Screening method of natural antibiotic-free drug for preventing and treating methicillin-resistant staphylococcus aureus
By screening and optimizing natural drug compositions, the drug resistance problem of methicillin-resistant Staphylococcus aureus was solved, efficient and economical antibacterial effects were achieved, and the combination of baicalin, emodin, and caffeic acid was screened out as the best solution.
Patent Information
- Application Number
- CN202510932997.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-08
- Publication Date
- 2025-10-10
AI Technical Summary
Existing technologies are difficult to effectively prevent and treat infections caused by methicillin-resistant Staphylococcus aureus, especially because it is resistant to multiple antibiotics, making clinical treatment difficult and costly.
By preparing bacterial culture medium, activating strains and preparing bacterial suspensions, preparing natural drug monomer solutions, determining minimum inhibitory concentrations and graded inhibitory indices, the ratio of natural drug monomer compositions was optimized, and the optimal combination of natural drugs such as baicalin, emodin, and caffeic acid was screened out. The microbroth dilution method and the checkerboard method were used for screening, and the mass ratio of baicalin, emodin, and caffeic acid was finally determined to be 2:1:4.
A rapid and low-cost screening method was provided, which verified that the combination of baicalin, emodin and caffeic acid had the best antibacterial effect against methicillin-resistant Staphylococcus aureus, and could achieve an inhibition rate of more than 95% within 4 hours at a low cost.
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Figure CN120758594A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological product preparation and application, and particularly relates to a screening method of a natural non-antibiotic drug for preventing and treating methicillin-resistant Staphylococcus aureus. BACKGROUND
[0002] Staphylococcus aureus is an important zoonotic pathogenic bacterium, which can cause soft tissue infection, respiratory infection, osteomyelitis and sepsis and various diseases in humans and animals. The high pathogenicity of Staphylococcus aureus is mainly due to its ability to adhere and produce toxins, immune escape related proteins and other various virulence factors.
[0003] Staphylococcus aureus is the main pathogenic microorganism causing mastitis in dairy cows. Staphylococcus aureus can produce toxins and enzyme substances, leading to inflammation of mammary gland tissue, and the toxins produced can cause irreversible damage to the udder of dairy cows, seriously affecting milk yield and milk quality.
[0004] The number of drug-resistant strains was small in the early development of antibiotics, and traditional antibiotics could be used to prevent and control Staphylococcus aureus infection. However, the non-standard use of antibiotics led to the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), which is a kind of Staphylococcus aureus resistant to methicillin antibiotics.
[0005] MRSA is one of the most important multi-drug resistant pathogens in the world, which is not only resistant to methicillin, but also has resistance to penicillin and cephalosporin antibiotics with similar structures. At the same time, it has different degrees of resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides and rifampicin, etc. At present, the main treatment methods for drug-resistant strains in clinical practice include screening of sensitive drugs or use of high-order antibiotics such as vancomycin and its derivatives, however, the lack of veterinary drugs further exacerbates the economic losses caused by MRSA.
[0006] A single antibacterial drug is difficult to cope with complex drug-resistant bacterial infections, and the development of a natural non-antibiotic drug for efficient prevention and control of drug-resistant bacteria has become an urgent need for livestock breeding industry. SUMMARY
[0007] The purpose of the present application is to provide a screening method of a natural non-antibiotic drug for preventing and treating methicillin-resistant Staphylococcus aureus and the screened drug composition, the screening method provided can screen the best drug combination and ratio, and the screened drug can be used as an alternative to antibiotic treatment, laying a foundation for drug screening and research and development for the treatment of methicillin-resistant Staphylococcus aureus infection.
[0008] The application provides a screening method of natural non-drug for preventing and treating methicillin-resistant Staphylococcus aureus, and comprises the following steps: 1) preparing a bacterial culture medium, As a specific embodiment, TSB liquid culture medium and TSA solid culture medium are prepared; 2) activating a bacterial strain and preparing a bacterial suspension The bacterial strain freeze-dried powder is dissolved in ultrapure water, and then coated on a TSA bacterial culture plate, and cultured in a 37 DEG C bacterial incubator for 24 hours; a typical colony is picked into TSB liquid culture medium, and cultured in a 37 DEG C bacterial incubator for 12-16 hours; the logarithmic phase bacterial liquid is adjusted to a concentration of 1.5*10 6 CFU / mL by using sterile liquid culture medium; 3) preparing a natural drug monomer solution The natural drug monomer is accurately weighed and dissolved into a 50mg / mL mother liquor; 4) determining a minimum inhibitory concentration and a fractional inhibitory concentration index The fractional inhibitory concentration index is determined by using a micro-broth dilution method and a chessboard method.
[0009] 5) optimizing a ratio of a natural drug monomer composition The natural drug monomers are divided into different factors, each factor is divided into different levels, according to a time-sterilization curve method, a drug combination is obtained according to an orthogonal ratio, mixed with the bacterial liquid, and cultured in a 37 DEG C bacterial incubator; bacterial samples are taken at 0 / 2 / 4 / 6 / 8 / 12 / 24 hours, then diluted and coated, cultured in a bacterial incubator for 24 hours, and then observed for colony growth, and counted; and the best composition is obtained according to the experimental results.
[0010] The application further provides a drug composition for resisting methicillin-resistant Staphylococcus aureus, which comprises baicalin, emodin and caffeic acid. Further, the mass ratio of the baicalin, emodin and caffeic acid is 2:1:4.
[0011] The application provides a method for rapidly screening a natural drug monomer composition for resisting methicillin-resistant Staphylococcus aureus by comprehensively using a minimum inhibitory concentration experiment and an orthogonal method, the method has low cost, short time consumption and simple operation. BRIEF DESCRIPTION OF DRAWINGS
[0012] Figure 1 Figure 1: Colony pictures of bacteria after co-culturing with nine drug combinations, wherein the pictures are photos of bacteria samples taken at 0, 2, 4, 6, 8, 12, 24 hours for plating; Figure 2 Figure 2: Colony pictures of bacteria at 2h after different drug combinations, wherein combination 1 has no bacterial growth, the bacterial growth of combination 2, combination 4, combination 6 and combination 8 is obviously reduced, and combination 3, combination 5, combination 7 and combination 9 still have bacterial growth; Figure 3 Figure 3: Colony pictures of bacteria at 4h after different drug combinations, wherein combination 1 has no bacterial growth, and the bacterial growth of other combinations is reduced, and combination 3, combination 7 and combination 9 still have bacterial growth; Figure 4 Figure 4: Colony pictures of bacteria at 6h after different drug combinations, wherein, except for combination 3, combination 7 and combination 9, the bacterial growth of other combinations is reduced or has individual small amount of bacterial growth; Figure 5 Figure 5: Colony pictures of bacteria at 8h after different drug combinations, wherein the bacterial growth of combination 3, combination 7 and combination 9 is reduced; Figure 6 Figure 6: Colony pictures of bacteria at 12h after different drug combinations, wherein combination 3 has a small amount of colonies, combination 7 and combination 9 only have individual colonies, and the bacterial growth of other combinations is reduced; Figure 7 Figure 7: Colony pictures of bacteria at 24h after different drug combinations, wherein the bacterial growth of nine combinations is reduced; Figure 8 Figure 8: Growth number line graph of methicillin-resistant Staphylococcus aureus after being treated by nine drug combinations for 0 / 2 / 4 / 6 / 8 / 12 / 24 hours. DETAILED DESCRIPTION
[0013] The screening method for preventing and treating methicillin-resistant Staphylococcus aureus provided by the present application comprises the following steps: 1. Preparation of bacterial culture medium 2. Activation of bacterial strain and preparation of bacterial suspension 3. Preparation of natural drug monomer solution 4. Determination of minimum inhibitory concentration and grading inhibitory index 5. Optimization of the ratio of natural drug monomer composition In step 1, the preparation of bacterial culture medium: TSB liquid medium and TSA solid medium are prepared according to the instructions, and after preparation, they are sealed and subjected to high-pressure sterilization treatment. After the TSB liquid medium is cooled to room temperature, it is stored in a 4°C refrigerator. The TSA solid medium is poured into a bacterial culture plate in a clean bench, and after solidification, it is sealed and stored in a 4°C refrigerator.
[0014] In step 2, the activated strain and bacterial suspension are prepared: the strain freeze-dried powder is taken out, dissolved in ultrapure water, and then coated on TSA bacterial culture plates for 24 hours of bacterial culture at 37 DEG C in a bacterial incubator, and typical colonies are picked into TSB liquid medium for 12-16 hours of bacterial culture at 37 DEG C in a bacterial incubator, and the logarithmic phase bacterial liquid is adjusted to 1.5x10 6 CFU / mL.
[0015] In step 3, the natural medicine monomer solution is prepared: the natural medicine monomers are accurately weighed and dissolved into 50mg / mL mother liquor respectively.
[0016] In step 4, the minimum inhibitory concentration is determined: the micro-broth dilution method and the chessboard method are used for graded inhibitory index determination: TSB liquid medium is used to vertically dilute A medicine and horizontally dilute B medicine on a 96-well plate, and an equal volume of bacterial liquid is added, mixed uniformly, and then cultured at 37 DEG C in a bacterial incubator for 18-24 hours, the experimental results are read, and the FICI value is calculated according to the formula.
[0017] In step 5, the ratio of the natural medicine monomer composition is optimized: the natural medicine monomers are divided into different factors, each factor is divided into different levels, according to the time-sterilization curve method, nine kinds of medicine combinations are obtained according to the orthogonal ratio, mixed with the bacterial liquid, and then cultured at 37 DEG C in a bacterial incubator, and the bacterial samples are taken at 0 / 2 / 4 / 6 / 8 / 12 / 24 hours, then diluted and coated, and then cultured in a bacterial incubator for 24 hours, and then the colony growth is observed and counted; according to the experimental results, the optimal medicine combination for resisting methicillin-resistant Staphylococcus aureus is baicalin: emodin: caffeic acid = 2:1:4.
[0018] The application will be described in detail below in combination with examples and drawings.
[0019] Example 1: method establishment
[0020] A method for establishing a "natural drug combination without drug resistance" for efficiently preventing and controlling methicillin-resistant Staphylococcus aureus, comprising the following steps: Step 1: screening natural medicine monomers with anti-Staphylococcus aureus effect.
[0021] Preparation of bacterial culture medium: the bacterial culture medium comprises TSB liquid medium and TSA solid medium; TSB liquid medium: take 3g of TSB powder and add 100mL of deionized water in a conical flask, and seal; TSA solid medium: take 3g of TSB powder and 1.5g of agar powder, add 100mL of deionized water in a conical flask, and seal; Put all the above bacterial culture media into a high-pressure steam sterilization pot and sterilize at 121 DEG C for 15 minutes. The liquid medium was stored in a 4°C refrigerator after cooling to room temperature, and the solid medium was poured into sterilized petri dishes after high-pressure sterilization, sealed after cooling and solidification, and stored in a 4°C refrigerator.
[0022] Bacterial activation and preparation of bacterial solution The glycerol bacterial freezing tube of Staphylococcus aureus (ATCC29213) was taken out from a -80°C refrigerator, and quickly thawed on ice or at room temperature; A small amount of glycerol bacterial solution was taken with a sterile inoculation loop, streaked on a TSA plate, and cultured in a 37°C bacterial incubator for 18-24 h; A single typical colony was picked and inoculated into 3-5 mL of TSB medium, and cultured in a 37°C bacterial incubator for 12-16 h; The concentration of the bacterial solution was adjusted to 0.5 McFarland turbidity by turbidimetry using TSB medium; 0.1 mL of the bacterial solution was taken and added to 9.9 mL of TSB medium to obtain a bacterial solution with a concentration of about 1×10 6 CFU / mL.
[0023] Preparation of natural medicine monomers The natural medicine monomer solution was prepared in a sterile clean bench. The mass of each natural medicine monomer was accurately weighed using an analytical balance, and the natural medicine monomer was dissolved in dimethyl sulfoxide (DMSO) and deionized water to prepare a mother liquor with a concentration of 50 mg / mL.
[0024] The different natural medicine monomer mother liquors were packaged in EP tubes and stored in a 4°C refrigerator.
[0025] Determination of the minimum inhibitory concentration of different natural medicine monomers on Staphylococcus aureus using microbroth dilution method Under sterile conditions, the natural medicine monomers were diluted horizontally and gradiently in TSB medium in a 96-well plate, and negative and positive controls were set up; A for baicalin, B for berberine, C for tanshinone, D for artemisinin, E for caffeic acid, F for chlorogenic acid, G for magnolol, and H for emodin; Columns 1-10 are the drug-containing bacterial culture media after dilution of the above drugs by a factor of 2 from left to right; Column 11 is the negative control, and 200 μL of TSB medium was added; column 12 is the positive control, and 100 μL of bacterial solution and 100 μL of TSB medium were added; Cultured in a 37°C bacterial incubator for 18-24 h; The minimum natural medicine monomer concentration corresponding to the clear hole is the minimum inhibitory concentration (MIC), which is 1.56 mg / mL for baicalin, 0.1 mg / mL for berberine, 2.5 mg / mL for tanshinone, 6.25 mg / mL for artemisinin, 1.56 mg / mL for caffeic acid, 6.25 mg / mL for chlorogenic acid, 12.5 mg / mL for magnolol, and 0.156 mg / mL for emodin.
[0026] Step two: screening natural medicine combinations for inhibiting methicillin-resistant Staphylococcus aureus.
[0027] 1. Preparation of bacterial culture medium: the bacterial culture medium includes TSB liquid medium and TSA solid medium, and the preparation is performed according to the operation steps of step 1.
[0028] 2. Activation of bacterial strain and preparation of bacterial suspension: Methicillin-resistant Staphylococcus aureus strain (ATCC 43300) was taken out from a -80°C refrigerator and purchased from North China Biological (BNCC), with strain number BNCC330041; After the surface of the ampoule was sterilized, it was opened in a safety cabinet, the top was burned with an alcohol lamp, and then sterile water was quickly dropped to break it, and then it was knocked with a pair of tweezers; 0.5 mL of sterile water was taken and injected into the freeze-dried tube, and after the bacterial powder was fully dissolved, 200 μL was injected into each TSA plate for uniform coating; The plate was placed in a 37°C bacterial incubator for 24 h; A suitable size and well-grown colony was selected and inoculated into 8 mL of TSB liquid medium, and shaken and cultured to the logarithmic growth phase; The bacterial concentration was adjusted to 0.5 McFarland turbidity with TSB bacterial culture medium; 0.1 mL of bacterial solution was taken and added to 9.9 mL of TSB medium to obtain a bacterial solution with a concentration of about 1×10 6 CFU / mL.
[0029] 3. Preparation of natural medicine monomer solution The natural medicine monomer solution was prepared in a sterile clean bench, the mass of each natural medicine monomer was accurately weighed with an analytical balance, dimethyl sulfoxide (DMSO) and deionized water were used to dissolve the natural medicine monomers, a drug mother liquor with a concentration of 50 mg / mL was prepared, and different natural medicine monomer mother liquors were packaged with EP tubes and stored at 4°C.
[0030] 4. Determination of minimum inhibitory concentration and fractional inhibitory index The drug combinations were designed by chessboard method, as shown in Table 1. Table 1: Combination of A drug and B drug A drug B drug Combination 1 Baicalin Caffeic acid Combination 2 Baicalin Emodin Combination 3 Emodin Caffeic acid Combination 4 Baicalin Berberine Combination 5 Berberine Emodin Combination 6 Berberine Caffeic acid Under sterile conditions, each natural medicine monomer was diluted with TSB medium at a ratio of 2:1. Medicine A was diluted at a ratio of 2:1 and marked as A1-A8. Medicine B was diluted at a ratio of 2:1 and marked as B1-B8. Then, different medicine monomer concentrations were mixed according to the format of Table 2. Table 2: A medicine and B medicine mixing sample table
[0031] The diluted medicine A and medicine B at a ratio of 2:1 were added to wells A1-H8 of a 96-well plate at 50 μL per well. 200 μL of TSB medium was added to each well in column 9 as a negative control. 100 μL of TSB medium and 100 μL of diluted bacterial solution were added to each well in column 10 as a positive control. 100 μL of medicine A diluted at a ratio of 2:1 was added to each well in column 11. 100 μL of medicine B diluted at a ratio of 2:1 was added to each well in column 12 as a determination of the minimum inhibitory concentration of A medicine and B medicine. 100 μL of diluted bacterial solution was added to each well in A1-H8 and A11-H12. The plate was incubated in a bacterial incubator at 37°C for 18-24 hours. When the negative control was clear and the positive control was turbid, the experimental results were valid. Parallel tests were performed three times. The minimum drug concentration corresponding to the clear well was determined as the minimum inhibitory concentration (MIC) by visual observation of columns 11-12. The well with the lowest concentration of bacterial growth was observed by visual observation of columns 1-8. The FICI value was calculated according to the formula.
[0032] FICI result determination: index ≤0.5 for synergistic effect, index 0.5-1 for additive effect, index 1-2 for irrelevant effect, and index >2 for antagonistic effect. As shown in Table 3, the combination of natural medicine monomers baicalin and emodin, emodin and caffeic acid, and baicalin and caffeic acid had additive effect. The combination of baicalin and berberine and berberine and emodin had synergistic effect. The combination of baicalin and berberine and berberine and emodin had irrelevant effect. Therefore, combinations 1, 2, and 3 were selected for subsequent experiments.
[0033] Table 3: FICI values of combinations of medicine monomers
[0034] 5. Method for preparing a combination of medicine monomers According to the minimum inhibitory concentration experiment described above, an L9(3 4 ) orthogonal table was used to design the experiment, as shown in Tables 4 and 5. The three factors are three drugs, factor A is baicalin, factor B is emodin, and factor C is caffeic acid; each factor has three levels, level 1 is 2MIC, level 2 is MIC, and level 3 is 1 / 2MIC; Table 4: L9(3 4 ) orthogonal design table
[0035] Table 5: Compound dose optimization table
[0036] Step three: bacteriostatic effect of different drug combinations Time-kill curve method (CLSI M26-A) was used; According to the above ratio, nine drug combinations were prepared, and the bacteriostatic effect was detected: 900 μL of the nine combinations of drug solution and 100 μL of bacterial solution (final concentration of 1×10 6 CFU / mL) were added to sterile test tubes; Immediately after mixing, sampling (labeled as T0) was performed; The remaining test tubes were placed in a 37℃ bacterial incubator for incubation; Sampling was performed at 2 / 4 / 6 / 8 / 12 / 24 hours, respectively; 100 μL of the sample was taken and added to 900 μL of TSB medium; Centrifugation: 3000g, 5min, discard the supernatant; Resuspend the bacterial pellet with 1 mL of TSB medium; Take 100 μL and spread on a TSA plate, and incubate in a 37℃ bacterial incubator for 24h; After incubation, use Image J to count the number of colonies; Table 6 shows the number of colonies of methicillin-resistant Staphylococcus aureus on TSA medium after 0, 2, 4, 6, 8, 12, and 24 hours of treatment with the nine different combinations of the three drugs; Table 6: Colony count table
[0037] The bacterial stock concentration was calculated according to the formula: ; Table 7 shows the bacterial stock concentration (CFU / mL) of methicillin-resistant Staphylococcus aureus after 0, 2, 4, 6, 8, 12, and 24 hours of treatment with the nine different combinations of the three drugs; Table 7: Bacterial stock concentration table
[0038] Figure 1 is a graph showing the growth of methicillin-resistant Staphylococcus aureus on TSA medium after 0 hours of treatment with the nine different combinations of the three drugs; Figure 2 is a graph showing the growth of methicillin-resistant Staphylococcus aureus on TSA medium after 2 hours of treatment with the nine different combinations of the three drugs; Figure 3 is a graph showing the growth of methicillin-resistant Staphylococcus aureus on TSA medium after 4 hours of treatment with the nine different combinations of the three drugs; Figure 4 is a graph showing the growth of methicillin-resistant Staphylococcus aureus on TSA medium after 6 hours of treatment with the nine different combinations of the three drugs; Figure 5 is a graph showing the growth of methicillin-resistant Staphylococcus aureus on TSA medium after 8 hours of treatment with the nine different combinations of the three drugs; Figure 6 is a graph showing the growth of methicillin-resistant Staphylococcus aureus on TSA medium after 12 hours of treatment with the nine different combinations of the three drugs; Figure 7 Figure 8 is a graph showing the growth of methicillin-resistant Staphylococcus aureus on TSA medium after 24 hours of treatment with the nine different combinations of the three drugs; According to the growth of methicillin-resistant Staphylococcus aureus on TSA medium, combination 1 had no bacterial growth at 2h; combinations 2, 4, and 6 had no bacterial growth at 6h; combinations 5 and 8 had no bacterial growth at 8h; combinations 3, 7, and 9 had no bacterial growth at 24h; According to the experimental results and the statistical analysis of the bacteriostatic rate in Table 8, the combinations that achieved a bacteriostatic and antibacterial effect of 95% or more within 4h were combinations 1, 2, 4, 6, and 8. If cost is not considered and the bacteriostatic and antibacterial efficiency is considered, the priority of the effects of the combinations is: combination 1, combination 6, combination 4, combination 8, and combination 2. If the cost price of the three drugs purchased from Macklin Reagent is considered, the priority is: combination 6, combination 8, combination 4, combination 2, and combination 1. Table 8: Statistical table of bacteriostatic rate
[0039] According to the experimental results and the ranking results, both combination 1 and combination 6 can achieve a bacteriostatic effect of 95% or more within 4h, but the cost of combination 6 is lower than that of the other combinations, so combination 6 is more recommended as the best. Since the horizontal combination of combination 6 is A2B3C1, the horizontal 1 is 2MIC, the horizontal 2 is MIC, and the horizontal 3 is 1 / 2MIC, the mass ratio of baicalin, emodin, and caffeic acid is 2:1:4. In summary, when the mass ratio of baicalin, emodin, and caffeic acid is 2:1:4, it has the best effect against methicillin-resistant Staphylococcus aureus.
Claims
1. A method for screening drug-resistant bacteria for naturally occurring drugs without resistance, characterized in that: The method comprises the following steps: 1) Preparation of bacterial culture medium 2) Activation of bacterial strains and preparation of bacterial suspension Take out the lyophilized powder of the strain, dissolve it in ultrapure water, and then spread it on TSA bacterial culture plates and culture it in a 37℃ bacterial incubator for 24 hours. Pick out typical colonies and add them to TSB liquid medium and culture them in a 37℃ bacterial incubator with shaking for 12-16 hours. 3) Preparation of natural drug monomer solution Accurately weigh the natural drug monomers and dissolve them into 50 mg / mL stock solutions; 4) Determination of minimum inhibitory concentration and graded inhibitory index The FICI value was determined by the graded inhibition index assay using the broth microdilution method and the checkerboard method; 5) Optimize the ratio of natural drug monomer compositions Natural drug monomers are divided into different factors, and each factor is divided into different levels. According to the time-kill curve method, a drug combination is obtained according to the orthogonal matching ratio. After mixing with the bacterial solution, the drug combination is cultured in a bacterial incubator at 37°C. Bacterial samples are taken at 0 / 2 / 4 / 6 / 8 / 12 / 24 hours, and then diluted and spread. After culturing in the bacterial incubator for another 24 hours, the colony growth is observed and statistically analyzed. Based on the experimental results, the optimal combination is obtained.
2. The method according to claim 1, wherein The method is for screening a pharmaceutical composition for resisting methicillin-resistant Staphylococcus aureus.
3. The method according to claim 1, wherein The step 1) is to prepare TSB liquid culture medium and TSA solid culture medium.
4. The method according to claim 1, wherein In the above 2), take the logarithmic phase bacterial solution and adjust the bacterial solution concentration to 1.5×10 6 CFU / mL.
5. A pharmaceutical composition for treating methicillin-resistant Staphylococcus aureus, characterized in that: The pharmaceutical composition is obtained by screening through the method described in claim 1.
6. The pharmaceutical composition according to claim 5, wherein The pharmaceutical composition contains baicalin, emodin and caffeic acid.
7. The pharmaceutical composition according to claim 6, wherein The mass ratio of baicalin, emodin and caffeic acid is 2:1:4.