Aquatic product source proteus vulgaris bacteriophage PF107 as well as composition and application thereof

Abstract

The invention belongs to the technical field of bacteriophages, and discloses an aquatic product source common proteus bacteriophage PF107, a composition thereof and application of the aquatic product source common proteus bacteriophage PF107, the preservation number of the aquatic product source common proteus bacteriophage PF107 is CGMCC No.46161, and the aquatic product source common proteus bacteriophage PF107 is preserved in the China General Microbiological Culture Collection Center on August 16, 2024, and the preservation number is CGMCC No.46161. The preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The bacteriophage PF107 has excellent splitting performance on common proteusbacillus vulgaris of various different aquatic products, is wide in splitting spectrum and high in titer, can be prepared into drugs, environment disinfectants, feed additives or aquatic product preservatives for preventing and treating the proteusbacillus vulgaris, can effectively prevent and treat propagation and secondary infection of the proteusbacillus vulgaris, and can be used for preparing medicines, environment disinfectants, feed additives or aquatic product preservatives for preventing and treating the proteusbacillus vulgaris. The death and culling rate of aquatic products can be effectively reduced, the positive rate of proteusbacillus vulgaris antibodies is reduced, the method is green and environment-friendly, and the problems of antibiotic residues and pathogenic bacterium drug resistance caused by use of antibiotics are avoided.

Description

Technical Field

[0001] The present invention relates to the field of phage counting, and particularly to an aquatic-source Proteus vulgaris phage PF107, a composition thereof, and applications thereof. Background Art

[0002] China is the world's largest producer and consumer of aquaculture products. The output of aquaculture products has increased from 524,000 tons in 1949 to 52.242 million tons in 2023, a 99.69-fold increase. However, for many years, due to the intensive development of aquaculture activities, the unorganized expansion of production scale, the blind pursuit of high density and large quantity production, and the realization of aquaculture growth through the unsustainable development of aquatic resources, the ecological environment pollution of aquaculture water bodies and the occurrence frequency of aquaculture animal diseases have gradually increased. With the rapid development of the national economy, the supply-demand relationship of aquatic products has tended to balance, and people's consumption concepts have also changed. More and more people have realized the important role of aquatic products in health and nutritional balance. People have put forward higher requirements for a good water ecological environment and high-quality aquaculture products.

[0003] Currently, it has been found that opportunistic pathogenic Proteus is a new threat to the aquaculture system and a potential hazard to the health of aquatic animals and the development of aquaculture. Proteus is a Gram-negative saprophytic bacterium widely distributed in nature, and it is distributed in soil, water, garbage, decaying organic matter, and the intestines of humans or animals. Proteus belongs to the Enterobacteriaceae family ( Enterobacteriaceae ), has flagella, no capsule, no spore, and is actively motile. Common groups include Proteus vulgaris, Proteus mirabilis, Morganella morganii, Proteus rettgeri, and Proteus penneri. Proteus can infect aquatic products and cause Proteus disease. Kumar et al. found that a Proteus hauseri can cause a large number of deaths of koi carp, and EL-Deen et al. found that Proteus vulgaris can cause a large number of deaths of crayfish and Nile tilapia. Sabyasachi Pattanayak et al. also first reported that a drug-resistant Proteus mirabilis can cause large-scale deaths of cultured carp in the Indian region. There are also reports in China of Proteus infections in tilapia, channel catfish, Chinese white shrimp, loach, and Procambarus clarkii (crayfish). Currently, the Proteus disease of aquatic products is mainly controlled by antibiotics. However, the use of antibiotics easily produces drug-resistant Proteus pathogenic bacteria, so it is necessary to find a safer and more reliable control method.

[0004] Bacteriophages are a type of virus that are obligate parasites or infect bacteria, and they are widely present in nature. Virulent bacteriophages can proliferate within sensitive host bacteria and finally lyse the host bacteria to release progeny bacteriophages, thereby achieving the effect of destroying the host bacteria. In terms of bactericidal effects, virulent bacteriophages have extensive applications, especially in the treatment of infections caused by "super bacteria" with extensive drug resistance, and good results have been achieved.

[0005] However, currently, there is relatively little research on Proteus bacteriophages in the aquaculture field, and there are no safe and effective Proteus vulgaris bacteriophages from aquatic sources that can be effectively applied to the prevention and control of Proteus diseases in aquaculture.

[0006] Therefore, the existing technology needs to be further improved. Summary of the Invention

[0007] In view of the above problems, the present invention provides an aquatic-source Proteus vulgaris bacteriophage PF107, its composition, and its applications. The aquatic-source Proteus vulgaris bacteriophage PF107 has broad-spectrum lytic activity against Proteus vulgaris from aquatic sources and can be used as an active ingredient to prepare drugs, feed additives, environmental disinfectants, or aquatic product preservatives for the prevention and control of Proteus infections. By adding this bacteriophage to water bodies or aquatic feeds, while solving the problem of Proteus infections in aquatic products, it can achieve the effects of purifying Proteus diseases in farms, improving the quality and production performance of aquatic products. To solve the above problems, the present application provides the following technical solutions: In a first aspect, the present application also provides an aquatic-source Proteus vulgaris bacteriophage ( Proteus vulgaris phage ) PF107, whose preservation number is CGMCC No. 46161.

[0008] This bacteriophage was isolated from diseased crayfish in a shrimp pond in Hubei Province and was preserved on August 16, 2024, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] In the present application, the bacteriophage PF107 includes mutant strains with a homology higher than 98% or 99% after point mutation, deletion mutation, or addition mutation and maintaining substantially the same bactericidal activity. Since bacteriophages are very prone to mutation during replication, the mutants of the above-mentioned bacteriophages are also within the scope of protection claimed in the present application. The sequence of the bacteriophage PF107 can be obtained by sequencing the biological material preserved in the present invention through known methods. For those skilled in the art, it does not require creative labor to screen out mutant strains with extremely similar traits to the bacteriophage provided by the present invention.

[0010] Experimental findings show that the aquatic-source Proteus vulgaris phage PF107 has excellent lysis performance against Proteus vulgaris from aquatic sources. The lysis rate of PF107 against 40 strains of Proteus vulgaris from aquatic sources (including fish, frogs, turtles, soft-shelled turtles, etc.) in this application is 82.5%. It has a wide lysis spectrum and great potential in the clinical on-site prevention and control of Proteus vulgaris.

[0011] In addition, the aquatic-source Proteus vulgaris phage PF107 also has good temperature stability and acid-base stability. The phage PF107 has relatively stable activity within the pH range of 5.0 - 10.0, and its titer can still remain at 10 9 PFU / mL. It still maintains its original activity after acting for 60 min within the range of 40 - 60 °C.

[0012] In a second aspect, the present application also provides a phage composition, which includes the aforementioned aquatic-source Proteus vulgaris PF107.

[0013] In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in the lysis spectra of different phages, and make complementary advantages, the above-mentioned aquatic-source Proteus vulgaris phage PF107 can be combined with other phages for use, so as to expand the bactericidal spectrum and improve the prevention and treatment effect of Proteus vulgaris diseases.

[0014] Preferably, the phage composition includes: the aforementioned Proteus vulgaris phage PF107 and Proteus vulgaris phage VB_PMIM_PF07. The preservation number of the Proteus vulgaris phage VB_PMIM_PF07 is CGMCC NO.23812, and its specific information can be found in the patent with the publication number CN116286678A.

[0015] In addition, the above-mentioned Proteus vulgaris phage PF107 can also be combined with other different types of phages (such as those that inhibit different pathogenic bacteria causing the same kind of diseases in aquatic products), and be used for the prevention and treatment of the same kind of diseases, such as Vibrio phages.

[0016] In a third aspect, the present application also provides the use of the above-mentioned aquatic-source Proteus vulgaris PF107 or the above-mentioned phage composition in the preparation of drugs, feed additives, environmental disinfectants or aquatic product preservatives for preventing and treating diseases caused by Proteus vulgaris infection.

[0017] Optionally, in the said use, the disease caused by Proteus vulgaris infection is aquatic Proteus vulgaris disease caused by Proteus vulgaris infection.

[0018] Optionally, the aquatic products include Procambarus clarkii (i.e., crayfish), loach, tilapia, channel catfish and other various fish.

[0019] The prevention and treatment include prophylaxis and therapy. As used herein, the term "prophylaxis" refers to all actions including inhibiting or delaying the disease by administering the composition. As used herein, the term "therapy" refers to all actions including improving or ameliorating the disease by administering the composition.

[0020] In a fourth aspect, the present application also provides a phage pharmaceutical preparation, the active ingredient of which comprises the aforementioned Proteus vulgaris PF107 derived from aquatic products or the aforementioned phage composition.

[0021] Optionally, in the phage pharmaceutical preparation, the form of the phage pharmaceutical preparation is a feed-mixing dosage form or a spraying dosage form.

[0022] The application method of the phage pharmaceutical preparation is as follows: adding phage as a therapeutic drug to water or feed, etc., can prevent and treat Proteus disease and improve the survival rate, etc.

[0023] Optionally, the phage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and characteristics of the administered active ingredient. In order to formulate the pharmaceutical composition into a liquid preparation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, glucose solution, maltodextrin solution, glycerol, ethanol, various culture media, etc. They can be used alone or in any combination thereof. According to needs, other conventional additives can be added, such as antioxidants, buffers, bacteriostatic agents, etc. When combined with diluents, dispersants, surfactants, binders, and / or lubricants, the composition of the present invention can also be prepared into oral dosage forms (such as aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as freeze-dried preparations.

[0024] In a fifth aspect, the present application also provides an aquatic feed additive or a water additive, the active ingredient of which comprises the aforementioned Proteus vulgaris PF107 derived from aquatic products.

[0025] Preferably, in the above feed additive, the concentration of phage is 10 8 PFU / mL or more.

[0026] By adding the above feed additive to water or mixing it with feed, feeding aquatic products such as fish and shrimp, thereby disinfecting and sterilizing the feed in the aquaculture farm, avoiding the spread of Proteus disease from the source, and effectively preventing and treating Proteus disease.

[0027] Optionally, the feed additive further contains other active ingredients for inhibiting or eliminating bacteria in water; the feed additive is in the form of a liquid dosage form, a powder dosage form or a solid dosage form, but is not limited to the above three dosage forms.

[0028] In a sixth aspect, the present application provides an environmental disinfectant, the active ingredient of which includes the aforementioned Proteus vulgaris PF107 of aquatic origin or the aforementioned phage composition.

[0029] Optionally, in the environmental disinfectant, the use concentration of the phage is 1×10 8 PFU / ml or more.

[0030] Optionally, the environmental disinfectant further contains other active ingredients for inhibiting or eliminating bacteria in the environment of an aquaculture farm.

[0031] The environments where the environmental disinfectant can be applied include aquaculture utensils, aquaculture water bodies, feed or aquaculture environments, and the aquaculture environments include breeding environments such as ponds or aquariums.

[0032] In a seventh aspect, the present application further provides the application of the environmental disinfectant in the disinfection of the environment of an aquaculture farm, and the application method is: the environmental disinfectant is used for disinfecting Proteus in aquaculture water bodies, aquaculture environments, feed or breeding utensils.

[0033] The application method includes, but is not limited to, disinfecting and decontaminating the water bodies and water distribution systems, aquaculture facilities, breeding utensils or other environmental surfaces in the aquaculture environment in the form of liquid immersion, spraying or combined use with a water-containing carrier, and disinfecting and preserving the feed against Proteus. This disinfectant can be used to replace antibiotics or traditional disinfection products, and it will not cause damage to humans and poultry.

[0034] In an eighth aspect, the present application further provides a fresh-keeping agent for aquatic products, which includes the aforementioned Proteus vulgaris PF107 of aquatic origin or the aforementioned phage composition.

[0035] By spraying the fresh-keeping agent for aquatic products on the surface of aquatic products or soaking the aquatic products in a solvent containing the fresh-keeping agent for aquatic products, the reproduction of Proteus in aquatic products can be inhibited, and the fresh-keeping effect can be achieved.

[0036] The present invention has the following beneficial effects: 1. The present invention provides an isolated aquatic-source Proteus vulgaris phage PF107, its composition and its applications. This Proteus vulgaris phage PF107 has excellent lysis performance against various aquatic-source Proteus vulgaris, with a broad lysis spectrum, high titer, good temperature stability and acid-base stability. It can be prepared into drugs for preventing and treating Proteus in aquatic products, environmental disinfectants, feed additives or aquatic product preservatives for application, which can not only effectively prevent and control the spread and secondary infection of Proteus, but also effectively reduce the mortality rate of aquatic products such as fish and crayfish and the positive rate of Proteus antibodies.

[0037] 2. The phage PF107 can be widely used as an environmental disinfectant in the aquaculture environment. It can not only significantly reduce the content of Proteus in the aquatic product breeding environment, thereby reducing the infection rate of Proteus, but also avoid the problems of antibiotic residues and pathogen drug resistance caused by the use of antibiotics. It can be promoted and applied as a new type of biological environmental disinfectant. The phage PF107 can also be used as a feed additive to disinfect and sterilize the water body and feed in aquaculture farms, avoiding the spread of Proteus from the source and effectively preventing and treating Proteus disease.

[0038] 3. The phage PF107 is obtained from nature and has the characteristics of safe use and environmental friendliness. It will not only cause pollution to the environment, but also harm beneficial bacteria in aquatic products or water bodies. In addition, this phage is easy to be industrially produced and has application advantages in industrial production, which can effectively reduce the production cost of phage-related products. Description of the Drawings Figure 1 It is the test result of the temperature stability of Proteus vulgaris phage PF107; Figure 2 It is the test result of the acid-base stability of Proteus vulgaris phage PF107; Figure 3 It is the test result of the one-step growth curve of Proteus vulgaris phage PF107; Figure 4 It is the electron micrograph of Proteus vulgaris phage PF107; Figure 5 It is the preservation effect of Proteus vulgaris phage PF107 on Procambarus clarkii at 4°C; Figure 6 It is the preservation effect of Proteus vulgaris phage PF107 on Procambarus clarkii at 25°C. Detailed Embodiments To make the technical means, creative methods, purposes, and effects achieved by the present invention easy to understand and use, the present invention will be described in detail and clearly below in combination with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to 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.

[0040] Example 1: Isolation and purification of a Proteus vulgaris phage for aquatic products 1. Experimental method: (1) Resuscitation culture of the host bacterium and preparation of the proliferation solution The host bacterium of the Proteus phage was isolated from the diseased Procambarus clarkii (i.e., crayfish) in a shrimp pond in Hubei Province and is a clinical strain of Proteus vulgaris. The isolated host bacterium was inoculated on an SS medium and cultured overnight at 37°C to obtain single colonies. A single colony was picked and cultured in 5 ml of nutrient broth medium in a shaker at 37°C and 170 rpm for 16 h to obtain a fresh Proteus bacterium solution, which was used as the host bacterium culture.

[0041] (2) Phage isolation and purification An appropriate amount of sludge sample from a diseased shrimp pond in Hubei Province was taken into a conical flask, an appropriate amount of broth medium was added, and a fresh Proteus bacterium solution was added. The mixture was shaken and cultured at 37°C and 170 rpm for 12 h, centrifuged at 11,000 rpm for 5 min, and then filtered through a 0.22 μm sterile microporous filter membrane to obtain a phage proliferation solution; The phage stock solution was diluted 10-fold serially. Appropriate gradient phage dilution solutions were taken and mixed evenly with Proteus for aquatic products one by one at a ratio of 1:1. After incubating at 37°C for 5 min, 200 μL of the mixed solution was aspirated and placed in the upper agar (agar concentration 0.7%), and after mixing, it was quickly poured onto the lower agar (agar concentration 1.5%) petri dish, shaken well and left standing until the medium solidified. After incubating in an inverted position in a 37°C incubator for 4 - 6 h, a double-layer plate with phage plaques formed was obtained.

[0042] Pick a single plaque from the double-layer agar medium with plaques formed and place it in 1 mL of NB broth. Incubate it in a constant-temperature shaker at 37°C and 170 rpm for about 30 min to obtain a phage leaching solution. Take the phage leaching solution and the corresponding host bacteria proliferation solution for plaque formation. Mix them evenly at a ratio of 1:1 (incubate at 37°C for 5 min), pipette 200 μL and place it on the upper agar. After mixing evenly, quickly pour it onto the lower agar petri dish, shake well and let it stand until the medium solidifies. Incubate it upside down in a 37°C incubator for 4 - 6 h, and then obtain a double-layer plate with plaques formed again. Use sterilized forceps to pick a single plaque from the double-layer medium with plaques formed and place it in 1 mL of LB broth. Incubate it in a constant-temperature shaker at 37°C and 170 rpm for about 30 min to obtain a phage leaching solution. Repeat the above steps 3 times to obtain a purified leaching solution of the cross-species lytic phage.

[0043] (3)Phage titer determination Take equal amounts of the purified phage leaching solution and the host bacteria proliferation solution and place them in 5 mL of liquid NB medium. Incubate them at 37°C and 170 rpm with shaking until the liquid becomes clear. Centrifuge the clear liquid at 11000 rpm for 10 min, take the supernatant, filter it through a 0.22 μm sterile microporous filter membrane to obtain a phage proliferation solution, and use the double-layer plate method to determine the titer of the newly isolated phage.

[0044] (4)Phage lysis spectrum determination Select 20 strains of Proteus isolated from aquatic products preserved in the laboratory (isolated from aquatic animals such as Procambarus clarkii and turtles mentioned above), and determine the lysis rate of the above-mentioned isolated and purified phages by the double-layer plate method to screen phages with higher lysis rates.

[0045] 2. Experimental results: As shown in the results of Table 1, according to the above experimental method, 5 strains of Proteus phages were screened using Proteus isolates from aquatic products, and they were sequentially numbered PF106 - PF110. All 5 strains of Proteus phages formed clear plaques on the double-layer agar medium plate, without a halo around them, and the edges were clearly visible, with a diameter of about 0.5 mm - 1 mm. The titers of these 5 phages were between 4.32×10 8 PFU / mL and 3.87×10 9 PFU / mL, and the lysis rates for Proteus were between 20% and 75%. According to the titer and the lysis rates for the two host bacteria, the phage PF107 with a higher titer and a higher lysis rate was selected as the subsequent experimental object.

[0046] Table 1 Titers of 5 strains of Proteus phages and lysis rates for Proteus (PFU / mL)

[0047] Example 2: Morphological Observation and Identification of Proteus phage PF107 and Determination of Multiple Biological Characteristics 1. Electron Microscopy Observation of Phage (1)Experimental Method Take 20 µL of phage sample and drop it on a copper grid with a carbon-coated film. Wait for it to precipitate naturally for 15 min. After slightly blotting it dry with filter paper, stain it with 2% (W / V) phosphotungstic acid (PTA) for 1 - 2 min, then slightly blot it dry with filter paper. After drying, observe and take pictures under a transmission electron microscope.

[0048] (2)Experimental Results and Analysis As Figure 4 shown, phage PF107 has a polyhedral head structure and a contractile tail. The head is 95 - 97 nm long and 91 - 93 nm wide, and the tail is about 143 - 145 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of phage PF107 in this application conforms to the characteristics of myoviruses and belongs to myoviruses.

[0049] 2. Temperature Stability (1)Experimental Method Respectively place the proliferated solution of Proteus phage PF107 at 5.0×109 PFU / mL at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃. For each temperature, there are three parallel samples. After incubating for 20 min, 40 min, and 60 min respectively, immediately cool the samples in an ice bath, and then use the double-layer plate method to detect the phage titer after different temperature treatments. Use temperature as the abscissa and the logarithm of phage titer as the ordinate to draw the thermal stability curve of PF107.

[0050] (2)Experimental Results and Analysis The results of the thermal stability curve are as Figure 1 shown. The results show that phage PF107 still maintains its original activity after acting for 60 min within 40 - 60℃; after acting at 80℃ for 20 min, the phage still contains 2.52×103 PFU / mL of active phage. Thus, it can be seen that Proteus phage PF107 has good temperature stability.

[0051] 3. Acid - Base Stability (1)Experimental Method Take three test tubes containing 4.5 mL of NB broth medium with different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) respectively. Place the test tubes containing the broth with different pH values in a water bath at 37°C for 30 min. After the temperature of the broth in the test tubes reaches stability, add 0.5 mL of phage PF107 at 3.0×109 PFU / mL respectively, shake well, and incubate in a constant temperature water bath at 37°C for 1 h, 2 h, and 3 h. After the specified time of action, add an appropriate amount of NaOH or HCl to the phage-containing mixture to adjust the pH to about 7.0 to terminate the action of acids and bases on the phage. Take each termination solution and use the double-layer plate method to determine the change in phage titer at different pH values and different action times. Use the lg value of phage titer as the vertical coordinate and pH value as the horizontal coordinate to draw the phage acid-base stability curve.

[0052] (2)Experimental results and analysis The results are as Figure 2 shown. In the range of pH 5.0 - 10.0, the titer of phage PF107 can still remain at 109 PFU / mL, and its activity is relatively stable; when acting for 3 h at pH 4.0, the titer is at 103 PFU / mL; when acting for 1 h at pH 11.0, the titer is at 106 PFU / mL. It can be seen that phage PF107 has strong stability in a relatively wide pH range and can resist a certain range of strong acid and strong base environments.

[0053] 4. Determination of the one-step growth curve (1)Experimental method Adjust the phage titer and bacterial concentration to a multiplicity of infection of 10. Take 1 mL each of the adjusted phage proliferation solution and bacterial solution, mix well (start timing at this time), incubate at 37°C for 5 min, centrifuge at 13000 rpm for 30 s, discard the supernatant, and then wash once with 5 mL of NB broth and centrifuge to discard the supernatant. Add 5 mL of NB broth medium preheated to 37°C to fully resuspend the precipitate, and quickly place it in an oscillating culture at 37°C and 170 rpm. Starting from 0 time, take out 200 μL each time, centrifuge at 11000 rpm for 5 min, take the supernatant, and use the double-layer plate method to determine the phage titer at different time points. Do 3 replicates for each gradient and calculate the average value. Use the infection time as the horizontal coordinate and the phage titer in the infection system as the vertical coordinate to draw the one-step growth curve, obtain the latent period and burst period of phage PF107, and calculate the burst size.

[0054] Burst size = total number of phages at the end of the phage burst / total number of bacteria at the beginning of the burst (2)Experimental results and analysis The results are as Figure 3As shown, after the phage PF107 infects the host bacterium, the latent period is about 0 - 30 min. Within 40 - 100 min after the phage infects the host bacterium, the number of phages increases sharply. After 100 min, the titer growth tends to be stable, and the titer can reach 109 PFU / mL at this time. It can be seen that the burst period of phage PF107 is about 60 min, and the burst size is 10 6 PFU / mL.

[0055] Determination of the lysis spectrum of Proteus vulgaris phage in Example 3 1. Experimental method Forty strains of Proteus vulgaris from aquatic sources (including fish, frogs, turtles, soft-shelled turtles, etc.) preserved in the laboratory were selected. Take the bacterial liquid from the cryopreservation tube and streak it on an SS plate for resuscitation, and then pick single colonies for proliferation culture to obtain fresh bacterial liquid. Prepare fresh PF107 phage proliferation liquid and vB_PmiM_PF07 phage proliferation liquid, and use the double-layer plate method to detect the lysis spectrum of the phage against 40 strains of Proteus vulgaris. The numbers of the 40 strains of Proteus vulgaris are BS23 - 42 and BD56 - 75.

[0056] 2. Experimental results and analysis The experimental results are shown in Table 2. The results show that Proteus vulgaris phage PF107 can lyse 35 out of 40 strains of Proteus vulgaris, with a lysis rate of 82.5%, and vB_PmiM_PF07 can lyse 24 strains, with a lysis rate of 60.0%. The results indicate that Proteus vulgaris phage PF107 has a broad lysis spectrum and has great potential in the prevention and control of aquatic Proteus diseases.

[0057] Table 2 Lysis spectra of PF107 and vB_PmiM_PF07 against 40 strains of Proteus vulgaris from different sources

[0058] Genome-wide analysis of Proteus vulgaris phage in Example 4 The genome of phage PF107 was extracted for whole-genome sequencing and sequence analysis, and the results are as follows: The full length of the PF107 genome is 161,373 kb, the G+C content is 31.05%, and the contents of bases C, G, A, and T are 14.57%, 16.48%, 35.03%, and 33.92% respectively. The online annotation results of the whole genome by RAST showed that the genome contains 249 open reading frames (ORFs), among which 85 ORFs have known functions and 164 ORFs are hypothetical protein expression sequences. Among these 85 open reading frames, 82 structural proteins were found, mainly including phage structural and packaging proteins (head protein, tail protein, tape measure protein, tail fiber protein, neck protein, capsid protein, large subunit of terminase, and small subunit of terminase, etc.), phage lysis-related proteins (lysozyme, perforin), and proteins related to DNA replication and modification (restriction endonuclease, DNA-binding protein, DNA polymerase, HNH endonuclease, and recombination protein, etc.). Analysis by PHASTEST showed that this genome does not contain lysogeny-related genes, and analysis by the online tool CGE server showed that this genome does not contain drug resistance genes and virulence genes.

[0059] In phage PF107: The gene sequence of the DNA polymerase protein is shown as sequence 1 in the sequence listing, and its protein sequence is shown as sequence 2; the gene sequence of the tail fibers protein related to phage host recognition is shown as sequence 3 in the sequence listing, and its protein sequence is shown as sequence 4; the gene sequence of the highly conserved large subunit of terminase protein is shown as sequence 5 in the sequence listing, and its protein sequence is shown as sequence 6.

[0060] Table 5 Gene Sequence Information Table

[0061] Example 5 Safety of Using Proteus vulgaris Phage on Procambarus clarkii 1. Experimental Method Thirty healthy Procambarus clarkii were selected and randomly divided into 3 groups, with 10 in each group, namely the control group, the spraying group, and the feeding group.

[0062] The Procambarus clarkii used was about 25 g per individual. Well water with sufficient aeration was used as the water source, the water temperature was maintained at about 25 °C, and the dissolved oxygen was above 5 mg / L. Aquatic plants and other shelters were set up, and they were temporarily raised for 5 days before the experiment and then placed in a 1-L aquarium for breeding.

[0063] In the spraying group and the feeding group, the PF107 phage proliferation solution was added respectively to make the final concentration of phage in the water body 5×109 PFU / ml. The control group was not treated with phage, and other treatments were the same.

[0064] Eight days after the start of the experiment, the growth status of Procambarus clarkii in each group was observed, and autopsy was performed to observe the lesions of the hepatopancreas and intestine.

[0065] 2. Experimental results and analysis The experimental results showed that there were no significant differences among the results of the control group, the mixing group, and the spraying group. During the feeding process, the mental state and feeding of Procambarus clarkii were normal, and no diseases or toxicity conditions were observed. After autopsy, the hepatopancreas and intestine were both normal. This result indicates that applying Proteus phage to aquatic products by spraying and mixing methods has high safety.

[0066] Example 6 Therapeutic effect of Proteus phage on Procambarus clarkii infected with Proteus vulgaris 1. Experimental method Eighty Procambarus clarkii with healthy growth and uniform quality were selected. The weight of each Procambarus clarkii was about 25 g. Well water with sufficient aeration was used as the water source, the water temperature was maintained at about 25 °C, and the dissolved oxygen was above 5 mg / L. Aquatic plants and other shelters were set up and the crayfish were temporarily cultured for 5 days before the experiment, and then placed in a 25-L aquarium for feeding.

[0067] The above-mentioned 80 Procambarus clarkii were randomly divided into four groups, with 20 in each group, namely the positive control group, the negative control group, the mixing group, and the spraying group.

[0068] Among them, in the positive control group, the mixing group, and the spraying group, a total of 0.04 ml of Proteus vulgaris bacterial solution at 5×107 CFU / tail was injected into the articular synovial membrane of Procambarus clarkii, and the negative control group was injected with 0.04 ml / tail of sterile normal saline. Within 1 h after challenge, the mixing group applied phage PF107 by mixing, and the spraying group applied the drug by spraying the phage proliferation solution. The final concentration of phage PF107 in the water body was controlled at 5×10 7 PFU / mL in the mixing group and the spraying group, while the positive control group and the negative control group did not use phage.

[0069] Eight days after the start of the experiment, the growth status of Procambarus clarkii in each group was observed, and autopsy was performed to observe the lesions of the hepatopancreas and intestine, and the positive rate of Procambarus clarkii infected with Proteus vulgaris in each group was counted.

[0070] 2. Experimental results and analysis The results are shown in Table 3. Compared with the positive control group, the mixing group and the spraying group could significantly reduce the infection of Proteus vulgaris, and the positive rates of Proteus vulgaris in the two groups were reduced by 85.00% and 80.00% respectively. This result indicates that phage PF107 can have an effective therapeutic effect on Procambarus clarkii infected with Proteus vulgaris, and both the mixing method and the spraying method can be selected for use.

[0071] Statistical Table of the Treatment of Proteus Infection in Shrimp with Proteus Phage

[0072] Example 7 Therapeutic Effect of Proteus on Tilapia Infected with Proteus vulgaris 1. Experimental Method: Before the experiment began, tilapia were acclimated for 2 weeks. Then, 60 healthy tilapia with uniform growth and quality were selected and randomly divided into four groups of 15 each, namely the positive control group, negative control group, feeding group, and spraying group.

[0073] Among them, the fish in the positive control group, feeding group, and spraying group were injected with 0.1 ml of Proteus bacterial solution at a concentration of 5×10 7 CFU / tail into the pectoral fin, and the negative control group was injected with 0.1 ml of normal saline. The positive control group and negative control group did not use phage. Phage was used within 1 h after infection. The final concentration of phage in the water body of the feeding group and spraying group was controlled at 5×10 7 PFU / ml.

[0074] During the experiment, the fish were fed in an apparent satiation manner, once at 8:30 and once at 17:00 every day. The water temperature was about 25 °C, the dissolved oxygen content was greater than 6 mg / L, and the ammonia nitrogen content was less than 0.04 mg / L.

[0075] Seven days after the start of the experiment, the mortality rate of tilapia in each group and the positive rate of Proteus infection in the internal organs were counted. The growth status and feeding situation of tilapia were observed, and the pathological changes of the internal organs were observed by dissection to count the positive rate of Proteus infection.

[0076] 2. Analysis of Experimental Results From the results in Table 4, it can be seen that the feeding and spraying methods have a therapeutic effect on tilapia infected with Proteus vulgaris, greatly improving the survival rate of tilapia and reducing the positive rate of Proteus.

[0077] Statistical Table of the Treatment of Proteus Infection in Tilapia with Proteus Phage

[0078] Example 8 Fresh-keeping Effect of Proteus Phage on Procambarus clarkii 1. Experimental Method (1) Preparation of Experimental Materials: In this experiment, the peeled tails of Procambarus clarkii (crayfish) were used, which were purchased from a supermarket in Chengyang District, Qingdao, Shandong. After grinding 1 g of sample tissue, it was added to NB medium for enrichment culture. Then, the proliferated liquid was taken for PCR test of Proteus, and negative results indicated that this product did not carry Proteus. In the laminar flow hood, the crayfish tails were placed in a culture dish, 75% disinfected alcohol was taken and sprayed onto the surface of the tails, and then irradiated with ultraviolet light for 30 min. After the alcohol completely volatilized, it was reserved for use.

[0079] (2)Insurance treatment The crayfish tails were divided into 3 groups: negative control group, positive control group and treatment group, with 2 replicates in each group. The temperature conditions were set at 4℃ and 25℃ respectively. Among them, 10 μL of normal saline was smeared on the surface of the crayfish tails in the negative control group; 2×10 6 CFU / mL of Proteus bacterial liquid was smeared on the surface of the crayfish tails in the positive control group and the treatment group respectively, so that the final concentration of Proteus on the crayfish tails was 1×10 5 CFU / g; in the treatment group, 10 μL of phage PF107 solution with a concentration of 1×10 9 PFU / mL was also smeared on the surface of the crayfish tails for treatment. Two hours after the experiment, the crayfish tails in each group were mixed and ground evenly, and colony counting was carried out on the SS medium plate, and the data were recorded.

[0080] 2. Analysis of experimental results The experimental results are as Figure 5 and Figure 6 shown. The concentration of Proteus in the positive control group increased under both 4℃ and 25℃ environments. The phage treatment group could effectively inhibit the reproduction of Proteus within 2 hours under the conditions of 4℃ and 25℃. Under the conditions of 4℃ and 25℃, the Proteus in the phage treatment group decreased by 3.8 and 3.7 (lgA, PFU / mL) respectively compared with the positive control group. The experimental results show that phage PF107 can effectively inhibit the growth of Proteus on the crayfish tails and can effectively remove Proteus.

[0081] Example 9 Storage stability 1. Experimental method The proliferated liquid of phage PF107 with a concentration of 3×10 9 PFU / mL was stored at 4℃, 18℃ and 30℃ respectively, and the phage titers at different temperatures were regularly measured at 1 month, 3 months, 6 months and 12 months after storage.

[0082] 2. Experimental results and analysis The titer of Proteus phage PF107 did not change significantly after being stored at 4°C for 12 months. The titer decreased by one gradient after being stored at 18°C for 12 months and decreased by two gradients after being stored at 30°C for 12 months, indicating that Proteus phage PF107 has good storage stability.

[0083] Table 5 Storage stability of Proteus phage PF107 (PFU / mL)

[0084] It can be understood that for those of ordinary skill in the art, equivalent substitutions or changes can be made according to the technical solution of the present invention and the inventive concept of the present invention, and all such changes or substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A strain of common Proteus phage from aquatic sources ( Proteusvulgaris phage ) PF107, characterized in that, Its deposit number is CGMCC No.46161.

2. A bacteriophage composition, characterized in that: Including the aquatic-derived Proteus vulgaris PF107 as described in claim 1.

3. Use of the aquatic-derived Proteus vulgaris PF107 as claimed in claim 1 or the bacteriophage composition as claimed in claim 2 in the preparation of drugs, feed additives, environmental disinfectants or aquatic product preservatives for preventing and treating diseases caused by Proteus infection.

4. The use according to claim 3, characterized in that: The disease caused by Proteus infection is aquatic Proteus disease caused by Proteus infection.

5. A bacteriophage pharmaceutical preparation, characterized in that: Its effective ingredients include the aquatic-derived Proteus vulgaris PF107 as claimed in claim 1 or the bacteriophage composition as claimed in claim 2.

6. The bacteriophage pharmaceutical preparation according to claim 5, characterized in that The bacteriophage drug preparation is in the form of a feed-mixing dosage form or a spraying dosage form.

7. An aquatic feed additive or water additive, characterized in that: Its effective ingredients include the aquatic-derived Proteus vulgaris PF107 as claimed in claim 1 or the bacteriophage composition as claimed in claim 2.

8. An environmental disinfectant, characterized in that The active ingredients include the aquatic Proteus vulgaris PF107 as claimed in claim 1 or the bacteriophage composition as claimed in claim 2; preferably, the concentration of the bacteriophage is 1×10 8 PFU / ml and above.

9. Use of the environmental disinfectant according to claim 8 in the environmental disinfection of aquaculture farms, characterized in that: The application method is as follows: the environmental disinfectant is used to disinfect aquaculture water, aquaculture environment, feed or feeding equipment against Proteus.

10. A preservative for aquatic products, characterized in that: It includes the aquatic-derived Proteus vulgaris PF107 as described in claim 1 or the phage composition as described in claim 2.

Images