CN115261334B

CN115261334B - Staphylococcus bacteriophage, bacteriophage preparation and application of bacteriophage preparation in preventing and treating diseases caused by staphylococcus infection

Info

Publication number: CN115261334B
Application number: CN202110752581.4A
Authority: CN
Inventors: 潘强; 任慧英; 孙虎芝; 刘广芹; 王翠; 魏凯
Original assignee: Qingdao Phagepharm Bio Tech Co ltd
Current assignee: Qingdao Phagepharm Bio Tech Co ltd
Priority date: 2021-07-02
Filing date: 2021-07-02
Publication date: 2023-06-27
Anticipated expiration: 2041-07-02
Also published as: CN115261334A

Abstract

The invention discloses a staphylococcus phage, a phage composition and application thereof in preventing and treating diseases of staphylococcus infection, the phage is named vB_SauM_SA34, the preservation number is CGMCC NO.19976, the phage is preserved in the China general microbiological culture Collection center of 05 month 15 in 2020, the phage has a wide cracking spectrum, a phage preparation combined with antibiotics can further widen the cracking spectrum, the preparation has flexible administration mode, no toxic or side effect and high safety, different administration modes have good treatment effect on swine exudative dermatitis, and compared with the single use of antibiotics, the survival rate of piglets is improved, and the phage and the preparation can be used for preparing medicines, environmental disinfectants, feeds, water additives and the like for preventing and treating diseases of staphylococcus infection.

Description

Staphylococcus bacteriophage, bacteriophage preparation and application of bacteriophage preparation in preventing and treating diseases caused by staphylococcus infection

Technical Field

The invention relates to the technical field of microorganisms, in particular to a staphylococcus bacteriophage and a bacteriophage preparation and application thereof in preventing and treating diseases of staphylococcus infection.

Background

Staphylococci are gram-positive bacteria, are conditional zoonotic pathogens, widely exist in the natural world and in the skin, nasopharyngeal mucosa and intestinal tracts of people and animals, and when the resistance of organisms is reduced or the skin mucosa is damaged, pathogenic bacteria can enter the mucosa, can cause the diseases of the organisms, and have great harm to the people and the animals. Exudative dermatitis in pigs is a disease caused by staphylococcal infection.

The exudative dermatitis of pigs, also called "oil skin disease", is frequently generated in suckling piglets after 5 days of age and weaned piglets after 3-5 weeks of age, and can cause infection and morbidity of whole-litter piglets in a short time. The disease can occur all the year round, but the incidence rate is irregular in the wet summer and autumn, the incidence rate is 10-70%, the death rate is usually 20-40%, and the growth of pigs and the economic benefit of pig farms are seriously affected. The common clinical symptoms of exudative dermatitis in pigs are depression of the spirit, coarse and messy hair and anorexia of the sick pigs. Redness, swelling, and spots or plaques of various depth colors from reddish brown to black appear on the whole body from the cheek, bleeding spots are seen by scraping the crust on the surface, oily secretions, and malodor develop. General lymph node swelling, hemorrhage, coronary fat hemorrhage, liver swelling and congestion after dissection. Some pigs show symptoms such as emaciation, diarrhea and the like. Swine exudative dermatitis is usually a death if not treated early.

The oozing dermatitis of the staphylococcus in pigs is usually treated by antibiotics, and common antibiotics include amoxicillin, ceftiofur, ofloxacin, enrofloxacin, ciprofloxacin, neomycin, gentamicin, kanamycin and the like. However, with the use of antibiotics, the emergence of staphylococcus strains with multiple drug resistance poses great difficulty in clinical treatment of swine exudative dermatitis, so that there is a need for an antibacterial substance capable of replacing antibiotics or auxiliary antibiotics to treat swine staphylococcus exudative dermatitis.

Phages are the largest number of biological populations in the biological kingdom and are also the largest viral populations known to date. Phages are classified into virulent phages, which invade host bacteria in a short time, rapidly replicate and lyse the host bacteria, and temperate phages, which are now widely used in the treatment of bacterial diseases.

Furthermore, studies have shown that when antibiotics are used in combination with phages, there is a synergistic effect (PAS effect): antibiotics can cause DNA loss-inducing reactions (SOS reactions) of bacteria even at Sub-bacteriostatic concentrations (Sub-Mic) to the bacteria, but the SOS reactions result in the bacteria pausing to divide and filiform. This state of the bacteria is more favorable for the replication of the phage because the SOS reaction destabilizes the cell wall of the bacteria, which is composed of peptidoglycans, which makes the bacteria more sensitive to lysozyme, perforin produced by the phage, thereby accelerating the entry of the phage into the bacteria. Although both antibacterial substances are used at the same time, a small probability of bacteria will develop resistance to both substances, and even resistance to both substances will result in reduced bacterial pathogenicity or reduced growth rate. Therefore, it is theoretically feasible to use the combination of antibiotics and phages to reduce bacterial resistance and increase the sterilization efficiency.

However, current staphylococcal phages for the prevention and treatment of swine staphylococcal exudative dermatitis are few and have limited lytic spectrum width, as in the applicant's 2017 application (issued publication number: CN 107779440B) discloses a staphylococcal phage SA1 with a lytic spectrum of 72% (lysis of 36 out of 50 staphylococci); furthermore, the use of a combination preparation of phage and antibiotics in this regard has not yet emerged.

Accordingly, there is a need for further improvements in the art.

Disclosure of Invention

Aiming at the problems, the invention provides a novel staphylococcus bacteriophage vB-SauM-SA34 with a broad-spectrum lysis and application thereof, and the bacteriophage has a strong lysis effect on staphylococcus causing swine exudative dermatitis, can be used for preparing medicines, environmental disinfectants, feeds, water additives and the like for preventing and treating diseases caused by staphylococcus infection, and solves the problems of antibiotic residue and pathogenic bacteria drug resistance caused by using antibiotics while solving the staphylococcus infection.

The technical scheme of the invention is as follows:

in a first aspect, the present invention provides a staphylococcus bacteriophage with broad spectrum and strong lytic property, which is isolated from sewage of a pig farm in Qingdao, and is named as vb_saum_sa34, and is preserved in the China general microbiological culture Collection center with a preservation address of 1 st, 3 rd, 05 month and 15 th in the Beijing, towards the yang area, and a preservation number of 19976.

The phage vB-SauM-SA34 has a polyhedral structure, a slender tail part, a phage head part of about 140-150nm and a width of about 120-130nm, and a contractile tail part of about 260-270nm, and the shape of the phage corresponds to the characteristics of the Myoglyceae and belongs to Myoglycetes according to the classification method of the International Commission for virus classification (ICTV).

In a second aspect, the present application also provides a phage composition comprising the staphylococcal phage vb_saum_sa34. In practical application, in order to further widen the lysis spectrum of the phage preparation, the difference of the lysis spectrums of different phages is fully exerted, advantage complementation is carried out, and the staphylococcal phage vB_SauM_SA34 and other phages can be combined for use, such as combined with SA1 which is the staphylococcal phage or other phages, for killing staphylococci in the environment and preventing and treating staphylococcal diseases. In addition, the staphylococcus phage vB_SauM_SA34 can be matched with other phages of different types (inhibiting different pathogenic bacteria which cause the same kind of diseases) for preventing and treating the same kind of diseases.

In a third aspect, the present application also provides a phage preparation whose active ingredients include the aforementioned staphylococcal phage vB_SauM_SA34 and antibiotics. The pharmaceutical preparation is in the form of percutaneous administration and oral administration.

Optionally, the antibiotic includes, but is not limited to: an antibiotic effective against staphylococci, such as amoxicillin, ceftiofur, cefoxitin, ofloxacin, enrofloxacin, ciprofloxacin, neomycin, gentamicin, kanamycin, penicillin, chloramphenicol, rifampin, azithromycin, norfloxacin, tetracycline, vancomycin, nitrofurantoin, sulfamethoxazole, trimethoprim, amikacin, netilmicin, erythromycin, and the like.

Preferably, the antibiotic is ceftiofur, e.g., a ceftiofur hydrochloride solution may be used. Experiments show that the phage preparation prepared by combining the staphylococcus phage vB-SauM-SA34 and ceftiofur has the best control effect on the swine exudative dermatitis, and the cracking spectrum is greatly widened. In addition, the ceftiofur hydrochloride solution in the phage composition provided by the invention has a broad-spectrum antibacterial effect and can play a role in preserving the phage composition.

Optionally, a pharmaceutically acceptable carrier is also included in the phage preparation. 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 administered active component. In order to formulate the pharmaceutical composition into a liquid formulation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, ringer's solution, buffered saline, albumin infusion, dextrose solution, maltodextrin solution, glycerol, ethanol, and various media. They may be used alone or in any combination thereof. Other conventional additives, such as antioxidants, buffers, bacteriostats, and the like, may be added if desired. When also combined with diluents, dispersants, surfactants, binders and/or lubricants, the compositions of the present invention can also be prepared in injection and oral dosage forms (e.g., aqueous solutions, suspensions and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilisates.

Preferably, the phage preparation provided by the invention uses NB liquid medium as a carrier for staphylococcal phage and ceftiofur pharmacy.

Depending on the age, weight, sex, condition and diet of the animal to be treated, the time of administration, route of administration, rate of excretion and a variety of factors of response sensitivity, the appropriate dosage of the phage preparation of the invention when administered, sprayed or otherwise administered may vary. It will be apparent to those skilled in the art that when a pharmaceutical composition is administered to a patient, the appropriate total daily dose can be determined by the attending physician or veterinarian with the appropriate medical judgment.

The ceftiofur hydrochloride solution in the phage preparation provided by the invention has sub-antibacterial concentration on the phage host bacteria: 2.5 μg/ml, and the ceftiofur hydrochloride solution in the phage composition still has partial antibacterial effect at the concentration of 2.5 μg/ml. Experiments prove that the ceftiofur hydrochloride solution can improve the titer of the staphylococcus phage when the concentration is sub-bacteriostatic.

The staphylococcus phage provided by the invention has a cracking effect on 224 strains in 256 staphylococcus of pig origin, and the cracking rate can reach 87.5%. When ceftiofur acts alone, the ceftiofur only has an inhibition effect on 109 strains in 256 strains of staphylococcus of pig origin, and the inhibition rate can reach 42.58 percent. The phage preparation composed of the two active ingredients has an inhibition effect on 241 strains in 256 strains of swine staphylococci, the inhibition rate can reach 94.14%, 17 strains of swine staphylococci which can not be cracked under the independent action of ceftiofur and phage can be cracked, so that the ceftiofur and the staphylococci have a synergistic effect, and the cracking range of the ceftiofur and the staphylococci is widened.

In a fourth aspect, the present application also provides the use of the staphylococcal phage, phage composition or phage preparation described above for the preparation of a medicament for the prevention and treatment of a disease caused by staphylococcal infection. The control includes prevention and treatment. The term "preventing" 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 improvement or improvement of the disease by administration of the composition.

Preferably, the staphylococcal infection disease includes swine exudative dermatitis.

In a fifth aspect, the present application also provides an environmental disinfectant, the active ingredient of which comprises the staphylococcal phage vb_saum_sa34, phage composition or phage preparation as described above. Based on the substances, the staphylococcus pathogenic bacteria disinfectant has excellent cracking performance, can be used as an environment disinfectant for killing pathogenic bacteria in pig farm environments. Preferably, the concentration of phage is 10 ⁸ PFU/ml.

Optionally, the environmental disinfectant also contains other active ingredients for the inhibition or elimination of viruses, bacteria in the environment; preferably, environments in which the environmental disinfectant may be applied include feed, water, and farming environments including tanks, floors, walls, manure, and litter.

In a sixth aspect, the present application further provides the use of the above-described environmental disinfectant for disinfecting a farm environment, in particular, by spraying, dipping, the environmental disinfectant disinfects a farming environment, including tanks, floors, walls, faeces and litter, a feeding implement. Such liquid soaking, spraying forms include, but are not limited to, detergents, disinfectants, decontaminating agents, and the like.

In a seventh aspect, the present application further provides a drinking water additive or a feed additive, wherein the active ingredients of the drinking water additive or the feed additive comprise the staphylococcus phage vb_saum_sa34 and the phage composition. Optionally, the potable water additive further comprises other active ingredients for viral, bacterial inhibition or elimination in water; the drinking water additive is in the form of liquid dosage form, powder dosage form or solid dosage form, but is not limited to the three dosage forms.

In an eighth aspect, the present application further provides a food preservative, which comprises the staphylococcal phage vb_saum_sa34 or phage composition as described above, and the staphylococcal preservative can be sprayed on the surface of meat or soaked in a solvent added with the food preservative to achieve the effect of inhibiting staphylococci in meat products and achieve the preservation effect.

In a ninth aspect, the present application also provides a detection kit comprising the aforementioned staphylococcal phage. Based on the lysis specificity of the staphylococcus phage vB-SauM-SA34 on host bacteria, the staphylococcus phage can be applied to rapid detection of staphylococcus, including but not limited to detection of staphylococcus in the form of test paper, test paper box and the like, or screening of target pathogenic bacteria in clinical samples, and the detection sensitivity is effectively ensured.

The invention has the following beneficial effects:

1. the staphylococcus phage vB-SauM-SA34 provided by the invention has a wide lysis spectrum, has a strong lysis effect on staphylococcus causing swine exudative dermatitis, can effectively prevent and treat staphylococcal diseases, and has the effect of preventing and treating staphylococcus exudative dermatitis. The phage can be used as active ingredient of environment disinfectant, and has good effect of sterilizing staphylococci in feed, environment, etc.

2. The present invention also provides a phage preparation consisting of a combination of staphylococcal phage vB_SauM_SA34 and an antibiotic, preferably ceftiofur. Through the synergistic effect of ceftiofur and the staphylococcus, the splitting range of phage vB-SauM-SA34 is further greatly widened, and the application value of the phage preparation is improved.

3. The phage preparation has various administration modes, can be used internally and externally, can directly kill bacteria at diseased places when being used for external administration compared with antibiotics injected by muscle, and is convenient and efficient. The phage composition provided by the invention can solve the problem of drug resistance caused by abuse of antibiotics, and the interaction of the staphylococcus phage and the antibiotics can promote the sterilization effect of the other side mutually.

4. The above phage preparation has no adverse effect on the mutual potency of staphylococcal phage and antibiotics, and the potency of staphylococcal phage vB_SauM_SA34 can be increased to 10 by synergistic effect with ceftiofur hydrochloride solution ⁹ PFU/ml. The two active ingredients can exist stably for a long time, can effectively kill staphylococcus biofilm and reduce staphylococcus residues in the environment.

Drawings

FIG. 1 is an electron microscope picture of phage vB_SauM_SA 34;

FIG. 2 is the results of a thermal stability test of phage vB_SauM_SA 34;

FIG. 3 shows the pH stability test results of phage vB_SauM_SA 34;

FIG. 4 is a one-step growth curve of phage vB_SauM_SA34.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

EXAMPLE screening and purification of phages and investigation of biological Properties

1. Screening and purification of phages

The phage is separated from sewage of a pig farm in Qingdao, phage is obtained by a double-layer plate method, single phage is obtained by repeatedly purifying a single plaque three times, the phage is named as vB_SauM_SA34, and host bacteria of the phage are ZB12.

2. Biological Properties of phages

(1) Electron microscopic observation of phage

A. The experimental method comprises the following steps: dropping 20 mu L of phage sample on a copper net with a carbon coating film, naturally precipitating for 15min, slightly sucking the phage sample with filter paper, then dyeing with 2% (W/V) phosphotungstic acid (PTA) for 1-2 min, slightly sucking the filter paper, and observing and photographing under a transmission electron microscope after the phage sample is dried.

B. Experimental results: the morphology is shown in figure 1, and the phage head is in a polyhedral structure and has a slender telescopic tail, the phage head is about 140-150nm long, the phage head is about 120-130nm wide, and the tail is about 260-270nm. According to the classification method of the International Commission on viral Classification (ICTV), the morphology of the phage in the study accords with the characteristics of the Myoglyceae, and belongs to the Myoglycetes. This phage was named vb_saum_sa34.

(2) Effect of temperature on phage vB_SauM_SA34

A. The experimental method comprises the following steps:

(1) will be added with 5.2×10 of the same volume ⁹ placing each container of pfu/mL phage proliferation liquid at 40deg.C, 50deg.C, 60deg.C, 70deg.C, 80deg.C, holding for 20min, 40min and 60min, sampling, cooling the samples in ice bath, diluting by 10 times, and measuring titer with proper dilution gradient. And drawing a thermal stability curve of the phage by taking the temperature as an abscissa and taking the logarithmic value of phage titer as an ordinate.

(2) Adding 4.5mL of NB broth with different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) into sterile test tubes, placing the test tubes into a water bath at 37deg.C, and adding 500 μl of 5.2X10 s after the temperature is stable ⁹ pfu/mL phage proliferation solution, mixing well, and water-bathing at 37 ℃ for 1h, 2h and 3h. Immediately after the completion of the action, a proper amount of 1mol/L HCl or NaOH was added to the mixture to give a pH of the mixture of about 7, and 10-fold dilution was performed, and the titer was measured by taking a proper dilution gradient, and 2 replicates were set for each pH test tube. And drawing a phage pH stability curve by taking the pH value as an abscissa and the logarithmic value of phage titer as an ordinate.

B. Experimental results and analysis

As shown in FIG. 2, the phage vB_SauM_SA34 still has high activity after being acted for 1h at 40-60 ℃, the activity is stable, and partial activity can still be kept within 20-40 min at 70 ℃. The phage has good thermal stability, and can meet the requirements of phage preparations for treating swine exudative dermatitis.

As shown in FIG. 3, the titer of phage vB_SauM_SA34 is basically unchanged in the pH range of 4-11, and part of activity can still be remained after the phage is acted for 3 hours under the condition of pH12, so that the phage has better acid and alkali resistance.

(3) Determination of phage cleavage spectra

A. Experimental materials: fresh phage multiplication solution vb_saum_sa34 and clinically isolated 256 staphylococci from different areas of eastern mountain, beijing, henna, northwest, guangxi farms were prepared. The 256 strains of staphylococcus of porcine origin each comprise one or more virulence genes, such as an exfoliative toxin, a leukocidal toxin, a toxin shock symptom toxin, an alpha hemolytic toxin, and the like.

B. The experimental method comprises the following steps:

(1) the split spectrum of phage was determined by double-layer plate method as follows: single colonies were picked up in a dish streaked with staphylococci, inoculated into 5ml NB liquid medium, and cultured with shaking at 180rpm in an air shaker at 37℃for 18h to give a single bacterial suspension. Mu.l of the bacterial suspension was mixed with 100. Mu.l of 3.4X10 g of the bacterial suspension ³ After pfu/mL phage incubation for 5min at 37 ℃, double-layer plates were prepared by adding to the upper agar, and after agar solidification, the plates were placed in an incubator at 37 ℃ for inversion culture overnight for observation.

(2) Comparison with existing phages: meanwhile, phage vB_SauM_SA34 and phage SA1 in patent CN107779440B of the company are subjected to splitting spectrum measurement, and splitting differences of two different staphylococcus phages are compared.

C. Experimental results and analysis:

the virulence gene information of 256 staphylococcus from swine and the lysis spectra of two staphylococcus phages are shown in table 1, and the results in table 1 show that phage vB_SauM_SA34 has a lysis effect on 224 staphylococcus from 256 staphylococcus, the lysis rate is 87.5%, and the broad lysis spectrum is provided. The bacteriophage SA1 has a lysis effect on 192 staphylococci in 256 staphylococci, and the lysis rate is only 75%; it is evident that the phage vB_SauM_SA34 has a significantly broader lysis spectrum for the 256 strains of Staphylococcus suis described above than for phage SA1.

TABLE 1 virulence gene information of 256 porcine staphylococci and staphylophage lysis spectra

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(4) One-step growth curve of staphylococcal phage vb_saum_sa34

1. The experimental method comprises the following steps:

phage multiplication solution with a multiplicity of infection of 10 and fresh host bacteria multiplication solution were each 1mL, mixed well (timing was started at this time), incubated at 37℃for 5min, centrifuged at 13000rpm for 30s, the supernatant was aspirated as much as possible with a micropipette, washed 1 time with 5mL NB broth (centrifuged at 13000rpm for 30 s), and the supernatant was discarded. The phage titer was measured by a double-layer plate method after 10-fold dilution of NB broth, 3 parallels were made, the results were averaged, the time of infection was taken as the abscissa, the titer of phage in the infection system was the ordinate, and a one-step growth curve was drawn to obtain the incubation period and burst period of phage vB_SauM_SA34.

Burst amount = total number of phage at end of burst/total number of bacteria at beginning of burst

2. Experimental results and analysis

The staphylococcus phage provided by the invention takes staphylococcus as a host, and a one-step growth curve discovers that the incubation period is 20min, the burst period is 90min and the burst amount is 85. Compared with phage SA1, vB_SauM_SA34 has short latency and burst period and large burst size.

(5) Lytic assay of phage vB_SauM_SA34 against non-host bacteria

1. Experimental method

The lysis experiment was performed by the double-plate method according to example one, by selecting 10 strains of E.coli, 10 strains of Salmonella, 10 strains of Pseudomonas aeruginosa, and the like.

2. Experimental results and analysis

None of the phage vB_SauM_SA34 recognizes the 30 non-host bacteria. The test phage has extremely strong host specificity and no damage to microbial community.

Example drug sensitivity test of Staphylococcus of porcine origin against ceftiofur

Sensitivity of 256 s.suis to ceftiofur drug was determined using a micro broth dilution method:

1. determination of Minimum Inhibitory Concentration (MIC) of ceftiofur hydrochloride solution

Obtaining single bacterial suspension of vB_SauM_SA34 host bacteria ZB12 by the method, measuring absorbance of bacterial liquid by using a spectrophotometer, detecting wavelength to 600nm, converting absorbance into bacterial concentration, and diluting bacterial liquid to 5×10 ⁵ CFU/ml. Ceftiofur hydrochloride solutions were diluted to 40960, 20480, 10240, 5120, 2560, 1280, 640, 320, 160, 80, 40, 20, 10, 5, 2.5, 1.25 and 0.625 μg/ml, respectively. MIC value determination: partitioning 96-well plates, and selecting 3 rows (such as A-C) as MIC determination regions for each groupRepeated 3 times. Adding 100 mu l of bacterial liquid into each row, and then adding 100 mu l of 12-dilution antibiotic diluent into each row of 1-12 holes respectively; setting a bacterial liquid control and repeating for 3 times, wherein the bacterial liquid control comprises 100 mu l of diluted bacterial liquid and 100 mu l of sterilized distilled water; a blank was set and repeated 3 times, and 100. Mu.l of physiological saline and 100. Mu.l of sterilized distilled water were added. OD of the culture was measured for 24 hours ₆₀₀ And calculating the light transmittance of the test hole.

Note that: the positive control and the blank control are all average values

When the transmittance of the test hole is higher than 95%, the minimum concentration reaching the standard is determined as the minimum antibacterial concentration of the ceftiofur hydrochloride solution. The test results confirm that the minimum inhibitory concentration of the ceftiofur hydrochloride solution is 5 mug/ml.

2. 256 pig-derived staphylococci drug susceptibility test:

(1) The experimental method comprises the following steps: obtaining single bacterial suspension from 256 strains of staphylococcus of swine origin according to the method, measuring absorbance of each bacterial strain by using a spectrophotometer, detecting wavelength to be 600nm, converting absorbance into bacterial concentration, and diluting each bacterial strain to 5×10 ⁵ CFU/ml. 100 mu.l of bacterial liquid and 100 mu.l of ceftiofur hydrochloride solution with the concentration of 5 mu.g/ml are added into each well of a 96-well plate, and the bacterial liquid is controlled to be 100 mu.l of diluted bacterial liquid and 100 mu.l of sterilized distilled water; a blank was set and repeated 3 times, and 100. Mu.l of physiological saline and 100. Mu.l of sterilized distilled water were added.

OD of the culture was measured for 24 hours ₆₀₀ And calculating the light transmittance of the test hole. When the transmittance of the test hole is higher than 95%, the staphylococcus strain is considered to be sensitive to the ceftiofur hydrochloride solution, and the ceftiofur hydrochloride solution has an inhibition effect on the staphylococcus strain.

(2) Experimental results and analysis:

through experiments, the ceftiofur hydrochloride solution has an inhibition effect on 109 strains in 256 strains of staphylococcus of swine origin, and the inhibition rate is 42.58%. Specific inhibition information is shown in table 1.

Example three phage preparation inhibition of staphylococci assay

1. The experimental method comprises the following steps: the inhibition rate of the phage preparation on 256 strains of staphylococcus of porcine origin was determined. In phage preparation, the volume ratio of vB_SauM_SA34 to ceftiofur hydrochloride solution is 1:1, vB_SauM_SA34 potency 1×10 ⁸ PFU/ml, ceftiofur hydrochloride solution concentration after mixing was 2.5. Mu.g/ml. The solvent was NB broth.

The split spectrum of phage was determined by double-layer plate method as follows: single colonies were picked up in a dish streaked with staphylococci, inoculated into 5ml NB liquid medium, and cultured with shaking at 180rpm in an air shaker at 37℃for 18h to give a single bacterial suspension. After incubating 100. Mu.l of the bacterial suspension with 100. Mu.l of phage preparation at 37℃for 5min, a double-layer plate was prepared by adding to the upper agar, and after the agar solidified, the plate was placed in an incubator at 37℃for inversion culture overnight to observe the results.

2. Experimental results and analysis:

the inhibition information of the phage preparation on 256 staphylococcus suis is shown in table 1. As shown in Table 1, the phage preparation has an inhibitory effect on 241 strains of Staphylococcus suis with an inhibition rate of 94.14% higher than that of the staphylococcal phage vB_SauM_SA34 alone and ceftiofur alone.

Second, as can be seen from the results of Table 1, the staphylococcal phage vB_SauM_SA34 in the phage preparation expands the respective lysis spectrum width by synergistic interaction with ceftiofur. Phage preparations specifically lyse 17 host bacteria (including ZB03, ZB08, ZB16, ZB29, ZB58, ZB73, ZB84, ZB88, ZB117, ZB122, ZB138, ZB152, ZB172, ZB182, ZB199, ZB211, ZB 239), while both the independently acting phages VB-SAUM-SA34 and ceftiofur have difficulty in lysing these host bacteria.

Example four sub antibacterial concentration ceftiofur hydrochloride solution to improve phage vB_SauM_SA34 potency test

1. The experimental method comprises the following steps:

the phage vB_SauM_SA34 of the host bacteria ZB12 inoculated in 5ml NB liquid medium, 37 degrees, 170rpm in an air shaker culture overnight; the control group was inoculated with 100. Mu.l each of vB_SauM_SA34 and its host strain ZB12 in a tube containing 5ml NB broth; test group in containing 5ml NB liquid medium test tube inoculation vB_SauM_SA34 and its host bacteria ZB12 each 100 u l after adding ceftiofur hydrochloride solution, to make the liquid ceftiofur hydrochloride solution concentration 2.5 u g/ml; in each of the test group and the control group, 1 test tube was inoculated with only the host strain ZB12 of vB_SauM_SA34. Test and control groups were co-incubated in an air shaker at 37℃at 170rpm until phage inoculated tubes were clear and transparent. The resulting phage solution was filtered and titers were determined using the double-layer plate method.

2. Experimental results and analysis: the results showed that the titer of phage vB_SauM_SA34 was increased by one titer after addition of a subbacteriostatic thifluzaine hydrochloride solution, and the titer of phage proliferation solution alone was 5.8X10 ⁸ PFU/ml, phage multiplication liquid titer is 7.2X10 after adding subbacteriostatic thifluzaine hydrochloride solution ⁹ PFU/ml。

Example safety test of five phages and antibiotic pharmaceutical formulations

1. The experimental method comprises the following steps: 20 Kunming mice of 18-20g of primary adults are selected and randomly divided into an experimental group and a control group (each group comprises male and female halves), 200 mu l of the phage preparation stock solution is injected intraperitoneally, 200 mu l of NB broth is injected into the control group, clinical changes of organs in the body are observed by sectioning after 7d, and liver, lung and spleen organ tissues are selected as sections for observing pathological changes.

2. Experimental results and analysis: through the sectioning examination, the lungs, the livers and the spleens of the mice injected with the phage preparation have no difference with the control group in performance, and the phage preparation does not cause obvious pathological changes to the lungs, the livers and the spleens of the mice from the view of tissue sections.

Example model therapeutic experiments with six phage preparations

1. The experimental method comprises the following steps: selecting 2 healthy piglets of 20 days old, carrying out local infiltration anesthesia on each male and female piglet by using 0.25% -0.5% procaine, cutting to deep fascia by using a 16mm cutter, cutting 4 incisions on the left and right sides of the spine, and subtracting the tissues in the incisions to form a comprehensive skin defect wound surfaceHemostatic, and 0.1ml ZB12 (bacterial amount 1×10) was added dropwise to the wound surface ⁹ CFU/ml) bandaging the wound surface with sterile gauze; the left side of the backbone is used as a phage preparation group, the right side is used as a control group, the phage preparation group is inoculated with 0.1ml of the phage preparation on a daily wound surface, the control group is inoculated with an equal amount of NB broth, the control group is continuously inoculated for 5 days, and the observation is carried out for 20 days.

2. Experimental results and analysis: after 10d of stopping, observation is carried out, and the result shows that black crusting appears on the wound surface of the phage preparation group, and the edge of the control group is red and swollen, and the wound surface has purulent secretion; after 20d of stopping, observation is carried out, and the wound surface of the phage preparation group is completely formed into black crusts to cover the wound surface, and the crusts are formed in a small range of the wound surface of the control group, but purulent secretion still oozes.

Example seven phage formulations treatment trials with different modes of administration

1. The experimental method comprises the following steps:

from the clinical collection of 60 exudative dermatitis pigs of 15-20 days of age, the test was randomly divided into 5 groups of 12, each group was set as a drug preparation intramuscular injection group, a drug preparation external application group, an antibiotic treatment group, a phage treatment group and a control group, and each group was treated separately in the following manner, and after stopping for 10d, observation was performed. The phage preparation used in the experiment was prepared in the same manner as in example 3, and the treatment method of each group was:

(1) the intramuscular injection of phage preparation is 1ml each day for each head of the intramuscular injection group of the drug preparation, and the continuous injection is carried out for 5 days;

(2) the phage preparation of 5-10ml is uniformly sprayed on the skin of the affected pigs every day for 5 days in the external group of the drug preparation;

(3) intramuscular injection (3 mg/kg) was performed daily using ceftiofur hydrochloride injection for 5 days in antibiotic treatment groups;

(4) phage treatment groups used phage solutions each evenly sprayed 5-10ml on the skin of diseased pigs for 5 days;

(5) the control group was not treated.

2. Experimental results and analysis:

(1) After 10d withdrawal, observations were made:

(1) pharmaceutical formulation intramuscular injection group: the skin of 83% pigs has crusting healing, and the dermatitis symptoms of the other 17% pigs are relieved;

(2) external group of pharmaceutical preparations: the crusting and healing of 92% of pig skin occur, and 8% of pig dermatitis symptoms are relieved;

(3) phage treatment group: skin crusting and healing occur in 75% of pigs, and dermatitis symptoms of the other 25% of pigs are relieved;

(4) antibiotic treatment group: 50% of dermatitis symptoms of pigs are relieved, the other 25% of dermatitis symptoms of pigs are not relieved, and 25% of pigs die;

(5) control group: 67% of pigs died and the remaining 33% had no relief from dermatitis symptoms.

(2) After 20d withdrawal, observations were made: the intramuscular injection group of the medicinal preparation and the external group of the medicinal preparation fully heal the skin; 92% of diseased pigs in the phage therapy group are healed by skin, 8% of pigs grow slowly and become stiff pigs; 58% of the diseased pigs in the antibiotic treatment group fully recover the skin, 25% of the pigs die, and 17% of the pigs slowly grow into stiff pigs; 75% of the control group diseased pigs die, and the other 25% of pigs grow slowly to become stiff pigs. The statistics of pig growth conditions shows that the treatment effect of the external phage preparation group and the intramuscular injection drug preparation group is obviously better than the effect of the phage treatment group and the antibiotic treatment group which are used independently.

EXAMPLE eight phage preparation environmental disinfection test

1. The experimental method comprises the following steps: in a personal pig farm in Qingdao, a plurality of barns 10 which have exploded out swine exudative dermatitis are randomly selected, and 20 sampling points are selected for each barn, such as a trough, a floor, a wall, feces, padding and the like. After sampling the 20 sampling points, respectively, immersing the samples in 1ml of physiological saline, respectively, and then coating 100 μl of the solution in MSA culture medium to calculate the staphylococcal content.

Using 1X 10 ⁸ PFU/ml phage preparation the selected barns were spray sterilized, 2L each, and after 24h sterilization were sampled at the same sampling points. After sampling and soaking in 1ml of physiological saline, 100. Mu.l of the solution is coated in MSA culture medium, and the grapes are calculatedContent of cocci.

2. Experimental results and analysis: as shown in the results of Table 2, the content of staphylococci is obviously reduced, and the clearance rate of the phage preparation to the staphylococci in the environment is more than 98%.

TABLE 2 Staphylococcus content in the environment of each barn before and after sterilization

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Claims

1. A staphylococcus bacteriophage is named as vB_SauM_SA34, and has a preservation number of CGMCC No.19976.

2. A phage composition comprising the staphylococcal phage of claim 1 and other phages.

3. A phage preparation comprising the staphylococcal phage of claim 1 and an antibiotic as active ingredients.

4. A phage preparation according to claim 3, characterized in that the pharmaceutical preparation is in the form of a transdermal or oral dosage form.

5. A phage preparation according to claim 3, wherein the antibiotic is ceftiofur.

6. The phage preparation of claim 5, wherein the ceftiofur is a liquid preparation.

7. A phage preparation according to claim 3, wherein the antibiotic is at a sub-bacteriostatic concentration.

8. The phage preparation according to claim 5, wherein the titer of staphylococcal phage is not less than 10 ⁹ PFU/ml。

9. Use of a staphylococcal phage according to claim 1, a phage composition according to claim 2 or a phage preparation according to claim 3 for the preparation of a medicament for the treatment of a disease associated with staphylococcal infection.

10. The use according to claim 9, wherein the disease of staphylococcal infection comprises swine exudative dermatitis.

11. A feed or drinking water additive comprising a staphylococcal phage according to claim 1 or a phage composition according to claim 2.

12. An environmental disinfectant comprising the staphylococcal phage of claim 1, the phage composition of claim 2 or the phage preparation of claim 4 as an active ingredient.

13. An environmental disinfectant according to claim 12, wherein the phage is used at a concentration of 1 x 10 ⁹ PFU/ml.

14. A food preservative comprising the staphylococcal phage vb_saum_sa34 of claim 1 or the phage composition of claim 2.