Veterinary pharmaceutical preparation and application thereof in preparation of veterinary product for preventing and treating intestinal diseases caused by increase of drug resistance of enterococcus faecium

Abstract

The invention provides a veterinary pharmaceutical preparation and application thereof in preparation of veterinary products for preventing and treating intestinal diseases caused by increase of drug resistance of enterococcus faecium. Baicalin contained in the pharmaceutical preparation can reduce or close an enterococcus faecium drug-resistant mutation selection window. Compared with the prior art, the veterinary pharmaceutical preparation has the following advantages that compared with single use of antibiotics, the MIC of the provided veterinary pharmaceutical preparation is reduced, and it is shown that the components in the veterinary pharmaceutical preparation have an obvious synergistic effect. It is found through determination of a drug-resistant mutation selection window that the MSW of antibiotic components in the veterinary drug preparation to enterococcus faecium is reduced. Compared with the use of traditional antibacterial drugs, the application has the advantages that enterococcus can be killed more efficiently, the efficacy of antibiotic drugs is enhanced, the use concentration of the antibacterial drugs is reduced, the MSW of the antibacterial drugs is effectively reduced, and the enrichment of drug-resistant bacteria is reduced.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to a veterinary drug preparation and its application in the preparation of veterinary products for preventing and treating intestinal diseases caused by increased drug resistance of Enterococcus faecium. Background Art

[0002] As intestinal symbiotic bacteria, enterococci can produce antibacterial substances such as organic acids and bacteriocins, inhibit the reproduction of pathogens, promote intestinal health, and play an important role in the treatment of intestinal diseases. The enterococcal genome has strong plasticity and usually lacks adaptive immune CRISPR-Cas loci. Under the selection pressure of antibiotics, it can easily acquire resistance genes of other bacteria and integrate them into chromosomes, spreading resistance genes by itself. The drug resistance of enterococci is complex. It can quickly acquire resistance to clinically used antimicrobial drugs through mobile genetic elements, and it is easy to be induced to produce new drug resistance. Therefore, it is resistant to common β-lactams and sulfonamides, and has low sensitivity to aminoglycosides, lincosamides and quinolones. The chemical structures of macrolides, lincosamides, and streptogramins are very different, but their antibacterial mechanisms are very similar, that is, they all act as protein synthesis inhibitors, inhibiting the extension of peptide chains by blocking the function of the peptidyl transferase center in the large subunit of the ribosome, and are collectively referred to as MLS antibiotics. Enterococci can express methylase through the erm resistance gene, which modifies the ribosomal A2058 site, making antibiotics unable to effectively inhibit bacterial protein synthesis, thus leading to MLS. B Class resistance.

[0003] In order to prevent and prevent the increase and spread of bacterial resistance, Han Zuilhof et al. proposed the mutant prevention concentration (MPC), and on this basis proposed the mutant selection window (MSW) theory to prevent bacterial resistance to antibiotics in vitro. According to the MSW theory, antibacterial drugs can be used in combination to reduce their MPC and reduce MSW at the same time. This is a relatively easy method to implement and is also a commonly used method of drug administration in clinical practice.

[0004] At present, although the combination of commonly used drugs can treat bacterial infections, it cannot prevent bacteria from developing drug resistance. Summary of the invention

[0005] In response to the above-mentioned technical limitations and the serious problem of drug resistance of Enterococcus faecium, the present application proposes a veterinary drug preparation and its application in the preparation of veterinary products for preventing and treating intestinal diseases caused by increased drug resistance of Enterococcus faecium. The baicalin contained in the veterinary drug preparation can narrow or close the selection window for drug-resistant mutations of Enterococcus faecium; it overcomes the deficiencies and defects mentioned in the background technology.

[0006] To achieve the above objectives, this application adopts the following technical solutions: The invention of the present application is to provide a veterinary drug preparation, wherein the active ingredients of the veterinary drug preparation are MLSB antibiotics and baicalin, and the weight ratio of the two is MLS B Antibiotic-like: Baicalin is (4-8): (1-4); the preferred ratio is 2:1.

[0007] Optionally, in the above-mentioned veterinary pharmaceutical preparation, the MLS B The antibiotic is selected from at least one of lincomycin, erythromycin and pristinamycin.

[0008] Optionally, the above-mentioned veterinary pharmaceutical preparation is a suspension, which is formed by dispersing the active ingredient, suspending agent, wetting agent and antioxidant in a dispersion medium.

[0009] Optionally, in the above-mentioned veterinary pharmaceutical preparation, the dispersing medium is selected from at least one of soybean oil, ethyl oleate, triacetin and isopropyl myristate.

[0010] Optionally, in the above-mentioned veterinary pharmaceutical preparation, the suspending agent is selected from a mixture of lecithin and beeswax, a mixture of hydrogenated castor oil and beeswax, polyvinyl pyrrolidone or hydrogenated castor oil.

[0011] Optionally, in the above-mentioned veterinary pharmaceutical preparation, the wetting agent is selected from at least one of Tween-80, Span-80 and 40-epoxy hydrogenated castor oil.

[0012] Optionally, in the above-mentioned veterinary pharmaceutical preparation, the antioxidant is selected from at least one of vitamin E, thiourea, butylated hydroxyanisole, sodium metabisulfite and propyl gallate.

[0013] Optionally, in the above-mentioned veterinary pharmaceutical preparation, the composition of the veterinary pharmaceutical preparation, by weight ratio, active ingredient: suspending agent: wetting agent: antioxidant: dispersase is (2-8): (0.1-2): (0.2-1.5: (0.01-1.2): (4-16).

[0014] The second invention of the present application is to provide a method for preparing the above-mentioned veterinary pharmaceutical preparation, comprising the following steps: S1. The suspending agent and the antioxidant are added to a portion of the dispersion medium and dissolved to obtain a liquid A; S2. The MLSB antibiotic, the wetting agent and the liquid A are successively added to a portion of the dispersion medium for grinding; S3. Continue to add the baicalin to the system of step S2, and use the dispersion medium to adjust the volume, and grind to obtain the veterinary drug preparation.

[0015] The third invention point of the present application is to provide the use of the above-mentioned veterinary pharmaceutical preparation in the preparation of veterinary products for preventing and treating intestinal diseases, wherein the intestinal diseases are intestinal diseases caused by increased drug resistance of Enterococcus faecium.

[0016] Compared with the prior art, this application has the following advantages: The present application provides a veterinary drug preparation and its application in the preparation of veterinary products for preventing and treating intestinal diseases caused by increased drug resistance of Enterococcus faecium. The baicalin contained in the veterinary drug preparation can narrow or close the drug resistance mutation selection window of Enterococcus faecium. When used alone, the minimum inhibitory concentration of erythromycin against Enterococcus faecium is 0.5-8 mg / mL, the minimum inhibitory concentration of lincomycin against Enterococcus faecium is 1-16 mg / mL, the minimum inhibitory concentration of prinamycin against Enterococcus faecium is 0.125-2 mg / mL, and the MIC of baicalin against Enterococcus faecium is 0.032-4 mg / mL. In the veterinary drug preparation (i.e. when antibiotics are used in combination with baicalin), the MIC of lincomycin, erythromycin and pristinamycin against Enterococcus faecium is reduced to the original 1 / 128MIC~1 / 4MIC, 1 / 1024MIC~1 / 4MIC and 1 / 32MIC~1 / 2MIC (FICI is 0.023~0.5, 0.027~0.5, 0.094~0.5 respectively; SBPI is 8~192, 6~1056, 3~48 respectively). This shows that there is an obvious synergistic effect between the components in the veterinary drug preparation of the present application. Through the determination of the drug-resistant mutation selection window, it was found that the MSW ranges of lincomycin, erythromycin and prinamycin for Enterococcus faecium were 0.475-4.8, 3.6-26 and 6.65-58, respectively, and the SI values ​​ranged from 2.5-9.6, 1.5625-5.2 and 8-64, respectively. The MSW of lincomycin, erythromycin and prinamycin for Enterococcus faecium was reduced by 70.4%, 65.18% and 68.50%, respectively. This shows that compared with the use of traditional antibacterial drugs, the present application can kill enterococci more efficiently, enhance the efficacy of antibiotic drugs, reduce the concentration of antibacterial drugs, and effectively reduce its MSW and reduce the enrichment of drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1In one embodiment of the present application, three different antibiotics were used alone (for comparison) and combined with baicalin to form a veterinary drug preparation and then used to test the effects on Enterococcus faecium MSW.

[0018] Figure 1 In the figure, the blue area is the drug-resistant mutation selection window of Enterococcus faecium. By comparing the changes in the size of the drug-resistant mutation selection window after using three different antibiotics alone and combining them with baicalin to make veterinary drug preparations, the impact of veterinary drug preparations on Enterococcus faecium is reflected. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0020] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0021] The design idea of ​​this application is: select Enterococcus faecium ATCC-19434 as the quality control strain and clinically isolated Enterococcus faecium S1-S10 as the experimental strain; select MLS B Antibiotics—lincomycin, erythromycin, and pristinamycin are experimental drugs.

[0022] 1. The minimum inhibitory concentration (MIC) of lincomycin, erythromycin, prinamycin and baicalin when used alone against Enterococcus faecium was determined by the two-fold dilution method. Based on this MIC concentration, the changes in the MIC of lincomycin, erythromycin and prinamycin against Enterococcus faecium after being combined with different concentrations of baicalin were determined. 2. The agar dilution method was used to determine the drug resistance mutation prevention concentration (MPC) of Enterococcus faecium when lincomycin, erythromycin and pristinamycin were used alone or prepared with baicalin as veterinary drug preparations. The MPC of lincomycin, erythromycin and pristinamycin against Enterococcus faecium when prepared with baicalin as veterinary drug preparations was determined based on the MIC of lincomycin, erythromycin and pristinamycin; 3. Calculate the size of the drug-resistant mutation selection window (MSW) of Enterococcus faecium when lincomycin, erythromycin and prinamycin are used alone or prepared with baicalin into veterinary drug preparations, as well as the SI value. Through the calculation of MSW, the effect of veterinary drug preparations on the MSW of Enterococcus faecium is finally determined.

[0023] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0024] Example 1 1. Source of strains: Enterococcus faecium ATCC-19434 was purchased from the American Type Culture Collection, and Enterococcus faecium S1-S10 were isolated from milk samples of cows with mastitis and stored in a -80°C refrigerator after biochemical identification and mass spectrometry detection.

[0025] 2. Main reagents and drugs: Enterococcus broth, Enterococcus chromogenic medium, Pfizer Enterococcus selective agar (PSE), Tryptone Soy Agar (TSA), Trypticase Soy Broth (TSB) were purchased from Qingdao Haibo Biotechnology Co., Ltd.; lincomycin, erythromycin and pristinamycin were purchased from Dalian Meilun Company; baicalin was purchased from Beijing Solebow Technology Co., Ltd.

[0026] 3. Experimental methods: 3.1 Determination of the minimum inhibitory concentration (MIC) of lincomycin, erythromycin and pristinamycin against Enterococcus faecium: The Enterococcus faecium cultured to the logarithmic growth phase was adjusted to a bacterial suspension of 0.5 McFarland units. Lincomycin, erythromycin and pristinamycin were used as test drugs, and the two-fold dilution method was used to determine the MIC of lincomycin, erythromycin and pristinamycin when used alone. Add 100 μl of MHB broth liquid culture medium to each 96-well plate. Take 100 μL of the diluted drug and add it to each well starting from the second column using the 2-fold dilution method to make the drug concentration a decreasing concentration gradient. Each group of drug concentrations was repeated 3 times. Use liquid culture medium to culture the strains required for the experiment overnight to ensure the activity of the strains. Different experiments require different bacterial solution concentrations. The concentration of this experiment is set to 1.0×10 5 CFU / mL, add 100μL of bacterial suspension to make the total volume of the solution in each well 200μL. This experiment set up a growth control group and a blank control group. Place the 96-well plate in a 37℃ constant temperature incubator for overnight culture. Judge according to the results of colony growth for 12 hours, and observe the growth of each Enterococcus faecalis with an inverted microscope. The judgment standard is that the concentration corresponding to the well without bacterial growth is the MIC value observed by naked eye. Repeat the test 3 times for each strain of bacteria. Note: Each time you add the drug or add the bacterial solution, you need to blow and mix it to ensure the accuracy of the experiment.

[0027] 3.2 Evaluation of the combined effects of lincomycin, erythromycin and pristinamycin with baicalin: The in vitro antibacterial effect of veterinary drug preparations prepared by lincomycin, erythromycin and prinamycin and baicalin was evaluated by the chessboard method, with the minimum inhibitory concentration of a single drug as a reference. The test drug concentration was diluted to the corresponding MIC of each drug to 1 / 1024MIC by the doubling dilution method. The concentration range of lincomycin, erythromycin and prinamycin in the well was 1 / 2-1 / 1024 times of their MIC against enterococci when used alone, and the concentration range of baicalin was controlled between 4MIC-1 / 128 times of their MIC against enterococci. 50 μL of the diluted drug solution of the two drugs from high concentration to low concentration was added to each well of the 96-well plate in a chessboard cross arrangement to ensure that each single drug concentration had a corresponding combined drug concentration, and the total volume of the two drugs was 100 μL. 100 μL of the diluted bacterial suspension in the logarithmic growth phase was added to each well to ensure that the final concentration of the bacterial solution was 1×10 5 CFU / mL, ensuring that the total volume of the solution in each test well is 200 μL. In addition, control wells, including growth control and blank drug control, were set up in the same 96-well plate and cultured at 37°C for 24 h. According to the MIC values ​​of the clinical isolates after single and combined use of drugs, the partial inhibitory concentration index, FIC (FIC=MIC when A drug is used in combination / MIC when A drug is used alone + MIC when B drug is used in combination / MIC when B drug is used alone) was calculated to judge the interaction between the two drugs: FIC≤0.5 is a drug synergistic effect.

[0028] 3.3 Determination of MPC of lincomycin, erythromycin and pristinamycin against Enterococcus faecium: The plate drug concentration gradient (1 to 16 times MIC) of lincomycin, erythromycin and prinamycin was set based on the MIC of lincomycin, erythromycin and prinamycin alone against Enterococcus faecium. Dissolve lincomycin, erythromycin and prinamycin in sterile double distilled water, and then filter and sterilize the obtained mother solution. According to the formula of TSA agar plate, prepare 100 ml of culture medium, disinfect it, add lincomycin, erythromycin and prinamycin after the culture medium cools to 50℃, shake the culture medium containing drugs and pour it on the plate. Take 100μL of bacterial suspension and evenly spread it on each drug-added culture medium. The standard is that there are no visible colonies on the drug-containing plate, which is recorded as the initial MPC value (MPCpr). If the MPCpr exceeds the concentration series, then the range of drug concentration used must be increased. Starting from the measured MPCpr value, the drug concentration was linearly reduced by 20% and the experiment was repeated referring to the MPCpr determination method. The lowest drug concentration at which no bacterial growth was observed was the precise MPC value of the strain for the antibiotic (SI=MPC / MIC, MSW=MPC-M1C).

[0029] 3.4 Determination of the MPC of lincomycin, erythromycin and prinamycin combined with baicalin against Enterococcus faecium: According to the method and procedure in 3.3, the MPC of different ratios of lincomycin, erythromycin and prinamycin combined with baicalin was determined. The MIC of the two drugs alone against enterococci was used as the benchmark, and 7 different ratio combinations were set (the ratio of lincomycin, erythromycin and prinamycin to baicalin was 1:1 to 64:64). The absence of colonies visible to the naked eye on the drug-containing plate was used as the standard, which was recorded as the initial MPC value of the combination of the two (MPCpr). If the MPCpr exceeds the concentration series, the range of drug concentrations used must be increased. Starting from the measured MPCpr value, the drug concentrations of lincomycin, erythromycin and prinamycin were linearly reduced by 20%, and the experiment was repeated according to the MPCpr determination method. The lowest drug concentration at which no bacterial growth was observed was the accurate MPC value of the two drugs combined against the strain (SI=MPC / MIC, MSW=MPC-M1C).

[0030] 3.5 Results: Table 1 shows the selection window (MSW) of resistance mutations of Enterococcus faecium to three different antibiotics.

[0031] Table 1

[0032] Table 2 shows the minimum inhibitory concentration (MIC) of three different antibiotics and baicalin for Enterococcus faecium after being made into veterinary drug preparations.

[0033] Table 2

[0034] In Table 2 , the MIC values ​​of different strains to three antibiotics are shown, and BAI shows the MIC values ​​of the strains to baicalin.

[0035] Table 3 shows the effects of three different antibiotics used alone and when the antibiotics and baicalin are prepared into veterinary pharmaceutical preparations on Enterococcus faecium MSW.

[0036] Table 3

[0037] In Table 3, each value is the distribution area of ​​the MIC, MPC, and MSW of Enterococcus faecium in the line graph when different antibiotics are used alone and when they are prepared into veterinary drug preparations with baicalin.

[0038] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A veterinary pharmaceutical preparation, characterized in that: The active ingredient of the veterinary pharmaceutical preparation is MLS B Antibiotics and baicalin; the weight ratio of the two is MLS B Antibiotic-like: Baicalin is (4-8): (1-4); the preferred ratio is 2:

1.

2. A veterinary pharmaceutical preparation according to claim 1, characterized in that: The MLS B The antibiotic is selected from at least one of lincomycin, erythromycin and pristinamycin.

3. A veterinary pharmaceutical preparation according to claim 2, characterized in that: The veterinary drug preparation is a suspension, which is formed by dispersing active ingredients, suspending agents, wetting agents and antioxidants in a dispersion medium.

4. A veterinary pharmaceutical preparation according to claim 3, characterized in that: The dispersant is selected from at least one of soybean oil, ethyl oleate, triacetin and isopropyl myristate.

5. A veterinary pharmaceutical preparation according to claim 3, characterized in that: The suspending agent is selected from a mixture of lecithin and beeswax, a mixture of hydrogenated castor oil and beeswax, polyvinyl pyrrolidone or hydrogenated castor oil.

6. A veterinary pharmaceutical preparation according to claim 3, characterized in that: The wetting agent is selected from at least one of Tween-80, Span-80 and epoxyethylene-40-hydrogenated castor oil.

7. A veterinary pharmaceutical preparation according to claim 3, characterized in that: The antioxidant is selected from at least one of vitamin E, thiourea, butylated hydroxyanisole, sodium metabisulfite and propyl gallate.

8. A veterinary pharmaceutical preparation according to any one of claims 1 to 7, characterized in that: In the composition of the veterinary pharmaceutical preparation, the weight ratio of active ingredient: suspending agent: wetting agent: antioxidant: dispersase is (2-8): (0.1-2): (0.2-1.5: (0.01-1.2): (4-16).

9. The method for preparing the veterinary pharmaceutical preparation according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. adding the suspending agent and the antioxidant to part of the dispersion medium and dissolving them to obtain liquid A; S2. The MLS B The antibiotic, the wetting agent and the liquid A are successively added into a part of the dispersion medium for grinding; S3. Continue to add the baicalin into the system of step S2, and use the dispersion medium to adjust the volume, and obtain the veterinary drug preparation after grinding.

10. Use of the veterinary pharmaceutical preparation according to any one of claims 1 to 8 in the preparation of veterinary products for preventing and treating intestinal diseases, characterized in that: The intestinal disease is an intestinal disease caused by increased drug resistance of Enterococcus faecium.

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