Ultra-wide host spectrum klebsiella pneumoniae bacteriophage and application thereof

Abstract

The invention discloses an ultra-wide host spectrum klebsiella pneumoniae bacteriophage and application thereof, and belongs to the technical field of biology. According to the invention, a novel klebsiella pneumoniae bacteriophage is separated and identified from a chicken farm sewage water sample, and the preservation number of the novel klebsiella pneumoniae bacteriophage is CCTCC (China Center for Type Culture Collection) NO: M 20242916. Tests show that the phage can be used for splitting 25 capsular serotypes such as KL25, KL47, KL64 and the like of ST11 type klebsiella pneumoniae, and has an ultra-wide host spectrum. Moreover, the bacteriophage is remarkable in antibacterial effect and good in biological safety, can be widely applied to preparation of drugs and environmental modifier products aiming at human and animal klebsiella pneumoniae, and provides a safe and efficient alternative antibody treatment scheme for human and animals.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an ultra-wide host spectrum Klebsiella pneumoniae phage and application thereof. Background Art

[0002] Klebsiella pneumoniae type ST11 is a highly resistant and virulent bacterium and an important clonal type of carbapenem-resistant Klebsiella pneumoniae (CRKP). This strain is very prevalent in hospital-acquired infections in China, especially in intensive care units (ICUs). Klebsiella pneumoniae type ST11 can produce carbapenemase KPC-2, which makes it resistant to carbapenem antibiotics. In addition, ST11 is highly virulent and can cause serious infections, including pneumonia and sepsis.

[0003] In recent years, the evolution and spread of ST11 Klebsiella pneumoniae has attracted widespread attention, among which KL25, KL47 and KL64 are the main K types of these strains. Studies have shown that some subclones of ST11 (such as ST11-KL64) have gradually replaced other subclones in the clinic and become dominant clones. Due to its high drug resistance and high virulence, ST11 Klebsiella pneumoniae poses a serious threat to public health. At present, colistin and ceftazidime / avibactam (CAZ / AVI) are the main options for treating infections caused by this bacterium, but drug resistance is also emerging. Infections caused by these drug-resistant bacteria are extremely difficult to treat, and there are limited effective antibiotics available. Improper use of antibiotics not only fails to effectively control infections, but may also lead to further spread of drug resistance, making infections more likely to break out in settings such as hospitals, increasing medical costs and patient mortality. Therefore, it is very important to strengthen monitoring and prevention and control measures for ST11 Klebsiella pneumoniae.

[0004] Phages are a type of virus. Like other viruses, their survival and production depend on the host. They have a high degree of host specificity and will not destroy the normal flora. Since the discovery of phages in 1915, researchers and clinicians have quickly recognized that they can be used as potential therapeutic agents for the treatment of bacterial infections. Most of the currently isolated phages have a narrow host range, targeting only one species, one strain or one serotype, which is difficult to cover multiple serotypes of Klebsiella pneumoniae infection and prevention, limiting the widespread application of phages. Therefore, it is of great significance to obtain some polyvalent phages with a wide host range. At present, some methods have been developed to expand the host range of phages. One is to synthesize phage libraries. However, before designing engineered phage libraries, it is necessary to sequence the whole phage genome and identify the host range determining region in the tail fiber protein or tail spike; the other is reasonable genetic engineering or recombination. However, the synthetic polyvalent phages rely on phages that can infect different hosts, and there are problems such as high technical difficulty in broadening the host spectrum, high cost, long cycle, and difficulty in meeting actual needs. In addition, the problem can be solved to a certain extent through the formulation of phage cocktails, but it faces the problem of greatly increased production costs. Using phage evolution, polyvalent phages can also be obtained. Almost all evolved wide-host-range phages can only infect species of the same genus, and the direction of evolution is uncontrollable. Few scientists have expanded the host range of phages to multiple bacterial genera or multiple serotypes through evolution.

[0005] Therefore, a broad-spectrum phage that can simultaneously lyse different serotypes of Klebsiella pneumoniae can solve the problem of pathogenic and drug-resistant Klebsiella pneumoniae infections in clinical practice and is worthy of further isolation and research. Summary of the invention

[0006] The purpose of the present invention is to provide a Klebsiella pneumoniae phage with an ultra-wide host spectrum and its application to solve the problems existing in the above-mentioned prior art. The phage can lyse 25 serotypes of Klebsiella pneumoniae, such as KL25, KL47, KL64, etc., has an ultra-wide host spectrum, and can have a significant antibacterial effect, good biosafety, and can be used as an active ingredient in products such as drugs and environmental improvers, providing a safe and efficient alternative antibiotic treatment plan for humans and animals.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an ultra-wide host spectrum Klebsiella pneumoniae bacteriophage PKP-80, whose preservation number is CCTCC NO: M 20242916, the preservation time is December 27, 2024, the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0009] The present invention also provides the use of the ultra-wide host spectrum Klebsiella pneumoniae phage in the preparation of medicines for preventing and treating diseases infected by Klebsiella pneumoniae.

[0010] Optionally, the Klebsiella pneumoniae infection disease includes diseases caused by different serotypes of Klebsiella pneumoniae, including KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161 and KL177.

[0011] The present invention also provides the use of the ultra-wide host spectrum Klebsiella pneumoniae phage in preparing a drug for lysing Klebsiella pneumoniae.

[0012] Optionally, the lysed Klebsiella pneumoniae includes strains of different serotypes, including KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161 and KL177, which lyse Klebsiella pneumoniae.

[0013] The present invention also provides the use of the ultra-wide host spectrum Klebsiella pneumoniae phage in preparing an improving agent for preventing and controlling the spread of Klebsiella pneumoniae in sewage.

[0014] Optionally, the prevention and control of the spread of Klebsiella pneumoniae in sewage includes preventing and controlling the spread of different serotype strains of Klebsiella pneumoniae, including KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161 and KL177 in sewage.

[0015] The present invention also provides a bactericidal agent for Klebsiella pneumoniae, comprising the ultra-wide host spectrum Klebsiella pneumoniae phage.

[0016] The present invention also provides a medicine for preventing and treating diseases caused by Klebsiella pneumoniae infection, comprising the ultra-wide host spectrum Klebsiella pneumoniae phage.

[0017] The present invention also provides an improver for preventing and controlling the spread of Klebsiella pneumoniae in sewage, comprising the ultra-wide host spectrum Klebsiella pneumoniae phage.

[0018] The present invention discloses the following technical effects:

[0019] (1) The Klebsiella pneumoniae phage PKP-80 of the present invention can effectively eliminate and inhibit the proliferation of Klebsiella pneumoniae, and can be used to fight Klebsiella pneumoniae infection. The highest titer of Klebsiella pneumoniae phage PKP-80 is 3.4×10 9 PFU / mL.

[0020] (2) The Klebsiella pneumoniae phage PKP-80 of the present invention has a low MOI of 10 -2 , the one-step growth curve has a short incubation period and a large burst volume.

[0021] (3) The Klebsiella pneumoniae phage PKP-80 of the present invention has good thermal stability and still maintains high activity after being exposed to 70°C.

[0022] (4) The titer of the Klebsiella pneumoniae phage PKP-80 of the present invention remains basically stable after 2 hours in the pH range of 3 to 11, and the titer will only decrease significantly when the pH is <3 or pH>11.

[0023] (5) The genome of the Klebsiella pneumoniae phage PKP-80 of the present invention is a linear double-stranded DNA with a total length of 166462 bp and a GC content of 39.64%. It has 267 open reading frames (ORFs) containing perforin and lysozyme encoding genes, and no drug resistance genes and virulence genes were detected. Based on the phylogenetic tree constructed based on the DNA polymerase, terminase large subunit and major capsid protein amino acid sequences of the phage, it was found that PKP-80 has a close relationship with Klebsiellaphage vB_Kpn529046-KEN25_1.

[0024] (6) The Klebsiella pneumoniae phage PKP-80 of the present invention has good safety and effectiveness in the mouse infection phage therapy model.

[0025] (7) The Klebsiella pneumoniae phage PKP-80 of the present invention has a bactericidal effect on up to 25 capsular serotypes of Klebsiella pneumoniae pathogens, has a significant antibacterial effect, and has good biosafety. It can be used as an active ingredient in products such as drugs and environmental improvers, providing a safe and efficient alternative antibiotic treatment for humans and animals infected with Klebsiella pneumoniae. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is the plaque morphology of bacteriophage PKP-80;

[0028] Figure 2 This is the transmission electron microscopy observation result of bacteriophage PKP-80;

[0029] Figure 3 This is the result of determining the optimal infection multiplicity MOI of bacteriophage PKP-80;

[0030] Figure 4 The results of one-step growth curve determination of bacteriophage PKP-80;

[0031] Figure 5 The results of thermal stability test of bacteriophage PKP-80;

[0032] Figure 6 The pH stability test results of bacteriophage PKP-80;

[0033] Figure 7 This is the complete genome map of bacteriophage PKP-80;

[0034] Figure 8 This is the result of phylogenetic tree analysis of bacteriophage PKP-80;

[0035] Fig. 9 The comparative genome analysis results of bacteriophage PKP-80;

[0036] Fig.10 To determine the in vitro bactericidal effect of bacteriophage PKP-80 on ST11 multiple serotypes of Klebsiella pneumoniae (ST11-KL25, ST11-KL47, ST11-KL64);

[0037] Fig.11 The results of the safety and efficacy test of bacteriophage PKP-80 in treating mouse infection models of Klebsiella pneumoniae with different serotypes ST11-KL25 (A), ST11-KL47 (B), and ST11-KL64 (C). DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1 Isolation, purification and identification of bacteriophage PKP-80

[0044] Take a sewage sample from a chicken farm and isolate bacteriophages with Klebsiella pneumoniae as the host bacteria. After the water sample was left to stand at 4°C overnight, impurities were filtered out, centrifuged at 6000×g for 10 minutes, and then sterilized by filtration through a 0.22μm filter membrane. 10mL of water sample was mixed with an equal volume of 2×LB liquid culture medium, and then 100μL of host bacteria was added, and cultured at 37°C and 150rpm. After centrifugation, 0.22μm filter membrane was used for sterilization to obtain a crude phage extract. The double-layer plate method was used to verify whether clear and transparent plaques could be formed to identify whether the phage was isolated. Then the double-layer agar plate method was used to purify the phage until the plaques on the plate were of consistent size and morphology; the phage titer was determined by the double-layer plate method.

[0045] The bacterial morphology of the purified phage PKP-80 is as follows: Figure 1 As shown in the figure, the plaques are round and uniform in size. The plaques are clear and transparent with neat edges and excellent translucency. The phage titer can reach 3.4×109 PFU / mL, showing the characteristics of phage with efficient lysis.

[0046] Example 2 Host spectrum determination of bacteriophage PKP-80

[0047] The host spectrum of phage PKP-80 was determined by the spot method with 84 serotypes of Klebsiella pneumoniae strains, with 5 μL of each phage, and the formation of obvious plaques was observed to determine whether it had lytic properties.

[0048] Table 1 Host spectrum of Klebsiella pneumoniae phage PKP-80

[0049]

[0050] The host spectrum range results of the above-mentioned phage PKP-80 are shown in Table 1. The phage can simultaneously lyse 25 serotypes of Klebsiella pneumoniae, including KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161, and KL177, and has an ultra-wide host lysis characteristic.

[0051] Example 3 Transmission electron microscopy observation of bacteriophage PKP-80

[0052] Soak the double-layer agar containing phages in SM buffer for about 6 hours, centrifuge at 6000×g for 10 minutes, and filter with a 0.22μm filter to sterilize. The obtained phage solution is centrifuged at 3000×g for 20 minutes using a 100kD ultrafiltration tube to concentrate the phages. Take 10μL of the phage concentrate and drop it into a 200-mesh copper mesh, absorb the excess liquid and let it dry naturally; negatively stain with 2% phosphotungstic acid, and observe the phage morphology with a transmission electron microscope (JEM-1400FLASH) after drying.

[0053] like Figure 2 As shown, the phage head under transmission electron microscopy is a polyhedral structure, with a head length of about 110nm, a width of 90nm, a tail length of about 120nm, and a total length of 330nm. According to the latest classification of viruses in 2023ICTV, PKP-80 belongs to Viruses; Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes; Straboviridae; Tevenvirinae; iaodavirus; unclassified Jiaodavirus.

[0054] The above-mentioned Klebsiella pneumoniae bacteriophage PKP-80 was deposited in the China Center for Type Culture Collection on December 27, 2024, with the deposit number CCTCC NO: M20242916; the deposit address is: Wuhan University, Wuhan, China.

[0055] Example 4 Determination of biological characteristics of bacteriophage PKP-80

[0056] (1) Determination of the optimal multiplicity of infection (MOI)

[0057] Phage PKP-80 was inoculated with host bacteria KP2322 (1×10 8 CFU / mL at different multiplicity of infection (10, 1, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 The mixed culture was centrifuged at 5000×g for 10 min and sterilized by filtration through a 0.22 μm filter membrane. The phage filtrate was diluted 10-fold and the phage titer was determined using the double-layer agar plate method.

[0058] The results are as follows Figure 3 As shown, when MOI is 10 -2 When the phage titer was the highest, it was 3.4×10 9 PFU / mL. Therefore, it is believed that the optimal infection multiplicity MOI of Klebsiella pneumoniae PKP-80 is 10 -2 This result indicates that PKP-80 has a very efficient bactericidal effect when infecting the host Klebsiella pneumoniae.

[0059] (2) One-step growth curve determination

[0060] With MOI = 10 -2 Add phage PKP-80 and KP2322 host bacteria (10 7 CFU / mL) and mixed, let stand at 37℃ for 20min, centrifuge at 10000×g for 5min at 4℃ to remove the unadsorbed phages in the supernatant; add 20mL LB liquid culture medium to resuspend the bottom precipitate, shake and culture at 37℃ and 160rpm for 120min, take samples every 10min to measure the phage titer. Record the results and draw the one-step growth curve of phage.

[0061] The results are as follows Figure 4As shown, the incubation period of bacteriophage PKP-80 is about 10 min, the lysis period is about 80 min, the burst volume exceeds 170 PFU / cell, and it enters the plateau period after about 90 min.

[0062] (3) Determination of thermal stability of bacteriophage PKP-80

[0063] 500 μL of phage stock solution was incubated at different temperatures (40°C to 90°C) for 30 min and 60 min, and the titer was determined by the double-layer agar method with the host bacteria KP2322. The temperature stability was as follows: Figure 5 shown.

[0064] In terms of the phage's tolerance to the environment, it remains stable in the range of 40°C to 60°C. When the temperature exceeds 60°C, the phage titer begins to decrease. When the temperature is higher than 70°C, the titer decreases rapidly and is completely inactivated at 80°C.

[0065] (4) pH stability determination

[0066] 500 μL of phage stock solution was incubated at different pH (2-13) for 1 h, and the titer was determined by double-layer agar method with host bacteria KP2322. Figure 6 As shown. When the pH value is between 3 and 11, the titer remains stable. This indicates that bacteriophage PKP-80 has good stability, which provides a basis for the application of bacteriophage PKP-80 in treatment.

[0067] In summary, phage PKP-80 has a lower optimal infection multiplicity (10 -2 ), can achieve micro-amount, efficient sterilization. Its incubation period is short and its burst volume is high, proving that the phage has strong replication and lysis capabilities in the host. The phage has a high tolerance range to temperature (still active at 70°C) and pH value (3-11). This provides good biochemical conditions for the preparation of biological antibacterial agents using phage.

[0068] Example 5 Bioinformatics Analysis of Bacteriophage PKP-80

[0069] (1) Completed genome map of bacteriophage PKP-80

[0070] The purified phage stock solution was used to extract phage DNA and then sent to Shanghai Paisono Biotechnology Co., Ltd. for whole genome sequencing. The DNA sequence of phage PKP-80 was uploaded to the National Center for Biotechnology Information (NCBI) in FASTA format, and the phages were compared online using BLASTn (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to retrieve similar phages. The open reading frames (ORFs) of phage-encoded genes were predicted using the RAST (https: / / rast.nmpdr.org / ) online gene annotation website. The predicted encoded amino acid sequence was entered into NCBI's BLASTp to perform a functional search of the predicted gene. Virulence factors and antibiotic resistance were predicted in the Virulence Factor Database (VFDB, http: / / www.mgc.ac.cn / VFs / ) and the Antibiotic Resistance Gene Database (ARDB, https: / / card.mcmaster.ca / ). CGview (https: / / proksee.ca / ) was used to draw the whole genome map of phage.

[0071] The genome was found to be circular double-stranded DNA (dsDNA) by high-throughput sequencing, with a total length of 166462 bp and a GC content of 39.64%. No virulence genes, drug resistance genes, or lysogeny-related genes were found in the genome by annotation comparison. The whole genome map is available at Figure 7 .

[0072] (2) Phage PKP-80 genome analysis and phylogenetic analysis

[0073] In order to reveal the relationship between bacteriophage PKP-80 and other bacteriophages, an evolutionary tree was constructed using the highly conserved sequence of the large subunit of the terminase. The amino acid sequence encoded by the large subunit of the terminase of PKP-80 was entered into BLASTp of NCBI, and the top 10 sequences with the highest homology were selected according to the search results and compared using ClustalW in MEGA11.0 software.

[0074] Phage PKP-80 and Klebsiella phage vB_Kpn529046-KEN25_1 belong to the same branch and have a close relationship.

[0075] (3) Analysis of host spectrum characteristics

[0076] The whole genome of PKP-80 was compared by BLAST (BLAST: Basic Local Alignment Search Tool (nih.gov)), and the genomes of phages with homology exceeding 95% were selected and downloaded to construct a whole genome evolutionary tree. The data was then uploaded to phagescope (PhageScope is an online bacteriophage database, https: / / phagescope.deepomics.org / ) for cluster analysis and comparative genome analysis.

[0077] Compared with neighboring bacteriophages, PKP-80 has prominent packaging, assembly, infection, and immunity-related gene modules, which can also explain why it has an ultra-wide host spectrum.

[0078] In summary, genomic analysis showed that it has no virulence genes, drug-resistance genes, and lysogenic genes, which shows the safety of its application at the cellular and genetic levels. It has the advantages of high stability and high safety, and provides basic theoretical support for phage preparations for responding to Klebsiella pneumoniae-related infections.

[0079] Example 6 Phage PKP-80 inhibits the growth of different serotypes of ST11 Klebsiella pneumoniae in vitro

[0080] Take several 40mL test tubes and add 18mL LB culture medium to each. Add 2mL of a mixture of bacteriophage PKP-80 and different ST11 serotypes of Klebsiella pneumoniae (ST11-KL25, ST11-KL47, ST11-KL64) to each test group (MOI = 0.01) and mark them. The control group only added the test strain culture as a positive control. The test tubes were cultured at 37°C and 200rpm. Take 500μL of culture from each tube every hour into an EP tube and detect for 3 to 4 hours. After vortexing, use a spectrophotometer to measure the OD of the co-culture solution of the host bacteria and phage. 600 The growth curve of the host bacteria at different MOIs was plotted.

[0081] Depend on Fig.10 It can be seen that when phage PKP-80 was co-cultured with different serotypes of ST11 Klebsiella pneumoniae (ST11-KL25, ST11-KL47, ST11-KL64), phage PKP-80 had a strong inhibitory effect on their growth, which was significantly different from the positive control group.

[0082] In summary, phage PKP-80 has a good bactericidal effect on different serotypes of ST11 Klebsiella pneumoniae (ST11-KL25, ST11-KL47, ST11-KL64). This provides solid data support for the preparation of phage preparations to deal with Klebsiella pneumoniae-related infections.

[0083] Example 7 Safety and efficacy evaluation of bacteriophage PKP-80 in a mouse infection model

[0084] SPF BALB / c female mice aged 6 to 8 weeks were selected and randomly divided into 3 groups, each containing 8 mice. Mice were injected intraperitoneally with MLD (10 7 CFU / mouse) dose of ST11-KL25, ST11-KL47, and ST11-KL64 Klebsiella pneumoniae. One hour after injection, the phage treatment group was given a phage treatment dose of MOI=1. The negative control group used sterile PBS as a substitute during both the challenge and treatment. The positive control group was treated with an equal amount of sterile PBS after intraperitoneal injection of the challenge. The survival of the mice was recorded every 12 hours.

[0085] like Fig.11 As shown, the attack bacteria MLD (10 7 After the phage treatment group was exposed to pathogenic ST11-KL25, ST11-KL47, and ST11-KL64 Klebsiella pneumoniae (CFU / mouse), all untreated mice died within 24 hours. The survival rate of mice in the phage treatment group was significantly improved. 7 After being treated with phage PKP-80 at a dose of PFU (MOI=1), the survival rate of mice within 7 days increased to 87.5% to 100%, indicating that the phage has a good therapeutic effect and greatly improves the survival rate of mice.

[0086] The above experimental data show that phage PKP-80 showed its safety and effectiveness in the treatment effectiveness test of Klebsiella pneumoniae mouse infection model with different serotypes ST11-KL25, ST11-KL47, and ST11-KL64, providing real experimental data for the prevention and control of Klebsiella pneumoniae-related infections by phage PKP-80 and laying a solid foundation for the clinical application of phages.

[0087] In summary, the present invention provides a potentially safe, stable and efficient bacteriophage PKP-80, which has an ultra-wide host spectrum lysis characteristic and can simultaneously lyse 25 serotypes of Klebsiella pneumoniae strains, including KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161 and KL177. The phage titer can reach up to 3.4×10 9 PFU / mL, and high titer phage PKP-80 can be obtained through simple culture, which is convenient for industrial production of phage preparations and their use and storage. The phage has a low optimal infection multiplicity (10 -2 ), with the characteristics of trace and high efficiency. Its incubation period is short, and the burst volume can reach 170PFU / cell, which proves that the phage has strong replication ability and cell lysis ability in the host. The phage has a strong tolerance to temperature and is still active at 70°C. The phage has a wide tolerance range for pH (pH=3~11). In terms of safety, genome analysis shows that it has no virulence genes, drug resistance genes, and lysogenic genes, etc., showing the safety of its application at the cellular and genetic levels. Animal clinical trials have shown that phage PKP-80 is safe and highly protective. Therefore, phage PKP-80 has the advantages of high titer, high stability, and high safety. It provides a basis for the research and development of phage preparations for the treatment of various serotypes of Klebsiella pneumoniae infections. Phage PKP-80 or its culture is used as an active ingredient, alone or in combination to make biological preparations. It can be used to prepare biological antibacterial preparations in order to serve as an active ingredient for drugs, environmental improvers and other products, providing safe and efficient alternative antibiotic treatment options for humans and animals, and has good application prospects.

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A Klebsiella pneumoniae bacteriophage with an ultra-wide host spectrum, characterized in that: Its deposit number is CCTCC NO: M 20242916.

2. Use of the ultra-broad host spectrum Klebsiella pneumoniae phage according to claim 1 in the preparation of a drug for preventing and treating diseases infected by Klebsiella pneumoniae.

3. The use according to claim 2, characterized in that The Klebsiella pneumoniae infection diseases include diseases caused by different serotype strains of Klebsiella pneumoniae, including KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161 and KL177.

4. Use of the ultra-wide host spectrum Klebsiella pneumoniae phage according to claim 1 in preparing a drug for lysing Klebsiella pneumoniae.

5. The use according to claim 4, characterized in that The lytic Klebsiella pneumoniae includes different serotype strains of KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161 and KL177 of lytic Klebsiella pneumoniae.

6. Use of the ultra-wide host spectrum Klebsiella pneumoniae phage as claimed in claim 1 in preparing an improver for preventing and controlling the spread of Klebsiella pneumoniae in sewage.

7. The use according to claim 6, characterized in that The method comprises preventing and controlling the spread of Klebsiella pneumoniae in sewage, including preventing and controlling the spread of different serotype strains of KL3, KL12, KL19, KL22, KL23, KL24, KL25, KL38, KL43, KL47, KL57, KL60, KL63, KL64, KL70, KL81, KL112, KL114, KL116, KL123, KL128, KL145, KL152, KL161 and KL177 of Klebsiella pneumoniae in sewage.

8. A bactericide for Klebsiella pneumoniae, characterized in that: The invention comprises the ultra-wide host spectrum Klebsiella pneumoniae phage according to claim 1.

9. A drug for preventing and treating diseases caused by Klebsiella pneumoniae, characterized in that: The invention comprises the ultra-wide host spectrum Klebsiella pneumoniae phage according to claim 1.

10. An improver for preventing and controlling the spread of Klebsiella pneumoniae in sewage, characterized in that: The invention comprises the ultra-wide host spectrum Klebsiella pneumoniae phage according to claim 1.

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