Traditional Chinese medicine compound preparation containing escherichia coli bacteriophage dcp514 and application thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses a traditional Chinese medicine compound preparation containing an Escherichia coli bacteriophage DCP514 and application. The Escherichia coli bacteriophage DCP514 has a preservation number of CCTCC NO: M 2024321, can efficiently kill pathogenic variants of Escherichia coli in a poultry breeding environment, has the characteristics of strong lyticity, good temperature tolerance, environmental friendliness, and the like, and has the advantages of safety, no residue, and no side effect. The bacteriophage can be used alone or in combination with a traditional Chinese medicine extract to enhance the bacteriostatic effect, and has a good application prospect in the biological control of Escherichia coli.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a traditional Chinese medicine compound preparation containing Escherichia coli bacteriophage DCP514 and its application. Background Technology

[0002] Pathogenic Escherichia coli (EEC) is a type of extraintestinal pathogenic Escherichia coli (ExPEC) that can cause colibacillosis in poultry. The onset of colibacillosis is not seasonal, and chickens at all stages can be infected, especially chicks. E. coli is excreted in feces, and fecal contamination of eggshells can lead to increased embryo mortality and a higher proportion of weak chicks. E. coli can also infect through drinking water, feed, air, cages, and other vectors via the digestive tract, respiratory tract, and umbilical cord, causing inflammation of the cloaca and oviduct. Depending on the specific type, it can cause various symptoms, including E. coli septicemia, air sacculitis, granulomas, perihepatitis, peritonitis, oviductitis, synovitis, and omphalitis. In recent years, with the vigorous development and promotion of large-scale intensive farming, the economic impact of pathogenic E. coli on livestock and poultry farming has become increasingly apparent, even threatening the healthy and sustainable development of animal husbandry.

[0003] Bacteriophages are viruses that can specifically infect bacteria and are widely found in various environments, such as feces, soil, water sources, and even in humans and animals. Bacteriophages specifically target bacteria as hosts, causing bacterial death. They exhibit strict host specificity, invading only one or a few types of bacteria. Therefore, bacteriophages are not infectious to humans, plants, or animals, nor do they contaminate the environment, making them relatively safe.

[0004] Many Chinese medicinal herbs have antibacterial and antiviral effects, with broad-spectrum antibacterial activity. They inhibit both Gram-positive (G+) and Gram-negative (G-) bacteria and are often used in traditional Chinese veterinary medicine for clearing heat and detoxifying.

[0005] Based on the antibacterial effects of bacteriophages and traditional Chinese medicine in the above-mentioned existing technologies, the applicant screened an Escherichia coli bacteriophage DCP514 and found that the bacteriophage has a synergistic effect when used in combination with traditional Chinese medicine preparations. Summary of the Invention

[0006] The purpose of this invention is to provide a traditional Chinese medicine compound preparation containing Escherichia coli phage DCP514, wherein the compound preparation includes Escherichia coli phage DCP514.

[0007] Another object of the present invention is to provide the use of the above-mentioned compound preparation in a medicament for the prevention or treatment of diseases caused by Escherichia coli.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] The applicant isolated a strain of Escherichia coli phage DCP514 from a water sample from a chicken farm in Hubei Province. The phage was deposited at the China Center for Type Culture Collection (CCTCC) on February 26, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2024321 and classification name: Escherichia coli phage DCP514.

[0010] A traditional Chinese medicine compound preparation containing Escherichia coli phage DCP514 comprises Escherichia coli phage DCP514 stock solution and traditional Chinese medicine extract; the ratio of Escherichia coli phage DCP514 stock solution to traditional Chinese medicine extract is 1-3:1-3, by volume; the potency of the Escherichia coli phage DCP514 stock solution is 1×10⁻⁶. 7 The concentration of the herbal extract is 1 mg / mL, and the preservation number is CCTCC NO: M 2024321. The extract is a water extract of 1-3 g of Coptis chinensis, 1-3 g of Lonicera japonica, 1-3 g of Isatis indigotica and 1-3 g of Glycyrrhiza uralensis.

[0011] Preferably, the herbal extract is an aqueous extract of the mixture of the above components in a ratio of 1:1.

[0012] In the above-mentioned important compound preparation, preferably, the volume ratio of the Escherichia coli phage DCP514 stock solution to the traditional Chinese medicine extract is 3:1.

[0013] The scope of protection of this invention also includes:

[0014] The application of the above-mentioned traditional Chinese medicine compound preparations in drugs for the prevention or treatment of diseases caused by Escherichia coli;

[0015] The application of the above-mentioned traditional Chinese medicine compound preparation in the preparation of Escherichia coli antibacterial agents.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] The bacteriophage in this invention has high fermentation efficiency, is a virulent bacteriophage, and is a natural bactericidal substance. It has a short incubation period in the host, a large burst volume, good lysis effect, and good antibacterial effect. The bacteriophage preparation of this invention has high stability, better resistance to acids, alkalis, and high temperatures, and can ensure its survival time in different environments, providing a wider range of application environments for bacteriophage preparations. The composite preparation formed by mixing this bacteriophage with traditional Chinese medicine extracts can enhance the bactericidal ability of the bacteriophage and avoid the development of resistance in Escherichia coli. Attached Figure Description

[0018] Figure 1 Morphage morphology of Escherichia coli phage DCP514 plaque. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific implementation examples, but the scope of protection of the present invention is not limited to the scope described in the embodiments. Unless otherwise specified, the technical solutions described in the present invention are conventional technologies; the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0020] Example 1:

[0021] Isolation and purification of E. coli bacteriophages:

[0022] Six water samples (20 mL each) were collected from near a chicken farm in Hubei Province. After centrifugation at 5000 rpm for 10 minutes, the supernatant was filtered and sterilized. The filtrate was then mixed with an equal volume of 2×TSB liquid culture medium and 1 mL of E. coli culture in the logarithmic growth phase (10...). 8 The sample was thoroughly mixed with cfu / mL and incubated overnight at 37°C and 180 rpm to enrich the phages. The enriched sample was centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm microporous membrane to obtain a filtrate containing phages. 100 μL of the filtrate was thoroughly mixed with 300 μL of the host *E. coli* culture and allowed to stand for 15 min to allow for complete binding to the receptors on the bacterial surface. The mixture was then added to 4 mL of TSB semi-solid agar medium cooled to 50°C, mixed well, and immediately spread onto a solidified TSA plate. After the agar solidified, the plate was inverted and incubated at 37°C for 8-12 h, and the growth of phage plaques was observed. On a double-layer agar plate where plaques have formed, large, clear plaques are picked up with a sterile pipette tip, desorbed by shaking in 1 mL of SM solution, and then sterilized through a 0.22 μm microporous membrane to obtain phage filtrate. This filtrate is inoculated into 5 mL of TSB liquid medium, and 100 μL of the corresponding host *E. coli* culture is added and mixed well. The plate is incubated overnight at 37°C and 180 rpm. After centrifugation at 5000 rpm for 10 min, the supernatant is collected and filtered through a bacterial filter membrane. The morphology of the plaques is observed using the double-layer agar plate method. After repeating the operation 3-5 times, plaques with consistent shape and size can be obtained.

[0023] Finally, a bacteriophage strain was obtained and named bacteriophage DCP514. This bacteriophage was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on February 26, 2024. The accession number is CCTCC NO: M2024321, and the classification name is Escherichia coli phage DCP514.

[0024] This screening experiment yielded a batch of virulent Escherichia coli phages. Phage DCP543 was used as a control Escherichia coli phage, and its fermentation titer, pH tolerance, and temperature tolerance were similar to those of Escherichia coli phage DCP514.

[0025] Example 2:

[0026] Determination of Escherichia coli phage titers at different multiplicity of infection and different infection times

[0027] A single colony of host *E. coli* was picked and inoculated into a test tube containing 3 mL of TSB culture medium. The culture was incubated overnight at 37°C with shaking at 180 rpm to obtain a host bacterial suspension. The bacterial suspension was transferred to 10 mL of TSB culture medium at a ratio of 1:100 and incubated at 37°C with shaking at 180 rpm until the early logarithmic growth phase. Bacteriophage DCP514 and its host *E. coli* were diluted to specific concentrations and counted. The samples were then cultured together at different multiples of infection (MOI = number of phages / number of bacteria), and TSB liquid medium was added to ensure the total volume of each tube was the same. The samples were incubated at 37°C with shaking at 180 rpm for 6 hours, and the phage titer was determined. Double replicates were performed at each point, and the average value was taken. The MOI that produced the highest phage titer was considered the optimal MOI. The experiment was repeated three times.

[0028] The results are shown in Table 1. At MOI = 0.03, phage DCP514 reached its highest titer (4.6 × 10⁻⁶) after 6 hours of culture. 10 PFU / mL).

[0029] Table 1. Titer of Escherichia coli bacteriophages at different infection multiplici.

[0030]

[0031]

[0032] Example 3:

[0033] pH stability test of Escherichia coli bacteriophage

[0034] Add 9 mL of TSB culture medium at different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) to each sterile bacterial bottle. Place the bacterial bottles in a 25°C constant temperature water bath. After temperature equilibration, add 1 mL of 1×10⁻⁶ TSB medium. 9 Pure phage culture medium of pfu / mL was incubated at 25℃ for 4 hours. Samples were taken at the 4th hour, appropriately diluted, and phage titers were determined using the double-layer plate method. Double-sample culture was performed at each point, and the average value was taken. The experiment was repeated three times.

[0035] The results are shown in Table 2. The titer of Escherichia coli phage DCP514 did not change significantly after 4 hours of treatment in the pH range of 3-11, and it still had a relatively high titer after 4 hours.

[0036] Table 2 pH stability of Escherichia coli phage DCP514

[0037]

[0038] Example 4:

[0039] Temperature stability test of Escherichia coli bacteriophage

[0040] Take several sterile 50mL centrifuge tubes and add 45mL of 1.5×10 to each tube. 10 After PFU / mL TSB was applied, the sample tubes were placed in a constant temperature water bath at the appropriate temperature. Once the temperature equilibrated, 5 mL of pure phage culture medium was added, and the tubes were incubated at 4℃, 25℃, 37℃, 50℃, 60℃, and 70℃ for 24 h. After the incubation period, the sample tubes were removed and immediately placed in an ice bath to cool. After appropriate dilution, the phage titer was determined using the double-layer plate method. Double-sample culture was performed at each point, and the average value was taken. The experiment was repeated three times.

[0041] The results are shown in Table 3. In the experimental group, Escherichia coli phage DCP514 survived well at temperatures below 50℃, still had a high titer after being bathed in a water bath at 60℃ for 1 hour, and had good stability at 4-37℃, allowing for long-term storage.

[0042] Table 3 Temperature stability of DCP514

[0043]

[0044] Example 5:

[0045] bacteriophage fermentation preparation

[0046] A single colony of host *E. coli* was picked and inoculated into a test tube containing 3 mL of TSB culture medium. The culture was incubated at 37°C and 180 rpm for 12 h to obtain a host bacterial suspension. The bacterial suspension was transferred to 500 mL of TSB culture medium at a ratio of 1:100 and incubated at 37°C and 180 rpm until the early logarithmic growth phase, at which point the bacterial suspension concentration was determined. The fermentation preparation system was 6 L, and the fermentation medium was TSB medium. Inoculation was performed using the flame inoculation method, with bacteriophages and host *Salmonella* added to the fermentation medium at the optimal multiplicity of infection (MOI). Sterile air was introduced during fermentation, and 3‰ antifoaming agent was added. The fermentation time was 12 h. Every 2 h from the start of fermentation, 20 mL of fermentation broth was collected from the sampling port into a sterile container, centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm microporous membrane to obtain a filtrate containing bacteriophages. The titer of the filtrate was determined, following the method described in Example 2. After fermentation is complete, the entire mixture of phage and host bacteria is taken out from the sampling port and transferred to a sterile container. It is centrifuged at 6000 rpm for 15 min, and the supernatant is filtered through a vacuum pump into a sterile filter device to obtain the phage fermentation broth, which is then stored at 4°C.

[0047] As shown in Table 4, the titer of bacteriophage DCP514 increased significantly after 6 hours of fermentation, reaching 5.5 × 10⁻⁶. 10 PFU / mL. Therefore, this phage can be prepared on a large-scale industrial basis using fermentation, which is short in fermentation time, highly efficient, and cost-effective. The fermentation stock solution can be diluted to the required concentration with phage SM buffer or water for use in the following experiments.

[0048] Table 4 Fermentation dynamics of Escherichia coli phage DCP514

[0049]

[0050] Example 6:

[0051] Assay of the lysis range of Escherichia coli by DCP514 bacteriophage

[0052] The above-mentioned Escherichia coli phage DCP514 fermentation broth was diluted with sterile water to a titer of approximately 1×10⁻⁶. 7 The pfu / mL dilution buffer was used for subsequent experiments, and the phage lysis profile was determined using the droplet method. Fifty single clones of drug-resistant *E. coli* from 15 different serotypes were picked and inoculated into centrifuge tubes containing 3 mL TSB. The tubes were incubated at 37°C and 180 rpm for 8 hours to prepare bacterial suspensions for each strain. 300 μL of each bacterial suspension was mixed with TSB semi-solid medium and plated onto prepared TSA plates. 5 μL of phage DCP514 culture medium was then added dropwise to each plate. After air drying, the plates were incubated at 37°C for 8–12 hours, and the results were observed. The experiment was repeated three times.

[0053] The results are shown in Table 5. Escherichia coli phage DCP514 was able to lyse 49 out of 50 drug-resistant Escherichia coli strains, including all 15 serotypes, with a lysis rate of 98%. This indicates that Escherichia coli phage DCP514 has a broad host spectrum and can effectively lyse various serotypes of Escherichia coli in poultry farming.

[0054] Table 5 Results of DCP514 lysis spectrum determination of Escherichia coli bacteriophage.

[0055]

[0056]

[0057]

[0058] "-" indicates no lysis; "+" indicates slight lysis with blurred lysis spots; "++" indicates lysis with relatively clear lysis spots; "+++" indicates lysis with very clear lysis spots.

[0059] Example 7:

[0060] Verification of the bactericidal effect of different ratios of Chinese herbal extracts on Escherichia coli

[0061] Different proportions of Coptis chinensis, Lonicera japonica, Isatis indigotica, and Glycyrrhiza uralensis were divided into 4 groups, as follows:

[0062] Group 1 includes 2g of Coptis chinensis, 2g of Lonicera japonica, 2g of Isatis indigotica, and 2g of Glycyrrhiza uralensis;

[0063] Group 2 includes 3g of Coptis chinensis, 3g of Lonicera japonica, 1g of Isatis indigotica, and 1g of Glycyrrhiza uralensis.

[0064] Group 2 includes 1g of Coptis chinensis, 1g of Lonicera japonica, 3g of Isatis indigotica, and 3g of Glycyrrhiza uralensis.

[0065] Group 2 includes 3g of Coptis chinensis, 1g of Lonicera japonica, 3g of Isatis indigotica, and 1g of Glycyrrhiza uralensis.

[0066] Soak the solids in distilled water at a ratio of 1:10 for 2 hours, mix, heat to a boil over high heat, then simmer over low heat for 1 hour. Filter through gauze and filter paper, collect the filtrate, concentrate to a 1 mg / mL extract, and store at 4°C. Take four 30 mL aliquots of TSB liquid culture base and place them in 50 mL centrifuge tubes, adding 1 × 10⁻⁶ TSB solution to each. 8 0.1 mL of CFU / mL Escherichia coli CVCC3026 suspension was added to each group, along with 2 mL of traditional Chinese medicine extract. The mixtures were incubated at 37℃ and 200 rpm, and their OD values ​​were measured. 600 The values ​​were measured over a total of 24 hours.

[0067] As shown in Table 5, the herbal extract prepared by combining Coptis chinensis, Lonicera japonica, Isatis indigotica, and Glycyrrhiza uralensis in a ratio of 1:1:1:1 has the best antibacterial effect.

[0068] Table 6. In vitro antibacterial test of different proportions of traditional Chinese medicine extracts against Escherichia coli.

[0069]

[0070]

[0071] Example 8

[0072] In vitro synergistic antibacterial test of Escherichia coli phage DCP514 and traditional Chinese medicine extracts against Escherichia coli

[0073] Take 5g each of Coptis chinensis, Lonicera japonica, Isatis indigotica, and Glycyrrhiza uralensis, and soak them thoroughly in distilled water at a ratio of 1:10 for 2 hours. Mix the ingredients, heat to a boil over high heat, then simmer over low heat for 1 hour. Filter the mixture through gauze and filter paper, collect the filtrate, concentrate it to a concentration of 1mg / mL, and store it at 4℃. Take eight 30mL TSB liquid culture media and place them in 50mL centrifuge tubes. Add 1×10⁻⁶ TSB liquid culture media to each tube. 8 0.1 mL of CFU / mL E. coli CVCC3026 suspension was used, and the groups were then set as follows:

[0074] Group 1 was treated with 2 mL of TSB liquid culture medium as a blank control group;

[0075] Group 2 was supplemented with 2 mL of the above-mentioned herbal extract;

[0076] Group 3 was added with a potency of 1×10 7 2 mL of E. coli phage DCP514 proliferation solution with pfu / mL;

[0077] Group 4 was added with a potency of 1×10 7 1.5 mL of E. coli phage DCP514 proliferation solution with pfu / mL and 0.5 mL of traditional Chinese medicine extract;

[0078] Group 5 contains a potency of 1×10 7 1 mL of pfu / mL Escherichia coli phage DCP514 proliferation solution and 1 mL of traditional Chinese medicine extract;

[0079] Group 6 contains a potency of 1×10 7 0.5 mL of E. coli phage DCP514 proliferation solution (pfu / mL) and 1.5 mL of traditional Chinese medicine extract.

[0080] Group 7 added with a potency of 1×10 7 2 mL of E. coli phage DCP543 proliferation medium with pfu / mL;

[0081] Group 8 added with a potency of 1×10 7 One mL of pfu / mL E. coli phage DCP543 proliferation medium and one mL of traditional Chinese medicine extract were incubated at 37℃ and 200 rpm, and their OD values ​​were measured. 600 The values ​​were measured over a total of 24 hours.

[0082] The results are shown in the table below. The bacteriophage DCP514 combined with traditional Chinese medicine extracts showed greater killing power against Escherichia coli than the bacteriophage alone, indicating that the traditional Chinese medicine extracts can significantly enhance the bactericidal ability of bacteriophage DCP514 against the host bacteria (groups 4-6), with the 3:1 combination showing the best bactericidal effect. However, the bactericidal effect of bacteriophage DCP543 combined with traditional Chinese medicine extracts did not show a significant improvement, indicating that bacteriophage DCP514 has greater application value.

[0083] Table 7. In vitro synergistic antibacterial test of bacteriophages and traditional Chinese medicine extracts against Escherichia coli.

[0084]

[0085]

[0086] In summary, the present invention provides a bacteriophage with excellent characteristics, which, when used in combination with traditional Chinese medicines such as Coptis chinensis, honeysuckle, Isatis indigotica, and licorice, can not only have the effects of clearing heat and detoxifying, nourishing blood and promoting blood circulation, but also enhance the bactericidal ability of the bacteriophage, effectively preventing or treating diseases caused by Escherichia coli. The Escherichia coli bacteriophage preparation of the present invention has good application prospects in the treatment of animal diseases.

Claims

1. A traditional Chinese medicine compound preparation containing Escherichia phage DCP514, characterized in that: It is composed of a diluted fermentation broth of *Escherichia coli* phage DCP514 and an extract obtained from a decoction of traditional Chinese medicine; the volume ratio of the diluted fermentation broth of *E. coli* phage DCP514 to the extract obtained from the decoction of traditional Chinese medicine is 1~3:1~3; the potency of the diluted fermentation broth of *E. coli* phage DCP514 is 1×10⁻⁶. 7 The concentration of pfu / ml of the Escherichia coli phage DCP514 is CCTCC NO: M 2024321; the concentration of the decoction extract of the traditional Chinese medicine is 1 mg / mL, which is a water extract of 1-3g Coptis chinensis, 1-3g Lonicera japonica, 1-3g Isatis indigotica and 1-3g Glycyrrhiza uralensis.

2. The traditional Chinese medicine compound preparation according to claim 1, wherein the decoction extract of the traditional Chinese medicine is an aqueous extract of 2g Coptis chinensis, 2g Lonicera japonica, 2g Isatis indigotica and 2g Glycyrrhiza uralensis.

3. In the traditional Chinese medicine compound preparation according to claim 1, the volume ratio of the diluted Escherichia coli phage DCP514 fermentation broth to the decoction extract of the traditional Chinese medicine is 3:

1.

4. The application of the traditional Chinese medicine compound preparation according to claim 1 in the preparation of an in vitro antibacterial agent for Escherichia coli.

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