CN115505576A - Vibrio harveyi phage V-YDF132 and application thereof - Google Patents

Abstract

The invention discloses a Vibrio harveyi phage V-YDF132 and application thereof. The phage is preserved in Guangdong province microorganism culture collection center in 2022, 7/05/month, and the preservation number is GDMCC No:62599-B1. The new virulent vibrio harveyi phage V-YDF132 is obtained by screening, has strong host specificity, and has strong cracking and killing effects on vibrio harveyi. The phage is insensitive to chloroform, has high thermal stability and pH stability, has strong tolerance to temperature below 60 ℃, can keep high cracking activity at the pH value of 5-11, and has the optimal infection complex number of 0.1-0.01. The Vibrio harveyi phage V-YDF132 provided by the invention can be widely applied to various links which are easy to cause loss due to Vibrio harveyi infection in the aquaculture process, daily disinfection of the aquaculture environment and other aspects.

Description

Vibrio harveyi phage V-YDF132 and application thereof

Technical Field

The invention belongs to the technical field of microbial control, and particularly relates to a Vibrio harveyi bacteriophage V-YDF132 and application thereof.

Background

Vibrio harveyi (vibiro haveyi) is a gram-negative and luminous marine bacterium, widely distributed in a warm marine environment near shore, is one of normal flora of marine biota, is recognized only at the end of the 20 th century, is an important pathogenic bacterium of aquaculture animals, and mainly causes infectious diseases of shrimps and fishes. The vibrio harveyi has wide infection range, can infect various aquaculture animals, such as weever, epinephelus coioides, turbot, oyster, large yellow croaker and the like, and particularly, the vibrio harveyi is the main pathogenic bacteria of vibriosis of the large yellow croaker. Vibrio harveyi causes symptoms such as eyeball protrusion, muscle ulceration, visceral necrosis and the like, causes death of the disease and causes huge loss to the aquaculture industry.

Due to the rapid shift of the aquaculture mode from extensive cultivation to intensive cultivation with high density, bacterial and fungal infections are often encountered during the cultivation process, which leads to the failure of batch cultivation. Although people can reduce the risk of bacterial infection by various means, such as maintaining the water quality of the aquaculture water body, controlling the temperature and salt conditions of the aquaculture water body, reducing the aquaculture density, improving the sanitary conditions of the aquaculture environment and the like, the preventive measures have little effect and cannot meet the expectation of people. Therefore, the dependence on antibiotics and other chemical drugs is continuously improved, and the abuse of the antibiotics in the form leads to a large amount of antibiotic residues in the cultured meat products on one hand, and people unconsciously take the antibiotics by eating the meat products to cause food safety problems; on the other hand, the residue of antibiotics in the environment or in the animal body not only adversely affects the aquatic animals and plants and the environment, but also selects many drug-resistant bacteria, and the treatment of such bacteria becomes more difficult when the bacteria are infected again.

In order to overcome the problems, phage therapy is expected to become a reliable means, and specific phage can be added into a culture environment to prevent and control bacterial infection, so that antibiotics are prevented from being added into feed for a long time, accumulation of the antibiotics in the environment is reduced, and generation of drug-resistant bacteria is reduced. The problems show that a new replacement strategy is also urgently needed for disease control in the field of aquaculture, so that phage treatment can provide a new replacement measure for disease control of aquaculture, the cost is low, the aquaculture cost is greatly reduced, the aquaculture yield is increased, meanwhile, the phage treatment process is green and environment-friendly, the defects of antibiotic control can be overcome if reasonable use is adopted, the aquaculture environment is greatly improved, and the quality of aquatic products is improved. For example, patent CN111676197A discloses a vibrio harveyi phage vB _ KaS _ PK08, patent CN111705041A discloses a vibrio harveyi phage vB _ KaS _ PK22, which has strong lysis effect on vibrio harveyi, but the environmental suitability such as pH stability and the like needs to be further improved, and the currently disclosed vibrio harveyi phage are still few and are difficult to meet the requirements of practical application.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a novel virulent Vibrio harveyi phage V-YDF132 with strong cracking and killing effects on Vibrio harveyi.

The second purpose of the invention is to provide the application of the novel virulent Vibrio harveyi phage V-YDF132.

The above object of the present invention is achieved by the following technical solutions:

the invention separates and screens a new virulent vibrio harveyi phage (Vibriophage) V-YDF132, wherein the phage V-YDF132 is preserved in Guangdong province microbial culture collection center (GDMCC) at 7 and 05 months in 2022, and the preservation number is GDMCC No:62599-B1. Phage V-YDF132 was shown using a projection electron microscope to have a regular icosahedral head with a diameter of about 69nm, a tail length of about 160nm, and a soft and flexible tail, identified as a virus of the family filoviridae (sipoviridae) according to the ICTV classification standard; . The phage V-YDF132 has high host specificity on Vibrio harveyi and has strong cracking and killing effects on Vibrio harveyi; and the V-YDF132 has good thermal stability and pH stability, can keep higher titer at 60 ℃ and pH value of 4-12, has short incubation period and can explode at 20min after infection. Therefore, the Vibrio harveyi V-YDF132 obtained by screening can efficiently kill and crack the Vibrio harveyi V-YDF132, has strong applicability, and can be used as an excellent biological antibacterial means to be applied to the Vibrio harveyi infection in aquaculture.

Further, the bacteriophage V-YDF132 has the capacity of remarkably degrading Vibrio harveyi V-YDF132. Therefore, the application of the bacteriophage V-YDF132 in killing and cracking Vibrio harveyi V-YDF132 is within the protection scope of the invention.

Further, the application of the bacteriophage V-YDF132 in preparing products for killing and cracking Vibrio harveyi V-YDF132 also falls within the protection scope of the invention. The product may be a disinfectant or other form of biological product that is involved in the control of Vibrio harveyi.

Further, the invention also provides application of the phage V-YDF132 in preparation of a medicine for preventing and treating vibrio harveyi infectious diseases.

Preferably, the Vibrio harveyi infectious disease is a disease caused by infection of an aquaculture animal with Vibrio harveyi.

Further preferably, the aquaculture animal comprises one or more of fish, crustaceans, shellfish, frogs or turtles.

Further, the Vibrio harveyi includes one or both of V-YDF132 and YF.42.

Further preferably, the Vibrio harveyi phage V-YDF132 is directed predominantly against Vibrio harveyi V-YDF132.

A medicine for preventing and treating Vibrio harveyi infectious diseases comprises the Vibrio harveyi bacteriophage V-YDF132.

Preferably, the concentration of the Vibrio harveyi phage V-YDF132 is 1X 10 ⁸ ～1×10 ⁹ PFU/mL。

Preferably, pharmaceutically acceptable auxiliary materials are also included.

Based on the above, the method for preventing and controlling the vibrio infection of aquatic animals by using the vibrio harveyi phage V-YDF132 to kill and crack vibrio harveyi also belongs to the protection scope of the invention.

Preferably, the Vibrio harveyi phage V-YDF132 has an optimal multiplicity of infection of between 0.1 and 0.01.

In order to control the better and more stable effect, the degradation or treatment conditions using the lysis solution are preferably controlled as follows: the temperature is 30-60 deg.C, and the pH is preferably 4-12.

Further, when the Vibrio harveyi is killed and cracked or the infection of the aquatic animals is prevented and controlled, the bacteriophage V-YDF132 is used at the temperature of 30-60 ℃, the pH value is 4-12, and the infection complex number is 0.1-0.01.

Further preferably, the bacteriophage V-YDF132 is used at the temperature of 30-40 ℃ and the pH value of 5-9, and the infection complex number of the schizomycete liquid is 0.01.

Most preferably, the bacteriophage V-YDF132 is used at a temperature of 40 ℃ and a pH of 6 with a multiplicity of infection of 0.1.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a new virulent vibrio harveyi phage V-YDF132, wherein the phage V-YDF132 is preserved in Guangdong province microorganism strain preservation center at 7 and 05 months in 2022, and the preservation number is GDMCC No:62599-B1. The bacteriophage V-YDF132 has high host specificity on Vibrio harveyi and has strong cracking and killing effects on Vibrio harveyi; and the V-YDF132 has good thermal stability and pH stability, can keep higher titer at 60 ℃ and pH value of 4-12, and has short incubation period and explosive amount of 298PFU/cell. The Vibrio harveyi V-YDF132 can efficiently kill and crack the Vibrio harveyi V-YDF132, has strong applicability, and can be used as an excellent biological antibacterial means to be applied to the Vibrio harveyi infection in aquaculture.

Drawings

FIG. 1 is a photograph of a plate culture of the Vibrio harveyi phage V-YDF132 in example 2.

FIG. 2 is a TEM photograph of Vibrio harveyi phage V-YDF132 in example 4.

FIG. 3 is a graph showing the results of the chloroform sensitivity test of the Vibrio harveyi phage V-YDF132 in example 5.

FIG. 4 is a graph showing the results of the heat stability experiment of the Vibrio harveyi phage V-YDF132 in example 6.

FIG. 5 is a graph showing the results of the pH stability experiment of Vibrio harveyi phage V-YDF132 in example 7.

FIG. 6 is a graph showing the results of an experiment for the optimal multiplicity of infection of the Vibrio harveyi phage V-YDF132 in example 8.

FIG. 7 is a graph showing the one-step growth of the Vibrio harveyi phage V-YDF132 in example 10.

FIG. 8 is a graph showing the effect of Vibrio harveyi phage V-YDF132 on infection of Vibrio harveyi YDF132 in example 11.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Vibrio harveyi YDF132 (YDF.132), YF.42 are deposited at the laboratory of the department of aquatic biology medicine of the oceanic institute of southern China university of agriculture.

LB broth culture medium: 10g tryptone, 5g Yeast, 10g sodium chloride, plus ddH ₂ O to 1000mL, adjusted to pH 7.2 with 5mol/L NaOH (ca. 0.2 mL), and sterilized at 121 ℃ for 30min.

SM buffer solution: purchased from Regen Biotechnology Ltd, beijing.

2216E Medium: purchased from Qingdao Haibo Biotechnology, inc.

Example 1 isolation of Vibrio harveyi phage V-YDF132

Vibrio harveyi YDF132 was inoculated into 100mL of LB broth plus 2% NaCl and shaken to log phase at 28 ℃ until use. The bacterial liquid is subpackaged into 2mL sterile EP tubes and kept at 4 ℃ for later use. Taking a water sample of a farm, adding the remaining 98mL of bacterial liquid into the water sample of the farm, shaking uniformly, placing in an incubator at 28 ℃ for 24h, and shaking occasionally. After the culture is finished, 10mL culture solution is centrifuged at 10000rpm for 5min, the supernatant is filtered by a 0.22 mu m filter, residual bacteria in the filtrate are removed to obtain phage stock solution, and the phage stock solution is stored at 4 ℃ for later use. Separating by spot method, sucking 200 μ L host bacteria liquid, uniformly coating on NA plate, and standing for 20min; then 20 mu L of phage stock solution is sucked, 3-5 drops of phage stock solution are carefully dripped on the NA plate, the NA plate is cultured for 6-24 h at 28 ℃, and whether the plaque appears on the plate is observed in real time. If the plaque appears on the plate, it indicates that there may be a corresponding Vibrio harveyi bacteriophage in the water sample. And verifying the phage by using a double-layer plate method. If a single plaque appears, the phage is confirmed to be obtained.

Example 2 purification of Vibrio harveyi phage V-YDF132

The phage was purified using SM buffer using a double-plate method. If more than one form of plaque is present on the bilayer, it is purified separately. Specific operation of purification: selecting the largest, transparent and different-shape plaque, moving the plaque by using a 1mL sterile filter element gun head, blowing the plaque into 1mL SM buffer solution, oscillating for 15s, and placing the plaque in a refrigerator at 4 ℃ overnight; taking out the phage SM solution the next day, and diluting the phage SM solution by 10 times with sterile normal saline to obtain a proper concentration gradient; sequentially adding 100 mu L of host liquid in logarithmic phase and 100 mu L of phage diluent into a screw-cap centrifuge tube filled with 5mL of LB broth semi-solid culture medium (agar concentration is 0.65%) cooled to about 50 ℃ along the tube wall, screwing the tube cap, horizontally rotating and uniformly mixing, then quickly pouring the mixture into a prepared NA solid culture medium (agar concentration is 1.5%), rotating the plate to uniformly spread the mixture, and standing for 15min; the solidified double-layer plate is placed in a constant temperature incubator at 28 ℃ upside down to be cultivated for 6-24 h; after the plaques appear, the plaques with the largest diameter and transparent centers are taken out from the double-layer flat plate by using a 1mL filter element gun head, blown into 1mL SM buffer solution, shaken for 15s, placed overnight at 4 ℃, and continuously manufactured into the double-layer plate. Repeating the steps for 3-5 times until the plaques with consistent sizes are obtained, namely completing the purification. The purified phage bi-layer plate is shown in FIG. 1, and the V-YDF132 plaque is seen to be about 2 mm in diameter, bright and translucent in the center, and halo around.

Example 3 potency assay for Vibrio harveyi phage V-YDF132

Diluting the phage solution filtered by the sterile filter head with the fixed number 0.22 by different times by using SM buffer solution, mixing the diluted phage solution with the logarithmic phase Vibrio haveyi YDF132 bacterial solution to prepare a double-layer plate, and culturing the double-layer plate at the constant temperature of 28 ℃ overnight to grow the plaques. The plaques on the double-layer plate are counted, and the titer is calculated by selecting the plate with the plaque number of 30-300. And (4) converting the number of the plaques on the plate with the corresponding dilution times to obtain the titer of the phage. Such as dilution 10 of Vibrio harveyi phage V-YDF132 solution ^-7 The two-layer plate with 100 plaques on the back has the titer of 1 x 10 for the Vibrio harveyi phage V-YDF132 liquid ⁹ PFU/ml. The titer of the Vibrio harveyi phage V-YDF132 was determined to be 2.38 x 10 ⁸ PFU/ml。

Example 4 Electron microscopy morphological Observation of Vibrio harveyi phage V-YDF132

Firstly, centrifuging the cultured phage lysate liquid at the rotation speed of 4000r/min for 15min (4 ℃), and filtering the supernatant through a 0.22/sterile filter head to obtain phage liquid. BacteriophageAdding RNase A enzyme and Dnase I enzyme into the solution until the final concentrations are respectively 1 degree and mL, and standing at room temperature for 30min; adding NaCl (solid state) to a final concentration of 1M, and carrying out ice bath for 1h after dissolving the NaCl; 8300r/min for 10min (4 ℃), transferring the supernatant into a new centrifuge tube and measuring the volume of the centrifuge tube; adding solid polyethylene glycol (PEG 8000) to final concentration of 100mg/mL, dissolving in shaking table at room temperature, and standing in ice water mixture overnight after solid polyethylene glycol is dissolved; centrifuging at 10000r/min for 15min (4 deg.C) the next day, discarding the supernatant, recovering the precipitate, washing with PBS for 3 times, and resuspending the precipitate with SM buffer. After obtaining the purified phage particles through the steps, taking the purified phage 10 with the liquid weight (more than 10) ⁹ PFU/mL) was stained with 2% uranium acetate and its morphological characteristics were observed using a transmission electron microscope. An electron microscope morphology chart of the phage V-YDF132 is shown in FIG. 2, which shows that the phage V-YDF132 has a regular icosahedron head with the diameter of about 69nm, the tail is about 160nm long, and the tail is soft and flexible, and the phage V-YDF132 belongs to the family of Long-tailed viruses (Siphoviridae) according to the virus classification standard of ICTV; the phage is preserved in Guangdong province microorganism culture collection center (GDMCC) at 7/05 of 2022, and the classified name number is VibriophageV-YDF132, and the preservation number is GDMCC No:62599-B1, preservation No. 100 of Miehe Zhou city, guangzhou, guangdong province.

Example 5 chloroform sensitive detection of Vibrio harveyi phage V-YDF132

Experiment setup 3 parallel controls, to 1mL phage suspension respectively adding 30 body suspension, 300 suspension of chloroform mix, appropriate mixing, room temperature under dark place 30min,5000n after centrifugation for 5min. 1mL of host bacterium liquid in logarithmic phase and 5mL of LB broth semi-solid medium (agar concentration 0.65%) at about 50 ℃ are quickly mixed, evenly poured on an NA plate, after the plate is solidified, 10 of the centrifuged upper aqueous phase part is taken and titrated on the plate, the edge of the plate is sealed by a sealing film, the plate is positively placed in an incubator at 28 ℃ for culture, and whether the plaque size of an experimental group is different from that of a control group is observed. The double-layer plate for detecting the chloroform sensitivity of the phage V-YDF132 is shown in figure 3, and the size of a plaque formed by the phage treated by chloroform and the phage of a control group is not different, so that the phage is not sensitive to chloroform and has no lipid structure.

Example 6 detection of the thermal stability of Vibrio harveyi phage V-YDF132

Placing 500 μ L phage liquid in 1.5mL EP tube, and allowing to act in water bath at 37 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, and 80 deg.C for 20min,40min, and 60min; immediately taking out the phage after the time is over, placing the phage on ice for ice bath cooling, determining the phage titer by adopting a double-layer plate method, and repeating the steps for 3 times; the thermal stability was judged according to the titer of the phage at different temperatures. The result of the thermal stability of the phage V-YDF132 is shown in FIG. 4, and it can be seen that the phage V-YDF132 has high thermal stability, strong tolerance to a temperature below 60 ℃ and very sensitive to a high temperature above 70 ℃.

Example 7 detection of pH stability of Vibrio harveyi phage V-YDF132

Adding LB culture medium (pH is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) with different pH 900 μ L into 11 2mL centrifuge tubes, placing the centrifuge tubes into a water bath kettle at 37 deg.C, adding 100 μ L of phage liquid after temperature balance, performing phage titer detection, and repeating for 3 times; and judging the pH stability of the phage according to the titer of the phage under different pH values. The pH stability result of the phage V-YDF132 is shown in figure 5, and the phage V-YDF132 has a wider application range to the pH value, still has higher lytic activity within the pH value of 4-12, and can keep higher lytic activity within the pH value of 5-11.

Example 8 determination of the optimal multiplicity of infection of the Vibrio harveyi phage V-YDF132

Adding 50 phage and host bacteria liquid into a 001 bacterium liquid culture medium of 900 samples according to the proportion that the MOI is 100, 10,1,0.1,0.01,0.001,0.0001 and 0.00001 respectively, leading the total volume to be 1mL, and oscillating at 37 ℃ for 4-6 h until the mixture becomes clear; centrifuging at 12000r/min for 3min, and filtering with 0.220r filter membrane. Titers were determined by the double-plate method, with the highest titer corresponding to the MOI, i.e., the optimal multiplicity of infection. The optimal complex infection results of the phage V-YDF132 are shown in FIG. 6, and it can be seen that the efficiency of V-YDF132 for infecting the host is very small when the complex infection is 0.1 and 0.01, and the efficiency of the phage for infecting the host is substantially saturated when the complex infection is less than 0.01, so that the optimal complex infection of V-YDF132 is between 0.1 and 0.01.

Example 9 host Range detection of Vibrio harveyi phage V-YDF132

In the experiment, 5 strains of bacteria stored in a laboratory are selected for phage host range detection. Namely, different vibrios growing to logarithmic phase are mixed with LB semisolid pre-cooled by heating (about 50 ℃) to prepare a double-layer flat plate, after the upper layer semisolid is solidified, 10 mu L of bacteriophage V-YDF132 filtered by a sterile filter membrane of 0.22 mu m is dripped, and the flat plate is placed in an incubator at 28 ℃ overnight to observe whether plaques appear. If so, indicating that the host can be lysed by the phage V-YDF 132; if not, this indicates that the host is not able to be lysed by phage V-YDF132. The results of the host range detection of V-YDF132 are shown in Table 1, and in the detected host, V-YDF132 can crack two strains of Vibrio harveyi (Vibiro harveyi) including YDF.132, but can not crack Vibrio harveyi such as Photobacterium damselae (Photobacterium damselae), vibrio alginolyticus (Vibrio algolyticus) and Aeromonas hydrophila (Aeromonas hydrophila) and has stronger host specificity.

TABLE 1 detection results of bacteriophage V-YDF132 lysis of different hosts

Note: "+" indicates that the host bacterium is sensitive to the phage; "-" indicates that the host is not susceptible to phage.

Example 10 one-step growth Curve assay of Vibrio harveyi phage V-YDF132

The titer of the Vibrio harveyi phage V-YDF132 phage determined in example 3 was determined by adding phage liquid and host bacteria at the optimum MOI ratio, standing at 28 ℃ for 10min, centrifuging at 8000r/min for 1min, washing 3 times with SM buffer to remove unadsorbed phage, and mixing the precipitate in a 10ml centrifuge tube. Immediately, the mixture was subjected to shaking culture (150 rpm) at 28 ℃ and the timer was started. Sampling 100uL at 0min, sampling every 10min later, and taking 70min totally, immediately detecting titer of the sample taken each time by using a double-layer flat plate method, paralleling 3 times at each time point, wherein the abscissa is infection time, and the ordinate is phage titer, and drawing a one-step growth curve graph. The one-step growth curve of the Vibrio harveyi phage V-YDF132 is shown in FIG. 7, and it can be seen that the incubation period for the Vibrio harveyi phage V-YDF132 to cleave the Vibrio harveyi YDF132 is about 20min, and the burst size is 298PFU/cell.

Example 11 measurement of the Bactericidal Effect of Vibrio harveyi phage V-YDF132

Picking single colony from TCBS solid plate cultured by streak overnight and inoculating in 2216E liquid culture medium, culturing at 28 deg.C and 120rmp to early log phase; measuring the concentration of bacterial liquid and diluting to 10 ⁸ Centrifuging at 8000 Xg for 5min at 4 deg.C for CFU/mL, discarding supernatant, and resuspending in equal volume of 2216E culture medium; taking a cell culture plate, adding 100 mu L of heavy suspension bacteria liquid into each hole, then respectively adding 100 mu L of bacteriophage diluent to enable the multiplicity of infection to be 0.1, simultaneously taking Doxycycline (100 mg/L) as a positive control group, and setting 3 groups in parallel for each group; adding 100 mu L of 2216E culture medium into 100 mu L of the resuspended bacterial liquid as a blank control group; the cell culture plate is placed statically at 28 ℃ for culture, the light absorption value at 600nm is detected by using an enzyme-labeling instrument every 1h, and data are recorded. The bactericidal effect of the Vibrio harveyi phage V-YDF132 is shown in FIG. 8, the V-YDF132 infects the host Vibrio harveyi YDF132 very effectively, and almost no bacteria grow after infection.

Claims (10)

1. Vibrio harveyi phage (Vibriophage) V-YDF132, wherein the phage was deposited at 7/05.2022 in the Guangdong province Collection of microorganisms with the deposit number GDMCC No:62599-B1.

2. Use of the bacteriophage V-YDF132 of claim 1 for lysing and killing vibrio harveyi.

3. Use of the bacteriophage V-YDF132 according to claim 1, in the preparation of a product for lysing and killing Vibrio harveyi.

4. The use of the bacteriophage V-YDF132 of claim 1 in the preparation of a medicament for the prevention or treatment of vibrio harveyi infectious disease.

5. The use of claim 4, wherein the Vibrio harveyi infectious disease is a disease caused by infection of an aquaculture animal with Vibrio harveyi.

6. Use according to claim 5 wherein the aquaculture animal comprises one or more of fish, crustaceans, shellfish, frogs or turtles.

7. The use of any one of claims 2 to 5, wherein the Vibrio harveyi is one or both of the YDF.132 strain or YF.42 strain.

8. A medicament for preventing and treating Vibrio harveyi infectious diseases, comprising Vibrio harveyi phage V-YDF132 of claim 1.

9. The drug according to claim 8, wherein the Vibrio harveyi phage V-YDF132 has a concentration of 1X 10 ⁸ ～1×10 ⁹ PFU/mL。

10. The medicament of claim 8, further comprising a pharmaceutically acceptable excipient.

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