Application of combination of dihydroartemisinin and ciprofloxacin hydrochloride in preparation of vibrio parahaemolyticus inhibitor

Abstract

The invention discloses application of dihydroartemisinin combined with ciprofloxacin hydrochloride in preparation of a vibrio parahaemolyticus inhibitor, the sensitivity of ciprofloxacin hydrochloride-resistant vibrio parahaemolyticus to ciprofloxacin hydrochloride can be increased by combined use of ciprofloxacin hydrochloride and dihydroartemisinin, and in the process of killing bacteria, the use amount of ciprofloxacin hydrochloride is reduced, so that the sensitivity of ciprofloxacin hydrochloride-resistant vibrio parahaemolyticus is improved. The ciprofloxacin hydrochloride and dihydroartemisinin composition is a composition of 64 [mu] g / mL of dihydroartemisinin and 16 [mu] g / mL of ciprofloxacin hydrochloride, and has the advantages that the ciprofloxacin hydrochloride and dihydroartemisinin composition has a good treatment effect on infection caused by vibrio parahaemolyticus, and meanwhile, the use amount of the antibiotic ciprofloxacin hydrochloride is reduced; green and healthy culture of aquatic products is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, and in particular to application of dihydroartemisinin combined with ciprofloxacin hydrochloride in the preparation of a Vibrio parahaemolyticus inhibitor. Background Art

[0002] Vibrio parahaemolyticus is a Gram-negative halophilic bacterium widely distributed in seawater environments. It can tolerate a high range of salinity (0.5-9.5%), pH (7.6-9.0) and temperature (7-43°C). It easily attaches to marine plankton and spreads through ocean currents, causing disease in a variety of aquatic economic animals such as fish and shrimp. In recent years, acute hepatopancreatic necrosis disease (AHPND), with Vibrio parahaemolyticus as the main pathogen, has swept the global shrimp farming industry, causing serious economic losses. In my country, the disease is mainly caused by Vibrio parahaemolyticus carrying PirA and PirB virulence factors (Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease, Vp AHPND ) is also a nationwide epidemic, usually breaking out in the late stage of shrimp larvae or the early stage of growth of young shrimp, with a mortality rate of up to 100%, seriously endangering the development of my country's shrimp farming industry. In addition, Vibrio parahaemolyticus is also present in large quantities in seafood. Eating raw or undercooked food containing Vibrio parahaemolyticus may cause gastroenteritis, and is an important zoonotic pathogen. Therefore, strengthening the effective control of Vibrio parahaemolyticus is not only related to the healthy farming of shrimp, but also directly affects the quality and safety of aquatic products.

[0003] In aquaculture, the prevention and control of Vibrio mainly relies on drugs represented by antibiotics. However, the irregular use of antibiotics, such as abuse and overuse, not only directly promotes the generation, spread and accumulation of resistance genes, but also aggravates the formation of bacterial resistance, and finally leads to the emergence of multi-drug resistant bacteria. This phenomenon has become increasingly prominent in aquaculture environments. Public awareness of the dangers caused by excessive exposure to antibiotics is increasing, prompting people to look for more environmentally friendly and safer drugs. Therefore, many studies are devoted to finding new alternative drugs to address the problem of drug resistance. Nature is considered to be an important potential source for drug discovery. For decades, plant-derived chemicals have attracted the attention of many scientists due to their structural diversity, fewer side effects and greater acceptance. Antibiotics and plant-derived monomers may have similar or completely different antibacterial mechanisms, and their combination can be used to treat a variety of diseases. Combination therapy can not only significantly reduce the possibility of drug resistance, but also improve the overall treatment effect. In view of the serious harm caused by Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis, to the shrimp farming industry, there is an urgent need to develop a combination of antibiotics and plant-derived monomers with clear technical effects to provide an efficient, safe and low-toxic drug solution for the prevention and treatment of acute hepatopancreatic necrosis of shrimp. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an application of dihydroartemisinin combined with ciprofloxacin hydrochloride in the preparation of a Vibrio parahaemolyticus inhibitor. The composition can significantly reduce the usage of ciprofloxacin hydrochloride, improve the sensitivity of Vibrio parahaemolyticus to ciprofloxacin hydrochloride, and effectively kill Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis of shrimp.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] The present invention provides application of dihydroartemisinin combined with ciprofloxacin hydrochloride in preparing a Vibrio parahaemolyticus inhibitor.

[0007] The present invention also provides application of the ciprofloxacin hydrochloride and dihydroartemisinin composition in improving the sensitivity of Vibrio parahaemolyticus to ciprofloxacin hydrochloride.

[0008] Preferably, the mass ratio of ciprofloxacin hydrochloride to dihydroartemisinin in the composition is 1:2-4.

[0009] The present invention also provides a medicine for treating acute hepatopancreatic necrosis of shrimp. The medicine comprises a composition of ciprofloxacin hydrochloride and dihydroartemisinin. The mass ratio of ciprofloxacin hydrochloride to dihydroartemisinin in the composition is 1:2-4.

[0010] Preferably, the composition is a composition of 64 μg / mL of dihydroartemisinin and 16 μg / mL of ciprofloxacin hydrochloride.

[0011] Compared with the prior art, the advantages of the present invention are: the present invention discloses for the first time the use of dihydroartemisinin combined with ciprofloxacin hydrochloride in the preparation of Vibrio parahaemolyticus inhibitors, and the dihydroartemisinin in the composition is an active metabolite of artemisinin drugs and a natural medicine. Ciprofloxacin hydrochloride is a broad-spectrum antibacterial drug of the fluoroquinolone class, which interferes with the replication, transcription and repair process of bacterial DNA by inhibiting the activity of DNA gyrase and topoisomerase IV, thereby achieving an antibacterial effect. Experiments have shown that compared with the use of ciprofloxacin hydrochloride or dihydroartemisinin alone, the combination of ciprofloxacin hydrochloride and dihydroartemisinin can reduce the amount of ciprofloxacin hydrochloride used and improve the survival rate of shrimp infected with Vibrio parahaemolyticus causing acute hepatopancreatic necrosis. It shows that the combined use of ciprofloxacin hydrochloride and dihydroartemisinin can enhance the efficacy of the drug, reduce the amount of antibiotic ciprofloxacin hydrochloride used, avoid the problem of bacterial resistance caused by long-term and large-scale use of antibiotics in shrimp production, reduce the safety risks in the breeding process of food animals, and promote the green and healthy breeding of aquatic animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0013] Figure 1 The effects of dihydroartemisinin, ciprofloxacin hydrochloride, and the combination of dihydroartemisinin and ciprofloxacin hydrochloride on the growth performance of Vibrio parahaemolyticus DX190406;

[0014] Figure 2 The bactericidal effects of dihydroartemisinin, ciprofloxacin hydrochloride, and the combination of dihydroartemisinin and ciprofloxacin hydrochloride on Vibrio parahaemolyticus DX190406;

[0015] Figure 3 The effects of dihydroartemisinin, ciprofloxacin hydrochloride and the combination of dihydroartemisinin and ciprofloxacin hydrochloride on biofilm formation of Vibrio parahaemolyticus DX190406;

[0016] Figure 4 The present invention is to investigate the effects of dihydroartemisinin, ciprofloxacin hydrochloride, and the combination of dihydroartemisinin and ciprofloxacin hydrochloride on the survival rate of Penaeus vannamei infected with Vibrio parahaemolyticus DX190406. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with reference to the accompanying drawings.

[0018] 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 application description and examples are exemplary only.

[0019] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0020] 1. Experimental drugs

[0021] Ciprofloxacin hydrochloride (abbreviated as CIP): effective content ≥ 98%, purchased from Beijing Solebow Technology Co., Ltd.; ciprofloxacin hydrochloride was prepared with deionized water into a storage solution with an initial concentration of 5120 μg / mL, filtered with a sterile syringe and a sterile 0.22 μm filter membrane, and stored at -80°C for use.

[0022] Dihydroartemisinin (abbreviated as DHA): effective content ≥ 98%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; dihydroartemisinin was prepared with dimethyl sulfoxide (DMSO) into a storage solution with an initial concentration of 50,000 μg / mL and stored at 4°C for use.

[0023] 2. Culture medium

[0024] The culture media used in the experiment included: 2216E liquid culture medium and 2216E agar culture medium, which were purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.;

[0025] Preparation of 2216E solid culture medium (1000mL system): weigh 52.4g 2216E agar medium, add 1000mL distilled water, mix well, autoclave at 121℃ for 15min, cool to 50℃, pour into a plate and cool at room temperature;

[0026] Preparation of 2216E liquid culture medium (1000 mL system): Weigh 37.4 g of 2216E liquid culture medium, add 1000 mL of distilled water, mix well, sterilize with high pressure steam at 121°C for 15 min, and leave to cool for use.

[0027] 3. Experimental strains

[0028] Four strains of Vibrio parahaemolyticus were isolated from Penaeus vannamei farms in various parts of Zhejiang Province in 2019, causing large-scale deaths of shrimp, showing typical symptoms of acute hepatopancreatic necrosis of shrimp. Specific information is shown in Table 1.

[0029] Table 14 Information on strains of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis

[0030]

[0031] Take the original bacterial solution of Vibrio parahaemolyticus grown to the logarithmic growth phase in Table 1, and adjust the concentration of the bacterial solution to 0.5 McFarland turbidity according to the McFarland turbidimetric method. Then dilute it 100 times with 2216E liquid culture medium to obtain a concentration of about 1×10 6 CFU / mL of Vibrio parahaemolyticus bacterial solution.

[0032] Example 1. Minimum inhibitory concentration (MIC) of ciprofloxacin hydrochloride against 4 strains of Vibrio parahaemolyticus.

[0033] 1. Experimental method: Take a 96-well microplate, inoculate 100 μL of 2216E liquid culture medium in each well from the 2nd to the 11th column, inoculate 200 μL of 2216E liquid culture medium in the 12th column, inoculate 100 μL of 512 μg / mL ciprofloxacin hydrochloride solution in each well from the 1st and 2nd columns, and dilute it twice from the 2nd column to the 10th column. Inoculate 100 μL of 512 μg / mL ciprofloxacin hydrochloride solution in each well from the 2nd column to the 10th column. 6 CFU / mL of Vibrio parahaemolyticus liquid. Column 11 is the positive control, column 12 is the negative control, incubated at 28°C for 18h. Read the positive and negative control wells, the negative control wells are clear, and the positive control wells are turbid. The MIC of a drug against bacteria is the lowest drug concentration that inhibits visible bacterial growth.

[0034] 2. Experimental results: As shown in Table 2, it can be seen that the MIC of the four strains of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis is 64μg / mL. According to the CLSIM45 document, if the MIC of Vibrio genus to ciprofloxacin hydrochloride is ≥4μg / mL, it can be determined as ciprofloxacin hydrochloride resistant bacteria.

[0035] Table 2 Minimum inhibitory concentration of ciprofloxacin hydrochloride against 4 strains of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis

[0036]

[0037] As shown in Table 2, the four strains of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis are all resistant to ciprofloxacin hydrochloride and have strong drug resistance.

[0038] Example 2: Minimum inhibitory concentration (MIC) of dihydroartemisinin against 4 strains of Vibrio parahaemolyticus.

[0039] 1. Experimental method: Take a 96-well microplate, inoculate 100 μL of 2216E liquid culture medium in each well from the 2nd to the 11th column, inoculate 200 μL of 2216E liquid culture medium in the 12th column, inoculate 100 μL of 512 μg / mL dihydroartemisinin solution in each well of the 1st and 2nd columns, and dilute it twice from the 2nd column to the 10th column. Inoculate 100 μL of 512 μg / mL dihydroartemisinin solution in each well from the 2nd column to the 10th column. 6 CFU / mL of Vibrio parahaemolyticus liquid. Column 11 is the positive control, column 12 is the negative control, incubated at 28°C for 18h. Read the positive and negative control wells, the negative control wells are clear, and the positive control wells are turbid. The MIC of a drug against bacteria is the lowest drug concentration that inhibits visible bacterial growth.

[0040] 2. Experimental results: As shown in Table 3, when dihydroartemisinin was used alone, the MIC of the four strains of Vibrio parahaemolyticus that caused acute hepatopancreatic necrosis was ≥256 μg / mL.

[0041] Table 3 Minimum inhibitory concentration of dihydroartemisinin against four strains of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis

[0042]

[0043] The experimental results in Table 3 show that dihydroartemisinin alone can inhibit the growth of bacteria, but the antibacterial effect of dihydroartemisinin alone is poor.

[0044] Example 3: Antibacterial effect of the combined use of dihydroartemisinin and ciprofloxacin hydrochloride on 4 strains of Vibrio parahaemolyticus.

[0045] 1. Experimental method: According to the CLSIM27-A3 drug sensitivity test standard, the checkerboard method was used to detect the antibacterial activity of the combination of dihydroartemisinin and ciprofloxacin hydrochloride against Vibrio parahaemolyticus. The specific steps are as follows:

[0046] (1) Take a 96-well microplate and inoculate 100 μL of each well at a concentration of approximately 1×10 6 CFU / mL of Vibrio parahaemolyticus liquid and 100 μL of drug solution (a combination of dihydroartemisinin and ciprofloxacin hydrochloride in different mass ratios) were used to perform an antibacterial activity experiment of dihydroartemisinin and ciprofloxacin hydrochloride against Vibrio parahaemolyticus in a 96-well microplate according to the checkerboard method, and incubated at 28°C for 18 hours;

[0047] (2) The MIC value of each well was calculated, and the graded inhibitory concentration index (FIC) was further calculated using the following formula: FIC index = MIC (ciprofloxacin hydrochloride combined with ciprofloxacin) / MIC (ciprofloxacin hydrochloride alone) + MIC (dihydroartemisinin combined with dihydroartemisinin) / MIC (dihydroartemisinin alone). The criteria for judging the FIC index were: FIC index ≤ 0.5 for synergistic effect; 0.5-1 for additive effect; 1-2 for irrelevant effect; and ≥ 2 for antagonistic effect.

[0048] 2. Experimental results: As shown in Table 4, the combination of 16 μg / mL ciprofloxacin hydrochloride + 64 μg / mL dihydroartemisinin can significantly inhibit Vibrio parahaemolyticus DX190406, with an FIC index of 0.5; the combination of 16 μg / mL ciprofloxacin hydrochloride + 32 μg / mL dihydroartemisinin can significantly inhibit Vibrio parahaemolyticus DX210401, with an FIC index of 0.375; the combination of 16 μg / mL ciprofloxacin hydrochloride + 64 μg / mL dihydroartemisinin can significantly inhibit Vibrio parahaemolyticus DX220801, with an FIC index of 0.5; the combination of 16 μg / mL ciprofloxacin hydrochloride + 64 μg / mL dihydroartemisinin can significantly inhibit Vibrio parahaemolyticus DX230702, with an FIC index of 0.5.

[0049] Table 4 Minimum inhibitory concentration and graded inhibitory concentration index of ciprofloxacin hydrochloride and dihydroartemisinin combined

[0050]

[0051]

[0052] The above results show that the combined use of dihydroartemisinin and ciprofloxacin hydrochloride has antibacterial activity against 4 strains of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis, and the usage of ciprofloxacin hydrochloride has decreased significantly, indicating that dihydroartemisinin can increase the sensitivity of Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis to ciprofloxacin hydrochloride, and dihydroartemisinin combined with ciprofloxacin hydrochloride has an antibacterial sensitization effect on Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis.

[0053] Example 4: Effect of the combination of dihydroartemisinin and ciprofloxacin hydrochloride on the growth performance of Vibrio parahaemolyticus DX190406.

[0054] Taking Vibrio parahaemolyticus DX190406 as an example, the effect of the combination of dihydroartemisinin and ciprofloxacin hydrochloride on bacterial growth was evaluated.

[0055] 1. Experimental method: Take a 96-well microplate and inoculate 100 μL of the first column with a concentration of about 1×10 6CFU / mL of Vibrio parahaemolyticus DX190406 bacterial solution and 100 μL of drug combination solution (final concentration of ciprofloxacin hydrochloride 16 μg / mL, dihydroartemisinin 64 μg / mL); the second column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial solution and 100 μL of 32 μg / mL ciprofloxacin hydrochloride solution (final concentration of ciprofloxacin hydrochloride was 16 μg / mL); the third column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial solution and 10 10 μL of 128 μg / mL dihydroartemisinin solution (final concentration of dihydroartemisinin is 64 μg / mL) was added to the 4th column, 100 μL of Vibrio parahaemolyticus DX190406 bacterial solution and 100 μL of 2216E liquid culture medium were inoculated as a positive control group; 100 μL of 2216E liquid culture medium and 100 μL of drug combination solution (final concentration of ciprofloxacin hydrochloride 16 μg / mL, dihydroartemisinin 64 μg / mL) were added to the 5th column, as a negative control group. Incubate at 28°C for 24 hours, and measure the absorbance at a wavelength of 600 nm every 1 hour.

[0056] 2. Experimental results: Figure 1 As shown, compared with the use of ciprofloxacin hydrochloride and dihydroartemisinin alone, under the treatment of the combination of ciprofloxacin hydrochloride and dihydroartemisinin, the bacteria did not proliferate significantly within 24 hours (absorbance ≈ 0.25, similar to the results of the negative control group). After 3 hours, the number of bacteria in the two experimental groups using the two drugs alone gradually increased (the absorbance gradually increased). The absorbance of the experimental group using ciprofloxacin hydrochloride alone reached 0.9 at 24 hours, and the results were similar to those of the positive control group (absorbance ≈ 0.9), indicating that the use of 16μg / mL ciprofloxacin hydrochloride alone had no inhibitory effect on bacteria. The absorbance of the experimental group using dihydroartemisinin alone reached 0.5 at 24 hours, which was greater than that of the negative control group, but also significantly less than that of the positive control group, indicating that dihydroartemisinin at a final concentration of 64μg / mL can partially inhibit bacteria. In summary, the combination of 64 μg / mL of dihydroartemisinin and 16 μg / mL of ciprofloxacin hydrochloride can completely inhibit the growth of Vibrio parahaemolyticus DX190406 within 24 hours.

[0057] Example 5: Test on the bactericidal ability of the combination of dihydroartemisinin and ciprofloxacin hydrochloride against Vibrio parahaemolyticus DX190406

[0058] 1. Experimental method: Take a 96-well microplate and inoculate 100 μL of the first column with a concentration of about 1×10 6CFU / mL of Vibrio parahaemolyticus DX190406 bacterial solution and 100 μL of drug combination solution (final concentration of ciprofloxacin hydrochloride 16 μg / mL, dihydroartemisinin 64 μg / mL); the second column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial solution and 100 μL of 32 μg / mL ciprofloxacin hydrochloride solution (the final concentration of ciprofloxacin hydrochloride was 16 μg / mL); the third column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 The fourth column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial solution and 100 μL of 2216E liquid culture medium, incubated at 28°C for 24 hours, and 100 μL of the treated culture medium was taken at 3, 6, 9, 12, and 24 hours, respectively, and spread on 2216E solid culture medium. After 12 hours, the number of bacteria on the plate was counted.

[0059] 2. Experimental results: Figure 2 As shown, after treatment with a combination of 64 μg / mL of dihydroartemisinin and 16 μg / mL of ciprofloxacin hydrochloride, the number of bacteria decreased rapidly, and 3 hours after treatment, the number of bacteria had reached 0. When 16 μg / mL of ciprofloxacin hydrochloride was used to treat bacteria, there was no significant change in the number of bacteria compared with the control group without any drug treatment, and it had no killing effect on bacteria. When 64 μg / mL of dihydroartemisinin was used to treat bacteria, the decrease in the number of bacteria was less than 3 log at each time point of treatment compared with the control group without any drug treatment. 10 CFU / mL, no obvious bactericidal effect. In summary, the combination of 64 μg / mL of dihydroartemisinin and 16 μg / mL of ciprofloxacin hydrochloride can completely kill Vibrio parahaemolyticus DX190406 within 3 hours, and has a good bactericidal effect.

[0060] Example 6. Effect of the combination of dihydroartemisinin and ciprofloxacin hydrochloride on biofilm formation of Vibrio parahaemolyticus DX190406.

[0061] 1. Experimental method: Take a 96-well microplate and inoculate 100 μL of the first column with a concentration of about 1×10 6CFU / mL of Vibrio parahaemolyticus DX190406 bacterial liquid and 100 μL of drug combination solution (final concentration of ciprofloxacin hydrochloride 16 μg / mL, dihydroartemisinin 64 μg / mL); the second column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial liquid and 100 μL of 32 μg / mL ciprofloxacin hydrochloride solution (the final concentration of ciprofloxacin hydrochloride was 16 μg / mL); the third column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial liquid and 100 μL of 128 μg / mL dihydroartemisinin solution (the final concentration of dihydroartemisinin was 64 μg / mL); the fourth column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial liquid and 100 μL of 2216E liquid culture medium, and incubated at 28°C for 24 hours. The bacterial solution in each well of the microplate was sucked off, and the wells were gently rinsed three times with phosphate buffer (PBS, pH = 7.2-7.4), and fixed in a 55°C drying oven for 30 minutes; 200 μL of 1% crystal violet solution was added to each well, incubated at room temperature for 15 minutes, the crystal violet solution in each well of the microplate was sucked off, and the wells were gently rinsed three times with sterile PBS, and fixed in a 55°C drying oven for 30 minutes. 200 μL of 75% ethanol was added to each well to dissolve the crystal violet. The dissolved crystal violet in each well was transferred to the corresponding well of a new 96-well microplate, and the absorbance at 595 nm was measured to calculate the biomass of the biofilm. The experiment was repeated three times.

[0062] 2. Experimental results: Figure 3 As shown, Vibrio parahaemolyticus DX190406 can produce biofilms, and the absorbance of the biofilm produced within 24 hours after treatment with crystal violet is 2.228±0.037; after the bacteria are treated with dihydroartemisinin and ciprofloxacin hydrochloride, the strain can still produce biofilms, and the absorbance values ​​corresponding to the amount of biofilm produced are 2.227±0.024 and 2.184±0.025, respectively, which are not significantly different from the amount of biofilm produced by bacteria without drug treatment. After the bacteria are treated with a combination of 64μg / mL dihydroartemisinin and 16μg / mL ciprofloxacin hydrochloride, the amount of biofilm produced by the bacteria is 1.197±0.038 after crystal violet staining, which is significantly lower than that of bacteria without drug treatment. In summary, the combination of dihydroartemisinin and ciprofloxacin hydrochloride can significantly reduce the biofilm formation of Vibrio parahaemolyticus DX190406, which may be one of the reasons for the increased sensitivity of the bacteria to ciprofloxacin hydrochloride.

[0063] Example 7: Effect of a combination of dihydroartemisinin and ciprofloxacin hydrochloride on the survival rate of Penaeus vannamei infected with Vibrio parahaemolyticus DX190406

[0064] 1. Experimental method: Vannamei shrimp (size: about 3.5 g / tail) were cultured in 200L PVC plastic barrels. The water was changed every day and the water temperature was maintained at 28±1℃, pH 8.0±0.01, and salinity 32‰. Healthy Vannamei shrimp were injected with Vibrio parahaemolyticus strain DX190406 (median lethal concentration of 6.67×10 7 CFU / mL (36 hours)] to establish an artificial infection model. 750 shrimps were divided into 5 groups, with 3 replicates in each group and 50 shrimps in each replicate. Except for the blank control group, the shrimps in the other 4 groups were injected with 6.67×10 5 CFU of Vibrio parahaemolyticus DX190406. After infection, the experimental shrimps were immediately fed with feeds containing different drugs according to the grouping, specifically including feeds containing ciprofloxacin hydrochloride (16 mg / kg), dihydroartemisinin (64 mg / kg), and drug combination (16 mg / kg ciprofloxacin hydrochloride + 64 mg / kg dihydroartemisinin). The blank control group and the infection group were fed with feeds without any experimental drugs. The feed feeding amount was 5% of the shrimp body weight, and it was fed once every 12 hours for 7 consecutive days. The mortality was recorded on the 0th, 0.5th, 1st, 3rd, 5th, and 7th days after infection.

[0065] 2. Experimental results: Figure 4 As shown, after artificial infection of Vibrio parahaemolyticus DX190406, the cumulative mortality rate of Penaeus vannamei within 7 days was 100%. After infection, after feeding 16 mg / kg ciprofloxacin hydrochloride and 64 mg / kg dihydroartemisinin, the shrimp still suffered serious death, with mortality rates of 71% and 61% on the 7th day respectively; after feeding feed containing 16 mg / kg ciprofloxacin hydrochloride and 64 mg / kg dihydroartemisinin, the cumulative mortality rate on the fifth day was 20%, and no more deaths occurred on the sixth and seventh days, and the mortality situation was controlled. It can be seen that feeding feed containing a certain proportion of dihydroartemisinin and ciprofloxacin hydrochloride can significantly reduce the mortality rate of Penaeus vannamei infected with Vibrio parahaemolyticus and obtain good therapeutic effects.

[0066] In summary, the present invention has experimentally verified that the combined use of ciprofloxacin hydrochloride and dihydroartemisinin can increase the sensitivity of ciprofloxacin hydrochloride-resistant Vibrio parahaemolyticus to ciprofloxacin hydrochloride, and reduce the amount of ciprofloxacin hydrochloride used in the process of killing bacteria. To further verify its combined antibacterial effect, the present invention uses Vibrio parahaemolyticus DX190406 as an experimental object, analyzes the effect of the composition of ciprofloxacin hydrochloride and dihydroartemisinin on the time growth curve, time killing curve and biofilm formation of bacteria, and the results show that the composition of ciprofloxacin hydrochloride and dihydroartemisinin can significantly kill Vibrio parahaemolyticus DX190406 and inhibit the formation of biofilm; animal experimental results show that the composition of ciprofloxacin hydrochloride and dihydroartemisinin significantly reduces the mortality rate of Penaeus vannamei after infection with Vibrio parahaemolyticus DX190406. Therefore, the ciprofloxacin hydrochloride and dihydroartemisinin composition disclosed in the present invention has a good therapeutic effect on infections caused by Vibrio parahaemolyticus, while reducing the usage of the antibiotic ciprofloxacin hydrochloride and promoting green and healthy aquaculture.

[0067] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. Application of dihydroartemisinin combined with ciprofloxacin hydrochloride in the preparation of Vibrio parahaemolyticus inhibitors.

2. Application of a combination of ciprofloxacin hydrochloride and dihydroartemisinin in improving the sensitivity of Vibrio parahaemolyticus to ciprofloxacin hydrochloride.

3. Use of the ciprofloxacin hydrochloride and dihydroartemisinin composition of claim 2 in improving the sensitivity of Vibrio parahaemolyticus to ciprofloxacin hydrochloride, characterized in that: The mass ratio of ciprofloxacin hydrochloride to dihydroartemisinin in the composition is 1:2-4.

4. A drug for treating acute hepatopancreatic necrosis of shrimp, characterized in that: The medicine comprises a combination of ciprofloxacin hydrochloride and dihydroartemisinin.

5. The drug for treating acute hepatopancreatic necrosis of shrimp according to claim 4, characterized in that: The composition is a composition of 64 μg / mL of dihydroartemisinin and 16 μg / mL of ciprofloxacin hydrochloride.

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