Escherichia coli bacteriophage PEC-36 with cross-species host characteristic and application thereof

Abstract

The invention discloses an Escherichia coli bacteriophage PEC-36 with cross-species host characteristics and application of the Escherichia coli bacteriophage PEC-36, and belongs to the technical field of biology. The Escherichia coli bacteriophage PEC-36 is separated from sewage of a pig factory, can simultaneously split O8: H7, O21: H34, O23: H16, O25: H4, O81: H27 and H25 serotype strains of Escherichia coli and fowl typhoid, London, swine cholera and other serotype strains of salmonella, and has a cross-species splitting characteristic. Experiments prove that the bacteriophage has the advantages of high titer, high stability, high safety and the like, and can remarkably inhibit the growth of escherichia coli and salmonella, so that a technical support is provided for research and development of bacteriophage preparations for treating infection related to escherichia coli and salmonella; the invention provides a safe and efficient antibiotic alternative scheme for treating human and animal Escherichia coli and salmonella related infection, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an Escherichia coli phage PEC-36 with cross-species host characteristics and its application. Background Art

[0002] Phages are a type of virus. Like other viruses, their survival and production depend on hosts, and they have a high degree of host specificity and do not destroy normal flora. Since phages were discovered in 1915, researchers and clinicians have quickly recognized their potential as therapeutic agents for treating bacterial infections. Currently, most isolated phages have a narrow host range, targeting only one species, one strain, or one serotype, which limits the widespread application of phages. Therefore, it is of great significance to obtain some polyvalent phages with a broad host range. Currently, some methods have been developed to expand the host range of phages. One is to synthesize a phage library. However, before designing an engineered phage library, it is necessary to sequence the entire phage genome and identify the host range-determining regions in the tail fiber protein or tail spike. Another is rational genetic engineering or recombination. However, the synthesized polyvalent phages rely on phages that can infect their respective different hosts. Using phage evolution can also obtain polyvalent phages. Almost all evolved broad-host-range phages can only infect species within the same genus, and the direction of evolution is uncontrollable. In addition, through phage cocktail formulation, the problem can be solved to a certain extent, but it faces a substantial increase in production costs. Currently, few scientists have expanded the host range of phages to multiple bacterial genera or multiple serotypes through evolution.

[0003] In today's globalized ecological environment and closely connected food chain system, the spread and infection of microorganisms have become key factors that cross species boundaries and affect public health security. Escherichia coli and Salmonella, as two widely distributed foodborne pathogenic bacteria in nature, pose a significant threat to the health of animals and humans, which has attracted great attention and in-depth research in the scientific community, the agricultural field, and the medical and health industry.

[0004] Escherichia coli is one of the normal flora members in the intestines of humans and animals. Most strains are harmless, but certain specific serotypes, such as enterohemorrhagic Escherichia coli (EHEC), have powerful pathogenic capabilities. After animals are infected with pathogenic Escherichia coli, they often exhibit symptoms such as diarrhea, dehydration, loss of appetite, and even death, seriously affecting the economic benefits and animal welfare of the livestock industry. In densely populated farming areas, once an Escherichia coli epidemic breaks out, it may spread rapidly, causing a large number of livestock to get sick or die, resulting in huge economic losses. Even more seriously, these infected animals can act as carriers and transmitters of Escherichia coli, passing the germs to humans through the food chain. After humans are infected with Escherichia coli, it may cause various diseases such as intestinal infections, urinary tract infections, and septicemia. Among them, enterohemorrhagic Escherichia coli infection can lead to serious complications such as hemorrhagic colitis and hemolytic uremic syndrome (HUS), posing a great threat to the life and health of children, the elderly, and those with weakened immune systems.

[0005] Salmonella is also a common foodborne pathogen that can infect a variety of animals, including poultry, livestock, and pets. Animal products contaminated with Salmonella, such as meat, eggs, and milk, are important sources of human infection. After animals are infected with Salmonella, they show symptoms such as fever, diarrhea, vomiting, and listlessness. Young animals are particularly susceptible, and the mortality rate is relatively high after infection. For humans, Salmonella infection can cause diseases such as typhoid fever, paratyphoid fever, and non-typhoid Salmonella infection. Typhoid fever and paratyphoid fever are relatively severe, accompanied by symptoms such as persistent fever, abdominal pain, and hepatosplenomegaly. If not treated promptly, they may lead to serious complications such as intestinal perforation and intestinal hemorrhage, even endangering life. Non-typhoid Salmonella infection usually causes acute gastroenteritis. Although most patients can recover on their own, in children, pregnant women, the elderly, and those with weakened immune systems, it may also develop into severe systemic infections.

[0006] With the growth of the global population, the acceleration of urbanization, the transformation of food production and processing methods, and the frequent international trade, the spread and diffusion of Escherichia coli and Salmonella show an increasingly complex trend. The extension and globalization of the food supply chain enable contaminated food to cross geographical boundaries in a short time, widely spreading the germs, greatly increasing the risk of epidemic outbreaks and the difficulty of prevention and control.

[0007] With the widespread use of antibiotics in medical, agricultural, and livestock industries, these two types of bacteria have gradually evolved antibiotic resistance. Escherichia coli has developed resistance to many common antibiotics, such as aminoglycosides, third-generation cephalosporins, fluoroquinolones, etc. Some strains of Escherichia coli have also shown multidrug resistance. The resistance of Salmonella to various commonly used antibiotics is also increasing. For antibiotics such as sulfonamides and streptomycin, the resistance rates to Salmonella are at relatively high levels in many regions. Taking Brazil as an example, more than half of the Salmonella strains have developed resistance to sulfonamides and streptomycin, one-third are resistant to tetracycline and gentamicin, and 7% of the strains have started to develop resistance to cephalosporins. In addition, strict laws and regulations on reducing, restricting, and eliminating the use of antibiotics have been formulated both domestically and abroad in terms of antibiotic usage norms. Therefore, there is an urgent need to find a new and effective antibacterial method to prevent and treat Escherichia coli and Salmonella infections.

[0008] Therefore, broad-spectrum bacteriophages that can simultaneously lyse Salmonella and Escherichia coli can solve the problem of mixed infections of pathogenic Escherichia coli and Salmonella in clinical practice and are worthy of further isolation and research. Summary of the Invention

[0009] The objective of the present invention is to provide an Escherichia coli bacteriophage PEC-36 with cross-species host characteristics and its applications to solve the problems existing in the above-mentioned prior art. This bacteriophage has a bactericidal effect on Escherichia coli and Salmonella of multiple serotypes, has a significant antibacterial effect, and has good biosafety. It can be widely used in the preparation of bacteriophage drugs against Escherichia coli and Salmonella in humans and animals, as well as in the preparation of products such as feed additives, food additives, and environmental improvers to prevent and control Escherichia coli and Salmonella contamination, providing a safe and efficient alternative antibiotic treatment plan.

[0010] To achieve the above objective, the present invention provides the following solutions:

[0011] The present invention provides an Escherichia coli bacteriophage (Escherichia colibacteriophage) PEC-36 with cross-species host characteristics. The preservation number of the bacteriophage is CCTCC NO: M 20242917, the preservation date is December 27, 2024, and the preservation unit is the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan, China.

[0012] The present invention also provides the application of the Escherichia coli bacteriophage in the preparation of an antibacterial agent for Salmonella and / or Escherichia coli.

[0013] The present invention also provides the application of the Escherichia coli bacteriophage PEC-36 in the preparation of a drug for treating diseases caused by Salmonella and / or Escherichia coli.

[0014] The present invention also provides the use of the Escherichia coli phage PEC-36 in the preparation of feed additives, food additives or environmental improvers.

[0015] The present invention also provides a bacteriostatic agent for Salmonella and / or Escherichia coli, wherein the bacteriostatic agent uses the Escherichia coli bacteriophage PEC-36 as an effective component.

[0016] The present invention also provides a medicine for preventing and treating diseases caused by Salmonella and / or Escherichia coli, wherein the medicine uses the Escherichia coli phage PEC-36 as an effective ingredient.

[0017] The invention also provides a feed additive, which uses the Escherichia coli bacteriophage PEC-36 as an effective ingredient.

[0018] The invention also provides a food additive, which uses the Escherichia coli bacteriophage PEC-36 as an effective ingredient.

[0019] The invention also provides an environmental improver, which uses the Escherichia coli phage PEC-36 as an effective component.

[0020] The principle of phages in preventing and controlling Escherichia coli and Salmonella: phages complete the infection of host cells and their own proliferation through five steps: "adsorption", "injection", "synthesis", "assembly" and "release". The first step is "adsorption", that is, the tail of the phage attaches to the host cell wall, opens a gap in the bacterial cell wall through the action of enzymes, the tail sheath shrinks, exposing the tail axis, and extends into the cell wall, "injecting" the head DNA into the host cell, and its protein shell remains outside the wall. After the phage DNA enters the bacterial cell, it will cause a series of changes: the bacterial DNA synthesis stops, the enzyme synthesis is also inhibited, and the phage gradually controls the cell metabolism. The phage cleverly uses the physiological environment of the host (bacterial) cell to replicate and "synthesize" the DNA and proteins of the progeny phage in large quantities, and "assemble" into complete phage particles. After the phage matures, in the latent period, the lytic enzymes that dissolve the host cell wall gradually increase, prompting the cell to lyse, thereby "releasing" a large number of progeny phages.

[0021] The present invention discloses the following technical effects:

[0022] (1) The Escherichia coli phage PEC-36 of the present invention can effectively eliminate and inhibit the proliferation of Escherichia coli and Salmonella, and can be used to resist Escherichia coli and Salmonella infection. The potency of Escherichia coli phage PEC-36 is 7.7×10 9 PFU / mL.

[0023] (2) The Escherichia coli phage PEC-36 of the present invention has a relatively low MOI of 10 -3 , with a short latent period and a large burst size in the one-step growth curve.

[0024] (3) The Escherichia coli phage PEC-36 of the present invention has good thermal stability and still maintains high activity after being treated at 70 °C for 1 h.

[0025] (4) The titer of the Escherichia coli phage PEC-36 of the present invention remains basically stable within the range of pH 3 - 12 after 2 h, and the titer will only decrease significantly when pH < 3 or pH > 12.

[0026] (5) The genome of the Escherichia coli phage PEC-36 of the present invention is linear double-stranded DNA, with a full length of 56,547 bp and a GC content of 45.69%. It has 84 open reading frames (ORFs), including genes encoding perforin and lysozyme, and no drug resistance genes and virulence genes are detected. Based on the phylogenetic tree constructed from the amino acid sequences of the phage DNA polymerase, terminase large subunit and major capsid protein, it is found that PEC-36 has a relatively close genetic relationship with Salmonella phage 8-19 and is a phage of the Myoviridae family.

[0027] (6) The Escherichia coli phage PEC-36 of the present invention has good safety and effectiveness in both the Galleria mellonella infection phage therapy model and the mouse infection phage therapy model.

[0028] (7) The Escherichia coli phage PEC-36 of the present invention has a bactericidal effect on pathogenic bacteria such as Escherichia coli and Salmonella, with a significant antibacterial effect and good biosafety, providing a safe and efficient alternative antibiotic treatment plan for humans and animals infected with Escherichia coli and Salmonella.

[0029] (8) The Escherichia coli phage PEC-36 of the present invention can be used as an active ingredient in products such as drugs, feed additives, food additives, and environmental improvers, providing a safe and efficient alternative antibiotic treatment plan for humans and animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 It is the plaque morphology of phage PEC-36;

[0032] Figure 2 Transmission electron microscopy observation results of phage PEC-36;

[0033] Figure 3 Determination results of the optimal multiplicity of infection (MOI) of phage PEC-36;

[0034] Figure 4 Determination results of the one-step growth curve of phage PEC-36;

[0035] Figure 5 Determination results of the thermal stability of phage PEC-36;

[0036] Figure 6 Determination results of the pH stability of phage PEC-36;

[0037] Figure 7 Genome map of phage PEC-36;

[0038] Figure 8 Phylogenetic tree analysis diagram of phage PEC-36;

[0039] Figure 9 Comparative genomic analysis diagram of phage PEC-36;

[0040] Figure 10 Determination of the in vitro bactericidal effect of phage PEC-36 against Escherichia coli EC539 (A) and Salmonella choleraesuis SE638 (B);

[0041] Figure 11 Determination of the in vitro bactericidal effect of phage PEC-36 against multiple Salmonella serotypes (Salmonella gallinarum SE187, Salmonella london SE3473, Salmonella gallinarum SE339);

[0042] Figure 12 Determination of the therapeutic efficacy of phage PEC-36 on the Galleria mellonella infection model of Escherichia coli EC539 (A) and Salmonella SE638 (B) strains;

[0043] Figure 13 Determination of the therapeutic efficacy of phage PEC-36 on the mouse infection model of Escherichia coli EC539 (A) and Salmonella SE638 (B) strains. Detailed implementation manners

[0044] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0048] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0049] Example 1 Isolation, Purification, and Identification of Bacteriophage PEC-36

[0050] Take the sewage water sample from a certain pig farm and isolate bacteriophages using 161 strains of pathogenic Escherichia coli as host bacteria. After the water sample is left standing at 4°C overnight to filter impurities, it is centrifuged at 6000×g for 10 min and then filtered through a 0.22 μm filter membrane to sterilize. Mix 10 mL of the water sample with an equal volume of 2×LB liquid medium, then add 100 μL of the host bacteria, and culture with shaking at 37°C and 150 rpm. After centrifugation, it is filtered through a 0.22 μm filter membrane to sterilize to obtain a crude bacteriophage extract. Use the double-layer plate method to verify whether clear and translucent plaques can be formed to identify whether bacteriophages are isolated. Then use the double-layer agar plate method to purify the bacteriophages until the plaque sizes and morphologies on the plates are consistent; determine the bacteriophage titer by the double-layer plate method.

[0051] The bacterial morphology of the purified bacteriophage PEC-36 prepared above is as Figure 1 shown. The plaque is circular, with consistent size, clear and transparent, neat edges, excellent transparency. The bacteriophage titer can reach 7.7×10 9PFU / mL, presenting the characteristics of highly lytic phages.

[0052] Determination of the host spectrum of phage PEC-36 in Example 2

[0053] The host spectrum of phage PEC-36 was determined by the spot method for 161 strains (including 99 serotypes) of pathogenic Escherichia coli and 53 strains (including 15 serotypes) of pathogenic Salmonella isolated from diseased pigs and chickens. 5 μL of each phage was used, and whether obvious plaques were produced was observed to judge whether it was lytic.

[0054] Table 1 Determination of the host spectrum of Escherichia coli phage PEC-36

[0055]

[0056]

[0057] The results of the host spectrum determination of the above phage PEC-36 are shown in Table 1. This phage can simultaneously lyse strains of serotypes O8:H7, O21:H34, O23:H16, O25:H4, O81:H27, O153:H42, O161:H4, and H25 of Escherichia coli and strains of serotypes such as Salmonella gallinarum, London, and swine cholera of Salmonella, showing cross-species lysis characteristics.

[0058] Observation of phage PEC-36 by transmission electron microscopy in Example 3

[0059] The double-layer agar containing phages was soaked in SM buffer for about 6 h, centrifuged at 6000×g for 10 min, and then filtered through a 0.22 μm filter membrane to remove bacteria. The obtained phage solution was concentrated by centrifuging with a 100 kD ultrafiltration tube at 3000×g for 20 min. 10 μL of the phage concentrate was dropped onto a 200-mesh copper grid, and the excess liquid was aspirated and allowed to dry naturally; it was negatively stained with 2% phosphotungstic acid, and after drying, the morphology of the phages was observed with a transmission electron microscope (JEM-1400FLASH).

[0060] As Figure 2 shown, the phage head under the transmission electron microscope is a polyhedral structure, about 110 nm long and 60 nm wide, the tail is about 100 nm long, and the total length is 210 nm. According to the latest classification of viruses in 2023 ICTV, PEC-36 belongs to the Duplodnaviria Realm, Heunggongvirae Kingdom, Uroviricota Phylum, Caudoviricetes Class, Rosemountvirus Genus, unclassified Rosemountvirus. Morphologically, it is classified as a T5 phage.

[0061] The above-mentioned phage PEC-36 was identified as Escherichia coli bacteriophage, and was deposited in the China Center for Type Culture Collection on December 27, 2024, with the deposit number CCTCC NO: M 20242917, and the deposit address: Wuhan University, Wuhan, China.

[0062] Example 4 Determination of Biological Characteristics of Phage PEC-36

[0063] (1) Determination of the Optimal Multiplicity of Infection (MOI)

[0064] Phage PEC-36 and host bacterium EC539 (1×10 8 CFU / mL) were co-cultured for 6 h at different multiplicities of infection (1, 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 ). The mixed culture was centrifuged at 5000×g for 10 min and filtered through a 0.22 μm filter membrane to remove bacteria. The phage filtrate was serially diluted 10-fold, and the phage titer was determined using the double-layer agar plate method. The results are as Figure 3 shown. When the MOI was 10 -3 , the phage titer was the highest, reaching 7.7×10 9 PFU / mL. Therefore, the optimal multiplicity of infection (MOI) of Escherichia coli PEC-36 was considered to be 10 -3 . This result indicates that PEC-36 has a very efficient bactericidal effect when infecting the host Escherichia coli.

[0065] (2) One-step Growth Curve Determination

[0066] Phage PEC-36 and host bacterium EC539 (1×10 -3 CFU / mL) were added and mixed at an MOI of 10 7 . After standing at 37°C for 20 min, the mixture was centrifuged at 10000×g for 5 min at 4°C to remove the unadsorbed phage in the supernatant. 20 mL of LB liquid medium was added to resuspend the precipitate at the bottom of the tube, and the mixture was cultured with shaking at 160 rpm at 37°C for 120 min. Samples were taken every 10 min to determine the phage titer. The results were recorded and the one-step growth curve of the phage was plotted. The results are as Figure 4 shown. The latent period of phage PEC-36 was approximately 20 min, the lysis period was approximately 80 min, the burst size exceeded 223 PFU / cell, and the plateau phase was entered after approximately 100 min.

[0067] (3) Determination of the thermal stability of phage PEC-36

[0068] 500 μL of the original phage solution was incubated at different temperatures (40 °C to 90 °C) for 30 min and 60 min respectively, and the titer was determined by the double-layer agar method using the host bacterium EC539. The temperature stability is as Figure 5 shown. Regarding the tolerance of the phage to the environment, it remained stable in the range of 40 °C to 60 °C. When the temperature exceeded 60 °C, the phage titer began to decline. When the temperature was higher than 70 °C, the titer decreased rapidly and was completely inactivated at 80 °C.

[0069] (4) Determination of pH stability

[0070] 500 μL of the original phage solution was incubated at different pH values (2 to 13) for 1 h, and the titer was determined by the double-layer agar method using the host bacterium EC539. The pH stability is as Figure 6 shown. The titer remained stable when the pH value was between 3 and 12. It shows that phage PEC-36 has good stability, providing a basis for the application of phage PEC-36 in the treatment of Salmonella and / or Escherichia coli infections.

[0071] In summary, phage PEC-36 has a low optimal multiplicity of infection (10 -3 ), and can achieve micro-scale and high-efficiency sterilization. Its latent period is short and the burst size is high, proving the strong replication ability and lysis ability of the phage in the host. The phage has a high tolerance range to temperature (still active at 70 °C) and pH value (3 to 12), providing good biochemical conditions for the preparation of phage biological antibacterial agents.

[0072] Example 5 Bioinformatics analysis of phage PEC-36

[0073] (1) Complete genome map of phage PEC-36

[0074] The purified phage stock solution was subjected to phage DNA extraction and then sent to Personal Biotechnology Co., Ltd. in Shanghai for whole-genome sequencing. The DNA sequence of phage PEC-36 was uploaded to the National Center for Biotechnology Information (NCBI) in FASTA format, and the phage was aligned online through BLASTn (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to retrieve similar phages. The online gene annotation website RAST (https: / / rast.nmpdr.org / ) was used to predict the open reading frames (ORFs) of the phage-encoded genes. The predicted amino acid sequences were input into BLASTp of NCBI for functional retrieval of the predicted genes. The virulence factors and antibiotic resistance were predicted in the Virulence Factor Database (VFDB, http: / / www.mgc.ac.cn / VFs / ) and the Antibiotic Resistance Gene Database (ARDB, https: / / card.mcmaster.ca / ). The CGview (https: / / proksee.ca / ) was used to draw the whole-genome map of the phage.

[0075] The genome obtained by high-throughput sequencing was circular double-stranded DNA (dsDNA) with a full length of 56,547 bp and a GC content of 45.69%. No virulence genes, resistance genes, or lysogeny-related genes were retrieved in the genome through annotation alignment. The whole-genome map is shown in Figure 7 .

[0076] (2) Genome analysis and phylogenetic relationship analysis of phage PEC-36

[0077] To reveal the phylogenetic relationship between phage PEC-36 and other phages, an evolutionary tree was constructed using the sequence of the large subunit of terminase with high conservation. The amino acid sequence encoded by the large subunit of terminase of PEC-36 was input into BLASTp of NCBI, and sequences with higher homology (>95%) were selected according to the retrieval results for alignment using ClustalW in the MEGA 11.0 software.

[0078] As Figure 8 shown, phage PEC-36 and Salmonella phage 8-19 belong to the same branch and have a relatively close phylogenetic relationship. Salmonella phage 8-19 belongs to the Myoviridae family.

[0079] (3) Analysis of the broad host spectrum characteristic

[0080] The whole genome of PEC-36 was compared by BLAST (BLAST: Basic Local Alignment Search Tool (nih.gov)), and the genomes of phages with homology exceeding 95% were selected and downloaded to construct a whole-genome phylogenetic tree. Then the data was uploaded to phagescope (PhageScope is an online bacteriophage database, https: / / phagescope.deepomics.org / ) for clustering analysis and comparative genomic analysis.

[0081] As Figure 9 shown, compared with neighboring bacteriophages, phage PEC-36 has prominent gene modules related to lysis, packaging, assembly, infection, and immunity. Therefore, it can also explain the reason for its cross-species host characteristics.

[0082] In summary, genomic analysis shows that it has no virulence genes, drug resistance genes, or lysogenic genes, etc., indicating its application safety at the cellular and genetic levels, and having advantages such as high stability and high safety, which provides basic theoretical support for the preparation of phage preparations for dealing with Escherichia coli and Salmonella-related infections.

[0083] Example 6 In vitro inhibition of the growth of Escherichia coli and Salmonella by phage PEC-36

[0084] (1) In vitro inhibition of Escherichia coli and Salmonella by phage PEC-36 with different MOIs

[0085] Take 4 test tubes with a specification of 40 mL, and add 18 mL of LB medium to each. Add 2 mL of the mixed solution of phage PEC-36 and the host bacteria Escherichia coli EC539 and Salmonella choleraesuis SE638 (MOI = 1, 0.1, 0.01) to 3 tubes respectively, and label them. Only add the test strain bacterial solution to the 4th tube as a positive control. Incubate the 4 tubes at 37 °C and 200 rpm. Take 500 μL of the culture from each tube to an EP tube every hour for detection for 3 - 4 h. After vortex oscillation, measure the OD 600 value of the co-culture solution of the host bacteria and the phage with a spectrophotometer and record it. Draw a growth curve of the host bacteria under different MOIs.

[0086] As Figure 10 can be seen, phage PEC-36 has a strong inhibitory effect on the growth of Escherichia coli EC539 and Salmonella choleraesuis SE638 at different doses (MOI = 1, 0.1, 0.01), showing significant differences compared with the positive control group.

[0087] (2) In vitro inhibition of various Salmonella serotype pathogenic bacteria by phage PEC-36

[0088] Take multiple 40 mL test tubes and add 18 mL of LB medium to each. In the experimental groups, add 2 mL of the mixed solution (MOI = 0.1) of phage PEC-36 and different serotypes of Salmonella (Salmonella gallinarum SE187, Salmonella london SE3473, Salmonella gallinarum SE339) respectively, and label them. The control group only adds the test strain bacterial solution as a positive control. Place the test tubes in an incubator at 37 °C and 200 rpm for cultivation. Take 500 μL of the culture from each tube to an EP tube every hour and detect for 3 - 4 h. After vortex oscillation, measure the OD value of the co-culture solution of the host bacteria and the phage with a spectrophotometer and record it. Draw the growth curve of the host bacteria under different MOIs. 600 value and record. Draw the growth curve of the host bacteria under different MOIs.

[0089] It can be seen from Figure 11 the results that when phage PEC-36 is co-cultured with different serotypes of Salmonella, phage PEC-36 has a strong inhibitory effect on their growth, and there are significant differences compared with the positive control group.

[0090] In summary, phage PEC-36 can well inhibit the growth of Escherichia coli and Salmonella under different MOIs, and phage PEC-36 has a good bactericidal effect on various serotypes of Salmonella. This provides a solid foundation for the preparation of phage preparations to deal with Escherichia coli- and Salmonella-related infections.

[0091] Example 7 Evaluation of the safety and efficacy of phage PEC-36 in the greater wax moth infection model

[0092] Resuscitate greater wax moth larvae, randomly divide the larvae into groups with 6 larvae in each group (removing those with weak vitality). Adjust the EC539 bacterial solution concentration to 10 10 CFU / mL, adjust the SE638 bacterial solution concentration to 10 9 CFU / mL, and adjust the phage PEC-36 titer to 10 8 、10 7 、10 6 、10 5 PFU / mL. Take 10 μL of EC539 and SE638 bacterial solutions with different concentrations and inject them into the greater wax moth larvae as the experimental groups, with the dose being the MLD (minimum lethal dose). After 1 h of infection, take out the larvae and inject 10 μL of phage PEC-36 at each dilution (according to MOI = 1, 0.1, 0.01) into the larvae for phage treatment. Set negative and positive group controls respectively. Place all the larvae in a constant temperature incubator at 37 °C for light-avoiding cultivation. Record the survival status every 12 h until 7 days.

[0093] As Figure 12As shown, when the phage doses were MOI = 1, 0.1, and 0.01 respectively, the survival rates of the greater wax moth infected with Escherichia coli EC539 within 48 h were all 66.6%. When only PBS was used to treat the greater wax moth infected with Escherichia coli EC539, the survival rate of the greater wax moth within 48 h was only 0. The survival rates of the greater wax moth infected with Salmonella SE638 within 48 h were 50%, 66.6%, and 50% respectively. When only PBS was used to treat the greater wax moth infected with Salmonella, the survival rate of the greater wax moth within 48 h was only 0. The above experimental results indicate that phage PEC-36 can significantly improve the survival rates of the greater wax moth infected with Escherichia coli EC539 and Salmonella SE638.

[0094] In summary, phage PEC-36 demonstrated the safety and effectiveness of its prevention and treatment in the determination of the treatment effectiveness of the greater wax moth infection models with Escherichia coli EC539 and Salmonella SE638 strains, providing real experimental data for the prevention and control of Escherichia coli and Salmonella related infections by phage PEC-36 and laying a solid foundation for the clinical application of phage.

[0095] Example 8 Safety and effectiveness evaluation of phage PEC-36 in a mouse infection model

[0096] SPF-grade BALB / c female mice aged 6 to 8 weeks were selected and randomly divided into 3 groups evenly, with each group containing 8 mice. The mice were intraperitoneally injected with MLD of Escherichia coli EC539 (dose of 10 8 CFU / mouse), Salmonella SE638 (dose of 10 9 CFU / mouse). One hour after injection, the phage treatment groups were given phage treatment doses with MOI = 1 to the mice respectively. The negative control group used sterile PBS as a substitute both during bacterial challenge and treatment. The positive control group was treated with an equal volume of sterile PBS after intraperitoneal injection of bacterial challenge. The survival status of the mice was recorded every 12 hours.

[0097] As Figure 13 shown, after challenging with MLD (10 8 CFU / mouse) of pathogenic Escherichia coli EC539, all the untreated mice died within 36 hours. The phage treatment group significantly improved the survival rate of the mice. After treatment with a dose of 10 8 PFU (MOI = 1) of phage, the survival rate of the mice within 7 days was increased to 75%. After challenging with MLD (10 9 CFU / mouse) of pathogenic Salmonella SE638, all the untreated mice died within 36 hours. The phage treatment group significantly improved the survival rate of the mice. After treatment with 10 9After treatment with phage at a PFU (MOI = 1) dose, the survival rate of mice within 7 days has increased to 62.5%. This indicates that the phage has good therapeutic effects and greatly improves the survival rate of mice.

[0098] According to the above results, it can be seen that phage PEC-36 demonstrated its safety and effectiveness in the determination of the therapeutic efficacy of the mouse infection models of Escherichia coli EC539 and Salmonella SE638 strains, providing real experimental data for the prevention and control of Escherichia coli and Salmonella-related infections by phage PEC-36 and laying a solid foundation for the clinical application of phages.

[0099] In summary, the present invention provides a potentially safe, stable, and highly efficient phage PEC-36, which has cross-species lysis characteristics and can simultaneously lyse Salmonella of serotypes such as Escherichia coli O8:H7, O21:H34, O23:H16, O25:H4, O81:H27, H25, and Salmonella serotypes such as Salmonella gallinarum, London, and swine cholera. The phage titer can reach 7.7×10 9 PFU / mL, and a high-titer phage PEC-36 can be obtained through simple cultivation, facilitating the industrial production of phage preparations and their use and storage. The phage has a relatively low optimal multiplicity of infection (10 -3 ), with the characteristics of high efficiency at low doses. Its latent period is short, and the burst size can reach 223 PFU / cell, demonstrating the strong replication ability and cell-lysing ability of the phage in the host. The phage has strong tolerance to temperature and remains active at 70°C. The phage has a wide pH tolerance range (pH = 3 - 12). In terms of safety, genomic analysis shows that it has no virulence genes, drug resistance genes, or lysogenic genes, etc., indicating its safety in application at the cellular and genetic levels. Animal clinical trials show that phage PEC-36 has safety and high protective efficacy. Therefore, phage PEC-36 has advantages such as high titer, high stability, and high safety. It provides technical support for the research and development of phage preparations for the treatment of Escherichia coli and Salmonella-related infections. Using phage PEC-36 or its culture as an active ingredient, alone or in combination, to prepare a biological preparation can be used as an effective ingredient for the preparation of biological antibacterial preparations for products such as drugs, feed additives, food additives, and environmental improvers, providing a safe and efficient alternative antibiotic treatment plan for humans and animals and having good application prospects.

[0100] The embodiments described above are only for describing the preferred mode of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Escherichia colibacteriophage PEC-36 with cross-species host characteristics, characterized in that: The deposit number of the bacteriophage is CCTCC NO: M 20242917.

2. Use of the Escherichia coli phage PEC-36 as claimed in claim 1 in the preparation of an antibacterial agent for Salmonella and / or Escherichia coli.

3. Use of the Escherichia coli phage PEC-36 as claimed in claim 1 in the preparation of a medicament for treating diseases caused by Salmonella and / or Escherichia coli.

4. Use of the Escherichia coli phage PEC-36 as claimed in claim 1 in the preparation of feed additives, food additives or environmental improvers.

5. An antibacterial agent for Salmonella and / or Escherichia coli, characterized in that: The antibacterial agent uses the Escherichia coli phage PEC-36 described in claim 1 as an active ingredient.

6. A drug for preventing and treating diseases caused by Salmonella and / or Escherichia coli, characterized in that: The drug uses the Escherichia coli phage PEC-36 described in claim 1 as an active ingredient.

7. A feed additive, characterized in that: The effective ingredient is the Escherichia coli phage PEC-36 described in claim 1.

8. A food additive, characterized in that: The effective ingredient is the Escherichia coli phage PEC-36 described in claim 1.

9. An environmental improver, characterized in that: The effective ingredient is the Escherichia coli phage PEC-36 described in claim 1.

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