Pseudomonas aeruginosa phage, microbial preparation and application thereof

Abstract

The invention belongs to the technical field of microbiology and agricultural application thereof, and particularly relates to a pseudomonas aeruginosa phage, a microbial preparation and application thereof. The pseudomonas aeruginosa bacteriophage provided by the invention belongs to a long-tail bacteriophage. The compound shows an excellent killing and destroying effect on host multi-drug-resistant pseudomonas aeruginosa, also has splitting activity on other seven clinical drug-resistant pseudomonas aeruginosa, is wide in antibacterial spectrum, and has extremely high application potential in the field of pseudomonas aeruginosa prevention and treatment drugs. Besides, the pseudomonas aeruginosa bacteriophage pPA.S2. XXXSZ disclosed by the invention has strong environmental tolerance, and can play a synergistic role with antibiotics, so that the antibacterial durability is greatly enhanced, and the use dosage of the antibiotics is further reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and its agricultural applications, and particularly relates to a Pseudomonas aeruginosa phage, a microbial preparation and their applications. Background Art

[0002] Pseudomonas aeruginosa is a common conditional Gram-negative pathogenic bacterium. At present, a variety of different subtypes of Pseudomonas aeruginosa have been identified, which frequently appear in clinical infection cases, pose a serious threat to immunocompromised populations, and also cause many problems in animal groups. In the field of livestock and poultry breeding, Pseudomonas aeruginosa is extremely easy to contaminate the breeding environment, and livestock and poultry are often infected through contact with contaminated water sources, feeds, or inhalation through the respiratory tract. Pseudomonas aeruginosa is also extremely easy to contaminate hospital environments, medical devices, and various humid places, and often causes infections through wound contact, inhalation through the respiratory tract, etc. According to statistics, the number of diseases caused by Pseudomonas aeruginosa infection in the animal kingdom every year is considerable, seriously affecting animal health.

[0003] Currently, the prevention and treatment of Pseudomonas aeruginosa infection mainly rely on antibiotics. However, Pseudomonas aeruginosa is a multi-drug resistant conditional pathogenic bacterium, which can develop resistance to antibiotics such as aminoglycosides, quinolones, and β-lactams, and clinically highly resists the treatment of antibiotics including penicillin, cephalosporins, aztreonam, cefazolin-tazobactam, etc. The unreasonable use of antibiotics for a long time has led to the increasingly serious problem of drug resistance of Pseudomonas aeruginosa. The continuous emergence of drug-resistant strains has greatly reduced the effectiveness of traditional antibiotics in the treatment of Pseudomonas aeruginosa infection, and significantly increased the clinical treatment difficulty.

[0004] As a type of virus that can specifically infect specific bacteria, according to morphological characteristics, phages are mainly divided into tailed phages and tailless phages, among which tailed phages account for up to 96% of the known phage species. The structure of phages is relatively simple, mainly composed of a protein coat and internal genetic material. As one of the highly potential alternatives to antibiotics, phages have their unique advantages, such as extremely wide sources, relatively simple isolation processes, relatively low production costs, good stability, and high specificity for host bacteria. Phage therapy, as an emerging treatment method, has shown great clinical application value and broad development prospects in the field of prevention and treatment of Pseudomonas aeruginosa infection.

[0005] However, in the current situation, the Pseudomonas aeruginosa phages screened from the natural environment are still lacking in species resources, and many deficiencies are exposed when dealing with the host resistance evolution of Pseudomonas aeruginosa and complex and changeable environmental factors. Therefore, in-depth screening of novel Pseudomonas aeruginosa phages is of crucial practical significance for enriching relevant species resources and improving the overall prevention and treatment level of Pseudomonas aeruginosa infection. Summary of the Invention

[0006] To solve the problems existing in the prior art, one of the objectives of the present invention is to provide a Pseudomonas aeruginosa phage, which belongs to the Siphoviridae family, has excellent environmental stability and a relatively broad antibacterial spectrum.

[0007] Another objective of the present invention is to provide a microbial preparation containing the Pseudomonas aeruginosa phage provided by the present invention, which can be used as a drug, feed additive, cleaner, and / or disinfectant, and has a highly efficient and persistent killing and destructive ability against Pseudomonas aeruginosa.

[0008] The third objective of the present invention is to provide an application of a microbial preparation. The Pseudomonas aeruginosa phage microbial preparation provided by the present invention can be used in combination / complex with antibiotics to exert a synergistic effect and has a more persistent antibacterial ability.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A Pseudomonas aeruginosa phage, named Pseudomonas aeruginosa phage pPA.S2XXXSZ, with a preservation number of CCTCC M 2025676, a preservation date of April 2, 2025, and preserved at the China Center for Type Culture Collection, located in Wuhan, China.

[0011] A microbial preparation containing the above-mentioned Pseudomonas aeruginosa phage.

[0012] In the specific embodiments of the present invention, the content of Pseudomonas aeruginosa phage in the above microbial preparation is ≥10 1 PFU / mL.

[0013] The specific embodiments of the present invention verify that the use of the Pseudomonas aeruginosa phage of the present invention alone can produce an antibacterial effect, and the combined use with antibiotics can produce a more persistent antibacterial effect and can reduce the dosage of antibiotics. As an example, the microbial agent of the present invention also contains an antibiotic, and the content of the antibiotic is <5% - 50% of the recommended antibacterial content of a single antibiotic; as an example, the antibiotic can be selected from amikacin.

[0014] Optionally, the antibiotic is amikacin, the content of Pseudomonas aeruginosa phage in the microbial preparation is 10 4 ~10 7 PFU / mL, and the content of amikacin is 0.5 - 64 μg / mL; preferably, the content of amikacin is 4 - 32 μg / mL.

[0015] By way of illustration, the above-mentioned microbial preparation can be used alone or in combination with antibiotics to prevent and / or treat Pseudomonas aeruginosa contamination and / or infection. It is particularly suitable for clinically drug-resistant Pseudomonas aeruginosa infections. Specific Pseudomonas aeruginosa strains exhibit multi-drug resistance characteristics and show high resistance to antibiotics such as meropenem, gentamicin, ciprofloxacin, amikacin, aztreonam, polymyxin, and ceftazidime (the minimum killing time far exceeds the duration required for normal sensitive strains) and multi-drug resistance.

[0016] Specifically, the above-mentioned microbial preparation can be used to prepare drugs for preventing and / or treating Pseudomonas aeruginosa infection, or to prepare cleaning agents or disinfectants for preventing and controlling Pseudomonas aeruginosa contamination, or to prepare feed additives.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention belongs to the Siphoviridae and is a novel Pseudomonas aeruginosa phage;

[0019] (2) The Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention has good killing and destructive ability against the host bacterium Pseudomonas aeruginosa resistant 2, and can also lyse other 7 clinically drug-resistant Pseudomonas aeruginosa strains to varying degrees, having the potential to be used as a drug for preventing and controlling Pseudomonas aeruginosa;

[0020] (3) The latent period of the Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention is 45 min, and the burst period is 45 - 145 min. It can replicate in large numbers and lyse host cells, efficiently releasing progeny phages, which is conducive to the rapid proliferation and spread of the phage population. Its burst size is about 65 PFU / mL, indicating that it has strong bactericidal or host bacterium growth control ability and has the potential to be developed into an antibacterial preparation;

[0021] (4) The Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention has strong environmental tolerance. When treated in a 70 °C water bath for 60 min, it still has activity. In the pH range of 5 - 10, its activity remains stable, and chloroform has no effect on it, indicating that the phage capsid has good stability and tolerance to organic solvents.

[0022] (5) The Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention can cooperate with antibiotics to have a more persistent antibacterial ability and reduce the dosage of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0024] Figure 1 is the plaque picture of Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention;

[0025] Figure 2 is the transmission electron micrograph of Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention;

[0026] Figure 3 is the optimal multiplicity of infection graph of Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention;

[0027] Figure 4 is the one-step growth curve graph of Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention;

[0028] Figure 5 is the schematic diagram of the influence of temperature on the activity of Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention;

[0029] Figure 6 is the schematic diagram of the influence of pH on the activity of Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention;

[0030] Figure 7 is the schematic diagram of the influence of chloroform on the activity of Pseudomonas aeruginosa phage pPA.S2.XXXSZ of the present invention;

[0031] Figure 8 is the schematic diagram of the bactericidal effect of Pseudomonas aeruginosa phage pPA.S2.XXXSZ in the culture medium of the present invention;

[0032] Figure 9 is the schematic diagram of the combined antibacterial effect of Pseudomonas aeruginosa phage pPA.S2.XXXSZ and amikacin of the present invention. Detailed implementation manners

[0033] The following further elaborates on the above content of the present invention through examples, but this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0034] Strains, reagents, and culture media involved in the examples:

[0035] Both the Pseudomonas aeruginosa strain resistant to 2 antibiotics for screening Pseudomonas aeruginosa phages and the Pseudomonas aeruginosa strains used for analyzing the host spectrum of the screened Pseudomonas aeruginosa phages are clinical drug-resistant strains, and their direct source is the strains preserved in the laboratory. Among them, the host strain Pseudomonas aeruginosa resistant to 2 antibiotics shows significant resistance to amikacin, with a minimum inhibitory concentration (MIC) value as high as 64 μg / mL; its resistance to gentamicin is even more prominent, with the MIC value soaring to 160 μg / mL; it shows extremely high resistance to meropenem, with the MIC value > 1024 μg / mL, and the MIC value for colistin is 1.25 μg / mL.

[0036] Other Pseudomonas aeruginosa strains used for host spectrum screening show multi-drug resistance characteristics, and exhibit high resistance (the minimum killing time far exceeds the time required for normal sensitive strains) and multi-drug resistance to antimicrobial drugs such as meropenem, gentamicin, ciprofloxacin, amikacin, aztreonam, polymyxin, and ceftazidime.

[0037] 0.7% LB semi-solid medium (400 mL): Weigh 4.0 g of peptone, 2.0 g of yeast extract, 4.0 g of NaCl, and 2.8 g of agar powder, add ddH2O to 400 mL, and autoclave at 121 °C for 20 min.

[0038] LB solid medium (400 mL): Weigh 4.0 g of peptone, 2.0 g of yeast extract, 4.0 g of NaCl, and 6.0 g of agar powder, add 400 mL of ddH2O, autoclave at 121 °C for 20 min, cool to 50 °C, pour the plates, and after cooling and solidifying, invert and set aside.

[0039] SM buffer, PEG8000, NaOH, HCL, NaCl, and amikacin powder are all commercially available.

[0040] Example 1 Isolation and purification of Pseudomonas aeruginosa phage pPA.S2.XXXSZ

[0041] 1) The sample was collected from the sewage in Xinxiang City, Henan Province. The sewage sample was centrifuged at 8000 rpm / min for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm filter membrane. Take 600 μL of the filtrate, add 100 μL of the host bacterial solution, and then add 5 mL of LB medium, and place it in a constant temperature shaker for 12 h. The next day, centrifuge the above culture at 8000 rpm / min for 10 min, and filter and sterilize the supernatant through a 0.22 μm filter membrane to obtain a filtrate containing phages;

[0042] 2) Streak the host strain Pseudomonas aeruginosa resistant to 2 antibiotics on the LB solid medium, and after culturing overnight, pick a single colony and inoculate it into 5 mL of LB broth medium, and culture it with shaking at 37 °C for 12 h as the host bacterial solution for standby;

[0043] 3) Mix 100 μL of the above-prepared host bacterial solution with 8 mL of LB semi-solid medium at about 50 °C in a 15 mL sterile centrifuge tube, pour it onto the solid medium, and divide the petri dish into three areas after it dries. Take 10 μL of the above phage-containing filtrate and drop it into two areas of the petri dish respectively, and drop 10 μL of PBS into the other area as a blank control. After natural drying, invert it and culture it in an incubator at 37 °C for 8 h, and observe whether plaque formation occurs in the area where the phage is dropped. If plaque formation occurs, it proves the existence of phage;

[0044] 4) If plaque formation occurs, serially dilute the above phage-containing filtrate 10-fold with LB liquid medium. Take 10 -4 ~10 -7 Dilute 100 μL of the dilution and mix it with 100 μL of the host bacterial solution respectively, let it stand for 20 min, add 8 mL of 0.7% LB semi-solid medium at about 50 °C, mix well and pour it onto the LB solid medium plate. Observe the growth of plaques after culturing at 37 °C for 12 h. Pick a single transparent and uniform-sized plaque into a 1.5 mL centrifuge tube containing LB broth medium and vortex to mix well. Dilute 100 μL of the above solution 10-fold again, take 100 μL of the dilution and culture it with the same volume of the host bacterial solution using the double-layer plate method. Repeat this process about 3 - 5 times to obtain phages with uniform plaque size, store them at 4 °C as the phage preservation solution for later use.

[0045] The plaque results are as Figure 1 shown. This phage can form transparent plaques with clear and regular edges on the solid LB medium plate.

[0046] Example 2 Amplification and Concentration of Pseudomonas aeruginosa Phage pPA.S2.XXXSZ

[0047] 1) Take 100 μL of the phage preservation solution reserved in Example 1 and 100 μL of the host bacterial solution reserved in Example 1 in a centrifuge tube, let them act for 15 min, add 5 mL of LB liquid medium, culture at 37 °C for 12 h, centrifuge at 4 °C and 8000 rpm for 10 min, take the supernatant, and filter it through a 0.22 μm filter membrane. The filtrate is the phage stock solution;

[0048] 2) PEG concentration: Add NaCl to the phage stock solution to a final concentration of 1 mol / L and ice-bath for 2 h, centrifuge at 4 °C and 10000 rpm / min for 20 min, take the supernatant and add PEG8000 with a final concentration of 10%, ice-bath for 16 h, then centrifuge at 4 °C and 12000 rpm / min for 15 min, carefully discard the supernatant, invert it for 5 min to drain the liquid, add 500 μL of SM buffer to resuspend the precipitate, and obtain the purified and concentrated phage stock solution.

[0049] Transmission Electron Microscopy Observation of Pseudomonas aeruginosa Phage pPA.S2.XXXSZ in Example 3

[0050] For the electron microscopy observation of the concentrated phage stock solution in Example 2: 10 μL of the concentrated phage stock solution in Example 2 was dropped on a copper grid, allowed to naturally precipitate for 2 - 3 min, the excess liquid was blotted with filter paper, 10 μL of 2% phosphotungstic acid (PTA, 2% w / v) was dropped for staining, and after drying at room temperature, it was observed using a transmission electron microscope; the observation results are as Figure 2 shown: Phage pPA.S2.XXXSZ belongs to the Siphoviridae family, with an icosahedral head structure. The head diameter is approximately 65.7 ± 1.25 nm, and the tail length is approximately 129 ± 1.29 nm.

[0051] The phage was self - named pPA.S2.XXXSZ and deposited in the China Center for Type Culture Collection with the deposit number CCTCC M 2025676.

[0052] Determination of the Optimal Multiplicity of Infection of Pseudomonas aeruginosa Phage pPA.S2.XXXSZ in Example 4

[0053] The host bacterial solution of Pseudomonas aeruginosa resistant 2 reserved in Example 1 was cultured to the logarithmic phase, and phage pPA.S2.XXXSZ with multiplicities of infection (MOI) of 100, 10, 1, 0.1, 0.01, and 0.001 were added respectively. They were co - cultured at 37°C for 6 h. The culture solution was centrifuged at 8000 r / min for 10 min, and the supernatant was filtered through a 0.22 - μm filter membrane. The phage titers at different multiplicities of infection were determined by the double - layer plate method, and the optimal MOI of the phage was determined. The detection results are as Figure 3 shown, and the optimal multiplicity of infection of phage pPA.S2.XXXSZ is 10.

[0054] Determination of the One - Step Growth Curve of Pseudomonas aeruginosa Phage pPA.S2.XXXSZ in Example 5

[0055] The host bacterial solution (Pseudomonas aeruginosa multi - drug resistant strain No. 2) reserved in Example 1 was mixed with the purified and concentrated phage stock solution preserved in Example 2 (the multiplicity of infection was approximately 0.1). After incubation at 37°C for 15 min, it was centrifuged at 7000 rpm / min for 5 min, and the supernatant was discarded. The precipitate was resuspended with 5 mL of LB broth medium and quickly placed in a shaker at 37°C for shaking culture. Starting from 0 min, 200 μL of the culture was taken every 10 min, centrifuged at 4°C and 10000 rpm / min for 2 min to remove bacteria, and the supernatant was serially diluted 10 - fold to an appropriate concentration. The phage titer was determined by the double - layer plate method. With the sampling time as the abscissa and the logarithm of the phage titer as the ordinate, a one - step growth curve was plotted. The results of the one - step growth curve are as Figure 4Shown as follows: The latency period of Pseudomonas aeruginosa phage pPA.S2.XXXSZ infecting the host bacteria is 45 min, and it enters the stationary phase after 145 min.

[0056] Example 6 Temperature and pH Tolerance Experiments of Pseudomonas aeruginosa Phage pPA.S2.XXXSZ

[0057] The purified and concentrated phages reserved in Example 2 were respectively placed in environments of 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C. Samples were taken every 20 min until 60 min. The purified and concentrated phage stock solution preserved in Example 2 was diluted 10-fold serially and then the double-layer plate method was used to determine its titer. The detection results are as Figure 5 Shown as follows: This phage can still maintain a high titer at 30 °C, 40 °C, and 50 °C. When under the condition of 60 °C water bath for 60 min, the phage titer began to decline. When under the condition of 80 °C water bath for 20 min, the phage was inactivated.

[0058] Take 12 sterile 1.5 mL centrifuge tubes, and add 100 μL of the purified and concentrated phage stock solution reserved in Example 2 and 900 μL of LB broth medium with pH values of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. Incubate at 37 °C for 1 h, and then use the double-layer plate method to determine the titer of the phage after treatment. The detection results are as Figure 6 Shown as follows: Pseudomonas aeruginosa phage pPA.S2.XXXSZ is active and stable in an environment with a pH value of 5 - 10. When the pH is lower than 5 or higher than 10, the phage is completely inactivated.

[0059] Example 7 Chloroform Sensitivity Experiment of Pseudomonas aeruginosa Phage pPA.S2.XXXSZ

[0060] The purified and concentrated phage stock solution reserved in Example 2 was mixed with chloroform at different ratios, and the final volume was 1 mL (chloroform volume / total volume = 0%, 10%, 20%, 30%, 40%, 50%). Shake well and incubate at 37 °C for 2 h, and then use the double-layer plate method to determine the titer of the phage after treatment. The detection results are as Figure 7 Shown as follows: Chloroform has no effect on Pseudomonas aeruginosa phage pPA.S2.XXXSZ.

[0061] Example 8 Host Range Analysis of Pseudomonas aeruginosa Phage pPA.S2.XXXSZ

[0062] Twenty-four clinically drug-resistant bacteria preserved in the laboratory were selected as host bacteria (the numbers are shown in Table 1), and the host range of Pseudomonas aeruginosa phage pPA.S2.XXXSZ was analyzed. The specific operation is as follows: Take 100 μL of the logarithmic growth phase culture broth of 24 strains of bacteria into a 15 mL sterile centrifuge tube, add 8 mL of LB semi-solid medium at about 50 °C, invert and mix well, pour it onto the LB medium plate, and wait for it to solidify. Then, take 10 μL of the purified and concentrated phage stock solution of Pseudomonas aeruginosa phage pPA.S2.XXXSZ preserved in Example 2 and drop it on the surface of the strain plate, using PBS as a negative control. After drying, invert it and culture it at 37 °C for 12 h. If a plaque is produced, it is recorded as "+", otherwise it is recorded as "-". The results are shown in Table 1: Pseudomonas aeruginosa phage pPA.S2.XXXSZ can lyse the host Pseudomonas aeruginosa drug-resistant 2, and can also lyse 7 other clinically drug-resistant Pseudomonas aeruginosa strains.

[0063] Table 1

[0064]

[0065] Example 9 Bactericidal effect of Pseudomonas aeruginosa phage pPA.S2.XXXSZ in the medium

[0066] Adjust the OD of the host bacterial solution Pseudomonas aeruginosa drug-resistant 2 reserved in Example 1 with LB broth medium 600 to 0.6 (the viable bacteria count is about 1×10 8 CFU / mL). Mix the purified and concentrated phage stock solution pPA.S2.XXXSZ reserved in Example 2 with the host bacterial solution in liquid LB medium at the multiplicity of infection of 100, 10, 1, 0.1, and 0.01 respectively, and place it in a constant temperature shaker at 37 °C for culture. Set a control group, and replace the phage solution with an equal amount of sterile PBS in the control group. Measure the absorbance of the culture at 600 nm every 1 h, and draw an in vitro antibacterial curve with time as the abscissa and OD 600 value as the ordinate.

[0067] The results of the phage bactericidal experiment are as Figure 8 shown. The phages with 5 different MOIs all have antibacterial effects on bacteria. In a short time, when the phage MOI is 100, the antibacterial effect is the best.

[0068] Example 10 Combined antibacterial effect of Pseudomonas aeruginosa phage pPA.S2.XXXSZ and amikacin

[0069] Adjust the OD of the host bacterial solution Pseudomonas aeruginosa drug-resistant 2 reserved in Example 1 with LB broth medium 600 to 0.6 (the viable bacteria count is about 1×10 8 CFU / mL), and keep it on ice for standby.

[0070] Take a sterile 96-well plate and add 50 μL of ice-bathed culture to each well (except for the 11th column) in a laminar flow hood. Add 50 μL of the original phage solution with a titer of 1×10 9 PFU / mL to the first row, add the original phage solution diluted with a 10-fold concentration gradient to the second row, with the concentration decreasing from top to bottom. Add 50 μL of LB broth to the last row. Add the original antibacterial drug solution diluted with a 2-fold concentration gradient to each column of wells, with the concentration decreasing from left to right. Add the antibacterial drug to the first column, add 50 μL of LB broth to the 10th column, add 150 μL of LB broth to the 11th column as a negative control, and add 50 μL of bacterial solution and 100 μL of LB to the 12th column as a positive control.

[0071] After the above system is configured, a combination of phages with different concentrations and antibacterial drugs with different concentrations in a checkerboard form is obtained. The last row is for the antibacterial drug to inhibit bacteria alone (the third well is the MIC value of the antibiotic), and the 10th column is for the phage to inhibit bacteria alone. Incubate the 96-well plate at 37 °C for 12 h, measure the absorbance at 600 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and draw a heat map of the bacterial growth situation (the color changing from green to red indicates more bacteria in the well).

[0072] Select amikacin as the antibacterial drug and verify the antibacterial performance of the combination of phage pPA.S2.XXXSZ and amikacin according to the above scheme. The results are as Figure 9 shown: The minimum inhibitory concentration of amikacin to inhibit bacteria alone is 64 μg / mL; when the phage titer is 10 4 ~10 7 PFU / mL in the case of the combination of phage pPA.S2.XXXSZ and amikacin, the dosage of amikacin can be reduced to 50%, or even 5%, of the dosage for inhibiting bacteria alone.

[0073] This experiment proves that the combined application of phage and amikacin can not only enhance the bactericidal effect, but also reduce the dosage of antibiotics, thus playing a certain role in replacing antibiotics.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Pseudomonas aeruginosa phage, characterized in that, It is named pPA.S2.XXXSZ, and its deposit number is CCTCC M2025676.

2. A microbial preparation, characterized in that, It contains the Pseudomonas aeruginosa phage as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The content of the Pseudomonas aeruginosa phage is ≥ 10 1 PFU / mL.

4. The microbial preparation according to claim 2 or 3, characterized in that, It also includes an antibiotic.

5. The microbial preparation according to claim 4, wherein The content of the antibiotic is < 5% - 50% of the recommended antibacterial content of a single antibiotic.

6. The microbial agent according to claim 5, wherein, The antibiotic is selected from amikacin.

7. The microbial preparation according to claim 6, characterized in that, The content of Pseudomonas aeruginosa phage in the microbial preparation is 10 4 ~10 7 PFU / mL, and the content of amikacin is 0.5 - 64 μg / mL; Preferably, the content of amikacin is 4 - 32 μg / mL.

8. Use of the microbial preparation according to any one of claims 2 to 7, characterized in that, It is used for preparing a drug for preventing and / or treating Pseudomonas aeruginosa infection, or for preparing a cleaner or disinfectant for preventing and controlling Pseudomonas aeruginosa contamination, or for preparing a feed additive.

9. Use of the microbial preparation according to claim 2 or 3, characterized in that, It is used for jointly preventing and / or treating Pseudomonas aeruginosa contamination and / or infection with an antibiotic.

10. The application according to claim 8 or 9, characterized in that, The Pseudomonas aeruginosa is selected from clinically identified Pseudomonas aeruginosa drug-resistant strains.

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