A bacteriophage GaoH6-13E capable of lysing Escherichia coli and Salmonella simultaneously and its evolution method and application

Abstract

The present invention relates to a bacteriophage GaoH6-13E capable of simultaneously lysing Escherichia coli and Salmonella, and an evolution method and application thereof, belonging to the field of biotechnology; the deposit number of the bacteriophage GaoH6-13E is CGMCC NO.46187; the bacteriophage GaoH6-13E obtained by the present invention can be used to prepare related bacteriophage products, applied to the treatment of bacterial infections caused by Escherichia coli and Salmonella, or used for the prevention, control and elimination of Escherichia coli disease or Salmonellosis; the host spectrum of the bacteriophage is expanded by using the evolution method of artificially expanding the host spectrum of the present invention, so that the evolved bacteriophage GaoH6-13E has the ability to simultaneously lyse Escherichia coli and Salmonella, shows a wide lysis spectrum and a high lysis rate, and successfully improves the effectiveness of the bacteriophage in clinical applications.

Description

Technical Field

[0001] The invention relates to a bacteriophage GaoH6-13E capable of simultaneously lysing Escherichia coli and Salmonella, an evolution method and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Phages are a type of virus. Like other viruses, their survival and production depend on the host. They are highly host-specific and do not destroy normal flora. Since the discovery of phages in 1915, researchers and clinicians have quickly recognized that they can be used as potential therapeutic agents for the treatment of bacterial infections. Most of the phages currently isolated have a narrow host range, targeting only one species or one strain, which limits the widespread application of phages. Therefore, it is of great significance to obtain some polyvalent phages with a wide host range. At present, some methods have been developed to expand the host range of phages. One is to synthesize phage libraries. However, before designing engineered phage libraries, it is necessary to sequence the whole phage genome and identify the host range-determining region in the tail fiber protein or tail spike; the other is reasonable genetic engineering or recombination. However, the synthetic polyvalent phages rely on phages that can infect different hosts. Polyvalent phages can also be obtained using phage evolution. Almost all evolved wide-host-range phages can only infect species of the same genus. Few scientists have expanded the host range of phages to multiple bacterial genera through evolution.

[0003] Phages that can simultaneously lyse E. coli and Salmonella have broad application prospects. According to estimates by the World Health Organization, at least 600 million people worldwide have been infected with foodborne diseases, and 420,000 people die from foodborne diseases each year. Both E. coli and Salmonella belong to the Enterobacteriaceae family and are major foodborne pathogens. Pathogenic E. coli can cause severe gastroenteritis and is the most common cause of acute renal failure in children. Salmonella is the main pathogen of bacterial food poisoning worldwide and can cause severe intestinal diseases such as diarrhea, abdominal cramps, nausea, etc. In addition to infecting humans, these pathogens can also infect many animals, including mammals, birds, reptiles, fish, amphibians and insects. Therefore, a broad-spectrum phage that can simultaneously lyse Salmonella and E. coli can solve the problem of mixed infection of pathogenic E. coli and Salmonella in clinical practice, and is worthy of further separation and research. Summary of the invention

[0004] The object of the present invention is to provide a bacteriophage GaoH6-13E capable of simultaneously lysing Escherichia coli and Salmonella, and an evolution method and application thereof, so as to solve the problems existing in the prior art as mentioned above.

[0005] The technical solution provided by the present invention is as follows:

[0006] The invention discloses a bacteriophage GaoH6-13E capable of simultaneously lysing Escherichia coli and Salmonella, which has been submitted for bacterial strain preservation.

[0007]

Biological Deposit Instructions

[0008] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0009] Storage address: Beijing, China

[0010] Date of deposit: September 18, 2024

[0011] Deposit number: CGMCC NO.46187

[0012] Classification name: Escherichia coli phage Escherichia coli phage

[0013] The present invention also discloses an evolution method of the bacteriophage GaoH6-13E, comprising the following steps: firstly separating and culturing the purified bacteriophage with Escherichia coli; E. coli 6-13. Salmonella Salmonella 169 mixed culture, and then incubation evolution.

[0014] Furthermore, the above-mentioned evolution method of bacteriophage GaoH6-13E specifically comprises the following steps:

[0015] (1) The isolated and purified phage was mixed with Escherichia coli E. coli 6-13. Salmonella Salmonella 169. Mix with LB liquid medium, incubate and evolve on a shaker, centrifuge and filter to obtain the supernatant;

[0016] (2) Mix the supernatant with Salmonella Salmonella 169. LB liquid medium was mixed in a centrifuge tube and incubated on a shaker to enrich bacteriophages capable of infecting Salmonella, and the supernatant was obtained by centrifugation and filtration;

[0017] (3) Steps (1) and (2) are repeated to obtain a polyvalent phage that can successfully infect Escherichia coli and Salmonella, that is, the phage GaoH6-13E that can simultaneously lyse Escherichia coli and Salmonella.

[0018] The present invention also discloses the use of the bacteriophage GaoH6-13E in preparing medicines for treating diseases caused by Salmonella and / or Escherichia coli.

[0019] The invention also discloses a bactericidal composition for preventing and controlling Salmonella and Escherichia coli, comprising the above-mentioned bacteriophage GaoH6-13E capable of simultaneously lysing Escherichia coli and Salmonella.

[0020] The invention also discloses the application of the bacteriophage GaoH6-13E in livestock and poultry breeding.

[0021] Furthermore, the livestock and poultry are chickens.

[0022] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0023] The bacteriophage GaoH6-13E obtained by the present invention can be used to prepare related phage products, which can be applied to the treatment of bacterial infections caused by Escherichia coli and Salmonella, or to the prevention, control and elimination of Escherichia coli disease or Salmonellosis; the host spectrum of the phage is expanded by using the evolutionary method of artificially expanding the host spectrum of the present invention, so that the phage has the ability to lyse Escherichia coli and Salmonella at the same time, showing a wide lysis spectrum and a high lysis rate, and successfully improving the effectiveness of the phage in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The bacteriophage GaoH6-13E of the present invention is in Escherichia coli E. coli Photographs of plaques on the double-layer plates of 6-13;

[0025] Figure 2 The bacteriophage GaoH6-13E of the present invention is used to Salmonella Photo of plaques on the double-layer plate of 169;

[0026] Figure 3 This is an electron microscope photo of the morphology of the bacteriophage GaoH6-13E of the present invention;

[0027] Figure 4 The one-step growth curve measurement result of the bacteriophage GaoH6-13E of the present invention;

[0028] Figure 5 The temperature stability test results of the bacteriophage GaoH6-13E of the present invention are as follows;

[0029] Figure 6 The pH stability test results of the bacteriophage GaoH6-13E of the present invention are as follows;

[0030] Figure 7 is the genome map of the bacteriophage GaoH6-13E of the present invention;

[0031] Figure 8 The results are the test results of the bacteriophage GaoH6-13E of the present invention on the bactericidal effect of Escherichia coli and Salmonella at 4°C;

[0032] Fig. 9The results are the test results of the bacteriophage GaoH6-13E of the present invention on the bactericidal effect of Escherichia coli and Salmonella at room temperature. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] In the following examples, the culture medium, reagents, and related equipment and materials used include:

[0035] LB (Luria broth) liquid medium (1 L): 10 g peptone, 5 g yeast powder, 10 g NaCl, add distilled water to 1 L, and autoclave at 121°C for 20 min.

[0036] LB solid medium (1L): 10g peptone, 5g yeast powder, 10g NaCl, 20g agar powder, add distilled water to 1L, sterilize at 121℃ for 20min, cool to 50℃ and pour into the plate. After solidification, turn it upside down for later use.

[0037] Phage buffer: 8.766 g NaCl, 6.057 g Tris, 1.64836 g MgCl2-6H2O, 0.2 g anhydrous CaCl2, add distilled water to 1 L, adjust pH to 7.5, and autoclave at 121°C for 20 min.

[0038] Equipment and materials: vertical pressure steam autoclave LDZX-50KBS (Shanghai Shen'an Medical Machinery Factory), full-temperature oscillating incubator (Suzhou Peiying Experimental Equipment Co., Ltd.), Xiangyi desktop high-speed refrigerated centrifuge TGL-16M (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), ultra-low temperature refrigerator (Changhong Meiling Co., Ltd.), clean bench (Suzhou Purification Equipment Co., Ltd.), micropipette (Eppendorf, Germany), culture dish, disposable syringe and 0.22 μm filter, centrifuge tube, Erlenmeyer flask, etc.

[0039] Example 1 Preparation of bacterial solution

[0040] Plate streak culture of E. coli E. coli 6-13 and Salmonella Salmonella 169, and then inoculated single colonies into LB liquid culture medium and cultured in a constant temperature shaking incubator at 37°C for 12-16h to obtain Escherichia coli E. coli 6-13 and Salmonella Salmonella 169 of bacterial solution. After the cultured bacterial solution was diluted stepwise, its concentration was determined by the plate spreading method. The result was 10 8 CFU / mL. After passing the test, store it in a 4℃ refrigerator for later use.

[0041] Example 2 Isolation and purification of bacteriophage

[0042] 5 g of sludge from a chicken farm and 10 mL of the Escherichia coli obtained in Example 1 were added. E. coli 6-13 bacterial solution (10 8 CFU / mL) was added to 100 mL LB liquid medium, placed at room temperature for 30 min, and cultured overnight at 37°C with shaking at 200 rpm. After centrifugation at 6000 rpm for 10 min at 4°C, the supernatant was filtered using a 0.45 µm cellulose acetate filter, and 10 µL of the filtrate was dropped on the surface of an LB agar plate covered with 5 mL of 0.7% water agar containing 500 μL of E. coli. E.col i 6-13 and 500 μL phage buffer. Incubate the plate at 37 °C to visualize plaques. Use a sterile inoculation loop to pick out a single transparent plaque and elute it into 100 μL phage buffer. Repeat this purification process five times until the phage morphology on the plate is uniform.

[0043] Example 3 Phage host expansion evolution and phage suspension preparation

[0044] 1. Phage host expansion evolution

[0045] In a 50 mL centrifuge tube, the phage obtained in Example 2 (10 8 PFU / mL) and 1 mL of the Escherichia coli obtained in Example 1 E. coli 6-13 bacterial solution (10 8 PFU / mL), 1 mL of the Salmonella obtained in Example 1 Salmonella 169 bacterial solution (10 8 PFU / mL) and 8 mL LB liquid medium were mixed and incubated on a shaker at 37°C for 48 h (evolution pool). The supernatant was obtained by centrifugation at 3500 g for 10 min at 4°C and filtration through a 0.22 μm filter. The supernatant was mixed with 1 mL Salmonella Salmonella 169 and 8 mL of LB liquid medium were mixed in a 50 mL centrifuge tube and incubated at 37 °C on a shaker for 24 h to enrich the phages that can infect Salmonella (enrichment pool). Afterwards, the supernatant was obtained by centrifugation again. The double-layer plate method was used to evaluate the supernatant in the enrichment pool against Escherichia coli. E. coli 6-13 and Salmonella Salmonella The infection capacity and PFU of 169 were measured. The above steps were repeated repeatedly. After 13 cycles, a multivalent phage that can infect Escherichia coli and Salmonella was successfully obtained and named as phage GaoH6-13E. Figure 1It was shown that bacteriophage GaoH6-13E was expressed in Escherichia coli E. coli Obvious plaques were shown on the double-layer plates of 6-13; Figure 2 The results showed that bacteriophage GaoH6-13E can inhibit the growth of Salmonella Salmonella The double-layer plates of 169 showed obvious plaques.

[0046] 2. Preparation of bacteriophage GaoH6-13E suspension

[0047] 100 μL of bacteriophage GaoH6-13E and 100 μL of host bacteria Escherichia coli were mixed. E. coli BL21 (10 8 CFU / mL) and allowed to stand at room temperature for 20 minutes to promote the infection and adsorption of bacteriophage GaoH6-13E to host bacterial cells as much as possible. Then, the mixed liquid was added to 100 mL of LB liquid culture medium and cultured at 37°C with shaking at 180 rpm for 5 hours. Finally, the obtained phage culture liquid was centrifuged at 3500g for 10 minutes and its titer was determined by double-layer plate method. The titer was 10 8 PFU / mL, the qualified phage suspension was placed in a 4℃ refrigerator for later use.

[0048] Example 4 Electron microscopic morphological observation of bacteriophage GaoH6-13E

[0049] 20 μL of the bacteriophage GaoH6-13E suspension (10 8 PFU / mL) was dropped on a copper grid. After natural precipitation for 30 minutes, excess liquid was removed from the side with filter paper and negatively stained with 20 μL 2% phosphotungstic acid. After staining for 10 minutes, the staining liquid was removed from the side with filter paper. After the sample was dried, the morphology of the bacteriophage was observed by transmission electron microscopy. The results are shown in Figure 3 As shown, the phage GaoH6-13E shows a head with a diameter of 70 nm and a short and almost invisible tail. According to the latest classification standards of the International Virus Taxonomy Organization, this phage can be classified as an autophagy family.

[0050] Example 5 Biological characteristics of bacteriophage GaoH6-13E

[0051] 1. One-step growth curve determination of bacteriophage GaoH6-13E:

[0052] 1 mL of the Escherichia coli obtained in Example 1 E. coli 6-13 bacterial solution (10 9 CFU / mL) and 1 mL of the suspension of bacteriophage GaoH6-13E obtained in Example 3 (10 8PFU / mL) to make MOI (multiplicity of infection) reach 0.1, and incubate at 37°C for 10 min. The mixture is centrifuged at 8000 rpm for 5 min at 4°C, the supernatant is removed, and the precipitate is resuspended in 1 mL of fresh LB medium to remove free phages. After elution twice, the suspension is placed in a shaker at 37°C, sampled every 20 min, and centrifuged at 5000 rpm for 2 min. The phage GaoH6-13E titer of the supernatant is determined by the double-layer plate method. Each independent experiment is repeated 3 times, and the average value is taken. The sampling time is used as the horizontal axis and the logarithm of the phage titer is used as the vertical axis to draw a one-step growth curve. The results are shown in Figure 2. Figure 4 As shown in the figure, the incubation period of bacteriophage GaoH6-13E is about 5 minutes, and the burst volume is 267 PFU / cell, indicating that the bacteriophage GaoH6-13E has the characteristics of short incubation period and strong lytic ability.

[0053] 2. Thermal stability determination of bacteriophage GaoH6-13E:

[0054] Take 6 1.5 mL centrifuge tubes and add 200 μL of the bacteriophage GaoH6-13E suspension (10 8 PFU / mL), and then continuously treated at -70℃, 4℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 1h, and then quickly cooled on ice. After gradient dilution, double-layer plates were spread to determine the titer of phage GaoH6-13E at each temperature.

[0055] The results are as follows Figure 5 As shown, phage GaoH6-13E has strong thermal stability. After induction at -70℃, 4℃, 30℃, 40℃, and 50℃ for about 1h, the titer of phage GaoH6-13E remained stable, the activity of phage GaoH6-13E decreased significantly at 60℃, and lost its activity at 70℃.

[0056] 3. Determination of pH stability of bacteriophage GaoH6-13E:

[0057] Prepare phage buffers with different pH values ​​(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Mix 900 μL of buffer with 100 μL of the phage GaoH6-13E suspension (10 8 The mixture was mixed with 4% paraformaldehyde (PFU / mL) and incubated at 37°C on a shaker for 2 h. The dilution was then performed in series and the titer of bacteriophage GaoH6-13E was measured by the double-layer plate method.

[0058] The results are as follows Figure 6As shown, phage GaoH6-13E exhibited excellent stability in the pH range of 3-11 and was inactivated at pH 1, 2, and 12.

[0059] Example 6 Whole genome sequencing and genome sequence analysis of bacteriophage GaoH6-13E

[0060] The genome of bacteriophage GaoH6-13E was extracted with phenol-chloroform-isoamyl alcohol and resuspended in ddH2O. Genome sequencing was performed based on the Illumina Hiseq PE250 platform. The clean reads obtained were assembled by SPAdes v3.14.1. The integrity of the bacteriophage GaoH6-13E genome was assessed using CheckV v0.8.1. The phage repeat ends were determined by phage PhageGT. Virsorter v1.0.3 was then used to predict the ORF (Open Reading Frame) of the phage genome and generate GBK files. Based on local BLAST, the functional genes in the ORFs were annotated using the NR database. The bacteriophage GaoH6-13E genome was compared with the NCBI reference database by local BLAST comparison. The reference genome that was highly similar to the phage GaoH6-13E genome was calculated for ANI by FastANI. The construction of the phage genome map was completed by Geneious Prime.

[0061] According to NCBI BLAST-N, the genomes of some phages were similar to that of phage GaoH6-13E. Based on the ANI calculation results, we selected the closest relative phage, Escherichia coli phage P694 (NC_028863.1), for comparative genome analysis and found that the ANI was less than 95%, indicating that phage GaoH6-13E is a novel phage.

[0062] The genome of bacteriophage GaoH6-13E is a double-stranded linear DNA molecule with a length of 40765 bp and a 111 bp direct redundant terminal repeat sequence. The G+C content of bacteriophage GaoH6-13E is 48.31%. Figure 7 It was shown that the genome of bacteriophage GaoH6-13E has a total of 50 ORFs, of which 6 ORFs encode structural proteins and belong to the phage structure and packaging module, 10 proteins are related to DNA metabolism and belong to the DNA metabolism module, and 13 ORFs encode other functional proteins.

[0063] Example 7 Determination of host spectrum of bacteriophage GaoH6-13E

[0064] The strains isolated from sick chicken samples in the laboratory were selected ( Salmonella 169. Salmonella 190, Salmonella 177. Salmonella 195. Salmonella 178. Salmonella 188. Salmonella 218, Salmonella 160, E. coli 1-3. E. coli 4-1, E. coli 5-2, E. coli 5-4, E. coli 6-11, E. coli 6-12, E. coli 6-13, E. coli BL21) was used as the host bacteria. The preserved strain was streaked onto a LB solid plate and cultured at 37°C for 24 h. A single colony was inoculated into LB liquid medium and shake-cultured at 37°C for 10 h to revive the strain.

[0065] The host range of bacteriophage GaoH6-13E was determined by double-layer plate spotting. First, 500 μL of the above-mentioned different Escherichia coli (10 8 PFU / mL) or Salmonella (10 8 PFU / mL) was mixed with 500 μL of phage buffer and 5 mL of water agar, and then the mixture was quickly poured into the LB solid medium at the bottom. After cooling, 10 μL of the phage GaoH6-13E suspension obtained in Example 3 (10 8 PFU / mL) and cultured in a 37°C incubator overnight. Finally, observe whether plaques appear on the plate. If plaques are formed, it indicates that the phage GaoH6-13E can lyse the corresponding bacteria. The degree of lysis is evaluated according to the transparency of the plaques. "-" means no lysis; "+" means lysis but not transparent; "++" means lysis and transparency.

[0066] The above method was used to test the lysis ability of bacteriophage GaoH6-13E on the above 16 different strains of Escherichia coli and Salmonella, and the results are shown in Table 1. Among the 16 bacterial strains, bacteriophage GaoH6-13E successfully lysed 15 strains, and the lysis rate reached 93.75%. This shows that the bacteriophage GaoH6-13E can lyse Escherichia coli and Salmonella at the same time, has a relatively wide lysis spectrum, and has good application prospects.

[0067] Table 1 Lysis results of bacteriophage GaoH6-13E against Escherichia coli and Salmonella

[0068]

[0069] Example 8 Safe fermentation production of bacteriophage GaoH6-13E

[0070] 1. Preparation of fermentation seed solution:

[0071] Host bacteria Escherichia coli E. coli Preparation of BL21 seed solution: Because bacteriophage GaoH6-13E has the ability to lyse non-pathogenic Escherichia coli E. coli BL21 ability. We selected Escherichia coli E. coli BL21 was used as the host for phage production. E. coli The BL21 strain was inoculated into 10 mL of LB liquid medium and cultured at 37°C for 6 hours. After the culture was completed, the host bacteria were inoculated into 300 mL of LB liquid medium at a 3% inoculation rate and cultured at 37°C for another 6 hours. Subsequently, the inoculation rate was inoculated into 1000 mL of LB liquid medium at a 3-4% inoculation rate and cultured at 37°C for 6 hours. After the cultured host bacteria seeds were gradient diluted, the concentration was determined by the spread plate method, and the result was 10 8 CFU / mL. After passing the test, store it in a 4℃ refrigerator for later use.

[0072] 2. Production of bacteriophage GaoH6-13E:

[0073] The scale of the phage fermentation production system was 20 L, and the fermentation medium used was LB medium. According to the infection multiplicity of 0.1, 3 mL of phage GaoH6-13E suspension (10 8 PFU / mL) and Escherichia coli E. coli BL21 seed solution 30mL (10 8 CFU / mL). During the fermentation process, filtered sterile air was continuously introduced, and 3% defoamer was added. The fermentation time was set to 4h. After the fermentation started, 10mL of fermentation broth was taken every 1h and placed in a 50mL sterile centrifuge tube. The bacteria were removed by centrifugation at 4℃ 6000g for 15min. After discarding the precipitate, the supernatant was filtered through a 0.22μm polyethersulfone filter membrane for further sterilization to obtain the fermented phage GaoH6-13E. Finally, the phage buffer was used for multiple dilution and its titer was determined. The titer data are shown in Table 2.

[0074] Table 2 Titer determination of bacteriophage GaoH6-13E fermentation production

[0075]

[0076] Example 9 Dual bactericidal effect of bacteriophage GaoH6-13E on Escherichia coli and Salmonella

[0077] Add 3 mL of whole milk to a 10 mL sterile centrifuge tube and add 250 μL of E. coli E. coli 6-13 bacterial solution (10 8 CFU / mL), 250μL Salmonella Salmonella 169 bacterial solution (10 8 CFU / mL) and 500 μL of bacteriophage GaoH6-13E suspension (10 8 PFU / mL), and then placed in 4℃ and room temperature environments after thorough mixing. 500μL of phage buffer was used to treat contaminated milk as a control. 100μL of the mixed system was taken at 0h, 2h, 4h, 6h, 10h, and 24h, and the CFU of bacteria in the system was measured using LB solid plates. The results are shown in Figure 8 As shown in the figure, under 4°C, the number of live bacteria in milk with phage buffer and two bacteria remained relatively stable within 24 hours without significant changes, while the number of live bacteria in milk with phage GaoH6-13E and two bacteria decreased significantly from 2 hours to 24 hours. Fig. 9 As shown, at room temperature, the number of live bacteria in the control group with the addition of phage buffer and two bacteria increased slowly, while the number of live bacteria in the experimental group with the addition of phage GaoH6-13E and two bacteria decreased significantly at 0-2h, and the number of live bacteria was always lower than that in the control group within 2-10h. It can be seen that the number of live bacteria decreased after the addition of phage GaoH6-13E, whether at 4°C or at room temperature, indicating that phage GaoH6-13E has a significant inhibitory and bactericidal effect on both Escherichia coli and Salmonella in milk.

[0078] Example 10 Evaluation of the clinical efficacy of bacteriophage GaoH6-13E in treating mixed infection of Escherichia coli and Salmonella in chickens

[0079] 72 healthy chicks aged 3 days were selected and divided into 3 groups, namely, the challenge group (negative control), the experimental group, and the antibiotic group (positive control). Each group was further divided into 3 parallel groups, with 8 chicks in each group. E. coli 6-13 and Salmonella Salmonella 169 was used as the pathogen to infect chicks. The challenge method was gavage with a dose of 0.2 mL of E. coli E. coli 6-13 bacterial solution (10 8 CFU / mL) and 0.2mL Salmonella Salmonella 169 bacterial solution (10 8 CFU / mL) mixture, and 0.4 mL of phage buffer was administered 2 h after the challenge. 8CFU / mL). In addition to the need for virus challenge, the antibiotic group was gavaged with 0.4mL of enrofloxacin solution (1.25mg / mL) 2h after virus challenge. The number and survival rate of chicks were counted after 2 weeks of the experiment.

[0080] The results are shown in Table 3. The survival rate of the chicks in the experimental group reached 83.33%, which was much higher than that in the antibiotic group. This indicates that bacteriophage GaoH6-13E can effectively treat the mixed infection of Escherichia coli and Salmonella in chickens.

[0081] Table 3 Clinical effect of bacteriophage GaoH6-13E in treating mixed infection of Escherichia coli and Salmonella in chickens

[0082]

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A bacteriophage GaoH6-13E that can lyse Escherichia coli and Salmonella simultaneously, characterized in that: It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 18, 2024, with the deposit address in Beijing, China, and its deposit number is: CGMCC NO.46187; the classification name is: Escherichia coli phage ( Escherichia coli phage ).

2. Use of the bacteriophage GaoH6-13E capable of simultaneously lysing Escherichia coli and Salmonella as claimed in claim 1 in the preparation of a drug for treating diseases caused by Salmonella and / or Escherichia coli.

3. A bactericidal composition for preventing and controlling Salmonella and Escherichia coli, characterized in that: The invention comprises the bacteriophage GaoH6-13E as claimed in claim 1, which can simultaneously lyse Escherichia coli and Salmonella.

4. Use of the bacteriophage GaoH6-13E capable of simultaneously lysing Escherichia coli and Salmonella as claimed in claim 1 in the preparation of a drug for treating diseases caused by Salmonella and / or Escherichia coli in livestock and poultry breeding.

5. The use according to claim 4, characterized in that: The livestock and poultry are chickens.

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