Proteus mirabilis bacteriophage PF05, and bacteriophage composition and application thereof

Abstract

The invention belongs to the technical field of bacteriophages, and discloses a proteus mirabilis bacteriophage PF05, a bacteriophage composition thereof and application of the proteus mirabilis bacteriophage PF05, the proteus mirabilis bacteriophage PF05 is preserved in China General Microbiological Culture Collection Center (CGMCC) on March 22, 2024, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.9687. The preservation number is CGMCC (China General Microbiological Culture Collection Center) The bacteriophage is resistant to high temperature, wide in lysis spectrum, high in storage stability and good in gastric acid tolerance, can keep activity in the stomach for a long time, and has a good prevention and treatment effect on infection of proteus mirabilis in the intestines and the stomach. The compound can be prepared into medicines, environment disinfectants, feeds, water additives and the like for preventing and treating proteus mirabilis for application, not only effectively prevents and treats propagation of proteus mirabilis, but also has the effects of reducing the death rate of chicken flocks, reducing digestive tract infection of the chicken flocks, avoiding diarrhea of the chicken flocks and improving the resistance of the chicken flocks, and has a very good market application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of bacteriophages, and in particular to a strain of Proteus mirabilis bacteriophage PF05, a bacteriophage composition thereof and application thereof. Background Art

[0002] As a branch of the Enterobacteriaceae family, the genus Proteus mirabilis mainly includes species such as Proteus vulgaris, Proteus mirabilis, Proteus myxofaciens, Proteus penneri and Proteus hauseri. Proteus mirabilis is a major pathogen of the genus Proteus.

[0003] Proteus mirabilis belongs to the family Enterobacteriaceae and the genus Proteus. The bacteria are carried at a high rate in humans, poultry, and livestock, and are widely distributed in animal feces, soil, and sewage. Proteus mirabilis can infect a variety of animals such as chickens, pigs, and goats, and is a common conditional pathogen in clinical practice. When Proteus mirabilis infects poultry, it mainly causes diarrhea, bacteremia, urinary tract infections, etc. Chicken Proteus mirabilis disease is an acute infectious disease of chickens of different ages caused by pathogenic Proteus mirabilis. The main clinical features are sepsis, watery diarrhea, and paralysis of one or both legs. It mainly harms chicks under 7 weeks of age, with high morbidity and mortality rates. The recovered chicks grow slowly, and the egg production of breeder hens will decrease, causing serious economic losses to the poultry industry.

[0004] my country's aquaculture industry is developing rapidly, mainly in the form of large-scale intensive aquaculture, which means that the spread of livestock and poultry diseases will bring great economic losses. The research and development of bacterial vaccines is difficult and the effect is poor, which makes bacterial diseases one of the important factors restricting the development of the aquaculture industry. At present, the aquaculture industry mainly relies on antibiotics to treat Proteus mirabilis disease. At present, the abuse and misuse of antibiotics in the clinical field and the aquaculture industry have led to the emergence of drug resistance in Proteus mirabilis, which is transmitted to humans through the food production chain. The drug-resistant Proteus mirabilis produced in the clinic can also be spread to the environment and animals in various ways. Proteus mirabilis can also increase its resistance to antibiotics by mutating itself and acquiring drug-resistant genes. Drug-resistant Proteus mirabilis brings huge challenges to clinical treatment and animal production, which arouses people's attention and vigilance; in addition, the abuse of antibiotics also leads to drug residue problems in animals, which affects human health. Therefore, in order to improve production efficiency and safety, bacteriophages have become the focus of attention for replacing antibiotics to treat animal diseases.

[0005] Bacteriophage is a general term for a class of viruses that can infect bacteria, fungi, spirochetes and other microorganisms. As early as 2006, the US FDA approved Listeria monocytogenes-specific phage as a new food preservative. Traditional methods for killing foodborne pathogens mainly include physical methods (high temperature, etc.) and chemical methods (preservatives, etc.), but they affect the shape of food; bacteriophage, as a natural bactericidal agent, can be used in various links such as food collection, production and processing, storage, and transportation, and is an ideal method for dealing with pathogenic bacteria contamination in the food production chain. Compared with antibiotics, bacteriophages have several potential advantages: first, they only target target bacteria, reducing damage to the host's natural flora and are harmless to humans and animals; at the same time, bacteriophages are widely distributed and have the advantages of strong self-reproduction ability and high specificity. In recent years, bacteriophages have been widely used in the prevention and treatment of human and animal diseases, agricultural production, and foodborne infection prevention and control. In particular, as an alternative or complementary treatment to antibiotics, great progress has been made in controlling the spread of pathogens and treating drug-resistant bacterial infections. As drug-resistant strains continue to emerge and the development of new antimicrobial drugs is slow, bacteriophages have once again attracted people's attention as a means of treating infectious diseases.

[0006] Based on the above advantages of bacteriophages, new bacteriophages have been gradually developed as environmental disinfectants or drugs and have been applied in some fields, such as the prevention and treatment of Proteus mirabilis. For example, the patent with publication number CN 116286678A applied by the applicant in 2023 discloses a strain of Proteus phage vB_PmiM_PF07 (abbreviated as: PF07), which can effectively prevent and treat diseases caused by Proteus mirabilis infection.

[0007] However, the currently available Proteus mirabilis phage resources are not only scarce, but also have poor stability during storage and application, which not only increases the preservation cost but also greatly affects its application effect. The existing technology needs to be further improved. Summary of the invention

[0008] In view of the above problems, the present invention provides a Proteus mirabilis phage PF05, a phage composition thereof and applications thereof. The Proteus mirabilis phage PF05 has high storage stability and strong acid resistance, and can be used as an active ingredient to prepare drugs, environmental disinfectants, feed additives and poultry water additives for preventing and treating Proteus mirabilis, thereby being used for the prevention and treatment of Proteus mirabilis.

[0009] To solve the above problems, this application provides the following technical solutions:

[0010] In a first aspect, the present application provides a Proteus mirabilis bacteriophage PF05, characterized in that its deposit number is CGMCC No.45896.

[0011] Proteus mirabilis phage PF05 was isolated from chicken feather contaminants in broiler farms in Shandong Province. It has a wide lysis spectrum, high temperature resistance and high storage stability.

[0012] The phage was deposited at the General Microbiology Center of the 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. 45896.

[0013] In the present application, bacteriophage PF05 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 PF05 can be obtained by sequencing the biological materials deposited according to the present invention by known methods. For those skilled in the art, it does not require creative labor to screen out mutants with extremely similar properties according to the bacteriophage provided by the present invention.

[0014] The mirabilis phage PF05 has good stability and acid and alkali resistance. The phage still maintains its original activity after being exposed to 60°C for 3 hours, and its potency is maintained at 1.20×10 6 PFU / mL, which shows that phage PF05 has strong thermal stability. The phage can maintain stable activity in the pH range of 4.0-11.0, and its titer can still be maintained at 10 after 3 hours of action at pH 2.0. 9 PFU / mL, and it still maintained a certain activity after 1 hour at pH 13, which shows that the phage has strong acid and alkali resistance and can be used in certain acid and alkali environments.

[0015] The lysis spectrum of the Proteus mirabilis phage PF05 is wide, and it has strong lysis performance on the host bacteria. Experiments have shown that the total lysis rate of the phage PF05 on 200 strains of Proteus mirabilis from different sources stored in the laboratory reached 94.00%; and for Proteus mirabilis from different sources, the lysis rate of 82 strains of Proteus mirabilis from chickens reached 98.78%, the lysis rate of 58 strains of Proteus mirabilis from ducks was 96.55%, and the lysis rate of 34 strains of Proteus mirabilis from pigeons was 91.18%; the lysis rate of Proteus mirabilis from pets was slightly lower, only 76.92%. It can be seen that the lysis spectrum of the phage is wide and the application range is wide.

[0016] The storage stability of Proteus mirabilis phage PF05 is high, and its potency remains stable after being stored at room temperature for 12 months, which can effectively reduce the storage cost and provide quality assurance for later clinical applications and storage. In addition, phage PF05 has a strong tolerance to gastric juice and can be fed through drinking water or feed addition. After entering the gastrointestinal tract of animals, it can still maintain a high phage activity, more effectively blocking oral infection of Proteus mirabilis and reducing Proteus mirabilis colonization in the intestine.

[0017] Based on the above excellent biological characteristics, bacteriophage PF05, as a new microorganism with application prospects, can be promoted and used as an antibiotic alternative in various applications for the prevention and control of Proteus mirabilis.

[0018] In a second aspect, the present application also provides a phage composition, which includes the Proteus mirabilis phage PF05 as described above.

[0019] In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in lysis spectrum of different phages, and complement each other's advantages, the above-mentioned Proteus mirabilis phage PF05 can be used in combination with other phages, such as being used in combination with other Proteus mirabilis phages to broaden the lysis spectrum, improve the killing of Proteus mirabilis in the breeding environment, and be used for better prevention and treatment of Proteus mirabilis infection. In addition, the above-mentioned Proteus mirabilis phage PF05 can also be combined with other different types of phages (suppressing different pathogens that cause the same type of disease) for the prevention and treatment of the same type of disease.

[0020] Optionally, the phage composition comprises: Proteus mirabilis phage PF05 and Proteus mirabilis sphage vB_PmiM_PF07 with a deposit number of CGMCCNO.23812. Proteus mirabilis sphage vB_PmiM_PF07 is disclosed in the patent with a publication number of CN116286678A.

[0021] In a third aspect, the present application also provides the use of the above-mentioned Proteus mirabilis phage PF05 or the aforementioned phage composition in the preparation of drugs, feed additives, environmental disinfectants or food preservatives for preventing and treating diseases caused by Proteus mirabilis infection.

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

[0023] Preferably, the disease caused by Proteus mirabilis infection includes: Proteus mirabilis disease in chickens, and the Proteus mirabilis includes chicken-derived Proteus mirabilis.

[0024] In a fourth aspect, the present application also provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Proteus mirabilis phage PF05 or the aforementioned phage composition.

[0025] Preferably, the bacteriophage pharmaceutical preparation further comprises other antibacterial or bactericidal active ingredients; the pharmaceutical preparation is in the form of an oral dosage form, an external dosage form or a parenteral dosage form.

[0026] The application method of the bacteriophage drug preparation is: adding bacteriophage PF05 or its bacteriophage composition as a therapeutic drug to chicken drinking water or feed, or administering it orally, subcutaneously or intramuscularly to chickens, thereby preventing and treating chicken Proteus mirabilis disease and improving the survival rate of chickens.

[0027] Optionally, the phage pharmaceutical preparation also includes a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" used herein refers to a carrier or diluent that does not cause significant stimulation to an organism and does not eliminate the biological activity and characteristics of the active component administered. In order to prepare the pharmaceutical composition as a liquid preparation, a pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, glucose solution, maltodextrin solution, glycerol, ethanol, various types of culture media, etc. They can be used alone or in any combination thereof. If necessary, other conventional additives can be added, for example, antioxidants, buffers and antibacterial agents, etc. When also combined with diluents, dispersants, surfactants, adhesives and / or lubricants, the composition of the present invention can also be prepared into injections and oral dosage forms (for example, aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilized agents.

[0028] In a fifth aspect, the present application further provides a feed additive or an animal water additive, which comprises the aforementioned Proteus mirabilis phage PF05 or the aforementioned phage composition. Preferably, the feed additive or animal water additive is a chicken feed additive or a chicken water additive.

[0029] The method for using the feed additive or animal water additive is as follows: by adding the above-mentioned animal drinking water additive or feed additive to animal water or mixing it with feed, feeding the chickens, thereby disinfecting the water and feed in the chicken farm, avoiding the spread of Proteus mirabilis disease from the source, and effectively preventing and controlling Proteus mirabilis.

[0030] Preferably, in order to further improve the antibacterial effect, the feed additive or animal water additive also contains other safe 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.

[0031] In a sixth aspect, the present application further provides an environmental disinfectant, the active ingredient of which includes the aforementioned Proteus mirabilis phage PF05 or the aforementioned phage composition. Preferably, in the environmental disinfectant, the concentration of the phage is 10 8 PFU / mL or above.

[0032] Preferably, in order to improve the disinfection effect, the environmental disinfectant also contains other active ingredients for inhibiting or eliminating bacteria in the environment.

[0033] Specifically, environments where environmental disinfectants can be applied include feed, water and breeding environments, and the breeding environment includes chicken houses, chicken cages, waterers, troughs and other feeding tools, feces and bedding.

[0034] In a seventh aspect, the present application also provides the use of the environmental disinfectant in the disinfection of chicken slaughterhouses, chicken product processing workshops or chicken breeding environments, for preventing and controlling the contamination of chicken Proteus mirabilis in these environments.

[0035] The breeding environment includes: chicken houses, feeding tools (such as chicken cages, waterers, troughs, etc.), feces and bedding, etc.

[0036] The application method includes, but is not limited to, disinfecting and decontaminating the water distribution system, breeding facilities, feeding equipment or other environmental surfaces in the application environment by liquid immersion, spraying, and combined use with an aqueous carrier, and disinfecting and preserving feed. This disinfectant can be used to replace antibiotics or traditional disinfection products, and it will not cause harm to humans and poultry.

[0037] In an eighth aspect, the present application further provides a chicken product preservative, the active ingredient of which includes the aforementioned Proteus mirabilis phage PF05 or the aforementioned phage composition. The chicken product preservative is applied by spraying the chicken product preservative solution on the surface of the chicken product, or soaking the chicken product in a solvent containing the chicken product preservative, which can inhibit the reproduction of Proteus mirabilis phage in the chicken product and achieve a preservation effect.

[0038] The present invention has the following beneficial effects:

[0039] 1. The present invention provides a self-isolated Proteus mirabilis phage PF05, which is not only resistant to high temperatures and has a wide lysis spectrum, but also has strong storage stability and good gastric acid tolerance, and can remain active in the stomach for a long time, and plays a good preventive and therapeutic role in the infection of Proteus mirabilis in the stomach and intestines. Therefore, the Proteus mirabilis phage PF05 can be prepared into drugs, environmental disinfectants, feed and water additives, etc. for preventing and treating Proteus mirabilis for application, which not only effectively prevents and treats the spread of Proteus mirabilis disease, but also has the effects of reducing the mortality rate of chickens, reducing digestive tract infections in chickens, avoiding diarrhea in chickens, and improving the resistance of chickens.

[0040] 2. The long-term storage stability of the bacteriophage PF05 is good, and the potency remains stable after being stored at room temperature for one year. Therefore, the bacteriophage has the advantage of a long shelf life, which can effectively reduce the cost of use and storage, and provide quality assurance for later clinical application and storage.

[0041] 3. The bacteriophage PF05 is obtained from nature, has high safety, is green and environmentally friendly, and has no toxic side effects. It will not only not pollute the environment, but also will not harm the beneficial bacteria in animals. In addition, the bacteriophage has a high explosive volume and strong reproductive ability, which is convenient for large-scale development and production, and effectively reduces the production cost of the bacteriophage product.

[0042] Biological Deposit Description

[0043] Proteus phage PF05 was deposited on March 22, 2024 at the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number CGMCC No.45896. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a plaque image of bacteriophage PF05;

[0045] Figure 2 This is an electron microscope image of bacteriophage PF05;

[0046] Figure 3 The results of the thermal stability test of bacteriophage PF05;

[0047] Figure 4 The pH stability test results of bacteriophage PF05;

[0048] Figure 5 This is the one-step growth curve of bacteriophage PF05. DETAILED DESCRIPTION

[0049] 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.

[0050] Example 1 Isolation and Screening of Proteus mirabilis Phage PF05

[0051] 1. Resuscitation culture of host bacteria and preparation of proliferation medium

[0052] Twenty strains of Proteus mirabilis (F23-1~F23-20) preserved in our laboratory were selected, and the freezing solution was dipped into a sterilized inoculation loop and streaked on SS culture medium for recovery. The culture was cultured in a constant temperature incubator at 37℃ for 18-24h to obtain a single colony; a single colony was picked and inoculated into 5mL of NB broth, and cultured at 37℃ with shaking at 170rpm / min for 16h to obtain a fresh Proteus mirabilis bacterial solution.

[0053] 2. Phage isolation and purification

[0054] Take appropriate amount of chicken feathers, feces and other samples from multiple farms in Shandong Province into a bubble sample bottle, add appropriate amount of broth culture medium, add the above 20 strains of Proteus mirabilis, put the mixed solution into 37℃170rpm / min shaking culture for 12h, centrifuge at 11000rpm for 5min, and then filter with a 0.22μm sterile microporous filter membrane to obtain phage proliferation liquid;

[0055] The phage proliferation liquid was diluted at a 10-fold ratio, and appropriate gradient phage dilution liquid was mixed with 20 strains of Proteus mirabilis at a ratio of 1:1. After incubation at 37°C for 5 minutes, 200 μL of the mixture was placed on the upper agar (agar concentration of 0.7%), and after mixing, it was quickly poured onto the lower agar (agar concentration of 1.5%) plate, shaken and placed flat until the culture medium solidified, and placed in a 37°C incubator for inverted culture for 4 to 6 hours to obtain a double-layer plate with plaque formation.

[0056] 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 / min for about 30 minutes to obtain a phage extract. Take the phage extract and the corresponding plaque-forming Proteus mirabilis (hereinafter referred to as the host bacteria) proliferation liquid 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, mix it and quickly pour it on the lower agar 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 to 6 hours, and then obtain a double-layer plate with plaques formed again. Use sterilized tweezers to pick a single plaque from the double-layer culture medium where plaques are formed and place it in 1 mL of LB broth, and culture it in a constant temperature shaker at 37°C, 170 rpm / min for about 30 minutes to obtain a phage extract. Repeat the above steps 3 times to obtain a purified phage extract.

[0057] 3. Phage titer determination

[0058] 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 / min with shaking until the liquid becomes clear. Centrifuge the clear liquid at 11000 rpm for 10 min, take the supernatant, and filter it with a 0.22 μm sterile microporous filter membrane to obtain phage proliferation liquid, and use the double-layer plate method to determine the titer of the newly isolated phage.

[0059] 4. Screening of Proteus mirabilis phages with long-term stability

[0060] (1) Experimental subjects:

[0061] Newly isolated Proteus mirabilis phage and other phages, including: Clostridium perfringens phage PMQ06 (see patent publication number CN111690620A), Escherichia coli phage BP7 (see patent announcement number CN103289963B), Proteus mirabilis phage PF07 (see patent publication number CN 116286678A), and Salmonella phage PC127 (see patent publication number CN 116083374A).

[0062] (2) Experimental methods:

[0063] The initial concentration of each phage was adjusted to 2.00 × 10 9 PFU / mL, stored at room temperature (ventilated and cool place, about 37°C) for 1 year, and stored at room temperature for 2 weeks, 1 month, 3 months, 6 months, 9 months, and 12 months, the potency of each phage was measured to compare the long-term storage stability of all phages.

[0064] 5. Experimental results and analysis

[0065] (1) Eight strains of Proteus mirabilis phage were screened using the 20 strains of Proteus mirabilis mentioned above, and they were numbered PF / SD2023012201 to PF / SD2023012208, respectively.

[0066] The eight strains of Proteus mirabilis phages all formed clear plaques on the double-layer agar medium plate, surrounded by a halo with clearly visible edges, with a diameter of about 0.5 mm to 1.5 mm; the titer was between 2.00×10 9 PFU / mL-8.00×10 10 PFU / mL.

[0067] By comparing the long-term storage stability of these eight phages, it was found that after one year of storage at room temperature, phage PF / SD2023012201 (PF05 for short) had the highest titer and a broad lysis spectrum, as well as better stability. Therefore, the Proteus mirabilis phage PF05 with the best overall performance was selected (results shown in Table 1) and used in the experiments of subsequent examples.

[0068] Table 1 Titers and lysis spectra of 8 newly isolated Proteus mirabilis phages stored at room temperature for one year

[0069]

[0070] (2) Figure 2 As shown, compared with other existing phage types (PMQ06, BP7 and PC127) and the aforementioned Proteus mirabilis phage PF07, the storage time of Proteus mirabilis phage PF05 at room temperature is significantly longer than other phages, and its long-term storage stability is the highest. This feature provides quality assurance for the later clinical application and preservation of the phage, and also effectively reduces its use and preservation costs.

[0071] Table 2 Changes in titer of different phages stored at room temperature for one year (PFU / mL)

[0072]

[0073] Example 2 Morphological observation and identification of Proteus mirabilis bacteriophage PF05

[0074] 1. Experimental methods

[0075] Take 20 μL of the bacteriophage PF05 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.

[0076] 2. Experimental results and analysis

[0077] like Figure 2 As shown, the bacteriophage PF05 has a polyhedral head structure and a non-contractile tail. The head is 56-60nm wide and 64-68nm long, and the tail is about 120-150nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the phage morphology of the present application conforms to the characteristics of the long-tailed bacteriophage family and belongs to the long-tailed phage.

[0078] Example 3 Whole genome analysis of Proteus mirabilis phage PF05

[0079] The genome of phage PF05 was extracted, and whole genome sequencing and sequence analysis were performed. The results are as follows:

[0080] (1) The genome of PF05 is 161439 bp in length, with a G+C content of 31.05%, an A+T content of 68.95%, and base C, G, A, and T contents of 16.48%, 14.57%, 33.91%, and 35.04%, respectively. The online annotation results of the whole genome RAST showed that the genome contained 262 open reading frames (ORFs). Among these 262 open reading frames (ORFs), 93 structural proteins were found, mainly including the structure and packaging proteins of the phage (capsid protein, capsid and scaffold protein, tail spike, structural protein, phage fiber protein, topoisomerase large subunit, etc.), phage lysis-related proteins (phage lysin), DNA replication and modification-related proteins (DNA polymerase, DNA helicase, DNA binding protein, endonuclease, etc.), and other functional proteins (phage repressor protein, dual infection immunity protein, etc.). At the same time, among the 262 ORFs, 244 start codons were ATG, 4 start codons were GTG, 1 start codon was ACA, 1 start codon was GGA, 2 start codons were GGT, 1 start codon was GCG, 1 start codon was CTG, 1 start codon was GGC, 1 start codon was GCA, 1 start codon was AGG, 1 start codon was GGG, and 4 start codons were TTG. The software tRNAscan-SE analysis showed that the genome contained tRNA genes. 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.

[0081] (2) In the genome of bacteriophage PF05: the gene sequence of the tail fiber protein protein related to phage host recognition is shown in Sequence 1 in the sequence listing; the gene sequence of the topoisomerase large subunit protein is shown in Sequence 2 in the sequence listing; the sequence of the DNA polymerase gene is shown in Sequence 3 in the sequence listing; the gene sequence of the lysin gene related to cleavage ability is shown in Sequence 4 in the sequence listing. For specific relevant information, see Table 3 below.

[0082] Table 3 Gene sequence information

[0083]

[0084] Example 4 Determination of the Optimal Multiplicity of Infection (MOI) of Proteus mirabilis Phage PF05 against Proteus mirabilis 1. Experimental Methods

[0085] Pick a single Proteus mirabilis colony and inoculate it into 5 ml NB broth medium, culture it at 37°C, 170 rpm for 12 to 16 h to obtain the bacterial solution, and determine the bacterial concentration by the pouring method; detect the phage titer by the double-layer plate method; add the phage titer and host bacteria concentration in a ratio of 1:1, 0.1:1, 0.01:1, 0.001:1, 0.0001:1, 0.00001:1, 0.000001:1, 0.0000001:1 to a test tube containing 5 mL NB broth, and culture it at 37°C, 170 rpm with shaking for 8 h to 12 h, until the liquid becomes clear from turbid. Take an appropriate amount of clear liquid and centrifuge it at 11000 rpm / min for 10 min. Take the supernatant and filter it through a 0.22 μm sterile microporous filter membrane. Determine the phage titer in the filtrate by the double-layer plate method. The MOI (number of phages / number of bacteria) with the highest phage titer is the optimal multiplicity of infection of the phage.

[0086] 2. Experimental results

[0087] The results in Table 4 show that when the MOI was 0.00001, the titer of PF05 reached the highest level, which was 5.50×10 11 PFU / mL, that is, the optimal MOI is 0.00001; this provides data reference for the later large-scale industrial production of products, and also shows that the phage has application advantages in industrial production.

[0088] Table 4 The titer of Proteus mirabilis phage PF05 at different infection multiplicity

[0089]

[0090]

[0091] Example 5 Temperature stability of Proteus mirabilis bacteriophage PF05

[0092] 1. Experimental methods:

[0093] 5.0×10 10 The PFU / mL of the Proteus mirabilis phage PF05 proliferation solution was placed at 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃, and three parallel samples were made at each temperature. After being placed for 20min, 40min, and 60min, the samples were immediately cooled in an ice bath after the reaction ended, and then the phage titer at different temperatures was detected by the double-layer plate method. The thermal stability curve of phage PF05 was drawn with temperature as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0094] 2. Experimental results and analysis

[0095] The results are as follows Figure 3 As shown in the results, it can be seen that the phage PF05 still maintains its original activity after being exposed to temperatures within 40℃ to 60℃ for 60 minutes; the titer of the phage is maintained at 1.0×10 6 PFU / mL; after the phage was exposed to 80℃ for 20 min, the titer was maintained at 1.20×10 6 PFU / mL; after being exposed to 90℃ for 20min, the phage PF05 still maintained a certain activity. It can be seen that the Proteus mirabilis phage PF05 has strong heat resistance and can adapt to higher temperature environments.

[0096] Example 6 pH stability of Proteus mirabilis bacteriophage PF05

[0097] 1. Experimental methods

[0098] 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 of phage PF05 proliferation solution, mix well and act in a 37℃ water bath for 1h, 2h, and 3h. After the action is over, immediately add an appropriate amount of 1mol / L HCl or NaOH to the mixture to make the pH value of the mixture about 7, dilute it 10 times, take a suitable dilution gradient to determine the titer, and set 3 parallel samples for each pH value test tube. Draw the phage pH stability curve with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0099] 2. Experimental results and analysis

[0100] The results are as follows Figure 4 As shown in the results, it can be seen that the titer of phage PF05 is maintained at 10 in the pH range of 4.0 to 11.0. 10 PFU / mL, stable activity; at pH 2.0 for 3h, the potency is 10 9 PFU / mL, able to resist a certain strong acid environment, at pH 1.0, it was not completely inactivated after 1 hour of action; after 1 hour of action at pH 12, the titer of the phage was 10 8 PFU / mL, and it still maintains a certain activity after 1h at pH13; therefore, bacteriophage PF05 has strong stability in a wide pH range and can adapt to a certain range of strong acid and strong alkaline environments.

[0101] Example 7 One-step growth curve of Proteus mirabilis phage PF05

[0102] 1. Experimental methods

[0103] Take 1 mL of phage proliferation liquid and host bacteria proliferation liquid with a multiplicity of infection of 10, mix 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 / min 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 / min shaking culture, take out 150μL 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 phage PF05, and calculate the outbreak volume.

[0104] Outbreak volume = total number of phages at the end of the outbreak / total number of bacteria at the beginning of the outbreak

[0105] 2. Experimental results and analysis

[0106] The one-step growth curve of bacteriophage PF05 is shown in Figure 5 As shown in the figure, after phage PF05 infects the host bacteria, the phage lysis cycle is about 60 minutes, the incubation period is about 10 minutes, and the phage outbreak period is about 50 minutes; after 60 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 PF05 was 86.

[0107] Example 8 Determination of the lysis spectrum of Proteus mirabilis phage PF05

[0108] 1. Experimental Materials

[0109] Host bacteria: 200 strains of Proteus mirabilis from different sources preserved in the laboratory were selected (82 strains of Proteus mirabilis from chicken, 58 strains of Proteus mirabilis from duck, 34 strains of Proteus mirabilis from pigeon, and 26 strains of Proteus mirabilis from pets).

[0110] 2. Experimental methods:

[0111] (1) Select 200 strains of Proteus mirabilis from different sources stored in the laboratory and streak them on SS plates to revive them. Pick a single colony for proliferation and culture to obtain fresh bacterial liquid. Prepare the proliferation liquid of bacteriophage PF05 and the bacterial liquid of 200 strains of Proteus mirabilis separately, and use the double-layer plate method to detect the lysis spectrum of the phage;

[0112] (2) Comparison of lysis performance with existing phages: The lysis spectrum of the above 200 strains of Proteus mirabilis was measured by using phage PF07 (chicken source) (disclosed in patent publication number CN116286678A) to compare the lysis differences of the two strains of Proteus mirabilis phages PF05 and PF07 on the above 200 strains of Proteus mirabilis;

[0113] 3. Experimental results and analysis

[0114] 3.1 Comparative analysis of the lysis effects of three phages on 200 strains of Proteus mirabilis from different sources

[0115] The following conclusions can be drawn from the experimental results in Table 5:

[0116] (1) For the 200 strains of Proteus mirabilis from different sources, the Proteus mirabilis phage PF05 can lyse 188 of them, with a total lysis rate of 94.00%; this shows that phage PF05 has the characteristics of wide lysis spectrum and strong lysis performance. Specifically, phage PF05 can lyse 81 of 82 strains of Proteus mirabilis from chicken, with a lysis rate of 98.78%; 56 of 58 strains of Proteus mirabilis from duck, with a lysis rate of 96.55%; 31 of 34 strains of Proteus mirabilis from pigeon, with a lysis rate of 91.18%; 20 of 26 strains of Proteus mirabilis from pets, with a lysis rate of 76.92%. Detailed results are shown in Table 5.

[0117] (2) Of the 200 strains of Proteus mirabilis from different sources, phage PF07 was able to lyse 165 of them, with a total lysis rate of 82.50%. Specifically, the lysis rate of phage PF07 against 82 strains of Proteus mirabilis from chicken was 87.80%; the lysis rate against 58 strains of Proteus mirabilis from duck was 84.48%; the lysis rate against 34 strains of Proteus mirabilis from pigeon was 88.24%; and the lysis rate against 26 strains of Proteus mirabilis from pets was 53.85%.

[0118] In summary, the lysis rate of Proteus mirabilis phage PF05 for the 200 strains of Proteus mirabilis from different sources used in this application is significantly higher than that of phage PF07; and for the Proteus mirabilis selected in patent CN 116286678A, the lysis rate of phage PF05 is consistent with that of phage PF07.

[0119] The above results indicate that phage PF05 has the characteristics of a wide lysis spectrum and better lysis effect, and can effectively prevent and control infections caused by Proteus mirabilis from different sources.

[0120] Table 5 Lysis spectra of 2 phages against 200 strains of Proteus mirabilis

[0121]

[0122]

[0123]

[0124] Table 6 Lysis spectra of two phages against 49 strains of Proteus mirabilis

[0125]

[0126] Example 9 Tolerance experiment of bacteriophage PF05 in simulated gastric acid

[0127] 1. Experimental methods

[0128] (1) Preparation of artificial gastric juice: Take 16.4 ml of dilute hydrochloric acid, add 800 ml of deionized water, stir thoroughly, adjust the pH value to 2.5, make up to 1 L with deionized water, sterilize at 121°C for 15 min, cool to room temperature, add 10 g of pepsin, and mix to fully dissolve.

[0129] (2) Take 1 mL of phage PF05, phage PF07 (from CN116286678A), and PF / SD202301203, PF / SD202301204 proliferation solution and add them to 150 mL conical flasks, and then add 20 mL of the aforementioned artificial gastric juice. Among them, PF03 and PF04 are chicken-derived Proteus mirabilis phages isolated and preserved by our laboratory, and are only used as controls for comparative analysis in this experiment.

[0130] (3) The conical flask was placed in a constant temperature incubator at 37°C and cultured at 90 rpm / min for 2 h. After 0.5 h, 1 h, and 2 h, 1 mL of the mixed solution was sampled and diluted to a suitable gradient in 9 mL of PBS buffer. The phage titer was determined by the double-layer plate method, and the survival rate was calculated.

[0131] Survival rate = (number of phages after artificial gastric juice treatment for a fixed time / number of phages at 0 h of artificial gastric juice treatment) × 100%

[0132] 2. Experimental results and analysis

[0133] The results are shown in Table 7. It can be seen from the results that after 2 hours of exposure to artificial gastric juice, the titer of phage PF05 was maintained at 3.25×10 9 PFU / mL, the activity is stable. After 2 hours of exposure to artificial gastric juice, the titer of bacteriophage PF07 increased from 5.00×10 9 The PFU / mL decreased to 2.10×10 2 PFU / mL; while the titer of bacteriophage PF / SD202301203 increased from 5.00×10 9 The PFU / mL decreased to 2.35×10 0 PFU / mL; PF / SD202301204 was exposed to artificial gastric juice for 2 hours, and the titer increased from 5.00×10 9 The PFU / mL decreased to 6.80×10 2 PFU / mL, the stability of phage PF05 in gastric juice was much higher than that of the other three phages.

[0134] The above experimental results show that bacteriophage PF05 has a strong tolerance in simulated gastric fluid and can enter the stomach through oral feeding to exert an antibacterial effect.

[0135] Table 7 Phage titer changes in artificial gastric juice at different times (PFU / mL)

[0136]

[0137] Example 10 Safety Test of Bacteriophage PF05

[0138] 1. Experimental methods

[0139] Twenty 4-week-old SPF female Kunming mice were randomly divided into two groups: the phage group and the control group. The phage group was orally administered with 0.5 mL of phage PF05 proliferation solution (1×10 10 PFU), and the control group was orally administered with the same dose of sterile saline. The mice were reared under the same conditions, and autopsied after 2 weeks to observe the lesions of the heart, liver, spleen, lung, kidney, brain, and intestines. During the rearing process, their spirit and feeding status were observed.

[0140] 2. Experimental results and analysis

[0141] No disease or toxicity symptoms were observed in the phage group and the control group, and the mental state and feeding were normal. After autopsy, the tissues, organs and intestines of each group were observed to be normal, which shows that phage PF05 is safe and reliable and has no adverse effects on the animal body.

[0142] Example 11 Environmental disinfection test of Proteus mirabilis bacteriophage PF05

[0143] 1. Experimental methods

[0144] A chicken farm in Shandong Province selected two 14-day-old chick houses and tested the content of Proteus mirabilis in the environment of each house before disinfection; the experimental group used phage preparations as phage disinfectants for disinfection, and the content of phage PF05 in the phage disinfectant was 10 7 PFU / mL, spraying volume is 10mL / m 2 The control group was disinfected with peracetic acid (1:500 dilution) at a spray volume of 10 mL / m 2 . After 30 minutes of disinfection, the content of Proteus mirabilis in the environment was tested again.

[0145] Detection method: A total of 5 points, including the center of the building and 4 corners, were used as test points. Two SS agar plates were placed at each point for sampling. After sampling, the culture dishes were placed in a 37°C constant temperature incubator. After 18 to 24 hours of incubation, the number of bacterial colonies in each culture dish was recorded.

[0146] According to the Ostwald formula, the total number of colonies is C = 50000N / AT, where C is the total number of colonies per cubic meter (CFU / m 3 ); N: number of colonies per dish; A: area of ​​culture dish (cm 2 ); T: sampling time (min).

[0147] 2. Experimental results and analysis

[0148] As shown in Table 8, after disinfection with bacteriophage PF05, the elimination rate of Proteus mirabilis in the chicken house was 89.39%; after disinfection with peracetic acid disinfectant, the elimination rate of Proteus mirabilis in the chicken house was 65.13%; this shows that the disinfecting effect of bacteriophage PF05 on Proteus mirabilis in the chicken house environment is significantly better than that of peracetic acid, and it can be promoted and applied as a new biological environmental disinfectant.

[0149] Table 8 Proteus mirabilis colony counts before and after disinfection with bacteriophage PF05 and peracetic acid disinfectant

[0150]

[0151] Example 12 Experiment on the preventive and therapeutic effect of bacteriophage PF05 on Proteus mirabilis infection in chicks

[0152] 1. Experimental methods

[0153] 90 SPF chicks were randomly divided into 3 groups, 30 in each group, namely, blank group, bacterial attack group, and phage group. All chickens were fed and watered freely and raised in an isolator. The bacterial attack group and phage group were orally administered 0.2 mL / bird, 5 × 10 7 CFU / mL of Proteus mirabilis was administered in drinking water, and the blank group was given an equal amount of normal saline orally; the phage group was given 0.2 mL / animal, 5×10 8 PFU / mL Proteus mirabilis bacteriophage PF05 was administered in drinking water for 3 consecutive days, and then the bacteria were attacked 3 days later. The drinking water was administered again 2 hours after the attack. The administration was continued for 20 consecutive days, and the incidence and mortality of the chicks were counted.

[0154] 2. Experimental results

[0155] The results in Table 9 show that all the chicks in the infection group became ill after 10 days, with a large number of chicks experiencing watery diarrhea, depression, and reduced feed intake, and the mortality rate was 53.33%; the blank group showed no symptoms of illness or death during the entire feeding period; the incidence rate in the phage group was only 10.00%, with individual chickens experiencing depression and diarrhea, but no deaths; the results indicate that Proteus mirabilis phage PF05 as a feed additive can effectively prevent and control Proteus mirabilis infection during breeding.

[0156] Table 9 Morbidity and mortality of chickens in each group

[0157]

[0158]

[0159] 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 bacteriophage PF05, characterized in that: Its deposit number is CGMCC No.45896.

2. A bacteriophage composition, characterized in that: It comprises the Proteus mirabilis phage PF05 as described in claim 1.

3. The bacteriophage composition according to claim 2, characterized in that Also includes: The Proteus mirabilis phage vB_PmiM_PF07 with the deposit number CGMCCNO.23812.

4. Use of the Proteus mirabilis phage PF05 as claimed in claim 1 or the phage composition as claimed in claim 2 or 3 in the preparation of a drug, feed additive, environmental disinfectant or food preservative for preventing and treating diseases caused by Proteus mirabilis infection.

5. A phage pharmaceutical preparation, the active ingredient of which comprises the Proteus mirabilis phage PF05 as claimed in claim 1 or the phage composition as claimed in claim 2 or 3.

6. A feed additive or animal water additive, characterized in that: The method comprises the Proteus mirabilis phage PF05 as claimed in claim 1 or the phage composition as claimed in claim 2 or 3.

7. An environmental disinfectant, characterized in that: The active ingredients include the Proteus mirabilis phage PF05 as described in claim 1 or the phage composition as described in claim 2 or 3; preferably, it also contains other active ingredients for inhibiting or eliminating bacteria in the environment.

8. Use of the environmental disinfectant as claimed in claim 7 in the disinfection of a chicken slaughterhouse, a chicken product processing workshop or a chicken breeding environment.

9. A chicken product preservative, characterized in that: The active ingredient thereof comprises the Proteus mirabilis phage PF05 as claimed in claim 1 or the phage composition as claimed in claim 2 or 3.

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