Phage capable of reducing drug resistance of vibrio parahaemolyticus and application thereof

Abstract

The application belongs to the field of microbial technology application in aquaculture industry, and discloses a bacteriophage capable of reducing drug resistance of Vibrio parahaemolyticus and application thereof. A Vibrio parahaemolyticus bacteriophage is separated by the applicant in an aquaculture pond in Rizhao, Shandong, and the preservation number of the bacteriophage is CCTCC NO: CCTCC M 2024945. The Vibrio parahaemolyticus bacteriophage is applied to the biological control process of water product vibrio, has the advantages of being capable of cooperating with a disinfectant, reducing the risk of drug resistance gene transmission, having a wide lysis spectrum, high sterilization efficiency, high fermentation efficiency, low production cost, and effective effect on various vibrios accounting for a large proportion in water products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology application in aquaculture industry, and particularly relates to a bacteriophage capable of reducing drug resistance of Vibrio parahaemolyticus and application thereof. BACKGROUND

[0002] Vibrio parahaemolyticus can cause acute hepatopancreatic necrosis disease (AHPND) in shrimp, which leads to great economic losses. Shrimp larvae with AHPND can have a mortality rate of up to 100% within 10-35 days after stocking. Therefore, research on Vibrio disease prevention and control technology is of great significance to the sustainable development of aquaculture industry.

[0003] Vibrio parahaemolyticus can cause fish surface hemorrhage, tail rot, abalone foot white spot, and acute hepatopancreatic necrosis disease in shrimp. In addition, Vibrio parahaemolyticus is also an important foodborne pathogen of humans, and consumption of contaminated raw seafood can cause acute gastroenteritis with diarrhea. Vibrio parahaemolyticus isolated from various sources has high resistance to single or multiple antibiotics. Under optimal conditions, Vibrio parahaemolyticus is a fast-growing pathogen that can form a biofilm and is difficult to remove from the food chain.

[0004] Antibiotics are the main means for preventing and treating Vibrio disease in early aquaculture production, and also play a huge role in the prevention and control of Vibrio disease. However, the selective pressure caused by the widespread use of antibiotics leads to the occurrence, persistence and spread of drug-resistant bacteria, reducing the effectiveness of drug control, and also causing great hidden dangers to the environment and food safety. At present, the available antibiotic agents in aquaculture include only 13 drugs, including thiamphenicol powder, florfenicol powder, florfenicol injection, flumequine powder, enrofloxacin powder, doxycycline hydrochloride powder, vitamin C phosphate magnesium ciprofloxacin hydrochloride premix, ciprofloxacin hydrochloride berberine hydrochloride premix, neomycin sulfate powder, sulfisoxazole sodium powder, compound sulfadimidine powder, compound sulfadimidine powder, and compound sulfamethoxazole powder. However, with the increase of drug resistance, tetracycline and oxytetracycline, which can be used to treat AHPND, have been restricted.

[0005] The use of antibiotics to treat bacterial diseases can kill normal and beneficial bacteria, often leading to an imbalance in the body's bacterial flora, leading to the growth of other resistant pathogenic bacteria and the occurrence of diseases. The extensive use of antibiotics, even misuse, inevitably leads to problems such as drug residues in aquatic products and bacterial resistance. Therefore, it is urgent to develop safe and effective alternatives to antibiotics.

[0006] Bacteriophages are highly efficient and specific in their lytic properties. They can target and eliminate specific strains of harmful bacteria when combating bacterial diseases. Once living bacteria are eliminated by phages, the phages lose their activity and do not remain in the animal's body, posing no significant risk to the aquatic environment or human health. Summary of the Invention

[0007] In order to address the drug resistance of various aquatic pathogens and the economic losses to the aquaculture industry, the present invention aims to provide a bacteriophage GRNVPP42 that can reduce the drug resistance of Vibrio parahaemolyticus. The deposit number of the bacteriophage is: CCTCC NO: CCTCC M 2024945.

[0008] Another object of the present invention is to provide an application of Vibrio parahaemolyticus phage GRNVPP42.

[0009] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0010] The applicant isolated a Vibrio parahaemolyticus phage GRNVPP42 in an aquaculture pond in Rizhao, Shandong. The phage was sent to the China Center for Type Culture Collection for preservation on May 15, 2024, address: Wuhan University, Wuhan, China; classification name: Vibrio parahaemolyticus phage (Vibrio parahaemolyticus phage) GRNVPP42, preservation number is CCTCC NO: M 2024945.

[0011] A phage complex bacterial agent comprises Vibrio parahaemolyticus phage GRNVPP42 and antibiotics.

[0012] In the above-mentioned composite bacterial agent, preferably, the antibiotic is florfenicol, doxycycline or enrofloxacin.

[0013] Application of the Vibrio parahaemolyticus phage GRNVPP42 or the above-mentioned phage complex bacterial agent in the preparation of a Vibrio parahaemolyticus antibacterial agent. Application of the Vibrio parahaemolyticus phage GRNVPP42 or the above-mentioned phage complex bacterial agent in the preparation of a drug for treating or preventing Vibrio parahaemolyticus infection.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1) The phage GRNVPP42 has a high fermentation rate. At the optimal multiplicity of infection (MOI) of 0.001, the titer can reach 3.7×10 after 7 h of fermentation. 11 PFU / mL, providing a theoretical basis for industrial fermentation.

[0016] 2) The bacteriophage GRNVPP42 has a broad bactericidal spectrum, with a lysis rate of up to 95% against 60 strains of clinically pathogenic Vibrio parahaemolyticus, including 10 phage receptors.

[0017] 3) Bacteriophage GRNVPP42 can act synergistically with antibiotics. Experiments have shown that it has a certain effect on reducing the MIC of 25 out of 28 clinically isolated drug-resistant aquatic Vibrio, indicating that it can reduce the resistance of Vibrio parahaemolyticus and reduce the risk of drug residues and the spread of drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The present invention discloses a phage plaque pattern of the Vibrio parahaemolyticus phage GRNVPP42.

[0019] Figure 2 The pH stability of the Vibrio parahaemolyticus phage GRNVPP42 of the present invention is reflected.

[0020] Figure 3 This is a stability diagram of the Vibrio parahaemolyticus phage GRNVPP42 at different temperatures according to the present invention.

[0021] Figure 4 This is a diagram showing the effect of the bacteriophage GRNVPP42 of the present invention in preventing and controlling the content of Vibrio parahaemolyticus in shrimp farming water. DETAILED DESCRIPTION

[0022] To make the technical means, creative features, objectives and effects of the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art, and the reagents or materials described, unless otherwise specified, are all sourced from commercial sources.

[0023] Example 1:

[0024] Isolation and Purification of Vibrio parahaemolyticus Phage GRNVPP42

[0025] Ten water samples of 50 mL each were collected from an aquaculture pond in Rizhao, Shandong Province. The samples were centrifuged at 6000 rpm / min for 10 min, and the supernatant was taken and sterilized with a 0.22 μm filter. 5 mL of the treated supernatant was mixed with 0.5 mL of Vibrio parahaemolyticus (concentration of 1×10 7 CFU / mL) were mixed evenly, and after static adsorption for 15 minutes, the mixture was placed in a 28°C incubator and shaken at 150 rpm / min for fermentation overnight. The fermented liquid was centrifuged at 6000 rpm / min for 10 minutes, the supernatant was taken, and sterilized with a 0.22 μm filter to obtain the seed filtrate. The filtrate was diluted 10 times, and 700 μL of each concentration dilution of the filtrate was taken and mixed with 300 μL of Vibrio parahaemolyticus (concentration of 5×10 8CFU / mL or so) was mixed evenly, and after 15 min of static adsorption, the mixture was mixed evenly with 2% sodium chloride TSB semi-solid (containing 0.65% agar), and was poured on a 2% sodium chloride TSA bottom plate. After solidification, the culture dish was placed in a 37°C incubator for overnight culture. The titer of the phage in the culture dish was recorded, and a bright plaque on the culture dish was picked and shaken in 1 mL SM solution to desorb, and then was filtered through a 0.22 μm microporous filter to obtain a phage filtrate. The phage filtrate was inoculated into 5 mL of 2% sodium chloride TSB liquid medium, 100 uL of corresponding host Vibrio parahaemolyticus bacterial solution was added and mixed evenly, and was statically adsorbed for 15 min. After overnight culture at 37°C and 150 rpm / min, the supernatant was obtained by centrifugation at 5000 rpm / min for 10 min, and was filtered through a bacterial filter. The double-layer plate method was used to observe the plaque morphology. After 3-5 repeated operations, plaques with consistent shape and size were obtained.

[0026] The plaques formed by the obtained Vibrio parahaemolyticus phage GRNVPP42 and the host bacteria were bright, round, and 1 mm in diameter. Figure 1 The phage GRNVPP42 was sent to the China Center for Type Culture Collection on May 15, 2024, and the classification and naming are as follows: Vibrio parahaemolyticus phage GRNVPP42, address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M 2024945.

[0027] Example 2:

[0028] Vibrio parahaemolyticus phage GRNVPP42 lytic range experiment on different receptors

[0029] The double-layer drop method was used to determine the lytic spectrum of the phage. An EP tube containing 3 mL of 2% sodium chloride TSB was prepared, 60 strains of clinically pathogenic Vibrio parahaemolyticus containing 10 phage receptors were obtained by 37°C constant temperature and 200 rpm / min shaking for 7 h, and pure bacterial solution was obtained. A sufficient amount of TSA bottom plate was prepared, 0.9 ml of bacterial solution was mixed evenly with 6 ml of 2% sodium chloride TSB semi-solid medium (containing 0.65% agar) at about 50°C, and was slowly spread on the TSA bottom plate. After the plate medium was solidified to room temperature, 7 μL of Vibrio parahaemolyticus phage GRNVPP42 solution (titer 1×10 9 PFU / ml) was dropped on the center of the double-layer plate, and after the solution was naturally air-dried, it was placed in a 37°C constant temperature biochemical incubator for culture for about 7 h. The results were observed, and each group of experiments was repeated 3 times.

[0030] The results are as follows: Figure 2As shown, the Vibrio parahaemolyticus phage GRNVPP42 has a high lysis rate, and has a lysis effect on 57 of 60 strains of water-borne pathogenic Vibrio parahaemolyticus, with a lysis rate of 95%. It has great application potential in the prevention and killing of pathogenic bacteria with multiple phage receptors in aquaculture.

[0031] Table 1 shows the lysis range of Vibrio parahaemolyticus phage GRNVPP42 on water-borne pathogenic bacteria

[0032]

[0033] Example 3:

[0034] MIC determination of Vibrio parahaemolyticus phage GRNVPP42 on drug-resistant Vibrio

[0035] The drug resistance of 64 clinically isolated Vibrio parahaemolyticus strains was determined using the agar diffusion method recommended by the World Organization for Animal Health. First, three drug-sensitive paper strips were selected, including florfenicol, doxycycline, and enrofloxacin. Fresh bacterial liquid of the isolated strains was spread on MH agar plates, and sterile forceps were used to attach the drug-sensitive strips to the surface of the culture medium. The plates were incubated at 37°C for 24 hours. According to the determination results, the strains were divided into sensitive, intermediate (moderately drug-resistant), and drug-resistant strains. The drug resistance was statistically analyzed. It was found that 28 of the 64 Vibrio parahaemolyticus strains were multi-drug resistant bacteria (results shown in Table 2 below), with a multi-drug resistance rate of 43.75%.

[0036] Antibiotic and phage synergistic bacteriostatic experiments were conducted on the 28 multi-drug resistant Vibrio strains. The drug resistance was determined using the broth dilution method in the CLSI document. Three drugs were selected, including florfenicol, doxycycline, and enrofloxacin. Vibrio parahaemolyticus strains were recovered in TSB medium, and the OD was determined to be 0.8. Fresh bacterial liquid of the isolated strains was diluted to 1×10 5 cfu / ml for standby.

[0037] The experiment was set up in two groups, and the broth dilution method recommended by CLSI was used. Group 1 only added antibiotic drugs (all added at a first hole concentration of 512 ug / ml, then diluted by multiples, and the MIC value of the antibiotic was measured). Group 2 was a mixture of antibiotic (all added at a first hole concentration of 512 ug / ml, then diluted by multiples) and 1×10 8 pfu / ml GRNVPP42 phage 1:1 (volume ratio). The final MIC value of the antibiotic in the mixture was determined. Each group had three replicates, and the incubator was set at 37°C overnight.

[0038] Table 2 shows the minimum inhibitory concentration of antibiotics when used alone and when used in combination with phage. For each strain, group 1 is the minimum inhibitory concentration of Vibrio parahaemolyticus when antibiotics are used alone, and group 2 is the minimum inhibitory concentration of Vibrio parahaemolyticus when antibiotics are used in combination with phage GRNVPP42.

[0039] The results show that the Vibrio parahaemolyticus phage GRNVPP42 can effectively reduce the resistance of multidrug-resistant Vibrio parahaemolyticus to antibiotics, reduce the use of antibiotics, enhance the bactericidal effect, and reduce the risk of bacterial resistance transmission.

[0040] Table 2 Drug sensitivity test of three antibiotics and phage combinations

[0041]

[0042]

[0043] Example 4:

[0044] Determination of the titer of Vibrio parahaemolyticus phage GRNVPP42 at different multiplicity of infection

[0045] Prepare the host bacteria of Vibrio parahaemolyticus phage GRNVPP42, Vibrio parahaemolyticus GRNVP006, prepare an EP tube filled with 3mL 2% sodium chloride TSB, pick a single colony of Vibrio parahaemolyticus GRNVP006 and inoculate it into the culture medium, shake and culture at 37℃ for 8h to obtain the host bacteria liquid. Inoculate the liquid into 100mL TSB medium at an inoculum volume of 1% and wait until Vibrio parahaemolyticus GRNVP006 grows to the initial logarithmic phase (concentration 1×10 8 CFU / mL) were then inoculated with the Vibrio parahaemolyticus phage GRNVPP42 in the culture medium at varying multiplicity of infection (MOI; MOI = number of phages / number of bacteria), with triplicate experiments per group. Cultures were shaken at 150 rpm in a 28°C shaker. A portion of the culture medium was removed every hour and centrifuged at 8000 rpm for 10 minutes. The supernatant was collected and the phage titer was determined. The culture was terminated after 12 hours.

[0046] The experimental results are shown in Table 1 below. When MOI = 0.001, the titer can reach 3.7×10 11 PFU / mL, providing a theoretical basis for industrial fermentation.

[0047] Table 3 Optimal multiplicity of infection of bacteriophage GRNVPP42

[0048] Phage concentration (PFU / mL) Host bacteria concentration Multiplicity of infection Titer (PFU / mL) <![CDATA[1.0×10 9 ]]> <![CDATA[1.0×10 8 ]]> 10 <![CDATA[5.8×10 8 ]]> <![CDATA[1.0×10 8 ]]> <![CDATA[1.0×10 8 ]]> 1 <![CDATA[4.9×10 9 ]]> <![CDATA[1.0×10 7 ]]> <![CDATA[1.0×10 8 ]]> 0.1 6.2 x 10 9 ]]> <![CDATA[1.0×10 6 ]]> <![CDATA[1.0×10 8 ]]> 0.01 <![CDATA[2.5×10 10 ]]> 1.0 x 10 5 ]]> <![CDATA[1.0×10 8 ]]> 0.001 3.7 x 10 11 ]]> <![CDATA[1.0×10 4 ]]> <![CDATA[1.0×10 8 ]]> 0.0001 <![CDATA[9.1×10 10 ]]>

[0049] Example 5:

[0050] pH Stability Test of Vibrio parahaemolyticus Phage GRNVPP42

[0051] Take sterile bacterial bottles and add 9 mL of 2% sodium chloride TSB medium with different pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. Then place the above bacterial bottles in a constant temperature water bath at 25°C. After the temperature is balanced, add 1 mL of pure phage culture solution (initial titer: 5×10 9 PFU / mL) and allowed to stand at 25°C for 15 minutes. After 2 hours, samples were taken and appropriately diluted, and the phage titer was determined using the double-layer plate method. Duplicate culture tubes were cultured at each point and the average value was obtained. The experiment was repeated three times.

[0052] The results are as follows Figure 2 As shown, the titer of Vibrio parahaemolyticus phage GRNVPP42 did not change significantly when treated in the pH range of 4-12 for 2 h, and changed slightly when treated in the pH range of 3 for 2 h.

[0053] Example 6:

[0054] Temperature Stability Test of Vibrio parahaemolyticus Phage GRNVPP42

[0055] Take several sterile 50mL centrifuge tubes, add 45mL TSB to each tube and place it in a constant temperature water bath at the corresponding temperature. After the temperature is balanced, add 5mL of phage pure culture solution (initial titer: 5×10 9 PFU / mL) were incubated at 40°C, 50°C, 60°C, and 70°C for 20, 40, and 60 minutes. After incubation, the sample tubes were removed and immediately cooled in an ice bath. After appropriate dilution, the phage titer was determined using the double-layer plate method. Duplicate cultures were performed at each point, and the average value was calculated. The experiment was repeated three times.

[0056] The results are as follows Figure 3 As shown, the Vibrio parahaemolyticus phage GRNVPP42 in the experimental group can survive more easily at temperatures below 60°C and can be stored for a long time at temperatures below 40°C.

[0057] Example 7:

[0058] Bacteriophage GRNVPP42 for the control of Vibrio parahaemolyticus in shrimp aquaculture water

[0059] The experiment was conducted at a whiteleg shrimp farm in Rizhao City, Shandong Province. The shrimp weighed an average of 7±0.5g. The water temperature was 26°C and the salinity was 25%. All experimental groups were fed at a fixed time and place.

[0060] The experiment was divided into 4 groups:

[0061] Group 1 was the blank control group, without any treatment;

[0062] Group 2 was the doxycycline group, which was added at 0.1%;

[0063] The third group was the bacteriophage GRNVPP42 group, using a final concentration of 1×10 4 pfu / ml. (i.e. dilute according to the volume of aquaculture water so that the final concentration of phage is 1×10 4 pfu / ml).

[0064] In the 4th group of doxycycline and phage GRNVPP42, doxycycline was added at 0.05% and the final concentration was 1×10 4 pfu / ml of bacteriophage GRNVPP42;

[0065] Three pools with similar breeding density and conditions were randomly selected for each group. Before the experiment, the Vibrio content was tested by coating TCBS plates and the initial number of Vibrio was recorded. After three consecutive days of use according to the above groups, the Vibrio content was counted according to the same counting rules. After the end of the medication, pool water samples were taken every 16 hours for a total of five tests, and the Vibrio content in each group was counted.

[0066] The results are as follows Figure 4 , it can be seen that Treatment 2 was initially effective for a short period of time, but then the Vibrio population rebounded, reaching the same level as the control group around 48 hours. However, throughout the experiment, the Vibrio population in the phage-treated group maintained a downward trend. This suggests that GRNVPP42 phage can be used as an effective biological agent to control Vibrio in actual aquaculture.

Claims

1. A phage complex agent, which is a Vibrio parahaemolyticus phage ( Vibrio parahaemolyticus phage ) GRNVPP42 and an antibiotic, the phage deposit number is CCTCC NO: M 2024945, and the antibiotic is florfenicol, doxycycline or enrofloxacin.

2. Use of the bacteriophage complex bacterial agent according to claim 1 in the preparation of a Vibrio parahaemolyticus antibacterial agent.

3. Use of the phage complex bacterial agent according to claim 1 in the preparation of a medicament for treating or preventing Vibrio parahaemolyticus infection.

Images