Novel split phage and application thereof

Abstract

The invention discloses a novel split phage and application thereof, and relates to the technical field of biology. The splitting type phage is preserved in Guangdong Microbial Culture Collection Center on April 24, 2025, the preservation address is the 5th floor of building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 66026-B1. The bacteriophage can be used for effectively splitting escherichia coli, and particularly shows remarkable splitting capacity on escherichia coli producing extended-spectrum beta-lactamase. The invention provides important technical support for developing novel antibacterial drugs and treatment schemes aiming at escherichia coli, and shows wide application prospect and potential value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a novel lytic bacteriophage and application thereof. Background Art

[0002] Escherichia coli is an important symbiotic microorganism in the human and animal intestines, and its population dynamics have a key impact on host health. When dysbiosis leads to excessive proliferation of pathogenic Escherichia coli or abnormal expression of toxin-producing strains, it may induce multiple pathological processes including gastroenteritis and hemolytic uremic syndrome. With the global spread of antibiotic resistance, the inhibitory effect of traditional antimicrobial drugs on drug-resistant Escherichia coli has been significantly weakened, and clinical treatment faces severe challenges. Such drug-resistant strains not only prolong the course of the disease and increase treatment costs, but are also more likely to form transmission chains in special environments such as medical institutions, significantly increasing the risk of cross-infection.

[0003] As a class of naturally occurring viruses, bacteriophages have the ability to accurately recognize and lyse specific bacteria. Their unique targeted bactericidal mechanism has attracted widespread attention in the biomedical field in recent years. Among them, Escherichia coli phages, as a class of viruses that specifically infect Escherichia coli, have shown significant advantages in controlling pathogenic Escherichia coli infections and have important research and application value. With the increasingly serious problem of antibiotic resistance, phage therapy, as a natural and specific antibacterial strategy, is gradually becoming a new treatment option for combating multidrug-resistant bacterial infections. Based on this, the development of new and efficient Escherichia coli phages, the optimization of their stability, the expansion of their antibacterial spectrum, and especially the enhancement of their ability to lyse multidrug-resistant Escherichia coli strains have become key scientific issues that need to be urgently addressed in the current biomedical field. Summary of the Invention

[0004] The present invention aims to provide a novel lytic bacteriophage and its application to address the aforementioned problems of the prior art. This bacteriophage is capable of lysing Escherichia coli, particularly those producing extended-spectrum β-lactamases. This provides technical support for the development of new antimicrobial drugs and antibacterial strategies for E. coli inhibition, and has promising application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a lytic phage, which is Escherichia coli phage vB_EcoS_GZMU_EI9; the Escherichia coli phage vB_EcoS_GZMU_EI9 was deposited in the Guangdong Provincial Microbiological Culture Collection on April 24, 2025, with the deposit address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number being GDMCC No: 66026-B1.

[0007] The present invention also provides use of the above-mentioned lytic bacteriophage in the preparation of a drug for preventing and / or treating Escherichia coli infection.

[0008] The present invention also provides the use of the above-mentioned lytic bacteriophage 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 drug for preventing and / or treating Escherichia coli infection, wherein the active ingredient includes the above-mentioned lytic bacteriophage.

[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 Escherichia coli.

[0014] The present invention also provides an Escherichia coli bactericide, the active ingredient of which includes the above-mentioned lytic bacteriophage.

[0015] Furthermore, the E. coli fungicide is in the form of a spray, powder, gel, granule or freeze-dried agent.

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

[0017] The present invention discovered a novel lytic Escherichia coli phage, vB_EcoS_GZMU_EI9, which exhibits typical lytic phage properties. The phage exhibits stable titers at pH values ​​between 4 and 12 and temperatures between 4 and 50°C, with the highest titer reaching 1.35 × 10 9 The phage can effectively lyse Escherichia coli, especially those producing extended-spectrum β-lactamases. This invention provides important technical support for the development of new antibacterial drugs and treatment options for E. coli, demonstrating broad application prospects and potential value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 is the morphological diagram of bacteriophage;

[0020] Figure 2 is an electron micrograph of phage particles;

[0021] Figure 3 This is a phylogenetic analysis diagram constructed based on the distance method of the whole genome sequence;

[0022] Figure 4 This is the result of thermal stability test;

[0023] Figure 5 This is the pH stability test result diagram;

[0024] Figure 6 This is the result of the optimal multiplicity of infection determination;

[0025] Figure 7 The antibacterial effect test results at different MOIs after 7h and 12h are shown;

[0026] Figure 8 The results of cleavage kinetics determination;

[0027] Figure 9 This is the one-step growth curve of phage.

[0028] Figure 10 This is the result of phage inhibition biofilm assay. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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 exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1 Isolation, identification and preservation of bacteriophage

[0035] 1. Isolation and purification of bacteriophage

[0036] Water samples were collected from Guangdong Huihang Technology Co., Ltd. in China. The water samples were first centrifuged (5000 rpm, 10 min) to remove most impurities. The liquid samples were then filtered using a 0.22 μm filter membrane for sterile filtration to remove bacteria and other macromolecules.

[0037] E. coli strain was used as host bacteria to isolate phage. The treated sample and host bacteria were inoculated into LB liquid medium and cultured in a shaking incubator at 37°C until the OD 600 =0.6 to ensure growth of the host bacteria. Mix the filtered liquid sample with the host bacteria, add liquid culture medium, and incubate overnight at 37°C in a shaker. Centrifuge the culture to remove bacterial debris, collect the supernatant (i.e., the liquid containing the phage), and filter through a 0.22 μm sterile filter membrane to remove bacteria.

[0038] The presence of target phage in the sample was detected by spot test enrichment solution. The phage sample to be tested was spotted on the host bacterial culture plate and incubated overnight at 37°C. The appearance of transparent lysis spots was observed at the spot location. These transparent spots are the lysis areas formed after the phage infected the bacteria. The phage was purified using the double-layer agar plate method until the plaque size on the plate was uniform. After multiple purifications, a lytic phage (Escherichia coli phage) vB_EcoS_GZMU_EI9 (hereinafter referred to as E19) was obtained. The phage morphology is as follows: Figure 1 shown.

[0039] 2. Phage titer detection

[0040] The phage titer is calculated by the double-layer agar plate method. Pour the bottom agar into the plate, solidify it, and wait for it to cool completely. Dilute the phage solution in a gradient manner, take the dilution and mix it with the logarithmic host bacterial solution, incubate it at 37°C for 15 minutes, mix it with 0.7% semi-solid LB agar and spread it on top of the bottom plate, and culture it at 37°C overnight. If the phage infects bacteria, the bacteria will be lysed to form transparent lysis spots (plaques). Select a 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 titer of the phage can reach up to 10 9 PFU / mL or above.

[0041] 3. Phage identification

[0042] (1) Observation using a transmission electron microscope (TEM)

[0043] In order to observe the morphology of the bacteriophage, the phosphotungstic acid negative staining method was used. First, the activated phage liquid was added dropwise to a 400-mesh carbon film copper mesh and allowed to stand for 2 minutes to allow the phage to be fully adsorbed on the copper mesh. Next, the excess liquid around the copper mesh was gently absorbed with filter paper, and 1% phosphotungstic acid solution was added dropwise to the copper mesh for negative staining. The staining was performed for 1 minute, and the excess phosphotungstic acid solution was again absorbed with filter paper. The copper mesh was allowed to dry naturally at room temperature. A Hitachi transmission electron microscope was used for observation, and the acceleration voltage was set to 80kV. The structural dimensions of the phage were measured using Image J software. Figure 2 As shown, phage E19 is a long-tailed phage with a non-retractable tail and an icosahedral head with a head length of 61±1 nm, a tail length of 110±1 nm, and a tail width of 7.8±0.3 nm.

[0044] (2) Phage genome analysis

[0045] The phylogenetic tree was constructed using the distance method with 1000 bootstrap replicates using MEGA 11.0.13 software, as shown in the figure. Figure 3As shown. According to the standards of the International Committee on Taxonomy of Viruses (ICTV), when the genome sequence similarity between two phages is less than 95%, they should be classified as different species. A comparative analysis of the similarity of phage E19 with other phages using VIRDIC showed that E19 had the highest sequence similarity with Escherichia phage vB_EcoM_SHAK7858 (Genbank ID: OR594183.1), with a genome similarity of 78.9%. Referring to the classification guidelines of BAVS, viruses with nucleotide sequence similarity exceeding 50% can be classified into the same genus. New species differ from existing species at the nucleotide level by more than 5%, that is, the similarity does not exceed 95%. Given that the genome similarity of E19 with other phages in the Kagunavirus genus falls within the range of greater than 50% and less than 95%, E19 was determined to be a new species of the genus Kagunavirus.

[0046] 4. Preservation of bacteriophages

[0047] Bacteriophage (Escherichia coli phage) vB_EcoS_GZMU_EI9 was deposited in the Guangdong Provincial Microbial Culture Collection on April 24, 2025. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 66026-B1.

[0048] Example 2 Phage host spectrum determination

[0049] The host range of phage E19 was determined by testing different 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 are shown in the table. Phage E19 had a lysis rate of 42.37% (25 / 59), including 13 extended-spectrum β-lactamase-producing Escherichia coli strains (labeled as ESBL-producing in Table 1).

[0050] Table 1 Phage host spectrum determination against 59 strains of Escherichia coli ("+" means lysis, "-" means no lysis)

[0051]

[0052] Example 3 Biological characteristics of bacteriophage E19

[0053] 1. Thermal stability

[0054] 100 μL of phage solution was added to 900 μL of sterile LB liquid medium, and different temperature conditions were set (4°C, 37°C, 50°C, 60°C, 70°C, and pre-cooled or pre-heated corresponding buffer). After the mixture was allowed to stand at the specified temperature for 1 hour, the phage titer was detected using the double-layer agar plate method. The results are as follows Figure 4 As shown, phage E19 maintained a relatively high titer in the range of 4°C-50°C, the titer decreased at 60°C, and the phage was inactivated above 70°C.

[0055] 2. pH stability

[0056] Add hydrochloric acid or sodium hydroxide solution to sterile LB liquid culture medium to adjust the pH value to 1 to 13, and filter it with a 0.22μm microporous filter membrane to sterilize it. Add 100μL of phage solution to 900μL of LB liquid culture medium with different pH values, incubate at 37℃ for 1 hour, and determine the phage titer using the double-layer agar plate method. The results are as follows Figure 5 As shown, phage E19 can maintain activity in the pH range of 2-12.

[0057] 3. Optimal multiplicity of infection

[0058] To determine the optimal infection multiplicity of the phage, dilute the host bacterial suspension in the logarithmic growth phase to 1 × 10 7 CFU / mL, and infected with phages of different titers. The phage titers at different multiplicities of infection (MOI) were detected using a double-layer agar plate method. The experimental results are shown in Figure 6 As shown, the optimal infection multiplicity of phage E19 is 0.01, and the titer reaches 1.35×10 9 PFU / mL.

[0059] 4. Cracking kinetics

[0060] The logarithmic phase host bacteria and phage solution were mixed at a ratio of 1:1, and 7 groups of different infection multiplicities (1000, 100, 10, 1, 0.1, 0.01, 0.001) were prepared and added to 96-well plates. The logarithmic phase host bacteria solution was used as the control group and placed in a shaking incubator at 37°C and 220 rpm. Samples were taken every 1 hour to measure the OD 600 The experiment was repeated 3 times. Figure 7-Figure 8 As shown in the figure, the absorbance of the pure bacterial solution in the control group increased rapidly. At the 4th hour, the host bacteria reached the logarithmic phase, which was similar to the OD of each phage group. 600 The values ​​were statistically different, and the host bacteria entered the stable period after 14 hours. The phage could inhibit the growth of E. coli for 7 hours, and the inhibition ability was strong. However, after 7 hours, E. coli developed phage resistance, and the phage group strain grew rapidly, with an OD of about 16 hours. 600The value was the same as that of the control group, and resistance appeared earlier, indicating that the Escherichia coli had a stronger ability to develop resistance to bacteriophage.

[0061] 5. One-step growth curve

[0062] The phage solution was mixed with the logarithmic phase bacterial solution at an MOI of 0.01 and co-cultured at 37°C on a shaker for 15 minutes. The mixture was then centrifuged for 10 minutes, the supernatant discarded, and the pellet resuspended in LB liquid medium and washed. The mixture was then cultured on a shaker at 37°C and 220 rpm. Samples were taken every 10 minutes to determine the phage titer. Figure 9 It can be seen that the incubation period of bacteriophage E19 is about 30 minutes, the outbreak period starts at 30-40 minutes, and the plateau period begins at about 110 minutes. The final phage concentration is maintained at 10 9 PFU / mL or so.

[0063] 6. Phage inhibition of biofilm

[0064] The crystal violet staining method was used to determine the inhibition of biofilm. Take the logarithmic phase host bacteria and phage solution in a ratio of 1:1 and mix them evenly and add them to a 96-well plate with an MOI of 100, 10, and 1. Use an equal amount of logarithmic phase bacterial solution as the positive control group (PC) and an equal amount of LB culture solution as the blank control (NC). Culture them in a 37°C incubator for 24 hours to form a biofilm, with three replicates per well. After culture, discard the culture solution in the wells and wash them once with 200μL PBS to remove floating bacteria. After washing, air dry, add 200μL 1% crystal violet solution to stain for 30 minutes, discard and wash three times, dry and add anhydrous ethanol to decolorize for 5 minutes, transfer the eluate to a new sterile 96-well plate, and use a multifunctional enzyme reader to determine the OD value at a wavelength of 595nm. The results are as follows. Figure 10 As shown in the figure, the OD values ​​of all phage groups were statistically significantly different from those of the PC group, indicating that phage E19 effectively inhibited the formation of host biofilm.

[0065] 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 lytic bacteriophage, characterized in that The lytic phage is Escherichia coli phage vB_EcoS_GZMU_EI9; the Escherichia coli phage vB_EcoS_GZMU_EI9 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 24, 2025, with the deposit address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 66026-B1.

2. Use of the lytic bacteriophage according to claim 1 in the preparation of a medicament for preventing and / or treating Escherichia coli infection.

3. Use of the lytic bacteriophage according to claim 1 in the preparation of an Escherichia coli bactericide.

4. The use according to claim 2 or 3, characterized in that The Escherichia coli is a β-lactamase-producing Escherichia coli.

5. A drug for preventing and / or treating Escherichia coli infection, characterized in that: The active ingredient comprises the lytic bacteriophage according to 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 Escherichia coli.

9. An Escherichia coli bactericide, characterized in that The active ingredient comprises the lytic bacteriophage according to claim 1.

10. The Escherichia coli bactericide according to claim 9, characterized in that The Escherichia coli bactericide is in the form of a spray, a powder, a gel, a granule or a freeze-dried agent.

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