Vibrio parahaemolyticus bacteriophage with wide lysis spectrum, bacteriophage composition thereof and application of bacteriophage composition

Abstract

The invention belongs to the technical field of bacteriophages, and discloses a wide-splitting-spectrum vibrio parahaemolyticus bacteriophage, a bacteriophage composition and application thereof, the wide-splitting-spectrum vibrio parahaemolyticus bacteriophage is named as vibrio bacteriophage PG422, the preservation number is CGMCC No.46164, and the wide-splitting-spectrum vibrio parahaemolyticus bacteriophage is preserved in China General Microbiological Culture Collection Center on August 16, 2024. The bacteriophage PG422 has broad-spectrum bactericidal ability to vibrio, has excellent splitting performance to various different pathogenic vibrio, such as vibrio parahaemolyticus, vibrio alginolyticus, vibrio harveyi and vibrio cholerae, is safe to use, can be used as an active component to prepare a pharmaceutical preparation, a water purifying agent and a disinfectant, is used in various applications of preventing and controlling vibrio infection, and has broad application prospects. The morbidity of vibriosis of aquatic products such as shrimp seeds is effectively controlled, and the yield of the aquatic products is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriophages, in particular to a Vibrio parahaemolyticus phage with a wide lysis spectrum, a phage composition thereof and application thereof. Background Art

[0002] Vibrio disease is one of the most serious infectious diseases affecting aquatic animals in estuarine and marine environments worldwide. A variety of Vibrio pathogens can cause various aquatic Vibrio diseases. Common Vibrio pathogens include Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus and Vibrio cholerae.

[0003] Among them, Vibrio parahaemolyticus is a Gram-negative, halophilic, thermophilic bacterium that thrives in warm climates in the ocean or estuary. Vibrio parahaemolyticus can survive in marine sediments and re-enter the water body when the temperature rises, thereby increasing the chance of infection by Vibrio parahaemolyticus. Vibrio parahaemolyticus is currently reported to be the most common pathogenic Vibrio that can cause human infection besides Vibrio cholerae. Eating food contaminated by Vibrio parahaemolyticus can lead to gastrointestinal infection. In addition, Vibrio parahaemolyticus is also a very important pathogenic bacterium for aquatic animals. In the context of the continuous development of intensive aquaculture, acute hepatopancreatic necrosis of shrimp caused by Vibrio parahaemolyticus has caused a large number of deaths in farmed shrimp. The bacteria can also cause sea cucumber rot, vibriosis in shellfish and large yellow croaker, causing huge economic losses to the aquaculture industry.

[0004] Among the Vibrio species, Vibrio harveyi is generally prevalent in summer, and its main targets of infection are shrimps, black grouper, etc. The symptoms caused by Vibrio harveyi vary, and generally include anorexia, bulging eyes, and bleeding on the body surface. In more severe cases, there will be symptoms such as loss of scales, skin and muscle ulcers, and spleen swelling.

[0005] Vibrio alginolyticus can cause disease in large yellow croaker, grouper, shellfish, etc., and can even cause white spot disease in Japanese shrimp and coral bleaching; the main symptoms of diseased fish are decreased motility, gray skin, loose scales, ulcers on the body surface, etc., and after dissection, congestion and water filling symptoms in the liver and digestive tract will be found. Vibrio cholerae can cause diseases and death in river crabs, grass carp, shrimps, etc.; Vibrio cholerae disease mainly causes eye rot in shrimps and crabs during the grow-out period.

[0006] At present, cephalosporins, tetracyclines, and quinolones are still the first choice antimicrobial agents for treating severe cases of vibriosis. The aquaculture industry also relies on the use of antibiotics to prevent and treat bacterial infections in fish and other animals; however, the widespread use of antibiotics in clinical, agricultural, and aquaculture has led to the development of multiple antibiotic resistance in Vibrio parahaemolyticus, and drug-resistant bacteria may be spread through food and other means, posing a potential threat to public health. In addition, the problem of bacterial resistance will also give rise to the emergence of multi-drug resistant "super bacteria". In 2020, the Ministry of Agriculture and Rural Affairs and 10 other ministries and commissions jointly issued the "Several Opinions on Accelerating the Green Development of the Aquaculture Industry", which clearly pointed out the implementation of a reduction in the use of aquaculture drugs. Since 2021, the development of alternative products to antibiotics has gradually become the focus of attention in the aquaculture industry.

[0007] Different from traditional probiotics, natural plant extracts and other alternative products, bacteriophages are the only antibiotic alternatives with direct bactericidal effects. After combining with the host bacteria, bacteriophages rapidly proliferate and lyse them in the host body, and they are green and safe to use. Based on their application advantages, bacteriophages are bound to become the development trend and research hotspot of alternative antibiotics in the future. Bacteriophages are viruses that can invade bacteria and other microorganisms, and they decompose bacterial cell walls by encoding endolysins. Bacteriophages must parasitize living bacteria to survive and replicate and reproduce, but they can specifically infect host bacteria and cause the death of host bacteria. Phage therapy has the following potential advantages over antibiotic therapy: First, phages are highly host-specific and only infect the corresponding pathogens, which reduces their threat to other bacteria in the environment and animals, and the therapeutic effect is not affected by bacterial resistance; second, phages multiply rapidly in an exponential form, and they can multiply exponentially with the proliferation of host bacteria and play a role in the entire process of bacterial infection, which is a very important advantage; in addition, phages are host-dependent and only act at the site of bacterial infection, and will be degraded or quickly metabolized after lysing the target bacteria; finally, phages are widely present in the natural environment, with very rich sources, and are easy to separate from the host bacterial habitat, with a short R&D cycle and low cost. Therefore, the study of phages provides new ideas for the prevention and treatment of Vibrio infections, and is of great significance for the development of new approaches and new preparations for the prevention and treatment of Vibrio diseases.

[0008] Based on the above advantages of bacteriophages, various bacteriophages have been gradually developed as pharmaceutical preparations or environmental disinfection and have been applied in some fields, including some Vibrio phages. For example, the patent with publication number CN 111172119B applied by the applicant in 2020 discloses a Vibrio parahaemolyticus phage PG07, which can effectively prevent and treat Vibrio parahaemolyticus infection.

[0009] However, the lysis spectrum of currently available Vibrio phages is relatively narrow, and the types of Vibrio that can be effectively lysed are relatively limited. There is no phage that has high lysis performance against multiple Vibrio species. Existing phage products are difficult to be widely used in infections with various types of Vibrio pathogens; therefore, the existing technology needs to be further improved. Summary of the invention

[0010] In response to the above problems, the present invention provides a broad-lytic spectrum Vibrio phage PG422, a phage composition thereof and applications thereof. The broad-lytic spectrum Vibrio parahaemolyticus phage PG422 has a high efficiency in lysing multiple different types of Vibrio, and can be used as an active ingredient to prepare pharmaceutical preparations, water purifiers, and aquatic product disinfectants, and used in various applications for preventing and controlling Vibrio infections.

[0011] To solve the above problems, this application provides the following technical solutions:

[0012] In a first aspect, the present application provides a broad lytic spectrum Vibrio phage, which is named Vibrio phage PG422, and its deposit number is CGMCC No.46164.

[0013] The phage was deposited at the General Microbiology Center of the China Culture Collection Administration on August 16, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46164.

[0014] It was observed under an electron microscope that the Vibrio phage PG422 has a polyhedral head structure and a contractile tail. The head is 50-53nm in size and the tail is about 190nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage PG422 conforms to the characteristics of the Long-tailed Bacteriophage family and belongs to the Long-tailed phage.

[0015] Vibrio phage PG422 is a phage with a wide lysis spectrum and strong lysis performance. Experiments show that its total lysis rate for 200 strains of Vibrio from different sources and multiple species preserved in the laboratory is 92.00%; among them, the lysis rates for Vibrio parahaemolyticus (61 strains), Vibrio alginolyticus (63 strains) and Vibrio harveyi (36 strains) are relatively high, all above 90.00%; and the lysis rate for Vibrio cholerae is above 80.00%. Compared with the existing Vibrio cholerae phage vB-VchS-PR02 (see patent publication number CN 111363723A for details), the lysis rate (85.00%) of Vibrio phage PG422 for 40 strains of Vibrio cholerae is significantly higher than the total lysis rate (75.00%) of vB-VchS-PR02. Compared with the existing Vibrio parahaemolyticus phage PG07 (see patent with publication number CN 111172119A), the total lysis rate of PG422 to 61 strains of Vibrio parahaemolyticus (95.08%) is significantly higher than the total lysis rate of PG07 (85.25%). Compared with the existing Vibrio alginolyticus phage PJ115 (see patent with publication number CN 113201505A), the total lysis rate of PG422 to 63 strains of Vibrio alginolyticus (93.65%) is significantly higher than the total lysis rate of PJ115 (85.71%), and the total lysis rate of PG422 to 36 strains of Vibrio harveyi (91.67%) is significantly higher than the total lysis rate of PJ115 (77.78%). It can be seen that the Vibrio phage PG422 has excellent lysis performance for a variety of Vibrio hosts, a wide lysis spectrum, and a wide range of applications.

[0016] The phage PG422 also has strong temperature resistance and acid and alkali resistance. Experiments have shown that the phage PG422 maintains its original activity after being exposed to 40℃~60℃ for 1 hour, and still maintains a high activity after being exposed to 70℃ for 1 hour. In addition, the activity of the phage is stable in the pH range of 4.0~10.0, and its titer is 10 after being exposed to pH 3.0 for 3 hours. 8 PFU / mL; it maintained a high activity after 3 hours at pH 11, indicating that the phage has strong acid and alkali resistance.

[0017] In the present application, phage PG422 includes mutants with point mutations, deletion mutations or addition mutations having a homology higher than 98% or 99% and maintaining substantially the same bactericidal activity. Since phages are very susceptible to mutations during replication, mutants of the above phages are also within the scope of protection claimed in the present application. The sequence of phage PG422 can be obtained by sequencing the biological materials deposited according to the present invention by known methods. For those skilled in the art, it does not require creative labor to screen out mutants with extremely similar properties according to the phage provided by the present invention.

[0018] In a second aspect, the present application also provides a phage composition, which includes the aforementioned Vibrio parahaemolyticus phage PG422.

[0019] In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in lysis spectrum of different phages, and complement each other's advantages, the above-mentioned Vibrio phage PG422 can be used in combination with other phages, such as PJ115 (see patent publication number CN 113201505A), which is also a Vibrio phage, to further broaden the lysis spectrum, improve the killing of Vibrio in water or food, and better prevent and treat Vibrio infections such as shrimp seedlings.

[0020] Optionally, the phage composition further includes: one or more of: Vibrio cholerae phage vB-VchS-PR02, Vibrio parahaemolyticus phage PG07, and Vibrio alginolyticus phage PJ115.

[0021] In a third aspect, the present application also provides the use of the above-mentioned Vibrio phage or the aforementioned phage composition in the preparation of pharmaceutical preparations, water purifiers, and aquatic product disinfectants for preventing and treating aquatic product diseases caused by Vibrio infection.

[0022] The control includes prevention and treatment. The term "prevention" herein refers to all actions including suppressing or delaying the disease by administering the composition. The term "treatment" herein refers to all actions including improving or ameliorating the disease by administering the composition.

[0023] Optionally, in the application, the Vibrio includes: Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cholerae. Preferably, the diseases caused by the Vibrio infection include Vibrio parahaemolyticus disease, Vibrio alginolyticus disease, Vibrio harveyi disease, Vibrio cholerae disease and the like.

[0024] In a fourth aspect, the present application further provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Vibrio parahaemolyticus phage PG422 or the aforementioned phage composition.

[0025] Preferably, the content of bacteriophage PG422 in the reagent is not less than 10 8 PFU / mL. Further preferably, the bacteriophage pharmaceutical preparation also includes other antibacterial or bactericidal active ingredients,

[0026] The application method of the bacteriophage drug preparation is: using the bacteriophage or its combination as a therapeutic drug to act on shrimp fry or water by various methods such as spraying, mixing, etc. The bacteriophage drug preparation is in the form of liquid dosage form, powder dosage form or solid dosage form, but is not limited to the above three dosage forms.

[0027] Optionally, the bacteriophage drug preparation further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not eliminate the biological activity and properties of the administered active ingredient.

[0028] In a fifth aspect, the present application further provides a feed additive, the active ingredient of which includes: the aforementioned Vibrio parahaemolyticus phage or the aforementioned phage composition. The feed additive containing the Vibrio phage PG422 can be added to feed for mixing and then fed to aquatic products.

[0029] In a sixth aspect, the present application also provides a water purifier, the active ingredients of which include the aforementioned Vibrio parahaemolyticus phage or the aforementioned phage composition; preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in water environments. Preferably, the titer of the Vibrio phage is 5×10 4 PFU / mL or above.

[0030] The present application also provides an application of the water purifier, wherein the application method is: directly adding the aforementioned Vibrio parahaemolyticus phage or the aforementioned phage composition to the water; preferably, the concentration of the phage in the water is not less than 5×10 4 PFU / mL.

[0031] The water purifier is used for purifying Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi or Vibrio cholerae in water.

[0032] The use of the water purifier can control the reproduction of Vibrio in aquaculture water and prevent the spread of Vibrio from the source. The purifier can be used to replace antibiotics or traditional disinfection products, and it will not cause harm to humans and animals, and also avoids the pollution of antibiotics to the environment.

[0033] In a seventh aspect, the present application also provides a disinfectant for aquatic products, the active ingredients of which include the aforementioned Vibrio parahaemolyticus phage or the aforementioned phage composition. Preferably, the disinfectant for aquatic products also contains other active ingredients for inhibiting or eliminating bacteria in aquatic products.

[0034] The above disinfectant can be used to treat fresh aquatic products by spraying or soaking to inhibit or eliminate Vibrio in the aquatic products, thereby preventing the human body from being infected through the spread of the aquatic products. Preferably, the concentration of bacteriophage is 10 7 PFU / mL or above.

[0035] The present invention has the following beneficial effects:

[0036] 1. The present invention provides a Vibrio parahaemolyticus phage PG422 with a wide lysis spectrum and strong high-temperature storage stability. The phage not only has a high titer, but also exhibits a strong lysis ability against Vibrio, and can lyse a variety of different Vibrio such as Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio hallii, showing the characteristics of a wide lysis spectrum; in addition, the above-mentioned Vibrio phage can be stored at a high temperature of 37°C for at least 1 month, has strong activity stability, and can ensure a longer duration of effectiveness when used in actual field applications to prevent and control Vibrio infections.

[0037] 2. Based on its excellent biological characteristics, bacteriophage PG422 can be prepared into pharmaceutical preparations, disinfectants and water purifiers for the prevention and treatment of Vibrio. It can not only effectively prevent the spread of various Vibrio diseases such as Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio hominis, but also more effectively control the incidence of Vibrio diseases in shrimp seedlings and increase the output of aquatic products.

[0038] 3. The bacteriophage PG422 in the present invention is isolated from natural water bodies and has the characteristics of safe use, green and environmental protection, and will not cause pollution to water bodies. In addition, the bacteriophage has a high explosive volume and strong reproductive ability, and has application advantages in industrial production, which can effectively reduce the production cost of the bacteriophage-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a plaque image of bacteriophage PG422;

[0040] Figure 2 This is an electron microscope image of bacteriophage PG422;

[0041] Figure 3 The results of the thermal stability test of bacteriophage PG422 are shown;

[0042] Figure 4 The pH stability test results of bacteriophage PG422;

[0043] Figure 5 is the one-step growth curve of bacteriophage PG422;

[0044] Figure 6 The effect of bacteriophage PG422 in water purification test;

[0045] Figure 7 This is the result of applying the Vibrio phage PG422 in Example 12 to the disinfection of aquatic products. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0047] Example 1 Isolation and Screening of Vibrio parahaemolyticus Phage

[0048] 1. Resuscitation culture of host bacteria and preparation of proliferation medium

[0049] Eight strains of Vibrio parahaemolyticus preserved in our laboratory were selected, and the Vibrio freezing solution was dipped into a sterilized inoculum stick and streaked on TCBS medium for recovery. The culture was incubated at 37°C incubator for 16-18 hours to obtain a single colony; a single colony was picked and inoculated into 5 mL of 2216E broth, and cultured at 37°C with shaking at 170 rpm / min for 16-24 hours to obtain a fresh Vibrio solution.

[0050] 2. Phage isolation and purification

[0051] Take an appropriate amount of sewage sample collected from Qingdao area, add it to an appropriate amount of 2216E culture medium, add 10 strains of Vibrio, put the mixed solution into 37℃170rpm / min shaking culture for 12h, centrifuge at 11000rpm for 5min, and then filter it with a 0.22μm sterile microporous filter membrane to obtain phage proliferation liquid;

[0052] The phage proliferation liquid was diluted at a 10-fold ratio, and appropriate gradient phage dilution liquid was mixed with 8 strains of Vibrio at a 1:1 ratio. After incubation at 37°C for 5 minutes, 200 μL of the mixture was placed on the upper agar, and after mixing, it was quickly poured onto the 2216E lower plate, shaken and placed flat until the culture medium solidified, and placed in a 37°C incubator for inverted culture for 4 to 6 hours to obtain a double-layer plate with plaque formation.

[0053] Pick a single plaque from the double-layer agar medium where plaques are formed, and place it in 1 mL of PBS and culture it in a constant temperature shaker at 37°C, 170 rpm / min for about 30 minutes to obtain a phage extract. Take the phage extract and mix it evenly with the corresponding plaqued Vibrio proliferation liquid at a ratio of 1:1, incubate at 37°C for 5 minutes, draw 200 μL and place it on the upper agar, mix it and quickly pour it on the 2216E lower plate, shake it evenly and place it flat until the culture medium solidifies, place it in a 37°C incubator and culture it upside down for 4 to 6 hours, and then obtain a double-layer plate with plaques formed again. Use sterilized tweezers to pick a single plaque from the double-layer culture medium where plaques are formed and place it in 1 mL of PBS, and culture it in a constant temperature shaker at 37°C, 170 rpm / min for about 30 minutes to obtain a phage extract. Repeat the above steps 3 times to obtain the purified phage extract.

[0054] 3. Phage titer determination

[0055] Take an equal amount of purified phage extract and host bacteria proliferation liquid in 5 mL of liquid 2216E medium, and culture at 37°C, 170 rpm / min with shaking until the liquid becomes clear. Centrifuge the clear liquid at 11000 rpm for 10 min, take the supernatant, and filter it with a 0.22 μm sterile microporous filter membrane to obtain phage proliferation liquid, and use the double-layer plate method to determine the titer of the newly isolated phage.

[0056] 5. Screening of 37℃ Temperature-resistant Vibrio phages

[0057] The newly isolated Vibrio parahaemolyticus phages were adjusted to a uniform initial concentration (5.00×10 8 PFU / mL), and after being stored in a high temperature environment at 37°C for 1 month, the phage titer change was determined. At the same time, other existing phages were selected for synchronous high temperature detection to compare the phage titer changes. Other existing phages are: Vibrio cholerae phage PR02 (see patent publication number CN111363723A), Vibrio parahaemolyticus phage PG07 (see patent publication number CN111172119A).

[0058] 6. Experimental results and analysis

[0059] 6.1 According to the above experimental method, 8 Vibrio isolates were used to screen 3 Vibrio phages, numbered PG421, PG421 and PG423. These 3 Vibrio phages all formed clear plaques on the double-layer culture medium plate, with clearly visible edges and a diameter of about 0.5mm to 1.0mm; the titers of the 3 phages were 5.00×10 8 PFU / mL, 8.70×10 9 PFU / mL, 3.30×10 10 PFU / mL.

[0060] 6.2 The results of the 37°C temperature stability test are shown in Table 1. From the results, it can be seen that among all the experimental phages, Vibrio phage PG422 is the best Vibrio parahaemolyticus phage with the best comprehensive performance such as lysis rate and high temperature stability. After being stored in a high temperature environment at 37°C for one month, the titer remained basically unchanged and maintained at 3.20×10 8 , while other phages were basically inactivated or mostly inactivated.

[0061] The good high-temperature stability of the Vibrio phage PG422 provides a guarantee for later field clinical applications, avoids the influence of high temperature in the natural environment, and also effectively reduces the cost of use and storage.

[0062] Table 1 Stability test of three newly isolated Vibrio phages

[0063]

[0064] Example 2 Morphological Observation and Identification of Vibrio parahaemolyticus Phage PG422

[0065] 1. Experimental methods

[0066] Take 20 μL of phage sample and drop it on a copper mesh with a carbon-coated film. Wait for it to precipitate naturally for 15 minutes. After blotting it with filter paper, stain it with 2% (W / V) phosphotungstic acid (PTA) for 1 to 2 minutes. After blotting it with filter paper, observe and photograph it under a transmission electron microscope.

[0067] 2. Experimental results and analysis

[0068] like Figure 2 As shown, the bacteriophage PG422 has a polyhedral head structure and a contractile tail. The head has a diameter of 50-53nm and the tail is about 190nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the phage morphology of the present application conforms to the characteristics of the long-tailed bacteriophage family and belongs to the long-tailed phage.

[0069] Example 3 Whole genome analysis of Vibrio parahaemolyticus phage PG422

[0070] The genome of bacteriophage PG422 was extracted, and whole genome sequencing and sequence analysis were performed. The results are as follows:

[0071] (1) The PG422 genome is 76765 bp in length, with a G+C content of 48.72% and an A+T content of 51.28%. The contents of C, G, A, and T are 25.74%, 22.98%, 23.54%, and 27.74%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 109 open reading frames (ORFs). Among these 109 open reading frames (ORFs), there are 8 structural proteins, mainly including the structure and packaging proteins of the phage (capsid protein, capsid and scaffold protein, tail spike, structural protein, phage fiber protein, topoisomerase large subunit, etc.), phage lysis-related proteins (phage lysin), DNA replication and modification-related proteins (DNA polymerase, DNA helicase, DNA binding protein, endonuclease, etc.), and other functional proteins (phage repressor protein, dual infection immunity protein, etc.). At the same time, among the 109 ORFs, 98 start codons were ATG, 8 start codons were GTG, 1 start codon was GCC, 1 start codon was CCG, and 1 start codon was CGT. The software tRNAscan-SE analyzed that the genome contained tRNA genes. The online tool CGE server analyzed that the genome did not contain drug resistance genes and virulence genes. The PHASTER analysis showed that the genome did not contain lysogeny-related genes.

[0072] (2) In the genome of bacteriophage PG422, the gene sequence of the tail fiber protein related to phage host recognition is shown in Sequence 1 of the sequence listing; the sequence of the DNA polymerase gene is shown in Sequence 2 of the sequence listing; the sequence of the lysin gene related to cleavage ability is shown in Sequence 3 of the sequence listing. The relevant information is specifically shown in Table 2 below.

[0073] Table 2 Gene sequence information

[0074]

[0075] Example 4 Determination of the Optimal Multiplicity of Infection (MOI) of Vibrio parahaemolyticus Phage PG422 against Vibrio

[0076] 1. Experimental methods

[0077] Pick a single colony of Vibrio parahaemolyticus PG422 and inoculate it into 5 ml 2216E liquid culture medium, culture at 37°C and 170 rpm for 16 to 19 hours to obtain bacterial solution, and determine the concentration of the Vibrio by pouring method; the phage titer was detected by double-layer plate method. The phage titer and host bacteria concentration were added to the test tube containing 5 mL 2216E liquid culture medium in the ratio of 1:1, 0.1:1, 0.01:1, 0.001:1, 0.0001:1, 0.00001:1, 0.000001:1, 0.0000001:1, and 0.0000001:1, respectively, and cultured at 37°C and 170 rpm for 4 to 12 hours, until the liquid changed from turbid to clear. Take an appropriate amount of clear liquid and centrifuge it at 11000 rpm / min for 10 min. Take the supernatant and filter it through a 0.22 μm sterile microporous filter membrane. Determine the phage titer in the filtrate by the double-layer plate method. The MOI (number of phages / number of bacteria) with the highest phage titer is the optimal multiplicity of infection of the phage.

[0078] 2. Experimental results

[0079] The results are shown in Table 3. When the MOI was 0.001, the PG422 titer reached a maximum of 1.07×10 11 PFU / mL, therefore, the optimal MOI of phage PG422 is 0.001, and this conclusion provides data reference for the later large-scale industrial production of products. During production, phage PG422 can achieve the highest reproduction yield with a small initial inoculation amount, which is conducive to its large-scale industrial production.

[0080] Table 3 The titer of Vibrio phage PG422 at different infection multiplicity

[0081] MOI PG422 (PFU / mL) Host bacteria (CFU / mL) PG422 proliferation titer (PFU / mL) 1 <![CDATA[10 9 ]]> <![CDATA[10 9 ]]> <![CDATA[1.20×10 9 ]]> 0.1 <![CDATA[10 8 ]]> <![CDATA[10 9 ]]> <![CDATA[9.75×10 9 ]]> 0.01 <![CDATA[10 7 ]]> <![CDATA[10 9 ]]> <![CDATA[4.70×10 10 ]]> 0.001 <![CDATA[10 6 ]]> <![CDATA[10 9 ]]> <![CDATA[1.07×10 11 ]]> 0.0001 <![CDATA[10 5 ]]> <![CDATA[10 9 ]]> <![CDATA[4.30×10 10 ]]> 0.00001 <![CDATA[10 4 ]]> <![CDATA[10 9 ]]> <![CDATA[3.60×10 10 ]]>

[0082] Example 5 Temperature stability of Vibrio parahaemolyticus phage PG422

[0083] 1. Experimental methods:

[0084] 2.00×10 10 PFU / mL Vibrio phage PG422 proliferation solution was placed at 40℃, 50℃, 60℃, 70℃, and 80℃, and three parallel samples were made at each temperature. After being placed for 20min, 40min, and 60min, the samples were immediately cooled in an ice bath after the reaction ended, and then the phage titer at different temperatures was detected by the double-layer plate method. The PG422 phage thermal stability curve was drawn with temperature as the horizontal axis and the logarithm of the phage titer as the vertical axis.

[0085] 2. Experimental results and analysis

[0086] like Figure 3The results showed that the titer of phage PG422 was relatively stable after 60 min at a temperature between 40°C and 60°C. After 60 min at 70°C, the titer of the phage was maintained at 9.25×10 6 PFU / mL; after being exposed to 80℃ for 20min, it still maintained a certain activity. It can be seen that Vibrio phage PG422 has strong heat resistance and can adapt to higher temperature environments.

[0087] Example 6 pH stability of Vibrio parahaemolyticus phage PG422

[0088] 1. Experimental methods

[0089] Take three sterile test tubes and add 4.5 mL of NB broth with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Then place the test tubes in a 37°C water bath. After the temperature stabilizes, add 500 μL of 2.00×10 10 PFU / mL of phage PG422 proliferation solution, mix well and act in a 37℃ water bath for 1h, 2h, and 3h. After the action is over, immediately add an appropriate amount of 1mol / L HCl or NaOH to the mixture to make the pH value of the mixture about 7, dilute it 10 times, take a suitable dilution gradient to determine the titer, and set 3 parallel samples for each pH value test tube. Draw the phage pH stability curve with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0090] 2. Experimental results and analysis

[0091] The results are as follows Figure 4 As shown in the results, it can be seen that the titer of phage PG422 is maintained at 10 in the pH range of 4.0 to 10.0. 10 PFU / mL, stable activity; at pH 3.0 for 3h, the potency is 10 8 PFU / mL; after being exposed to pH 12.0 for 1 hour, the phage still maintained a certain activity; therefore, phage PG422 has strong stability in a wide pH range and can adapt to a certain range of strong acid and strong alkaline environments.

[0092] Example 7 One-step growth curve of Vibrio parahaemolyticus phage PG422

[0093] 1. Experimental methods

[0094] Take 1 mL of phage PG422 proliferation fluid and Vibrio parahaemolyticus host bacteria proliferation fluid with a multiplicity of infection of 10, mix thoroughly (start timing at this time), incubate at 30°C for 5 min, centrifuge at 13000 rpm for 30 s, use a micropipette to remove the supernatant as much as possible, wash once with 5 mL of 2216E liquid culture medium (centrifuge at 13000 rpm / min for 30 s), and discard the supernatant. Use preheated 2216E liquid to suspend the precipitate (total volume is 5 mL) and mix thoroughly, quickly place it in a 37°C shaker and culture it at 170 rpm / min, take out 150 μL at time 0 and every 5 minutes, centrifuge it at 10000 rpm for 1 minute, make a 10-fold dilution with NB broth, and use the double-layer plate method to measure the phage titer. Make 3 parallels and take the average value of the results. Draw a one-step growth curve with the infection time as the horizontal axis and the titer of the phage in the infection system as the vertical axis to obtain the incubation period and outbreak period of phage PG422, and calculate the outbreak volume.

[0095] Outbreak volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak

[0096] 2. Experimental results and analysis

[0097] The results are as follows Figure 5 As shown in the results, after phage PG422 infects the host bacteria, the phage lysis cycle is about 80 minutes, the incubation period is about 20 minutes, and the phage outbreak period is about 60 minutes; after 80 minutes, the number of phages remains basically unchanged and enters a stable period, at which time the titer can reach 10 10 PFU / mL, the burst amount of bacteriophage PG422 was 91.

[0098] Example 8 Determination of the lysis spectrum of Vibrio parahaemolyticus phage PG422

[0099] 1. Experimental Materials

[0100] Host bacteria: A total of 200 strains of Vibrio used in the lysis spectrum determination examples in three patents (Patent with Publication No. CN 111363723A, Patent with Publication No. CN 111172119A, and Patent with Publication No. CN 113201505A) were selected, including 61 randomly selected strains of Vibrio parahaemolyticus (Patent with Publication No. CN 111172119A), 63 randomly selected strains of Vibrio alginolyticus and 36 strains of Vibrio harveyi (Patent with Publication No. CN 113201505A), and 40 strains of Vibrio cholerae (Patent with Publication No. CN111363723A).

[0101] 2. Experimental methods:

[0102] (1) Select 200 Vibrio strains from different sources and resuscitate them in TCBS medium. Pick a single colony for proliferation and culture to obtain fresh bacterial liquid. Prepare phage proliferation liquid PG422 and 200 Vibrio strains to detect the phage lysis spectrum using the double-layer plate method;

[0103] (2) Comparison of lysis performance with existing phages: The lysis spectra of the above 200 strains of Vibrio were simultaneously measured using the bacteriophages PR02 (see patent publication number CN 111363723A), PG07 (see patent publication number CN 111172119A) and PJ115 (see patent publication number CN 113201505A) in the applicant's existing published patents, and the differences in lysis performance of the Vibrio parahaemolyticus phage PG422 of the present application and the three existing Vibrio phages on the above 200 strains of Vibrio were compared.

[0104] 3. Experimental results and analysis

[0105] 3.1 Comparative analysis of the lysis effects of four phages on 200 strains of Vibrio from different sources

[0106] (1) The Vibrio parahaemolyticus phage PG422 of the present application can lyse 184 of the above 200 strains of Vibrio from different sources, with a total lysis rate of 92.00%. Among them, the lysis rate of Vibrio parahaemolyticus was the highest, reaching 95.08% (58 / 61).

[0107] (2) The existing Vibrio cholerae phage PR02 (see publication number CN 111363723A) can lyse 98 of the above 200 strains of Vibrio from different sources, with a total lysis rate of 49.00%; among them, the lysis rate against Vibrio cholerae is the highest, reaching 75.00% (30 / 40), while the lysis rate of Vibrio phage PG422 against Vibrio cholerae is 85% (34 / 40).

[0108] (3) Vibrio parahaemolyticus phage PG07 (see publication number CN 111172119A) can lyse 138 of the above 200 strains of Vibrio from different sources, with a total lysis rate of 69.00%. Among them, the lysis rate of Vibrio parahaemolyticus was the highest, reaching 85.25% (52 / 61).

[0109] (4) Vibrio alginolyticus phage PJ115 (see publication number CN 113201505A) can lyse 145 of the above 200 strains of Vibrio from different sources, with a total lysis rate of 72.50%. Among them, the lysis rate of Vibrio alginolyticus was the highest, reaching 85.71% (54 / 63).

[0110] The above results show that compared with phages PR02, PG07 and PJ115, phage PG422 has a wider lysis spectrum against Vibrio, and has excellent lysis ability against Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cholerae.

[0111] 3.2 Analysis of the lysis effect of bacteriophage PG422 on various Vibrio species

[0112] (1) From the results in Table 5, it can be seen that for the 40 strains of Vibrio cholerae in Table 4, phage PG422 can lyse 34 of them, with a lysis rate of 85.00%, which is significantly higher than the total lysis rate of Vibrio cholerae phage PR02 (75.00%).

[0113] (2) From the results in Table 5, it can be seen that for the 61 strains of Vibrio parahaemolyticus in Table 4, Vibrio phage PG422 can lyse 58 of them, with a lysis rate of 95.08%, which is significantly higher than the lysis rate of PG07 (85.25%).

[0114] (3) From the results in Table 5, it can be seen that for the 99 strains of Vibrio in Table 4 (63 strains of Vibrio alginolyticus and 36 strains of Vibrio harveyi), the Vibrio phage PG422 can lyse 92 of them, with a lysis rate of 92.93%; specifically, the lysis rate of phage PG422 for 63 strains of Vibrio alginolyticus is 93.65%, and the lysis rate for 36 strains of Vibrio harveyi is 91.67%. The Vibrio alginolyticus phage PJ115 can lyse 82 of the 99 strains of Vibrio, with a lysis rate of 82.83%, among which the lysis rate of phage PG422 for 63 strains of Vibrio alginolyticus is 85.71%, and the lysis rate for 36 strains of Vibrio harveyi is 77.78%.

[0115] From the above, it can be seen that the total lysis rate of Vibrio phage PG422 for 200 strains of Vibrio from different sources used in this embodiment is much higher than that of existing phages PR02, PG07 and PJ115. At the same time, for the lysis effects of four host bacteria, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cholerae, the lysis rates of Vibrio phage PG422 are also higher than those of Vibrio phage PR02, PG07 and PJ115, respectively. This result shows that Vibrio phage PG422 has the characteristics of wide lysis spectrum and better lysis effect, and can effectively prevent and control the infection of various types of Vibrio from different sources, with a wide range of applications and great potential.

[0116] Table 4 Detailed lysis spectra of 4 Vibrio phages against 200 Vibrio strains

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Table 5 Lysis spectrum of 2 Vibrio phages against 200 Vibrio strains

[0125]

[0126]

[0127] Example 9 Safety Test of Vibrio parahaemolyticus Phage PG422

[0128] 1. Experimental methods

[0129] Select 2-3 grams of healthy whiteleg shrimp and fast for 12 hours. Set up a blank group and a phage group. Phage group 1: Add Vibrio phage to the water in a certain proportion, and the final concentration of phage is 5.00×10 4 PFU / mL. Phage group 2: The phage was mixed evenly with shrimp feed. The titer of the phage after mixing with the feed was 5.00×10 8 PFU / g. Blank group: no treatment. All shrimp fry were raised under the same conditions. At the end of the experiment, the mortality of shrimp fry in each experimental group and the incidence of hepatopancreas were counted.

[0130] 2. Experimental results and analysis

[0131] The shrimps in the two phage groups and the blank group grew well, and the hepatopancreas of the shrimps in the phage group was consistent with that in the blank group, without abnormal phenomena such as necrosis. This shows that Vibrio phage PG422 is safe and reliable, and has no adverse effects on the animal body.

[0132] Example 10 Application of Vibrio parahaemolyticus phage PG422 in treating shrimp infected with Vibrio

[0133] 1. Experimental methods

[0134] Healthy white shrimp weighing about 5g were randomly divided into three groups: the bacterial attack group, the phage group, and the blank group. Each group had 50 shrimps and fasted for 12 hours. The bacterial attack group and the phage group were injected with Vibrio at a dose of 5×10 6CFU / shrimp; the blank group was injected with an equal volume of saline. The phages were mixed evenly with feed at a volume-to-mass ratio of 3% and fed 1 hour after the challenge. The mortality of shrimps in each group within 48 hours after the challenge was recorded, and the protection rate of the phages was calculated.

[0135] 2. Experimental results and analysis

[0136] The experimental results are shown in Table 6. The survival rate of white shrimp in the phage group reached 88%, which is much higher than that in the challenge group. This shows that compared with the challenge group, the mortality rate of the phage group was greatly reduced, and feeding feed containing phages can effectively improve the survival rate of shrimp. The protection rate of the phage group against shrimp infected with Vibrio can reach 87.23%, which shows that phages can effectively prevent and control Vibrio infection when added to feed as a pharmaceutical preparation.

[0137] Table 6 The effect of bacteriophage on the prevention and treatment of Vibrio vannamei disease in white shrimp

[0138] Grouping Number of deaths mortality rate Protection rate Blank Group 0 0 - Bacteria attack group 47 94.00％ - Phage Group 6 12.00％ 87.23％

[0139] Example 11 Application of Vibrio parahaemolyticus phage PG422 in water purification

[0140] 1. Experimental methods

[0141] A shrimp fry farm in Dongying was selected for field experiment. The shrimp fry farm had suffered from severe Vibrio infection in the early stage. Two treatment groups were designed, namely the control group and the phage group. The phage groups were set in parallel. Phage PG422 was added to the shrimp ponds to a final concentration of 5×10 4 PFU / mL; the number of Vibrio in the water was detected at 0h, 2h, 6h, 12h, 24h and 48h after adding bacteriophage.

[0142] 2. Experimental results and analysis

[0143] The results are shown in Table 7 and Figure 6 As shown in Figure 2, after 24 hours of treatment with phage group 1 and phage group 2, the content of Vibrio in the water had dropped to 10 1 CFU / mL, which was much lower than that of the control group (7.23×10 5 ), which shows that Vibrio phage PG422 can significantly reduce the number of live bacteria in aquaculture water and can be promoted and used as a water Vibrio purifier with a fast water purification speed.

[0144] Table 7 Vibrio residual in bacteriophage PG422 water purification test (CFU / mL)

[0145] time Blank Group Phage Group 1 Phage Group 2 0h <![CDATA[7.85×10 5 ]]> <![CDATA[7.85×10 5 ]]> <![CDATA[7.85×10 5 ]]> 2h <![CDATA[7.60×10 5 ]]> <![CDATA[5.60×10 4 ]]> <![CDATA[6.35×10 4 <!-- 17 -->]]> 6h <![CDATA[8.00×10 5 ]]> <![CDATA[1.52×10 3 ]]> <![CDATA[1.88×10 3 ]]> 12h <![CDATA[7.50×10 5 ]]> <![CDATA[3.20×10 2 ]]> <![CDATA[3.35×10 2 ]]> 24h <![CDATA[7.23×10 5 ]]> <![CDATA[2.35×10 1 ]]> <![CDATA[1.97×10 1 ]]> 48h <![CDATA[7.65×10 5 ]]> <![CDATA[2.08×10 1 ]]> <![CDATA[2.20×10 1 ]]>

[0146] Example 12 Application of Vibrio parahaemolyticus phage PG422 in the disinfection of aquatic products

[0147] 1. Experimental methods

[0148] Using simulated contaminated salmon meat as a model, the inhibitory effect of bacteriophage PG422 on Vibrio in salmon was determined. Fresh salmon meat was collected and cut into 5cm×5cm×1cm slices, first soaked in sodium hypochlorite for 15 minutes, removed and drained, rinsed with sterile water 5 times, 3 minutes each time, and drained for later use.

[0149] The salmon meat of the experimental group and the control group were first inoculated with Vibrio parahaemolyticus suspension (1×10 6 CFU / mL) for 10 min, then the phage group was treated with 1×10 7 Soak in PG422 phage suspension with PFU / mL for 10 minutes, remove and drain, and put into a fresh-keeping bag. All salmon fillets were placed at 25°C for constant temperature storage. Take out salmon fillets at 0h, 2h, 4h, 6h, 12h, 24h, and 48h, respectively, set up parallel groups, detect the number of Vibrio in them, and compare the antibacterial effect of Vibrio phage.

[0150] 2. Experimental results and analysis

[0151] The results are as follows Figure 7 As shown in the figure, after 4 hours of storage, the number of Vibrio colonies detected in the salmon of the control group increased by 1 order of magnitude compared with the initial value, while the total number of Vibrio detected in the phage group decreased by 5 orders of magnitude; after 24 hours of storage, the number of Vibrio detected in the salmon of the phage group was 10 5 CFU / g, much lower than the control group (10 9 CFU / g (p<0.01). The results show that Vibrio phage PG422 can inhibit the growth of Vibrio in salmon fillets, providing a reference for the application of Vibrio phage in the prevention and control of Vibrio infection in the field of aquatic products.

[0152] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A Vibrio parahaemolyticus bacteriophage with a broad lytic spectrum, characterized in that: It was named Vibrio parahaemolyticus phage PG422, and its deposit number is CGMCC No.46164.

2. A bacteriophage composition, characterized in that: It comprises the Vibrio parahaemolyticus phage PG422 as claimed in claim 1.

3. Use of the Vibrio parahaemolyticus phage according to claim 1 or the phage composition according to claim 2 in the preparation of a pharmaceutical preparation, a water purifier or aquatic product disinfectant for preventing and treating aquatic product diseases caused by Vibrio infection.

4. The use according to claim 3, characterized in that: The Vibrio include: Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio harveyi and Vibrio cholerae.

5. A bacteriophage pharmaceutical preparation, characterized in that: The active ingredient comprises the Vibrio parahaemolyticus phage according to claim 1 or the phage composition according to claim 2.

6. A feed additive, characterized in that: include: The Vibrio parahaemolyticus phage according to claim 1 or the phage composition according to claim 2.

7. A water purifier, characterized in that: The active ingredients include the Vibrio parahaemolyticus phage as claimed in claim 1 or the phage composition as claimed in claim 2; preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in aquatic environments.

8. The water purifier according to claim 7, characterized in that: The titer of Vibrio phage is 5×10 4 PFU / mL or above.

9. The use of the water purifier according to claim 8, characterized in that: The application method is: directly adding the Vibrio parahaemolyticus phage as claimed in claim 1 or the phage composition as claimed in claim 2 into the water body.

10. A disinfectant for aquatic products, characterized in that: The active ingredient comprises the Vibrio parahaemolyticus phage according to claim 1 or the phage composition according to claim 2.

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