Engineered bacteriophage targeting drug-resistant gram-negative bacteria, screening method and application

Abstract

The invention relates to an engineered bacteriophage targeting drug-resistant gram-negative bacteria, a screening method and application. The engineered phage is Escherichia phage M13, the preservation time is February 11, 2025, the preservation address is China Center for Type Culture Collection (CCTCC), Wuhan University, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC No: M 2025196. The invention also provides application of the engineered bacteriophage in preparation of medicines for treating and diagnosing multi-drug-resistant gram-negative bacterium infection. The invention also provides a kit for diagnosing or treating multi-drug-resistant gram-negative bacterium infection, and the kit comprises the bacteriophage. The invention also provides a screening method of the engineered bacteriophage. The invention solves the problem that the natural bacteriophage is targeted to single species of bacteria and is difficult to cope with common mixed infection of multiple drug-resistant bacteria clinically.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an engineered bacteriophage targeting drug-resistant Gram-negative bacteria, a screening method and an application thereof. Background Art

[0002] In the field of medical and biological research, bacterial infections, especially multidrug-resistant organisms (MDR) infections, have become a major threat to global public health security. According to statistics from the World Health Organization (WHO), the number of deaths caused by drug-resistant bacteria infections worldwide has exceeded 700,000 per year, and it is expected that this number may rise to 10 million by 2050. The widespread use and improper abuse of traditional antibiotics have led to the continuous increase of bacterial resistance, which has significantly reduced the therapeutic effect of existing antibiotics and has led to the dilemma of "no drugs available" in clinical practice.

[0003] Currently, the treatment of multidrug-resistant bacterial infections mainly relies on the research and development of new antibiotics. However, the research and development of new antibiotics is long (usually takes 10-15 years), the investment is large (the average research and development cost exceeds 1 billion US dollars), and the speed at which bacteria develop resistance is much faster than the speed of new drug development. This situation has prompted researchers to turn their attention to the development of alternative antibacterial strategies. Among them, phage therapy, as an antibacterial method with a century-old history, has regained attention due to its unique antibacterial mechanism.

[0004] Bacteriophages are a type of virus that can specifically infect bacteria. They do this by recognizing specific receptors on the surface of bacteria, and then proliferate in the bacteria, eventually causing the bacteria to lyse and die. Compared with antibiotics, bacteriophages have the following advantages: (1) High specificity, targeting only specific bacteria without destroying normal flora; (2) Self-replication ability, allowing them to proliferate at the site of infection; (3) Not prone to cross-resistance; and (4) No toxic side effects on organisms. However, natural bacteriophages still have many limitations in practical applications: First, their host range is too narrow, and they can usually only infect bacteria of a specific genus, making it difficult to cope with common mixed infections of multidrug-resistant bacteria in clinical practice.

[0005] Therefore, seeking a new antibacterial agent that can overcome the limitations of natural phages while maintaining their advantages has become a technical problem that needs to be urgently solved in the current medical and biological research fields. Summary of the invention

[0006] The purpose of the present invention is to provide an engineered phage targeting drug-resistant Gram-negative bacteria, a screening method and application, so as to solve the problem that natural phages target bacteria of a single genus and are difficult to cope with the common mixed infection of multi-drug resistant bacteria in clinical practice.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: An engineered phage targeting drug-resistant Gram-negative bacteria, wherein the engineered phage is Escherichia phage M13. Preservation time: February 11, 2025. Preservation address: China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Preservation number: CCTCC No:M 2025196.

[0008] It has been proven through experiments that the above engineered phage can achieve precise targeting of multi-drug resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, and can be applied to the diagnosis and treatment of multi-drug resistant Gram-negative bacterial infections. It effectively solves the problem that natural phages target single-species bacteria and are difficult to cope with the clinically common mixed infections of multi-drug resistant bacteria.

[0009] The present invention also provides an application of the engineered phage in the preparation of a drug for treating multi-drug resistant Gram-negative bacterial infections.

[0010] Preferably, the application of the engineered phage as a targeting vector in the preparation of a drug for treating multi-drug resistant Gram-negative bacterial infections to enhance the targeting property.

[0011] The present invention also provides an application of the engineered phage in the preparation of a product for diagnosing multi-drug resistant Gram-negative bacterial infections.

[0012] Preferably, the engineered phage is used as a targeting vector in the preparation of a product for treating multi-drug resistant Gram-negative bacterial infections to enhance the targeting property.

[0013] Preferably, the multi-drug resistant Gram-negative bacteria are at least one of multi-drug resistant Klebsiella pneumoniae (MDR-Kp), multi-drug resistant Pseudomonas aeruginosa (MDR-Pa), and multi-drug resistant Acinetobacter baumannii (MDR-Ab).

[0014] The present invention also provides a kit for diagnosing or treating multi-drug resistant Gram-negative bacterial infections, comprising the phage described above.

[0015] The present invention also provides a screening method for the engineered phage, comprising the following steps: S1. Using the Ph.D.-7 phage display peptide library kit, mixing whole cells of multi-drug resistant Klebsiella pneumoniae (MDR-Kp) with the phage library in PBS buffer, incubating to obtain bound phages; S2. Washing and centrifuging the bound phages, adding elution buffer to disrupt the binding between the phages and the target cells to obtain a mixture; S3. Add Tris-HCl buffer to the mixture to obtain eluted phages; S4. Infect the eluted phages with host Escherichia coli and amplify according to the infection method of M13 phage to obtain amplified phages; S5. Bio-pan the multi-drug resistant Pseudomonas aeruginosa (MDR- Pa ) and multi-drug resistant Acinetobacter baumannii (MDR- Ab ) respectively according to the methods of S1 to S4 to obtain the engineered phages.

[0016] The screening method of the engineered phages of the present invention can quickly screen out phages that bind to multi-drug resistant bacteria by using the Ph.D.-7 phage display peptide library kit. Through fine operations in multiple steps, such as incubation, washing, centrifugation, elution, and amplification, etc., the screening efficiency is effectively improved, making the whole process more efficient. This method is specifically for screening multi-drug resistant bacteria, such as multi-drug resistant Klebsiella pneumoniae (MDR-Kp), multi-drug resistant Pseudomonas aeruginosa (MDR-Pa), and multi-drug resistant Acinetobacter baumannii (MDR-Ab), etc. This makes the screened phages highly targeted and specific, capable of accurately recognizing and binding to a variety of multi-drug resistant Gram-negative bacteria, overcoming the deficiency of natural phages targeting single-species bacteria, and providing a powerful tool for subsequent treatment and research. The preparation method is relatively simple. Through multiple rounds of bio-panning and verification, stable and highly targeted engineered phages can be obtained, with good repeatability and operability. They can be used as diagnostic reagents for quickly and accurately detecting multi-drug resistant bacteria; they can also be used as therapeutic agents to play a therapeutic role by targeting and killing multi-drug resistant bacteria. In addition, these phages can also be used to study the pathogenic mechanism and drug resistance mechanism of multi-drug resistant bacteria, providing strong support for the development of new antibacterial drugs. The engineered phages can be used as bacterial targets and have broad application prospects in the diagnosis, treatment of mixed infections of multi-drug resistant Gram-negative bacteria, and microbial ecology research, etc.

[0017] Preferably, in the S1, the incubation temperature is 37 °C and the incubation time is 1 h.

[0018] Preferably, the volume ratio of the whole cells of multi-drug resistant Klebsiella pneumoniae (MDR-Kp) to the phage library is 1 mL:100 μL.

[0019] Preferably, in the S2, the bound phages are washed with TBST solution; the elution buffer is 0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA.

[0020] Preferably, in the S3, it also includes concentrating and purifying the amplified engineered phage particles by 20% PEG / 2.5 M NaCl.

[0021] Preferably, in S5, the obtained engineered phage is added with an equal volume of sterile glycerol and stored at -20°C.

[0022] Preferably, the screening method of the engineered phage comprises the following steps: S1. Using the Ph.D.-7 Phage Display Peptide Library Kit (New England Biolab, UK), take 1 mL of whole cells of multidrug-resistant Klebsiella pneumoniae (MDR-Kp) and mix it with 100 μL of the phage library (about 2×10 9 clones) in PBS buffer, and incubate at 37°C for 1 h to obtain the bound phage. S2. Wash the bound phage 10 times with TBST (TBS + 0.1% [v / v] Tween-20) to remove the unbound phage, centrifuge to obtain the washed bound phage, and then add 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA) to the washed bound phage and act at room temperature for 10 minutes to disrupt the binding interaction between the centrifuged whole cells and the bound phage to obtain a mixture. S3. Add 50 μL of 1 M Tris-HCl buffer (pH 9.1) to the mixture to neutralize the mixture and obtain the eluted phage. S4. According to the infection method of M13 phage, amplify the eluted phage in the host Escherichia coli ER2738 (OD 600≈0.5) to obtain the amplified phage. S5. Respectively adopt a similar procedure for multidrug-resistant Pseudomonas aeruginosa (MDR- Pa ) or multidrug-resistant Acinetobacter baumannii (MDR- Ab ) to obtain the engineered phage.

[0023] Among them, in each round of biopanning, the selected phages are verified and sequenced by the Beijing Genomics Institute (China). The amplified phage particles are concentrated and purified by 20% PEG / 2.5 M NaCl. The finally obtained phage is added with an equal volume of sterile glycerol and stored at -20°C.

[0024] The beneficial effects of the present invention are as follows: The engineered phage targeting drug-resistant Gram-negative bacteria of the present invention rapidly screens out phages that bind to multi-drug resistant bacteria by using the Ph.D.-7 phage display peptide library kit, and screens for multi-drug resistant bacteria such as multi-drug resistant Klebsiella pneumoniae (MDR-Kp), multi-drug resistant Pseudomonas aeruginosa (MDR-Pa), and multi-drug resistant Acinetobacter baumannii (MDR-Ab). This enables the screened phages to have high targeting and specificity, capable of precisely identifying and binding to a variety of multi-drug resistant Gram-negative bacteria, overcoming the deficiency of natural phages targeting single-species bacteria, and thus being able to be better applied to the diagnosis and treatment of related diseases. The screened engineered phages have broad application prospects in the biomedical field and have extensive promotion and practical value in the field of microbial technology. Brief Description of the Drawings

[0025] Figure 1 It is a flowchart of the screening method for engineered phages; Figure 2 It is a transmission electron micrograph of phage Escherichia phage M13; Figure 3 It is a sequencing result diagram of the phages after three rounds of screening; Figure 4 It is for Cy5-labeled targeting phages and MDR- Kp 、MDR- Pa and MDR- Ab Affinity verification result diagram; Figure 5 It is a fluorescence intensity result diagram of the lungs. Detailed Embodiments

[0026] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention and not for limiting the protection scope of the present invention.

[0027] Example 1 As Figure 1 shown, a screening method for engineered phages includes the following steps: S1. Using the Ph.D.-7 phage display peptide library kit (New England Biolab, UK), take 1 mL of whole cells of multi-drug resistant Klebsiella pneumoniae (MDR-Kp) and 100 μL of phage library (about 2×10 9The clones were mixed in PBS buffer and incubated at 37 °C for 1 h to obtain the bound phages; S2. The bound phages were washed 10 times with TBST (TBS + 0.1% [v / v] Tween-20) to remove the unbound phages, centrifuged to obtain the washed bound phages. Subsequently, 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA) was added to the washed bound phages and allowed to act at room temperature for 10 minutes to disrupt the binding interaction between the whole cells after centrifugation and the bound phages, obtaining a mixture; S3. 50 μL of 1 M Tris-HCl buffer (pH 9.1) was added to the mixture to neutralize the mixture, obtaining the eluted phages; S4. According to the infection method of M13 phage, the eluted phages were amplified in the host Escherichia coli ER2738 (OD 600 ≈ 0.5) to obtain the amplified phages; S5. Take 1 mL of multidrug-resistant Pseudomonas aeruginosa (MDR- Pa ), and mix it with the phages obtained in the first round of screening (i.e., the amplified phages obtained in S4) in PBS buffer, incubate at 37 °C for 1 hour, wash 10 times with TBST (TBS + 0.1% [v / v] Tween-20) to remove the unbound phages. 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA) was added to the washed bound phages, allowed to act at room temperature for 10 minutes, and 150 μL of 1 M Tris-HCl buffer (pH 9.1) was added to neutralize the mixture, obtaining the eluted phages.

[0028] The eluted phages were amplified in the host Escherichia coli ER2738 (OD600 ≈ 0.5). The amplified phages were concentrated and purified by 20% PEG / 2.5 M NaCl. The amplified phages were added with an equal volume of sterile glycerol and stored at -20 °C; S6. Take 1 mL of multidrug-resistant Acinetobacter baumannii (MDR- Ab),(Mix with the phages obtained from the second round of screening (i.e., the amplified phages obtained from S5) in PBS buffer, incubate at 37 °C for 1 hour, wash 10 times with TBST (TBS + 0.1% [v / v] Tween-20) to remove unbound phages. Add 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA) to the washed bound phages, incubate at room temperature for 10 minutes, and add 150 μL of 1 M Tris-HCl buffer (pH 9.1) to neutralize the mixture to obtain eluted phages. Amplify the eluted phages in the host Escherichia coli ER2738 (OD600 ≈ 0.5). Concentrate and purify the amplified phages by 20% PEG / 2.5 M NaCl. Add an equal volume of sterile glycerol to the amplified phages and store at -20 °C.)

[0029] Among them, in each round of biopanning, the selected phages were verified and sequenced by the Beijing Genomics Institute (China). The amplified phage particles were concentrated and purified by 20% PEG / 2.5 M NaCl. The finally obtained phages were added with an equal volume of sterile glycerol and stored at -20 °C.

[0030] The obtained engineered phages were verified and sequenced by the Beijing Genomics Institute (China), and the binding peptide sequence of the obtained phages was Trp-Ser-Leu-Gly-Thr-Tyr-Gly (WSLGYTG).

[0031] The Escherichia phage M13 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on February 11, 2025, with the deposit number: CCTCC No: M2025196.

[0032] Example 2 Identification of strains 1) Morphological observation of the phage Escherichia phage M13 Perform transmission electron microscopy analysis on the Escherichia phage M13 screened in Example 1, and the results are as Figure 2 shown.

[0033] From Figure 2 the observation, it can be seen that the phage Escherichia phage M13 screened in Example 1 has an elongated filamentous morphology and no other impurities.

[0034] 2) Molecular identification of the strain The Escherichia phage M13 screened in Example 1 was verified and sequenced by the Beijing Genomics Institute (China) for the selected phages. The sequencing results of the phages screened three times are as Figure 3 shown. The final binding peptide sequence of the Escherichia phage M13 obtained was Trp-Ser-Leu-Gly-Thr-Tyr-Gly (WSLGYTG). As a binding peptide sequence screened by phage display technology, WSLGYTG has broad application prospects.

[0035] Detection and analysis Verification of targeting ability The specific operation is as follows: The engineered phage screened in Example 1 for targeting multi-drug resistant Gram-negative bacteria was labeled with Cy5. Similarly, non-target phages obtained from the peptide display library without specific targeting ability were also labeled with Cy5 as a control. Both phages were stirred overnight to ensure effective binding to the fluorescent dye. Dialysis was carried out for 3 days at room temperature using a dialysis bag with a molecular weight cut-off of 2000 Da to purify the Cy5-labeled phages and prepare them for the experiment. To evaluate the targeting specificity and binding affinity of these phages, the test bacterial strains including MDR- Kp 、MDR- Pa and MDR- Ab were incubated with the engineered phage and non-target phage respectively. The mixtures were co-incubated for 30 minutes in the dark, and then washed three times with sterile PBS buffer to remove unbound or excess phages. Then, the bacteria-phage complexes were resuspended in 1 mL of sterile PBS buffer, and 10 μL of each suspension was dropped on a microscope slide. The co-localization of the Cy5-labeled phages and target bacteria was observed and analyzed by confocal laser scanning microscopy (CLSM). The results are as Figure 4 shown.

[0036] Analysis from Figure 4 showed the targeting affinity of the engineered phage screened in Example 1 for three different bacteria (MDR-Kp, MDR-Ab, MDR-Pa). In the images of the targeting phages, the phages were labeled with Cy5 and targeted to the bacteria, showing red fluorescence, indicating that the engineered phage screened in Example 1 successfully recognized and bound to the bacterial surface. This affinity was not obvious in the control group of non-target phages. This indicates that the engineered phage screened in Example 1 can specifically recognize and bind to the target bacteria.

[0037] In vivo targeting verification test of polymyxin B prodrug nanoparticles and targeting phage complex Experimental design: To verify the lesion targeting of polymyxin B prodrug nanoparticles and targeted phage complex in a mouse pneumonia infection model, the polymyxin B prodrug nanoparticles-targeted phage complex was fluorescently labeled for tracking and evaluating its distribution and targeting in the mouse model.

[0038] Experimental procedures: First, prepare the polymyxin B prodrug and its nanoparticles, and then mix and react the polymyxin B prodrug nanoparticles with phages to form the polymyxin B prodrug nanoparticles-targeted phage complex. Next, fluorescently label the polymyxin B prodrug nanoparticles-targeted phage complex and the polymyxin B prodrug nanoparticles with a fluorescent dye. Among them, the nanoparticles were labeled with Cy5 to ensure that both of the former have traceable fluorescent signals. Select a certain number of healthy mice to establish a pneumonia infection model.

[0039] Among them, the preparation methods of the polymyxin B prodrug and its nanoparticles, and the polymyxin B prodrug nanoparticles-targeted phage complex include the following steps: S1. Take 0.3 mL of lecithin solution and 0.3 mL of a mixture of DSPE-PEG2000-Mal (distearoyl phosphatidylethanolamine-polyethylene glycol 2000-maleimide) / DSPE-PEG2000 (wherein, in the mixture, the mass ratio of DSPE-PEG2000-Mal to DSPE-PEG2000 is 1:1, the organic solvent is ethanol, and the concentration is 30 mg / mL) and add them to 15 mL of deionized water. Stir at 65 °C for 30 minutes to obtain a mixed solution; S2. In a 50 mL round-bottom flask under nitrogen protection, dissolve 1.11 g (4.70 mmol) of PBAP (4-(hydroxymethyl)phenylboronic acid pinacol ester) in 10 mL of anhydrous dichloromethane, then add 1.59 g (71.50 mmol) of CDI (carbonyldiimidazole), and react at room temperature for 30 minutes. After the reaction, wash with 10.00 mL of water, extract to obtain the organic phase, then extract three times with 10.00 mL of saturated sodium chloride solution, dry the organic phase with anhydrous sodium sulfate, and concentrate by rotary evaporation to obtain the activated PBAP, that is, PBAP-CDI; S3. Dissolve 1.58 g (6.03 mmol) of activated PBAP, 1.19 g (0.86 mmol) of PMB (polymyxin B), and 1.10 g (24.04 mmol) of DMAP (4-dimethylaminopyridine) in 20.00 mL of anhydrous DMSO (dimethyl sulfoxide). Deoxygenate by purging with nitrogen three times and react at room temperature for 24 hours. After the reaction, a precipitate is obtained. Mix the precipitate with deionized water, centrifuge, and discard the supernatant to obtain a solid. Lyophilize the obtained solid to obtain the prodrug PPMB; S4. Ultrasonically dissolve 50 mg of the prodrug PPMB in 5 mL of methanol to obtain a PPMB methanol solution; S5. Under vigorous stirring, drop the PPMB methanol solution into the mixed solution prepared in S1, stir at room temperature for 2 hours to obtain a first mixed solution; Remove the organic solvent and excess aqueous phase from the first mixed solution by vacuum distillation to obtain activated maleimide (MAL)-PNP (i.e., polymyxin B prodrug nanoparticles); S6. Mix the activated maleimide (MAL)-PNP (i.e., polymyxin B prodrug nanoparticles) with the targeted phage (Escherichia phage M13), and stir overnight at pH 7.4 so that maleimide specifically covalently binds to the thiol group at the end of the phage through a click reaction to form a stable disulfide bond, thereby obtaining a complex of polymyxin B prodrug nanoparticles and the targeted phage TPNP.

[0040] Randomly divide the infected mice into two groups. One group receives treatment with the complex of polymyxin B prodrug nanoparticles and the targeted phage (TPNP) (treatment group), and the other group receives treatment with polymyxin B prodrug nanoparticles (control group). 6 hours after infection, intravenously inject 1 mg / kg of the complex of polymyxin B prodrug nanoparticles and the targeted phage into the mice in the treatment group through the tail vein, and administer the drug once every 12 hours for a total of two administrations. Administer the same dose and the same number of times of polymyxin B prodrug nanoparticles to the mice in the control group. At the same time points after administration, use in vivo imaging technology to perform fluorescence imaging on the mice to track the distribution of the fluorescence signal in the body, especially the enhancement of fluorescence in the lungs. The results are as Figure 5 shown.

[0041] Analysis from Figure 5 shows that the fluorescence intensity of the treatment group is significantly higher than that of the control group, indicating that the complex of polymyxin B prodrug nanoparticles and the targeted phage successfully accumulates in the lungs, further proving that the complex of polymyxin B prodrug nanoparticles and the targeted phage has precise targeting.

[0042] In summary, for the engineered phage targeting drug-resistant Gram-negative bacteria of the present invention, by using the Ph.D.-7 phage display peptide library kit, phages that bind to multi-drug resistant bacteria were quickly screened out. Through multiple rounds of screening against multi-drug resistant bacteria, such as multi-drug resistant Klebsiella pneumoniae (MDR-Kp), multi-drug resistant Pseudomonas aeruginosa (MDR-Pa), and multi-drug resistant Acinetobacter baumannii (MDR-Ab), etc., the screened phages have high targeting and specificity, can accurately identify and bind to a variety of multi-drug resistant Gram-negative bacteria, overcoming the deficiency of natural phages targeting single-species bacteria, and thus can be better applied to the diagnosis and treatment of related diseases. The screened engineered phages have broad application prospects in the biomedical field and have extensive promotion and practical value in the field of microbial technology.

[0043] The above embodiments are only the preferred embodiments of the present invention, and the protection scope is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. An engineered phage targeting drug-resistant Gram-negative bacteria, characterized in that, The engineered phage is Escherichia phage M13. Preservation time: February 11, 2025. Preservation address: China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Preservation number: CCTCC No: M2025196.

2. Use of an engineered phage as claimed in claim 1 in the preparation of a medicament for treating multi-drug resistant Gram-negative bacterial infections.

3. The use according to claim 2, wherein the engineered phage is used as a targeting vector in the preparation of a medicament for diagnosing multi-drug resistant Gram-negative bacterial infections.

4. The application according to claim 2 or claim 3, characterized in that, The multi-drug resistant Gram-negative bacteria are at least one of multi-drug resistant Klebsiella pneumoniae (MDR- Kp ), multi-drug resistant Pseudomonas aeruginosa (MDR- Pa ), and multi-drug resistant Acinetobacter baumannii (MDR- Ab ).

5. A kit for diagnosing or treating multi-drug resistant Gram-negative bacterial infections, characterized in that, Comprising the phage as claimed in claim 1.

6. A screening method for an engineered phage as described in claim 1, characterized in that, Comprising the following steps: S1. Use the Ph.D.-7 phage display peptide library kit. Take the whole cells of multidrug-resistant Klebsiella pneumoniae (MDR- Kp ) and mix them with the phage library in PBS buffer, and incubate to obtain the bound phages; S2. Washing the bound phage, centrifuging, and adding an elution buffer to disrupt the binding of the phage to the target cells to obtain a mixture. S3. Adding Tris-HCl buffer to the mixture to obtain eluted phage. S4. Infecting the host Escherichia coli with the eluted phage and amplifying according to the infection method of M13 phage to obtain amplified phage. S5. Respectively perform biopanning on multidrug-resistant Pseudomonas aeruginosa (MDR- Pa ) and multidrug-resistant Acinetobacter baumannii (MDR- Ab ) according to the methods of S1 to S4 to obtain the engineered phage.

7. The screening method according to claim 6, wherein In S1, the incubation temperature is 37°C and the incubation time is 1 h.

8. The screening method according to claim 6, characterized in that The volume ratio of the multi-drug resistant Klebsiella pneumoniae (MDR- Kp ) whole cells to the phage library is 1 mL: 100 μL.

9. The screening method according to claim 6, characterized in that, In S2, the bound phage is washed with TBST solution; the elution buffer is 0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA. And / or, in S3, it further includes concentrating and purifying the amplified engineered phage particles by 20% PEG / 2.5 M NaCl.

10. The screening method according to claim 6, characterized in that, In S5, the obtained engineered phage is added with an equal volume of sterile glycerol and stored at -20°C.

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