Klebsiella pneumoniae bacteriophage and application thereof

Abstract

The present invention provides a Klebsiella pneumoniae bacteriophage. The invention also provides compositions of the bacteriophage and methods of treating Klebsiella pneumoniae infection with the bacteriophage or compositions.

Description

Technical Field

[0001] The present invention relates to the field of microbiology. In particular, the present invention relates to Klebsiella pneumoniae phages and combinations thereof. Background Art

[0002] Klebsiella pneumoniae is one of the common opportunistic pathogens in clinical practice, causing more than 95% of Klebsiella-related diseases. When the body's immunity decreases, Klebsiella pneumoniae can cause a series of pathological changes, such as pneumonia, mastitis, metritis, meningitis, and even sepsis. The conventional treatment for Klebsiella pneumoniae infection is to use carbapenem antibiotics, such as imipenem, meropenem, etc. However, with the abuse of antibiotics and the spread of drug resistance in the aquaculture industry and clinics, carbapenem-resistant Klebsiella pneumoniae has been widely distributed around the world, and some strains even have multidrug resistance, becoming one of the most difficult Gram-negative bacteria to treat in the world. The continuous emergence of drug-resistant bacteria has caused great difficulties in the selection of clinical treatment drugs, and the development of new antibacterial drugs is imminent.

[0003] Bacteriophages are viruses that use bacteria as hosts and exist in any environment where bacteria exist. Phage replication in host bacteria will eventually lead to bacterial cell lysis. In the current bacterial resistance crisis, phage therapy has become one of the most promising therapies for drug-resistant bacteria. Compared with antibiotics and other antibacterial products, phages have unique advantages, including strong specificity, proliferation, and high safety.

[0004] Therefore, in order to treat Klebsiella pneumoniae infections (including drug-resistant strains), it is necessary to isolate phages that use Klebsiella pneumoniae as a host. Summary of the invention

[0005] In a first aspect, the present invention provides a Klebsiella pneumoniae phage, the genome of the phage comprising SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher identity with SEQ ID NO: 1 or 2. In some embodiments, the phage is a phage deposited with CGMCC No. 45591 or CCTCC M20231558 or a progeny thereof.

[0006] In a second aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a bacteriophage, wherein the genome of the bacteriophage comprises SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity with SEQ ID NO: 1 or 2.

[0007] In some embodiments, the composition comprises a first phage and a second phage, wherein the genome of the first phage comprises SEQ ID NO: 1, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, and the genome of the second phage comprises SEQ ID NO: 2, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 2.

[0008] In some embodiments, the phage is selected from one or both of the phages deposited as CGMCC No. 45591 and CCTCC M 20231558, or progeny thereof.

[0009] In some embodiments, the pharmaceutical composition comprises a bacteriophage deposited under CGMCC No. 45591 and CCTCC M 20231558 or a progeny thereof.

[0010] In a third aspect, there is provided a use of the bacteriophage or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease associated with Klebsiella pneumoniae infection in a subject in need thereof.

[0011] In some embodiments, the Klebsiella pneumoniae is a drug-resistant bacterium. In some embodiments, the disease is selected from pneumonia, mastitis, metritis, meningitis and sepsis caused by Klebsiella pneumoniae. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The inhibition of Klebsiella pneumoniae strains KPK (left panel) and KPM (right panel) by the phage cocktail of the invention is shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] Although various embodiments and aspects of the present invention are shown and described herein, it is obvious to those skilled in the art that these embodiments and aspects are provided only by way of example. Without departing from the present invention, those skilled in the art will appreciate that many modifications, changes and substitutions are possible. It should be understood that the various alternatives of the embodiments of the present invention described herein may be used to implement the present invention.

[0015] I. Definition

[0016] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar to or equivalent to those described herein can be used to implement the present invention. The following definitions are provided to facilitate the understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0017] As used herein, the term "bacteriophage" refers to a virus that uses bacteria as a host and replicates therein. The bacteriophage is composed of a protein capsid and nucleic acid (DNA or RNA, single-stranded or double-stranded, circular or linear), and is about 20-200nm in length. The bacteriophage binds to the host by recognizing specific receptors on the surface of bacteria, and has strict specificity. The large-scale reproduction of bacteriophages in bacterial cells will eventually lead to bacterial cell lysis, thereby killing the bacterial cells. Bacteriophages parasitize in living bacteria and have strict host specificity, which depends on the molecular structure and complementarity of the bacteriophage adsorption structure and the receptor surface receptor of the recipient bacteria. Bacteriophages are the most common and widely distributed group among viruses, and bacteriophages are usually present in some environments where bacterial communities exist.

[0018] The term "identity" or "sequence identity" is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (excluding additions or deletions) to achieve optimal alignment of the two sequences. Identity is usually expressed as a percentage. For example, the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window sequence, and the result is multiplied by 100 to calculate the percentage to obtain the identity of the sequence.

[0019] In order to compare sequences, a sequence is usually used as a reference sequence to compare it with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence according to the program parameters.

[0020] As used herein, a "comparison window" includes a segment with reference to any one of a plurality of consecutive positions, wherein after the two sequences are optimally aligned, a sequence can be compared to a reference sequence of the same number of consecutive positions. For example, the comparison window includes about or at least about 20, 50, 100, 1000, 2500, 5000, 7500, 10000, 15000, 20000, 25000, 30000 or more consecutive positions.

[0021] Methods for sequence alignment are well known in the art, for example, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0022] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25: 3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215: 403-410 (1990), respectively. Those skilled in the art will appreciate that software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

[0023] The "lysis" of bacteria by phages generally means that the phages lyse the bacterial cells from the inside out after assembling inside the bacterial cells. In this process, the phages complete a complete cycle of first replicating and proliferating and then lysing the bacteria. It should be understood that the lysis also includes external lysis. "External lysis" means that the phages do not enter the bacterial cells and directly lyse the bacteria. There is no replication and proliferation of phages during the external lysis process.

[0024] Herein, the expressions "first phage" and "second phage" are only used to distinguish different phages, and are not intended to limit the order.

[0025] Herein, "progeny" of a phage refers to a phage obtained by subculturing or culturing the original phage.

[0026] The terms "solution" and "suspension" of bacteriophage are used interchangeably to refer to a mixture of bacteriophage particles dispersed in a liquid medium.

[0027] As used herein, "isolating" a bacteriophage means separating the bacteriophage from at least some components with which it is associated (e.g., components in an environmental sample). "Isolated" bacteriophage includes (1) a bacteriophage that has been separated from at least some components with which it is associated when originally produced (whether naturally or in an experimental setting), and / or (2) a bacteriophage that has been artificially produced, prepared, purified, and / or manufactured, such as using artificial culture conditions, such as (but not limited to) growing on plates and / or in fermenters.

[0028] As used herein, "purifying" a phage includes (1) separating the phage from impurities (e.g., components from an environmental sample), and / or (2) separating different phage clones to obtain single clones. A "purified" phage can be greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% (e.g., by weight) pure.

[0029] Existing phage purification is performed by a double-layer plate method. The "double-layer plate method" generally includes (1) preparing a lower solid culture medium, (2) mixing phage and host bacteria with a melted culture medium (e.g., a semi-solid culture medium), (3) transferring the mixture to the lower solid culture medium to form a double-layer plate, (4) incubating the double-layer plate, and (5) obtaining purified phage from the plaque. The double-layer plate method is complex to operate and cannot be used for high-throughput screening.

[0030] The inventors have discovered that phages can be purified by plate streak separation. As used herein, the terms "plate streak separation" and "streak separation method" are used interchangeably and refer to a method for purifying microorganisms, comprising diluting the microorganisms by streaking in a plurality of predetermined areas on a solid culture medium plate to obtain a single clone of the microorganism. The streak separation method is commonly used to separate bacteria and yeast. Surprisingly, the inventors have discovered that the streak separation method can also be used to purify phages.

[0031] As used herein, "pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that facilitate administration of active agents to subjects and absorption thereof, and can be included in the compositions of the present invention without causing significant adverse toxicological effects to patients. Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, water, physiological saline solutions, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, flavoring agents, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and pigments, etc. These preparations can be sterilized and, if necessary, can be mixed with adjuvants that do not react adversely with bacteriophages, such as preservatives, stabilizers, wetting agents, emulsifiers, osmotic pressure regulators, buffers, colorants, and / or aromatic substances, etc.

[0032] "Patient" or "subject in need thereof" refers to a human or non-human animal, such as a non-human mammal, who has or may have the indicated condition. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bush babies, monkeys, and apes), rabbits, dogs (e.g., companion dogs, service dogs, or working dogs), horses (e.g., race horses and working horses), cats (e.g., domestic cats), livestock (e.g., pigs, cattle, donkeys, mules, bison, goats, camels, and sheep), and deer.

[0033] The terms "subject," "patient," "individual," and the like are not intended to be limiting and are generally interchangeable. That is, an individual described as a "patient" does not necessarily have a disease but may simply be seeking medical advice.

[0034] As used herein, "treating" a condition, disease, or disorder, or symptoms associated with a condition, disease, or disorder refers to methods for obtaining beneficial or desired results, including clinical results. Beneficial or desirable clinical results may include, but are not limited to, alleviating or ameliorating one or more symptoms or conditions, reducing the extent of a condition, disorder, or disease, stabilizing the state of a condition, disease, or disorder, preventing the development of a condition, disease, or disorder, preventing the spread of a condition, disease, or disorder, delaying or slowing the progression of a condition, disease, or disorder, delaying or slowing the onset of a condition, disease, or disorder, alleviating or relieving the condition, disease, or disorder state, and eliminating the condition, disease, or disorder state, whether partially or completely. "Treatment" may also mean temporarily inhibiting the progression of a condition, disease, or disorder, temporarily slowing the progression of a condition, disease, or disorder, although in some cases, it involves permanently stopping the progression of a condition, disease, or disorder.

[0035] As used herein, the terms "treat" and "prevent" are not intended to be absolute terms. It should be understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or symptoms of a disease or condition. Treatment may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of a disease or condition.

[0036] As used herein, the term "therapeutically effective amount" and the like refers to an amount of a drug / composition sufficient to alleviate a condition. For example, administration of a therapeutically effective amount of a drug will increase or decrease a given parameter by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.

[0037] 2. Klebsiella pneumoniae phages and their combinations

[0038] The invention discloses a Klebsiella pneumoniae phage, which can lyse multiple Klebsiella pneumoniae strains.

[0039] In some embodiments, the genome of the phage comprises SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity to SEQ ID NO: 1 or 2.

[0040] In some embodiments, the phage is a phage deposited under CGMCC No. 45591 or CCTCC M 20231558.

[0041] Those skilled in the art will recognize that the progeny phages obtained by subculturing the above-mentioned deposited phages will have the same or similar biological activity as the deposited ones. Therefore, the phages of the present invention also include the progeny of the phages deposited under CGMCC No. 45591 or CCTCC M 20231558.

[0042] In order to broaden the host spectrum to be treated and reduce the possibility of bacterial resistance to phages, the present invention also provides a combination of phages, which comprises two phages of the present invention.

[0043] In some embodiments, the combination comprises a first phage and a second phage, wherein the genome of the first phage comprises SEQ ID NO: 1, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, and the genome of the second phage comprises SEQ ID NO: 2, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 2.

[0044] In some embodiments, the combination comprises a phage deposited as CGMCC No. 45591 or a progeny thereof, and a phage deposited as CCTCC M 20231558 or a progeny thereof.

[0045] 3. Pharmaceutical Compositions and Kits

[0046] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the bacteriophage or a combination of bacteriophages of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0047] In some embodiments, the composition comprises a bacteriophage whose genome comprises SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity to SEQ ID NO: 1 or 2.

[0048] In some embodiments, the composition comprises a first phage and a second phage, wherein the genome of the first phage comprises SEQ ID NO: 1, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, and the genome of the second phage comprises SEQ ID NO: 2, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 2.

[0049] In some embodiments, the pharmaceutical composition comprises one or two selected from the bacteriophages deposited as CGMCC No. 45591 and CCTCC M20231558, or progeny thereof.

[0050] In some embodiments, the composition comprises a bacteriophage deposited as CGMCC No. 45591 or a progeny thereof, and a bacteriophage deposited as CCTCC M 20231558 or a progeny thereof.

[0051] The present invention also provides a kit comprising the bacteriophage of the present invention.

[0052] In some embodiments, the kit comprises a bacteriophage whose genome comprises SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity to SEQ ID NO: 1 or 2.

[0053] In some embodiments, the kit comprises a first phage and a second phage, wherein the genome of the first phage comprises SEQ ID NO: 1, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, and the genome of the second phage comprises SEQ ID NO: 2, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 2.

[0054] In some embodiments, the kit comprises one or both of the bacteriophage deposited as CGMCC No. 45591 and CCTCC M 20231558, or progeny thereof.

[0055] In some embodiments, the kit comprises a bacteriophage deposited as CGMCC No. 45591 or a progeny thereof, and a bacteriophage deposited as CCTCC M 20231558 or a progeny thereof.

[0056] Those skilled in the art will appreciate that the kit may be packaged as a single product or as multiple products.

[0057] 4. Treatment and prevention of Klebsiella pneumoniae infection

[0058] The present invention provides a method for treating or preventing a disease associated with Klebsiella pneumoniae infection in a subject in need thereof, comprising administering to the subject a bacteriophage of the present invention or a combination thereof (eg, using a kit of the present invention) or a pharmaceutical composition of the present invention.

[0059] The present invention also provides use of the bacteriophage of the present invention or a combination thereof, the pharmaceutical composition of the present invention or the kit of the present invention in the preparation of a medicament for treating a disease associated with Klebsiella pneumoniae infection in a subject in need thereof.

[0060] The bacteriophage of the present invention or a combination thereof, the pharmaceutical composition of the present invention or the kit of the present invention is used for treating a disease associated with Klebsiella pneumoniae infection in a subject in need thereof.

[0061] In some embodiments, the Klebsiella pneumoniae is a drug-resistant bacterium. In some embodiments, the disease is selected from pneumonia, mastitis, metritis, meningitis and sepsis caused by Klebsiella pneumoniae.

[0062] In some embodiments, the subject is administered at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or more of each of the phage particles. In some embodiments, at least 10 6 PFU, 10 7 PFU, 10 8 PFU, 10 9 PFU, 10 10 PFU, 10 11 PFU, 10 12 PFU, 10 13 PFU, 10 14 PFU or more of each of the bacteriophage. In some embodiments, at least 10 4 / kg, 10 5 / kg, 10 6 / kg, 10 7 / kg, 10 8 / kg, 10 9 / kg, 10 10 / kg, 10 11 / kg, 10 12 / kg or more of each of the phage particles. In some embodiments, at least 10 6 PFU / kg, 10 7 PFU / kg, 10 8 PFU / kg, 10 9 PFU / kg, 10 10 PFU / kg, 10 11 PFU / kg, 10 12 PFU / kg, 10 13 PFU / kg, 10 14 PFU / kg or more of each of said bacteriophages.

[0063] V. Beneficial Effects of the Invention

[0064] The present invention provides a bacteriophage that can lyse Klebsiella pneumoniae, providing an effective method for treating antibiotic-resistant bacterial infections. The host strains of the bacteriophages of the present invention complement each other, so the combination of multiple bacteriophages of the present invention (bacteriophage cocktail therapy) can solve the potential problems of narrow host spectrum of single phages and easy resistance of bacteria. In addition, the bacteriophages of the present invention, when used in combination, show an effect superior to that of a single phage, indicating that the bacteriophages of the present invention do not interfere with each other and achieve unexpected synergy.

[0065] 6. Preservation of bacteriophages

[0066] The bacteriophage KPA22S21Q3 of the present invention was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on June 16, 2023, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number is CGMCC No.45591; the bacteriophage KPK22S70Q4 was deposited in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with the address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC M 20231558. Example:

[0067] The following examples are only used to illustrate the present invention to help those skilled in the art understand the technical solutions of the present invention, and are not intended to limit the scope of the present invention in any way.

[0068] Example 1. Isolation of bacteriophages using Klebsiella pneumoniae as host

[0069] In this study, two clinically isolated Klebsiella pneumoniae strains, KPA and KPK (donated from Huashan Hospital Affiliated to Fudan University), were used as host bacteria to isolate and identify phages from samples derived from pig farm wastewater or hospital wastewater.

[0070] Specifically, the Klebsiella pneumoniae strain was cultured in LB liquid culture medium at 37°C to the logarithmic growth phase (OD600=0.5); 5 mL of 2X LB liquid culture medium, 5 mL of sewage sample filtered by a 0.45 μm PES water filter (purchased from Millipore), and 75 μL of fresh liquid culture of Klebsiella pneumoniae in the logarithmic phase were added to a 15 mL centrifuge tube, and incubated at 37°C, 180 rpm for 12-16 hours; after incubation, centrifuged at 8,000 g at room temperature for 10 minutes, and 1 ml of supernatant was collected in a 1.5 mL centrifuge tube to obtain a phage stock solution; the phage stock solution was diluted with SM buffer at 1:10 and 1:10 0, then 10 μL of the stock solution and the dilution were dropped onto the LB agar plate and separated by streaking, and after drying, 1 mL of fresh liquid culture of Klebsiella pneumoniae in the logarithmic phase was added, the plate was rotated to make the bacterial solution contact the entire surface, and then the remaining liquid was removed, and the plate was dried in a biosafety cabinet and incubated in a 37°C incubator; the plate was observed on the next day, and if there were plaques, a 200 μL pipette tip was inserted under the agar to dip two plaques of different shapes, and resuspended in 200 μL SM buffer, and then 10 μL of the resuspended liquid was drawn to repeat the streaking separation until a single plaque of the same shape and uniform size was obtained on the plate; 50 μL of the resuspended liquid of the single plaque was taken, and 5 ml of OD 600 = 0.1 of fresh bacterial solution, incubate at 37°C, 180 rpm for 4-6 hours, centrifuge at 8,000g for 5 min at room temperature, and filter the supernatant using a 0.22 μm PES water filter (purchased from Millipore) to obtain the phage stock solution.

[0071] The two strains of bacteriophages KPA22S21Q3 and KPK22S70Q4 using Klebsiella pneumoniae as hosts were obtained by the above separation method. After sequencing, the genome of bacteriophage KPA22S21Q3 has a nucleotide sequence of SEQ ID NO: 1, and the genome of bacteriophage KPK22S70Q4 has a nucleotide sequence of SEQ ID NO: 2.

[0072] Example 2. Determination of host spectrum of bacteriophage

[0073] In this example, 115 clinically isolated Klebsiella pneumoniae strains (obtained from Huashan Hospital Affiliated to Fudan University) were used to determine the host spectrum of the two phages isolated in Example 1 using the spot method.

[0074] The liquid culture of the strain was evenly spread on a solid LB plate. After it was slightly dry, 5 μL of the phage suspension was dropped in the center of different bacterial coating areas. After incubation at 37°C overnight, the appearance of plaques was observed. If a translucent area (or a large number of plaques that could not be measured) appeared in the droplet area, the strain was considered to be a sensitive host of the corresponding phage, recorded as "+++"; if a lighter bacterial lawn grew in the transparent area of ​​the droplet area, the strain was considered to be moderately sensitive to the phage, recorded as "++"; if only a few or a dozen plaques appeared, the strain was considered to be a suspected host (the possible reason was "external lysis" or too low plaque formation rate), recorded as "+"; strains rated as moderately sensitive ("++") and sensitive ("+++") were identified as hosts of phages.

[0075] As shown in Table 1 , the host spectrum of the two phage strains was synergistic and complementary, and after integration, 38 (33%) of the 115 clinical isolates could be lysed.

[0076] Table 1. Phage host spectrum of two strains of Klebsiella pneumoniae

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[0080] Example 3. Evaluation of the antibacterial efficacy of phage cocktail

[0081] In this example, based on the synergistic and complementary characteristics of the host spectra of the two isolated phage strains, the two phage strains were combined into a phage cocktail, and their ability to inhibit Klebsiella pneumoniae in vitro was further determined in vitro.

[0082] The absorbance of the logarithmic phase liquid culture of Klebsiella pneumoniae strains KPK and KPM was adjusted to OD 600 =0.1 (corresponding to a concentration of approximately 3×10 7 CFU / mL). The suspensions of the two phage strains in LB were mixed in equal volumes to obtain a total concentration of 1×10 8 PFU / mL (5×10 per phage strain) 7 PFU / mL) of phage mixture. 100 μL OD 600 = 0.1 KPK / KPM bacterial solution, and then add 100 μL of a total concentration of 1×10 8PFU / mL of phage mixture (MOI = 3) (phage cocktail group, 3 replicates); at the same time, a blank group (only 200 μL LB was added), a control group (100 μL of bacterial solution was added to 100 μL LB), and a single phage group (100 μL of 1×10 8 The microplate reader was set to 37°C and the OD was measured every 10 minutes. 600 , and the 96-well plate was shaken for 30 seconds before the measurement. The absorbance data was sorted out after the set time was stopped, and the inhibition curve was drawn with time as the horizontal axis and the absorbance value as the vertical axis.

[0083] like Figure 1 As shown, compared with the positive control group, the single phage KPK22S70Q4 can inhibit bacterial growth, but the bacteria can still proliferate. Unexpectedly, for the KPK strain, the phage cocktail of the present invention almost completely inhibited the growth of the strain within the experimental period (65 hours), showing an excellent antibacterial effect; for the KPM strain, although the bacteria in the phage cocktail group began to grow rapidly around 14 hours, the subsequent bacterial growth inhibition showed that the phage was still working, and the bacterial growth was controlled again after 20 hours and lasted until 36 hours, and throughout the experimental period (65 hours), the bacterial growth of the phage cocktail group was significantly lower than that of the control group and the single phage group. Figure 1 The inhibition curves show that the phage cocktail of the present invention has a significant inhibitory effect on the Lebsiella strains KPK and KPM. In addition, as shown in Table 1, KPA22S21Q3 cannot lyse the Lebsiella strains KPK and KPM. Figure 1 The results showed that the inhibition of the phage cocktail containing KPK22S70Q4 and KPA22S21Q3 on the Lebsiella strains KPK and KPM was significantly higher than that of using a higher concentration of KPK22S70Q4 alone, indicating that there is a synergistic effect between KPK22S70Q4 and KPA22S21Q3.

[0084] Phages may interfere with each other. If the phages that interfere with each other are combined, the antibacterial effect will be weaker than that of a single phage. The above experimental results show that the effect of the phage cocktail of the present invention is better than that of a single phage, which shows that the phages of the present invention do not interfere with each other when used in combination.

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Claims

1. A Klebsiella pneumoniae phage, the genome of which comprises SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity to SEQ ID NO: 1 or 2.

2. The bacteriophage according to claim 1, which is a bacteriophage deposited as CGMCC No. 45591 or CCTCC M 20231558 or a progeny thereof.

3. A pharmaceutical composition comprising a therapeutically effective amount of a Klebsiella pneumoniae phage, the genome of which comprises SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity to SEQ ID NO: 1 or 2.

4. The pharmaceutical composition of claim 3, comprising a first bacteriophage and a second bacteriophage, wherein the genome of the first bacteriophage comprises SEQ ID NO: 1, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, and the genome of the second bacteriophage comprises SEQ ID NO: 2, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO:

2.

5. The pharmaceutical composition of claim 3, wherein the bacteriophage is selected from one or both of the bacteriophages deposited under CGMCC No. 45591 and CCTCC M20231558, or progeny thereof.

6. The pharmaceutical composition according to any one of claims 3 to 5, comprising the bacteriophage deposited under CGMCC No. 45591 and CCTCC M20231558 or its progeny.

7. Use of the bacteriophage of claim 1 or 2 or the pharmaceutical composition of any one of claims 3 to 6 in the preparation of a medicament for treating a disease associated with Klebsiella pneumoniae infection in a subject in need thereof.

8. The use according to claim 7, wherein the Klebsiella pneumoniae is a drug-resistant bacterium.

9. The use according to claim 7 or 8, wherein the disease is selected from pneumonia caused by Klebsiella pneumoniae, mastitis, metritis, meningitis and sepsis.

10. A kit for treating a disease associated with Klebsiella pneumoniae infection in a subject in need thereof, comprising a Klebsiella pneumoniae phage, the phage's genome comprising SEQ ID NO: 1 or 2, or a nucleotide sequence having 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity to SEQ ID NO: 1 or 2.

11. The kit of claim 10, comprising a first bacteriophage and a second bacteriophage, wherein the genome of the first bacteriophage comprises SEQ ID NO: 1, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, and the genome of the second bacteriophage comprises SEQ ID NO: 2, or a nucleotide sequence 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO:

2.

12. The kit of claim 10, wherein the bacteriophage is selected from one or both of the bacteriophages deposited under CGMCC No. 45591 and CCTCC M20231558, or progeny thereof.

13. The kit of claim 12, comprising the bacteriophage deposited under CGMCC No. 45591 and CCTCC M 20231558 or its progeny.

14. The kit according to any one of claims 10 to 13, wherein the Klebsiella pneumoniae is a drug-resistant bacterium.

15. The kit according to any one of claims 10 to 14, wherein the disease is selected from pneumonia caused by Klebsiella pneumoniae, mastitis, metritis, meningitis and sepsis.

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