM). Detailed Implementation
[0030] The following specific embodiments of the present invention will provide a detailed and comprehensive description of the technical solutions of the present invention. It should be noted that the provided embodiments represent only a part of the present invention, and not all of it. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0031] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods; unless otherwise specified, the materials and reagents used are commercially available reagents and materials, and the commercially available raw materials are used to remove insoluble matter and other impurities by general filtration methods.
[0032] Example 1: Determination of minimum inhibitory concentration
[0033] The quality control strains 25922 and bla NDM-5 Positive strains were streaked onto their respective agar media (conventional agar media) and incubated at 37°C for 16–18 h. Single colonies were picked and inoculated into 500 μL of MH broth and cultured at 37°C with shaking at 180 rpm until the logarithmic growth phase was reached for later use.
[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 corresponding concentration of tea polyphenols and meropenem to the wells in the first column. Mix thoroughly using a pipette, then aspirate 100 μL to the next well. Repeat this process until the last well, then aspirate and discard 100 μL. Dilute the logarithmic growth phase bacterial culture with MH broth at a ratio of 1:100, and add 100 μL of the culture to each well. Additionally, add negative and positive controls to each 96-well plate. Incubate at 37°C for 16–18 h before interpreting the results.
[0035] As shown in Table 1, meropenem for bla NDM-5 The minimum inhibitory concentrations (MICs) of tea polyphenols against *Escherichia coli* and *Klebsiella pneumoniae* were 128 μg / mL and 256 μg / mL, respectively; NDM-5 The minimum inhibitory concentrations for 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 for positive strains
[0037] Example 2: Micro-broth checkerboard method
[0038] This invention uses bla NDM-5Positive Escherichia coli (DP01) and Klebsiella pneumoniae (AH2786K) (deposited in the Pharmacology Laboratory of the College of Veterinary Medicine, South China Agricultural University, and commercially available) were used as test strains. Antimicrobial susceptibility testing was performed using the micro-broth checkerboard method to determine the susceptibility of tea polyphenols combined with meropenem to B. pylori. NDM-5 Combined Inhibition Index (FICI) of positive strains.
[0039] The test strain was inoculated into MH broth and cultured at 37°C and 180 rpm until the logarithmic growth phase. It was then diluted 100-fold with MH broth to obtain a concentration of 1×10⁻⁶. 6 The CFU / mL bacterial culture concentration should be prepared for future use.
[0040] For bla NDM-5 For positive Escherichia coli, add 50 μL of MH broth to each well in columns 1 to 8 of a 96-well plate. Add 50 μL of meropenem as drug A to each well in row 1. After mixing by pipetting, dilute the drug in a 2-fold serial order from high to low, 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, respectively. Tea polyphenols, as drug B, were serially diluted 2-fold in seven gradients. Then, 50 μL of meropenem was added to each well in a horizontally decreasing 2-fold concentration sequence. 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. The final results showed that tea polyphenols and meropenem had a significant effect on the efficacy of meropenem in drug B. 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 LMH broth to each well in columns 1 to 8 of a 96-well culture plate. Add 50 μL of meropenem as drug A to each well in row 1. After mixing by pipetting, dilute the drug in a 2-fold serial order from high to low in each well, 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, respectively. Tea polyphenols, as drug B, were serially diluted 2-fold in seven gradients. Then, 50 μL of meropenem was added to each well in a horizontally decreasing 2-fold concentration sequence. From right to left, the concentrations of meropenem in each well in each column were 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, respectively. The final results showed that tea polyphenols and meropenem had a significant effect on the efficacy of meropenem in treating B. NDM-5 The mass ratio of positive Klebsiella pneumoniae is in the range of 20:1 to 5:1.
[0042] Finally, add 100 μL of diluted bacterial solution to each well in columns 1–8, then incubate at 37°C for 18 h. The results are then evaluated and the FICI value is calculated. The combined drug susceptibility test results are based on the following formula: 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 bacillus bladder. NDM-5 The antibacterial effect of positive strains. Among them, the combined use of tea polyphenols and meropenem showed antibacterial activity against bacillus. NDM-5 The FICI value of positive Escherichia coli DP01 was 0.25 (≤0.5 indicates synergistic effect). Figure 1 (and Table 2), the combined use of tea polyphenols and meropenem on blavin NDM-5 The FICI value of Klebsiella pneumoniae AH2786K positive was 0.375 ( Figure 2 This indicates that the combined efforts of both are aimed at bla. NDM-5 Positive E. coli and bacillus NDM-5 All positive Klebsiella pneumoniae strains showed a significant synergistic effect.
[0044] Table 2. Drug sensitivity results when meropenem and tea polyphenols are used in combination.
[0045] Example 3: Enzyme Inhibition Assay
[0046] The effect of tea polyphenols on NDM-5 enzyme activity was investigated using a cefotaxime hydrolysis assay. NDM-5 is a metallo-β-lactamase that leads to bacterial resistance to various β-lactam antibiotics. Inhibition of NDM-5 can restore the activity of these antibiotics.
[0047] The experimental procedures were as follows: NDM-5 protein was diluted to an appropriate concentration with HEPES buffer, and then different concentrations of tea polyphenols (0-39 μg / mL) were added. The total volume was set at 200 μL and placed in a 96-well plate. After incubation at 37°C for 20 min, cefotaxime was added to a final concentration of 25 μM, and incubation was continued at 37°C for another 20 min. 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 was 18.13 μg / mL.
[0049] Example 4: Time-sterilization curve
[0050] This experimental method utilizes time-bactericidal curves to evaluate the changes in the effects of antimicrobial drugs on bacterial growth and killing over time.
[0051] The experimental treatment was as follows: The strain was cultured in LB liquid medium to the logarithmic growth phase, centrifuged and washed, then resuspended in PBS, and the bacterial concentration was adjusted to approximately 1×10⁻⁶. 7 CFU / mL. Bacteria were inoculated into test tubes containing single drugs (meropenem or tea polyphenols) and combination drugs, with a blank control group (no drugs added). Each group was divided into three replicates. The culture was incubated at 37°C with shaking, and samples were taken at 0, 2, 4, 6, 8, 10, and 24 h. At each time point, 100 μL of bacterial culture was taken, serially diluted tenfold, and plated onto LB agar plates. After incubation at 37°C for 18–24 hours, the colony count (CFU / mL) was counted. A bactericidal curve was plotted with time on the x-axis and the logarithm of colony count on the y-axis to evaluate the bactericidal effect of the combination drugs at different time points.
[0052] Conclusion: The combined use of tea polyphenols and meropenem has an effect on blavin. NDM-5 Positive Escherichia coli ( Figure 4 ) and bla NDM-5 positive Klebsiella pneumoniae ( Figure 5 This demonstrates a good synergistic bactericidal effect.
[0053] Example 5: Infection model of the large wax moth
[0054] Healthy, uniformly sized larvae of the large wax moth were randomly divided into seven groups of ten larvae each: a blank control group, a model group (treated with sterile PBS), a 5 mg / kg meropenem treatment group, a 10 mg / kg meropenem treatment group, a 50 mg / kg tea polyphenol treatment group, a combined 5 mg / kg meropenem and 50 mg / kg tea polyphenol treatment group, and a combined 10 mg / kg meropenem and 50 mg / kg tea polyphenol treatment group. Sterile PBS was used to prepare both tea polyphenols and meropenem, followed by sterilization through a sterile filter membrane. Bacterial suspensions were injected into the abdomen of the large wax moths using a microinjector, 10 µL per larva, resulting in a bacterial concentration of approximately 1 × 10⁻⁶. 5 CFU / mL, with the blank control group receiving no bacterial suspension or drugs. One hour after infection, appropriate drugs were injected according to the groups: the model group received PBS, the single-drug treatment group received a single drug, and the combination drug group received a combination of drugs, with an injection volume of 10 µL for each group. All large wax moths were housed in a constant temperature incubator (28℃, dark conditions) for 5 days. The number of surviving individuals in each group was recorded daily, and survival curves were plotted to statistically analyze the survival differences between different treatment groups.
[0055] Conclusion: Treatment with tea polyphenols in combination with meropenem significantly improved the efficacy of bladder function. NDM-5 Survival rate of the large wax moth infected with positive Escherichia coli ( ) Figure 6 ).
[0056] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.
Claims
1. The application of tea polyphenols combined with meropenem in the preparation of drugs against carbapenem-resistant bacteria, characterized in that, The carbapenem-resistant bacteria are of animal origin. bla NDM-5 When used in combination with positive Escherichia coli, the concentration of tea polyphenols is 1250 μg / mL and the concentration of meropenem is 128 μg / mL.
2. The application according to claim 1, characterized in that, Take tea polyphenols and meropenem in the specified proportions, prepare a solution, and mix thoroughly.