Compound preparation capable of reducing drug resistance of pathogenic bacteria to florfenicol and preparation method thereof

Abstract

The invention discloses a compound preparation capable of reducing drug resistance of pathogenic bacteria to florfenicol and a preparation method of the compound preparation, and belongs to the technical field of medicines. The compound preparation is prepared from a composition of florfenicol and potassium clavulanate, and the composition is prepared from the following components in parts by weight: 2 to 60 parts of florfenicol and 0.5 to 15 parts of potassium clavulanate. The invention also provides an application of the potassium clavulanate in preparation of a product for reducing the antibacterial activity of pathogenic bacteria on florfenicol, and an application of the potassium clavulanate in preparation of a product for reducing the drug resistance of the pathogenic bacteria on florfenicol. Results of the embodiment of the invention show that the potassium clavulanate can reduce the drug resistance of the florfenicol and enhance the antibacterial activity of the florfenicol, and the compound preparation provided by the invention is simple to prepare, easily available in raw materials, suitable for industrial large-scale production and wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a compound preparation capable of reducing the resistance of pathogenic bacteria to florfenicol and a preparation method thereof. Background Art

[0002] In the technical field where modern medicine and the breeding industry are closely intertwined, florfenicol (FF), as a key component of the amide alcohol broad-spectrum antibiotics, exhibits a unique pharmacological mechanism of action. Its core principle of action is to accurately inhibit the activity of peptidyl transferase, thereby cutting off the synthesis chain of bacterial proteins, and thus achieving efficient antibacterial efficacy. In the 1990s, florfenicol was successfully approved for marketing, and stood out with its low toxicity and other characteristics, greatly reducing the potential negative effects of drugs on the physiological functions of livestock bodies, giving farmers a wider range of choices. Florfenicol has a broad antibacterial spectrum and can effectively respond to infections caused by both Gram-positive and Gram-negative bacteria. In addition, florfenicol has the characteristics of rapid absorption. Once applied, it can efficiently penetrate the body's tissue barriers and quickly reach the target site to release the drug's efficacy; at the same time, it has a fast excretion rate, a short withdrawal period, and will not accumulate in the animal body. These excellent properties are highly consistent with the rigid demand for rapid turnover in modern animal husbandry, so florfenicol has become an important choice for many breeding companies in the layout of disease prevention and control strategies.

[0003] In aquaculture, the frequency of various bacterial diseases is increasing day by day, causing huge economic losses. Florfenicol has injected a shot in the arm for the stability and vigorous development of aquaculture with its excellent antibacterial activity. However, with the passage of time, the difficulties of florfenicol in its application have gradually surfaced. At present, the abuse of florfenicol in aquaculture is serious, and illegal application is everywhere, which accelerates the development of bacterial resistance. In the past, conventional doses could easily kill pathogens, but now even if the dose is increased, the effect is not satisfactory. These have not only caused the breeding costs to rise exponentially, but also made the disease prevention and control system on the verge of collapse, and the entire aquaculture industry is mired in a quagmire.

[0004] Therefore, how to effectively reduce bacterial resistance and better exert the antibacterial activity of florfenicol through scientific and reasonable medication strategies, innovative drug combination schemes or new drug dosage form improvements is a technical problem that urgently needs to be solved in this field. Summary of the invention

[0005] The purpose of the present invention is to provide a compound preparation and a preparation method thereof that can reduce the resistance of pathogens to florfenicol, so as to solve the problems existing in the above-mentioned prior art. Potassium clavulanate can greatly reduce drug resistance and enhance the antibacterial activity of florfenicol. The florfenicol and potassium clavulanate compound powder provided by the present invention can well treat various disease infections of aquatic animals.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A compound preparation capable of reducing the resistance of pathogens to florfenicol, the compound preparation comprising a combination of florfenicol and potassium clavulanate.

[0008] Furthermore, the composition comprises 2-60 parts of florfenicol and 0.5-15 parts of potassium clavulanate in parts by weight.

[0009] Furthermore, the composition comprises 5-15 parts of florfenicol and 2.5-7.5 parts of potassium clavulanate in parts by weight.

[0010] Furthermore, the pathogenic bacteria include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii and drug-resistant Citrobacter freundii.

[0011] Technical Solution 2: A compound powder of florfenicol and potassium clavulanate for aquaculture, wherein the raw materials, measured by weight, include 2-60 parts of florfenicol, 0.5-15 parts of potassium clavulanate and 25-97.5 parts of starch.

[0012] Preferably, by weight, the florfenicol is 10-50 parts, the potassium clavulanate is 2.5-12.5 parts, and the starch is 37.5-87.5 parts.

[0013] Preferably, the compound preparation comprises a pharmaceutical preparation and a feed additive.

[0014] Furthermore, the dosage form of the compound preparation includes powder.

[0015] Furthermore, the preparation method of the compound powder comprises the following steps: firstly mixing the florfenicol and potassium clavulanate, then adding the starch to mix, and sieving to obtain the compound preparation.

[0016] Technical solution three: Use of the compound preparation or the compound powder in enhancing the antibacterial ability of aquatic animals against drug-resistant pathogens.

[0017] Technical Solution 4: Use of the compound preparation or the compound powder in the preparation of medicines for treating aquatic animal diseases, wherein the diseases are diseases caused by infection with drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii or drug-resistant Citrobacter freundii.

[0018] Technical Solution 5: Use of potassium clavulanate in the preparation of a product that reduces the antibacterial activity of pathogens against florfenicol, wherein the pathogens include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas freundii and drug-resistant Citrobacter freundii.

[0019] Technical Solution 6: Use of potassium clavulanate in the preparation of a product for reducing the resistance of pathogens to florfenicol, wherein the pathogens include resistant Aeromonas hydrophila, resistant Aeromonas freundii and resistant Citrobacter freundii.

[0020] The present invention discloses the following technical effects:

[0021] The reason for the drug resistance of florfenicol is that the amide bond hydrolase in bacteria can hydrolyze the amide bond of florfenicol, resulting in reduced drug efficacy. Therefore, the bacterial drug resistance can be reduced by inhibiting the activity of amide bond hydrolase, thereby improving the antibacterial activity of florfenicol. Potassium clavulanate is an amide bond hydrolase inhibitor that can bind to most amide bond enzymes to form an irreversible conjugate, thereby improving the antibacterial activity of antibiotics. In order to reduce bacterial resistance and better exert the antibacterial activity of florfenicol, the present invention prepares a compound preparation of florfenicol and potassium clavulanate, and uses it for the treatment of aquatic animal diseases. The results show that the compound preparation can well treat aquatic animal disease infections. In short, potassium clavulanate can greatly reduce the drug resistance of florfenicol and enhance the antibacterial activity of florfenicol; the compound preparation provided by the present invention is simple to prepare, the raw materials are easy to obtain, and it is suitable for industrial large-scale production. Therefore, it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a chessboard pattern diagram. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The preparation method of the compound powder of florfenicol and clavulanate potassium is as follows: 2-60 parts of florfenicol, 0.5-15 parts of clavulanate potassium, and 25-97.5 parts of starch are used for standby. Specifically comprising the following steps: the sifted florfenicol and clavulanate potassium are placed in a mixer and fully mixed, the sifted starch is added to the mixer, and then sieved to obtain the compound powder. Preferably, according to the weight parts, 10-50 parts of florfenicol, 2.5-12.5 parts of clavulanate potassium, and 37.5-87.5 parts of starch.

[0030] In a specific embodiment, the effect of the compound powder in treating bacterial infection diseases in aquatic animals was verified by a challenge experiment using yellow catfish as the experimental subject. The drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii and drug-resistant Citrobacter freundii used in the embodiment of the present invention are all derived from clinical isolation, all isolated from yellow catfish, and their species have been confirmed by molecular biology verification, and the place of isolation is Wuhan City, Hubei Province.

[0031] The reagents and biological materials involved in the embodiments of the present invention are as follows:

[0032] Potassium clavulanate (61177-45-5) was purchased from Jiangxi Ruiweier Biotechnology Co., Ltd., with a content of 99%; Florfenicol (73231-34-2) was purchased from Shandong Guobang Pharmaceutical Co., Ltd., raw material, with a content of 98%;

[0033] The 16S sequence of the drug-resistant Aeromonas hydrophila is:

[0034]

[0035] The 16S sequence of the drug-resistant Aeromonas veronii is:

[0036]

[0037] The 16S sequence of drug-resistant Citrobacter freundii is:

[0038]

[0039] Unless otherwise specified, other experimental materials are conventional materials in this field and can be purchased through conventional channels.

[0040] Example 1 Potassium clavulanate increases the bactericidal ability of florfenicol against drug-resistant Aeromonas hydrophila

[0041] 1. The minimum inhibitory concentration of potassium clavulanate and florfenicol against drug-resistant Aeromonas hydrophila was determined by microdilution method. First, 200 μL of drug working solution was added to the preset maximum drug concentration well, and then 100 μL of sterilized MH broth was added to the remaining wells. Then, 100 μL of drug working solution was added to the second well from the maximum drug concentration well, and 100 μL was added to the third well after repeated blowing 5 times to mix. Then, 100 μL was added to the third well, and multiple dilutions were made in turn, and the 100 μL liquid aspirated from the last well was discarded. At this time, each well contained 100 μL of drug-containing broth, and the drug concentration was 512 μg / mL to 0.002 μg / mL.

[0042] 2. Chessboard method for determining the effect of combined use of clavulanate potassium and florfenicol

[0043] See the chessboard pattern diagram Figure 1 . Specifically, a 96-well bacterial culture plate was taken, and the Y1 row and X1 column were the rows and columns for drug A, potassium clavulanate, and drug B, florfenicol, respectively. 100 μL of standard drug solution with a concentration of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of the Y1 row; 100 μL of standard drug solution with a concentration of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of the X1 column. 50 μL of standard solution with a concentration of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of the Y2-7 row; 50 μL of standard solution with a concentration of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of the X2-7 column. 100 μL of bacterial suspension was added to each well except well X1Y1, and 200 μL of bacterial suspension was added to well X1Y1. At this time, the total volume of the liquid in the 96-well microplate is 200 μL. Put the 96-well microplate with the sample into a constant temperature incubator (28°C) for 24-48 hours. The lowest concentration of the clear well is the MIC for combined drug use. The results are determined by the equivalent midpoint method: each tube along the 0-point angular bisector is an equivalent midpoint tube. The lowest concentration of sterile growth is read as point 1. The point on the X-axis corresponding to this point is MICA, and the point on the Y-axis is MICB. The partial concentration inhibition index (FIC) is used as the basis for the judgment of the combined drug sensitivity test, and the calculation formula is as follows:

[0044]

[0045] Among them, an FIC index ≤ 0.5 indicates a synergistic effect; 0.5 < FIC index ≤ 1 indicates an additive effect; 1 < FIC index ≤ 2 indicates an irrelevant effect; and FIC index > 2 indicates an antagonistic effect.

[0046] Results: The MIC of clavulanate potassium against drug-resistant Aeromonas hydrophila was 64 μg / mL, and the MIC of florfenicol against drug-resistant Aeromonas hydrophila was 16 μg / mL. After the combination of clavulanate potassium and florfenicol, the lowest concentration without bacterial growth was: 16 μg / mL for clavulanate potassium and 8 μg / mL for florfenicol. After calculation, the FIC index was 0.75, showing an additive effect.

[0047] Example 2 Clavulanate potassium enhances the bactericidal ability of florfenicol against drug-resistant Aeromonas veronii

[0048] The determination method was the same as that in Example 1.

[0049] Results: The MIC of clavulanate potassium against drug-resistant Aeromonas veronii was 32 μg / mL, and the MIC of florfenicol against drug-resistant Aeromonas veronii was 16 μg / mL. After the combination of clavulanate potassium and florfenicol, the lowest concentration without bacterial growth was: 4 μg / mL for clavulanate potassium and 4 μg / mL for florfenicol. After calculation, the FIC index was 0.375, showing a synergistic effect.

[0050] Example 3 Clavulanic acid enhances the bactericidal ability of florfenicol against drug-resistant Citrobacter freundii

[0051] The determination method was the same as that in Example 1.

[0052] Results: The MIC of clavulanate potassium against drug-resistant Citrobacter freundii was 64 μg / mL, and the MIC of florfenicol against drug-resistant Citrobacter freundii was 8 μg / mL. After the combination of clavulanate potassium and florfenicol, the lowest concentration without bacterial growth was: 32 μg / mL for clavulanate potassium and 1 μg / mL for florfenicol. After calculation, the FIC index was 0.625, showing an additive effect.

[0053] Example 4 Clinical trial

[0054] 150 Pelteobagrus fulvidraco were randomly divided into five groups, with 30 in each group.

[0055] Blank control: Without virus challenge and treatment;

[0056] Negative control: Challenged with drug-resistant Aeromonas veronii from Pelteobagrus fulvidraco without treatment;

[0057] Treatment group 1: Challenged with drug-resistant Aeromonas veronii from Pelteobagrus fulvidraco and treated with florfenicol at a dose of 15 mg / kg, twice a day for three consecutive days;

[0058] Treatment group 2: The yellow catfish-derived drug-resistant Aeromonas veronii was used for challenge and potassium clavulanate was given for treatment at a dose of 15 mg / kg, twice a day for three consecutive days;

[0059] Treatment Group 3: The yellow catfish-derived drug-resistant Aeromonas veronii was used for infection, and the animals were treated with a combination powder of florfenicol and clavulanate potassium at a dose of 15 mg / kg (calculated as florfenicol), twice a day for three consecutive days.

[0060] After 7 days of treatment, the mortality rate of each group of yellow catfish was calculated.

[0061] The preparation method of the florfenicol and potassium clavulanate compound powder comprises the following steps: 20 parts of florfenicol, 5 parts of potassium clavulanate and 75 parts of starch are prepared for standby use, and the preparation method specifically comprises the following steps: sifting the florfenicol and potassium clavulanate separately, mixing them in a mixer, adding the sifted starch into the mixer, and sifting again to obtain the compound powder.

[0062] Table 1 Test results

[0063] Group Number of dead animals mortality rate Blank control group 1 3.33％ Negative control group 30 100％ Treatment Group 1 (Flofenicol) 25 83.33％ Treatment Group 2 (potassium clavulanate) 24 80.00％ Treatment group 3 (florfenicol and clavulanate potassium compound powder) 1 3.33％

[0064] According to the mortality results of each group in Table 1, the compound powder prepared by florfenicol and clavulanate potassium can be well used to treat disease infections in aquatic animals.

[0065] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A compound preparation capable of reducing the resistance of pathogens to florfenicol, characterized in that: The compound preparation comprises a combination of florfenicol and potassium clavulanate.

2. The compound preparation according to claim 1, characterized in that: In parts by weight, the composition comprises 2-60 parts of florfenicol and 0.5-15 parts of potassium clavulanate.

3. The compound preparation according to claim 1, characterized in that: In parts by weight, the composition comprises 5-15 parts of florfenicol and 2.5-7.5 parts of potassium clavulanate.

4. The compound preparation according to claim 1, characterized in that: The pathogenic bacteria include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii and drug-resistant Citrobacter freundii.

5. A compound powder of florfenicol and potassium clavulanate for aquaculture, characterized in that: The raw materials include 2-60 parts of florfenicol, 0.5-15 parts of potassium clavulanate and 25-97.5 parts of starch by weight.

6. The compound powder according to claim 5, characterized in that: The preparation method of the compound powder comprises the following steps: firstly mixing the florfenicol and potassium clavulanate, then adding the starch to mix, and then sieving to obtain the compound preparation.

7. Use of the compound preparation according to any one of claims 1 to 4 or the compound powder according to claim 5 or 6 in enhancing the antibacterial ability of aquatic animals against drug-resistant pathogens.

8. Use of the compound preparation according to any one of claims 1 to 4 or the compound powder according to claim 5 or 6 in the preparation of a medicament for treating aquatic animal diseases, characterized in that: The disease is a disease caused by infection with drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii or drug-resistant Citrobacter freundii.

9. Use of potassium clavulanate in the preparation of a product for reducing the antibacterial activity of florfenicol against pathogenic bacteria, characterized in that: The pathogenic bacteria include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii and drug-resistant Citrobacter freundii.

10. Use of potassium clavulanate in the preparation of a product for reducing the resistance of pathogens to florfenicol, characterized in that: The pathogenic bacteria include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veronii and drug-resistant Citrobacter freundii.

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