Klebsiella pneumoniae bacteriophage vBKpnPGZMUVR401 and application thereof

Abstract

The invention discloses a klebsiella pneumoniae bacteriophage vBKpnPGZMUVR401 and application thereof, and relates to the technical field of biology. The klebsiella pneumoniae bacteriophage vBKpnPGZMUVR401 is preserved in Guangdong Microbial Culture Collection Center on October 14, 2024, the preservation address is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 65265-B1. The bacteriophage is stable in titer under the environment that the pH is 4-10 and the temperature is 4-50 DEG C, the titer can be up to 1011 pfu / mL or above, and the bacteriophage can split klebsiella pneumoniae and particularly can strongly split carbapenem-resistant klebsiella pneumoniae (CRKP). The invention provides a technical support for developing a novel antibacterial drug and an antibacterial scheme for inhibiting klebsiella pneumoniae, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401 and an application thereof. Background Art

[0002] In recent years, antibiotic resistance has increased dramatically, with drug-resistant bacteria causing over 700,000 deaths annually. Klebsiella pneumoniae (KP), a widespread Gram-negative bacterium, is one of the most common Gram-negative opportunistic pathogens, commonly causing pneumonia, bloodstream infections, and other nosocomial infections. Carbapenems are atypical β-lactam antibiotics with the broadest spectrum of antimicrobial activity and the highest antimicrobial activity currently used in clinical practice. They are considered the last line of defense for treating drug-resistant Gram-negative infections. With the widespread clinical use of carbapenems, the isolation rate of carbapenem-resistant K. pneumoniae (CRKP) has climbed annually. Among the pathogens and drug combinations responsible for the most drug-resistant deaths in 2019, carbapenem-resistant K. pneumoniae and third-generation cephalosporin-resistant K. pneumoniae led the pack. To address the outbreaks and rapid spread of CRKP in hospitals, the development of new antimicrobial agents and antimicrobial regimens is urgently needed.

[0003] Phage therapy, the use of bacteriophages to treat bacterial infections, is a targeted approach against these pathogens. Unlike traditional antibiotics, phages offer advantages such as low R&D costs, ease of cultivation, high bactericidal efficiency, and ease of genetic modification. Their high specificity minimizes impact on beneficial microbiota. Given the threat of severe infections associated with CRKP, the research and application of phage therapy has become a hot topic and holds great promise. Summary of the Invention

[0004] The present invention aims to provide a Klebsiella pneumoniae phage strain vB_KpnP_GZMU_VR401 and its applications to address the aforementioned problems of the prior art. This phage is capable of lysing Klebsiella pneumoniae, particularly carbapenem-resistant Klebsiella pneumoniae (CRKP), thereby providing technical support for the development of new antibacterial drugs and antimicrobial strategies to inhibit Klebsiella pneumoniae and promising 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_KpnP_GZMU_VR401, which was deposited in the Guangdong Provincial Microbiological Culture Collection on October 14, 2024, with the deposit address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number being GDMCC No: 65265-B1.

[0007] The present invention also provides use of the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401 in preparing a drug for preventing and / or treating Klebsiella pneumoniae infection.

[0008] The present invention also provides use of the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401 in preparing 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 includes the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401.

[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 pneumonia bactericide, the active ingredient of which includes the above-mentioned Klebsiella pneumonia phage vB_KpnP_GZMU_VR401.

[0015] Furthermore, the Klebsiella pneumoniae fungicide is in the form of a spray, powder, gel, granule or lyophilized agent.

[0016] The present invention discloses the following technical effects:

[0017] The present invention discovered a novel Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401, 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 titer of up to 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 solutions for inhibiting Klebsiella pneumoniae, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 is the morphology of bacteriophage;

[0021] Figure 2 This is the maximum likelihood (ML) phylogenetic analysis diagram based on the whole genome sequence;

[0022] Figure 3 is an electron micrograph of phage particles;

[0023] Figure 4 This is the result of thermal stability test;

[0024] Figure 5 This is the pH stability test result diagram;

[0025] Figure 6 This is the result of the optimal multiplicity of infection determination;

[0026] Figure 7 Figure 1 is the result of cleavage kinetics determination;

[0027] Figure 8 The graph shows the antibacterial effect test results at different MOIs after 12 hours. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] Example 1: Acquisition of strains and isolation and identification of bacteriophages

[0034] 1. Strain acquisition

[0035] Strains were obtained from clinical samples, and species identification and drug sensitivity testing were performed using Vitek2. A total of 52 Klebsiella pneumoniae strains were obtained and used as host bacteria to isolate phages.

[0036] 2. Isolation and purification of bacteriophage

[0037] The lake water and sewage mixture was collected from Guangzhou Medical University and the Second Affiliated Hospital of Guangzhou Medical University in Guangdong, China. The mixture was centrifuged at 5000 rpm for 10 minutes and filtered through a 0.22 μm microporous membrane to remove large impurities and most bacteria. The host bacterial strain was inoculated into LB liquid medium and cultured in a shaking incubator at 37°C until the bacterial solution OD 600 =0.6. Take 10mL of filtrate and 1mL of host bacterial solution, add 5mL of triple-concentration LB nutrient broth, and co-cultivate overnight in a constant temperature shaker at 37°C. After centrifugation, take the supernatant and filter it through a 0.22μm microporous filter membrane to obtain the first enrichment solution. Repeat the above operation to obtain the second enrichment solution. The enrichment solution is tested for the presence of plaques by spot test. The phage is purified using the double-layer agar plate method until the plaque size on the plate is uniform. After 8 purifications, a lytic phage Klebsiella phage vB_KpnP_GZMU_VR401 (hereinafter referred to as VR401) is obtained. The plaques are round and transparent, with consistent morphology, size and clarity, as shown in the figure. Figure 1 shown.

[0038] 3. Phage titer determination

[0039] The double-layer plate method was used to determine the phage titer. The phage solution was diluted in a gradient manner, and 100 μL of the diluted phage solution was mixed with the logarithmic phase host bacteria at a ratio of 1:1. After incubation at 37°C for 15 minutes, the 0.7% semi-solid LB agar was evenly spread on top of the 1.5% solid LB agar pre-laid on the culture dish, and placed in a 37°C incubator for overnight culture. Several translucent plaques were visible on the plate, and plates with 30-300 plaques in the field of view were selected for counting. The titer (PFU / mL) = number of plaques × 10 × dilution factor. The results showed that phage VR401 had typical lytic phage characteristics, and the titer of this phage could reach up to 10 11 pfu / mL or above.

[0040] 4. Phage Whole Genome Analysis

[0041] Single viral genome sequencing was performed on the phage, and the whole phage genome was sequenced using the Illumina sequencing platform to obtain raw offline data. Reads with low sequencing quality values ​​were filtered, and high-quality reads were retained to obtain Clean Data. Genome assembly was performed using Megahit, and the assembly effect was evaluated using Checkv. The virulence factors and antibiotic resistance genes in the phage genome were compared with the VFDB database (http: / / www.mgc.ac.cn / VFs / ) and the CARD database (https: / / card.mcmaster.ca / ). The results showed that the genome size of phage VR401 was 42944bp (as shown in Table 1), with a GC content of 54.1%. The genome had no virulence genes or antibiotic resistance genes, indicating the safety of the phage at the genetic level.

[0042] Table 1 Sequence of bacteriophage VR401 genome

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[0055] According to the standards of the International Committee on Taxonomy of Viruses (ICTV), when the genomic sequence similarity of two phages is less than 95%, they should be classified as different species. In the NCBI database, BLAST was used to compare the similarity of phage VR401 with other phages. The results showed that the sequence similarity with Klebsiella phage phiKpS2 (Genbank number: NC_047857.1) under the genus Drulisvirus was the highest, showing 94.17% identity and 84% query coverage. Referring to the classification guidelines of BAVS, viruses with a nucleotide sequence similarity of more than 50% can be classified as the same genus. Therefore, VR401 can be determined as a new species of the genus Drulisvirus. Using MEGA 11.0.13 software, the phylogenetic tree constructed by the maximum likelihood method with 1000 bootstrap repetitions is as follows. Figure 2 .

[0056] 5. Preservation of bacteriophages

[0057] Bacteriophage VR401 was deposited in the Guangdong Provincial Microbial Culture Collection on October 14, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65265-B1.

[0058] Example 2 Phage host spectrum determination

[0059] The host range of bacteriophage VR401 was determined by testing different bacterial strains using a spot assay. 100 μL of logarithmic-phase host bacteria was mixed with 0.7% semisolid LB agar and plated onto a Petri dish to create a strip plate. 10 μL of phage solution was then dropped onto the plate and incubated overnight at 37°C. The appearance of plaques indicated that the phage infected the corresponding host bacteria. The results, shown in Table 2, showed that phage VR401 had a lysis rate of 13.5% (7 / 52) and was able to strongly lyse one carbapenem-resistant Klebsiella pneumoniae (CRKP) strain.

[0060] Table 2 Phage host spectrum determination of 52 strains of Klebsiella pneumoniae ("++" means strong lysis, "+" means lysis, "-" means no lysis)

[0061]

[0062]

[0063] Example 3 Biological characteristics of bacteriophage

[0064] 1. Morphological observation of bacteriophage

[0065] The negative staining method of phosphotungstic acid was used for observation. The activated phage VR401 was attached to a 400-mesh carbon film copper grid and allowed to stand for adsorption. After 2 minutes, the residual liquid around the copper grid was absorbed with filter paper. 2% phosphotungstic acid was dropped on the copper grid for staining for 1 minute. The stain was absorbed with filter paper, and the grid was washed twice with deionized water and allowed to stand for drying. A Hitachi electron microscope was used for observation with an accelerating voltage of 80 kV. ImageJ was used to measure the structural size of the phage. Figure 3 As shown, VR401 belongs to the family of short-tailed bacteriophages, with an icosahedral head structure of 60±1.5 nm in length and a non-retractable tail of 16±0.4 nm in length and 12±3 nm in width.

[0066] 2. Thermal stability

[0067] 100 μL of phage solution was added to 900 μL of sterile LB liquid culture medium at different temperatures (different groups of buffer were pre-cooled or pre-heated at 4°C, 37°C, 50°C, 60°C, 70°C, and 80°C), the mixture was allowed to stand at the corresponding temperature for 1 hour, and the phage titer at different temperatures was detected using the double-layer agar plate method. The results are as follows Figure 4 As shown, the titer of phage VR401 is stable at 4-50°C, the titer begins to decrease at 60°C, and the phage is inactive above 70°C.

[0068] 3. pH stability

[0069] Add hydrochloric acid solution or sodium hydroxide solution to sterile LB liquid culture medium in advance to adjust the pH to 1 to 12, and filter sterilize with a 0.22 μm microporous filter membrane. Add 100 μL of phage solution to 900 μL of LB liquid culture medium with different pH values, incubate at 37°C for 1 hour, and use the double-layer agar plate method to detect the phage titer at different pH values. Figure 5 As shown, phage VR401 can maintain stable titer in an environment with a pH of 4-10.

[0070] 4. Optimal Multiplicity of Infection

[0071] To determine the optimal infection multiplicity of the phage, dilute the host bacterial suspension in the logarithmic phase to 1 × 10 8 CFU / mL, the host bacteria were infected with phages of different titers, and the titers of phages after infection with different MOI were detected using the double-layer agar plate method. Figure 6As shown, the optimal multiplicity of infection of bacteriophage VR401 is 0.01.

[0072] 5. Cracking kinetics

[0073] Take the logarithmic phase host bacteria and phage solution 1:1 and mix them evenly. Prepare 5 groups of different infection multiplicities (1, 0.1, 0.01, 0.001, 0.0001) and add them to 96-well plates. Use the same amount of logarithmic phase bacteria solution as the control group. Place them in a shaker at 37°C and 220 rpm for shaking culture. Take samples every 1 hour and measure the OD by spectrophotometry. 600 , lasting 12 hours, and the experiment was repeated 3 times. Figure 7 、 Figure 8 As shown in the control group, the absorbance of the pure bacterial solution increased rapidly, and the OD 600 The value reached 0.98 and entered the plateau phase. Compared with the control group, the phages under each MOI condition showed effective inhibitory effects on the growth of Klebsiella pneumoniae. At 12h, OD 600 The value was maintained at around 0.57, indicating that phage VR401 had good inhibition efficiency.

[0074] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401, characterized in that: The Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 14, 2024. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC No: 65265-B1.

2. Use of the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401 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_KpnP_GZMU_VR401 as claimed in claim 1 in the preparation of a Klebsiella pneumoniae bactericide.

4. The use 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 comprises the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401 described in claim 1.

6. The drug according to claim 5, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that The dosage form of the medicine is injection, powder, gel, granule or lyophilized agent.

8. The drug according to claim 5, characterized in that The drug also includes other active ingredients that have an antibacterial effect on Klebsiella pneumoniae.

9. A Klebsiella pneumoniae bactericidal agent, characterized in that: The active ingredient comprises the Klebsiella pneumoniae phage vB_KpnP_GZMU_VR401 described in claim 1.

10. The Klebsiella pneumoniae bactericidal agent according to claim 9, characterized in that: The Klebsiella pneumoniae bactericidal agent is in the form of a spray, a powder, a gel, a granule or a lyophilized agent.

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