ETEC phage and preparation method thereof

Abstract

The invention relates to the technical field of microbial technology and disease control, and discloses an ETEC phage and a preparation method thereof, the preservation number of the ETEC phage CQ615 is CGMCC No.46362, and the preservation time is December 30, 2024. According to the scheme, the bacteriophage CQ615 which is separated and purified in sewage by taking ETEC as host bacteria can be used for simultaneously splitting multiple serotypes of escherichia coli and has a relatively wide splitting spectrum; whole genome sequencing is carried out by extracting nucleic acid of the bacteriophage, and data are analyzed to find that the bacteriophage is free of non-virulence factors and drug-resistant factors and can become a potential selection mode for safe prevention and treatment of Escherichia coli; through verification of in-vivo and in-vitro experiments, the bacteriophage CQ615 can effectively reduce the number of ETEC bacteria in milk and lettuce, and can improve the survival rate of greater wax moths infected by ETEC in greater wax moth in-vivo experiments.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology and disease prevention technology, and in particular to an ETEC bacteriophage and a preparation method thereof. Background Art

[0002] Escherichia coli, also known as Escherichia coli, is a Gram-negative straight short rod, arranged singly or in pairs, facultatively anaerobic, grows well on ordinary nutrient agar plates and LB plates, and forms 2-3nm round, smooth, moist, grayish-white opaque colonies after culturing at 37°C for 24 hours. Escherichia coli is part of the normal intestinal flora, but it can also cause intestinal and extraintestinal diseases in humans. It is a conditional pathogen, and its pathogenic factors are mainly related to invasiveness, endotoxins, and enterotoxins. Under certain conditions, it can cause gastrointestinal infections or urethral infections in humans and animals, bringing certain harm to human production, life and health. According to different serotypes, virulence and symptoms caused, Escherichia coli that cause human diarrhea are divided into the following categories: enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), also known as Shiga toxin-producing E. coli (STEC), and enteroaggregative E. coli (EAEC).

[0003] Enterotoxigenic E. coli is the main cause of diarrhea in children and travelers in developing countries. According to the latest statistics from the World Health Organization (WHO), hundreds of millions of children suffer from diarrhea each year, and 30.4% of travelers also experience diarrheal symptoms. ETEC causes more than 128 million cases of diarrhea each year, resulting in more than 840,000 deaths. ETEC can produce virulence factors, including colonization factors (CF) and enterotoxins. Enterotoxins can be divided into heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST). These factors bind to specific receptors on epithelial cells and induce diarrhea. ETEC is also the main pathogen causing diarrhea in piglets. It colonizes in the epithelial cells of the small intestine through pili or non-pili adhesins, and continuously produces enterotoxins during proliferation. Enterotoxins can effectively act on epithelial cells, causing a large amount of water and electrolytes to enter the intestinal cavity, leading to diarrhea and dehydration in piglets. With the in-depth study of the main virulence factors of ETEC and its pathogenic mechanism, more and more new virulence factors and antigens have been discovered. So far, more than 25 different types of CF have been discovered and described.

[0004] Diarrhea caused by ETEC infection not only seriously threatens human health, but also causes huge economic losses to the global aquaculture industry. Currently, antibiotics play a vital role in preventing and treating ETEC infection. However, problems such as bacterial resistance, drug residues, and environmental pollution caused by long-term abuse of antibiotics have gradually aroused people's thinking. Seeking effective ways to prevent and treat ETEC infection has become the focus of global public health and food safety.

[0005] Due to the increase in antibiotic resistance, phage therapy is becoming a viable alternative to antibiotics. Bacteriophage (phage) is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes or spirochetes, which can cause lysis of some host bacteria. Phage individuals are tiny and do not have a complete cell structure. They are composed of proteins and nucleic acids and use the bacterial ribosomes, various factors, amino acids and energy production systems to achieve their own growth and proliferation. Once out of the host cell, the phage can neither grow nor replicate. Virulent phages replicate entirely through the lytic cycle, using bacteria to produce new phages before cell lysis and release. Temperate phages can enter the lysogenic cycle and form prophages, which are integrated into the bacterial chromosome or maintained outside the chromosome. In contrast, filamentous phages cause chronic infections and are continuously secreted from bacteria in the absence of lysates. Since phages are specific for the lysis of host bacteria, ETEC phages can only lyse ETEC or its corresponding serotype ETEC.

[0006] However, the types and numbers of phages discovered so far are still limited, and there are no phages that can target multiple types of E. coli at the same time. Summary of the invention

[0007] The present invention is intended to provide an ETEC phage and a preparation method thereof, aiming to provide an ETEC phage with strong lysis performance and good tolerance to physical and chemical factors, so as to solve the technical problem of limited types of existing phages.

[0008] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: an ETEC phage, the ETEC phage CQ615 has a deposit number of CGMCC No.46362, a deposit time of December 30, 2024, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, and is classified and named: Escherichia coli phage.

[0009] Preferably, as an improvement, the present scheme also provides a method for preparing ETEC bacteriophage, which is obtained by using sewage as the bacteriophage source and ETEC (O6:K15) as the host bacteria for separation and purification.

[0010] The ETEC phage CQ615 obtained in this protocol was isolated and purified from sewage from a sewage treatment plant in Linyi City, Shandong Province, China, using ETEC (O6:K15) as the host bacteria.

[0011] Preferably, as an improvement, the preparation method comprises the following steps:

[0012] (I) Isolation of ETEC bacteriophages: centrifuge a sewage sample, take the supernatant and filter to obtain a filtrate; mix the filtrate, host bacterial culture and LB liquid culture medium to obtain a proliferation liquid; centrifuge the proliferation liquid, take the supernatant and filter to obtain a phage enrichment liquid; culture the phage enrichment liquid by a double-layer plate method to obtain plaques;

[0013] (ii) Purification of phage: Pick up plaques and mix them with host bacterial solution and LB liquid culture medium for culture. Centrifuge the suspension obtained by culture and filter the supernatant to obtain the phage stock solution. After the phage stock solution is gradient diluted, the double-layer plate method is used to separate and purify the phage until the size and shape of the plaques are uniform, thereby obtaining ETEC phage CQ615.

[0014] Preferably, as an improvement, the present solution also provides an application of an ETEC bacteriophage, including the application of the above-mentioned ETEC bacteriophage CQ615 in the preparation of bactericidal drugs or drugs for preventing and treating infectious diseases caused by Escherichia coli.

[0015] Preferably, as an improvement, the present solution further provides a fungicide, wherein the active ingredient of the fungicide is ETEC bacteriophage CQ615.

[0016] Preferably, as an improvement, the present solution further provides a bactericidal drug, wherein the active ingredient of the bactericidal drug is ETEC bacteriophage CQ615.

[0017] Preferably, as an improvement, the present solution also provides a disinfectant, wherein the active ingredient of the disinfectant is ETEC bacteriophage CQ615.

[0018] Preferably, as an improvement, the present solution further provides a cleaning agent, wherein the active ingredient of the cleaning agent is ETEC bacteriophage CQ615.

[0019] Preferably, as an improvement, the present solution also provides a feed additive composition, wherein the active ingredient of the feed additive composition is ETEC bacteriophage CQ615.

[0020] Preferably, as an improvement, the present solution also provides a method for using the above-mentioned bactericide, bactericidal drug, disinfectant, detergent or feed additive composition, wherein the target pathogens during sterilization include Escherichia coli.

[0021] The principles and advantages of this solution are:

[0022] 1. In this scheme, the bacteriophage CQ615 isolated and purified from sewage using ETEC as the host bacteria can simultaneously lyse multiple serotypes of E. coli and has a wide lysis spectrum;

[0023] 2. This scheme extracts the nucleic acid of the bacteriophage and performs whole genome sequencing. After analyzing the data, it is found that the bacteriophage has no virulence factors and drug resistance factors, and has the potential to become a safe choice for the prevention and treatment of E. coli;

[0024] 3. This scheme has been verified through in vivo and in vitro experiments. Bacteriophage CQ615 can effectively reduce the number of ETEC bacteria in milk and lettuce. In the in vivo experiment of G. mellonella, it can increase the survival rate of G. mellonella infected with ETEC. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the colony expression of the bacteriophage CQ615 obtained in the example of the present invention in a plate.

[0026] Figure 2 This is a TEM image of bacteriophage CQ615 obtained in an example of the present invention.

[0027] Figure 3 The phage titers obtained by incubating the bacteriophage CQ615 obtained in the example of the present invention with host bacteria at different MOIs.

[0028] Figure 4 This is the one-step growth curve of bacteriophage CQ615 obtained in the example of the present invention.

[0029] Figure 5 This is the thermal stability curve of bacteriophage CQ615 obtained in the example of the present invention.

[0030] Figure 6 This is the acid-base stability curve of bacteriophage CQ615 obtained in the example of the present invention.

[0031] Figure 7 This is the genome map of bacteriophage CQ615 obtained in the example of the present invention (GC content is shown in black, GC skew+ and skew- are shown in light green and purple, respectively).

[0032] Figure 8 This is a phylogenetic tree of the bacteriophage CQ615 constructed based on the amino acid sequence of the DNA ligase in the present invention.

[0033] Fig. 9 This is the curve obtained by co-culturing the bacteriophage CQ615 obtained in the example of the present invention with milk and ETEC at 4°C.

[0034] Fig.10 This is the curve obtained by co-culturing the bacteriophage CQ615 obtained in the example of the present invention with milk and ETEC at 25°C.

[0035] Fig.11 This is the curve obtained by co-culturing the bacteriophage CQ615 obtained in the example of the present invention with lettuce and ETEC at 4°C.

[0036] Fig.12 This is the curve obtained by co-culturing the bacteriophage CQ615 obtained in the example of the present invention with lettuce and ETEC at 25°C.

[0037] Fig.13 This is the survival curve of G. mellonella inoculated with PBS, bacteriophage CQ615, and infected with different concentrations of ETEC O6:K15 bacterial solution after 48 hours of culture in the present invention.

[0038] Fig.14 This is the survival curve of the bacteriophage CQ615 obtained in the example of the present invention in inhibiting the G. mellonella infected with ETEC O6:K15.

[0039] Fig.15 The phage CQ615 obtained in the example of the present invention inhibits the survival status of G. mellonella infected with ETEC O6:K15 (A is the survival status of the PBS group after injection of PBS, B is the survival status of the S1 group after injection of ETEC+PBS, C is the survival status of the S2 group after injection of ETEC+MOI 10 phage, and D is the survival status of the S3 group after injection of ETEC+MOI 100 phage). DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0041] Program Overview

[0042] This scheme provides an ETEC phage, the ETEC phage CQ615 preservation number is CGMCC No.46362, and the preservation time is December 30, 2024.

[0043] This scheme also provides a method for preparing ETEC bacteriophage, which is obtained by using sewage as a bacterial source and ETEC (O6:K15) as a host bacteria for separation and purification.

[0044] The ETEC phage CQ615 obtained in this method is obtained by separation and purification of sewage from a sewage treatment plant in Linyi City, Shandong Province, China using ETEC (O6:K15) as the host bacteria. The preparation method specifically includes the following steps:

[0045] (I) Isolation of ETEC bacteriophages: centrifuge a sewage sample, take the supernatant and filter to obtain a filtrate; mix the filtrate, host bacterial culture and LB liquid culture medium to obtain a proliferation liquid; centrifuge the proliferation liquid, take the supernatant and filter to obtain a phage enrichment liquid; culture the phage enrichment liquid by a double-layer plate method to obtain plaques;

[0046] (ii) Purification of phage: Pick up plaques and mix them with host bacterial solution and LB liquid culture medium for culture. Centrifuge the suspension obtained by culture and filter the supernatant to obtain the phage stock solution. After the phage stock solution is gradient diluted, the double-layer plate method is used to separate and purify the phage until the size and shape of the plaques are uniform, thereby obtaining ETEC phage CQ615.

[0047] The present solution also provides an application of an ETEC bacteriophage, including the application of the above-mentioned ETEC bacteriophage CQ615 in the preparation of bactericidal drugs or drugs for preventing and treating infectious diseases caused by Escherichia coli.

[0048] This solution also provides a fungicide, the active ingredient of the fungicide is ETEC bacteriophage CQ615.

[0049] This scheme also provides a bactericidal drug, the active ingredient of which is ETEC bacteriophage CQ615.

[0050] This solution also provides a disinfectant, the active ingredient of the disinfectant is ETEC bacteriophage CQ615.

[0051] The scheme also provides a cleaning agent, wherein the active ingredient of the cleaning agent is ETEC bacteriophage CQ615.

[0052] The present invention also provides a feed additive composition, wherein the active ingredient of the feed additive composition is ETEC bacteriophage CQ615.

[0053] The present solution also provides a method for using the above-mentioned bactericide, bactericidal drug, disinfectant, detergent or feed additive composition, wherein the target pathogens during sterilization include Escherichia coli.

[0054] Example

[0055] 1. Isolation of ETEC phages:

[0056] Take 30mL of sewage sample in a 50mL centrifuge tube, centrifuge at 4℃, 4000rpm for 10min, filter with a 0.45um filter, and collect the filtrate for later use. Take 2.5mL of sewage filtrate, 1.25mL of ETEC bacterial solution (i.e., host bacterial solution) and mix with 5mL of LB liquid culture medium, shake and culture at 200rpm for 24h at 37℃ to proliferate the phage to obtain the proliferation liquid. Centrifuge the proliferation liquid at 4℃, 12000rpm for 10min, take the supernatant and filter with a 0.45um filter to obtain the phage enrichment liquid. Take 100μL of host bacterial solution and mix it with 5mL of LB semi-solid agar, pour it on LB solid culture medium (containing 1.5% agar) to prepare a double-layer plate, cool it down, use the vertical drop method to drop the phage enrichment liquid onto the double-layer plate, dry it, and invert it at 37℃ for 16-24h to observe whether plaques appear. If plaques appear, the ETEC phage is successfully isolated.

[0057] 2. Purification of phage

[0058] Use a sterile pipette to pick up the plaques on the double-layer plate and mix them with 100 μL of host bacterial solution and 5 mL of LB liquid culture medium for incubation. Cultivate at 37°C and 200 rpm for 6 to 12 hours. Centrifuge the suspension at 12,000 rpm for 10 minutes at 4°C, filter the supernatant with a 0.45 um filter, and collect the filtrate. The filtrate is the phage stock solution. After the phage stock solution is diluted 10 times in a gradient, the phage is separated and purified by the double-layer plate method until the size and shape of the plaques are uniform. The ETEC phage is named phage CQ615. The phage appears in the plate as follows: Figure 1 shown.

[0059] 3. Extraction and detection of crude phage particles

[0060] (I) Extraction

[0061] (1) Take 2 mL of host bacterial culture cultured to the logarithmic phase and 1 mL of phage suspension and inoculate them into 100 mL of liquid LB medium. Incubate at 37°C and 200 rpm with shaking for about 4 to 6 hours.

[0062] (2) Add DNase I and RNase A to the culture to a final concentration of 1 μg / ml each and incubate in a 37°C water bath for 30 min;

[0063] (3) Add 5.84 g NaCl, mix well to dissolve, place on ice for 1 h, centrifuge at 10,000 × g for 10 min, and collect the supernatant;

[0064] (4) Add solid PEG 8000 (polyethylene glycol) to a final concentration of 10%, shake thoroughly to dissolve, place on ice for 1 h or overnight to allow phage particles to precipitate, centrifuge at 4°C and 12,000 × g for 10 min, and discard the supernatant;

[0065] (5) Resuspend the precipitate with 2.5 ml of TM solution, add an equal volume of chloroform, shake for 30 seconds, centrifuge at 5000 × g for 10 minutes, and collect the upper aqueous phase;

[0066] (6) Extract once more with an equal volume of chloroform to obtain crude phage particles.

[0067] (ii) Transmission electron microscopy observation

[0068] (1) Adsorption: Use a pipette to draw 20 μL of the crude phage particle suspension and drop it onto the copper mesh for natural adsorption for 5 to 10 minutes. Then use a filter paper strip to absorb the excess droplets and let it dry slightly.

[0069] (2) Staining: Pipette 20 μL of 2% phosphotungstic acid solution and drop it onto the copper grid, and let it stand for 3 to 5 minutes;

[0070] (3) Drying: remove excess droplets with filter paper and dry under an incandescent lamp;

[0071] (4) Observation and photography: Observation and photography under a transmission electron microscope. The results are as follows: Figure 2 As shown, bacteriophage CQ615 displays a head with a non-contractible shortened tail.

[0072] 4. Phage titer determination

[0073] The purified phages were graded diluted, and the dilution multiples were selected with the number of phages on the double-layer plate between 30 and 300. The phage titer was calculated according to the formula, and the selected dilution multiples were used for three parallel experiments.

[0074] Phage titer (PFU / mL) = average number of plaques x dilution factor x 10

[0075] 5. Determination of phage host range

[0076] ETEC (O6: K15), EPEC (O111: K58), STEC (O157), EIEC (O143), EAEC, Escherichia coli standard strain (ATCC25922), and EHEC (O157: H7) were used as indicator bacteria, and the double-layer plate method was used to determine the phage host spectrum. 100 μL of the bacterial solution in the logarithmic phase was prepared with 5 mL of LB semi-solid agar. After the agar solidified, 5 μL of phage was vertically dripped, dried and cultured at 37°C for 16 to 24 hours, and the biofilm was observed for plaques. The presence of plaques indicates that the phage can lyse the bacteria. The results are shown in Table 1.

[0077] Table 1 Host bacterial spectrum of bacteriophage CQ615

[0078] Bacteria Type Strain number Serotype Plaque ETEC CICC 24190 O6:K15 + STEC CICC 10670 O157 + EIEC CICC 24188 O143 + E.coil ATCC 25922 - + EPEC CICC 24189 O111:K15 - EAEC CICC 24186 - - EHEC CICC 24187 O157:H7 -

[0079] Experimental data showed that the phage CQ615 isolated and purified by this scheme could produce plaques when co-cultured with ETEC (CICC 24190), STEC (CICC 10670), EIEC (CICC 24188), and E.coil (ATCC 25922), indicating that it has a wide lysis spectrum.

[0080] 6. Optimal infection multiplicity of phage

[0081] The ETEC bacterial solution was cultured to OD600 of 0.6, and 200 μL of the bacterial solution was mixed with 200 μL of phage suspension of different dilution concentrations to make the MOI of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1 and 10, and incubated at 37°C for 2 hours. After the culture, centrifuged at 4°C and 12000 r / min for 10 minutes, and the supernatant was taken for gradient dilution. The titer was determined by double-layer plate method at appropriate dilution multiples. A coordinate graph was drawn after 3 parallel experiments. The results are detailed in Figure 3 The experimental data showed that at an MOI of 0.1, phage CQ615 reached the highest titer.

[0082] 7. One-step growth curve of bacteriophage

[0083] Take 500 μL of host bacteria and phage at the ratio of the optimal infection multiplicity, incubate at 37°C for 15 min, centrifuge at 8000×g for 5 min, discard the supernatant, resuspend the precipitate with 1 mL of LB liquid culture medium, add the resuspended solution to 9 mL of LB liquid culture medium, and culture at 37°C in a shaking table. Sample 0.6 mL every 5 min for the first 20 min until 140 min. Repeat the experiment three times at each time point, centrifuge at 10000×g for 2 min, take the supernatant for gradient dilution, calculate the titer of phage at each time point, draw the one-step growth curve of phage and calculate its burst volume.

[0084] Burst volume (PFU / cell) = final titer of phage / initial number of bacteria

[0085] The results are shown in Figure 4 The one-step growth curve can be divided into the latent period, lytic period and stable period, which can quantitatively describe the growth of the phage and reflect the characteristics of the latent period and lytic amount of the phage. The experimental data showed that the latent period of the phage CQ615 isolated by this scheme was 5 minutes, and it entered the stable period at 100 minutes. In addition, the average burst size was 510 PFU / infected cell.

[0086] 8. Phage thermal stability test

[0087] Take 1mL of phage suspension at different temperatures (37℃, 50℃, 60℃, 70℃ and 80℃), take 200μL at 20min, 40min and 60min respectively, take 100μL of suspension after cooling, dilute 10 times, culture with double-layer plate method, determine phage titer, repeat three times as control. Plot the average value of phage titer as ordinate and temperature as abscissa to get the thermal stability of phage. See the results for details. Figure 5 .

[0088] Experimental data showed that bacteriophage CQ615 could tolerate normal temperature, and as the temperature increased, the phage titer gradually decreased and reached zero after incubation at 80°C for 60 min.

[0089] 9. Phage acid-base stability experiment

[0090] The LB liquid medium was adjusted to a pH of 1-14 with NaOH and HCl. 100 μL of the phage suspension was mixed with 100 μL of LB liquid medium with different pH values ​​and incubated at 37°C for 1 h. The phage titer was determined by the double-layer plate method. The experiment was repeated three times. The results are detailed in Figure 6 .

[0091] Experimental data showed that phage CQ615 survived at pH 6 to 10. Specifically, phage CQ615 showed good stability after continuous incubation for 1 h in the pH range of 6 to 12. However, its titer decreased significantly when the pH value was lower than 4 and higher than 11, especially when the pH value dropped from 10 to 11, the titer decreased by 5.95 log 10 PFU / mL.

[0092] 10. Phage genome sequencing and phylogenetic tree

[0093] This example was performed by high-throughput sequencing by Chengdu Life Baseline Technology Co., Ltd. The genome map of the amplified bacteriophage CQ615 is as follows: Figure 7 As shown, the genome of bacteriophage CQ615 is double-stranded DNA with a total length of 39709bp (the full sequence has been uploaded to NCBI, sequence number PQ010280) and a GC content of 48.6%. Genomic analysis showed that bacteriophage CQ615 belongs to the family of Autoviridae and encodes a total of 51 open reading frames (see Table 2 for details). Fifteen of the 51 ORFs (29.4%) were annotated as hypothetical proteins, and the remaining ORFs were annotated as functional proteins, including 5 functional modules: transcription, replication, envelope biogenesis, host lysis, and unknown function. It is worth noting that the phage lacks tRNA genes, drug resistance genes, and virulence factors.

[0094]

[0095]

[0096] Phylogenetic trees were constructed using conserved DNA ligase to determine the evolutionary relationship between phage CQ615 and other phages. Figure 8 The phylogenetic tree revealed a high degree of affinity between phage CQ615 and Escherichia phage N30, which, according to the International Committee on Taxonomy of Viruses (ICTV), belongs to the genus Teseptimavirus in the family Autographaviridae.

[0097] 11. In vitro and in vivo validation experiments of bacteriophage CQ615:

[0098] (1) In vitro experiments: To evaluate the effect of phage on ETEC (O6:K15), 9.8 ml of skim milk was added to a test tube and irradiated with ultraviolet light for 30 min, and then 100 μL of ETEC (O6:K15) was added to make the final concentration of 4 log CFU / ml. Then, 100 μL of phage was added to 9.8 mL of milk at an MOI of 10,000. The mixture was incubated at 4°C and 25°C for 48 h. The plate colony count method was used to detect bacterial growth at 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h. Three parallel tests were performed, and the results are detailed in the following. Fig. 9 and Fig.10 The experiment showed that in milk, treatment with bacteriophage CQ615 at an MOI of 10,000 resulted in a 1.48 log reduction in ETEC viable counts after 48 h of incubation at 4 °C. 10 CFU / mL. However, at 25°C, after treatment with bacteriophage CQ615 for 6 to 12 hours, the number of viable bacteria in milk infected with ETEC dropped below the detection limit, and after 48 hours of incubation, it decreased by 4.82 log 10 CFU / mL.

[0099] (2) For the lettuce experiment, clean lettuce leaves were prepared and cut into 1 cm × 1 cm squares, then washed once in PBS solution and soaked in 75% disinfectant alcohol for 10 min. They were then irradiated with ultraviolet light for 10 min. The treated lettuce was then placed on LB solid medium to confirm that no bacteria grew after the plate was incubated at 37 °C for 12 h. Clean lettuce leaves were placed in a 12-well plate, and 10 μL of 1 × 10 7 ETEC bacterial suspension, 10 μL PBS buffer was added to the control group, and 10 μL phage CQ615 was added to the other two groups, so that the MOI of the mixture was 10,000. All samples were cultured at 4°C and 25°C, and were taken out at 1, 3, 6, 12, 24 and 48 hours, respectively, and washed with 1 mL PBS. The experiment was performed in triplicate, and the results are detailed in Fig.11 and Fig.12 The experiment showed that at 4°C, bacteriophage CQ615 also had a significant antibacterial effect on lettuce, reducing the 10 CFU / cm 2 When incubated at 25°C for 3 to 24 hours, bacteriophage CQ615 reduced the viable count to below the detection limit after 48 hours of incubation, a reduction of 5.28 log 10 CFU / cm 2 .

[0100] (3) In vivo experiment on G. mellonella: G. mellonella larvae were used as a model to evaluate the therapeutic effect of bacteriophage CQ615 on ETEC (O6:K15). In order to determine the appropriate concentration of infectious bacteria, larvae weighing 250-350 mg were selected and divided into 6 groups, with 10 larvae in each group:

[0101] The control group was the blank control group, the PBS group was injected with 10 μL PBS, the T1 group was injected with 10 μL phage CQ615, and the T2-T4 groups were injected with 10 μL of different concentrations of ETEC (O6:K15), with the concentrations of 1×10 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5 CFU / mL. If the larvae turned black and did not move after being touched, they were considered dead. The survival status of the greater wax moth was observed and a survival curve was drawn. The results are as follows Fig.13 The data show that the injection concentration is 1×10 6 10 μL of bacteria with CFU / mL caused 50% mortality of G. mellonella after 48 hours.

[0102] Select 1×10 6 The bacterial concentration in CFU / ml was used for phage treatment evaluation. 40 larvae were divided into 4 groups:

[0103] The PBS group was injected with 10 μL PBS; the S1 to S3 groups were injected with 10 μL 1×10 6 CFU / mL ETEC (O6:K15) bacterial solution. One hour later, the S1 group was injected with 10 μL PBS (equivalent to the S1 group being injected with ETEC bacterial solution); the S2 and S3 groups were injected with 10 μL phage at MOI 10 and MOI 100, respectively. The treated larvae in each group were incubated in the dark at 37°C, and their survival rates were evaluated every 12 h. Survival curves were plotted. The results are detailed in Fig.14 and Fig.15 The data showed that different MOIs of bacteriophage CQ615 significantly increased the survival rate of G. mellonella after 48 h. In particular, the mortality rate at MOI = 100 was reduced from 50% to 10% (e.g. Fig.15 D).

[0104] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. An ETEC bacteriophage, characterized in that: The ETEC phage CQ615 has a deposit number of CGMCC No. 46362 and a deposit date of December 30, 2024.

2. A method for preparing ETEC bacteriophage, characterized in that: The ETEC phage according to claim 1 is obtained by using sewage as the phage source and ETEC (O6:K15) as the host bacteria for separation and purification.

3. The method for preparing an ETEC bacteriophage according to claim 2, characterized in that: The preparation method comprises the following steps: (I) Isolation of ETEC bacteriophages: centrifuge a sewage sample, take the supernatant and filter to obtain a filtrate; mix the filtrate, host bacterial culture and LB liquid culture medium to obtain a proliferation liquid; centrifuge the proliferation liquid, take the supernatant and filter to obtain a phage enrichment liquid; culture the phage enrichment liquid by a double-layer plate method to obtain plaques; (ii) Purification of phage: Pick up plaques and mix them with host bacterial solution and LB liquid culture medium for culture. Centrifuge the suspension obtained by culture and filter the supernatant to obtain the phage stock solution. After the phage stock solution is gradient diluted, the double-layer plate method is used to separate and purify the phage until the size and shape of the plaques are uniform, thereby obtaining ETEC phage CQ615.

4. An application of an ETEC bacteriophage, characterized in that: The invention comprises the use of the ETEC bacteriophage CQ615 as claimed in claim 1 or the ETEC bacteriophage CQ615 prepared by the method as claimed in any one of claims 2 to 3 in the preparation of bactericidal drugs or drugs for preventing and treating infectious diseases caused by Escherichia coli.

5. A bactericide, characterized in that: The active ingredient of the bactericide is the ETEC bacteriophage CQ615 described in claim 1.

6. A bactericidal drug, characterized in that: The active ingredient of the bactericidal drug is the ETEC bacteriophage CQ615 described in claim 1.

7. A disinfectant, characterized in that: The active ingredient of the disinfectant is the ETEC bacteriophage CQ615 described in claim 1.

8. A cleaning agent, characterized in that: The active ingredient of the cleaning agent is the ETEC bacteriophage CQ615 according to claim 1.

9. A feed additive composition, characterized in that: The active ingredient of the composition is the ETEC bacteriophage CQ615 described in claim 1.

10. A method for using any one of the bactericide according to claim 5, the bactericidal drug according to claim 6, the disinfectant according to claim 7, the cleaning agent according to claim 8 or the feed additive composition according to claim 9, characterized in that: The target pathogens for sterilization include Escherichia coli.

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