CN111363724B

CN111363724B - Novel bacteriophage, bacteriophage mixed preparation and application of novel bacteriophage and bacteriophage mixed preparation in medicine for preventing and treating hemorrhagic pneumonia of mink

Info

Publication number: CN111363724B
Application number: CN202010169791.6A
Authority: CN
Inventors: 潘强; 任慧英; 孙虎芝; 闫艳新; 袁嘉婧
Original assignee: Qingdao Phagepharm Bio Tech Co ltd
Current assignee: Qingdao Phagepharm Bio Tech Co ltd
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2021-11-09
Anticipated expiration: 2040-03-12
Also published as: CN111363724A

Abstract

The invention discloses a novel bacteriophage, a bacteriophage mixed preparation and application thereof in a medicine for preventing and treating hemorrhagic pneumonia of mink, wherein the novel bacteriophage is Klebsiella phage PH35 with the preservation number of CGMCC NO. 18858; the phage mixed preparation comprises the Klebsiella phage PH35 with the preservation number of CGMCC NO.18858 and the Pseudomonas aeruginosa phage PA69 with the preservation number of CGMCC NO.18855, has broad-spectrum sterilization effect, greatly widens the sterilization range, increases the sterilization activity and can greatly improve the cure rate of minks; the phage mixed preparation has no toxic or side effect and high safety through mouse tests, and is convenient for large-scale production; the phage mixture preparation has a good hydrophilic phase, is easy to prepare into spray liquid or injection, and can effectively kill Klebsiella pneumoniae and pseudomonas aeruginosa in the environment.

Description

Novel bacteriophage, bacteriophage mixed preparation and application of novel bacteriophage and bacteriophage mixed preparation in medicine for preventing and treating hemorrhagic pneumonia of mink

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a phage mixed preparation and application thereof in a medicine for preventing and treating hemorrhagic pneumonia of minks.

Background

Pseudomonas aeruginosa and Klebsiella are important zoonotic opportunistic pathogens responsible for hemorrhagic pneumonia in fur-bearing animals. The early hemorrhagic pneumonia of minks is mainly caused by pseudomonas aeruginosa infection, has rapid onset of disease and is difficult to control, and is endemic in a plurality of countries and regions, which causes the death of a large number of minks and causes serious economic loss. In recent years, the mixed pathogenic condition of pseudomonas aeruginosa and klebsiella is increased, the main clinical manifestations of the disease are mental depression, inappetence, septicemia and sudden death, and some minks have autopsy to find lung bleeding and lymph node abscess. The mink is infected by eating contaminated feed, and also infected with pseudomonas aeruginosa and klebsiella by animal feces and contaminated water. Therefore, the prevention and treatment of the Klebsiella and the Pseudomonas aeruginosa for infecting the fur-bearing animals becomes a key factor for the cultivation of the fur-bearing animals.

At present, penicillin drugs, cephalosporins and aminoglycoside antibiotics are the first choice drugs for treating hemorrhagic pneumonia of mink, but because of the abuse of antibiotic drugs in recent years and the natural drug resistance mechanism of pseudomonas aeruginosa and klebsiella, the pseudomonas aeruginosa and the klebsiella have generated drug resistance to most antibiotics, and some drugs are multiple drug resistance. The two types of bacteria are also very important in the infection of human pneumonia, and become one of the most important pathogenic bacteria of traditional Chinese medicine infection in recent years, and can cause infection of a plurality of parts of the whole body. The drug resistance of pseudomonas aeruginosa and klebsiella is serious in human beings, livestock and poultry, so that a safe and effective drug capable of replacing antibiotics is urgently needed to be found.

Phage is a virus that infects bacteria, a ubiquitous organism, and exists wherever bacteria are present. The targeting property of the phage is strong, once the surface of the pathogenic bacteria contains receptors recognized by the phage, the phage can be quickly adsorbed, after the specific bacteria are lysed, the phage can stop reproducing and be gradually discharged out of the body, and drug resistance can not be generated, so that the phage is the gram of the super bacteria. The phage has the advantages of wide existence, short development time, strong specificity, high proliferation speed, safety, effectiveness and no residue, and has great prospect in development as a substitute of antibiotics.

However, no safe and effective phage preparation with wide lysis spectrum is found at present and is used for preventing and treating hemorrhagic pneumonia of minks, so that further screening and research work of new phages is needed.

Disclosure of Invention

Aiming at the problems, the invention provides a phage PH35 capable of efficiently killing Klebsiella pneumoniae, a phage mixture preparation and application thereof, wherein the phage mixture preparation comprises the Klebsiella pneumoniae PH35 and the Pseudomonas aeruginosa phage PA69, has a wide cracking spectrum, can efficiently crack pathogenic Klebsiella pneumoniae and pathogenic Pseudomonas aeruginosa clinical strains separated from a mink farm, and provides a safe, effective and pollution-free phage product for environment disinfection of the mink farm.

The technical scheme of the invention is as follows:

on one hand, the invention provides a Klebsiella pneumoniae bacteriophage PH35 screened from sewage of a mink farm in Weifang in Shandong province, wherein the bacteriophage is preserved in the China general microbiological culture Collection center on 11 months and 04 days in 2019 with the preservation number of CGMCC NO. 18858; the phage is myotail phage, can form transparent plaques on a solid culture medium, has consistent shape and size, clear and regular edges and a diameter of 0.5 mm.

The invention also provides pseudomonas aeruginosa bacteriophage PA69 screened from sewage of a certain mink farm in Qingdao, Shandong province, wherein the bacteriophage is preserved in the China general microbiological culture Collection center on 11-4.2019 with the preservation number of CGMCC NO. 18855; PA69 is a long-tail phage. The phage can form transparent plaques on a solid culture medium, the shapes and the sizes are consistent, the edges are clear and regular, and plaques with the diameter of 0.5mm are formed after 6 hours of culture at 37 ℃.

The morphology of the klebsiella phage PH35 was observed under an electron microscope as: the head length is 90-100nm, the head width is 110-120nm, and the tail length is 120-130nm, belonging to the myotail phage family; the pseudomonas aeruginosa bacteriophage PA69 is a long-tail bacteriophage with the head length of 90-100nm, the width of 80-90nm, and the tail of 270-280 nm.

The two novel phages have good prevention and treatment effects on the hemorrhagic pneumonia of the minks, and can be respectively used for preparing medicines for preventing and treating the hemorrhagic pneumonia of the minks or environmental disinfectants for mink farms.

Further, the invention also provides a phage mixture preparation, which comprises the Klebsiella pneumoniae PH35 and the Pseudomonas aeruginosa phage PA 69. The phage mixed preparation formed by compounding the two phages has stronger cracking capability, wider cracking spectrum and wider application range, and makes up the limitation of a host spectrum when the phages are applied singly.

Optionally, in the phage mixture preparation, the ratio of the number of the Klebsiella pneumoniae phage PH35 to the number of the Pseudomonas aeruginosa phage PA69 in living bodies is 1: 1; the content of each bacteriophage is not less than 5 × 10⁹PFU/ml. The phage mixture thus formulated exhibited a better lytic effect.

Further, the phage cocktail formulation can further comprise one or more of a mutant of klebsiella phage PH35 and pseudomonas aeruginosa phage PA 69; the mutant has a homology of not less than 90% with the corresponding phage.

Since bacteriophages are very susceptible to mutations during replication, it is preferred that mutants of the aforementioned bacteriophages are also within the scope of the present application. Homology can be suitably determined by computer programs well known in the art, with mutants of bacteriophage PH35 and bacteriophage PA69 being at least 90% identical to the native sequence of the bacteriophage; and the mutant has substantially the same function of killing pathogenic bacteria as the original phage. More preferably, the mutants are 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to the native sequence of the respective phage. The sequences of the phages PH35 and PA69 can be sequenced according to the biological materials deposited according to the invention by known methods. The mutants of the two phages can be point, deletion or addition mutations, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases can be changed relative to the original phage sequence. It is not necessary for the skilled person to inventively work to select a mutant with a similar trait from the phages provided according to the invention.

Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active ingredient being administered. In order to formulate the pharmaceutical composition as a liquid formulation, a pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, glucose solution, maltodextrin solution, glycerol and ethanol. They may be used alone or in any combination thereof. Other conventional additives, for example, antioxidants, buffers, bacteriostats, and the like, may be added if desired. The composition of the present invention may also be prepared into injections (e.g., aqueous solutions, suspensions, and emulsions), or pills, capsules, granules, or tablets, when further combined with diluents, dispersants, surfactants, binders, and/or lubricants.

The invention also provides application of the phage mixture preparation in medicines for preventing and treating hemorrhagic pneumonia of minks. The phage mixture preparation can effectively prevent and treat hemorrhagic pneumonia of mink caused by infection of Klebsiella pneumoniae and pseudomonas aeruginosa. The term "prevention" is meant herein to include all actions that inhibit or delay the disease by administering the composition. The term "treatment" is meant herein to include all actions that result in an improvement or amelioration of the disease by administration of the composition.

The invention also provides a pharmaceutical composition or feed additive, which is characterized in that the effective components comprise the novel bacteriophage or the bacteriophage mixed preparation; preferably, the phage cocktail formulation further comprises phages for specific pathogenic bacteria of different species of bacteria.

Optionally, the pharmaceutical composition is formulated in the form of an oral administration dosage form, a topical dosage form, or an parenteral dosage form. In particular, the compositions of the present invention can be administered via the cutaneous, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, intranasal, and inhalation routes.

The invention also provides an environmental disinfectant, the effective component of which comprises the novel phage or phage mixed preparation; preferably, the concentration of each bacteriophage is 5X 10⁹PFU/ml or more.

Preferably, the environmental disinfectant further comprises other active ingredients for inhibiting or eliminating viruses and bacteria in the environment.

The invention also provides an application of the environmental disinfectant in the environmental disinfection of the farm, wherein the farm environment comprises feed, a water source, a trough, a cage, the ground, walls, excrement and padding. The application method of the environmental disinfectant comprises the following specific steps: the environment disinfectant is used in feed storing and treating workshops, utensils and breeding environments to prevent the pollution of pathogenic bacteria in the environment. For example, including but not limited to, disinfection and decontamination of water distribution systems, medical facilities, aquaculture facilities (troughs, etc.), public and private facilities, or other environmental surfaces (e.g., walls, bedding) by liquid immersion, spraying, use in combination with aqueous carriers, etc., can be effective in controlling the growth and activity of target bacteria. The liquid soaking, spraying forms include but are not limited to detergents, disinfectants, detergents, and the like.

The invention also provides a detection kit which comprises the novel bacteriophage PH35 or the bacteriophage mixed preparation. The above-mentioned phage or phage mixture preparation can be used by those skilled in the art to prepare a detection kit for detecting the host bacteria specifically infected therewith, and for detecting and controlling diseases caused by infection of the host bacteria, based on the present disclosure and common knowledge in the art.

The invention has the following beneficial effects:

1. two phages with strong lytic capacity are selected by the invention, namely the Klebsiella pneumoniae phage PH35 and the Pseudomonas aeruginosa phage PA69, and can be used for preventing and treating diseases caused by the infection of the Klebsiella pneumoniae or the Pseudomonas aeruginosa.

2. The invention also provides a phage mixture preparation for efficiently preventing and treating hemorrhagic pneumonia of minks, which is prepared by compounding Klebsiella pneumoniae PH35 and Pseudomonas aeruginosa phage PA69 as active ingredients and has broad-spectrum bactericidal effect; compared with single lytic pseudomonas aeruginosa, the bactericidal composition greatly widens the bactericidal range, increases the bactericidal activity, and can greatly improve the cure rate of minks.

3. The phage mixed preparation has no toxic or side effect and high safety through mouse tests, and is convenient for large-scale production; the phage mixture preparation has a good hydrophilic phase, is easy to prepare into spray liquid or injection, and can effectively kill Klebsiella pneumoniae and pseudomonas aeruginosa in the environment.

Drawings

FIG. 1 is an electron micrograph of bacteriophage PH 35;

FIG. 2 is an electron micrograph of phage PA 69;

FIG. 3 is a graph of the thermostability of bacteriophage PH 35;

FIG. 4 is a pH stability diagram of bacteriophage PH 35;

FIG. 5 is a graph showing the bactericidal effect of bacteriophage PH 35;

FIG. 6 is a graph of the thermostability of phage PA 69;

FIG. 7 is a pH stability diagram of bacteriophage PA 69;

FIG. 8 is a graph showing the bactericidal effect of the bacteriophage PA 69.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.

EXAMPLE 1 isolation and purification of phage culture

1. Isolated culture of host bacteria

In the examples, the host bacterium of the Klebsiella pneumoniae was a clinical strain of Klebsiella pneumoniae, and was obtained from a mink dead of Klebsiella pneumoniae infection in a mink farm in Weifang, Shandong province.

The host bacteria of the pseudomonas aeruginosa bacteriophage in the embodiment are clinical strains of pseudomonas aeruginosa, and are obtained from minks infected with pseudomonas aeruginosa in certain mink farms in Qingdao, Shandong province.

Inoculating host bacteria on 2% nutrient agar, culturing overnight, selecting single colony, inoculating in 5ml nutrient broth culture medium, and shake culturing at 37 deg.C and 170rpm for 12 hr as host bacteria culture.

2. Isolation culture of bacteriophage

The wastewater from mink farms was placed in a bubble vial, 200. mu.l of host bacteria was added to the bubble vial, and then the nutrient broth medium was added thereto, followed by shaking culture overnight in a shaker at 37 ℃ and 170 rpm. A part of the liquid after overnight culture was put into a 10ml centrifuge tube, centrifuged at 11000rpm for 10min, and the supernatant was collected and filtered through a 0.22 μm filter to form a phage stock solution.

190. mu.l of each well of a 96-well plate was addedNutrient broth culture medium, then adding 5 μ l of host bacteria and phage stock solution, respectively, each host bacteria being provided with a control group of 195 μ l of nutrient broth and 5 μ l of host bacteria; placing 96-well plate into 37 deg.C incubator, culturing for 5 hr, and measuring OD₆₀₀And calculating the OD value, wherein the OD value of the phage group is reduced by 50% compared with that of the control group, and then the phage is considered to be separated.

The liquid in the 96-well plate was removed, 100. mu.l of the liquid was taken with a pipette and added to 900. mu.l of nutrient broth, diluted 10-fold and diluted to 10^-6Then putting the bacterial strain and host bacteria into an incubator at 37 ℃ for incubation for 5min, and adsorbing the phage to the host bacteria. Preparing a culture dish of 2% nutrient agar and 0.7% upper-layer nutrient agar in advance by adopting a double-layer plate method, adding a phage and host bacterium mixed solution into the 0.7% upper-layer nutrient agar after 5min, quickly mixing uniformly, pouring the mixture on the culture dish of 2% nutrient agar, and putting the mixture into a 37 ℃ incubator for culture for 4-5h after solidification to obtain a single plaque.

3. Purification and multiplication of bacteriophage by spot-scratching

Taking 1ml of nutrient broth for later use, picking up the plaque on a culture dish by using sterilized tweezers, putting the picked plaque into 1ml of nutrient broth, putting the broth in a shaking table, culturing at 37 ℃ for 30min, then centrifuging at 11000rpm for 5min, taking the supernatant, obtaining single plaque by using a double-layer plate method, picking up the single plaque, and purifying for 3-5 times until the plaque with uniform size and shape appears on the culture dish.

Adding 100 μ l of host bacteria and phage spot-removing leachate into 5ml of nutrient broth, adding 100 μ l of host bacteria into another 5ml of nutrient broth as a control, simultaneously culturing in a shaker at 37 deg.C and 170rpm for 3-4h, and collecting phage proliferation solution after the mixed solution becomes clear. The phage proliferation solution was diluted 10 fold, titer was measured by double-plate method, and 3 replicates were prepared for each dilution. The Klebsiella phage forms transparent plaques with uniform size and shape, clear and regular edges and a diameter of 0.5 mm; the pseudomonas aeruginosa bacteriophage forms transparent plaques with consistent shape and size and clear and regular edges, and the plaques with the diameter of 0.5mm are formed by culturing at 37 ℃.

Example 2 morphological Observation of phages

The experimental method comprises the following steps: take 1X 10¹⁰Mu.l of the PFU/ml phage sample was dropped onto a microporous copper mesh, precipitated for 15min, and excess liquid was blotted off with filter paper. 15 μ l of 2% phosphotungstic acid (PTA) was dropped on the copper mesh, dyed for 5min, and excess dye solution was sucked off with filter paper, dried, observed with a transmission electron microscope and photographed.

As shown in the electron microscope image of FIG. 1, the Klebsiella phage has a head length of 90-100nm, a head width of 110-120nm, and a tail length of 120-130 nm. The form of bacteriophage PH35, defined by the International Committee for viral Classification (ICTV), corresponds to the characteristics of the Myoviridae family, is Myoviridae, and is designated PH 35.

As shown in the electron microscope picture of FIG. 2, the Pseudomonas aeruginosa phage has a head length of 90-100nm, a width of 80-90nm, a tail length of 270-280nm, and a long tail. The phage morphology, as defined by the International Committee for viral Classification (ICTV), was characterized by the long-tailed phage family, which was designated PA 69.

Example 3 Whole genome analysis of phages

Extracting the genome of the pseudomonas aeruginosa bacteriophage PA69, and performing whole genome sequencing to obtain:

the whole genome length of phage PA69 was: 45414bp, wherein in the genome, the gene sequence of tail protein related to the recognition of a phage host is shown in a sequence 1 in a sequence table, the gene sequence of a highly conserved terminal enzyme large subunit (terminating) is shown in a sequence 2 in the sequence table, the sequence of 2 DNA polymerase (DNA polymerase) genes is shown in a sequence 3-4 in the sequence table, and the gene sequence of lyase (lysozyme) related to the cleavage capability is shown in a sequence 5 in the sequence table. The specific information of the above genes is shown in Table 1 below.

TABLE 1 Gene sequence information Table of phage PA69

Example 4 biological characterization of bacteriophages

1. Biological characterization of phage PH35

(1) Detection of thermostability of bacteriophage

Mixing 1.3X 10¹⁰The PFU/ml bacteriophage PH35 proliferation solution is respectively treated in water bath at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C for 20min, 40min, and 60min, and each temperature is provided with two parallel groups. The titer of the phage was determined by the double-layer plate method.

The specific results are shown in FIG. 3, the titer remained at 1X 10 after the phage was exposed to 40 ℃ and 50 ℃ for 1h⁹PFU/ml; after 1h of action at 60 ℃, the temperature is still maintained at 1X 10⁸PFU/ml or more; the phage is basically inactivated after being acted at 70 ℃ for 40min, and is completely inactivated at 80 ℃. The thermal stability of PH35 was thus higher.

(2) Detection of pH stability of bacteriophage

Adding NB broth (4.5 ml) with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) into sterile test tube, placing three tubes in water bath at 37 deg.C, and adding 500 μ l 7.5 × 10 tubes after temperature stabilization⁹PFU/ml phage proliferation liquid, mixing uniformly at 37 deg.C in water bath for 1h, 2h, 3 h. After the reaction is finished, the pH value of the mixed solution is about 7 by adding a proper amount of HCl or NaOH into the mixed solution, and the titer of the phage is measured by a double-layer plate method.

The results are shown in FIG. 4, where a gradient was observed for the phage pH35 titer at pH5-12, with the titer at 10⁸Above PFU/ml, the activity decreases even completely inactivated at pH2-3 and pH 13. Indicating that PH35 is able to withstand certain weak acid and strong base environments.

(3) Optimal multiplicity of infection (MOI) assay for bacteriophages

The Klebsiella pneumoniae PH35 and the host bacterium Klebsiella pneumoniae are propagated according to a conventional method, the titer of the phages and the host bacterium is measured, and the phages PH35 and the host bacterium are appropriately diluted. 100 μ lPH35 and host bacteria were added to NB broth at a multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.0000001, respectively. The culture was shaken at 37 ℃ and 170rpm until the broth became clear, and the broth clarification time was recorded. Centrifuging at 11000rpm for 5min, and determining the titer of the phage by a double-layer plate method.

The optimal multiplicity of infection of the phage is 0.001, under which the titer of the progeny phage produced by infecting the host bacteria with the phage is 5.0X 10¹⁰PFU/ml, the phage titer was highest among 9 multiplicity of infection.

(4) In vitro lysis assay of bacteriophages

Adding Klebsiella pneumoniae H6 and bacteriophage PH35 according to a certain proportion, wherein the final concentration of the Klebsiella pneumoniae is 1 × 10⁸CFU/ml, final concentration of phage 1X 10⁹PFU/ml，1×10⁸PFU/ml，1×10⁷PFU/ml, the same amount of sterile broth as the phage was added to the control, and the broth and phage were mixed and cultured in an incubator at 37 ℃. And measuring OD values at regular intervals until the mixed solution becomes clear, and measuring the residual quantity of each group of bacteria after acting for a certain time by a coating plate method.

As shown in FIG. 5, the lysis effect of PH35 on Klebsiella pneumoniae is good, the lysis rate of 3 phages with different concentrations on the Klebsiella pneumoniae strain can reach more than 99.9%, and the time is different, but the better killing effect can be achieved within 4.5.

2. Biological characterization of phage PA69

(1) Detection of thermostability of bacteriophage

Will be 3X 10¹⁰The PFU/ml phage PA69 proliferation solution is respectively treated in water bath at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C for 20min, 40min, and 60min, and each temperature is provided with two parallel groups. The titer of the phage was determined by the double-layer plate method.

As shown in FIG. 6, the phage PA69 maintained substantially the original titer after 1h at 40 ℃, 50 ℃ and 60 ℃; the titer is reduced by 4 gradients after the phage is acted at 70 ℃ for 20min, so that the phage can endure a certain high-temperature environment, is stable at the temperature of 60 ℃ and below and is sensitive to the high temperature of more than 70 ℃.

(2) Detection of pH stability of bacteriophage

Adding NB broth (4.5 ml) with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) into sterile test tube, placing three tubes in water bath at 37 deg.C, and adding 500 μ l each after temperature stabilization 3×10¹⁰PFU/ml phage proliferation liquid, mixing uniformly at 37 deg.C in water bath for 1h, 2h, 3 h. After the reaction is finished, the pH value of the mixed solution is about 7 by adding a proper amount of HCl or NaOH into the mixed solution, and the titer of the phage is measured by a double-layer plate method.

The results are shown in FIG. 7: in the pH range of 5-11, the phage PA69 titer was hardly or slightly reduced, still at 8.5X 10⁹The adaptation range of the phage to pH is wider as seen by PFU/ml.

(3) Optimal multiplicity of infection (MOI) assay for bacteriophages

And multiplying the pseudomonas aeruginosa bacteriophage PA69 and the host bacterium pseudomonas aeruginosa according to a conventional method, measuring the titer of the bacteriophage and the host bacterium, and appropriately diluting the bacteriophage PA69 and the host bacterium. 100. mu.l each of PA69 and host bacteria were added to NB broth at a multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.0000001. The culture was shaken at 37 ℃ and 170rpm until the liquid became clear and the shake-up time was recorded. Centrifuging at 10000rpm for 5min, and measuring the titer of the phage by a double-layer plate method.

As a result, it was found that the optimum multiplicity of infection of the phage was 0.00001, under which the titer of progeny phage produced by infecting the host bacterium with the phage was 4.2X 10¹⁰PFU/ml, the phage titer was highest among 9 multiplicity of infection.

(4) In vitro lysis assay of bacteriophages

Adding Pseudomonas aeruginosa strain A17 and bacteriophage PA69 at a certain ratio to obtain a final concentration of 1.00 × 10⁸CFU/ml, final concentration of phage was 1.00X 10⁹PFU/ml、1.00×10⁸PFU/ml、1.00×10⁷PFU/ml and 1.00X 10⁶PFU/ml, the same amount of sterile broth as the phage was added to the control, and the broth and phage were mixed and incubated with shaking at 37 ℃ and 170rpm in a shaker. And measuring OD values at regular intervals until the mixed solution becomes clear, and measuring the residual quantity of each group of bacteria after acting for a certain time by a coating plate method.

The results are shown in FIG. 8, where it can be seen that: PA69 has good cracking effect on Pseudomonas aeruginosa strains, the cracking efficiency of 4 phages with different concentrations on the Pseudomonas aeruginosa strains can reach more than 99.5 percent, and the phages only have different time lengths, but can achieve better killing effect within 3 hours.

Example 5 determination of phage lysis Profile

Determination of the lysis Spectrum of the PH35 phage

The method for determining the lysis spectrum of the phage by adopting a double-layer plate method comprises the following steps: bacterial suspensions of host bacteria were obtained according to the method in example 1, and 73 Klebsiella pneumoniae were derived from the lungs of fur-bearing animals such as mink and fox dying from hemorrhagic pneumonia in regions such as Weifang, Jinan and Qingdao. Phage PH35 were plated on double plates with 73 Klebsiella pneumoniae, respectively, and cultured overnight in an inverted state.

The results show that the phage PH35 can crack 65 strains of the 73 Klebsiella pneumoniae serving as host bacteria, and the cracking rate reaches 89.04 percent when the cracking spectrum is determined.

(II) determination of the lysis Spectrum of the PA69 phages

The method for determining the lysis spectrum of the phage by adopting a double-layer plate method comprises the following steps: bacterial suspensions of host bacteria were obtained according to the method in example 1, and 123 strains of pseudomonas aeruginosa were derived from lungs of fur-bearing animals such as mink, fox and the like dead in hemorrhagic pneumonia in Qingdao, Weifang, sunshine, Dalian and the like. And (3) respectively paving the phage PA69 and 123 strains of pseudomonas aeruginosa on a double-layer flat plate, and performing inverted culture for 5-6 h.

The result shows that the bacteriophage PA69 can crack 111 strains in the lysis spectrum by using 123 strains of pseudomonas aeruginosa as host bacteria, and the lysis rate reaches 90.24%.

(III) determination of lysis Spectroscopy of phage cocktail preparation

73 Klebsiella pneumoniae (numbered H1-H73) and 123 pseudomonas aeruginosa (numbered A1-A123) which are stored in a laboratory are selected, and the lysis spectra of PH35 and PA69 are detected by a double-layer plate method, wherein the lysis spectra are specifically shown in the following table 2.

TABLE 2 lysis profiles of different phages

The experimental results show that: the phage mixture preparation has a lysis rate of 89.04% for 73 Klebsiella pneumoniae and 90.24% for 123 Pseudomonas aeruginosa, can simultaneously identify Klebsiella pneumoniae and Pseudomonas aeruginosa, and has maximized lysis capacity. The phage cocktail composition can make up the limitation that the phage is only effective to one pathogenic bacterium when being applied singly.

Example 6 safety test of phages

Selecting 40 healthy BALB/C mice with the weight of 18-20 g, dividing the mice into 3 experimental groups and 1 control group with each half of male and female, and respectively drenching the purified PA69 proliferation solution, the PH35 proliferation solution, the PA69 and the PH35 mixed preparation (200 mul 10 g)¹⁰PFU/ml) and physiological saline (200. mu.l), continuously drenched for 7d, observed the behavior of the mice, and examined the change of the organs of the mice by autopsy.

As a result, the behavior of the mice in the 3 experimental groups and the control group is not abnormal, and the organs such as the liver, the lung, the heart, the spleen, the kidney and the like which are subjected to the autopsy are normal and have no obvious difference with the control group.

Example 7 prevention and treatment experiment of bacteriophage on hemorrhagic pneumonia of mink

1. Bacteriophage PH35 for preventing and treating hemorrhagic pneumonia of mink caused by Klebsiella infection

(1) The experimental method comprises the following steps: 1-2 kg of healthy minks, 20 minks, 10 male minks and 10 female minks are selected and divided into 2 groups, and each group is half of a male mink and a female mink. Before test, collecting blood 300 μ l from hind interphalangeal vein of mink, determining phage in blood by double-layer plate method, and feeding 10g of mink with 1ml10 when no phage exists¹⁰PFU/ml PH35 mixed feed, 10g feed mixed with 1ml normal saline is fed to each mink in the control group, and after 1h feeding phage, 20 minks in the experimental group and the control group are fed according to 2X 10⁸CFU/single dose of the strain is injected intraperitoneally with the clinical isolate of the Klebsiella counteracting virus. Observe and record waterMink death was observed continuously for 7d, and minks suspected to be dead in hemorrhagic pneumonia were necropsied to verify death due to hemorrhagic pneumonia, and the mortality and protection rate were counted (see table 3).

TABLE 3 control Effect of phages on mink hemorrhagic pneumonia

(2) The experimental results show that: the mortality rate of the mink fed with the phage in advance due to hemorrhagic pneumonia is 20%, the protection rate of the phage is 80%, and the mortality rate of the control group is 70%, which shows that the phage PH35 has good control effect on the hemorrhagic pneumonia of the mink caused by Klebsiella.

2. Bacteriophage PA69 for preventing and treating hemorrhagic pneumonia caused by mink infected by pseudomonas aeruginosa

(1) The experimental method comprises the following steps: 1-2 kg of healthy minks, 20 minks, 10 male minks and 10 female minks are selected and divided into 2 groups, and each group is half of a male mink and a female mink. Before test, collecting blood 300 μ l from hind interphalangeal vein of mink, determining phage in blood by double-layer plate method, and feeding 10g and 1ml10 ml for each mink when no phage exists¹⁰PFU/ml PA69 feed, 10g feed mixed with 1ml normal saline for each mink in control group, and 1h phage feeding, and 4 × 10 for 20 minks in experimental group and control group⁸The clinical separated strain of pseudomonas aeruginosa is attacked by the intraperitoneal injection of CFU/dose. Mink deaths were observed and recorded, and minks suspected of hemorrhagic pneumonia deaths were necropsied for 7d, checked for deaths due to hemorrhagic pneumonia, and the mortality and protection rates were counted (see table 4).

TABLE 4 control Effect of phages on mink hemorrhagic pneumonia

(2) The result shows that the mortality rate of the mink fed with the phage in advance due to the hemorrhagic pneumonia is 20%, the protection rate of the phage is 80%, and the mortality rate of the control group is 80%, which shows that the phage has a good control effect on the hemorrhagic pneumonia of the mink caused by pseudomonas aeruginosa.

3. Phage mixed preparation for preventing and treating hemorrhagic pneumonia of mink caused by mixed infection of klebsiella and pseudomonas aeruginosa

(1) The experimental method comprises the following steps: 1-2 kg of healthy minks, 20 minks, 10 male minks and 10 female minks are selected and divided into 2 groups, and each group is half of a male mink and a female mink. Before test, collecting blood 300 μ l from hind interphalangeal vein of mink, determining phage in blood by double-layer plate method, and feeding 10g and 1ml10 ml for each mink when no phage exists¹⁰PFU/ml PH35 and PA69 feed, 10g feed mixed with 1ml normal saline is fed to each mink in the control group, and after 1h phage feeding, 20 minks in the experimental group and the control group are fed according to 4 x 10⁸CFU/Pseudomonas aeruginosa and 2X 10⁸The strain is clinically separated by the mixed bacterium liquid for attacking toxin by intraperitoneal injection of CFU/Klebsiella. Mink deaths were observed and recorded, and minks suspected of hemorrhagic pneumonia deaths were necropsied for 7d, checked for deaths due to hemorrhagic pneumonia, and the mortality and protection rates were counted (see table 5).

(2) The experimental results show that: the mortality rate of the mink fed with the phage in advance due to hemorrhagic pneumonia is 20%, the protection rate of the phage is 80%, and the mortality rate of the control group is 90%, which shows that the phage has good control effect on the hemorrhagic pneumonia of the mink caused by mixed infection of pseudomonas aeruginosa and klebsiella.

TABLE 5 control effect of phage cocktail on hemorrhagic pneumonia in mink

EXAMPLE 8 Disinfection Effect of phage cocktail formulation on Klebsiella pneumoniae and Pseudomonas aeruginosa in mink feed

The concentrations of Klebsiella pneumoniae and Pseudomonas aeruginosa in the overnight culture were adjusted to 1X 10, respectively⁸CFU/ml, uniformly spraying 1ml of mixed bacteria liquid by using a small-sized sprayerSpreading the mink on the surface of 100g of spread mink material to an area of about 0.5m²Then spraying the phage mixed with the preparation in an amount of 1ml and at a pH of 35 of 5X 10⁹PFU and PA69 are 5X 10⁹PFU, spraying for 1h, 2h, 3h, 4h and 5h, and detecting the number of the Klebsiella and the pseudomonas aeruginosa in the feed.

The detection result shows that the quantity of the Klebsiella pneumoniae and the Pseudomonas aeruginosa in the feed is obviously reduced after the phage is sprayed for 1 hour, and the quantity of the Klebsiella pneumoniae and the Pseudomonas aeruginosa is reduced to below 10CFU/g after 2 hours, which indicates that the phage can effectively kill the Klebsiella pneumoniae and the Pseudomonas aeruginosa in the mink feed.

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Sequence listing

<110> Qingdao Nonbert Biotechnology Ltd

<120> novel bacteriophage, bacteriophage mixed preparation and application thereof in medicine for preventing and treating hemorrhagic pneumonia of mink

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 648

<212> DNA

<213> Tail protein of phage vB _ PaeS _ PA69 (tail protein of phase vB _ PaeS _ PA69)

<400> 1

atgggtcttg aggtcgcaac gtatatcgat cagttggtgc ctacgaaccc taccggctct 60

gatctgaagt cctttgggga tgaccatatt cgcctcatca agagtgccat taagaatacc 120

ttccccaata ttaaccaagc tgtcaccgta acagcagccc aattgaactc agtcgcagat 180

accacccaat atgtgaaacc tgggatggtt atcatgtggg cgggtactct tgctcagatc 240

ccagcaggtt ggaaactttg taatggcgta gggacaacct ctaatggcat ccccgtacct 300

aacctcatcg gagcattccc ttggggtatt gatggtacat cccaaactgt aggtactcgg 360

ggtggtaacg ctaacatcgt atgggaaggt cttaccgagg gccacgctct tacgctagcc 420

caaatcccag cacacactca cacttggcgt acccgagggg ctaccaccct gactggttct 480

gctggtgatt ctggtgcgtt gactggtggt tctggtaacg ctgccaatac caacctagag 540

actggctctg ctggacaagg acaggcacac acccacgctg ttaagatcaa catgccattg 600

ggcaacatcc cgccgttctg tgctgtattc ttcattatta agaactga 648

<210> 2

<211> 1449

<212> DNA

<213> terminal enzyme Large subunit of bacteriophage vB _ PaeS _ PA69 (terminating large subunit of phase vB _ PaeS _ PA69)

<400> 2

atggataccc aagagcggtt gcgtaaccta gtcagagaat tggctgagcg gcagaagtac 60

ttccgcatga accagtacac cccctatggg tggcaagaga agttcattgc agcctcttcc 120

acttgtgctc agctactggc tatgactggc aaccgctgtg gtaagacata caccggggcc 180

ttcatcatgg cgtgtcacct taccggtcgc taccctgagt ggtggactgg aaggaagtat 240

gacaagccag tgaactgctg ggccgcaggg atctccacag atactactcg ggacattctt 300

cagtctgaac tactaggtga ttggaagaac cccgaggcat tcggcactgg gatgatcccc 360

aaagaggata tcgtaaagac tgaacgtagg gaaggcaaac ccggatgcgt ccaagctgta 420

atggtaaggc acgtatctgg gggtttgtcc tcgctaatct tcaaatctta cgaaatgtcc 480

caagacaaat tcatgggtac tgctattgac gtcatctggc ttgacgagga gtgccccaag 540

gacatttata cccagtgtgt aacccgaacg gctactactg ggggtattgt ctatctaacc 600

tttaccccag agcatggcct cacggaaatc gtgaaggact tcctccagga tcttaaacct 660

ggtcagttcc tgatccatgc aagctgggaa gatgccccac acctcagtcc agaagttaaa 720

gagcagctac tctcggtata ctctccagca gaacgcagga tgagggctga gggtattcct 780

atgctcggct ctggtgtagt cttccctatt ctggaagaga agttcgtatg tgagcctttt 840

gatatcccag atcacttcca taggataatt ggtatcgacc tagggtttga ccaccctaac 900

gctattgcct gtgtagcctg ggatgcagag aaggacaagt attacctgta tgatgaacga 960

agtgaatctg gcgaaaccct cggcatgcac gctgatgcca tctacctcaa gggtggtcac 1020

cagatccctg ttgtagtccc ccacgatgcg tttaagcatg atggggctac ctcaggtcgt 1080

cgcttcgtag acctcctgaa ggatgaccat aacctcaacg ttgtttatga acccttcagt 1140

aaccccccag gtcctgatgg taagcacggg ggtaactctg tagagtttgg tgtcaactgg 1200

atgcttactc gtatggagaa tggggatctc aaggtattca atacttgtac taacttcctc 1260

aaagaaatga agatgtacca ccgtaaagat ggtaagattg tggatcgtaa tgatgacatg 1320

atctcagcaa ctcggtatgc ccttctgatg gcttcccgtc acgctcgtcc cggtgctgta 1380

cgaaacagtg gatactacag gagtgatacc gcaaggttga tccctgattg gtttgggagt 1440

attgtctaa 1449

<210> 3

<211> 1638

<212> DNA

<213> DNA polymerase 1 of phage vB _ PaeS _ PA69 (DNA polymerase 1 of phase vB _ PaeS _ PA69)

<400> 3

gtggattgga gaaagtcact gttcgttgag cataaggtag ctgatatcat tagccgccag 60

agtaaacggg gggtctactt ccagactcaa cgggctaagt ggcttatcca cgtactcaac 120

gaacgaatcc tcaagattga cctagaggca gtcccccaga tgcctcccat gatcgtccaa 180

gcgggagcat tcagtaagcc attcctaaag agtggcctgc cgaacaaacg cttggtcgct 240

ctatggcaac gcttgggtca cttcgaggta tctggaccct ttacagcaat cgagtacaag 300

gactttgacc tgggtaaaac cgcaaggttc aaagactgga tgctgtctca aggctggata 360

cctgaccagt ggaatatcaa ggatattaca gtcggcactg atggcaagaa gctacgtgga 420

tccgatctga atggtgcctt gaaccgctac atcgaagacc tccgaagctc cccctctggg 480

ctactccgaa tgaagctcca gggaatcatc cctgggaaga ctacagtcgg tgaggtcaag 540

agaaagcttg agaaacaacg taaggttctc actacgccga agatgactga gacctccatg 600

gataccgtcc agggggatct tggtaagctt gtgatgcagc gaatggtttg ggctcaccga 660

cgctccctcc ttcagggatt agtcgagcag gtgaagccta atggacgcct agaggggagt 720

gctaacccct gtgcaacacc cacgggccgt atgagacacc gtgtagtagt taatatcccc 780

gctgctcgtt ctcccttcgg gcctgaaatc cgagggttgt tccaggggac tcctaatgct 840

ggtgaatgga aatggactgt cctccgccgc gacataggtg agaacgaaag ggtaaggccc 900

cacactaaca tcgtggaggt cctcaaaggc ggtaagtgga agacggtagg caagtaccga 960

gtatacgtcc cagcgaatca actgatcttc gtgggctatg acggtgctgg tctagagctt 1020

cggatgcttg cctcctacat tggcgaccca gactataccc gagaggttgt agatggtgac 1080

gtccatactg ccaaccaaat agccgcagga ttacctaccc gagacgatgc taagacgttc 1140

atctacgcct tcatctacgg tgctggtgat gccaaaattg ggcagatcat tggtggcacg 1200

agggccgatg gggctaggct gagggagcaa ttccttaagg ccaacccaga gctggctaag 1260

ctgattgaga gggttaagca ggaagccgaa aggggctacc ttgtaggact cgacggacgt 1320

aagctaacga tgcgccgtag tgagtctgga gaggtgatgg tccacaaggc attgaacacc 1380

ctcctccaag ctgctggtgc tattgtcatg aagtgggcaa tggtgatcct agatgagaag 1440

gtacgacggg ctggactcaa ggcgtggaag gttttggata tccacgatga aggccagtgg 1500

gagtgccatc ccgaggacct tgcgactctc cgtggattca tggagacttg tgtcaaggaa 1560

gctggggaaa tcttgggttg taactgtcct cttgccagtg attctatcgc aggtaggtcc 1620

tggtatgaca cacactga 1638

<210> 4

<211> 528

<212> DNA

<213> DNA polymerase 2 of phage vB _ PaeS _ PA69 (DNA polymerase 2 of phase vB _ PaeS _ PA69)

<400> 4

atgacgaagt acctgaggac gacgagtccc caatttgatt acgttatcta cgacctagag 60

ggtgatgccc tttacgataa cgtcacaagg ctttggtgcg ctgtccttgt ggatatccca 120

actcaggtag tccggggatt ccggcctgag gaaatggaga cattctacag gatcatcgcc 180

catgcaaagt tcgtggtcgg gcacaacatc cttgactacg acaacagggt ccttgagaaa 240

cttcatggta tcgtgattcc agaagaaagg tcttacgata ccttggtcgc ctcccgactc 300

acttggcctg accgtcctat gggacactcg ctgggagcct ggggtagatt cctgaagtgt 360

cacaagggtg acttcaacga cttctccaag ttctccgagg aaatgtttga gtattgccta 420

caggatgggg ttgttagtat ggcactcttc aactacctgc ttaaggttct cgggatgacc 480

tgggaggagc tagtagagtg gaggactggt tcttggctaa acgtgtga 528

<210> 5

<211> 372

<212> DNA

<213> lyase of phage vB _ PaeS _ PA69 (lysozyme of phase vB _ PaeS _ PA69)

<400> 5

ttggggtatt ggactggagg atacggccac ctacagcgtc ccggagagga tggacccatc 60

accctagccc gagcagagac ttggctagag aacgacagtc aggcagccta cgatgcagcc 120

caacggcaag tatctgagct gcctttttgc actccagaac tattcgatgc cctagtgagc 180

gtcaacttcc agcttggtac agcatggact aagaagttcc ccaagacgtg gaaccttctg 240

aaagccgggg agttcgacag agcagcttgg gaggccgagg atagtgcctg ggctaagcaa 300

accccagttc gcgtaaggga cctccaacga gccctttggc gagcatctac gataggcagc 360

acggtgactt aa 372

Claims

1. A novel bacteriophage which is a Klebsiella phage PH35 with the preservation number of CGMCC NO. 18858.

2. Use of the novel bacteriophage of claim 1 for the manufacture of a medicament for the prevention and treatment of hemorrhagic pneumonia in mink or an environmental disinfectant for mink farms.

3. A phage cocktail formulation comprising the Klebsiella phage PH35 of claim 1 and the Pseudomonas aeruginosa phage PA69 with a accession number of CGMCC NO. 18855.

4. The phage cocktail of claim 3, wherein the ratio of viable count of Klebsiella phage to Pseudomonas aeruginosa phage is 1: 1; the content of each bacteriophage is not less than 5 × 10⁹PFU/ml。

5. The phage cocktail formulation of claim 3, further comprising a pharmaceutically acceptable carrier.

6. The use of the phage mixture formulation of claim 3 in the preparation of a medicament for the prevention and treatment of hemorrhagic pneumonia in mink.

7. A pharmaceutical composition or feed additive characterized in that the active ingredient thereof comprises the novel bacteriophage according to claim 1 or the bacteriophage cocktail preparation according to any one of claims 3 to 5.

8. The pharmaceutical composition or feed additive according to claim 7, which is formulated in an oral administration form or an parenteral administration form.

9. An environmental disinfectant, characterized in that the effective ingredient comprises the novel bacteriophage of claim 1 or the bacteriophage cocktail formulation of any one of claims 3-5.

10. An environmental disinfectant according to claim 9 further comprising other active ingredients for inhibiting or destroying bacteria in an environment.

11. The use of an environmental disinfectant according to claim 9 for disinfecting a farm environment, wherein said farm is a mink farm, and the farm environment includes feed, water sources, tanks, floors, walls, manure, and bedding.

12. A test kit comprising the novel bacteriophage according to claim 1 or the bacteriophage cocktail according to any one of claims 3 to 5.