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

Abstract

The invention discloses an application of dihydroartemisinin combined with ciprofloxacin hydrochloride in preparing a Vibrio parahaemolyticus inhibitor. The combined use of ciprofloxacin hydrochloride and dihydroartemisinin can increase the sensitivity of ciprofloxacin hydrochloride-resistant Vibrio parahaemolyticus to ciprofloxacin hydrochloride, reduce the usage of ciprofloxacin hydrochloride in the process of killing bacteria, and the ciprofloxacin hydrochloride and dihydroartemisinin composition is a composition of 64 μg / mL dihydroartemisinin and 16 μg / mL ciprofloxacin hydrochloride. The advantage is that the ciprofloxacin hydrochloride and dihydroartemisinin composition has a good therapeutic effect on infections caused by Vibrio parahaemolyticus, reduces the usage of the antibiotic ciprofloxacin hydrochloride, and promotes green and healthy aquaculture.

Description

Technical Field

[0001] The present 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 that is 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 that causes acute hepatopancreatic necrosis disease, Vp AHPND ) is a nationwide epidemic, typically occurring in the late postlarval or early growth stages of juvenile shrimp. Mortality rates can reach 100%, severely jeopardizing the development of my country's shrimp farming industry. Furthermore, Vibrio parahaemolyticus is also found in high concentrations in seafood. Consuming raw or undercooked food containing V. parahaemolyticus can cause gastroenteritis, making it a significant zoonotic pathogen. Therefore, strengthening effective control of V. parahaemolyticus is crucial not only for the healthy farming of shrimp but also for the quality and safety of aquatic products.

[0003] In aquaculture, the control of Vibrio spp. primarily relies on antibiotics. However, improper use of antibiotics, such as misuse and overuse, not only directly contributes to the generation, spread, and accumulation of resistance genes but also exacerbates the development of bacterial resistance, ultimately leading to the emergence of multidrug-resistant bacteria. This phenomenon has become increasingly prominent in aquaculture settings. Growing public awareness of the dangers of excessive antibiotic exposure is prompting the search for more environmentally friendly and safer drugs. Consequently, extensive research is dedicated to identifying new alternatives to combat drug resistance. Nature is considered a key potential source for drug discovery. For decades, plant-derived chemicals have attracted the attention of numerous scientists due to their structural diversity, limited side effects, and increased tolerance. Antibiotics and plant-derived monomers can have similar or distinct antimicrobial mechanisms, and their combination can be used to treat a wide range of diseases. Combination therapy not only significantly reduces the likelihood of drug resistance but also improves overall therapeutic efficacy. Given 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, increase 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 use of dihydroartemisinin combined with ciprofloxacin hydrochloride in the preparation of a Vibrio parahaemolyticus inhibitor.

[0007] The present invention also provides an application of a 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, wherein the mass ratio of ciprofloxacin hydrochloride to dihydroartemisinin in the composition is 1:2-4.

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

[0011] Compared with existing technologies, the present invention offers the following advantages: It discloses for the first time the use of dihydroartemisinin combined with ciprofloxacin hydrochloride in the preparation of Vibrio parahaemolyticus inhibitors. The dihydroartemisinin in the combination is an active metabolite of artemisinin-based drugs and a natural medicine. Ciprofloxacin hydrochloride is a broad-spectrum fluoroquinolone antibiotic that inhibits DNA gyrase and topoisomerase IV, interfering with bacterial DNA replication, transcription, and repair, thereby achieving its antibacterial effect. Experiments have shown that, compared with using either ciprofloxacin hydrochloride or dihydroartemisinin alone, the combined use of ciprofloxacin hydrochloride and dihydroartemisinin can reduce the dosage of ciprofloxacin hydrochloride and improve the survival rate of shrimp infected with Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis. This suggests that the combined use of ciprofloxacin hydrochloride and dihydroartemisinin can enhance efficacy, reduce the dosage of the antibiotic ciprofloxacin hydrochloride, avoid bacterial resistance caused by long-term and excessive antibiotic use in shrimp production, reduce safety risks in food animal farming, and promote healthy and sustainable aquaculture. 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 described below 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 any 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 are shown;

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

[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 Litopenaeus vannamei infected with Vibrio parahaemolyticus DX190406. DETAILED DESCRIPTION

[0017] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[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 without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative 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 (CIP): effective content ≥ 98%, purchased from Beijing Solebow Technology Co., Ltd.; ciprofloxacin hydrochloride was prepared with deionized water to a stock solution with an initial concentration of 5120 μg / mL, filtered using a sterile syringe and a sterile 0.22 μm filter membrane, and stored at −80°C until use.

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

[0023] 2. Culture medium

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

[0025] Preparation of 2216E solid medium (1000 mL system): Weigh 52.4 g of 2216E agar medium, add 1000 mL of distilled water, mix well, and autoclave at 121°C for 15 min. When cooled to 50°C, 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, autoclave at 121°C for 15 min, and let cool until used.

[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 shrimp deaths and 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 stock solution of Vibrio parahaemolyticus grown to the logarithmic growth phase in Table 1 and adjust the concentration of the solution to 0.5 McFarland turbidity using the McFarland turbidimetric method. Then dilute it 100 times with 2216E liquid medium to obtain a concentration of approximately 1×10 6 CFU / mL of Vibrio parahaemolyticus liquid.

[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 medium in each well from column 2 to column 11, inoculate 200 μL of 2216E liquid medium in column 12, and inoculate 100 μL of 512 μg / mL ciprofloxacin hydrochloride solution in each well from column 1 and column 2. Dilute the solution twice starting from column 2 until column 10. Inoculate 100 μL of 512 μg / mL ciprofloxacin hydrochloride solution in each well from column 1 to column 11. 6 CFU / mL of Vibrio parahaemolyticus culture. Column 11 is the positive control, and column 12 is the negative control. Incubate at 28°C for 18 hours. Read the positive and negative control wells; the negative control wells should be clear, while the positive control wells should be 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, the MIC of all four strains of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis was 64 μg / mL. According to the CLSIM45 document, Vibrio species are considered resistant to ciprofloxacin hydrochloride if their MIC to ciprofloxacin hydrochloride is ≥4 μg / mL.

[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 medium in each well from column 2 to column 11, 200 μL of 2216E liquid medium in column 12, and 100 μL of 512 μg / mL dihydroartemisinin solution in each well from column 1 and column 2. Dilute the solution twice starting from column 2 until column 10. Inoculate 100 μL of 512 μg / mL dihydroartemisinin solution in each well from column 1 to column 11. 6 CFU / mL of Vibrio parahaemolyticus culture. Column 11 is the positive control, and column 12 is the negative control. Incubate at 28°C for 18 hours. Read the positive and negative control wells; the negative control wells should be clear, while the positive control wells should be 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 can inhibit the growth of bacteria when used alone, but the antibacterial effect of dihydroartemisinin when used alone is poor.

[0044] Example 3: Antibacterial effect of dihydroartemisinin combined with ciprofloxacin hydrochloride on four strains of Vibrio parahaemolyticus.

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

[0046] (1) Take a 96-well microplate and inoculate 100 μL of the solution into 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 at different mass ratios) were used to perform an antibacterial activity test of dihydroartemisinin and ciprofloxacin hydrochloride against Vibrio parahaemolyticus using a checkerboard method in a 96-well microplate. The cells were incubated at 28°C for 18 h.

[0047] (2) Calculate the MIC value of each well, and further calculate the graded inhibitory concentration index (FIC). The calculation formula is as follows: FIC index = MIC (ciprofloxacin hydrochloride combined) / MIC (ciprofloxacin hydrochloride alone) + MIC (dihydroartemisinin combined) / MIC (dihydroartemisinin alone). The judgment criteria of the FIC index are: FIC index ≤ 0.5 indicates synergistic effect; 0.5-1 indicates additive effect; 1-2 indicates irrelevant effect; ≥ 2 indicates 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 four strains of Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis, and the usage of ciprofloxacin hydrochloride is significantly reduced, 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 at a concentration of approximately 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 (final concentration of ciprofloxacin hydrochloride was 16 μg / mL); the third column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial liquid and 10 The cells were incubated at 28°C for 24 hours, with absorbance measured at 600 nm every hour.

[0056] 2. Experimental results: Figure 1 As shown, compared with the use of ciprofloxacin hydrochloride and dihydroartemisinin alone, there was no significant bacterial proliferation within 24 hours under the treatment of the combination of ciprofloxacin hydrochloride and dihydroartemisinin (absorbance ≈ 0.25, similar to the results of the negative control group). After 3 hours, the number of bacteria in the two experimental groups that used the two drugs alone gradually increased (absorbance gradually increased). The absorbance of the experimental group using ciprofloxacin hydrochloride alone reached 0.9 at 24 hours, which was similar to the result 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 dihydroartemisinin and 16 μg / mL ciprofloxacin hydrochloride can completely inhibit the growth of Vibrio parahaemolyticus DX190406 within 24 hours.

[0057] Example 5: Test of the bactericidal ability of a 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 at a concentration of approximately 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 The fourth column was inoculated with 100 μL of Vibrio parahaemolyticus DX190406 bacterial solution and 100 μL of 2216E liquid 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 medium. After 12 hours, the number of bacteria on the plate was counted.

[0059] 2. Experimental results: Figure 2 As shown in the figure, after treatment with a combination of 64 μg / mL dihydroartemisinin and 16 μg / mL ciprofloxacin hydrochloride, the bacterial count decreased rapidly, and 3 hours after treatment, the bacterial count was already close to 0. When the bacteria were treated with 16 μg / mL ciprofloxacin hydrochloride, there was no significant change in the bacterial count compared to the control group without any drug treatment, and it had no killing effect on the bacteria. When the bacteria were treated with 64 μg / mL dihydroartemisinin, the bacterial count decreased by less than 3 log at each time point compared to the control group without any drug treatment. 10 In summary, the combination of 64 μg / mL dihydroartemisinin and 16 μg / mL ciprofloxacin hydrochloride can completely kill Vibrio parahaemolyticus DX190406 within 3 hours, showing 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 at a concentration of approximately 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 suspension from each well of the microplate was aspirated, gently rinsed three times with phosphate-buffered saline (PBS, pH 7.2-7.4), and fixed in a 55°C drying oven for 30 minutes. 200 μL of a 1% crystal violet solution was added to each well and incubated at room temperature for 15 minutes. The crystal violet solution from each well of the microplate was aspirated, 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 biofilm biomass. The experiment was repeated three times.

[0062] 2. Experimental results: Figure 3 As shown, Vibrio parahaemolyticus DX190406 can produce biofilms. The biofilm produced within 24 hours after treatment with crystal violet produced an absorbance of 2.228±0.037. After treatment with dihydroartemisinin and ciprofloxacin hydrochloride, the strain was still able to produce biofilms. The corresponding absorbance values ​​for the biofilm produced were 2.227±0.024 and 2.184±0.025, respectively, which were not significantly different from the biofilm produced by bacteria without drug treatment. When the bacteria were treated with a combination of 64μg / mL dihydroartemisinin and 16μg / mL ciprofloxacin hydrochloride, the amount of biofilm produced, as measured by crystal violet staining, was 1.197±0.038, 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 Litopenaeus vannamei Infected with Vibrio parahaemolyticus DX190406

[0064] 1. Experimental method: Vannamei shrimp (size: about 3.5g / tail) were cultured in 200L PVC plastic barrels. The water was changed daily and 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)], an artificial infection model was established. 750 shrimp were divided into 5 groups, with 3 replicates in each group and 50 shrimp 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 shrimp were immediately fed with feed containing different drugs according to the group, including feed 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-only group were fed with feed without any experimental drugs. The feed was fed at a rate of 5% of the shrimp body weight, once every 12 hours for 7 consecutive days, and the mortality was recorded on days 0, 0.5, 1, 3, 5, and 7 after infection.

[0065] 2. Experimental results: Figure 4 As shown, after artificially infecting Litopenaeus vannamei with Vibrio parahaemolyticus DX190406, the cumulative mortality rate was 100% within 7 days. After infection, shrimp were fed 16 mg / kg of ciprofloxacin hydrochloride and 64 mg / kg of dihydroartemisinin, respectively, the shrimp still suffered severe mortality, with mortality rates of 71% and 61% on the seventh day, respectively. After feeding a feed containing 16 mg / kg of ciprofloxacin hydrochloride and 64 mg / kg of dihydroartemisinin, the cumulative mortality rate was 20% on the fifth day, and no more deaths occurred on the sixth or seventh days, indicating that the mortality rate was under control. This indicates that feeding a feed containing a combination of dihydroartemisinin and ciprofloxacin hydrochloride in a certain ratio can significantly reduce the mortality rate of Litopenaeus vannamei infected with Vibrio parahaemolyticus, achieving a good therapeutic effect.

[0066] In summary, the present invention has experimentally verified that the combined use of ciprofloxacin hydrochloride and dihydroartemisinin can increase the sensitivity of ciprofloxacin-resistant Vibrio parahaemolyticus to ciprofloxacin hydrochloride, reducing the amount of ciprofloxacin hydrochloride used in the process of killing bacteria. To further verify the combined antibacterial effect, the present invention used Vibrio parahaemolyticus DX190406 as the experimental subject and analyzed the effects of the combination of ciprofloxacin hydrochloride and dihydroartemisinin on the bacterial growth curve, time-kill curve, and biofilm formation. The results showed that the combination of ciprofloxacin hydrochloride and dihydroartemisinin can significantly kill Vibrio parahaemolyticus DX190406 and inhibit biofilm formation. Animal experimental results also showed that the combination of ciprofloxacin hydrochloride and dihydroartemisinin significantly reduced the mortality rate of white shrimp 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 persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. Use of dihydroartemisinin combined with ciprofloxacin hydrochloride in the preparation of Vibrio parahaemolyticus inhibitors, characterized in that: The mass ratio of the ciprofloxacin hydrochloride to the dihydroartemisinin is 1:2-4.

2. Use of a combination of ciprofloxacin hydrochloride and dihydroartemisinin in the preparation of a reagent for increasing the sensitivity of Vibrio parahaemolyticus to ciprofloxacin hydrochloride.

3. The use according to claim 2, 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 by: 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 combination of 64 μg / mL of dihydroartemisinin and 16 μg / mL of ciprofloxacin hydrochloride.

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