Proteus mirabilis bacteriophage PF09 and bacteriophage composition and application thereof

Abstract

The invention belongs to the technical field of bacteriophages, and discloses a proteus mirabilis bacteriophage PF09 and a bacteriophage composition and application thereof, the preservation number of the proteus mirabilis bacteriophage PF09 is CGMCC NO.45897, the proteus mirabilis bacteriophage PF09 is preserved in China General Microbiological Culture Collection Center (CGMCC) on March 22, 2024, the proteus mirabilis bacteriophage PF09 has a wide lysis spectrum, and the proteus mirabilis bacteriophage PF09 can be used for preparing a proteus The compound has a splitting property on proteusbacillus vulgaris of different sources, particularly has a specific splitting advantage on pet proteusbacillus vulgaris, can be used as an active ingredient for preparing medicines, feed additives and disinfectants for preventing and treating pet diseases caused by proteusbacillus vulgaris infection, provides a more effective prevention and control scheme for the problem of prevention and control of pet proteusbacillus vulgaris infection, and can be used for preventing and controlling the pet proteusbacillus vulgaris infection. And the problems of antibiotic residues and drug resistance of pathogenic bacteria caused by the use of antibiotics are also avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriophages, and in particular to a Proteus mirabilis bacteriophage PF09, a bacteriophage composition and applications thereof. Background Art

[0002] Proteus belongs to the Gram-negative bacteria of the Enterobacteriaceae family and is a common commensal bacteria of the gastrointestinal flora. Hauser first reported and identified Proteus in the late 19th century. Proteus can survive in a variety of environments, including soil, sewage and other environments. It is a multi-host pathogen and one of the most important intestinal conditional pathogens besides Escherichia coli. It is the main pathogen of urinary tract infection after Escherichia coli and one of the most common causes of catheter-associated urinary tract infection (CAUTI). The formation of kidney stones and bladder stones may be related to infection with Proteus mirabilis. It can infect a variety of animals including humans and cause different degrees of disease. At present, the harm of Proteus mirabilis to livestock and poultry such as chickens, pigs, and sheep, as well as wild animals, has been reported in China, but there are few studies on Proteus mirabilis in companion animals such as dogs and cats.

[0003] In order to prevent the adverse effects of bacterial resistance on the breeding industry and public health, my country promulgated relevant laws in 2020 to ban or restrict the use of antibiotics and actively seek appropriate alternatives to promote animal intestinal health. This has become an urgent problem that the poultry industry needs to solve. In the context of banning antibiotics, bacteriophages have received widespread attention due to their antibacterial effects.

[0004] Since bacteriophages are obligate parasites of host bacteria, they are highly specific and only destroy the corresponding host bacteria, have no effect on other bacteria, and die as the host bacteria are eliminated, so they have the advantages of being safe and harmless to use. When bacteriophages infect host bacteria, the depolymerases and lysins encoded by them can degrade polysaccharides and peptidoglycans, destroy the extracellular polymer structure, and lyse the biofilm.

[0005] At present, proteus phages have been developed one after another, but there is no phage that can be effectively used to prevent and treat proteus diseases in pets. Therefore, the prior art needs to be further improved. Summary of the invention

[0006] In view of the above problems, the present invention provides a Proteus mirabilis phage PF09 and a phage composition and application thereof. The Proteus mirabilis phage PF09 has the characteristics of high titer, acid and alkali resistance, and a wide lysis spectrum, and has a significant effect in preventing and controlling Proteus infection in pets. It can be used to prepare medicines, feed additives and disinfectants for preventing and treating pet diseases caused by Proteus infection, and is used to effectively prevent and control Proteus infection.

[0007] To solve the above problems, this application provides the following technical solutions:

[0008] In a first aspect, the present application provides a Proteus mirabilis phage Proteus phage, whose deposit number is CGMCC NO.45897.

[0009] The Proteus phage was isolated from pet feces samples in Shandong Province and was deposited in the General Microbiology Center of China Culture Collection Administration on March 22, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.45897.

[0010] In the present application, bacteriophage PF09 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 bacteriophages are very susceptible to mutations during replication, mutants of the above bacteriophages are also within the scope of protection requested in the present application. The sequence of bacteriophage PF09 can be obtained by sequencing the biological materials deposited according to the present invention using 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 bacteriophage provided by the present invention.

[0011] The miraculous Proteus phage PF09 has a wide lysis spectrum and has lysis properties for Proteus from different sources. The lysis rate of the 40 strains of Proteus from different sources in the experiment is as high as 90%, and the lysis performance of 30 strains of Proteus from pets is especially better, with a lysis rate of up to 93.33%, which is much higher than the existing Proteus phage VB_PMIM_PF07 (the lysis rate is only 66.67%, and the phage is disclosed in the patent with publication number CN116286678A). The experimental results show that the phage PF09 has a specific lysis advantage for pet Proteus and can be effectively used in the prevention and treatment of pet variant bacillus disease.

[0012] The mirabilis phage PF09 has good acid-base tolerance. In the pH range of 3.0 to 10.0, the titer of the mirabilis phage PF09 is maintained at 10 10 pfu / mL, stable activity. At pH 2.0 for 2h, the titer is 10 9 pfu / mL, and can resist a certain strong acid environment; after 3 hours at pH 11, the titer of the phage was 10 9 Therefore, bacteriophage PF09 has strong stability in acidic environment and can adapt to strong acid environment with low pH.

[0013] The mirabilis phage PF09 also has good high temperature tolerance. The titer of the mirabilis phage PF09 is stable at 40℃~60℃ for 1h, and the titer decreases by 4 gradients at 80℃ for 60min, indicating that it has high thermal stability. Therefore, the mirabilis phage PF09 has good environmental adaptability and has a certain tolerance to adverse environments such as high temperature and acid and alkali.

[0014] In a second aspect, the present application also provides a phage composition, which includes the aforementioned Proteus mirabilis phage PF09.

[0015] 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 complement each other's advantages, the Proteus mirabilis phage PF09 and other phages can be used in combination to expand the bactericidal spectrum and improve the prevention and control effect.

[0016] Preferably, the phage composition comprises: the aforementioned Proteus mirabilis phage PF09 and Proteus phage VB_PMIM_PF07. The deposit number of the Proteus phage VB_PMIM_PF07 is CGMCC NO.23812, and its specific information can be found in the patent publication number CN116286678A.

[0017] In addition, the above-mentioned Proteus mirabilis phage PF09 can also be combined with other different types of phages (inhibiting different pathogens that cause the same disease) for the prevention and treatment of the same type of disease, such as acute diarrhea caused by mixed infection of different pathogens.

[0018] In a third aspect, the present application also provides the use of the above-mentioned Proteus mirabilis phage PF09 or the above-mentioned phage composition in the preparation of drugs, feed additives and disinfectants for preventing and treating pet diseases caused by Proteus infection.

[0019] Preferably, pet Proteobacterium diseases include: secondary pyoderma, otitis externa, otitis media, etc.

[0020] 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.

[0021] In a fourth aspect, the present application further provides a phage medicine for pets or a phage health product for pets, the active ingredients of which include the aforementioned Proteus mirabilis phage PF09 or the aforementioned phage composition.

[0022] Optionally, the phage drug for pets or phage health products for pets also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant stimulation to the organism and does not eliminate the biological activity and characteristics of the administered active component. If necessary, other conventional additives may be added, for example, antioxidants, buffers, and antibacterial agents. When combined with a diluent, a dispersant, a surfactant, a binder, and / or a lubricant, the phage drug or phage health product for pets of the present invention may also be prepared into injections, parenteral dosage forms, oral dosage forms (e.g., aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilized agents.

[0023] Correspondingly, preferably, the phage medicine for pets or the phage health product for pets can be in the form of an oral dosage form, an external dosage form or a parenteral dosage form.

[0024] The application method of the phage medicine or phage health product for pets is to add it to pet drinking water or pet feed for feeding, or to administer it orally, subcutaneously, or intramuscularly to the pet. Any of the above methods can prevent and treat Proteus diseases and improve the survival rate of pets.

[0025] Preferably, the phage medicine or phage health product for pets also includes other antibacterial or bactericidal active ingredients.

[0026] In a fifth aspect, the present application also provides a pet feed additive or a pet water additive, which includes the aforementioned Proteus mirabilis phage PF09 or the aforementioned phage composition.

[0027] In practical applications, the pet feed additive or pet water additive can be mixed with feed or added to pet water for feeding, thereby disinfecting pet water and feed. Since the bacteriophage has good acid resistance and can survive in the environment of gastric acid, the above feeding method can effectively prevent and treat pet Proteus disease.

[0028] Optionally, the pet water additive or feed additive also contains other active ingredients for inhibiting or eliminating bacteria in water; the drinking water additive and feed additive are in the form of liquid dosage form, powder dosage form or solid dosage form, but are not limited to the above three dosage forms.

[0029] In a sixth aspect, the present application further provides a disinfectant, the active ingredient of which includes the aforementioned Proteus mirabilis phage PF09 or the aforementioned phage composition. Preferably, the concentration of phage PF09 in the disinfectant is 10 8 PFU / mL or above.

[0030] Preferably, other active ingredients for inhibiting or eliminating bacteria in the environment are also included.

[0031] In a seventh aspect, the present application also provides the use of the above disinfectant in the disinfection of a pet breeding environment, and the application method is: the disinfectant is sprayed or soaked to disinfect the pet breeding environment, pet feed, snacks or toys against Proteus. Pet breeding environment types include: pet breeding houses, pet hospitals, and home breeding environments, etc.

[0032] The pet breeding environment includes: pet cages, drinking fountains, feeding tools (food troughs and water troughs), and excrement, etc. The application method includes but is not limited to disinfecting and decontaminating the surface of the pet's living environment by liquid immersion, spraying, or combined use with an aqueous carrier (such as fumigation), and disinfecting and preserving feed. This disinfectant is safe to use and can be used to replace antibiotics or traditional disinfection products without causing harm to pets and humans.

[0033] In an eighth aspect, the present application also provides a Proteus detection kit, which includes the aforementioned Proteus mirabilis phage PF09. Based on the specific lysis of Proteus mirabilis phage PF09 to Proteus hosts, the phage can be used as an effective component of the detection kit for identifying Proteus in a sample, or for screening samples, which can effectively ensure the sensitivity of the detection. The detection kit is in the form of a test paper or a conventional kit.

[0034] The present invention has the following beneficial effects:

[0035] 1. The present invention provides a virulent Proteus mirabilis phage PF09 isolated from pet feces. The phage has a wide lysis spectrum and has lysis properties against Proteus from different sources, especially has a specific lysis advantage against Proteus in pets, and has high titer and acid and alkali resistance, and can effectively prevent and treat diseases caused by Proteus infection in pets. It can be used as an active ingredient to prepare drugs, feed additives and disinfectants for preventing and treating pet diseases caused by Proteus infection. In terms of the prevention and control of Proteus infection in pets, it can provide a variety of more effective prevention and control schemes, and also avoid the problems of antibiotic residues and pathogen resistance caused by the use of antibiotics.

[0036] 2. The Proteus mirabilis phage PF09 is isolated from nature and has the characteristics of safe use and green environmental protection. It will not pollute the environment, nor harm the beneficial bacteria in the pet's body. In addition, the phage has a high burst volume and strong reproductive ability, and has application advantages in industrial production, which can effectively reduce the production cost of the phage-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1This is a plaque image of Proteus mirabilis phage PF09;

[0038] Figure 2 This is an electron microscope image of Proteus mirabilis bacteriophage PF09;

[0039] Figure 3 The test results of pH stability of Proteus mirabilis bacteriophage PF09 are as follows;

[0040] Figure 4 The test results of temperature stability of Proteus mirabilis bacteriophage PF09;

[0041] Figure 5 This is the one-step growth curve of Proteus mirabilis phage PF09. DETAILED DESCRIPTION

[0042] 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.

[0043] Example 1 Isolation and purification of Proteus mirabilis bacteriophage PF09

[0044] 1. Experimental methods

[0045] (1) Resuscitation and cultivation of host bacteria

[0046] A strain of Proteus mirabilis preserved in this laboratory was selected, and the freezing liquid was dipped into a sterilized inoculum to streak on SS culture medium for recovery, and cultured in a constant temperature incubator at 37°C for 18 to 24 hours to obtain a single colony; a single colony was picked and inoculated into 5 mL of NB broth, and cultured at 37°C with shaking at 170 rpm / min for 16 hours to obtain a fresh Proteus mirabilis bacterial liquid.

[0047] (2) Isolation of bacteriophage

[0048] Take an appropriate amount of sewage, pet feces and other samples from Shandong area into a bubble sample bottle, add an appropriate amount of broth culture medium, add the above-mentioned activated Proteus mirabilis bacterial solution, put it into 37°C 170rpm / min shaking culture for 12h, centrifuge it at 11000rpm for 5min, and then filter it with a 0.22μm sterile microporous filter membrane to obtain a phage proliferation solution; dilute the phage stock solution by 10 times, take appropriate gradient phage dilution liquid and mix it with Proteus mirabilis bacterial solution in a ratio of 1:1, incubate it at 37°C for 5min, draw 200μL of the mixed solution and place it in the upper agar (agar concentration is 0.7%), mix it and quickly pour it onto the lower agar (agar concentration is 1.5%) plate, shake it and place it flat until the culture medium solidifies, place it in a 37°C incubator and culture it upside down for 4-6h to obtain a double-layer plate with plaque formation.

[0049] (3) Phage purification

[0050] Pick a single plaque from the double-layer agar medium where plaques are formed, and place it in 1 mL of NB broth and culture it in a constant temperature shaker at 37°C, 170 rpm for about 30 minutes to obtain a phage extract. Take the phage extract and the proliferation liquid of Proteus mirabilis (hereinafter referred to as the host bacteria) and mix them evenly in a ratio of 1:1 (incubate at 37°C for 5 minutes), draw 200 μL and place it on the upper agar plate, mix it evenly, and then quickly pour it on the lower agar 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, place it in 1 mL of NB broth, and culture it in a constant temperature shaker at 37°C, 170 rpm for about 30 minutes to obtain a phage extract. Repeat the above steps 3 times to obtain a purified phage extract.

[0051] (4) Phage proliferation and titer determination

[0052] Take an equal amount of purified phage extract and host bacteria proliferation liquid in 5 mL liquid NB medium, and culture at 37°C, 170 rpm 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.

[0053] 5. Experimental results and analysis

[0054] According to the above experimental method, a Proteus mirabilis isolate was used to screen a Proteus mirabilis phage, which was named PF09.

[0055] like Figure 1As shown in the figure, the Proteus mirabilis phage PF09 formed a clear plaque on the double-layer agar medium plate, with no halo around it and a clearly visible edge, with a diameter of about 1 mm. The titer of the phage was 3.2×10 10 PFU / mL.

[0056] Example 2 Morphological Observation and Identification of Proteus mirabilis Phage PF09

[0057] 1. Experimental methods

[0058] Copper mesh production and electron microscope observation:

[0059] 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.

[0060] 2. Identification results

[0061] Electron microscope photos Figure 2 As shown, the bacteriophage PF09 has a polyhedral head structure and a contractile tail. The head is 85-90 nm wide and 110-120 nm long, and the tail is about 125-130 nm 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 Myotailed Phage family and belongs to the Myotailed Phage.

[0062] Example 3 Whole genome analysis of Proteus mirabilis phage PF09

[0063] The genome of phage PF09 was extracted, and whole genome sequencing and sequence analysis were performed. The results are as follows:

[0064] (1) The genome is 161781 bp long, with a G+C content of 31.06%, and the base C, G, A, and T contents of 16.46%, 14.60%, 33.86%, and 35.09%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 250 open reading frames (ORFs). Among these 250 open reading frames (ORFs), structural proteins were found to mainly include phage structural and packaging proteins (tail protein, neck protein, head protein, capsid protein, binding protein, phage fiber protein, terminal enzyme large subunit, etc.), phage-like proteins, regulatory proteins, and other functional proteins. The online tool CGE server analysis showed 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.

[0065] (2) In the genome of the phage: the highly conserved gene sequence of the terminase large subunit is shown in Sequence 1 in Sequence Listing 1, and the sequence of the perforin protein gene associated with phage lysis is shown in Sequence 2 in Sequence Listing 1. The sequence detection of the above two conserved genes can be used to quickly identify the Proteus mirabilis phage PF09.

[0066] Table 1 Gene sequence information

[0067] type Start stop Function Sequence 1 Peg143 105976 104153 terminase large subunit Sequence 2 Peg125 93390 94013 holin

[0068] Example 4 Determination of the lysis spectrum of Proteus mirabilis phage PF09

[0069] 1. Experimental Materials

[0070] Host bacteria: 30 strains of Proteus isolated from pets in different regions of the country and 10 drug-resistant Proteus strains preserved in the laboratory were selected, totaling 40 pathogenic bacteria.

[0071] 2. Experimental methods:

[0072] The above 40 pathogenic strains were used to determine the lysis rate of bacteriophage PF09 against 40 strains of Proteus by the double-layer plate method. The detailed lysis spectrum information is shown in Table 2 below.

[0073] 3. Experimental results and analysis

[0074] From the experimental results of the fragmentation spectra in Table 2 and Table 3, we can see that:

[0075] (1) The mirabilis phage PF09 can lyse 36 of 40 strains of Proteus from different sources, with a lysis rate of up to 90%; the lysis rate of 30 of the 40 Proteus from pets was 93.33%. At the same time, the lysis spectrum of VB_PMIM_PF07 in the previous patent showed that the lysis rate of VB_PMIM_PF07 for Proteus from pets was 66.67%, indicating that the lysis rate of phage PF09 for Proteus from pets is higher, and it has a specific lysis advantage for them.

[0076] (2) The lysis rate of the bacteriophage PF09 of the present application on 10 strains of drug-resistant Proteus is 80%. At the same time, the lysis spectrum of VB_PMIM_PF07 in the previous patent on drug-resistant Proteus shows that the lysis rate of VB_PMIM_PF07 is 40%, which shows that the bacteriophage has a good lysis effect and broad spectrum on Proteus. In order to improve the use effect of the bacteriophage product, the bacteriophage PF09 and the bacteriophage VB_PMIM_PF07 used in the previous patent can be used in combination to improve the lysis spectrum of the bacteriophage product.

[0077] Table 2 Lysis spectrum of Proteus mirabilis phage PF09 against 30 strains of Proteus

[0078]

[0079]

[0080] Table 3 Lysis spectrum of Proteus mirabilis phage PF09 against 10 strains of multidrug-resistant Proteus

[0081] Serial number strain Strain name source Drug resistance PF09 VB_PMIM_PF07 31 F24-100 Proteus mirabilis birds Multidrug resistance + - 32 F24-101 Proteus mirabilis birds Multidrug resistance + + 33 F24-102 Proteus mirabilis birds Multidrug resistance - - 34 F24-103 Proteus mirabilis birds β-lactams + + 35 F24-104 Proteus vulgaris birds Multidrug resistance + - 36 F24-105 Proteus mirabilis birds Multidrug resistance + + 37 F24-106 Proteus mirabilis ox Multidrug resistance + + 38 F24-107 Proteus mirabilis ox Multidrug resistance - - 39 F24-108 Proteus vulgaris pig β-lactams + - 40 F24-109 Proteus mirabilis pig Multidrug resistance + -

[0082] Example 5 Lysis test of Proteus mirabilis phage PF09 on non-host bacteria

[0083] 1. Experimental methods

[0084] Ten strains of Escherichia coli, five strains of Staphylococcus aureus, five strains of Riemerella anatipestifer and five strains of Clostridium perfringens were selected, a total of 25 strains of different types of non-host bacteria, and a lysis spectrum determination experiment of Proteus mirabilis phage PF09 was carried out according to the lysis spectrum determination method in Example 4.

[0085] 2. Experimental results and analysis

[0086] The experimental results showed that no clear plaques were found in the double-layer plates of Proteus mirabilis phage PF09 and 25 strains of non-host bacteria, indicating that the phage could not recognize the above-mentioned 10 strains of Escherichia coli, 5 strains of Staphylococcus aureus, 5 strains of Proteus and 5 strains of Clostridium difficile. This shows that the test phage PF09 has extremely strong host specificity and has no damaging effect on the microbial community. Its specificity can be used to prepare detection kits.

[0087] Example 6 Tolerance of Proteus mirabilis phage PF09 to pH

[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 5×10 10 pfu / mL phage proliferation solution, mix well and incubate in a 37℃ water bath for 1h, 2h, and 3h. After the incubation, 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 an appropriate dilution gradient to determine the titer, and set 2 replicates for each pH value test tube. Draw the phage pH stability curve with pH as the horizontal axis and the logarithm of the phage titer as the vertical axis.

[0090] 2. Experimental results and analysis

[0091] from Figure 3 It can be seen that the titer of phage PF09 is maintained at 10 in the pH range of 3.0 to 10.0. 10 pfu / mL, stable activity; at pH 2.0 for 2h, the titer is 10 9 pfu / mL, and can resist a certain strong acid environment. It still maintains a certain activity at pH 1.0 for 3 hours. After 3 hours at pH 11, the titer of the phage is 10 9 pfu / mL, at pH 13 for 3h, the titer dropped to 10 2 pfu / mL, therefore, bacteriophage PF09 has strong stability in acidic environment and can adapt to strong acid environment with certain low pH.

[0092] Example 7 Temperature tolerance of Proteus mirabilis phage PF09

[0093] 3×10 10 The proliferation liquid of Proteus mirabilis phage PF09 with a concentration of PFU / mL was placed in a water bath at 40℃, 50℃, 60℃, 70℃, and 80℃ for 20min, 40min, and 60min, respectively, with two parallel groups set up at each temperature. The titer of the phage was determined by the double-layer plate method.

[0094] The results showed that the titer of Proteus mirabilis phage PF09 remained at 10 after exposure to 40℃, 50℃ and 60℃ for 1h. 10 PFU / mL; after 20min, 40min and 60min at 70℃, the phage titer decreased by 1, 2 and 3 orders of magnitude respectively. After 60min at 80℃, the titer decreased by 4 levels. The specific results are shown in Figure 4 , indicating that the thermal stability of Proteus mirabilis phage PF09 is relatively high.

[0095] Example 8 One-step growth curve of Proteus mirabilis phage PF09

[0096] 1. Experimental methods

[0097] Take 1 mL of the phage PF09 proliferation solution with a multiplicity of infection of 10 and the fresh proliferation solution of the host bacteria, mix them thoroughly (start timing at this time), incubate at 37°C for 5 minutes, centrifuge at 13000rpm for 30 seconds, use a micropipette to remove the supernatant as much as possible, wash once with 5 mL of NB broth (centrifuge at 13000rpm for 30 seconds), and discard the supernatant. Use preheated NB broth to suspend the precipitate (total volume is 5 mL) and mix thoroughly, quickly place it in a 37°C shaker with 170rpm shaking culture, take out 150 μL at time 0 and every 5 minutes, centrifuge at 10000rpm for 1 minute, dilute it 10 times with NB broth, and use the double-layer plate method to measure the phage titer. Make 3 parallels, take the average value of the results, use the infection time as the horizontal axis and the titer of the phage in the infection system as the vertical axis, draw a one-step growth curve, obtain the incubation period and outbreak period of the phage, and calculate the outbreak volume.

[0098] Outbreak volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak

[0099] 2. Experimental results and analysis

[0100] from Figure 5 The results show that after phage PF09 infects the host bacteria, the phage lysis cycle is about 65 minutes, the incubation period is about 10 minutes, and the phage outbreak period is about 60 minutes; after 75 minutes, the number of phages remains basically unchanged and enters a stable period, at which time the titer can reach 10 10 pfu / mL, and the burst amount of bacteriophage PF09 was 118.

[0101] Example 9 Determination of the Optimal Multiplicity of Infection (MOI) of Proteus mirabilis Phage PF09

[0102] 1. Experimental methods

[0103] Pick a single Proteus colony and inoculate it into 5 ml NB broth medium. Cultivate at 37°C and 170 rpm for 12 to 16 hours to obtain bacterial solution. Determine the bacterial concentration by pouring method. Adjust the bacterial solution concentration to 1×10 9 cfu / mL, 1×10 8 cfu / mL...1×10 5 cfu / mL. The concentration of phage isolated in Example 1 was adjusted to 1×10 5 ~1×10 7 PFU / mL. According to the ratio of the number of phages to the number of bacteria in Table 3, the phages and bacterial solution were added to the NB medium respectively, and the culture was propagated in a shaker at 37°C and 170 rpm until the liquid became clear, and the proliferation time was recorded.

[0104] Take an appropriate amount of clear liquid and centrifuge it at 11000rpm for 10min, take the supernatant, filter it with a 0.22μm sterile microporous filter membrane, and 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.

[0105] 2. Experimental results

[0106] The results in Table 4 show that the PF09 titer reached the highest when the MOI was 0.001:1, 1.3×10 11 PFU / mL, at this time the initial input of phage is small, and the highest reproduction yield can be achieved, which is conducive to large-scale industrial production and has advantages in batch industrial production.

[0107] Table 4 The titer of Proteus mirabilis phage PF09 at different infection multiplicities

[0108]

[0109] Example 10 Storage stability of Proteus mirabilis bacteriophage PF09

[0110] 1. Experimental methods

[0111] 3×10 10 The PFU / mL of Proteus mirabilis phage PF09 were stored at -80℃, 4℃, and 30℃, and the potency was tested regularly to explore its storage stability.

[0112] 2. Experimental results

[0113] The results are shown in Table 5. The potency of Proteus mirabilis phage PF09 remained unchanged when stored at -80°C for one year, decreased by one level when stored at 4°C for one year, decreased by one level when stored at 30°C for three months, and decreased by two levels in one year. This result shows that Proteus mirabilis phage PF09 has good storage stability.

[0114] Table 5 Potency information of Proteus mirabilis phage PF09 stored and tested under different conditions

[0115] -80℃ 4℃ 30℃ 1 month <![CDATA[3×10 10 PFU / mL]]> <![CDATA[3×10 10 PFU / mL]]> <![CDATA[2.1×10 10 PFU / mL]]> 3 months <![CDATA[3×10 10 PFU / mL]]> <![CDATA[2.8×10 10 PFU / mL]]> <![CDATA[5.2×10 9 PFU / mL]]> 6 months <![CDATA[2.8×10 10 PFU / mL]]> <![CDATA[2.5×10 10 PFU / mL]]> <![CDATA[3.3×10 8 PFU / mL]]> 12 months <![CDATA[2.0×10 10 PFU / mL]]> <![CDATA[4.2×10 9 PFU / mL]]> <![CDATA[1.4×10 8 PFU / mL]]>

[0116] Example 11 Clinical Use Effect Verification of Proteus mirabilis Phage PF09

[0117] 1. Experimental Animals

[0118] Thirty healthy pet dogs in a pet hospital were collected. All the pet dogs were small dogs under one year old, half of them were male and half were female. There was no Proteus in their feces.

[0119] 2. Experimental methods

[0120] (1) Thirty healthy pet dogs were randomly divided into three groups, each with 10 dogs (half male and half female), namely the phage treatment group, the Proteus challenge group, and the blank group. The dogs were raised separately, and each group was raised by a designated person to avoid cross contamination.

[0121] (2) Virus challenge treatment: Both the phage treatment group and the Proteus challenge group were gavaged with 0.1 mL of 10 9 CFU / mL of pathogenic Proteus PF09, and the blank group was orally administered with 0.1 mL PBS for isolation. 24 hours after the challenge, 3 mice in each group were randomly selected to collect feces to count the load of Proteus.

[0122] (3) Treatment: 24 hours after infection, the pet dogs in the phage treatment group were given drinking water for 3 hours every day (the final concentration of phage in drinking water was 1×10 8 PFU / mL) for 4 consecutive days. The challenge group and the blank group were fed normally, and the feces of each group of dogs were collected 24 hours after the treatment to calculate the bacterial load.

[0123] 2. Experimental results and analysis

[0124] The results are shown in Table 6. From the table, it can be seen that after 4 days of treatment by adding Proteus mirabilis phage PF09 to the drinking water 24 hours after the pets were challenged with Proteus, the Proteus load of the pet dogs in the phage treatment group had decreased by 6 levels. This result shows that the Proteus mirabilis phage PF09 can effectively treat diseases caused by Proteus in pets.

[0125] Table 6 Statistics of Proteus load in feces

[0126] Phage therapy group Anti-virus group Blank Group 24h after infection <![CDATA[1.3×10 8 (CFU / g)]]> <![CDATA[1.2×10 8 (CFU / g)]]> 0(CFU / g) 24h after treatment <![CDATA[3.7×10 2 (CFU / g)]]> <![CDATA[5.9×10 7 (CFU / g)]]> 0(CFU / g)

[0127] Example 12 Safety Test of Proteus mirabilis Phage PF09

[0128] 1. Experimental methods

[0129] Twenty 6-month-old beagle dogs were selected and divided into a phage group and a control group. The phage group was orally administered with 2 mL of 1×10 10 The proliferation liquid of PFU bacteriophage PF09 was fed and observed for 7 days. The control group was given the same dose of sterile saline orally. The heart, liver, spleen, lung, kidney, brain and intestinal lesions were observed by autopsy. The spirit and feeding status were observed during the feeding process.

[0130] 2. Experimental results and analysis

[0131] During the entire administration period, no disease or toxicity symptoms were observed in the phage group and the control group, and the mental state and feeding were normal. After detailed clinical autopsy observations, the main organs and intestines of the animals were normal in both the phage group and the control group. This shows that phage PF09 is highly safe and has no adverse effects on the animal body.

[0132] Example 13 Application of Proteus mirabilis phage PF09 as a disinfectant in pet breeding farms

[0133] 1. Experimental methods

[0134] In a dog farm, dogs often have skin infections, and the sterilization effect of chemical disinfectants is not obvious. This experiment is designed to use bacteriophage PF09 to spray disinfection on the farm.

[0135] The preparation method of the bacteriophage disinfectant is as follows: the aforementioned bacteriophage liquid and host bacteria are mixed in NB broth medium at a ratio of 1:1, cultured at 37°C and 170 rpm for 4 to 6 hours until the liquid is clear and bacterial fragments are visible. The clear liquid is filtered to remove bacterial fragments and residues, and the bacteriophage titer is determined to be 1×10 10 pfu / ml, the phage liquid was diluted 100 times, and the preparation titer was 1×10 8 pfu / mL of bacteriophage disinfectant, seal and store at 2-8℃ for future use.

[0136] The air disinfection method is used, and the operation is as follows:

[0137] Find two equally sized feeding houses and use the natural sedimentation method to sample the air in front and behind the hatchery. Take the central point and four corners as the test points, with a total of five points. The sampling point is 0.5m from the ground, and the four corners are 1m away from the wall. Place a 9cm diameter SS agar plate at each point. Use two SS agar plates at each sampling point before disinfection, open the lid of the culture dish, and sample for 10 minutes.

[0138] The farm's own fog line was used for disinfection. Experimental and control groups were set up. The experimental group was disinfected with bacteriophage preparations (10 mL / m 3 , phage titer 1×10 8 pfu / mL), and the control group was disinfected with benzalkonium bromide (benzalkonium bromide 1:25 dilution). After 30 minutes of disinfection, two SS agar plates were placed at each of the five sampling points, and the lid of the culture dish was opened for sampling for 10 minutes. The sampling culture dishes before and after disinfection were placed in a 37°C constant temperature incubator for 12 to 24 hours, and the cultured bacteria were counted.

[0139] The total number of colonies was counted according to Ostwald's formula: C = 50,000 N / AT, where C: total number of colonies per cubic meter (CFU / m3); N: number of colonies per dish; A: area of ​​the dish (cm2); and T: sampling time (min).

[0140] 2. Experimental results and analysis

[0141] As shown in Table 7, in the control group, after disinfection with benzalkonium bromide disinfectant, the elimination rate of Salmonella in the hatchery air was 56.76%; in the experimental group, after disinfection with bacteriophage PF09, the elimination rate of Proteus in the feeding house air was 72.27%. It can be seen that the environmental disinfection effect of bacteriophage PF09 is significantly better than the existing chemical disinfectant such as benzalkonium bromide, and it is safer to use.

[0142] Therefore, the above results show that bacteriophage PF09 has an excellent disinfecting effect on Proteus in pet houses, can significantly reduce Proteus in the environment, and can be promoted and applied as a new biological environmental disinfectant to protect the health of pets.

[0143] Table 7 Salmonella colony counts before and after disinfection with bacteriophage PF09 and benzalkonium bromide disinfectant

[0144]

[0145] 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 strain of Proteus mirabilis phage PF09, characterized in that: Its deposit number is CGMCC NO.45897.

2. A bacteriophage composition, characterized in that: The invention comprises the Proteus mirabilis phage PF09 as described in claim 1.

3. Use of the Proteus mirabilis phage PF09 as claimed in claim 1 or the phage composition as claimed in claim 2 in the preparation of drugs, feed additives and disinfectants for preventing and treating diseases caused by Proteus infection.

4. The use according to claim 3, characterized in that: The Proteus is pet-derived Proteus.

5. A phage medicine or phage health product for pets, wherein the active ingredient comprises the Proteus mirabilis phage PF09 as claimed in claim 1 or the phage composition as claimed in claim 2.

6. A pet feed additive or pet water additive, characterized in that: The method comprises the Proteus mirabilis phage PF09 as claimed in claim 1 or the phage composition as claimed in claim 2.

7. A disinfectant, characterized in that: The active ingredient comprises the Proteus mirabilis phage PF09 as claimed in claim 1 or the phage composition as claimed in claim 2.

8. Use of the disinfectant according to claim 7 in the disinfection of a pet breeding environment, characterized in that: The disinfectant can be used to disinfect the breeding environment, pet feed, snacks or toys from Proteus by spraying or immersing.

9. A Proteus detection kit, characterized in that: It comprises the Proteus mirabilis phage PF09 as described in claim 1.

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