Phage composition aiming at klebsiella pneumoniae as well as obtaining method and application thereof

Abstract

The invention relates to a method for obtaining a bacteriophage composition aiming at klebsiella pneumoniae, the bacteriophage composition aiming at klebsiella pneumoniae obtained by the method and application of the bacteriophage composition. The bacteriophage composition obtained according to the method disclosed by the invention can resist the evolution of the bacteriophage resistance of klebsiella pneumonia and solve the problem of the bacteriophage resistance of klebsiella pneumonia, so that the klebsiella pneumonia can be effectively inhibited for a long time, the effective prevention and control and treatment of klebsiella pneumonia are realized, and the application prospect is wide. Good application prospects and industrialization values are realized.

Description

Technical Field

[0001] The present invention relates to the technical field, and in particular to a method for obtaining a phage composition targeting Klebsiella pneumoniae, a phage composition targeting Klebsiella pneumoniae obtained by the method, and related products and applications thereof. Background Art

[0002] Drug resistance of pathogenic bacteria has become a global problem that threatens human health. Recently, phage therapy has received increasing attention as an important strategy to address bacterial resistance. The use of phages to prevent and treat bacterial infections has unique advantages. Phages have extremely high host specificity and can eliminate target bacteria without affecting the balance of the microecological system. Phages use host bacteria to reproduce, are highly efficient, and are easy to obtain. In the successful use of phages to treat drug-resistant bacterial infections, no obvious side effects of phages on the human body have been found.

[0003] Klebsiella pneumoniae is one of the most common pathogens and also one of the most concerned pathogens in the world. Carbapenem-resistant Klebsiella pneumoniae is listed in the list of the most threatening pathogens released by WHO. An important feature of Klebsiella pneumoniae is that it constantly acquires new genetic materials and forms new clinical features. Klebsiella pneumoniae not only acquires bla KPC ,bla NDM The most common types of Klebsiella pneumoniae are classical Klebsiella pneumoniae (cKp) and hypervirulent Klebsiella pneumoniae (hvKp). In the past 30 years, infections caused by hvKp have mainly occurred in the Asia-Pacific region and have been on the rise. In recent years, infections caused by hvKp have gradually increased outside Asia. cKp often causes nosocomial infections, while hvKp can cause multi-organ infections in healthy individuals. The most typical symptom is liver abscess, which has a high mortality rate. hvKp can infect multiple organs from the intestine. Generally speaking, cKp strains are multidrug-resistant, while hvKp strains are often sensitive to antibiotics. However, in recent years, carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) has appeared and become prevalent, bringing very severe challenges to clinical treatment and urgently needing new treatments.

[0004] However, the problem of "phage resistance" is prone to occur during phage treatment of Klebsiella pneumoniae, which has become a bottleneck affecting phage treatment. To address this problem, there is an urgent need in the art for a phage preparation and preparation method that can address the phage resistance problem of Klebsiella pneumoniae. Summary of the invention

[0005] Purpose of the Invention

[0006] In view of the problems existing in the prior art, the object of the present invention is to provide a method for obtaining a phage composition against Klebsiella pneumoniae, a phage composition against Klebsiella pneumoniae obtained by this method, and related products and applications thereof.

[0007] Solution

[0008] In order to achieve the above object, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a method for obtaining a phage composition against Klebsiella pneumoniae, characterized in that the method comprises the following steps:

[0010] (1) screening a phage that is sensitive to the target Klebsiella pneumoniae from a Klebsiella pneumoniae phage library as phage I;

[0011] (2) co-culturing the target Klebsiella pneumoniae with bacteriophage I to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage I, which is recorded as Klebsiella pneumoniae mutant strain A;

[0012] (3) further screening a phage that is sensitive to the Klebsiella pneumoniae mutant strain A obtained in step (2) from the Klebsiella pneumoniae phage library as phage II;

[0013] (4) co-culturing the Klebsiella pneumoniae mutant strain A obtained in step (2) with bacteriophage II to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage II, which is recorded as Klebsiella pneumoniae mutant strain B;

[0014] (5) screening a phage that is sensitive to the Klebsiella pneumoniae mutant strain B obtained in step (4) from the Klebsiella pneumoniae phage library as phage III;

[0015] (6) Combining bacteriophages I, II, and III to obtain the bacteriophage composition.

[0016] In a feasible embodiment, the target Klebsiella pneumoniae is K1, O1 antigen type Klebsiella pneumoniae, wherein:

[0017] Preferably, in step (1), the phage screened to be sensitive to the target Klebsiella pneumoniae is a phage sensitive to the Klebsiella pneumoniae K antigen;

[0018] Preferably, in step (3), the phage screened to be sensitive to the mutant strain I of Klebsiella pneumoniae obtained in step (2) is a phage sensitive to the O1 antigen of the target Klebsiella pneumoniae;

[0019] Preferably, in step (5), the phage screened to be sensitive to the Klebsiella pneumoniae mutant strain II obtained in step (4) is a phage sensitive to the O2 antigen of the target Klebsiella pneumoniae.

[0020] In a preferred embodiment, the target Klebsiella pneumoniae is K1, O1 antigen type Klebsiella pneumoniae, wherein the O1 antigen is composed of two parts, O1-antigen and O2-antigen, and the O1-antigen is located outside the O2-antigen; the method for obtaining a phage composition against the above Klebsiella pneumoniae comprises the following steps:

[0021] (1) screening out a phage specific to the K1 antigen of the target Klebsiella pneumoniae from a Klebsiella pneumoniae phage library as phage I;

[0022] (2) co-culturing the target Klebsiella pneumoniae with bacteriophage I to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage I, which is recorded as Klebsiella pneumoniae mutant strain A;

[0023] (3) constructing a mutant strain of the target Klebsiella pneumoniae which lacks the capsule and the O1 antigen O1-antigen part by gene knockout; using the mutant strain, screening out a phage specific to the O1 antigen O1-antigen part of the target Klebsiella pneumoniae from the above-mentioned Klebsiella pneumoniae phage library; further, screening out a phage with enhanced sensitivity to the Klebsiella pneumoniae mutant strain A obtained in step (2) as phage II from the obtained phage specific to the O1 antigen O1-antigen part of the target Klebsiella pneumoniae;

[0024] (4) co-culturing the Klebsiella pneumoniae mutant strain A obtained in step (2) with bacteriophage II to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage II, which is recorded as Klebsiella pneumoniae mutant strain B;

[0025] (5) constructing a mutant strain of the target Klebsiella pneumoniae which lacks the capsule and both the O1-antigen part and the O2-antigen part of the O1 antigen by gene knockout, and using the mutant strain to screen out a phage specific for the O1-antigen part of the target Klebsiella pneumoniae from the above-mentioned Klebsiella pneumoniae phage library; further, screening out a phage with enhanced sensitivity to the Klebsiella pneumoniae mutant strain B obtained in step (4) from the obtained phage specific for the O1-antigen part of the target Klebsiella pneumoniae as phage III;

[0026] (6) Combining bacteriophages I, II, and III to obtain a bacteriophage composition targeting the target Klebsiella pneumoniae.

[0027] In a preferred specific embodiment of the above method, the target Klebsiella pneumoniae is Klebsiella pneumoniae NK01067 (hereinafter referred to as "Kp067"), which belongs to the K1 and O1 antigen types, wherein the O1 antigen is composed of two parts, O1-antigen and O2-antigen, the O2-antigen is the base, directly connected to lipid A (Lipid A), and the O1-antigen is located outside the O2-antigen; the method for obtaining a phage composition against Klebsiella pneumoniae NK01067 comprises the following steps:

[0028] (1) by expressing the phage tail fiber protein (the tail fiber protein has a lytic enzyme activity and can specifically recognize and lyse different Kp capsule types), a phage specific for the K1 antigen of Kp067 was screened from the Klebsiella pneumoniae phage library as phage I;

[0029] (2) Kp067 was co-cultured with phage I to obtain a Klebsiella pneumoniae mutant strain resistant to phage I, which was designated as Klebsiella pneumoniae mutant strain A;

[0030] (3) constructing a mutant strain of Kp067 lacking the capsule and the O1 antigen O1-antigen part by gene knockout; using the mutant strain, screening phages specific to the O1 antigen O1-antigen part of Kp067 from the above-mentioned Klebsiella pneumoniae phage library; and screening phages with enhanced sensitivity to the Klebsiella pneumoniae mutant strain A obtained in step (2) as phage II from the obtained phages specific to the O1 antigen O1-antigen part of Kp067;

[0031] (4) co-culturing the Klebsiella pneumoniae mutant strain A obtained in step (2) with bacteriophage II to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage II, which is recorded as Klebsiella pneumoniae mutant strain B;

[0032] (5) constructing a mutant strain of Kp067 lacking the capsule and both the O1-antigen part and the O2-antigen part of the O1 antigen by gene knockout, and using the mutant strain to screen out phages specific to the O1-antigen part of Kp067 from the above-mentioned Klebsiella pneumoniae phage library; and, from the obtained phages specific to the O1-antigen part of Kp067, screening out phages with enhanced sensitivity to the Klebsiella pneumoniae mutant strain B obtained in step (4) as phage III;

[0033] (6) Phages I, II, and III are combined to obtain a phage composition against Klebsiella pneumoniae NK01067.

[0034] The above method for obtaining a phage composition against Klebsiella pneumoniae is discovered based on the following unexpected phenomenon:

[0035] When Klebsiella pneumoniae evolves resistance to phage A, it may become more susceptible to phage B; when Klebsiella pneumoniae develops resistance to phage B, it may simultaneously become more susceptible to phage C.

[0036] The above phenomenon was discovered accidentally by the inventors during their long-term research on phage therapy. Based on this discovery, the use of single, double or even triple cross-sensitivity can effectively resist the evolution of Klebsiella pneumoniae resistance, thereby developing a phage composition that can effectively inhibit Klebsiella pneumoniae for a long time.

[0037] In a second aspect, the present invention provides a phage composition against Klebsiella pneumoniae, wherein the phage composition is obtained by the method described in the first aspect above.

[0038] In a feasible embodiment, the phage composition includes: Klebsiella pneumoniae phage RCIP0041 with a preservation number of CGMCC:46174, Klebsiella pneumoniae phage RCIP0070 with a preservation number of CGMCC:46176, and Klebsiella pneumoniae phage RCIP0002 with a preservation number of CGMCC:46175; the taxonomic names of the above three phages are all Klebsiella phage (Klebsiella phage), which are all deposited in the General Microbiology Center of China National Culture Collection Administration, and the preservation date is August 23, 2024.

[0039] Preferably, the Klebsiella pneumoniae targeted by the phage composition is the Klebsiella pneumoniae NK01067 strain, which is a highly toxic and highly drug-resistant strain belonging to the K1 serotype and O1 antigen type, and carries the carbapenem resistance gene bla NDM-1 ;

[0040] Preferably, in the phage composition, the titer of each phage is 10 8 -10 10 PFU / mL, 10 8 -10 10 PFU / mL, 10 8 -10 10 PFU / mL.

[0041] In a third aspect, the present invention provides a phage against Klebsiella pneumoniae, wherein the phage is selected from the following: Klebsiella pneumoniae phage RCIP0041 with a deposit number of CGMCC: 46174, Klebsiella pneumoniae phage RCIP0070 with a deposit number of CGMCC: 46176, and / or Klebsiella pneumoniae phage RCIP0002 with a deposit number of CGMCC: 46175. The deposit information of the three phages is as described above.

[0042] Among them, phages RCIP0041, RCIP0070 and RCIP0002 are members of the genera Drulisvirus, Sugarlandvirus and Slopekvirus, respectively; transmission electron microscopy (TEM) morphological observations showed that phage RCIP0041 belongs to the Brachyviridae family, phage RCIP0002 belongs to the Myoviridae family, and phage RCIP0070 belongs to the Longiviridae family.

[0043] The above-mentioned Klebsiella pneumoniae phages were all screened from the Klebsiella pneumoniae phage library previously constructed by the inventor, and are all deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 23, 2024.

[0044] In a fourth aspect, the present invention provides a use of the bacteriophage composition as described in the second aspect or the bacteriophage as described in the third aspect in preparing a product for preventing and treating Klebsiella pneumoniae infection;

[0045] In a feasible embodiment, the product is a disinfectant, a cleaning agent or an additive thereof.

[0046] In a fifth aspect, the present invention provides use of the bacteriophage composition as described in the second aspect above or the bacteriophage as described in the third aspect above in the preparation of a medicament for preventing or treating a disease caused by Klebsiella pneumoniae.

[0047] In a feasible embodiment, the disease is pneumonia, urinary tract infection, meningitis, sepsis and liver abscess;

[0048] In a feasible embodiment, the drug is in a gastrointestinal dosage form, a respiratory dosage form or a topical dosage form.

[0049] In a sixth aspect, the present invention provides a bactericidal composition for preventing and treating Klebsiella pneumoniae infection, the bactericidal composition comprising an effective amount of the bacteriophage composition as described in the second aspect above or the bacteriophage as described in the third aspect above;

[0050] In a feasible embodiment, the bactericidal composition is a cleaning agent, a disinfectant or an additive thereof.

[0051] In a seventh aspect, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by Klebsiella pneumoniae, the pharmaceutical composition comprising an effective amount of the phage composition as described in the second aspect or the phage as described in the third aspect, and optionally a pharmaceutically acceptable excipient.

[0052] In an eighth aspect, the present invention provides a method for reducing or inhibiting the colonization or growth of Klebsiella pneumoniae on a non-biological surface, the method comprising: contacting the non-biological surface with the phage composition described in the second aspect, the phage described in the third aspect, or the bactericidal composition described in the sixth aspect.

[0053] In a ninth aspect, the present invention provides a method for preventing or treating a disease caused by Klebsiella pneumoniae, the method comprising: administering to a subject in need thereof a preventive or therapeutically effective amount of the phage composition as described in the second aspect above, the phage as described in the third aspect above, or the pharmaceutical composition as described in the seventh aspect above.

[0054] The "preventively or therapeutically effective amount" may vary depending on the subject of administration, the subject organ, symptoms, the method of administration, etc., and can be determined based on the doctor's judgment, taking into account the type of dosage form, the method of administration, the patient's age and weight, the patient's symptoms, etc.

[0055] Beneficial Effects

[0056] The phage composition obtained according to the method of the present invention can resist the evolution of phage resistance of Klebsiella pneumoniae and solve the phage resistance problem of Klebsiella pneumoniae, thereby being able to effectively inhibit Klebsiella pneumoniae for a long time, achieving effective prevention, control and treatment of Klebsiella pneumoniae, and having good application prospects and industrialization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0058] Figure 1 This is the in vitro inhibition curve of the phage composition of the present invention.

[0059] Figure 2It shows that the phage composition of the present invention can effectively reduce the infection of mice caused by Klebsiella pneumoniae NK01067, as described in Example 3; wherein, Figure A shows the survival rate curve of mice in different treatment groups, Figure B shows the fecal bacterial load of mice in different treatment groups, and Figure C shows the weight change curve of mice in different treatment groups.

[0060] Figure 3 The evolutionary path of phage-resistant strains is shown, as described in Example 4; wherein the "R" in the upper right corner of each phage on the right indicates that the bacteria are resistant to the phage, and the "S" indicates that the bacteria are sensitive to the phage.

[0061] The Klebsiella pneumoniae phage RCIP0041, Klebsiella pneumoniae phage RCIP0070 and Klebsiella pneumoniae phage RCIP0002 recorded in this article were all deposited in the General Microbiology Center of China National Culture Collection Administration on August 23, 2024. Their taxonomic names are all Klebsiella phage (Klebsiella phage), and their deposit numbers are CGMCC:46174, CGMCC:46176 and CGMCC:46175, respectively. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Unless explicitly stated otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0064] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0065] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0066] Example 1: Screening of phage compositions against Klebsiella pneumoniae NK01067 by the method of the present invention

[0067] In the early stage of this study, we collected 2,500 strains of Klebsiella pneumoniae from 42 hospitals in China, including 105 ST types and 55 KL types, and constructed a large and rich phage library containing 243 strains of Klebsiella pneumoniae phages. The following phages were all screened from this Klebsiella pneumoniae phage library.

[0068] As a representative example, in this embodiment, a phage composition targeting highly toxic and highly drug-resistant Klebsiella pneumoniae NK01067 was screened and obtained by the method of the present invention.

[0069] Klebsiella pneumoniae NK01067 (also referred to as Kp067 in this article) belongs to the K1 serotype, O1 antigen type, and carries the carbapenem resistance gene bla NDM-1 The O1 antigen is composed of two parts, O1-antigen and O2-antigen. The O2-antigen is the base, directly connected to lipid A, and the O1-antigen is located outside the O2-antigen.

[0070] The specific steps are as follows:

[0071] (1) by expressing phage tail fiber protein (tail fiber protein has lytic enzyme activity and can specifically recognize and lyse different Kp capsule types), a phage specific for the K1 antigen of Kp067 was screened from the above-mentioned Klebsiella pneumoniae phage library as phage I;

[0072] (2) Kp067 was co-cultured with phage I to obtain a Klebsiella pneumoniae mutant strain resistant to phage I, which was designated as Klebsiella pneumoniae mutant strain A;

[0073] (3) The Kp capsule of Kp067 is deleted by gene knockout; further, in the Kp capsule-deficient strain, the O1-antigen portion of the O1 antigen of Kp067 is deleted by gene knockout to construct a mutant strain in which the Kp capsule is deleted and the O1-antigen portion of the O1 antigen is deleted; using the mutant strain, phages specific to the O1-antigen portion of the O1 antigen of Kp067 are screened from the above-mentioned Klebsiella pneumoniae phage library; and, from the obtained phages specific to the O1-antigen portion of the O1 antigen of Kp067, phages with enhanced sensitivity to the Klebsiella pneumoniae mutant strain A obtained in step (2) are screened as phage II;

[0074] (4) co-culturing the Klebsiella pneumoniae mutant strain A obtained in step (2) with phage II to obtain a Klebsiella pneumoniae mutant strain resistant to phage II, which is recorded as Klebsiella pneumoniae mutant strain B;

[0075] (5) By means of gene knockout, the O2-antigen portion of the mutant strain lacking the Kp capsule and the O1-antigen portion of the O1 antigen in step (3) is lacking, thereby constructing a mutant strain lacking the Kp capsule and both the O1-antigen portion and the O2-antigen portion of the O1 antigen; using this mutant strain, phages specific to the O2-antigen portion of the O1 antigen of Kp067 are screened from the above-mentioned Klebsiella pneumoniae phage library; and, from the obtained phages specific to the O2-antigen portion of the O1 antigen of Kp067, phages with enhanced sensitivity to the Klebsiella pneumoniae mutant strain B obtained in step (4) are screened as phage III.

[0076] Through the above steps and based on the principle of comprehensive coverage of short-tail, long-tail and muscle-tail phages, we screened out a total of 19 phage combinations. Through further experimental screening, we obtained the phage composition of the present invention, which comprises: phages RCIP0041, RCIP0070 and RCIP0002; these phages belong to the genera Drulisvirus, Slopekvirus and Sugarlandvirus, respectively; transmission electron microscopy (TEM) morphological observation showed that the morphology of ΦRCIP0041 is a short-tail phage, the morphology of ΦRCIP0002 is a muscle-tail phage, and the morphology of ΦRCIP0070 is a long-tail phage.

[0077] Example 2: In vitro inhibitory effect of phage composition on Kp067

[0078] In this example, the in vitro antibacterial experiment was performed to detect the antibacterial effect of the phage composition of the present invention obtained by screening in Example 1 on Klebsiella pneumoniae NK01067 (abbreviated as Kp067, carrying bla NDM-1 , for K1 and O1 antigen types) in vitro inhibitory effect; the specific experimental method is as follows:

[0079] In vitro inhibition curve detection of phage cocktail combination: In order to evaluate the in vitro lysis ability of phage cocktail against Kp067 strain, an automatic growth curve analyzer (Bioscreen C, Helsinki, Finland) was used for growth curve detection. The specific experimental operation is as follows: Take 100 μL of bacterial culture solution with OD600 = 0.2 (approximately equal to 10 7CFU) were added to a 100-well plate (Honeycomb Microplate, Turku, Finland) and mixed with phage solution (single phage: 100 μL; two phage cocktails: 50 μL each; three phage cocktails: 33.3 μL each) to give a final multiplicity of infection MOI = 1.0. Bacteria were supplemented with SM buffer (Coolaber, Beijing, China) without phage as a control group. The values ​​under OD600 absorbance were measured every 1 h for 72 h, with shaking culture during the test. The experiment was repeated three times, and the inhibition curve was drawn based on the average of the three repetitions.

[0080] The results are as follows Figure 1 shown); Figure 1 The results showed that the phage composition of the present invention containing three phages almost completely inhibited bacterial regrowth compared with a single phage or a combination of two phages in the phage composition, and no obvious bacterial regrowth was observed within 72 hours of detection, indicating that the phage composition of the present invention can prevent or delay the development of resistance. Therefore, this cocktail combination of phages ΦRCIP0041, ΦRCIP0002 and ΦRCIP0070 shows special potential for therapeutic applications.

[0081] Example 3: The phage composition of the present invention can effectively reduce the infection of mice caused by Kp067

[0082] In this example, the inhibitory effect of the phage composition of the present invention obtained by screening in Example 1 on mouse infection caused by Kp067 was detected using a mouse infection model; the specific experimental method is as follows:

[0083] Construction of mouse infection model and phage cocktail treatment: A mouse model was established using 6-8 week old male BALB / c mice (Charles River Corp., Beijing, China). The mice were randomly divided into cages, five per cage, and the experiment was divided into three groups: a normal control group (i.e., a group not infected with Kp067), a model control group (i.e., a group infected with Kp067 only without phage cocktail treatment), and a phage cocktail treatment group, with 6 mice in each group. Then, 100 μL of 1×10 8 CFU / mL of Kp067 bacterial solution was orally inoculated into mice in the infection and treatment groups, while the control group was orally inoculated with only 100 μL PBS. 8PFU / mL of phage cocktail (RCIP0002, RCIP0041 and RCIP0070). During the 7-day observation period, the survival rate, percentage change in body weight and fecal bacterial load of the mice were monitored and recorded. To quantify the intestinal bacterial load, approximately 2 fresh fecal pellets were collected from each mouse and suspended in 1 mL of sterile PBS. The samples were thoroughly mixed until no precipitation was observed, and then centrifuged to obtain the supernatant for bacterial counts. For counting, the samples were serially diluted tenfold, and 10 μL of the dilution was inoculated on SCAI (Simmons Citrate agar medium containing 1% inositol) supplemented with ampicillin (25 μg / mL) and cultured overnight at 37°C to specifically screen for Klebsiella pneumoniae.

[0084] The survival rate curves, fecal bacterial load and body weight change curves of mice in different treatment groups are shown in Figure 2. Figure 2 As shown in Figures A, B, and C.

[0085] Depend on Figure 2 As shown in Figure A, during the 7-day observation period, the model control group mice that were not treated with the phage composition had a higher mortality rate (mortality rate as high as 67%); in contrast, no death was observed in either the phage composition treatment group or the normal control group.

[0086] Figure 2 Figure B in the figure shows the results of Kp067 abundance detection in the fecal samples of mice in different treatment groups, which shows that starting from the first day, the phage composition of the present invention can significantly reduce the abundance of Kp067 in the feces of mice (compared with the model control group); by the third day, Kp067 was almost undetectable in the fecal samples of mice in the phage treatment group (i.e., already below the minimum detection limit "LOD"), which was significantly lower than that in the model control group (about 10 5 CFU / g). It is noteworthy that there was only a small change in the percentage of body weight of mice between the phage-treated and non-infected groups ( Figure 2 c).

[0087] The above results fully demonstrate the efficacy of the phage composition of the present invention in significantly reducing bacterial load and alleviating infection in mice, thereby demonstrating its potential as a treatment against infection caused by CR-hvKp.

[0088] Example 4: Phage compositions of the present invention restrict the evolution of phage resistance of Kp067 through cross-sensitivity

[0089] In order to screen phage-tolerant strains, we used Kp067 as the starting strain. The strain was mixed with phage at MOI=10 and cultured until the bacterial culture fluid was obviously turbid. The potential tolerant phage colony was then diluted and streaked on a plate to screen out single colonies. The phage sensitivity of the tolerant strain was further verified by the double-layer soft agar plate method. In this way, the strain Kp067-M1 tolerant to phage ΦRCIP0041 was screened out; based on Kp067-M1, the strain Kp067-M2 tolerant to ΦRCIP0002 was screened out; based on Kp067-M2, the strain Kp067-M3 tolerant to ΦRCIP0070 was screened out.

[0090] In order to analyze the mechanism by which the phage composition of the present invention is not prone to resistance, we selected carbapenem-resistant, highly virulent, K1 and O1 antigenic type Kp067 as the host bacteria, conducted continuous evolution experiments, and presented the results using a spot plate experiment. The results are shown in Figure 3 .

[0091] like Figure 3 As shown, the experiment was performed under the selection pressure of three different phages; initially, Kp067 was co-cultured with ΦRCIP0041 to generate the phage-resistant mutant Kp067-M1. Interestingly, ΦRCIP0002 and ΦRCIP0070 produced more obvious plaques on Kp067-M1 compared to Kp067. Kp067-M1 was then co-cultured with ΦRCIP0002 to select the phage-resistant mutant Kp067-M2, which was still lysed by ΦRCIP0070. The last phage-resistant mutant, Kp067-M3, was obtained when Kp067-M2 was co-cultured with ΦRCIP0070. More interestingly, Kp067-M3 showed resistance to ΦRCIP0070, but regained sensitivity to ΦRCIP0002.

[0092] Therefore, during the above evolutionary process, two cross-sensitivity events occurred, which should be the main reason why this phage cocktail is not easy to produce resistance.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for obtaining a phage composition against Klebsiella pneumoniae, characterized in that: The method comprises the following steps: (1) screening a phage that is sensitive to the target Klebsiella pneumoniae from a Klebsiella pneumoniae phage library as phage I; (2) co-culturing the target Klebsiella pneumoniae with bacteriophage I to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage I, which is recorded as Klebsiella pneumoniae mutant strain A; (3) further screening a phage that is sensitive to the Klebsiella pneumoniae mutant strain A obtained in step (2) from the Klebsiella pneumoniae phage library as phage II; (4) co-culturing the Klebsiella pneumoniae mutant strain A obtained in step (2) with bacteriophage II to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage II, which is recorded as Klebsiella pneumoniae mutant strain B; (5) screening again from the Klebsiella pneumoniae phage library a phage that is sensitive to the Klebsiella pneumoniae mutant strain B obtained in step (4) as phage III; (6) Combining bacteriophages I, II, and III to obtain the bacteriophage composition.

2. The method according to claim 1, characterized in that: The target Klebsiella pneumoniae is K1 and O1 antigenic Klebsiella pneumoniae, wherein: In step (1), the phage screened to be sensitive to the target Klebsiella pneumoniae is a phage sensitive to the Klebsiella pneumoniae K1 antigen; And / or, in step (3), the phage screened to be sensitive to the mutant strain of Klebsiella pneumoniae resistant to phage I obtained in step (2) is a phage sensitive to the O1 antigen of the target Klebsiella pneumoniae; And / or, in step (5), the phage screened to be sensitive to the mutant strain of Klebsiella pneumoniae resistant to phage II obtained in step (4) is a phage sensitive to the O2 antigen of the target Klebsiella pneumoniae.

3. The method according to claim 1, characterized in that The target Klebsiella pneumoniae is K1 and O1 antigenic Klebsiella pneumoniae, and the method for obtaining a phage composition targeting the Klebsiella pneumoniae comprises the following steps: (1) screening out a phage specific to the K1 antigen of the target Klebsiella pneumoniae from a Klebsiella pneumoniae phage library as phage I; (2) co-culturing the target Klebsiella pneumoniae with bacteriophage I to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage I, which is recorded as Klebsiella pneumoniae mutant strain A; (3) constructing a mutant strain of the target Klebsiella pneumoniae that lacks the capsule and partially lacks the O1 antigen by gene knockout; Using the mutant strain, a phage specific to the O1 antigen O1-antigen portion of the target Klebsiella pneumoniae is screened out from the above-mentioned Klebsiella pneumoniae phage library; further, from the obtained phages specific to the O1 antigen O1-antigen portion of the target Klebsiella pneumoniae, a phage with enhanced sensitivity to the Klebsiella pneumoniae mutant strain A obtained in step (2) is screened out as phage II; (4) co-culturing the Klebsiella pneumoniae mutant strain A obtained in step (2) with bacteriophage II to obtain a Klebsiella pneumoniae mutant strain resistant to bacteriophage II, which is recorded as Klebsiella pneumoniae mutant strain B; (5) constructing a mutant strain of the target Klebsiella pneumoniae which lacks the capsule and both the O1-antigen part and the O2-antigen part of the O1 antigen by gene knockout, and using the mutant strain to screen out a phage specific for the O1-antigen part of the target Klebsiella pneumoniae from the above-mentioned Klebsiella pneumoniae phage library; further, screening out a phage with enhanced sensitivity to the Klebsiella pneumoniae mutant strain B obtained in step (4) from the obtained phage specific for the O1-antigen part of the target Klebsiella pneumoniae as phage III; (6) Combining bacteriophages I, II, and III to obtain a bacteriophage composition targeting the target Klebsiella pneumoniae.

4. The method according to claim 3, characterized in that The target Klebsiella pneumoniae is Klebsiella pneumoniae NK01067.

5. A phage composition against Klebsiella pneumoniae, characterized in that: The phage composition is obtained by the method according to any one of claims 1 to 4.

6. The bacteriophage composition according to claim 5, characterized in that The phage composition comprises: Klebsiella pneumoniae phage RCIP0041 with a deposit number of CGMCC:46174, Klebsiella pneumoniae phage RCIP0070 with a deposit number of CGMCC:46176, and Klebsiella pneumoniae phage RCIP0002 with a deposit number of CGMCC:46175.

7. The bacteriophage composition according to claim 6, characterized in that The Klebsiella pneumoniae targeted by the bacteriophage composition is the Klebsiella pneumoniae NK01067 strain; And / or, in the phage composition, the titer of each phage is 10 8 -10 10 PFU / mL, 10 8 -10 10 PFU / mL, 10 8 -10 10 PFU / mL.

8. A bacteriophage against Klebsiella pneumoniae, characterized in that: The bacteriophage is selected from the following: Klebsiella pneumoniae phage RCIP0041 with a deposit number of CGMCC:46174, Klebsiella pneumoniae phage RCIP0070 with a deposit number of CGMCC:46176, and / or Klebsiella pneumoniae phage RCIP0002 with a deposit number of CGMCC:46175.

9. Use of the bacteriophage composition according to any one of claims 5 to 7 or the bacteriophage according to claim 8 in the preparation of a product for preventing and treating Klebsiella pneumoniae infection; Optionally, the product is a disinfectant, a cleaning agent or an additive thereof.

10. Use of the bacteriophage composition according to any one of claims 5 to 7 or the bacteriophage according to claim 8 in the preparation of a medicament for preventing or treating a disease caused by Klebsiella pneumoniae; Preferably, the disease is pneumonia, urinary tract infection, meningitis, sepsis and liver abscess; Preferably, the drug is a dosage form for gastrointestinal administration, respiratory tract administration or local administration.

11. A bactericidal composition for preventing and treating Klebsiella pneumoniae infection, characterized in that: The bactericidal composition comprises an effective amount of the bacteriophage composition according to any one of claims 5 to 7 or the bacteriophage according to claim 8; Optionally, the bactericidal composition is a cleaning agent, a disinfectant or the aforementioned additives.

12. A pharmaceutical composition for preventing or treating a disease caused by Klebsiella pneumoniae, characterized in that: The pharmaceutical composition comprises an effective amount of the phage composition according to any one of claims 5 to 7 or the phage according to claim 8, and optionally a pharmaceutically acceptable excipient.

13. A method for reducing or inhibiting the colonization or growth of Klebsiella pneumoniae on a non-biological surface, characterized in that The method comprises contacting the non-biological surface with the bacteriophage composition of any one of claims 5 to 7, the bacteriophage of claim 8, or the bactericidal composition of claim 11.

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