Short-tailed virus T7 phage S11 for splitting escherichia coli and application of short-tailed virus T7 phage S11
By developing the T7 phage S11, the problem of diarrhea in calves caused by drug-resistant E. coli is difficult to prevent and treat, and efficient, safe and environmentally friendly treatment effects are achieved.
Patent Information
- Application Number
- CN202411887423.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-25
AI Technical Summary
The prior art is difficult to effectively prevent and treat diarrhea in calves caused by drug-resistant E. coli, and traditional antibiotic treatments have drug resistance problems, probiotic regulation effect is mild, and vaccine development cycle is long.
A bacteriophage S11 with strong specificity was developed, which can efficiently lyse E. coli. The bacteriophage S11 has the characteristics of high titer, good stability, strong broad spectrum, difficult to produce drug resistance, safe, environmentally friendly, fast and efficient.
Phage S11 can reproduce in large quantities in a short period of time, quickly control bacterial infection, shorten the treatment cycle, effectively prevent and treat diarrhea in calves, and avoid drug residues and drug resistance problems.
Smart Images
Figure CN119685269A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a short-tailed virus T7 bacteriophage S11 for lysing Escherichia coli and an application thereof. Background Art
[0002] Escherichia coli (E. coli) is the most common conditional pathogen in clinical practice, usually causing Escherichia coli disease in a variety of animals, mostly in young livestock and poultry, and the economic losses caused by the disease are hundreds of millions each year. Diarrhea in newborn calves is one of the important causes of calf death, and enterotoxigenic Escherichia coli is one of the main pathogens causing calf diarrhea. Calf diarrhea is a common disease in the process of calf breeding, accounting for about 80% of calf diseases, and is one of the main diseases causing calf growth retardation and death. Calf diarrhea can not only cause a high mortality rate, but also calves that have recovered will have low immunity, easy to stimulate respiratory diseases, slow growth and development, etc. Antibiotics were once the first choice for treating Escherichia coli disease in livestock and poultry breeding. However, with the long-term and widespread use of antibiotics, drug-resistant Escherichia coli has rapidly emerged and spread, and a large number of multi-drug-resistant and extensively drug-resistant strains have been reported in clinical practice. The global spread of antibiotic resistance has become a social public safety issue that threatens the health of humans and animals, and it is urgent to find new antibacterial agents to prevent and control the spread and infection of drug-resistant bacteria. Traditional antibiotic treatment: For a long time, antibiotics have been the main means of combating E. coli infection, controlling the disease by inhibiting the growth of bacteria or directly killing bacteria. However, with the widespread use of antibiotics, the problem of bacterial resistance has become increasingly serious. Probiotic regulation: Some probiotics are used to regulate intestinal flora to inhibit the growth of E. coli, but the effect of probiotics often varies from person to person and the effect is relatively mild. Vaccine development: Although vaccines against E. coli are under development, there is currently no commercialized vaccine. The vaccine development cycle is long and needs to be constantly updated to cope with bacterial mutations.
[0003] Bacteriophage is a virus that can specifically lyse bacteria. It has strict host specificity, can kill host cells without developing drug resistance, and has the advantages of exponential proliferation, wide distribution, and short development time. At present, 96% of the discovered bacteriophages belong to the order Caudovirales, which includes three families: Brachyuraviridae, Longuraviridae, and Myoviridae. A typical bacteriophage contains an icosahedral head, a hollow needle-like structure, a tail composed of an outer sheath, and a base composed of a tail filament and a tail needle.
[0004] In recent years, bacteriophages, as a new type of antimicrobial agent, have made great progress in the prevention and treatment of foodborne pathogenic bacteria infections, controlling the spread of pathogens, and treating drug-resistant bacterial infections. Phage therapy refers to the use of bacteriophages to treat bacterial diseases in humans or animals. Studies have reported that when bacteriophages were used to treat acute diarrhea caused by E. coli in animals, the effect of using bacteriophages to treat E. coli diarrhea in mice was better than that of antibiotics. Therefore, in recent years, due to the lack of vaccines and special drugs for the prevention and treatment of calf diarrhea in China, efforts have been made to increase the research and development and promotion of antibiotic products. Summary of the invention
[0005] To solve the above technical problems, the present invention proposes a short-tailed virus T7 phage S11 for lysing Escherichia coli and its application, which can specifically lyse enterotoxigenic Escherichia coli from calf diarrhea, and the phage S11 has high titer and good stability.
[0006] To achieve the above-mentioned purpose, the present invention provides a short-tailed virus T7 phage S11 for lysing Escherichia coli. The short-tailed virus T7 phage S11 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO.46209.
[0007] The present invention also provides the use of the short-tailed virus T7 bacteriophage S11 in preparing products for preventing and / or treating diarrhea or infection caused by enterotoxigenic Escherichia coli in young animals.
[0008] Preferably, the young animals are one or more of piglets, lambs or calves.
[0009] Preferably, the short-tailed virus T7 phage S11 prevents and / or treats diarrheal infections in young animals by highly specifically lysing enterotoxigenic Escherichia coli strains.
[0010] The present invention also provides a product for preventing and / or treating diarrhea or infection caused by enterotoxigenic Escherichia coli in young animals, comprising the short-tailed virus T7 phage S11.
[0011] Preferably, the total content of the short-tailed virus T7 bacteriophage S11 in the product is ≥ 2×10 9 PFU / mL.
[0012] Preferably, the products include medicines, feed additives, cleaning agents and disinfectants.
[0013] Preferably, the preparation forms of the product include a dosage form for gastrointestinal administration and a dosage form for parenteral administration.
[0014] The present invention also provides the use of the short-tailed virus T7 bacteriophage S11 in the preparation of a preparation for inhibiting enterotoxigenic Escherichia coli.
[0015] The present invention also provides a preparation for inhibiting enterotoxigenic Escherichia coli, comprising the short-tailed virus T7 phage S11.
[0016] Compared with the prior art, the present invention has the following advantages and technical effects:
[0017] The present invention has isolated a short-tailed virus T7 phage S11 (phage S11) with strong specificity and the ability to lyse drug-resistant Escherichia coli. The phage has the potential to be used in the prevention and treatment of calf diarrhea. The phage has a short incubation period, high lysis efficiency, and is resistant to low and high temperatures as well as acid and alkali. (1) Strong broad-spectrum: phage S11 has a high degree of specificity for highly pathogenic Escherichia coli strains and can accurately attack target bacteria without affecting other beneficial bacteria. (2) Not prone to drug resistance: Since the mechanism of action of phage S11 is different from that of antibiotics, it is difficult for bacteria to develop drug resistance to phage, thereby ensuring long-term treatment effects. (3) Safety and environmental protection: phage S11 is a naturally occurring microorganism that is harmless to the human body and the environment and does not produce problems such as drug residues. (4) High efficiency and rapidity: phage S11 can reproduce in large quantities in a short period of time, quickly control bacterial infections, and shorten the treatment cycle.
[0018] In conclusion, the bacteriophage S11 of the present invention specifically lyses Escherichia coli and has a high titer of 2×10 9 PFU / mL; good stability, the phage titer remained basically unchanged after being treated in a water bath at -20℃, 4℃, and 37℃ for 60 minutes, and there was no significant effect on the activity of phage S11 under pH 5-10 conditions; it has a broad-spectrum and efficient lysis effect on pathogenic Escherichia coli from calf diarrhea; it has a good therapeutic effect on the calf diarrhea model infected with artificial enterotoxigenic Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0020] Figure 1 The morphological characteristics of the short-tailed virus T7 bacteriophage S11, wherein A is the morphological diagram of the short-tailed virus T7 bacteriophage S11, and B is the transmission electron microscope diagram, with a scale of 200 nm;
[0021] Figure 2It is a biological characteristic determination diagram of short-tailed virus T7 bacteriophage S11, wherein A is a multiplicity determination diagram of short-tailed virus T7 bacteriophage S11, and B is a one-step growth curve diagram of short-tailed virus T7 bacteriophage S11;
[0022] Figure 3 This is a stability determination diagram of short-tailed virus T7 bacteriophage S11, where A is thermal stability and B is acid-base stability;
[0023] Figure 4 This is the annotated genome map of the short-tailed virus T7 bacteriophage S11;
[0024] Figure 5 This is the evolutionary phylogenetic tree of the short-tailed virus T7 phage S11. DETAILED DESCRIPTION
[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.
[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0030] Example 1
[0031] 1.1 Methods
[0032] 1.1.1 Strains and growth conditions
[0033] A total of 60 samples of sewage were collected from cattle farms around Daqing City, Heilongjiang Province. Each sample was treated as follows: 1 mL of the collected sewage was drawn with a sterile 1 mL syringe and transferred to a 1.5 mL micro reaction tube. Each sample of sewage was transferred to two tubes. The two tubes of sewage samples were placed in a high-speed centrifuge and centrifuged at 12000 r / min for 5 minutes. The supernatant of one tube of sewage sample was extracted with a pipette and transferred to a new 1.5 mL micro reaction tube and stored at -20°C. The liquid component of the other tube was poured out and placed on a clean bench. Irradiate with ultraviolet light 30 minutes in advance, turn off the ultraviolet lamp and light the alcohol lamp, fully burn the inoculation loop, dip a small amount of precipitate and streak it on MacConkey medium and ss medium, culture it in a constant temperature incubator at 37℃ overnight to isolate Escherichia coli in the environment, use a sterile inoculation loop to pick a single Escherichia coli colony grown on the plate and inoculate it into TSB liquid culture medium, culture it in a constant temperature shaker (37℃) overnight, and preserve the purified Escherichia coli (ECS11 bacterial liquid).
[0034] 1.1.2 Isolation of bacteriophage
[0035] Use a pipette to take 100 μL of the separated ECS11 bacterial solution and add it to 10 mL of 45°C agar. Shake well to mix, pour into a sterile glass dish, wait for the agar-ECS11 to completely solidify, centrifuge the supernatant stored in 1.1.1 at 12000 r / min for 5 min, pass the supernatant obtained by centrifugation through a 0.22 μm microporous filter membrane to obtain a filtrate, use a pipette to transfer 5 μL of the filtrate droplet to the solidified agar-ECS115, let it stand for 5 min, and then culture it in a 37°C constant temperature incubator for 6 h. If there are bacteriophages in the sewage sample, plaques will form on the surface of the agar culture medium.
[0036] 1.1.3 Phage purification
[0037] Use a sterile pipette tip to pick up a single plaque, and inject the sterile pipette tip into an EP tube filled with 1 mL of normal saline. Use a vortex shaker to vortex for 10 seconds, and then perform high-speed centrifugation at 12000r / min for 5 minutes. Take the supernatant after centrifugation, filter it through a 22μm microporous filter membrane to obtain the filtrate, repeat step 1.1.2 to obtain plaques, and repeat step 1.1.3 three times to obtain purified plaques of the same morphology and size.
[0038] 1.1.4 Phage morphology
[0039] The morphology of the phage was analyzed by transmission electron microscopy (TEM). The filtered high titer phage particles were dropped on a copper grid and negatively stained with 2% phosphotungstic acid. The excess liquid was aspirated and the grid was observed under a FEI T12 transmission electron microscope.
[0040] 1.1.5 Phage titer determination
[0041] Light an alcohol lamp in a clean bench that has been irradiated with ultraviolet light for 30 minutes in advance. Use a sterile gun tip to pierce a single phage plaque next to the lit alcohol lamp and insert it into 1000μL of saline for blowing and suction. After blowing and suctioning evenly, phage liquid is obtained. Pipette 100μL of phage liquid and add it to 900μL of saline. This liquid is a 10-fold diluted phage liquid. Continue to perform gradient dilution to obtain 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 Use a pipette to pipette 100 μL of the above 6 kinds of gradient diluted phage liquids into 10 mL of semi-solid culture medium melted at 50°C, and add 100 μL of ECS11 bacterial liquid in 1.1.1 to each, mix well and pour into the semi-solid agar medium, let it stand for 10 min.
[0042] 1x10 19 CFU / mL host E. coli solution and 1x10 12 PFU / mL phage solution was prepared according to the optimal infection multiplicity, incubated at room temperature for 100 minutes, and samples were taken every 10 minutes. The phage titer was determined by the double-layer agar plate method. A one-step growth curve was drawn with titer as the ordinate and culture time as the abscissa to determine the incubation period and lysis period, and the lysis amount was calculated according to the following formula:
[0043] Lysis amount (PFU / mL) = phage titer at the end of lysis: initial host E. coli bacterial concentration.
[0044] 1.2 Analysis of phage biological characteristics
[0045] 1.2.1 Determination of optimal phage infection multiplicity (MOI)
[0046] Take 100 μL of 1x10 10CFU / mL host Escherichia coli liquid, mix the phage with the host Escherichia coli liquid at a MOI of 0.001, 0.01, 0.1, 1, 10, 100, and 1000, add an equal volume of LB broth, culture on a constant temperature shaker for 6 hours, centrifuge at 10000g for 10 minutes, take the supernatant and filter it with a 0.22μm filter membrane, and determine the phage titer by double-layer agar plate method. The MOI with the highest phage titer at the end of culture is the optimal MOI.
[0047] 1.2.2 One-step growth curve drawing of bacteriophage
[0048] E. coli and isolated phage were mixed at the optimal MOI and placed in a shaker at 37°C. Every 10 minutes, 1 mL of the mixture was centrifuged at 10,000 r / min for 5 minutes, the supernatant was filtered with a 0.22 μm microporous filter, and the titer of the bacteria was determined by the double-layer agar plate method. The test was repeated 3 times at each time point.
[0049] Lysis amount = phage titer at the end of the outbreak / host bacteria concentration at the beginning of infection.
[0050] 1.2.3 Phage thermal stability
[0051] The phage liquid was divided into 4 groups, each with 6 mL, and placed in water baths at -20℃, 4℃, 37℃, 50℃, 60℃, and 80℃ respectively. 1 mL of the bacterial liquid was taken out every 10 minutes and diluted 10 times to prevent high temperature from affecting the experimental results. It was temporarily stored at 4℃. After all the sampling was completed, the liquid was taken out uniformly for multiple dilution and the phage titer was determined. Repeat 3 times.
[0052] 1.2.4 Phage pH stability.
[0053] Take 1 mL of bacterial liquid and add it to 9 mL of nutrient broth with different pH values of 1 to 14. Incubate at 37°C for 1 hour. Take it out and dilute it in multiples. Determine the phage titer using the method in 1.2.3. Repeat 3 times.
[0054] 1.3 Determination of bacterial host range
[0055] All other E. coli stored in the laboratory were revived and cultured to the logarithmic phase. 50 μL of bacterial solution was added to a small test tube and then 5 mL of semi-solid culture medium (50°C) was added. After mixing, the semi-solid culture medium was spread on the agar plate. After solidification, 2 to 3 drops of bacterial solution were added to the center. After drying, the plate was inverted and cultured at 37°C overnight. The next day, the center of each plate was observed to see if there was a bacterial plaque.
[0056] 1.4 Whole-genome analysis of bacteriophages
[0057] The whole genome of the extracted phage was sent to Beijing Novogene Technology Co., Ltd. for whole genome sequencing. Sequencing was performed using the umina NovaSeg platform, and the sequencing data were spliced using A5-MiSeg (http: / / arxiv.org / abs / 1401.5130) and SPAdes (http: / / cab.spbu.ru / ilesrelease3.12.0 / manual.html). GeneMarkS software (http: / / topaz.gatech.edu / ) was used to predict bacterial coding genes, RepeatMasker software was used to predict interspersed repeat sequences, and TRF was used to search for tandem repeat sequences in DNA sequences. The predicted genes were compared with the ARDB, CAZy, COG, GO, NR, and Swiss-Prot databases to obtain annotation information to find their drug resistance genes, carbohydrate-related enzyme family and other activities. The phage full gene sequence and the major capsid protein gene sequence were compared at NCBI, and the phage genome sequence with high homology was downloaded, and the phylogenetic tree was constructed using MEGA 11.0 software.
[0058] 2 Results
[0059] 2.1 Isolation and titer determination of bacteriophage
[0060] like Figure 1 As shown in Figure A, a virulent phage was isolated from untreated sewage from a hospital and named short-tailed virus T7 phage S11 (hereinafter referred to as phage S11). After purification using the double-layer plate method, a purified single phage was obtained. The plaque of phage S11 is round, transparent, and has no halo, with a diameter of about 1 to 2 mm. By calculation, it can be concluded that the titer of phage S11 is about 2×10 9 PFU / mL.
[0061] 2.2 Phage morphological observation
[0062] like Figure 1 As shown in B, bacteriophage S11 has a typical tadpole-like appearance, including a regular icosahedral head with a diameter of about 100 nm and a retractable spirally symmetrical tail. According to the classification rules of the ICTVdb database, bacteriophage S11 belongs to the Tevenvirinae subfamily of the Myoviridae family (Caudovirales).
[0063] 2.3 Determination of the optimal infection multiplicity of phage
[0064] like Figure 2As shown in A, when the infection multiplicity of phage S11 and host bacteria is 0.001, the number of progeny phages produced is the largest, with a titer of 2.6×10 10 PFU / mL, indicating that the optimal infection multiplicity of bacteriophage S11 is 0.001.
[0065] 2.4 One-step growth curve of bacteriophage and determination of burst size
[0066] Take 500 μL of the host E. coli liquid and phage liquid corresponding to the optimal infection multiplicity of 0.001, and measure the titer of phage at different times. Draw a growth curve with time as the horizontal axis and the logarithm of phage titer as the vertical axis. The results are as follows Figure 2 As shown in B, the incubation period of phage S11 is about 15 minutes, and the lysis period is about 60 minutes.
[0067] 2.5 Determination of phage stability
[0068] 2.5.1 Phage stability at different temperatures
[0069] This experiment tested the activity of bacteriophage S11 at -20℃, 4℃, 37℃, 50℃, 60℃ and 80℃ within 1 hour. Figure 3 As shown in A, after treatment in a water bath at -20°C, 4°C, and 37°C for 60 minutes, the bacterial titer remained basically unchanged; as the temperature increased, the titer of bacteriophage S11 decreased significantly after treatment in a water bath at 50°C and 60°C for 60 minutes, and the titer of bacteriophage S11 could not be detected after treatment in a water bath at 80°C for 60 minutes.
[0070] 2.5.2 Phage stability at different pH
[0071] The results of the test of the survival of bacteriophage S11 within 1 hour in an environment with a pH of 1 to 14 are as follows: Figure 3 As shown in B: Under conditions of pH 5-10, there is no significant effect on the activity of phage S11, and the phages all show high activity; under conditions of pH 4 or 11, the activity of the phages decreases slightly, but a large number of phages still survive; under conditions of pH <4 or pH>11, almost no surviving phages are detected.
[0072] 2.6 Phage host spectrum detection
[0073] 70 pathogenic Escherichia coli from calf diarrhea, 1 Pseudomonas aeruginosa, 1 Proteusbacillus vulgaris, 2 Klebsiella, and 4 Salmonella strains preserved in the Key Laboratory of Cattle Disease Control of Heilongjiang Province were all revived and cultured to the logarithmic phase, and lysis assays were performed using bacteriophage S11.
[0074] Table 1 Results of host spectrum detection of bacteriophage S11
[0075]
[0076] The results show that as shown in Table 1, bacteriophage S11 cannot lyse the above-mentioned Pseudomonas aeruginosa, Proteus, Klebsiella and Salmonella. However, it can lyse Escherichia coli with a lysis rate of 18.6% (13 / 70), including the identified serotypes O8 (n=2), O101 (n=4), O78 (n=3) and untyped Escherichia coli (n=4). The lysed Escherichia coli carry virulence genes including Irp-2 (GenBank Accession No. L18881), FyuA (GenBank Accession No. Z38065), K99 (GenBank Accession No. M35282), F41 (GenBank Accession No. M21788), STa (GenBank Accession No. KY581592) and Eaea (GenBank Accession No. U38618). This shows that bacteriophage S11 is specific to Escherichia coli and has a broad spectrum to different Escherichia coli strains.
[0077] 2.7 Phage genome analysis
[0078] The sequencing results showed that the total length of the bacteriophage S11 genome sequence was 40867bp. The GC content was 48.63%. From the distribution of gene length, it showed a diverse feature. Among them, there were 6 genes in 100-200bp, 11 genes in 200-300bp, 4 genes in 300-400bp, 6 genes in 400-500bp, 5 genes in 500-600bp, and 5 genes greater than or equal to 2000bp. This distribution of gene length may be related to the functional complexity of different genes.
[0079] Gene type and quantitative functional analysis of biological functions, cell structure, and molecular functions of bacteriophage S11: In terms of biological functions, bacteriophage S11 has 9 genes involved in cellular processes and metabolic processes, which reflects its influence and intervention ability on the physiological metabolism of host cells. The 3 genes involved in biological processes and the 3 genes involved in biological regulation of microbial processes jointly construct the biological regulation network of bacteriophages in host cells. At the same time, the 1 gene involved in negative regulation of cellular processes and response to stimuli provides an important guarantee for the phage to adapt to changes in the host cell environment. In terms of cell structure, the 12 genes that constitute the cell structure ensure the integrity of the phage's own structure. The 9 genes with catalytic activity and the 8 genes with binding function play a key role in the molecular functions of the phage, such as participating in the infection process of the phage to the host cell, the replication and transcription of nucleic acids, etc.
[0080] like Figure 4 The NR annotation results shown in the figure show that phage S11 has gene matches with a variety of organisms. The number of genes matching with Escherichia coli is as high as 26, which indicates that phage S11 has a close relationship with Escherichia coli at the genetic level, and may use these similar genes to achieve infection and replication in the process of infecting Escherichia coli. There are 5 matching genes with fungal phages, and a small number of gene matches with various organisms such as Massilia and Xylella fastidiosa, which reflects that phage S11 has complex genetic exchanges and interactions with different organisms during its evolution.
[0081] 2.8 Phage functional annotation
[0082] Using the BLAST nucleotide sequence alignment function of the NCBI database, the genomic DNA of phage S11 has a very high similarity with the genomic DNA of the phage of S. cerebroides (similarity 92.16%; coverage 89%; accession number: ON568193.1), which is the same as the BLAST nucleotide sequence alignment result, based on Figure 5 The phylogenetic tree of DNA polymerases shown shows that bacteriophage (PG) S11 and phages of Sedasidium are located in the same branch.
[0083] 3. Effect of bacteriophage S11 in treating calf diarrhea
[0084] Newborn calves were artificially infected with enterotoxigenic Escherichia coli HLJ20211027 from calf diarrhea (isolated from the intestines of newborn calves with diarrhea and dead calves with neurological symptoms at Mou Dairy Farm in Daqing City, Heilongjiang Province). Diarrhea occurred 24 hours after infection. Phage S11 was orally administered 6 hours after infection for 10 minutes each time. 11 PFU, continuous treatment for 3 days, the diarrhea was effectively controlled.
[0085] The bacteriophage S11 of the present invention has a high specificity for Escherichia coli, and can accurately identify and infect target bacteria without affecting the beneficial flora in the calf. Once the phage successfully infects the host bacteria, they can replicate rapidly in the bacteria and produce a large number of progeny phages, thereby effectively killing the bacteria. Unlike antibiotics, phages will naturally degrade after completing the bactericidal task and will not remain in the calf, avoiding the potential threat of drug residues to food safety and the environment. During the experiment, the stability of the phage was monitored. The results show that phage S11 can maintain good activity and stability under different environmental conditions (such as temperature, pH value, etc.), providing a guarantee for its practical application.
[0086] The whole genome of the bacteriophage S11 of the present invention is 40867bp long and contains 47 genes in total. In the annotation results obtained by comparing the ARDB, CAZy, COG, GO, NR, and Swiss-Prot databases, it is found that it contains GMB50 family members and GH23 family members, and GMB50 will attach to the GH23 family to play a role in cleaving chitin or peptidoglycan, thereby causing a gap in the bacterial cell wall. In the gene annotation obtained from the COG database, it was found that it contained a T7 phage lysozyme structure. Through the prediction results of the software EffectiveT3, it was found that it contained 2 proteins encoding the T3SS system. According to the transfection characteristics of the phage, it is inferred that the source of the ability of Escherichia coli to secrete exotoxins is likely to be related to phage infection, which makes it necessary to detect these genes that may lead to enhanced bacterial virulence when using phages to prevent the disease from being difficult to control. In the comparison results with the GO database, 1 gene related to nuclear chromosome composition, 3 genes related to DNA binding, 2 genes related to transcription, 4 genes related to DNA recombination, 2 genes related to nuclear structure, 2 genes related to enzymes related to cell wall synthesis, repair and degradation, 3 genes related to DNA helicase, 5 genes related to nucleoside binding, 6 genes related to DNA-dependent biological processes, 2 genes related to nuclease exonucleases, 1 gene related to viral capsid assembly, 1 gene related to ribonuclease activity, 1 gene related to coenzyme binding, and 1 gene related to pathogenesis were found. It can be seen that the survival mechanism of the bacteriophage S11 of the present invention focuses more on interfering with or promoting certain biological processes, which may make the process of virus lysing Escherichia coli complex but efficient; the comparison results with the NR database show that most of its proteins have homology with Escherichia coli proteins, which may be the result of the mutual promotion of evolution between bacteriophage and Escherichia coli in their respective different species. A total of 40 proteins were predicted in the Swiss-Prot database. Their protein functions mainly include destroying cell walls and cell membranes and injecting nucleic acids deep into cells in some way. When using the host for replication, related proteins are also involved in preventing the degradation of their own nucleic acids. These complex mechanisms undoubtedly enhance the lysis effect of the phage.
[0087] In summary, the present invention uses a double-layer agar plate method to isolate bacteriophage S11 from school sewage. It has good proliferation efficiency, short incubation period, cross-host lysis, and a wide range of temperature stability and acid-base stability. The optimal multiplicity of infection is 0.001, the incubation period is 15min, the lysis period is 60min, and bacteriophage S11 can survive at 60°C and pH 4.0-10.0. The full length of the bacteriophage S11 genome sequence is 40867bp. The GC content is 49.26%, which will have a good effect in treating calf diarrhea caused by highly pathogenic Escherichia coli. The bacteriophage S11 with a broad spectrum of hosts and strong specificity developed by the present invention has significant effects and broad application prospects in the treatment of calf diarrhea caused by Escherichia coli. It is expected to become an important means of treating calf diarrhea and even other bacterial infectious diseases, providing strong support for the healthy development of the breeding industry.
[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A short-tailed virus T7 bacteriophage S11 for lysing Escherichia coli, characterized in that: The short-tailed virus T7 phage S11 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 31, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO.46209.
2. Use of the short-tailed virus T7 phage S11 as claimed in claim 1 in the preparation of a product for preventing and / or treating diarrhea or infection caused by enterotoxigenic Escherichia coli in young animals.
3. The application according to claim 2, characterized in that: The young animals are one or more of piglets, lambs or calves.
4. The application according to claim 2, characterized in that: The short-tailed virus T7 bacteriophage S11 prevents and / or treats diarrhea infection in young animals by highly specifically lysing enterotoxigenic Escherichia coli strains.
5. A product for preventing and / or treating diarrhea or infection caused by enterotoxigenic Escherichia coli in young animals, characterized in that: It includes the short-tailed virus T7 phage S11 described in claim 1.
6. The product according to claim 5, characterized in that: The total content of the short-tailed virus T7 bacteriophage S11 in the product is ≥2×10 9 PFU / mL.
7. The product according to claim 5, characterized in that: The products include medicines, feed additives, cleaners and disinfectants.
8. The product according to claim 5, characterized in that: The preparation forms of the product include a dosage form for gastrointestinal administration and a dosage form for parenteral administration.
9. Use of the short-tailed virus T7 bacteriophage S11 as claimed in claim 1 in the preparation of a preparation for inhibiting enterotoxigenic Escherichia coli.
10. A preparation for inhibiting enterotoxigenic Escherichia coli, characterized in that: It includes the short-tailed virus T7 phage S11 described in claim 1.