Diarrhea-related multi-drug-resistant Escherichia coli phage and application thereof

Abstract

The invention relates to a diarrhea-related multi-drug-resistant coliphage and application thereof. In particular to a bacteriophage vBEcoMP66 screened from a multi-drug-resistant escherichia coli library, and the bacteriophage vBEcoMP66 is preserved in the China Center for Type Culture Collection on December 12, 2024, and the preservation number is CCTCC M 20242794. The bacillus subtilis has a splitting effect on multiple strains of multi-drug-resistant escherichia coli and piglet diarrhea pathogenic escherichia coli strains, and also has an obvious inhibition effect on bacteria in a biological membrane, and the inhibition capability is superior to the antibiotic level under the same condition. Besides, the bacteriophage has extremely low host resistance mutation frequency, the resistance mutation frequency of host bacteria can be effectively reduced, animal experiments show that the bacteriophage has the effects of preventing piglet diarrhea and reducing the level of inflammatory factors in vivo, and the bacteriophage has extremely high industrial value and wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a diarrhea-related multi-drug resistant Escherichia coli phage and its application. Background Art

[0002] Escherichia coli is one of the common pathogens causing diarrhea in piglets. Escherichia coli infection can lead to Escherichia coli diarrhea in piglets, which is a disease that seriously affects the growth and survival rate of piglets. Escherichia coli usually infects piglets through the digestive tract, and contaminated feed, drinking water, environment, and the nipples of sows may all become sources of infection. Escherichia coli diarrhea is mainly manifested as watery or mucous diarrhea, and may be accompanied by symptoms such as vomiting, dehydration, and weight loss in severe cases. If not treated in time, it may lead to the death of piglets. Among them, Enterotoxigenic Escherichia coli is the most common cause of diarrhea in humans and young livestock. It can cause infectious diarrhea in piglets, which is easy to spread and has a high fatality rate, posing a great threat to the breeding industry. The toxicity of ETEC is mainly related to the enterotoxin it produces and the adhesin related to the ability of the bacterial body to infect and adhere. For porcine ETEC, generally, the strains are classified based on the types of adhesins on the fimbriae. Currently, more than ten adhesins have been confirmed, mainly including K88, K99, F18, F41, and 987P, among which the K88 fimbrial adhesin has the widest prevalence. Newborn piglets are particularly sensitive to Escherichia coli due to their immature immune systems, especially piglets within 7 days after birth. Infected piglets may show intestinal distension, congestion, edema, and inflammatory reactions in their small intestines.

[0003] In recent years, due to the abuse of antibiotics, the problem of bacterial drug resistance has become increasingly serious, and various multi-drug resistant bacteria (MDR) have emerged, including multi-drug resistant Escherichia coli (MDR E. coli), whose main difference from ordinary Escherichia coli lies in their resistance to multiple antibiotics. The reason is that when using antibiotics, bacteria develop drug resistance through natural selection and genetic variation. Therefore, bacteria with stronger drug resistance survive and reproduce. In addition, drug resistance genes can be spread among bacteria through horizontal gene transfer (such as transformation, transduction, conjugation), and even different types of bacteria can acquire drug resistance. Due to their drug resistance, these bacteria may cause more serious infections, increasing hospitalization rates and mortality rates, posing a major threat to public health. Infections may lead to more serious diseases, such as urinary tract infections, sepsis, intestinal infections, etc., and treatment options are limited.

[0004] Currently, phage therapy has attracted increasing attention as a potential alternative therapy in veterinary and human medicine. Compared with traditional antibiotics, phage therapy has the advantages of strong self-replicating ability, high host specificity, safety, and low R & D cost, making it an ideal alternative to antibiotics. Multiple types of phage preparations that can be used to treat piglet diarrhea are known in the prior art. However, current research is only limited to focusing on the therapeutic effects of phages, while ignoring two major problems faced by current phage therapy: the occurrence of phage-resistant mutants and the ability to inhibit bacterial biofilms. Most bacteria in clinical infections exist in biofilms. In particular, some Escherichia coli strains that can form biofilms have high drug resistance and thus evade the attack of the immune system. Research shows that some phage genomes can encode and synthesize various specific enzymes, such as endolysin or lysin, depolymerase, and VAPGHs. These enzymes can degrade substances such as bacterial peptidoglycan, capsular polysaccharide, and lipopolysaccharide to assist phages in rapidly destroying the integrity of bacterial biofilms, which is one of the important mechanisms against bacterial biofilms. Summary of the Invention

[0005] In the present invention, multiple strains of diarrhea-related multidrug-resistant Escherichia coli phages vB_EcoM_P8, vB_EcoM_P54, vB_EcoM_P20, vB_EcoM_P48, and vB_EcoM_P66 were isolated and screened. Among them, vB_EcoM_P66 can infect most of these strains and was deposited at the China Center for Type Culture Collection on December 12, 2024, with the classification label: Escherichia phage vB_EcoM_P66 and the deposit number: CCTCC M 20242794.

[0006] Furthermore, the phages can maintain the integrity of their structure and function between 4 - 60 °C; the optimal pH of the phages is 5 - 11; the latent period of the phage growth is 20 min, the lysis period is approximately 50 min, and the burst size is 120 PFU / Cell.

[0007] The present invention also provides a phage preparation containing the phage vB_EcoM_P66.

[0008] Furthermore, the phage preparation also includes: phages vB_EcoM_P8 and vB_EcoM_P54, wherein the genome accession number of phage vB_EcoM_P8 is: PV390659.2 (https: / / www.ncbi.nlm.nih.gov / nuccore / PV390659), and the genome accession number of phage vB_EcoM_P54 is: PV390658.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / PV390658).

[0009] Furthermore, the mixing ratio of phages vB_EcoM_P66, vB_EcoM_P8 and vB_EcoM_P54 is: 1:1:1.

[0010] Furthermore, the phage preparation is a drug, feed additive, drinking water additive, environmental cleaner or disinfectant.

[0011] Furthermore, the dosage form of the phage preparation is a liquid preparation, powder preparation or oral solid preparation.

[0012] The present invention also provides a method for preparing the multi-drug resistant Escherichia coli phage vB_EcoM_P66. Inoculate the multi-drug resistant Escherichia coli in a liquid medium and culture it until the logarithmic growth phase, and then inoculate the multi-drug resistant Escherichia coli phage vB_EcoM_P66 for culture.

[0013] Furthermore, the culture temperature of the phage is 37 °C; the pH range for phage culture is 5 - 11; the culture time of the phage is 4 - 6 h.

[0014] The present invention also provides the application of the phage or the preparation in any of the following:

[0015] A1) Preparing a bactericide for diarrhea-related Escherichia coli or multi-drug resistant Escherichia coli;

[0016] A2) Preparing a feed additive, drinking water additive, environmental cleaner or disinfectant;

[0017] A3) Preparing a biological preparation for preventing or treating multi-drug resistant Escherichia coli infection;

[0018] A4) Preparing a product for preventing or treating piglet diarrhea;

[0019] A5) In vitro lysing pathogenic Escherichia coli or multi-drug resistant Escherichia coli;

[0020] A6) Preparing a product for preventing or treating piglet diarrhea caused by Escherichia coli of ST10, ST48, ST58, ST101 types or ETEC-K88, EPEC, EHEC.

[0021] The present invention also provides a method for disinfecting an environment with multi-drug resistant Escherichia coli. The phage vB_EcoM_P66 or the phage mixture preparation is configured into a liquid solution for spraying.

[0022] Beneficial effects:

[0023] 1. Through screening, the present invention obtained multiple strains of phages that can lyse multi-drug resistant Escherichia coli and the diarrheal pathogen ETEC-K88. Finally, the phage vB_EcoM_P66 that can infect most of the tested multi-drug resistant Escherichia coli strains was obtained. It was deposited at the China Center for Type Culture Collection on December 12, 2024, with the deposit number: CCTCC M20242794.

[0024] 2. The phage vB_EcoM_P66 can maintain the structural and functional integrity between 4 - 60 °C, with the optimal pH being 5 - 11, the optimal multiplicity of infection MOI = 0.1, the latent period being 20 min, the lysis period being approximately 50 min, the burst size being 120 PFU / Cell, and its growth is not affected by chloroform.

[0025] 3. The multiple strains of phages screened in the present invention show different resistance mutation frequencies to the host. Among them, the resistance mutation frequencies MF of vB_EcoM_P20 and vB_EcoM_P48 are both 1, that is, the host Escherichia coli is extremely likely to produce drug-resistant mutations, while the phage vB_EcoM_P66 shows a lower frequency of resistance mutations, and the resistance mutation frequency MF is only 0.033. Further, different cocktails were prepared from the multiple strains of phages screened to observe their resistance mutation frequencies. The results showed that when vB_EcoM_P20 and vB_EcoM_P48 were included, the resistance mutation frequency of bacteria to phages increased significantly. However, no matter what cocktail combination was used, as long as vB_EcoM_P66 appeared in the combination, the occurrence of the resistance mutation rate could be significantly reduced. Therefore, it can be used in combination with other phages to make a cocktail combination to achieve the effect of inhibiting the colonization of Escherichia coli and effectively preventing and controlling Escherichia coli-induced diarrhea in piglets.

[0026] 4. Mouse experiments confirmed that the phage prevention group could significantly inhibit the colonization of Escherichia coli K88 in mice and reduce the levels of inflammatory factors in the body. In piglet experiments, the phage prevention group could significantly inhibit the colonization of Escherichia coli K88 in the piglet intestine, and reduce the levels of inflammatory factors in the body (TNF-α / IL-6 / Zonulin), maintain the normal intestinal morphological structure, reduce the permeability of the small intestinal mucosa, and enhance the tight junction function of the piglet intestine. That is, it has the effects of preventing piglet diarrhea and reducing the levels of inflammatory factors in the body. Description of the Drawings

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the embodiments will be briefly introduced below. It should be understood that the following accompanying drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant accompanying drawings can also be obtained based on these drawings.

[0028] Figure 1 In which, A is the transmission electron microscopy morphology observation of phage vB_EcoM_P66, and B is the determination of the optimal multiplicity of infection of phage vB_EcoM_P66.

[0029] Figure 2 In which, A is the determination of the temperature stability of phage vB_EcoM_P66, B is the determination of the pH stability of phage vB_EcoM_P66, C is the determination of the chloroform sensitivity of phage vB_EcoM_P66, and D is the determination of the one-step growth curve of phage vB_EcoM_P66.

[0030] Figure 3 It is the determination of the ability of phage vB_EcoM_P66 to inhibit the biofilm of diarrhea strain ETEC-K88. In figure A: Using Escherichia coli K88 as the host bacterium, the ability of 5 different phages and the antibiotic enrofloxacin (FQ) to inhibit the formation of its biofilm under the same conditions was determined. Among them, vB_EcoM_P66 (the 5th plate from left to right) showed a strong effect on inhibiting the biofilm of Escherichia coli; B is the quantitative analysis of vB_EcoM_P66 and norfloxacin (FQ) in inhibiting the biofilm of Escherichia coli.

[0031] Figure 4 It is the antibacterial effect of phage vB_EcoM_P66 on diarrhea strain ETEC-K88 and multidrug-resistant Escherichia coli A240.

[0032] Figure 5 It is the determination of the resistance mutation frequency of diarrhea strain ETEC-K88 to phage vB_EcoM_P66.

[0033] Figure 6 It is the safety evaluation of phage vB_EcoM_P66 in mice: Mice fed with different titers of phage vB_EcoM_P66 showed no acute toxicity reaction.

[0034] Figure 7For the evaluation of the effectiveness of phages in mice, Figure A shows the changes in the bacterial abundance of Escherichia coli K88 in feces after prophylactic feeding of mice with phages at different titers for 7 days, and the bacterial colonization number of K88 can be effectively reduced; Figure B shows the observation of HE-stained tissue pathological sections of mice in different treatment groups (healthy control group, ETEC challenge group, phage prophylaxis group). Among them, the intestinal mucosal villi of the control group mice are intact and plump. In the ETEC treatment group, the villi are significantly swollen and broken, and the number of goblet cells is significantly reduced; it is suggested that the inflammation may be caused by the colonization of K88 in the mouse intestine. In the phage prophylaxis group, the goblet cells and intestinal villi basically return to normal without obvious swelling. This shows that the phages play a certain prophylactic and protective role; Figure C shows the changes in the expression levels of the inflammatory factor interleukin-6 in three groups of mice (healthy control group, ETEC challenge group, phage prophylaxis group) after prophylactic feeding of mice with phages at different titers for 7 days; Figure D shows the changes in the expression levels of the inflammatory factor - tumor necrosis factor TNF-α in three groups of mice (healthy control group, ETEC challenge group, phage prophylaxis group) after prophylactic feeding of mice with phages at different titers for 7 days.

[0035] Figure 8 For the evaluation of the effect of phages in the diarrhea of piglets infected with ETEC-K88, Figure A shows the changes in the expression levels of the inflammatory factor - tumor necrosis factor TNF-α in three periods (before challenge, after challenge, and at the time of sacrifice) of piglets in the ETEC challenge group; Figure B shows the changes in the expression levels of the inflammatory factor - tumor necrosis factor TNF-α in three periods (before challenge, after challenge, and at the time of sacrifice) of piglets in the phage prophylaxis group; Figure C shows the changes in the expression levels of the inflammatory factor - interleukin-6 in three periods (before challenge, after challenge, and at the time of sacrifice) of piglets in the ETEC challenge group; Figure D shows the changes in the expression levels of the inflammatory factor interleukin-6 in three periods (before challenge, after challenge, and at the time of sacrifice) of piglets in the phage prophylaxis group; Figure E shows the changes in the expression levels of the inflammatory factor Zonulin protein in three periods (before challenge, after challenge, and at the time of sacrifice) of piglets in the ETEC challenge group; Figure F shows the changes in the expression levels of the inflammatory factor Zonulin protein in three periods (before challenge, after challenge, and at the time of sacrifice) of piglets in the phage prophylaxis group; Figure G shows the changes in the bacterial abundance of Escherichia coli K88 in the feces of piglets in different treatment groups (healthy control group, ETEC challenge group, phage prophylaxis group); Figure H shows the changes in the bacterial abundance of Escherichia coli K88 in the jejunal contents of piglets in different treatment groups (healthy control group, ETEC challenge group, phage prophylaxis group).

[0036] Figure 9For the ileal tissue pathological changes and immunofluorescence staining of piglets in the healthy, ETEC group, and phage prevention group. Among them, Figures a, b, and c are the ileal tissues of the healthy control group, without swelling or fragmentation; the ileal tissues of the ETEC challenge group were significantly damaged, with pathological changes such as broken and swollen intestinal villi caused by inflammation and a decrease in the number of goblet cells. There were no obvious pathological changes in the ileal tissues of the phage prevention group, indicating that the phage successfully colonized in the intestine, effectively reducing the invasion of pathogenic bacteria, thus playing a certain protective role in the intestine; Figures d, e, and f are the ileal tissues of the healthy control group with stronger fluorescence intensity, clear cell membrane boundaries, and linear distribution. The fluorescence intensity of the ileal tissues in the ETEC challenge group was weakened, the density decreased, the linear structure was broken, and even disappeared, significantly downregulating the expression of Zo-1 protein. The fluorescence intensity of the ileal tissues in the phage prevention group was slightly weakened, but the structure remained intact.

[0037] Figure 10 For the determination results of the host range of phage vB_EcoM_P66.

[0038] Figure 11 For the plaque assay experiment to detect that vB_EcoM_P66 also had antibacterial effects on the other two pathogenic Escherichia coli (enteropathogenic Escherichia coli EPEC / enterohemorrhagic Escherichia coli EHEC) that caused diarrhea in piglets besides enterotoxigenic Escherichia coli ETEC. Specific implementation manner

[0039] The following examples are only used to more clearly illustrate the technical solutions of the present invention, so they are only used as examples and cannot be used to limit the protection scope of the present invention. It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should be the commonly understood meanings by those skilled in the art to which the present invention belongs. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0040] All the multidrug-resistant Escherichia coli used in this invention were provided by the College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province, and have been uploaded to the NCBI database with the project number PRJNA1044843. The ETEC-K88 used to construct the animal diarrhea model was the CVCC196 standard strain purchased from the National Veterinary Microbial Culture (Virus) Collection Center. All Escherichia coli grew aerobically on LB plates at 37°C. The ordinary LB medium was composed of 10 g of peptone, 10 g of yeast powder, 5 g of sodium chloride, made up to 1 L with deionized water, and the pH was adjusted to 7 with NaOH. After the above medium was prepared, it was autoclaved at 121°C for 30 minutes. The solid medium needed to add 15 g / L of agarose, and was cultured and streaked in a 37°C incubator for 18 - 24 hours, and then placed at room temperature for colony counting. The liquid medium needed to be shaken at a speed of 220 rmp in a 37°C shaker until the logarithmic growth phase.

[0041] Example 1 Isolation, Purification and Identification of Phage

[0042] 1. Isolation and Cultivation of Phage

[0043] Fecal samples were collected from farms (pigs / chickens) in Central and North China. An appropriate amount of about 15 - 20 g of feces was soaked overnight in 0.9% NaCl solution. The next day, it was centrifuged at 8,000 g at 4°C for 10 minutes, then filtered through a 0.45 μm cellulose filter membrane to remove bacteria, and further filtered through a 0.22 μm filter. After mixing 100 μL of the fecal filtrate with 900 μL of the logarithmic-phase bacterial culture, it was poured onto a single-layer LB plate and cultured overnight in a 37°C incubator. The next day, the obtained phage plaques were streaked and purified three times in the same host bacteria, and then amplified to prepare a working stock solution.

[0044] 2. Purification of Phage

[0045] The Limulus reagent method was used to detect the endotoxin content. The phage was filtered through a sterile nitrocellulose filter with a membrane pore size of 0.22 μm, which had the effect of removing part of the endotoxin. The endotoxin was further removed by ultrafiltration centrifugation. An ultrafiltration centrifugal tube with a membrane pore size of 100 kDa was selected for phage filtration. After re-detection, the result effectively reduced the endotoxin content. And by measuring the titer of the ultrafiltered phage, it was found that it concentrated the phage and the titer could be increased by one order of magnitude. Therefore, a higher purity phage can be obtained by the ultrafiltration method, and the phage can be effectively concentrated.

[0046] 3. Extraction of Phage Genomic DNA

[0047] The obtained high-concentration phage precipitate was suspended in 1 mL of pure water, 20 μL of Dnase I (3 mg / mL) and 10 μL of Rnase A (10 mg / mL) were added, and cultured overnight at 37°C. The next day, 20 μL of 2 mol / L ZnCl2 was added, incubated at 37°C for 5 min, after centrifugation, the precipitate was resuspended in 500 μL of TES buffer and incubated at 65°C for 15 min. Then 20 μL of proteinase k (20 mg / mL) was added, incubated at 50°C for 1 - 1.5 h, and then 60 μL of pre-cooled 3 mol / L CH3COOK (PH5.2) was added. After centrifugation, the supernatant was extracted with an equal volume (about 600 μL) of phenol / chloroform / isoamyl alcohol (25:24:1). After centrifugation, the supernatant was taken, and DNA was precipitated overnight with 2 / 3 - 1 volume of isopropanol (-20°C). It was washed once with 500 ul of 70% ethanol, the supernatant was discarded by centrifugation, and the residual ethanol was air-dried at room temperature. Finally, the DNA was dissolved in 40 - 60 μL of deionized water, the DNA concentration and purity were measured with Nanodrop, and 1 μg of DNA sample was taken for library construction and sequencing.

[0048] 4. Determination of the host range of phages

[0049] Plaque assay is a method for detecting the degree of virus infection. On a solid agar plate where host bacteria grow, phages can lyse bacteria to form transparent plaques. The spot test method was used to detect the host range of phages. The heated semi-solid LB medium was dispensed into centrifuge tubes at 6 - 8 mL. When the semi-solid medium cooled to about 40°C, 1 mL of the bacterial suspension was added, and it was mixed well by inverting up and down and poured into a petri dish containing the bottom solid LB medium. This is a double-layer plate. After the upper semi-solid medium solidified, 5 μL of the phage sample was spotted and air-dried. The petri dish was cultured in an incubator at 37°C for 12 - 24 h, and classified and judged according to the state of the spotted plaque: 0: no lysis, 1: there are some individual phages, 2: there is a large amount of turbidity in the spotted area, 3: the spotted area is completely lysed but there is a faint background, 4: complete lysis, and the background is transparent.

[0050] Result analysis: In this invention, multiple phages were isolated from the fecal samples of diarrheal dead piglets and sewage samples from their original living environment. Different ST-type multidrug-resistant Escherichia coli were used as hosts for screening, including ESBL-positive strains and diarrheal pathogenic strains. Double-stranded DNA viruses representing the main families of tailed phages were isolated, and their host bacteria were ETEC-K88 and A78, A369, A333, A431, A473, A240, A481, A323, A30, A374, A466. Among them, A333, A240, and A78 belong to the sequence types ST10 and ST58 of Escherichia coli, which are the main bacteria causing diarrheal infections in piglets.

[0051] The host range of the Escherichia coli phage library was evaluated using 30 different Escherichia coli strains, which covered 16 different sequence types (including diarrhea-related ST10, ST48, ST58, ST101), and the diarrhea pathogen ETEC-K88 (all strain information has been uploaded).

[0052] Through screening, multiple multi-drug resistant Escherichia coli phages vB_EcoM_P8, vB_EcoM_P54, vB_EcoM_P20, vB_EcoM_P48, and vB_EcoM_P66 related to diarrhea were obtained in the present invention. Among them, phage vB_EcoM_P66 can infect most of these strains ( Figure 10 ), and it was deposited at the China Center for Type Culture Collection on December 12, 2024, with the deposit number: CCTCC M 20242794.

[0053] Example 2 Analysis of Phage Biological Characteristics

[0054] 1. Observation of Phage Electron Microscopy Morphology

[0055] The enriched phage suspension was ultracentrifuged at 30000 r / min for 2 h. After discarding the supernatant, the phages were suspended with 0.1 M ammonium acetate for sample preparation. For a high-concentration phage sample, a copper mesh was placed on the suspension with forceps. After adsorption for about 10 min, sodium phosphotungstate was added for counterstaining, and then dried for transmission electron microscopy observation. The results are as Figure 1 shown in -A. After identification, phage vB_EcoM_P66 belongs to the order Caudovirales, the family Myoviridae, and the genus Tequatrovirus (T4) phage.

[0056] 2. Determination of the Optimal Multiplicity of Infection of Phages

[0057] Bacterial culture medium in the logarithmic growth phase was prepared, and the bacterial concentration was calculated by measuring the OD value. The phages were serially diluted with deionized water in advance according to the ratios of multiplicity of infection of 0.001, 0.01, 0.1, 1, 10, and 100. 5 mL of the measured-concentration bacterial culture medium was mixed with phages of different dilutions obtained at different multiplicities of infection, and cultured at 37 °C in a constant-temperature shaking incubator at 220 rpm for 3 h. After centrifugation, the supernatant was taken and filtered through a 0.22-μm microporous membrane to remove bacteria. The titer of the phages was determined by the double-layer plate method, and the highest titer was the optimal multiplicity of infection. The results are as Figure 1 shown in -B. The results showed that the optimal multiplicity of infection MOI of phage vB_EcoM_P66 was 0.1.

[0058] 3. Determination of the Temperature Stability of Phages

[0059] The phage solution was diluted to 10 8PFU / mL. Take 1 mL and put it into a test tube. Incubate it in a water bath or metal bath instrument at 4, 37, 50, 60, and 80 °C for 2 h, and determine the phage titer by the double-layer plate method. Set up three parallel experiments, plot the titer value on the vertical axis and the temperature on the horizontal axis. The results are as Figure 2 shown in -A. The results show that the phage vB_EcoM_P66 has the best activity at 37 °C and can maintain the structural and functional integrity between 4 and 60 °C.

[0060] 4. Determination of the pH stability of the phage

[0061] Prepare KAC solution with pH 4 and 5, PBS solution with pH 6, 7, and 8, and Gly-NaOH solution with pH 9 and 10. Dilute the phage solution to 10 9 PFU / mL. Take 100 μL of the phage sample and 900 μL of the buffer with different pH values and put them into a test tube to mix evenly and incubate for 2 h. Use the double-layer plate method to determine the phage titer. Set up three parallel experiments, plot the titer value on the vertical axis and the pH value on the horizontal axis.

[0062] The results are as Figure 2 shown in -B. The results show that the phage vB_EcoM_P66 can maintain its structural and functional integrity between pH 5 and 11.

[0063] 5. Chloroform sensitivity experiment

[0064] Add 0.1 mL of chloroform to 1 mL of the phage stock solution, wait for it to mix evenly and then let it stand for 15 min. Then take the upper aqueous phase part for ten-fold serial dilution, and use the single-layer plate method to detect the titer of the aqueous phase part and compare it with the original titer of the phage to observe the change in titer.

[0065] The results are as Figure 2 shown in -C. The results show that the phage vB_EcoM_P66 is not affected by chloroform.

[0066] 6. Determination of the one-step growth curve of the phage

[0067] The one-step growth curve can more intuitively and clearly describe the growth law of phages: Take 2 mL of the bacterial mixed solution in the logarithmic growth phase, centrifuge to discard the supernatant, resuspend it in 1 mL of fresh TSB liquid medium, and mix it with 1 mL of phages, with an MOI of 0.1. Let it stand for 10 min, centrifuge to discard the supernatant, and wash the precipitate with TSB medium 2 - 3 times repeatedly to remove unadsorbed phage particles. Resuspend the precipitate in 10 mL of fresh TSB liquid medium and culture it at 37 °C for 140 min. During the incubation period, collect 0.2 mL of the culture solution every 20 min, and determine the phage titer by the double-layer agar plate method. Finally, take the sampling time as the abscissa and the titer as the ordinate to plot the one-step growth curve of the phage, and determine the latent period, lysis period, and burst size of the phage infecting the host. The calculation is as follows: Burst size = phage titer in the stationary phase / host concentration at the initial stage of infection.

[0068] The results are as Figure 2 shown in -D. The results show that the latent period of the phage vB_EcoM_P66 is 20 min, the lysis period is about 50 min, and the burst size is about 120 PFU / Cell.

[0069] 7. Phage biofilm assay

[0070] Most bacteria in clinical infections exist in biofilms. In particular, Escherichia coli forming biofilms has high drug resistance and can evade the attack of the immune system, making it difficult to control. Phages cooperate with traditional antibiotics such as depolymerase and virion associated peptidoglycan hydrolases (VAPGHs). These enzymes can degrade substances such as bacterial peptidoglycan, capsular polysaccharide, and lipopolysaccharide to assist phages in quickly destroying the integrity of bacterial biofilms, which is one of the important mechanisms against bacterial biofilms.

[0071] Inoculate Escherichia coli in the logarithmic growth phase into 5 mL of LB medium and culture it at 37 °C for about 20 h until the bacterial concentration reaches OD 600= 1. Centrifuge to collect the bacterial cells, resuspend and wash the bacteria with LB + 0.5% glucose, and adjust the OD value again. Take 100 μl of the above dilution and add it to a 96-well plate. Additionally, add 100 μl of the phage simultaneously. Set up a control group and place it in an incubator at 37°C for static incubation for 24 h. The next day, gently wash away the floating bacteria with PBS. Add 200 μl of 0.1% crystal violet to the 96-well plate, incubate at 37°C, and then remove the crystal violet. After washing with PBS, add 200 μl of 95% ethanol and incubate on a shaker at 37°C until completely dissolved. Measure the absorbance at a wavelength of 600 nm. At the same time, referring to the procedures of Diskey and Perrot et al., the effect of the phage on the number of bacteria in the biofilm was measured. The 96-well plate was statically incubated in an incubator at 37°C for 24 h. After washing with PBS, use an appropriate pipette tip to blow and disrupt the biofilm of the bacteria in the wells. Finally, add it to pre-cooled PBS, perform serial dilutions, spread the bacteria, and conduct colony counting. Set up three parallel experiments.

[0072] Through measurement, it was found that among the multiple phages screened in the present invention, some phages (vB_EcoM_P8, vB_EcoM_P54, vB_EcoM_P48) showed no obvious inhibitory effect on the bacteria in the biofilm, while when the phage vB_EcoM_P66 was applied, it had an obvious inhibitory effect on the bacteria in the biofilm (as Figure 3 shown). Further, its antibacterial effect was compared with that of the antibiotic enrofloxacin (FQ) (both were the number of bacteria at a concentration of 10 -4 ). As Figure 3 -B shows, the antibacterial effect of the phage vB_EcoM_P66 of the present invention was significantly better than the antibiotic level under the same conditions.

[0073] 8. In vitro antibacterial experiment of phage

[0074] Measure the ability of the phage to inhibit the growth of the diarrhea strain ETEC-K88 and the multi-drug resistant Escherichia coli A240 in vitro. Centrifuge the bacterial suspension in the logarithmic growth phase, and wash the cell pellet 3 times with sterile PBS. Then resuspend the bacterial pellet in 100 μL of LB medium to a final concentration of 1×10 6 CFU / mL, and mix it with 100 μL of a single phage or a phage cocktail preparation of different combinations in a 96-well microplate at an MOI of 0.1. Place it in a microplate reader and incubate at 37°C and 220 rpm for 6 h. Measure the absorbance (OD 600 ) of the mixture at 600 nm every 1 h. Only add LB medium to the bacterial solution as a control group. Set up three parallel experiments.

[0075] The results are as Figure 4As shown, the phage vB_EcoM_P66 showed significant antibacterial effects against both the diarrhea strain ETEC-K88 and the multidrug-resistant Escherichia coli A240.

[0076] 9. Detection by phage double-layer plate spot method

[0077] Transfer the following target strains at a ratio of 1:100 (v / v) respectively: enterotoxigenic Escherichia coli ETEC, enteropathogenic Escherichia coli EPEC, and enterohemorrhagic Escherichia coli EHEC, and culture them in a shaker at 37°C until they grow to the logarithmic growth phase. Pour a layer of solid LB medium in a sterile petri dish, which is the single-layer plate; then pour 6 - 8 mL of semi-solid LB medium, and wait until it cools to about 40°C, and add the bacterial suspension, which is the double-layer plate. Take 2 - 5 μL of the phage stock solution and spot it on the double-layer plate, and culture it in an incubator at 37°C for 8 - 12 h, and observe whether the double-layer plate contains plaques. The positions with plaques are clearer or completely transparent compared to other positions.

[0078] The results are as Figure 11 shown. The results showed that vB_EcoM_P66 also had excellent antibacterial effects against the other two pathogenic Escherichia coli (enteropathogenic Escherichia coli EPEC / enterohemorrhagic Escherichia coli EHEC) that cause piglet diarrhea except for enterotoxigenic Escherichia coli ETEC.

[0079] 10. Determination of phage resistance

[0080] Evaluate the control effect of phages on multidrug-resistant Escherichia coli and diarrhea strain infections through in vitro experiments, and then explore the preventive or delaying effect of the cocktail therapy on phage-resistant mutants: Wash and resuspend Escherichia coli in the logarithmic phase with sterile PBS, and the concentration of the bacteria is about 1×10 8 CFU / mL. Continuously dilute the bacteria 10-fold with sterile PBS: 10 0 -10 -7 . Divide the dilution solutions of each gradient into two groups. The dilution solution of group A is used for counting the colonies of the original strain, and group B is the phage-resistant mutant strain group, that is, take 100 μm of the dilution solutions of 10 0 -10 -7 each and mix them with the phage solution, and spread them on the LB solid plate. The colonies grown on this plate are phage-resistant mutants. Incubate all the plates overnight in an incubator at 37°C, and count the colonies on the plates the next day. The generation frequency of phage-resistant mutants is the number of colonies in group B / the number of colonies in group A, and set up three parallel experiments.

[0081] Multiple phages screened in this invention show different resistance mutation frequencies to the host. Among them, the resistance mutation frequencies MF of vB_EcoM_P20 and vB_EcoM_P48 are both 1, that is, the resistance mutation rate of the host bacteria to the phage is 1:1, indicating that this phage easily causes mutations in bacteria, that is, the host Escherichia coli is extremely likely to produce drug-resistant mutations. However, phage vB_EcoM_P66 shows a lower frequency of resistance mutation. The results are as Figure 5 shown. The resistance mutation frequency MF of phage vB_EcoM_P66 is 0.033.

[0082] Furthermore, multiple phages screened were made into different cocktail combinations to observe their resistance mutation frequencies. The results showed that when vB_EcoM_P20 and vB_EcoM_P48 were included, the resistance mutation frequency of bacteria to the phage was significantly increased. However, no matter what cocktail combination it was, as long as vB_EcoM_P66 appeared in the combination, the occurrence of the resistance mutation rate could be significantly reduced. Among them, the cocktail combination of vB_EcoM_P8 (genome accession number: PV390659.2 (https: / / www.ncbi.nlm.nih.gov / nuccore / PV390659)), vB_EcoM_P54 (genome accession number: PV390658.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / PV390658)) and vB_EcoM_P66 was the best. This combination was selected, and the three phages were mixed in equal proportions to make a cocktail combination for subsequent animal experiments.

[0083] Example 3 Animal verification experiment

[0084] 1. Mouse experiment

[0085] 1.1 Establishment of a mouse diarrhea model: In the process of studying piglet diarrhea, the mouse model is an important method. Successfully establishing a diarrhea model is the basis of the experiment. By observing the preventive treatment effect of phages on the mouse diarrhea model, the effectiveness and safety of phages are evaluated.

[0086] Using ETEC-K88 to establish a mouse diarrhea model: Divide 6-week-old male BALB / C mice into three different groups. 1) Control group: Fed with ordinary mouse food and given free drinking water. 2) Phage prevention group: Fed with phage-treated mouse food and given free drinking water. 3) Challenge group: Fed with ordinary mouse food and given free drinking water. The mice in the phage prevention group ate 10 6Mouse food and drinking water soaked with phages at 8 PFU / mL. After 7 days of feeding, mice in the phage prevention and challenge groups were challenged with the ETEC-K88 strain. During the entire experiment, the body weight, mental state, and diarrhea status of each group of mice were strictly observed. All experimental mice were housed in the Experimental Animal Center of Wuhan National Bioindustry Base. The indoor temperature of the animal room was 18-25 °C, and the relative humidity was 70-80%. After 3 days of adaptive feeding, the mice were fasted and water-deprived for 4 h, then gavaged with 0.1 ml of streptomycin aqueous solution, and then fed with food and water. At the same time, timing started. After 20 h of gavage with streptomycin, the mice were fasted and water-deprived again for 3.5 h, and then gavaged with 0.2 ml of 3%

[0087] 1.2 Safety assessment of phages in mice: By successfully establishing a mouse diarrhea model using Escherichia coli ETEC-K88 (determining that 5×10 8 CFU / mL is the optimal infection concentration for modeling), the effectiveness and safety of phages in treating diarrhea mice were evaluated.

[0088] The experiment was divided into three groups, namely: 1) Control group: Normal feeding without any intervention; 2) Phage prevention group: After prophylactically feeding experimental mice with phages for 7 days, 3) Challenged with Escherichia coli K88; ETEC challenge group: After normal feeding for 7 days, challenged with Escherichia coli K88. First, the three phages in the cocktail combination were diluted at different ratios of 1:10 / 100 / 1000 / 10000, that is, the phage titer was 10 8 / 10 7 / 10 6 / 10 5PFU / mL. Next, the diluted phages with different titers were used to prophylactically feed mice by soaking mouse food for 7 days to evaluate the effects of phages on the efficacy and safety in mice. During the feeding period, mouse feces were collected daily and the number of viable phages in the feces was detected. We observed that phages with different titers could be detected in the feces from the first day to the seventh day when mice consumed phages, and the titer of phages increased with the increase of the number of days of administration. During this period, no abnormal manifestations were observed in the mice in the phage prophylaxis group, including food intake, body weight, and mental state, indicating that the phages were safe in mice and had no acute toxic effects. After 7 days of feeding, mice were intragastrically administered with K88 and the phages in the feces were detected. The results showed that the phage titer in the feces of the challenged mice increased significantly. This indicated that the diarrhea model was successfully constructed, and the prophylactically administered phages could successfully colonize in mice and their titer increased significantly after mouse diarrhea.

[0089] In addition, the expression levels of inflammatory factors such as TNF-α and IL-6 in the serum of each group of experimental mice and the pathological changes of the ileum tissue of mice were also detected ( Figure 7 ): Compared with the control group, the inflammatory factors in the challenged group of mice all increased, while the inflammatory factors in the phage prophylaxis group of mice decreased to varying degrees, and the antibacterial effect was the most significant at a titer of 1:1000 (10 6 PFU / mL). In addition, through the observation of the pathological sections of the ileum tissue, it was found that the intestinal mucosal villi of the control group of mice were complete and plump. The intestinal villi of the ETEC group of mice were significantly swollen and broken, and the number of goblet cells decreased, suggesting that it might be related to the reduced glycogen synthesis and slower absorption caused by the colonization of K88 in the mouse intestine and the resulting inflammation. The goblet cells and intestinal villi of the phage prophylaxis group of mice basically returned to normal and there was no obvious swelling, indicating that the prophylactic administration of phages to mice played a prophylactic and protective role.

[0090] In summary, the mouse experiment confirmed that the phage prophylaxis group could significantly inhibit the colonization of Escherichia coli K88 in mice and reduce the level of inflammatory factors in the body.

[0091] 2. Piglet experiment

[0092] 2.1 Establishment of piglet diarrhea model: 18 healthy weaned large three-way piglets at 14 days of age were raised in the animal breeding center of the College of Animal Science and Technology, Huazhong Agricultural University. They were randomly divided into 3 groups, with 6 piglets in each group. The control group: fed with ordinary diet and drank water freely. The phage prophylaxis group: consumed 10 6The diet and drinking water soaked with phage cocktail at PFU / mL. Challenge group: fed with normal diet and had free access to water. After 7 days of feeding, piglets in the phage prevention group and the challenge group were challenged with K88 strain. The challenge method was basically the same as the method of "establishment of mouse diarrhea model" mentioned above. The dosage of antibiotics and pathogenic bacteria for challenge was converted according to the body weight of piglets. The animal care and procedures used in this study were carried out according to the guidelines of the Chinese Animal Protection Association. The experimental pigs were fed in separate pens with the same equipment, and the piglets could freely eat and drink during the feeding period. The health status and diarrhea of piglets were observed and recorded regularly every day during the experimental period, and diseases were treated in time. During the experiment, the feces of piglets were observed every day and scored. 0 point: feces in strip or granular form; 1 point: soft feces, can form; 2 points: thick, unformed, feces and water not separated; 3 points: liquid, unformed, feces and water separated. Piglets were judged to have diarrhea when the fecal score was 2.

[0093] At the end of the experiment, the diarrhea index and diarrhea frequency of each group during the experimental period were counted. Diarrhea index (%) = 100 × total diarrhea score of piglets in each group during the experimental period / number of piglets in each group; Diarrhea frequency (%) = total number of diarrhea times / (total number of experimental pigs × number of experimental days) × 100%. On the first and last days of the experiment, each group of piglets was weighed. The piglets were fasted for 12 h before weighing, and the initial weight and final weight of the piglets were recorded. The feed consumption and mortality of piglets were recorded in detail during the whole experimental period, and the relevant indexes of piglet growth performance were calculated after the feeding experiment. At the same time, blood samples were collected from the anterior vena cava of each group of piglets, and the main inflammation-related immune factors such as TNF-α and IL-6 were detected by ELISA technology using a full-wavelength multifunctional microplate reader. On the last day of the experiment, after each group of piglets was sacrificed, the terminal ileum tissue was taken back, prepared into pathological sections by HE staining, and the pathological changes and immunofluorescence staining were observed. During the feeding period of piglets, fecal samples were collected from all groups every day, and the intestinal contents were collected on the day of sacrifice and stored at -80 °C. The abundance of K88 bacteria in feces was quantitatively detected by qPCR.

[0094] The results are as Figure 9As shown, the ileal mucosal villi of the piglets in the control group were complete and plump. In the ETEC group, there were inflammatory manifestations caused by K88 colonization in the intestine: obvious inflammatory cell infiltration in the piglets' intestine, severe damage to intestinal villi, and pathological changes such as cell necrosis. In the phage prevention group, the intestinal villi of the piglets were complete and there were basically no pathological changes, indicating that the phages were successfully colonized in the intestine and effectively reduced the invasion of pathogenic bacteria, playing a certain protective role in the piglets' intestine. The intestinal morphology of pigs, especially the length of villi and the depth of crypts, is an important indicator affecting pig intestinal health and growth performance. Further findings showed that the length of the ileal villi in the ETEC group of piglets was significantly shortened, the depth of crypts became higher, and the ratio of villus length to crypt depth decreased. The villus height of the piglets in the phage prevention group was significantly increased and the crypt depth became shallower, indicating that the phages effectively reduced the occurrence of intestinal inflammation, protected the intestinal barrier function, and provided mucosal immunity from non-host sources. In addition, Zonula occludens 1, Zo-1 is one of the important components constituting tight junctions. The down-regulation of its expression or the decrease in its activity will affect the formation of tight junctions between cells and hinder the intestinal mucosa from playing an important defense barrier function, thereby increasing the risk of intestinal infection caused by the penetration of harmful bacteria and toxins through the intestine into the blood. The expression of Zo-1 in the intestinal tract of piglets in different treatment groups was observed by immunofluorescence staining method ( Figure 8 ): The fluorescence intensity of the ileal tissue of the piglets in the control group was strong, and the cell membrane boundary was clear, showing a linear distribution. The fluorescence intensity in the ETEC group was weakened, the density decreased, and the linear structure was broken or even disappeared, significantly down-regulating the expression of Zo-1 protein. Although the fluorescence intensity in the phage prevention group was weakened to some extent, the structure remained intact. This again proved that the phages played an important protective and alleviating role in Escherichia coli-induced piglet diarrhea, maintained the normal morphological structure of the intestine, reduced the permeability of the small intestinal mucosa at the same time, and enhanced the tight junction function of the piglet intestine.

[0095] In summary, the phage prevention group can significantly inhibit the colonization of Escherichia coli K88 in piglets, reduce the levels of inflammatory factors in the body (TNF-α / IL-6 / Zonulin), maintain the normal morphological structure of the intestine, reduce the permeability of the small intestinal mucosa at the same time, and enhance the tight junction function of the piglet intestine.

[0096] The above specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all belong to the protection scope of the present invention.

Claims

1. A multi-drug resistant Escherichia coli phage vB_EcoM_P66 was deposited at the China Center for Type Culture Collection on December 12, 2024, with the deposit number: CCTCC M 20242794.

2. The phage according to claim 1, characterized in that, The phage can maintain the structural and functional integrity between 4 - 60 °C; the optimal pH of the phage is 5 - 11; the latent period of the phage growth is 20 min, the lysis period is about 50 min, and the burst size is 120 PFU / Cell.

3. A phage preparation containing any one of the phages described in claims 1 - 2.

4. The bacteriophage preparation according to claim 3, characterized in that, The phage preparation further includes: phages vB_EcoM_P8 and vB_EcoM_P54.

5. The bacteriophage preparation according to claim 3, characterized in that, The phage preparation is a drug, feed additive, drinking water additive, environmental cleaner or disinfectant.

6. The bacteriophage preparation according to claim 3, characterized in that, The dosage form of the phage preparation is a liquid preparation, powder preparation or oral solid preparation.

7. A method for preparing the multi-drug resistant Escherichia coli phage vB_EcoM_P66 according to any one of claims 1-2, characterized in that: Inoculate multi-drug resistant Escherichia coli into a liquid medium and culture it until the logarithmic growth phase, then inoculate any one of the multi-drug resistant Escherichia coli phages vB_EcoM_P66 described in claims 1 - 2 for culture.

8. The method according to claim 7, wherein The culture temperature of the phage is 37 °C; the pH range for phage culture is 5 - 11; the culture time of the phage is 4 - 6 h.

9. Use of any one of the phages described in claims 1 - 2 or any one of the preparations described in claims 3 - 6 in any of the following: A1) Preparation of a bactericide for diarrhea-related Escherichia coli or multi-drug resistant Escherichia coli; A2) Preparation of a feed additive, drinking water additive, environmental cleaner or disinfectant; A3) Preparation of a biological preparation for preventing or treating multi-drug resistant Escherichia coli infection; A4) Preparation of a product for preventing or treating piglet diarrhea; A5) In vitro lysis of pathogenic Escherichia coli or multi-drug resistant Escherichia coli; A6) Preparation of a product for preventing or treating piglet diarrhea caused by Escherichia coli of ST10, ST48, ST58, ST101 types or ETEC-K88, EPEC, EHEC.

10. A method for disinfecting an environment with multi-drug resistant Escherichia coli, characterized in that, Prepare a liquid solution of any one of the phages described in claims 1 - 2 or any one of the preparations described in claims 3 - 6 for spraying.

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