Escherichia coli bacteriophage GXEC2-19 and application thereof
The drug formulations and biological antibacterial agents prepared using Escherichia coli phage GXEC2-19 specifically lyse pathogenic Escherichia coli in poultry, solving the problem of prevention and control of pathogenic Escherichia coli infection in poultry and achieving efficient and safe environmental and feed disinfection effects.
Patent Information
- Application Number
- CN202511418176.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2025-11-18
AI Technical Summary
Existing technologies are insufficient to effectively control avian pathogenic Escherichia coli (APEC) infection, especially in intensive farming environments. Traditional antibiotic treatment strategies face the challenge of drug resistance and lack efficient control measures.
A strain of Escherichia coli phage GXEC2-19 is provided, which has strong antibacterial activity and can be used to prepare pharmaceutical preparations and biological antibacterial agents. It can be used to disinfect the breeding environment and equipment by soaking or spraying, and specifically lyse pathogenic Escherichia coli of chickens.
Escherichia coli phage GXEC2-19 can continuously and stably inhibit Escherichia coli, has high safety, is easy to formulate, and effectively kills contaminants in the environment and feed, providing a new treatment and disinfection method.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, specifically to an Escherichia coli bacteriophage GXEC2-19 and its applications. Background Technology
[0002] Avian pathogenic Escherichia coli (APEC) is a Gram-negative bacterium belonging to the genus *Escherichia coli* of the family Enterobacteriaceae. It is a major pathogen causing various diseases in poultry. This bacterium is widespread in poultry farming, causing serious economic losses to the global poultry industry, especially in intensive farming environments where APEC infection is more common. APEC is highly adaptable and pathogenic, with complex serotypes, and different strains exhibit varying virulence factors and drug resistance. APEC is transmitted primarily through vertical and horizontal transmission: vertical transmission refers to the transmission of bacteria from hens to their offspring via the yolk sac or oviduct, leading to infection in chicks during or after hatching; horizontal transmission occurs through contaminated feed, water, feces, air, and equipment within the flock. APEC infection can cause a variety of disease manifestations, including acute septicemia, air sacculitis, pericarditis, perihepatitis, salpingitis, and omphalitis. Infected chickens may exhibit symptoms such as lethargy, loss of appetite, difficulty breathing, diarrhea (white or green watery droppings), and abdominal distension, leading to high mortality rates in severe cases. Due to the increasing drug resistance of APEC (Australasian poultry), traditional antibiotic treatment strategies face challenges. Currently, prevention and control measures mainly include strengthening biosecurity, improving the farming environment, using vaccines, and exploring alternative antibiotic regimens such as probiotics, phage therapy, and immune enhancers. In addition, strict disinfection of hatching eggs and strict incubation management are also important means to reduce vertical transmission. APEC prevention and control not only affects the economic benefits of the poultry industry but also has significant implications for food safety and public health. With the development of genomics and molecular biology technologies, researchers are delving into the pathogenic mechanisms and drug resistance of APEC to develop more effective prevention and control strategies. In the future, through comprehensive prevention and control measures and the development of new vaccines, it is hoped that the threat of APEC to the poultry farming industry can be further reduced.
[0003] Bacterial phages are viruses that are widely distributed in nature and specifically kill host bacteria. They are characterized by their wide distribution, diverse species, and strong host specificity. Unlike antibiotics, which kill bacteria in a specific way, bacterial phages can rapidly proliferate, adsorb onto host bacteria, release lysins, or introduce their own lysing genes into the host bacteria's nucleic acid, causing bacterial death. Furthermore, they exhibit strict host specificity and do not affect the normal flora of humans or animals. Many studies have shown that bacterial phages can be used as a clinical agent to treat bacterial infections without harming the normal cells and flora of animals. In the current environment of constantly evolving multidrug-resistant bacteria and superbugs, bacterial phages open up new avenues for combating bacteria. At the same time, bacterial phages can be coupled with antibiotics to enhance their bactericidal effect. In recent years, the Ministry of Agriculture and Rural Affairs has banned the addition of antibiotics to animal feed. As a natural antibacterial agent, bacterial phages target and kill harmful bacteria, making them a natural bactericidal substance. Due to their highly efficient and specific bactericidal effects, bacterial phages have great potential for treating or preventing diseases. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides a strain of Escherichia coli bacteriophage GXEC2-19, which exhibits strong antibacterial activity and can be used to inhibit pathogenic Escherichia coli in birds. This provides a new treatment option for treating infections caused by pathogenic Escherichia coli in birds and offers a new disinfection method for environmental and feed contamination caused by pathogenic Escherichia coli in birds.
[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:
[0006] A strain of Escherichia phage GXEC2-19 was deposited on November 13, 2024, at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with accession number CCTCC NO: M 20242555.
[0007] The application of Escherichia coli phage GXEC2-19 as described above in the preparation of drugs for the prevention and treatment of avian pathogenic Escherichia coli phage infection.
[0008] A phage composition comprising Escherichia coli phage GXEC2-19 as described above.
[0009] A bacteriophage drug formulation, the active ingredient of which includes Escherichia coli bacteriophage GXEC2-19 as described above. The bacteriophage drug formulation further comprises a pharmaceutically acceptable carrier, and its dosage form is a solution, powder, gel, granule, or lyophilized form.
[0010] A biological antibacterial agent for disinfection in poultry farming, the active ingredient of which includes Escherichia coli phage GXEC2-19 as described above; the method of using the biological antibacterial agent is to disinfect the farming environment, feeding equipment and feed by soaking or spraying.
[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0012] The Escherichia coli phage GXEC2-19 of this invention can effectively lyse pathogenic Escherichia coli of chickens, including Escherichia coli phage DC05 and another strain of chicken-derived Escherichia coli. Furthermore, the Escherichia coli phage GXEC2-19 of this invention can provide long-lasting and sustained antibacterial activity, has high stability, good safety, and is easy to formulate into preparations. It has a good killing and control effect on environments and feeds contaminated with Escherichia coli phage.
[0013] Preservation Information
[0014] Escherichia phage GXEC2-19 was deposited at the China Center for Type Culture Collection (CCTCC) on November 13, 2024, with accession number CCTCC NO: M 20242555. Attached Figure Description
[0015] Figure 1 This is a phage plaque diagram of the Escherichia coli phage GXEC2-19 of this invention.
[0016] Figure 2 This is a transmission electron microscope image of the Escherichia coli bacteriophage GXEC2-19 of this invention.
[0017] Figure 3 This is a one-step growth curve of the Escherichia coli phage GXEC2-19 of the present invention.
[0018] Figure 4 This is the optimal infection multiplicity diagram for the Escherichia coli phage GXEC2-19 of this invention.
[0019] Figure 5 This is a temperature tolerance diagram of the Escherichia coli phage GXEC2-19 of the present invention.
[0020] Figure 6 This is a pH tolerance diagram of the Escherichia coli phage GXEC2-19 of this invention.
[0021] Figure 7 This is a diagram showing the biofilm clearance effect of different MOIs of the Escherichia coli phage GXEC2-19 of this invention.
[0022] Figure 8 This is an environmental antibacterial diagram of the Escherichia coli bacteriophage GXEC2-19 of this invention. Detailed Implementation
[0023] The following detailed description, in conjunction with the accompanying drawings, outlines specific embodiments. However, it should be understood that the scope of protection of this invention is not limited to these specific embodiments. Unless otherwise specified, all raw materials and reagents used in the examples are commercially available.
[0024] The whole genome sequencing of the Escherichia coli phage GXEC2-19 of this invention has been completed by Sangon Biotech (Shanghai) Co., Ltd. The sequence has been uploaded to the GenBank database with accession number GenBank PQ374922 and can be searched and downloaded from the website of the National Center for Biotechnology Information (NCBI).
[0025] The host bacteria used in the specific implementation method is DC05 (in Table 1) from chickens in Nanning, Guangxi. It was isolated from sewage samples from chicken farms and co-deposited with the Escherichia coli phage GXEC2-19 of this invention at the China Center for Type Culture Collection.
[0026] LB liquid medium: 10.0 g tryptone, 5.0 g yeast extracts, 10.0 g NaCl, and deionized water to a final volume of 1.0 L. Sterilize at 115°C for 20 min.
[0027] LB solid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 15 g agar powder, and deionized water to a final volume of 1.0 L. Sterilize at 115℃ for 20 min and pour into 100 mm × 100 mm Petri dishes for later use.
[0028] RNase A, DNase I, and PEG 8000 are commercially available.
[0029] Example 1
[0030] Isolation, preparation and purification of Escherichia coli bacteriophage GXEC2-19
[0031] 1. Isolation of bacteriophages from pathogenic Escherichia coli in birds
[0032] Samples were collected from wastewater in poultry farms. Peptone solution was added to the samples, and the mixture was shaken at room temperature for 2–4 h. The supernatant was collected, centrifuged at 5000 × g / min for 30 min, and filtered through a 0.22 μm filter. 1 mL of the supernatant was added to 50 mL of LB liquid medium, followed by 1 mL of host bacteria DC05. The mixture was then incubated overnight at 37 ℃ with shaking at 150 rpm (2 × LB liquid overnight culture). The next day, the overnight culture was centrifuged at 10000 × g / min at 4 ℃ for 30 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter to obtain the phage stock solution.
[0033] After streaking the host bacterium DC05 onto 1.2% LB solid medium and culturing overnight, single clones were picked and inoculated into 5 mL of LB liquid medium. The culture was then incubated at 37°C with shaking for 8 hours to obtain the host bacterium culture for later use.
[0034] Take 0.1 mL of the phage stock solution and dilute it 10 times. Take 0.1 mL of the diluted solution and mix it with 0.1 mL of the host bacteria DC05. Add about 5 mL of 0.6% LB solid medium, mix well, and quickly pour it onto the top layer of LB solid medium. Shake well and let it stand for 5 min to solidify. After anaerobic incubation at 37℃ for 12 h, observe the formation of phage plaques. Transparent plaques with a diameter of about 1 cm can be seen.
[0035] 2. Purification of Escherichia coli phage GXEC2-19
[0036] The host bacterium DC05 was streaked onto LB solid medium at -80℃ using glycerol and incubated at 37℃ for 16-20 hours. A single colony was inoculated into 5 mL of LB liquid medium and cultured at 37℃ with shaking at 200 rpm for 12-16 hours until the stationary phase. The colony was then transferred to fresh LB liquid medium at a 1:100 ratio and cultured at 37℃ with shaking at 200-250 rpm. The OD600 was monitored every 30 minutes. When the OD600 reached 0.4-0.6, the host bacterium DC05 was in the logarithmic growth phase. Take 2 mL of the host bacterial culture DC05 in the logarithmic phase, centrifuge, resuspend in 1 mL of LB liquid medium, add 0.1 mL of phage stock solution at MOI (multiple of infection) ratios of 1:1, 1:10, and 1:100 respectively; add 100 mL of LB liquid medium, and incubate at 37°C with shaking for 6-8 h. Centrifuge the resulting culture at 13000×g / min at 4°C for 30 min, collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to form phage lysis buffer.
[0037] Take 2 ml of the obtained phage lysate and add RNase A and DNase I sequentially to prepare a phage suspension with a final concentration of 1 μg / mL. Incubate at 37°C for 30 min. Add 9.3 g PEG 8000 and 5.8 g NaCl, shake until dissolved, and incubate on ice for 1 h. Centrifuge at 10000×g / min for 30 min at 4°C, and discard the supernatant. Add 5 mL SM buffer to thoroughly wash the tube wall and precipitate, and incubate at room temperature for 30 min. Add an equal volume of chloroform to extract PEG and cell debris from the phage suspension, and shake for 30 s. Centrifuge at 3000×g•min for 15 min at 4°C to recover the hydrophilic phase containing phage particles, obtaining purified phage. Store at 4°C for later use. Detect the purified phage using a double-layer plate assay, as follows: Figure 1 As shown: This phage forms clear and transparent plaques in LB solid medium, with no halo around it and clear and regular edges, which is typical of a lytic phage.
[0038] Example 2
[0039] Morphage morphology observation via transmission electron microscopy
[0040] Take the purified phage stored at 4℃ in Example 1 for later use, and observe it under an electron microscope. Add 20 μL of sample to a copper grid, wait for it to precipitate for 15 min, absorb the excess liquid with filter paper, stain with 2% phosphotungstic acid for 30 min, dry and observe under an electron microscope.
[0041] The results are as follows Figure 2 As shown, the phage has a symmetrical head with a diameter of approximately 90.32 ± 2 nm, a tail length of approximately 123 ± 2 nm, and a tail diameter of 8.53 ± 1 nm. This bacterium is named GXEC2-19.
[0042] Example 3
[0043] Detection of the lysis spectrum of Escherichia coli phage GXEC2-19
[0044] Lysis profiles were determined using 17 strains of *Escherichia coli*, 5 strains of *Salmonella enteritidis*, and 3 strains of *Typhalus murineis*. The selected strains were cultured overnight at 37°C to obtain overnight cultures of different bacteria. LB agar was divided into several regions, and overnight cultures of different bacteria were collected. 0.1 mL of the overnight culture was added to LB agar, spread evenly, and allowed to air dry. Then, 0.01 mL of purified bacteriophage (preserved at 4°C in Example 1) was added to each of the LB agar plates containing different bacteria. After air drying, the plates were inverted and incubated at 37°C for 12 hours. The lysis was then observed. The results are shown in Table 1: *Escherichia coli* bacteriophage GXEC2-19 lysed only the host DC05, indicating strong specificity of this bacteriophage.
[0045] Table 1 Host spectrum of Escherichia coli phage GXEC2-19
[0046] serial number strain name Place of origin of strain Animal source serotype Pyrolysis 1 DC05 Nanning, Guangxi Chicken source O2 + 2 HZDC01 Nanning, Guangxi Chicken source - 3 GXEC-01 Nanning, Guangxi Chicken source + 4 GXEC-02 Nanning, Guangxi Chicken source - 5 GXEC-03 Nanning, Guangxi Chicken source O157 - 6 GXEC-04 Nanning, Guangxi Chicken source - 7 GXEC-05 Nanning, Guangxi Chicken source - 8 GXEC-06 Nanning, Guangxi Chicken source O157 - 9 GXEC-07 Nanning, Guangxi Chicken source - 10 PD-1 Nanning, Guangxi Pig source - 11 PD-2 Nanning, Guangxi Pig source O114: K90 - 12 PD-3 Nanning, Guangxi Pig source - 13 PD-4 Nanning, Guangxi Pig source - 14 PD-5 Nanning, Guangxi Pig source - 15 PD-6 Nanning, Guangxi Pig source - 16 PD-7 Nanning, Guangxi Pig source O26: K60 - 17 PD-8 Nanning, Guangxi Pig source - 18 SF-BC01 Nanning, Guangxi Niu Yuan Typhoid Escherichia coli - 19 SF-BC02 Nanning, Guangxi Niu Yuan Typhoid Escherichia coli - 20 SF-BC03 Hengxian County, Guangxi Niu Yuan Typhoid Escherichia coli - 21 GXSE-A1 Nanning, Guangxi Chicken source Enteritis Escherichia coli - 22 GXSE-A2 Nanning, Guangxi Chicken source Enteritis Escherichia coli - 23 GXSE-A3 Nanning, Guangxi Chicken source Enteritis Escherichia coli - 24 GXSE-A4 Nanning, Guangxi Chicken source Enteritis Escherichia coli - 25 GXSE-A5 Nanning, Guangxi Chicken source Enteritis Escherichia coli -
[0047] Note: In Table 1, "+" indicates that the bacteriophage can lyse the strain, "_" indicates no lysis, and the serotype blank row indicates no detection.
[0048] Example 4
[0049] Determination of one-step growth curve of Escherichia coli phage GXEC2-19
[0050] The host bacterial culture prepared in Example 1 was mixed with an excess of purified bacteriophage stored at 4°C in Example 1 (MOI > 10 to ensure all bacteria and bacteriophages adsorbed). After incubating at 37°C for 15 min, the mixture was centrifuged at 12000 rpm for 1 min. The supernatant (unadsorbed bacteriophages) was discarded. The precipitate (mutually adsorbed bacteria and bacteriophage particles) was washed once with LB liquid medium. The precipitate was resuspended in 10 mL of preheated LB liquid medium and quickly placed in a shaker at 37°C. Starting from 0 min, 120 μL of culture was taken every 10 min, centrifuged at 4°C and 10000 rpm for 2 min to remove bacteria. The supernatant was diluted to an appropriate concentration (the appropriate concentration is the concentration that forms 30-300 plaques on LB solid medium). The bacterial titer was determined using a two-layer method, measured for 130 min, for a total of 18 samplings. A one-step growth curve was plotted with sampling time as the x-axis and the logarithm of the bacteriophage titer as the y-axis to obtain the phage latency, outbreak period, and outbreak size. The one-step growth curve results are shown below. Figure 3 As shown, its incubation period for infecting the host bacteria is relatively short (<20 min), while its outbreak period is relatively long (>60 min).
[0051] Example 5
[0052] Determination of the optimal multiple of infection (MOI) for Escherichia coli phage GXEC2-19 (MOI is the ratio of the number of phages to the number of host bacteria at the initial stage of infection).
[0053] Take the host bacterial culture prepared in Example 1 and adjust the concentration to 1×10⁻⁶. 9 CFU / mL was added to purified phage stored at 4℃ in Example 1 and host bacterial cultures prepared in Example 1, respectively, according to multiplicity of infection ratios of 1000, 100, 10, 1, 0.1, 0.01, and 0.001. LB broth was added to ensure a uniform total volume of the culture system. The cultures were incubated statically at 37℃ for 5 hours, centrifuged at 10000 rpm for 10 minutes, and the supernatant was collected and diluted to an appropriate concentration. The titer was determined using a two-layer chromatography method. The results are as follows: Figure 4 As shown, the optimal multiplicity of infection for Escherichia coli phage GXEC2-19 is 0.01.
[0054] Example 6
[0055] Determination of temperature tolerance of Escherichia coli phage GXEC2-19
[0056] The purified phage obtained in Example 1 and stored at 4°C for later use was diluted to approximately 10. 9 The PFU / mL was aliquoted into two sterile centrifuge tubes, 1 mL each. The centrifuge tubes were placed in constant temperature water baths at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃, respectively, and the potency was measured after 30 min and 60 min.
[0057] The results of the temperature tolerance test of Escherichia coli phage GXEC2-19 are as follows: Figure 5 As shown, the results indicate that Escherichia coli phage GXEC2-19 has good temperature tolerance and remains active at temperatures ranging from 30°C to 60°C.
[0058] Example 7:
[0059] Determination of pH tolerance of Escherichia coli phage GXEC2-19
[0060] Using LB liquid medium, the pH was adjusted to 1-12 with NaOH and HCl. 10 μL (10 μL) of purified phage with known potency obtained in Example 1, stored at 4°C for later use, was taken. 9 PFU / mL was added to 990 mL of peptone water with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), and the titer of phage in each centrifuge tube was determined after incubating at 37°C for 2 h.
[0061] The pH tolerance test results of Escherichia coli phage GXEC2-19 are as follows: Figure 6 As shown, Escherichia coli phage GXEC2-19 has good acid and alkali tolerance and maintains its activity at pH 4 to 12.
[0062] Example 8
[0063] The effect of Escherichia coli phage GXEC2-19 on the removal of pathogenic Escherichia coli biofilm in birds.
[0064] The concentration of the host bacterial culture prepared in Example 1 was adjusted to 1×10⁻⁶. 9CFU / mL, 500 μL was aspirated into a 24-well cell culture plate, with positive and negative controls set up. The negative control was LB liquid medium only, and the positive control was bacterial culture only without phage treatment. The cell plates with host bacterial culture were incubated at 37°C for 72 h. After the host bacteria were aspirated and discarded, the plates were washed three times with PBS. GXEC2-19 phage with MOIs of 1, 0.1, and 0.01 was added to the cell plates and incubated at 37°C for 12 h as the experimental groups. After incubation, the phage solution was aspirated, washed three times with PBS, fixed with 500 μL of methanol for 15 min, the methanol was aspirated, and the plates were dried. 500 μL of crystal violet staining solution was added for staining for 15 min, the crystal violet solution was aspirated, and 500 μL of methanol was added to dissolve the staining solution for 15 min. The OD590 of the staining solution was measured using a microplate reader.
[0065] like Figure 7 As shown, the OD590 of positive bacteria after 72 h of culture, measured by crystal violet staining, was 0.62, while that of negative bacteria was 0.11. The results indicate that, after staining with biofilms treated with three different MOIs, E. coli phage GXEC2-19 effectively eliminated biofilms produced by the host bacteria.
[0066] Example 9
[0067] Environmental disinfection effect of Escherichia coli phage GXEC2-19
[0068] Using a poultry farm as the experimental site, the host bacterial culture prepared in Example 1 was diluted to 1x10⁻¹. 4 CFU / mL and spray the alcohol evenly onto the ground (mL / m 2 Then, the purified phage stored at 4°C in Example 1 was adjusted to a concentration of 10. 9 PFU / mL, with the obtained concentration of 10 9 PFU / mL bacteriophages were sprayed onto the ground to kill bacteria (mL / m²). 2 Every hour, the number of ground-based host bacteria was detected using the plate count method, and the results are as follows: Figure 8 The result showed that the number of E. coli on the ground decreased to 10 after 1 hour. 3 After 2 hours, the number of E. coli on the ground decreased to 10 CFU, and after 3 hours, E. coli was almost undetectable on the ground, indicating that the E. coli phage GXEC2-19 of the present invention can effectively kill E. coli on the ground in the breeding environment.
[0069] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.
Claims
1. A strain of Escherichia phage GXEC2-19, characterized by: It is Escherichia coli phage GXEC2-19, with accession number CCTCC NO: M 20242555.
2. The use of the Escherichia coli phage GXEC2-19 as described in claim 1 in the preparation of drugs for the prevention and treatment of avian pathogenic Escherichia coli phage infection.
3. A bacteriophage composition, characterized in that: Includes the Escherichia coli bacteriophage GXEC2-19 as described in claim 1.
4. A bacteriophage drug formulation, characterized in that: Its active ingredient includes the Escherichia coli bacteriophage GXEC2-19 as described in claim 1.
5. The phage drug formulation according to claim 4, characterized in that: The phage drug formulation also includes a pharmaceutically acceptable carrier, and its dosage form is a solution, powder, gel, granule or lyophilized form.
6. A biological antibacterial agent for disinfection in poultry farming, characterized in that: The active ingredient includes the Escherichia coli bacteriophage GXEC2-19 as described in claim 1.
7. The biological antibacterial agent for disinfection in poultry farming according to claim 6, characterized in that: The method of using biological antibacterial agents is to disinfect the breeding environment, feeding equipment, and feed by soaking or spraying.