Novel splitting type escherichia coli bacteriophage vBEcoSGZMUE2010 and application thereof
By developing the new E. coli phage vB_EcoS_GZMU_E2010, the problem of antibiotic-resistant E. coli infection was solved, efficient lysis of E. coli was achieved, and the application basis for new antibacterial drugs and bactericides was provided.
Patent Information
- Application Number
- CN202510438191.8
- 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 effectiveness of existing antibiotics to fight E. coli infection has gradually decreased, and the transmission of drug-resistant E. coli has led to cross-infection in hospitals. Finding new and effective antibacterial strategies has become a hot topic of research.
A new lytic E. coli phage vB_EcoS_GZMU_E2010 was developed, which has high titer and stability, and can lyse E. coli, especially the ultra-wide-spectrum β-lactamase E. coli, for the preparation of drugs and bactericides.
The phage is stable in the specific pH and temperature ranges and can effectively cleave E. coli, especially ultra-wide-spectrum β-lactamase strains, providing technical support for novel antibacterial drugs and regimens.
Smart Images
Figure CN120272438A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly to a novel lytic Escherichia coli phage vB_EcoS_GZMU_E2010 and its application. Background Art
[0002] Escherichia coli is one of the common resident bacteria in the intestines of humans and animals. However, when its quantity is excessive or it produces specific toxins, it may cause serious infections, leading to a series of health problems such as gastrointestinal diseases and urinary tract infections. In recent years, due to the increasingly serious problem of antibiotic resistance, the effectiveness of traditional antibacterial drugs in combating Escherichia coli infections has gradually decreased, posing a great challenge to clinical treatment. Drug-resistant Escherichia coli not only increases the difficulty of treating infections but also expands the scope of infection transmission. Especially in the hospital environment, the transmission of drug-resistant Escherichia coli may lead to nosocomial cross-infection. Therefore, finding new and effective antibacterial strategies has become a research hotspot in the fields of medicine and microbiology today.
[0003] As viruses that can specifically infect and kill bacteria in nature, phages have received extensive attention in recent years due to their high targeting and bactericidal properties. Escherichia coli phages are phages that specifically infect Escherichia coli and can effectively eliminate pathogenic Escherichia coli, having broad application potential. As a natural and specific antibacterial treatment method, phage therapy is gradually becoming a potential solution for treating drug-resistant bacterial infections. Therefore, developing new Escherichia coli phages, improving the stability of phages, and enhancing their bactericidal activity against multiple drug-resistant Escherichia coli strains have become an important topic in current biomedical research. Summary of the Invention
[0004] The object of the present invention is to provide a novel lytic Escherichia coli phage vB_EcoS_GZMU_E2010 and its application to solve the problems existing in the above-mentioned prior art. This phage can lyse Escherichia coli, especially can strongly lyse Escherichia coli producing extended-spectrum β-lactamase, providing technical support for the development of new antibacterial drugs and antibacterial programs against Escherichia coli, and having good application prospects.
[0005] To achieve the above object, the present invention provides the following solution:
[0006] The present invention provides an Escherichia coli phage vB_EcoS_GZMU_E2010, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 27, 2024. The deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64685-B1.
[0007] The present invention also provides the use of the above-mentioned Escherichia coli phage vB_EcoS_GZMU_E2010 in the preparation of a medicament for preventing and / or treating Escherichia coli infection.
[0008] The present invention also provides the use of the above-mentioned Escherichia coli phage vB_EcoS_GZMU_E2010 in the preparation of an Escherichia coli bactericide.
[0009] Furthermore, the Escherichia coli is β-lactamase-producing Escherichia coli.
[0010] The present invention also provides a medicament for preventing and / or treating Escherichia coli infection, the active ingredient of which comprises the above-mentioned Escherichia coli phage vB_EcoS_GZMU_E2010.
[0011] Furthermore, the medicament also comprises pharmaceutically acceptable excipients.
[0012] Furthermore, the dosage form of the medicament is an injection, powder, gel, granule or lyophilized product.
[0013] Furthermore, the medicament also comprises other active ingredients having antibacterial effects on Escherichia coli.
[0014] The present invention also provides an Escherichia coli bactericide, the active ingredient of which comprises the above-mentioned Escherichia coli phage vB_EcoS_GZMU_E2010.
[0015] Furthermore, the dosage form of the Escherichia coli bactericide is a spray, powder, gel, granule or lyophilized product.
[0016] The present invention discloses the following technical effects:
[0017] The present invention discovers a novel lytic Escherichia coli phage vB_EcoS_GZMU_E2010, which has typical characteristics of lytic phages. The phage has stable titer in an environment with a pH of 4-10 and a temperature of 4-50 °C, and the highest titer can reach 10 9 PFU / mL or more, and it can lyse Escherichia coli, especially strongly lyse extended-spectrum β-lactamase-producing Escherichia coli.
[0018] The present invention provides technical support for the development of novel antibacterial drugs and antibacterial regimens for inhibiting Escherichia coli, and has good application prospects. Description of the Drawings
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying 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 accompanying drawings can be obtained based on these drawings.
[0020] Figure 1 It is a morphological diagram of a phage;
[0021] Figure 2 It is an electron micrograph of phage particles;
[0022] Figure 3 It is a phylogenetic analysis diagram constructed by the distance method based on the whole genome sequence;
[0023] Figure 4 It is a diagram of the results of thermal stability measurement;
[0024] Figure 5 It is a diagram of the results of pH stability measurement;
[0025] Figure 6 It is a diagram of the results of the determination of the optimal multiplicity of infection;
[0026] Figure 7 It is a diagram of the results of lysis kinetics measurement;
[0027] Figure 8 It is a diagram of the results of the detection of the antibacterial effect at different MOIs after 9 h;
[0028] Figure 9 It is a diagram of the results of the detection of the antibacterial effect at different MOIs after 15 h;
[0029] Figure 10 It is a diagram of the results of the determination of phage inhibition of biofilm. Detailed implementation manners
[0030] Now, various exemplary implementation manners of the present invention will 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.
[0031] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used 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 can be independently included or excluded from the range.
[0032] 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 this invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0033] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of the present invention are merely exemplary.
[0034] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.
[0035] Example 1 Isolation, Identification and Preservation of Bacteriophage
[0036] 1. Isolation and Purification of Bacteriophage
[0037] Lake water was collected from Guangzhou Medical University in Guangzhou, Guangdong, China. The water sample was first centrifuged (5000 rpm, 10 min) to remove most impurities, and the liquid sample was filtered using a 0.22 μm filter membrane for aseptic filtration to remove bacteria and other macromolecular substances.
[0038] Escherichia coli strains were used as host bacteria to isolate bacteriophages. The treated sample and the host bacteria were inoculated into LB liquid medium and cultured in a shaker at 37 °C until the OD of the bacterial liquid 600 = 0.6 to ensure the growth of the host bacteria. The filtered liquid sample was mixed with the host bacteria, added to the liquid medium, and co-cultured in a constant temperature shaker at 37 °C overnight. The culture solution was centrifuged to remove bacterial residues, and the supernatant (i.e., the liquid containing bacteriophages) was collected and filtered through a 0.22 μm aseptic filter membrane to remove bacteria.
[0039] The spot test enrichment solution was used to detect whether the target bacteriophage was present in the sample. The bacteriophage sample to be detected was spotted on the host bacteria culture plate and cultured overnight at 37 °C. Whether transparent lysis plaques appeared at the spotted positions was observed. These transparent spots were the lysis regions formed after the bacteriophage infected the bacteria. The double-layer agar plate method was used to purify the bacteriophage until the plaque sizes on the plate were uniform. After multiple purifications, a lytic bacteriophage Escherichia coli phage vB_EcoS_GZMU_E2010 (hereinafter simply referred to as E2010) was obtained. The morphology of the bacteriophage is asFigure 1 as shown
[0040] 2. Detection of phage titer
[0041] The phage titer was calculated by the double-layer agar plate method. Pour the bottom agar into the plate, and after solidification, wait until it has completely cooled. Dilute the phage liquid in gradients, take the diluted liquid and mix it with the host bacterial liquid in the logarithmic phase. After incubating at 37°C for 15 min, mix 0.7% semi-solid LB agar and pour it on top of the bottom plate, and culture overnight at 37°C. If the phage infects the bacteria, the bacteria will be lysed to form clear plaques. Select the plate with 30 - 300 plaques in the field of view for counting. Titer (PFU / mL) = number of plaques × 10 × dilution factor. The results showed that the highest titer of this phage could reach 10 9 PFU / mL or more.
[0042] 3. Phage identification
[0043] (1) Observation using transmission electron microscope (TEM)
[0044] To observe the morphology of the phage, the phosphotungstic acid negative staining method was adopted. First, add a drop of the activated phage liquid to a 400-mesh carbon film copper grid, and let it stand for 2 minutes to allow the phage to fully adsorb on the copper grid. Then, gently suck the excess liquid around the copper grid with filter paper, drop 1% phosphotungstic acid solution on the copper grid for negative staining, stain for 1 minute, and again use filter paper to suck off the excess phosphotungstic acid solution. Let the copper grid air-dry at room temperature. Observe using a Hitachi transmission electron microscope, and set the acceleration voltage to 80 kV. Measure the structural dimensions of the phage using Image J software. As Figure 2 shown, phage E2010 is a long-tailed phage, the tail is non-retractable, the head is an icosahedral structure, the head length is 61 ± 1 nm, the tail length is 110 ± 1 nm, and the tail width is 7.8 ± 0.3 nm.
[0045] (2) Whole-genome analysis of the phage
[0046] Perform whole-genome sequencing of the phage using the Illumina sequencing platform. Use Megahit for genome assembly and Checkv to evaluate the assembly effect. The results showed that the genome size of phage E2010 is 44255 bp, and the GC content is 50.81%. By comparing with the VFDB database (http: / / www.mgc.ac.cn / VFs / ) and the CARD database (https: / / card.mcmaster.ca / ), it was found that the genome of phage E2010 has no virulence genes and antibiotic resistance genes, indicating the safety of this phage at the gene level.
[0047] The phylogenetic tree constructed by the distance method using MEGA 11.0.13 software with 1000 bootstrap replicates is as Figure 3 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 E2010 and other phages was compared and analyzed using BLAST. The sequence similarity between E2010 and Escherichia phage vB_VIPECOMC04 (Genbank accession number: PQ423993.1) was the highest, with 94.78% identity and 65.00% 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 E2010 to other phages in the genus Kagunavirus falls within the range of greater than 50% and less than 95%, E2010 was determined to be a new species of the genus Kagunavirus.
[0048] 4. Preservation of phage
[0049] The phage (Escherichia coli phage) vB_EcoS_GZMU_E2010 was deposited in the Guangdong Provincial Culture Collection Center of Microorganisms on December 27, 2024. The deposit address is the 5th floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64685-B1.
[0050] Example 2 Determination of phage host spectrum
[0051] The host range of phage E2010 was determined by detecting different strains through the spot test. 100 μL of logarithmic-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 incubated overnight in a 37°C incubator. If plaques appeared, it indicated that the phage infected the corresponding host bacteria. The results are shown in the table. The lysis rate of phage E2010 was 13.56% (8 / 59), including 3 strains of Escherichia coli producing extended-spectrum β-lactamase (marked as producing ESBL in Table 1).
[0052] Table 1 Determination of the host spectrum of phage against 59 strains of Escherichia coli ("√" indicates strong lysis, "×" indicates no lysis)
[0053]
[0054]
[0055] Biological Characteristics of Bacteriophage in Example 3
[0056] 1. Thermal Stability
[0057] Add 100 μL of phage liquid to 900 μL of sterile LB liquid medium, and set different temperature conditions (4 °C, 37 °C, 50 °C, 60 °C, 70 °C, and pre-cool or pre-heat the corresponding buffer). After allowing the mixture to stand at the specified temperature for 1 hour, detect the phage titer using the double-layer agar plate method. The results are as Figure 4 shown. Bacteriophage E2010 maintains a relatively high titer in the range of 4 °C - 50 °C, the titer decreases at 60 °C, and the phage is inactivated above 70 °C.
[0058] 2. pH Stability
[0059] Add hydrochloric acid or sodium hydroxide solution to sterile LB liquid medium to adjust the pH value to the range of 1 to 12, and filter and sterilize using a 0.22 μm microporous filter membrane. Add 100 μL of phage liquid to 900 μL of LB liquid medium with different pH values, incubate at 37 °C for 1 hour, and measure the phage titer using the double-layer agar plate method. The results are as Figure 5 shown. Bacteriophage E2010 can maintain a relatively high titer within the pH range of 4 - 10, but when the pH value is 3, the phage titer decreases significantly.
[0060] 3. Optimal Multiplicity of Infection
[0061] To determine the optimal multiplicity of infection of the phage, dilute the host bacterial liquid in the logarithmic growth phase to 1×10 6 CFU / mL, and infect with phages of different titers. Detect the phage titer at different multiplicities of infection (MOI) using the double-layer agar plate method. The experimental results are as Figure 6 shown. The optimal multiplicity of infection of bacteriophage E2010 is 0.001, and the titer reaches 7.57×10 9 PFU / mL.
[0062] 4. Lysis Kinetics
[0063] Mix the host bacteria in the logarithmic phase and the phage liquid in a 1:1 ratio, and prepare 7 groups with different multiplicities of infection (1000, 100, 10, 1, 0.1, 0.01, 0.001), and add them to a 96-well plate. Use the host bacterial liquid in the logarithmic phase as the control group, place it in a shaker at 37 °C and 220 rpm for culture, sample every 1 hour, and measure the OD 600 value, and repeat the experiment 3 times. The results are as Figures 7 - 9As shown, the absorbance of the pure bacterial solution in the control group increased rapidly. At the 9th hour, the host bacteria reached the logarithmic phase, and there was a statistical difference in the OD 600 value compared with each phage group. After 12 hours, the host bacteria entered the stationary phase. In the phage infection groups, the growth of Escherichia coli was effectively inhibited under various MOI conditions, and the OD 600 value was stable at about 0.45, indicating that phage E2010 had a good inhibitory effect.
[0064] 5. Effect of phage on inhibiting biofilm
[0065] The crystal violet staining method was used to determine the inhibition of biofilm. The logarithmic-phase host bacteria and phage solution were mixed evenly at a ratio of 1:1 and added to a 96-well plate. The MOI was 100, 10, and 1. An equal amount of logarithmic-phase bacterial solution was used as the positive control group (PC), and an equal amount of LB culture medium was used as the blank control (NC). The mixture was cultured in an incubator at 37 °C for 24 hours to form a biofilm, and each well was replicated three times. After incubation, the culture medium in the wells was discarded, and the wells were washed twice with 200 μL of PBS to remove planktonic bacteria. The biofilm was fixed with methanol solution for 15 minutes, then discarded and air-dried. Then, 100 μL of 1% crystal violet solution was added and stained for 30 minutes, after which it was discarded and washed twice. After drying, 5 minutes of decolorization was carried out with absolute ethanol, and the eluate was transferred to a new sterile 96-well plate. The OD value at a wavelength of 595 nm was measured using a multifunctional microplate reader. The results were as Figure 10 shown. The OD values of all phage groups and the PC group were significantly different statistically. The inhibition rate of each phage group on biofilm was greater than 50%, indicating that phage E2010 effectively inhibited the formation of the host biofilm.
[0066] The embodiments described above are only descriptions of the preferred embodiments 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 solutions of the present invention should fall within the protection scope determined by the claims of the present invention.
Claims
1. An Escherichia coli phage vB_EcoS_GZMU_E2010, characterized in that, The Escherichia coli phage vB_EcoS_GZMU_E2010 was deposited at the Guangdong Microbial Culture Collection Center on December 27, 2024. The deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64685-B1.
2. Use of the Escherichia coli phage vB_EcoS_GZMU_E2010 according to claim 1 in the preparation of a drug for preventing and / or treating Escherichia coli infection.
3. Use of the Escherichia coli phage vB_EcoS_GZMU_E2010 according to claim 1 in the preparation of an Escherichia coli bactericide.
4. The application according to claim 2 or 3, characterized in that, The Escherichia coli is β-lactamase-producing Escherichia coli.
5. A drug for preventing and / or treating Escherichia coli infection, characterized in that, The active ingredient includes the Escherichia coli phage vB_EcoS_GZMU_E2010 according to claim 1.
6. The drug according to claim 5, characterized in that, The drug further includes pharmaceutically acceptable excipients.
7. The drug according to claim 6, characterized in that, The dosage form of the drug is injection, powder, gel, granule or lyophilized product.
8. The medicament according to claim 5, characterized in that, The drug further includes other active ingredients having an antibacterial effect on Escherichia coli.
9. A bactericide for Escherichia coli, characterized in that, The active ingredient includes the Escherichia coli phage vB_EcoS_GZMU_E2010 according to claim 1.
10. The Escherichia coli bactericide according to claim 9, characterized in that, The dosage form of the Escherichia coli bactericide is spray, powder, gel, granule or lyophilized product.