Phage capable of splitting multi-drug-resistant klebsiella pneumoniae and application

Abstract

The invention relates to the technical field of microorganisms, in particular to a bacteriophage capable of splitting multi-drug-resistant klebsiella pneumoniae and application of the bacteriophage. The phage is sent to China Center for Type Culture Collection for preservation, the classification name of the phage is Klebsiella pneumoniae phage JNP-KP-001, the preservation number is CCTCC NO: M 20242558, the preservation date is November 14, 2024, and the preservation address is Wuhan University, Wuhan, China. The klebsiella pneumoniae bacteriophage JNP-KPN-001 provided by the invention can still effectively inhibit the replication of ST11 type klebsiella pneumoniae under the condition that the infection complex number MOI is 0.00001, the temperature and acid-base tolerance range is wide, cell tests prove that the bacteriophage has small toxic and side effects and high safety, and the klebsiella pneumoniae bacteriophage JNP-KPN-001 can be used for preparing the klebsiella pneumoniae bacteriophage. The bacteriophage can provide an effective method for body drug-resistant ST11 type klebsiella pneumoniae infection, hospital environment disinfection, prevention and the like.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a bacteriophage capable of lysing multi-drug resistant Klebsiella pneumoniae and its application. Background Art

[0002] Klebsiella pneumoniae ( Klebsiella pneumoniae Klebsiella pneumoniae (KP) is an important clinical pathogen with high pathogenicity and multidrug resistance potential. ST11 Klebsiella pneumoniae is one of the most important clinical pathogens in China and the main type of nosocomial infection. However, due to the extensive use of antibiotics, multidrug-resistant Klebsiella pneumoniae has emerged. Its multidrug resistance is becoming an increasingly serious global problem, posing a huge challenge to clinical treatment.

[0003] Bacteriophage is a virus that can specifically infect bacteria. It is a natural antibacterial substance that can produce proteases that degrade bacterial surface polysaccharides. The principle of phage therapy is that phages can specifically infect and lyse host bacteria to achieve the effect of treating diseases caused by the infection of the host bacteria. Phage therapy is currently used in the treatment of Klebsiella pneumoniae infections. However, due to the host specificity of phages, their antibacterial spectrum is too narrow. Therefore, personalized treatment must be made for each case, and a single treatment cannot be adopted. At present, there are no phage preparations specifically for ST11 Klebsiella pneumoniae in phage therapy. Therefore, the continuous isolation of new phages and the analysis of their basic biological properties and antibacterial potential are the primary prerequisites for the development of phage biopharmaceuticals to adapt to the diversity of bacterial species and the new species produced by continuous mutations. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the present invention provides a bacteriophage that can lyse multi-drug resistant Klebsiella pneumoniae and its application. The bacteriophage has been sent to the China Center for Type Culture Collection for preservation and is classified and named as: Klebsiella pneumoniae phage ( KlebsiellaThe phage is a phage of Klebsiella pneumoniae (KPN-001), with a deposit number of CCTCC NO: M 20242558, a deposit date of November 14, 2024, and a deposit address of Wuhan University, Wuhan, China. The phage can effectively lyse multidrug-resistant ST11 Klebsiella pneumoniae. By detecting the growth curve of ST11 Klebsiella pneumoniae infected with Klebsiella pneumoniae phage JNP-KPN-001, it was found that Klebsiella pneumoniae phage JNP-KPN-001 can still effectively inhibit the replication of ST11 Klebsiella pneumoniae when the multiplicity of infection MOI is 0.00001. Compared with other phages that can lyse ST11 Klebsiella pneumoniae, the Klebsiella pneumoniae phage JNP-KPN-001 can achieve the same lysis effect as other phages at a lower MOI value, has a wide range of temperature and acid-base tolerance, and can provide new technical means for the treatment of multidrug-resistant Klebsiella pneumoniae infections.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect of the present invention, a bacteriophage capable of lysing multi-drug resistant Klebsiella pneumoniae is provided. The bacteriophage has been sent to the China Center for Type Culture Collection for preservation and is classified and named as: Klebsiella pneumoniae phage ( Klebsiella phage) JNP-KPN-001, the deposit number is CCTCC NO: M 20242558, the deposit date is November 14, 2024, and the deposit address is: Wuhan University, Wuhan, China.

[0006] In the present invention, the bacteriophage is the Klebsiella pneumoniae phage JNP-KPN-001 obtained by screening and separation from the sewage treatment station of Qingchun Campus of the First Affiliated Hospital of Zhejiang University School of Medicine in Hangzhou.

[0007] In the present invention, the bacteriophage is a short-tailed phage with a head size of about 70 nm. The bacteriophage can withstand a high temperature of 50°C, and the optimal pH of the bacteriophage is 4-10. Under the condition of pH 12, it can still maintain some activity. The bacteriophage can effectively inhibit the growth of its host bacteria within the range of infection multiplicity (MOI) of 0.001-0.00001.

[0008] The second aspect of the present invention provides the use of the above-mentioned bacteriophage in the preparation of a product for treating multidrug-resistant Klebsiella pneumoniae infection.

[0009] Furthermore, the multidrug-resistant Klebsiella pneumoniae is ST11 type Klebsiella pneumoniae.

[0010] In a third aspect, the present invention provides a preparation comprising the above-mentioned bacteriophage.

[0011] Furthermore, the preparation can be used for the disinfection and prevention of drug-resistant Klebsiella pneumoniae infection and hospital environment.

[0012] Furthermore, the content of bacteriophage in the preparation is 1×10 8 ~1×10 10 PFU / mL, preferably 1×10 9 PFU / mL.

[0013] Furthermore, the dosage form of the preparation is any one of a liquid preparation, a freeze-dried preparation, and a tablet preparation.

[0014] Furthermore, the preparation also contains pharmaceutically acceptable excipients or carriers.

[0015] In a fourth aspect, the present invention provides use of the above-mentioned bacteriophage in the preparation of an environmental disinfectant or an environmental cleaner.

[0016] Specifically, it is to control the contamination of ST11 Klebsiella pneumoniae in the space environment.

[0017] Furthermore, the phage content was 1×10 8 ~1×10 10 PFU / mL, preferably 10 9 PFU / mL.

[0018] In a fifth aspect, a method for killing Klebsiella pneumoniae in a space environment is provided, the method comprising spraying the above-mentioned bacteriophage or the above-mentioned preparation into the space environment.

[0019] One or more of the above technical solutions have the following beneficial effects: The present invention provides a new phage of Klebsiella pneumoniae, which can still effectively inhibit the replication of ST11 Klebsiella pneumoniae when the infection multiplicity MOI is 0.00001. Compared with other phages that can lyse ST11 Klebsiella pneumoniae, the Klebsiella pneumoniae phage JNP-KPN-001 can achieve the same lysis effect as other phages at a lower MOI value, and has a wide range of temperature and acid-base tolerance. Cell experiments have confirmed that the phage has low toxic and side effects and high safety. The phage can provide an effective method for drug-resistant Klebsiella pneumoniae infection, hospital environment disinfection, prevention, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1This is a schematic diagram of the Klebsiella pneumoniae phage JNP-KPN-001 infecting ST11 type Klebsiella pneumoniae plaque in Example 1 of the present invention; Figure 2 This is a transmission electron micrograph of the Klebsiella pneumoniae phage JNP-KPN-001 in Example 3 of the present invention; Figure 3 This is a schematic diagram of the growth curve of ST11 type Klebsiella pneumoniae after being infected with Klebsiella pneumoniae phage JNP-KPN-001 in Example 4 of the present invention; Figure 4 This is a schematic diagram of the effect of temperature on the titer of Klebsiella pneumoniae phage JNP-KPN-001 in Example 5 of the present invention; Figure 5 This is a schematic diagram of the effect of pH on the titer of Klebsiella pneumoniae phage JNP-KPN-001 in Example 5 of the present invention; Figure 6 This is a schematic diagram of the cytotoxicity of Klebsiella pneumoniae phage JNP-KPN-001 to THP1 and A549 in Example 6 of the present invention; Figure 7 This is a schematic diagram of the killing effect of Klebsiella pneumoniae phage JNP-KPN-001 on Klebsiella pneumoniae in the environment in Example 7 of the present invention, wherein ** means p<0.01; Figure 8 This is the phylogenetic evolutionary tree of Klebsiella pneumoniae phage JNP-KPN-001 in Example 8 of the present invention. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology in the art are usually followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0024] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0025] The strains, reagents and culture media involved in the embodiment are: The host bacteria used in the experiment were all ST11 Klebsiella pneumoniae, which were strains isolated from patient samples clinically. They were identified as ST11 Klebsiella pneumoniae after genome sequencing and preserved in the Jinan Microecology and Biomedicine Provincial Laboratory.

[0026] The bacteriophage JNP-KPN-001 in the embodiment was isolated and screened from the sewage treatment station of the Qingchun Campus of the First Affiliated Hospital of Zhejiang University School of Medicine in Hangzhou, and was classified and named as Klebsiella pneumoniae phage ( Klebsiella phage) JNP-KPN-001, deposited in China Center for Type Culture Collection on November 14, 2024, with the accession number CCTCC NO: M20242558, and the deposit address: Wuhan University, Wuhan, China.

[0027] The culture medium formula used in the embodiment is as follows: LB liquid medium (1L): peptone 10 g, yeast powder 5 g, NaCl 10 g, ddH 2 Dilution: 1 L, adjust pH to 7.0, and sterilize by high pressure at 121°C for 20 min.

[0028] 0.75% LB semi-solid medium (1L): peptone 10 g, yeast powder 5 g, NaCl 10 g, agar powder 7.5 g, add ddH 2 O to 1 L, adjust the pH to 7.0, and sterilize by autoclave at 121°C for 20 min.

[0029] 1.5% LB solid medium (1L): peptone 10 g, yeast powder 5 g, NaCl 10 g, agar powder 15 g, add ddH 2 O to 1L, adjust pH to 7.0, sterilize at 121℃ for 20 min, cool to 50℃, pour into a plate, cool and solidify, and then invert for later use.

[0030] SM Buffer, DNaseI, RNase A, PEG8000, and phosphotungstic acid (PTA, 2% w / v) were commercially available.

[0031] Example 1 Isolation of Klebsiella pneumoniae phage JNP-KPN-001 Isolation method: The samples were collected from the sewage treatment station of Qingchun Campus of the First Affiliated Hospital of Zhejiang University School of Medicine in Hangzhou in January 2023 for phage isolation. The samples were centrifuged at 12000 g for 2 minutes, and the supernatant after centrifugation was filtered with a 0.22 μm microporous filter as a standby phage suspension. 50 μL of ST11 Klebsiella pneumoniae and 200 μL of phage suspension were added to 6 mL LB medium. Oscillating culture was carried out at 220 rpm in a 37°C shaker. After 12 hours of culture, the culture was centrifuged at 12000 g for 2 minutes, and the supernatant after centrifugation was filtered with a 0.22 μm microporous filter. Take 100 μL of the supernatant and use the double-layer agar plate method to detect whether the phage suspension contains phages that lyse ST11 Klebsiella pneumoniae.

[0032] Purification method: After the double-layer agar plate was incubated at 37°C for 12-24 hours, the upper agar with plaques was picked up with a sterile pipette tip and placed in SM buffer. The agar was vortexed for 2-5 minutes to fully dissolve the phage in the agar into the SM buffer. The supernatant was filtered with a 0.22 μm microporous filter and the phage stock solution was diluted 10 times with SM buffer. 10 -2 , 10 -4 , 10 -6 , 10 -8 Mix 50 μL of the dilution with 50 μL of ST11 Klebsiella pneumoniae in the logarithmic phase, let it stand for 15 min, add 3.5 mL of 0.75% semi-solid LB medium at about 45°C, mix evenly, spread evenly on the pre-prepared solid LB plate, and observe the growth of plaques after culturing at 37°C for 12 h. Pick a single transparent plaque without halo and uniform size into an EP tube filled with SM Buffer. Repeat this process about 10 times to obtain phages with uniform plaque size, store at 4°C for later use.

[0033] The above-mentioned spare phages were tested by double-layer plate method, and the results were as follows Figure 1 As shown, the phage infects Klebsiella pneumoniae to form a transparent plaque circle with a diameter of about 0.5-1.0 cm in the middle, surrounded by a large semi-transparent circle with clear and regular edges, which is a typical lytic phage. It is named Klebsiella pneumoniae phage JNP-KPN-001.

[0034] Example 2 Amplification and Purification of Klebsiella Pneumoniae Phage JNP-KPN-001 Take 0.1 mL of the Klebsiella pneumoniae phage JNP-KPN-001 prepared in Example 1 and 0.1 mL of the bacterial solution of ST11 Klebsiella pneumoniae (logarithmic growth phase), mix them in a test tube and let them stand for 15 min, add 10 mL of LB liquid culture medium, culture at 37°C for 6 h, centrifuge at 4°C and 12000 rpm for 20 min, take the supernatant, filter with a 0.22 μm filter membrane, and the filtrate is the phage suspension.

[0035] The double-layer plate method was used to detect the phage titer: the above phage suspension was diluted 10 times in a gradient, 0.1 mL of the phage dilution solution of each gradient was taken and mixed with 0.1 mL of the host bacterial solution, and then spread on a double-layer agar plate. The plate was cultured at 37°C for about 12 h, and the plaques were counted on each agar plate. The plate with about 30-300 plaques was selected. The initial phage concentration calculated according to the dilution multiple was the phage titer. The phage titer (PFU / mL) = dilution multiple × number of plaques × 10, and the phage titer was 1 × 10 9 -1×10 10 PFU / mL.

[0036] Example 3 Observation of the morphology of Klebsiella pneumoniae phage JNP-KPN-001 under electron microscope The phage obtained in Example 2 was observed under an electron microscope. The upper agar layer with plaques was picked up with a sterile gun tip and placed in sterile deionized water. The mixture was vortexed for 2-5 minutes to fully dissolve the phage in the agar in the deionized water. The mixture was centrifuged at 12000 g for 20 minutes to allow the bacterial fragments and agar blocks to settle. The supernatant was taken and dripped onto a copper mesh. After natural drying, 5% phosphotungstic acid staining solution was dripped for negative staining. The staining time was about 15 minutes. After drying, the phage morphology was observed under a 120 kV transmission electron microscope. The observation results are shown in FIG. Figure 2 As shown, the head size of Klebsiella pneumoniae phage JNP-KPN-001 is about 70 nm, and a small amount of tail filaments can be seen.

[0037] The applicant named the phage Klebsiella pneumoniae phage JNP-KPN-001 and sent it to the China Center for Type Culture Collection for preservation on November 14, 2024. The preservation number is CCTCC NO: M 20242558. The preservation address is: Wuhan University, Wuhan, China.

[0038] Example 4 Growth curve of Klebsiella pneumoniae phage JNP-KPN-001 infecting ST11 Klebsiella pneumoniae (infection multiplicity) The host bacteria ST11 pneumoniae in the logarithmic growth phase were counted, and the Klebsiella pneumoniae phage JNP-KPN-001 and the host bacteria ST11 pneumoniae were mixed in LB liquid culture medium at a multiplicity of infection of 0.001, 0.0001, and 0.00001, respectively, and cultured in a shaking incubator at 37°C at a speed of 220 rpm. During the 12-hour culture, the OD600 value was measured every 1 hour. The measurement results are shown in Figure 3 As shown, phage JNP-KPN-001 can still effectively inhibit the growth of multidrug-resistant ST11 Klebsiella pneumoniae at an infection multiplicity of 0.00001, and can be used as a candidate phage preparation for the treatment of pneumonia caused by ST11 Klebsiella pneumoniae.

[0039] Example 5 Temperature and pH tolerance of Klebsiella pneumoniae phage JNP-KPN-001 Add 50 μL of ST11 Klebsiella pneumoniae culture in logarithmic phase and 50 μL of Klebsiella pneumoniae phage JNP-KPN-001 suspension purified in Example 1 to 6 mL of LB medium. Incubate at 37°C in a shaker at 220 rpm. After 12 hours of culture, centrifuge the culture at 12000 g for 2 minutes, and filter the supernatant after centrifugation using a 0.22 μm microporous filter to obtain a mixed phage suspension.

[0040] Take 6 sterile EP tubes, add 0.5 mL of the mixed phage suspension mentioned above to each tube, incubate at -20℃, 4℃, 37℃, 50℃, 60℃ and 70℃ for 1 hour, cool to room temperature, and use the double-layer agar plate method to determine the phage activity. The results are as follows: Figure 4 As shown, it can withstand a high temperature of 50°C for one hour without affecting its activity.

[0041] Take 7 portions of 0.1 mL of the above phage suspension and add them to LB liquid culture medium with pH values ​​of 2, 4, 6, 7, 8, 10, and 12, respectively, and incubate at 37°C for 1 hour. Then, the phage activity change is determined using the double-layer agar plate method. Figure 5 As shown, in the pH range of 4-10, its activity is not affected; at pH 12, it can still maintain some activity; when pH = 2, the phage is completely inactivated. Therefore, the optimal pH of Klebsiella pneumoniae phage JNP-KPN-001 is 4-10.

[0042] Example 6 Cytotoxicity of Klebsiella pneumoniae phage JNP-KPN-001 to THP1 and A549 cells THP1 cells and A549 cells were inoculated into 96-well plates (THP1 and A549 cell culture medium was 1640 medium containing 10% fetal bovine serum and 1% double antibody), and 5×10 THP1 cells were inoculated into each well. 4 A549 cells were seeded in each well at 1×10 4 After culturing at 37° C. for 12 hours, the Klebsiella pneumoniae phage JNP-KPN-001 purified in Example 1 was diluted with 1640 cell culture medium to obtain 10 5 , 10 6 , 10 7 The phage suspension was added to THP1 and A549 cells, and the 1640 cell culture medium without phage was used as the control group. After 24 hours of co-culture, the cytotoxicity of JNP-KPN-001 phage to THP1 and A549 cells was detected by CCK8 detection kit. Figure 6 As shown, Klebsiella pneumoniae phage JNP-KPN-001 had no cytotoxic effect on the above two cell types.

[0043] Example 7 Klebsiella pneumoniae phage JNP-KPN-001 for controlling Klebsiella pneumoniae ST11 contamination in the environment In the sterile P2 laboratory, two 1 m2 floors were selected and the ST11 Klebsiella pneumoniae liquid with an OD600 of 0.2-0.3 was evenly sprayed on the two floors. After 2 h, the Klebsiella pneumoniae phage JNP-KPN-001 suspension (1×10 8 ~1×10 10 PFU / mL) was sprayed on one of the floors, and the control floor was sprayed with saline. After 12 hours, a sample was taken from the floor with a sterile cotton swab and dissolved with 1 mL of saline. The sampled Klebsiella pneumoniae was counted using the dilution plate method. Figure 7 As shown, the spray application of Klebsiella pneumoniae phage JNP-KPN-001 can significantly reduce the CFU of Klebsiella pneumoniae in the environment (P < 0.01).

[0044] Example 8 Phage whole genome sequencing and bioinformatics analysis 10 mL of ST11 Klebsiella pneumoniae liquid with OD600 between 0.2 and 0.3 was mixed with 50 μL of bacteriophage JNP-KPN-001 (titer 10 9 -10 10The cells were mixed and cultured for 12 h, and the bacterial precipitate was removed by centrifugation at 12,000 g for 10 minutes. The supernatant was filtered twice through a 0.22 μm PVDF membrane and centrifuged at 150,000 × g for 4 hours. The precipitate was extracted using a phage DNA extraction kit (brand: NORGEN Biotek, catalog number: 46800) and then sequenced by the Illumina sequencing platform. The data was assembled using Spades software after quality control, and the assembled sequence was annotated using Prokka. The Blast tool was used to align the sequence of the terminase large subunit of the phage, and the phage terminase large subunit sequence with the highest similarity to it was downloaded from the NCBI library. The evolutionary tree was drawn using MEGA 11.0 software. The phylogenetic evolutionary tree of Klebsiella pneumoniae phage JNP-KPN-001 is shown in the figure. Figure 8 As shown, JNP-KPN-001 and Kp11 UP042311.1 are in the same branch and are short-tailed phages.

Claims

1. A bacteriophage capable of lysing multidrug-resistant Klebsiella pneumoniae, characterized in that: The phage has been sent to the China Center for Type Culture Collection for preservation and is classified and named as: Klebsiella pneumoniae phage ( Klebsiella phage) JNP-KPN-001, the deposit number is CCTCC NO: M 20242558, the deposit date is November 14, 2024, and the deposit address is: Wuhan University, Wuhan, China.

2. Use of the bacteriophage according to claim 1 in the preparation of a product for treating multidrug-resistant Klebsiella pneumoniae infection.

3. The use according to claim 2, characterized in that The multidrug-resistant Klebsiella pneumoniae is ST11 type Klebsiella pneumoniae.

4. A preparation comprising the bacteriophage according to claim 1.

5. The preparation according to claim 4, characterized in that The preparation is one of a liquid preparation, a freeze-dried preparation and a sheet preparation.

6. The preparation according to claim 4, characterized in that The preparation further comprises pharmaceutically acceptable excipients.

7. The preparation according to claim 4, characterized in that The content of Klebsiella pneumoniae phage JNP-KPN-001 in the preparation is 1×10 8 ~1×10 10 PFU / mL.

8. Use of the bacteriophage according to claim 1 in preparing an environmental disinfectant or an environmental cleaner.

9. The environmental disinfectant or environmental cleaner according to claim 8, characterized in that The phage content was 1×10 8 ~1×10 10 PFU / mL.

10. A method for killing Klebsiella pneumoniae in a space environment, characterized in that: The method comprises spraying the bacteriophage according to claim 1 or the preparation according to any one of claims 4 to 7 into the space environment.

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