Application of quinoline in prevention and treatment of vibrio parahaemolyticus

Abstract

The application discloses application of quinoline in prevention and treatment of Vibrio parahaemolyticus. The application studies bacteriostatic activity of quinoline on Vibrio parahaemolyticus, and further explores influence of the quinoline on growth inhibition rate of Vibrio parahaemolyticus, biofilm formation, and influence of the quinoline on prevention and treatment effects of Vibrio parahaemolyticus on Neocaridina denticulata. The result proves that the quinoline can effectively prevent and treat Vibrio parahaemolyticus disease of the Neocaridina denticulata.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of quinoline in the prevention and control of Vibrio parahaemolyticus. Background Technology

[0002] Vibrio parahaemolyticus is a Gram-negative bacterium widely distributed in coastal and estuarine waters worldwide, and is one of the major foodborne pathogens in my country. Besides its negative effects on humans after consumption, Vibrio parahaemolyticus can also cause vibriosis in farmed aquatic animals, leading to acute hepatopancreatic necrosis in farmed shrimp and other aquaculture products. This often results in rapid infection and mortality in the early stages of aquaculture, causing significant losses to the aquaculture industry. Currently, research on bacterial diseases caused by Vibrio parahaemolyticus mainly focuses on using existing antibiotics. However, the extensive use of antibiotics not only damages the ecological environment but also leads to antibiotic resistance in Vibrio parahaemolyticus.

[0003] Quinoline is an important class of nitrogen-containing heterocyclic compounds with antibacterial and anticancer biological activities. It has been widely used in the production of various drugs, such as nicotinic acid and hydroxyquinoline drugs, the pesticide 8-hydroxyquinolineone, and systemic fungicides. In addition, its low toxicity, high efficiency, environmental friendliness, and easy degradation have made it a mainstream research area for highly effective, low-toxicity, and green pesticides in the field of crop disease control. To date, quinoline and its derivatives have not been used for the prevention and control of diseases in aquaculture. Summary of the Invention

[0004] One of the objectives of this invention is to provide the application of quinoline in the prevention and treatment of Vibrio parahaemolyticus, and to provide the aquaculture industry with a class of drugs that can replace antibiotics and are low in toxicity and highly effective against vibrio diseases.

[0005] The second objective of this invention is to provide an aquaculture drug preparation, the active ingredient of which is quinoline.

[0006] This invention investigated the antibacterial activity of quinoline against Vibrio parahaemolyticus, and further explored the effects of quinoline on the growth inhibition rate and biofilm formation of Vibrio parahaemolyticus, as well as the preventive and therapeutic effects of quinoline on Vibrio parahaemolyticus in New Zealand shrimp. The results showed that quinoline can effectively prevent and control Vibrio parahaemolyticus disease in New Zealand shrimp. Attached Figure Description

[0007] Figure 1 The results show the antibacterial activity of quinoline against Vibrio parahaemolyticus.

[0008] Figure 2 The results of SEM observation of the effects of quinoline on the morphology of Vibrio parahaemolyticus.

[0009] Figure 3OD of biofilm after treatment with different time and different quinoline concentrations 575 The statistical analysis results.

[0010] Figure 4 These are the results of confocal microscopy (CLMS) observations of Vibrio parahaemolyticus biofilms. Detailed Implementation

[0011] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0012] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0013] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0014] Example 1: Detection of the antibacterial activity of quinoline against Vibrio parahaemolyticus

[0015] 5 μL of Vibrio parahaemolyticus was inoculated into 5 mL of LB liquid medium (Table 1) and cultured until OD500. 600 Centrifuge at 12000 r / min and 4℃ for 5 min, collect the precipitate, wash three times with sterile ddH2O, and then add 5 mL of sterile ddH2O to prepare a bacterial suspension. Add 5 μL of the above bacterial suspension to each of 5 mL LB liquid medium containing 0, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 μg / mL quinoline. Measure the OD using Nanodrop after 0, 1, 2, 4, 8, 16, and 24 h. 600 The value of .

[0016] Table 1. Formulation of liquid LB medium (1L)

[0017]

[0018] Note: Adjust the pH to 7.0 with 1 mol / L NaOH, and bring the volume to 1 L. Autoclave at 121℃ for 20 min, and store at 4℃.

[0019] After 24 hours of incubation, quinoline completely inhibited the growth of Vibrio parahaemolyticus at concentrations of 1,000, 2,000, and 5,000 μg / mL, with inhibition rates of 21%, 35%, and 64% at concentrations of 100, 200, and 500 μg / mL, respectively. However, a concentration of 10 μg / mL had no significant effect on the growth of Vibrio parahaemolyticus.

[0020] The results are as follows Figure 1 As shown, quinoline has a significant inhibitory effect on Vibrio parahaemolyticus. After culturing for 0, 1, 2, 4, 8, 16, and 24 hours, 1000 μg / mL quinoline completely inhibited bacterial growth (IC50). 50 =234.5 μg / mL). After treatment with 0, 100, 200, and 500 μg / mL quinoline, the growth inhibition rate of Vibrio parahaemolyticus increased linearly with the presence of quinoline.

[0021] Example 2: Effect of quinoline on the integrity of Vibrio parahaemolyticus biofilm formation

[0022] Vibrio parahaemolyticus was cultured at 200 rpm in 5 mL LB liquid medium containing 0, 100, or 200 μg / mL quinoline until OD200. 600 =1.2, take 1 mL of culture medium from each of the tubes and place it into three 1.5 mL EP tubes. Centrifuge at 4 °C and 6000 r / min for 5 min to obtain bacterial cell pellets. Wash the pellets three times with sterile water. Add 200 μL of sterile water to each of the three EP tubes and mix well to prepare bacterial suspensions. Take three clean glass slides and label them "0 μg / mL", "100 μg / mL", and "200 μg / mL" respectively. Add 10 μL of bacterial suspension and 30 μL of PFA fixative to the center of each slide and mix well. Allow them to air dry. After drying, dehydrate with 65%, 75%, 85%, 95%, and 100% ethanol (15 min for each gradient treatment). Finally, observe the bacterial growth after treatment with 0, 100, and 200 μg / mL quinoline using a scanning electron microscope.

[0023] The results are as follows Figure 2 As shown, in the untreated control group, Vibrio parahaemolyticus cells aggregated and formed a biofilm, and a large amount of secretory structures were observed covering the gaps between bacterial cells. However, in bacterial cells treated with 100 and 200 μg / mL quinoline, the formation of biofilms was reduced, indicating that the compound inhibited the formation of Vibrio parahaemolyticus biofilms.

[0024] Example 3: Detection of the effect of quinoline on the biofilm formation process of Vibrio parahaemolyticus using a UV spectrophotometer

[0025] 5 μL of Vibrio parahaemolyticus was inoculated into 5 mL of LB liquid medium and cultured until OD600 = 1.2. The culture was centrifuged at 10000 rpm for 5 min at 4 °C to obtain a bacterial pellet. 5 mL of sterile water was added, and the pellet was thoroughly mixed to prepare a bacterial suspension. 30 μL of the bacterial suspension was added to each of 3 mL of LB liquid medium containing 0, 200, and 500 μg / mL quinoline, respectively. The suspensions were incubated at 28 °C, and biofilm formation was assessed at 0, 2, 4, 6, and 8 days. The supernatant was removed from the tubes, and 6 mL of 10% crystal violet solution was added to each tube. The tubes were incubated at room temperature for 1 h. The crystal violet solution was then removed from the tubes, and the tubes were washed 6 times with sterile water and air-dried at room temperature. Add 3 mL of the mixed solution (40% methanol + 10% glacial acetic acid + 50% ddH2O) to each test tube, mix thoroughly by pipetting, and then measure the OD575 value using a NanodropOne ultra-micro UV spectrophotometer. The biofilm thickness should also be measured after 4 days of static incubation.

[0026] The results are as follows Figure 3 As shown, compared with the untreated control group, the biofilms of Vibrio parahaemolyticus treated with 200 and 500 μg / mL quinoline showed significant reductions after 2, 4, 6, and 8 days of culture. 200 μg / mL quinoline reduced the biofilm formation by 55.3%, 38.3%, 18.4%, and 16.4%, respectively, while 500 μg / mL quinoline reduced it by 89.6%, 81.8%, 82.3%, and 69.6%, respectively.

[0027] Example 4: Detection of the effect of quinoline on Vibrio parahaemolyticus biofilm formation using confocal laser scanning microscopy

[0028] 1 μL of Vibrio parahaemolyticus was inoculated into 1 mL of LB liquid medium containing 0, 100, and 200 μg / mL quinoline, respectively, and incubated statically at 28°C. The culture was then centrifuged at 10,000 rpm for 5 min at room temperature, washed three times with sterile water, and 50 μL of calcium fluorescent white dye was added to each medium. The biofilms were stained for 1 min under dark conditions. Each medium was then washed three more times with sterile water, and 100 μL of sterile water was added to each medium. The mixture was thoroughly mixed to prepare a suspension, and the biofilm formation was observed using a confocal microscope.

[0029] The results are as follows Figure 4 As shown, the Vibrio parahaemolyticus biofilm in the control group exhibited a densely distributed structure. However, the bacterial biofilm content after quinoline treatment was low and showed uneven dispersion. The inhibitory effect was directly proportional to the concentration of quinoline, and almost no biofilm structure was observed after using 200 μg / mL quinoline.

[0030] Example 5: The preventive effect of quinoline on *Neomyxodon multidentatus*.

[0031] Based on data from preliminary experiments, 40 *Neomyces multidentatus* shrimp were used in each group, totaling 5 groups. The shrimp were pretreated with 200 μg / mL quinoline for 2.5 h. The tanks were cleaned, the water changed, and each group was divided into 5 portions. These portions were then immersed in different concentrations of *Vibrio parahaemolyticus* for 20 min, 40 min, 60 min, and 120 min, respectively. After immersion, the shrimp were removed and placed in clean water. Observations were continued for 2 days, and the shrimp's condition and vitality were recorded. The results are shown in Table 2.

[0032] Table 2. Analysis of the preventive effect of quinoline on Vibrio parahaemolyticus disease in New Zealand shrimp.

[0033]

[0034]

[0035] It can be seen that when *Neotrichum multidentatus* pretreated with 200 μg / mL quinoline was subsequently infected with different concentrations of *Vibrio parahaemolyticus*, quinoline had a good preventive and therapeutic effect when the OD value of *Vibrio parahaemolyticus* was below 1.5.

[0036] Example 6: Therapeutic effect of quinoline on *Neomyxodon multidentatus*

[0037] This study aimed to observe and record whether quinoline could reduce the mortality rate of *Neotrichum multidentatus* infected with *Vibrio parahaemolyticus*. *Neotrichum multidentatus* shrimp were temporarily housed in 5L glass tanks for 5 days to acclimatize to laboratory conditions for further investigation. Fifty *Neotrichum multidentatus* shrimp were placed in water containing *Vibrio parahaemolyticus* (OD value 1.9) for 1 hour. The tanks were then cleaned to remove the bacterial suspension, the water was changed, and 200 μg / mL quinoline was added to the tank for treatment at 20 min, 40 min, 60 min, and 120 min. After each treatment, the shrimp were removed and placed in clean water for continued observation and recording. The control group was treated with *Vibrio parahaemolyticus* (OD = 1.9) for 1 hour without 200 μg / mL quinoline and was directly placed in clean water for observation. The results are shown in Table 3.

[0038] Table 3. Analysis of experimental results of quinoline treatment on *Neotrichum multidentatus*.

[0039]

[0040] *The control group was not treated with 200 μg / mL quinoline and was directly placed in purified water for observation.

[0041] This shows that 200 μg / mL quinoline has a good therapeutic effect on *Neotrichum multidentatus* infected with *Vibrio parahaemolyticus*. Without quinoline treatment, the mortality rate would be 23.3%. However, after quinoline treatment, the mortality rate was significantly reduced.

Claims

1. Application of quinoline in the preparation of aquaculture products for the prevention and treatment of Vibrio parahaemolyticus.

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