Drug-resistant klebsiella pneumoniae bacteriophage vBKpnSYPH3 and application thereof

Abstract

The invention relates to a drug-resistant klebsiella pneumoniae bacteriophage vBKpnSYPH3 and application thereof, and belongs to the technical field of microorganisms, the bacteriophage is preserved in the China Center for Type Culture Collection on May 28, 2025, and the preservation number is CCTCC No.20251208; the bacteriophage disclosed by the invention is capable of efficiently cracking a special and refractory pathogen klebsiella pneumoniae small colony mutant with drug resistance to the Erwinin, an in-vitro experiment proves that the bacteriophage can completely remove host bacteria in a short time, and the bacteriophage can be used for treating SCV infection with failure of treatment by existing antibiotics, especially Erwinin, and has a good application prospect. The invention provides a brand-new and effective treatment choice, and is expected to overcome the bottleneck problem in clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically, it relates to a drug-resistant Klebsiella pneumoniae bacteriophage vB_KpnS_YPH3 and its applications. Background Technology

[0002] Klebsiella pneumoniae is an important opportunistic pathogen that frequently causes hospital-acquired infections and community-acquired infections, such as pneumonia, bloodstream infections, urinary tract infections, and abdominal infections, posing a significant threat, especially to immunocompromised patients. Small colony variants (SCVs) are a special type of bacterial phenotypic variation characterized by slow growth and tiny colonies; they are usually associated with persistent infection, biofilm formation, and increased resistance to multiple antibiotics, leading to difficult clinical treatment and a high recurrence rate. Eracycline is a novel broad-spectrum tetracycline antibiotic commonly used to treat infections caused by multidrug-resistant Gram-negative bacteria, including Klebsiella pneumoniae. However, with its increased clinical use, the emergence and spread of drug-resistant strains (especially eracycline-resistant Klebsiella pneumoniae, ER-KP) has become a serious public health challenge. For ER-KP, especially its SCV phenotype, conventional antibiotic treatment regimens often fail. SCVs exhibit intrinsic resistance to multiple antibiotics and readily form biofilms, further hindering antibiotic penetration and leading to chronic, refractory infections. Developing new antibiotics is a long and costly process, and bacteria can quickly develop new resistance.

[0003] Phage therapy is an alternative therapy that utilizes bacteriophages to specifically lyse bacteria and treat bacterial infections. It offers advantages such as high host specificity, self-replication, the ability to penetrate biofilms, and low likelihood of causing dysbiosis. Isolating bacteriophages with highly efficient lytic capabilities against specific drug-resistant strains is an effective strategy for addressing antibiotic resistance. Currently, there are no reports on bacteriophages specifically targeting "eiracycline-resistant Klebsiella pneumoniae small colony mutants" and their applications. This invention aims to fill this gap by providing a bacteriophage capable of efficiently lysing such multidrug-resistant, highly persistent bacteria, offering a new biological agent for solving clinical challenges. Summary of the Invention

[0004] To overcome the problems existing in the background art, the present invention provides a drug-resistant Klebsiella pneumoniae phage vB_KpnS_YPH3 and its application. The phage vB_KpnS_YPH3 is deposited at the China Center for Type Culture Collection on May 28, 2025, with accession number CCTCCNo.20251208.

[0005] The present invention also provides the use of the bacteriophage vB_KpnS_YPH3 in the preparation of medicaments for the prevention and treatment of diseases caused by drug-resistant Klebsiella pneumoniae infection.

[0006] The present invention also provides a phage composition comprising phage vB_KpnS_YPH3.

[0007] The present invention also provides a phage drug formulation, the drug formulation comprising the above-described phage or the above-described phage composition.

[0008] This invention also provides the application of bacteriophage vB_KpnS_YPH3 in in vitro inhibition or elimination of elastocycline-resistant Klebsiella pneumoniae small colony mutants.

[0009] The beneficial effects of this invention are: The bacteriophage of this invention can efficiently lyse a special and difficult-to-treat pathogen, "eiracycline-resistant Klebsiella pneumoniae small colony mutant". In vitro experiments have shown that the bacteriophage can completely eliminate the host bacteria in a short time. It provides a new and effective treatment option for SCV infection that has failed the treatment of existing antibiotics, especially eiracycline, and is expected to overcome the bottleneck problem in clinical treatment.

[0010] The bacteriophages of this invention can serve as a green alternative to traditional chemical disinfectants for biodecontamination in hospital environments and high-risk areas. Attached Figure Description

[0011] Figure 1 This is the phage vB_KpnS_YPH3 morphology of the present invention; Figure 2 This is the phage vB_KpnS_YPH3 gene map of the present invention; Figure 3 This is the colony morphology on KPN11 blood agar plates in Example 2 of the present invention; Figure 4 This describes the antibacterial effect of the bacteriophage of the present invention on KPN11; Figure 5 These are the phage susceptibility test results of the present invention; Figure 6 This describes the phage lysis of kpn11 as described in this invention. Detailed Implementation

[0012] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0013] To illustrate the present invention more clearly, the following embodiments will be described in detail.

[0014] Example 1 Acquisition and biological characteristics of drug-resistant Klebsiella pneumoniae phage vB_KpnS_YPH3 (1) Collect 8000g of untreated sewage from the sewage treatment plant of the First People's Hospital of Yunnan Province, centrifuge for 10min, take the supernatant, and then filter it through a 0.22μm membrane filter to obtain the clear liquid for later use.

[0015] (2) Take 100 μl of the filtered liquid and put it into a sterile sputum cup. Add 100 μl of 0.5 McFarland units of fresh bacterial culture. Mix the two thoroughly and let it stand at room temperature for 10 min. Then add 6 mL of melted sterile 0.5% LB agar medium. Shake the mixture thoroughly and pour it onto the upper layer of a 2% LB agar plate. Let it stand to solidify and then incubate at 37°C overnight. Observe the appearance of phage plaques on the LB plate the next day.

[0016] (3) For plates with plaques, use a sterile pipette tip to pick up a single plaque and inoculate it into 5 mL of fresh host bacterial culture medium (the host is Klebsiella pneumoniae kpn0692, which was isolated from sputum samples from clinical patients). Incubate at 37°C with shaking at 160 r / min for 8 h to amplify the cultured phage. Then centrifuge at 8000 g for 10 min and collect the supernatant. Filter the supernatant through a 0.22 μm membrane filter. Dilute the filtrate 10-fold with physiological saline and perform a plaque formation test as described in step (2) above. Repeat the experiment three times to purify the phage.

[0017] (4) The purified phage solution was serially diluted 10-fold, and 100 μL of each dilution was taken to determine the phage titer using the double-layer agar plate method. Plates with 30-300 phage plaques were selected to estimate the phage titer. The titer (PFU / mL) = number of phage plaques × phage solution dilution factor × 10. The undiluted phage stock solution after titer determination was used for phage morphological observation and whole genome sequencing. Another portion of the phage stock solution was mixed with sterile glycerol (final glycerol concentration of 20%) and stored at -80℃.

[0018] Transmission electron microscopy observation of bacteriophages: Purified and titer-tested bacteriophages (i.e., the bacteriophages obtained in step (4) above) were used for morphological observation. The morphology of the bacteriophages was observed by transmission electron microscopy (TEM), and the specific steps were as follows: Prepare 2% uranium acetate staining solution for later use. Take 10 μl of the purified bacteriophage suspension and drop it onto a copper grid (supported by a carbon film). Let it stand for 5 minutes to ensure sample adhesion. Use filter paper to absorb the excess liquid. Add 10 μl of 2% uranium acetate staining solution to the sample. After staining for two minutes, gently absorb the excess staining solution with filter paper. Allow it to dry naturally at room temperature. Place the processed sample under a transmission electron microscope and observe it using an accelerating voltage of 80 kV (as shown in the attached image). Figure 1 As shown in the image, this bacteriophage belongs to the order Caudata and the family Longtail Phageidae. Its head has an icosahedral symmetry structure with a diameter of about 100 nm, and its tail is a long tail of about 150 nm.

[0019] The bacteriophage was deposited at the China Center for Type Culture Collection on May 28, 2025, with accession number CCTCC No. 20251208.

[0020] Bacteriophage whole genome sequencing: Phage genome extraction was performed using the PEG precipitation method of the Shanghai Yuanye Biotechnology Phage Genomic DNA Extraction Kit, with slight modifications based on relevant literature. The specific steps are as follows: Take 12 ml of the purified and titer-detected phage fluid (culture medium from step (4)) and transfer it to a 15 ml centrifuge tube. Centrifuge at 8000×g for 10 min to remove bacterial fragments. Collect 10 ml of supernatant, add 5 μL LNaseA and 10 μL LNaseI, mix well, and incubate at 37°C for 30 min.

[0021] Add 4 mL of phage precipitate to the supernatant, mix thoroughly and ensure complete dissolution, let stand in an ice bath for 1 hour, then centrifuge at 10000×g for 20 min at 4°C. Discard the supernatant, add 1 mL of SMbuffer to wash the tube wall and precipitate, and transfer to a new tube.

[0022] Add 40 μL of phage lysis buffer to a new tube and incubate at 68 °C for 15 min. Add an equal volume of protein removal buffer, mix gently, and centrifuge at 12,000 × g for 5 min.

[0023] Mix the resulting supernatant with the ethanol solution (final ethanol concentration 70%) and centrifuge at 8000×g for 8 min at 4°C. Discard the supernatant and repeat this washing step.

[0024] Air-dry the DNA naturally, add an appropriate amount of TE buffer, and store at -40°C.

[0025] The extracted bacteriophage whole genome was sent to Shanghai Paiseno Biotechnology Co., Ltd., and genome sequencing was performed using the Llumina Novased platform. Sequencing data were assembled using the A5-MiSeg platform, and gene-coding proteins were predicted using Gene Marks, while non-coding RNAs were predicted using the Rfam database. Functional annotation of protein-coding genes was performed using the NR, egg-NOG, KEGG, and GO databases. The genome diagram is shown below. Figure 2 As shown, the genome of this bacteriophage is a linear double-stranded DNA, with a total length of approximately 112,001 bp. The GC content is 44.72%, and it contains 157 open reading frames (ORFs), with a total ORF length of 103,980 bp.

[0026] Example 2: Identification of host bacteria and drug susceptibility testing Strain source and identification: The host bacterium used in this invention is Klebsiella pneumoniae kpn11, which was isolated from the urine of a clinical patient and identified as Klebsiella pneumoniae by Gram staining and mass spectrometry.

[0027] Validation of the small colony mutant phenotype: The isolated strain was streaked onto blood agar plates and incubated upside down at 37°C for 12 hours. Observation revealed that the strain formed small, non-pigmented colonies, consistent with the typical characteristics of a small colony mutant. After subculturing to fresh culture medium, the small colony mutant phenotype remained stable, as shown in the results. Figure 3 As shown.

[0028] Epracycline resistance susceptibility test: The microbroth dilution method was used, following the Clinical Laboratory Standards Institute (CLSI) M100 guidelines. Experimental group: Epracycline standard was serially diluted in rows AC of a 96-well plate (drug concentrations in wells 1-11 were 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg / ml, volume 100 μl). Well 12 served as a growth control without epracycline: 200 μl of 0.5 McFarland concentration Kpn11 bacterial culture. Then, 100 μl of 0.5 McFarland concentration (1*10⁻⁶) was added to each well from wells 1-11. 8 CFU / ml) kpn11 MH culture medium was prepared, with row D serving as a blank control, and only 200 μl of blank MH broth added to each well; row AD was prepared with 20 μl of antimicrobial susceptibility indicator (blue, which turns pink when infective bacteria) added to each well, and then incubated at 37℃ for 12 hours to read the minimum inhibitory concentration. Results are as follows. Figure 4As shown, the blank group is blue, while the experimental group and growth control are both pink, indicating that KPN11 is resistant to iracycline. Simultaneously, a paper disc susceptibility test was performed. A 0.5% McFarland concentration of KPN11 bacterial suspension was spread onto an LB agar plate, and iracycline discs were placed on the medium. The plates were then incubated overnight at 37°C, and the results were observed. The results are as follows: Figure 5 As shown, the diameter of the inhibition zone was 14.1 mm < 15 mm, indicating that KPN11 is resistant to iracycline.

[0029] Example 3: Lysis of Klebsiella pneumoniae phage vB_KpnS_YPH3 on kpn11 The purified phage Klebsiella pneumoniae phage vB_KpnS_YPH3 solution from step (4) of Example 1 was serially diluted 10-fold with physiological saline. 100 μl of each dilution of phage solution was placed in a sterile sputum cup, and 100 μl of 0.5 McFarland units of fresh Kpn11 bacterial solution was added (a single Kpn11 colony on blood agar was inoculated into 30 ml of liquid LB and cultured at 37°C and 160 rpm for 12 h with shaking, then adjusted to 0.5 McFarland units using a turbidimeter for later use). The two solutions were thoroughly mixed and left at room temperature for 10 min.

[0030] Then add 6 mL of melted sterile 0.5% LB agar medium. After thoroughly mixing, immediately pour the mixture onto the top layer of a 2% LB agar plate. After allowing it to solidify, incubate overnight at 37°C. Observe the appearance of phage plaques on the LB plate the next day. Select plates with 30-300 phage plaques and estimate the phage titer (PFU / mL) = number of phage plaques × phage dilution factor × 10. The results are as follows: Figure 6 As shown, at a phage dilution of 10... 5 At different concentration gradients, clear and independent translucent phage plaques were observed on the double-layer plates, indicating that the phage had a good lysis effect on kpn11.

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A drug-resistant Klebsiella pneumoniae phage vB_KpnS_YPH3, characterized in that, The bacteriophage vB_KpnS_YPH3 is deposited at the China Center for Type Culture Collection (CCTCC) on May 28, 2025, with accession number CCTCCNo.20251208.

2. The use of the bacteriophage vB_KpnS_YPH3 as described in claim 1 in the preparation of medicaments for the prevention and treatment of drug-resistant Klebsiella pneumoniae infection.

3. A bacteriophage composition, characterized in that, Includes the bacteriophage vB_KpnS_YPH3 as described in claim 1.

4. A bacteriophage drug formulation, wherein the active ingredient comprises the bacteriophage as described in claim 1 or the bacteriophage composition as described in claim 3.

5. The application of the bacteriophage vB_KpnS_YPH3 as described in claim 1 in in vitro inhibition or elimination of eracycline-resistant Klebsiella pneumoniae small colony mutants.

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