Klebsiella pneumoniae bacteriophage vBKpnSGZMUVR402 and application thereof
By developing Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402, the problem of resistance to Klebsiella pneumoniae was solved, providing efficient Klebsiella pneumoniae lysing ability, achieving effective treatment of drug-resistant strains, and having good application prospects.
Patent Information
- Application Number
- CN202510438265.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art is difficult to effectively deal with the drug resistance problem of Klebsiella pneumoniae, especially the increased resistance to broad-spectrum antibiotics, which leads to the ineffectiveness of traditional antibiotic treatment, and drug-resistant strains spread rapidly in environments such as intensive care units, and lack effective alternative treatment options.
A Klebsiella pneumoniae bacteriophage vB_KpnS_GZMU_VR402 was developed, which can maintain high titer at pH 4-10 and temperature 4-50°C. It has strong cleavage ability especially for carbapenem-resistant Klebsiella pneumoniae (CRKP), and is used to prepare drugs and fungicides to prevent and treat Klebsiella pneumoniae infection.
The phage has excellent performance in stability and efficacy, with a titer of up to 1011pfu/mL. It can effectively lyse Klebsiella pneumoniae, especially carbapenem-resistant strains, and provides technical support for new antibacterial drugs and antibacterial solutions.
Smart Images
Figure CN120272439A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 and its application. Background Art
[0002] Klebsiella pneumoniae is one of the common pathogenic bacteria causing human infections. Especially in the population of immunocompromised patients, it is an important pathogen causing diseases such as pneumonia, urinary tract infections, and bloodstream infections. In recent years, with the widespread use of antibiotics, the problem of drug resistance in Klebsiella pneumoniae has become increasingly serious. In particular, the resistance to broad-spectrum antibiotics has increased, posing a huge challenge to clinical treatment. This drug resistance has gradually rendered traditional antibiotic treatments ineffective, and the spread of drug-resistant strains has also made the public health problem increasingly severe. In the 2024 World Health Organization list of priority bacterial pathogens, carbapenem-resistant and third-generation cephalosporin-resistant Klebsiella pneumoniae are listed as "critical priority" pathogens. Globally, the drug resistance of Klebsiella pneumoniae continues to expand. Especially in environments such as intensive care units, the spread rate of drug-resistant strains is rapid, further exacerbating the antibiotic resistance crisis. Therefore, how to address the drug resistance problem of Klebsiella pneumoniae and develop new alternative treatment options has become an urgent problem to be solved in the current public health field.
[0003] Phage therapy, as an alternative treatment to antibiotics, has received extensive attention in recent years. Phages are viruses that parasitize bacteria in nature and can specifically infect and lyse host bacteria. Although certain progress has been made in the research on phages against Klebsiella pneumoniae, it still faces a series of challenges such as a lack of phage resources, difficulties in screening and identification, and clinical translational applications. Therefore, developing a new type of Klebsiella pneumoniae phage with strong stability and significant efficacy is of great significance for filling this technological gap and cracking the treatment dilemma of drug-resistant bacteria. Summary of the Invention
[0004] The object of the present invention is to provide a Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 and its application to solve the problems existing in the above-mentioned prior art. This phage can lyse Klebsiella pneumoniae, especially can strongly lyse carbapenem-resistant Klebsiella pneumoniae (CRKP), thus providing technical support for the development of new antibacterial drugs and antibacterial regimens against Klebsiella pneumoniae and having good application prospects.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] The present invention provides a Klebsiella pneumoniae phage (Klebsiella phage) vB_KpnS_GZMU_VR402, wherein the Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 14, 2024, with a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC No: 65266-B1.
[0007] The present invention also provides use of the Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 in preparing a drug for preventing and / or treating Klebsiella pneumoniae infection.
[0008] The present invention also provides the use of the Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 in the preparation of a Klebsiella pneumoniae bactericide.
[0009] Furthermore, the Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae.
[0010] The present invention also provides a drug for preventing and / or treating Klebsiella pneumoniae infection, wherein the active ingredient comprises the above-mentioned Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402.
[0011] Furthermore, the medicine also includes pharmaceutically acceptable excipients.
[0012] Furthermore, the dosage form of the drug is injection, powder, gel, granule or lyophilized agent.
[0013] Furthermore, the drug also includes other active ingredients that have an antibacterial effect on Klebsiella pneumoniae.
[0014] The present invention also provides a Klebsiella pneumoniae bactericide, wherein the active ingredient comprises the Klebsiella pneumoniae bacteriophage vB_KpnS_GZMU_VR402.
[0015] Furthermore, the Klebsiella pneumoniae bactericidal agent is in the form of a spray, a powder, a gel, a granule or a lyophilized agent.
[0016] The present invention discloses the following technical effects:
[0017] The present invention discovered a novel Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402, which has typical lytic phage characteristics. The phage has a stable titer at a pH of 4-10 and a temperature of 4-50°C, with a maximum titer of 10 11 pfu / mL or above, it can lyse Klebsiella pneumoniae, especially carbapenem-resistant Klebsiella pneumoniae (CRKP).
[0018] The present invention provides technical support for the development of new antibacterial drugs and antibacterial regimens against Klebsiella pneumoniae, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0020] Figure 1 It is a morphological diagram of a phage;
[0021] Figure 2 It is a phylogenetic analysis diagram based on the distance method of the whole genome sequence;
[0022] Figure 3 It is an electron micrograph of phage particles;
[0023] Figure 4 It is a diagram of the results of thermal stability determination;
[0024] Figure 5 It is a diagram of the results of pH stability determination;
[0025] Figure 6 It is a diagram of the results of lysis kinetics determination;
[0026] Figure 7 It is a diagram of the detection results of the antibacterial effect at different MOIs after 8 h;
[0027] Figure 8 It is a diagram of the detection results of the antibacterial effect at different MOIs after 12 h;
[0028] Figure 9 It is a diagram of the results of the inhibitory effect of phage on biofilm. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0029] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.
[0030] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0031] Unless otherwise specified, all 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 pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0032] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.
[0033] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.
[0034] Example 1 Isolation and Identification of Bacteriophage
[0035] 1. Isolation and Purification of Bacteriophage
[0036] Lake water and sewage mixture were collected from Dafushan Forest Park and Central Lake Park in Panyu District, Guangzhou City, Guangdong Province, China. After centrifugation, larger impurities and most bacteria were removed using a 0.22 μm microporous filter membrane. Using Klebsiella pneumoniae as the host bacterium, the strain was inoculated into LB liquid medium and cultured in a shaker at 37 °C until the OD of the bacterial liquid 600= 0.6. Take 10 mL of the filtered solution and 1 mL of the host bacterium solution, add 5 mL of triple-concentration LB nutrient broth, and co-culture overnight in a constant temperature shaker at 37 °C. After centrifugation, take the supernatant and filter it through a 0.22 μm microporous filter membrane to obtain the first enrichment solution. Repeat the above operation to obtain the secondary enrichment solution, and detect the presence of phage plaques by spot testing the enrichment solution. Use the double-layer agar plate method to purify the phage until the phage plaques on the plate are of uniform size. After 8 times of purification, a lytic phage Klebsiella phage vB_KpnS_GZMU_VR402 (hereinafter referred to as VR402) was obtained. The phage plaques are round and transparent, and there is a semi-transparent outer halo around the edge of the phage plaque, as Figure 1 .
[0037] 2. Phage titer determination
[0038] Use the double-layer plate method to determine the phage titer. Gradient dilute the phage solution, take 100 μL of the diluted phage solution and mix it with the host bacterium in the logarithmic phase at a ratio of 1:1. After incubating at 37 °C for 15 min, mix 0.7% semi-solid LB agar evenly and pour it on top of the 1.5% solid LB agar pre-laid on the culture dish, and place it in a 37 °C incubator overnight. Several translucent phage plaques can be seen on the plate. Select the plate with 30 - 300 phage plaques in the field of view for counting. The titer (PFU / mL) = the number of phage plaques × 10 × dilution factor. The results show that the highest titer of phage VR402 can reach 10 11 pfu / mL or more.
[0039] 3. Phage whole-genome analysis
[0040] Perform single-virus genome sequencing on the phage, and use the Illumina sequencing platform for phage whole-genome sequencing. After obtaining the sequencing data, perform quality control on the sequencing data quality, that is, evaluate and remove the low-quality data to ensure the credibility of the subsequent analysis results. After removing host contamination, use Megahit to assemble the high-quality reads. Compare the virulence factors and antibiotic resistance genes in the phage genome with the VFDB database (http: / / www.mgc.ac.cn / VFs / ) and the CARD database (https: / / card.mcmaster.ca / ). The results show that the phage VR402 genome size is 46396 bp, the GC content is 48.0%, and there are no virulence genes and antibiotic resistance genes in the genome, indicating the safety of the phage at the gene level. Use PhageGE (https: / / jason-zhao.shinyapps.io / PhageGE_Update / ) to predict the phage lifestyle, and VR402 can be classified as a lytic phage.
[0041] The phylogenetic tree constructed by the distance method using MEGA 11.0.13 software with 1000 bootstrap replicates is as follows Figure 2 shown. According to the criteria of the International Committee on Taxonomy of Viruses (ICTV), when the genomic sequence similarity between two phages is less than 95%, they should be classified as different species. In the NCBI database, the similarity between phage VR402 and other phages was compared and analyzed by BLAST. VR402 had the highest sequence similarity with Klebsiella phage VLCpiS13b (Genbank accession number: NC_071155.1), with 94.16% identity and 49.00% query coverage. Referring to the classification guidelines of BAVS, those with nucleotide sequence similarity exceeding 50% in the virus population can be classified into the same genus, and new species differ from existing species by more than 5% at the nucleotide level, that is, the similarity is not more than 95%. Given that the similarity of the genome of VR402 with other phages in the genus Roufvirus all falls within the range of greater than 50% and less than 95%, VR402 was determined to be a new species of the genus Roufvirus.
[0042] 4. Preservation of phage
[0043] Phage VR402 was deposited in the Guangdong Provincial Culture Collection of Microorganisms on October 14, 2024. The deposit address is the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65266 - B1.
[0044] Example 2 Determination of phage host spectrum
[0045] The host range of phage VR402 was determined by detecting different strains through the spot test. 100 μL of log-phase host bacteria was mixed with 0.7% semi-solid LB agar and spread on a culture dish to prepare a bacteria-containing plate. 10 μL of phage liquid was dropped on the surface of the plate and cultured overnight in an incubator at 37°C. If plaques appeared, it indicated that the phage infected the corresponding host bacteria. The results are shown in Table 1. The lysis rate of phage VR402 was 15.4% (8 / 52).
[0046] Table 1 Determination of the host spectrum of phage against 52 strains of Klebsiella pneumoniae ("++" indicates strong lysis, "+" indicates lysable, "-" indicates non-lysis)
[0047]
[0048] Example 3 Biological characteristics of phage
[0049] 1. Morphological observation of phage
[0050] Observation was carried out using phosphotungstic acid negative staining method. The activated phage VR402 was attached to a 400-mesh carbon film copper grid, allowed to stand for adsorption, and after 2 min, the residual liquid around the copper grid was blotted with filter paper. 2% phosphotungstic acid was dropped on the copper grid for staining for 1 min, the staining solution was blotted with filter paper, washed twice with deionized water, and allowed to stand and dry. Observation was performed using a Hitachi electron microscope, the accelerating voltage was set at 80 kV, and the structural dimensions of the phage were measured using ImageJ. As Figure 3 shown, VR402 is a long-tailed phage with a non-retractable tail, a tail length of 238.2 ± 0.5 nm, a tail width of 10.8 ± 0.3 nm, and an icosahedral head structure with a head length of 62 ± 0.5 nm.
[0051] 2. Thermal stability
[0052] To evaluate the effect of different temperatures on the phage titer, 100 μL of phage solution was added to 900 μL of sterile LB liquid medium pretreated at different temperatures, and allowed to stand for 1 hour at temperature conditions of 4°C, 37°C, 50°C, 60°C, and 70°C respectively. The titer was determined using the double-layer agar plate method, and the experimental results are as Figure 4 shown, the phage VR402 maintained relatively stable activity in the temperature range of 4°C to 60°C, and its titer could reach 10 10 PFU / mL or above. In a high-temperature environment of 70°C, the phage VR402 lost its activity.
[0053] 3. pH stability
[0054] To evaluate the effect of different pH values on the phage titer, hydrochloric acid solution or sodium hydroxide solution was added to sterile LB liquid medium in advance to adjust the pH value to different ranges from 1 to 12, and the medium was filtered through a 0.22 μm microporous membrane to remove miscellaneous bacteria. 100 μL of phage solution was added to 900 μL of LB liquid medium with different pH values and incubated at 37°C for 1 hour. The titer was determined using the double-layer agar plate method, and the experimental results are as Figure 5 shown, the phage VR402 could maintain relatively stable titer in an environment with a pH value of 4 to 10, and the titer could reach 10 10 PFU / mL or above, indicating that it has good stability and activity within this pH range.
[0055] 4. In vitro antibacterial activity
[0056] The multiplicity of infection (MOI) was adjusted to 1000, 100, 10, 1, 0.1, 0.01, and 0.001 respectively to evaluate the efficiency of phage infection. By comparing OD 600To evaluate the inhibitory effect on bacterial growth for 12 h, and the experiment was repeated 3 times. The host bacteria in the logarithmic phase were mixed evenly with the phage solution at a ratio of 1:1 and added into a 96-well plate. An equal amount of bacteria in the logarithmic phase was used as the control group, and they were cultured in a shaker at 37 °C with an oscillation speed of 220 rpm. The results are as Figures 6 - 8 shown. All phage treatment groups showed good antibacterial activity.
[0057] 5. Inhibition of biofilm
[0058] The crystal violet staining method was used to judge the inhibition of biofilm. The host bacteria in the logarithmic phase were mixed evenly with the phage solution at a ratio of 1:1 and added into a 96-well plate. The MOIs were 100, 10, and 1. An equal amount of bacteria in the logarithmic phase was used as the positive control group (PC), and an equal amount of LB culture medium was used as the blank control (NC). They were cultured in an incubator at 37 °C for 24 h to form a biofilm, and each well was replicated three times. After incubation, the culture solution in the wells was discarded, and the wells were washed twice with 200 μL of PBS to remove planktonic bacteria. They were fixed with methanol solution for 15 minutes. After discarding and air-drying, 100 μL of 1% crystal violet solution was added for staining for 30 minutes. After discarding and washing twice, they were air-dried and then decolorized with absolute ethanol for 5 minutes. The eluate was transferred to a new sterile 96-well plate, and the OD value at a wavelength of 595 nm was measured using a multifunctional microplate reader. The results are as Figure 9 shown. The OD values of all phage groups and the PC group were significantly different statistically. The biofilm inhibition rates at MOIs of 100, 10, and 1 were 69.3%, 72.4%, and 72.6% respectively, indicating that phage VR402 effectively inhibited the formation of the host biofilm.
[0059] The embodiments described above are only used to describe the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. A Klebsiella phage vB_KpnS_GZMU_VR402, characterized in that, The Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 was deposited at the Guangdong Microbial Culture Collection Center on October 14, 2024. The deposit address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65266-B1.
2. Use of the Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 as claimed in claim 1 in the preparation of a medicament for preventing and / or treating Klebsiella pneumoniae infection.
3. Use of the Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 as claimed in claim 1 in the preparation of a Klebsiella pneumoniae bactericide.
4. The application according to claim 2 or 3, characterized in that, The Klebsiella pneumoniae is carbapenem-resistant Klebsiella pneumoniae.
5. A drug for preventing and / or treating Klebsiella pneumoniae infection, characterized in that, The active ingredient includes the Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 as claimed in claim 1.
6. The medicament according to claim 5, wherein The medicament further includes pharmaceutically acceptable excipients.
7. The drug according to claim 6, characterized in that, The dosage form of the medicament is injection, powder, gel, granule or lyophilized product.
8. The medicament according to claim 5, wherein The medicament further includes other active ingredients having an antibacterial effect on Klebsiella pneumoniae.
9. A bactericide for Klebsiella pneumoniae, characterized in that, The active ingredient includes the Klebsiella pneumoniae phage vB_KpnS_GZMU_VR402 as claimed in claim 1.
10. The Klebsiella pneumoniae bactericide according to claim 9, characterized in that, The dosage form of the Klebsiella pneumoniae bactericide is spray, powder, gel, granule or lyophilized product.