A bacteriophage for lysing salmonella and Escherichia coli and its application

Abstract

The present invention relates to the field of biological bacteriophage technology, and discloses a bacteriophage for lysing Salmonella and Escherichia coli, and the bacteriophage is named Salmonella phage GRNPK. The bacteriophage is a virulent bacteriophage, has a strong lysis effect on both Salmonella and Escherichia coli, and belongs to the Demerecviridae family; the bacteriophage forms small and transparent plaques on a double-layer agar plate; has high stability, is active under pH 2-12 and under conditions of -20°C-70°C; has strong infectivity, and is cultured for 12 hours under the condition of an optimal multiplicity of infection (MOI) of 0.001, and has a titer of 1.1×10 12 PFU / mL. High-throughput sequencing showed that the phage GRNPK did not encode any lysogeny, toxin, drug resistance and virulence factor-related genes. In vitro experiments showed that GRNPK could effectively inhibit the growth of Salmonella and Escherichia coli and delay the bacteria from developing resistance to the phage. Animal test results showed that the phage could effectively reduce the number of Enteritidis Salmonella in the intestines of chicks.

Description

Technical Field

[0001] The invention relates to the technical field of biological bacteriophages, in particular to a bacteriophage for lysing salmonella and Escherichia coli and application thereof. Background Art

[0002] Salmonella and pathogenic Escherichia coli are common foodborne pathogens that are widely found in poultry, livestock and other animals. Human infection with Salmonella and pathogenic Escherichia coli comes from a variety of sources, usually from eating contaminated food or drinking contaminated water, as well as direct contact with infected people or animals and their surroundings. Among them, meat, eggs, dairy products and vegetables are the most seriously contaminated by Salmonella or Escherichia coli, and are also the main sources of human infection with Salmonella or Escherichia coli. Compared with other foodborne pathogens, foodborne diseases caused by Salmonella and pathogenic Escherichia coli are at the forefront, which poses a huge challenge to global public health security.

[0003] The clinical manifestations of salmonellosis and pathogenic E. coli are complex and varied, and are extremely harmful to young livestock and poultry, mainly including asymptomatic carriers, pullorum, fowl typhoid, fowl paratyphoid, gastroenteritis, endocarditis, osteomyelitis, colitis, bloody diarrhea, abortion in pregnant females, decreased egg production in poultry, and bacteremia; in severe cases, they can lead to the death of livestock and poultry. Human infection with Salmonella or E. coli often leads to gastroenteritis, bloody diarrhea, food poisoning, etc.

[0004] At present, the use of antimicrobial drugs is an important means to prevent and treat Salmonella and Escherichia coli infections, but the frequent and irrational use of antimicrobial drugs has led to the rapid development of bacterial antibiotic resistance and the decline of animal immune function. A research report from the European Center for Disease Prevention and Control (ECDC) and the European Food Safety Authority (EFSA) showed that between 2013 and 2022, a significant increase in the resistance rate of Enteritidis Salmonella to ciprofloxacin was observed in 12 countries. Between 2021 and 2022, the resistance of Salmonella and commensal Escherichia coli isolated from livestock and poultry to ampicillin, tetracycline and sulfonamides was moderate to very high. These resistant Salmonella and Escherichia coli strains reduce the efficacy of common antimicrobial drugs or even make them ineffective. Due to the large number of Salmonella and Escherichia coli serotypes and the increasingly serious drug resistance, the prevention and control of Salmonella or Escherichia coli faces severe challenges. Therefore, it is urgent to research and develop a safe, effective and economical antibiotic alternative to target Salmonella and Escherichia coli infections in animal production.

[0005] Bacteriophage is a prokaryotic virus that can infect bacteria and replicate itself. It was published and named by Canadian microbiologist d'Herelle in 1917. Unlike the bactericidal mechanism of antibiotics, bacteriophages also have bactericidal effects on antibiotic-resistant bacteria. As early as the beginning of the 20th century, scientists realized that bacteriophages may have the potential to treat bacterial diseases. In 1919, d'Herelle successfully used bacteriophages to treat chickens infected with Salmonella; then, in 1921, phages were used to successfully treat five patients with bacillary dysentery. Since 2006, bacteriophage products have been authorized by the U.S. Food and Drug Administration (FDA) for use in human food, and there are several products that control Salmonella and E. coli contamination, such as Salmonelex TM and Ecolicide PX TM , has been certified as Generally Recognized as Safe (GRAS). Phages are widely present in nature due to their remarkable characteristics, including specific infection of target bacteria, rapid self-reproduction, huge numbers, rich diversity, and as an important component of the intestinal microbial community of humans and animals, phages are relatively safe, making them considered as a potential antibacterial means to combat antibiotic resistance.

[0006] CN12680423A discloses a broad-spectrum Escherichia coli phage isolate that can lyse four types of bacteria, namely, Escherichia coli, Salmonella, Enterobacter cloacae, and Shigella, and its application in sterilization and antibacterial, which is used to prevent and control Escherichia coli, Salmonella, Enterobacter cloacae, and Shigella contamination in food production and processing, and animal production. CN113583971A discloses a Salmonella phage that can simultaneously lyse Escherichia coli, aiming to prevent and control the problem of Salmonella infection in poultry farms. At present, the research on phages mainly focuses on phages that infect a single genus of pathogenic bacteria, while there are fewer studies on virulent phages that can infect pathogenic bacteria of different genera and have a wide lysis spectrum, as well as their biological characteristics and genomic characteristics. It is worth noting that, similar to antibiotics, bacteria are prone to evolve resistance to phages, which leads to a decrease in the antibacterial effect of phages. The above-mentioned technology does not study the effect of long-term application of a single phage on the development of phage resistance in bacteria. In addition, phages that infect a single pathogen are not ideal for the prevention and control of mixed infections of multiple pathogens in livestock and poultry farms. Therefore, it is urgent to study new virulent phages that infect Salmonella and Escherichia coli across species, develop efficient phage antibacterial preparations with stable bactericidal effects and delay the development of phage resistance in bacteria, provide more possibilities for the prevention and control of mixed infections of Salmonella and Escherichia coli in livestock and poultry, and provide high-quality phage resources for phage resource libraries. Based on this, the present invention proposes a phage that lyses Salmonella and Escherichia coli and its application to solve the above problems. Summary of the invention

[0007] 1. Technical issues to be resolved

[0008] In view of the deficiencies in the prior art, the present invention provides a bacteriophage for lysing Salmonella and Escherichia coli and its application. The bacteriophage can be used alone or in combination with other bacteriophages to replace the use of antibiotics in the prevention and control of drug-resistant Salmonella or Escherichia coli infections in livestock and poultry, solve the problem of ineffective antibiotics due to the development of pathogen resistance in livestock and poultry farming, and provide new solutions to the long-term prevention and control of Salmonella and Escherichia coli and mixed infections of multiple pathogens.

[0009] (II) Technical solution

[0010] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a bacteriophage for lysing Salmonella and Escherichia coli, the bacteriophage being deposited in the China Type Culture Collection of Wuhan University, Wuhan City, Hubei Province, China on July 22, 2024, with a deposit number of CCTCC NO: M 20241606, and the bacteriophage being named Salmonella phage GRNPK.

[0011] Preferably, the Salmonella includes at least one of Salmonella pullorum, Salmonella fowlty, Salmonella typhimurium, monophasic Salmonella typhimurium, Salmonella Arizona, Salmonella Enteritidis, Salmonella Derby, Salmonella Kentucky, Salmonella Typhi, Salmonella Paratyphi A, Salmonella Paratyphi B, Salmonella Javana, Salmonella Indiana, Salmonella Rissen, Salmonella Potsdam, Salmonella Livingstone, Salmonella London, Salmonella Wertavreden, Salmonella Arizona, and Salmonella Brundenloop.

[0012] Preferably, the Escherichia coli includes at least one of enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), and extraintestinal pathogenic Escherichia coli (ExPEC).

[0013] Preferably, the phage survives at pH 2-12 and temperature -20°C-70°C, and the phage is cultured for 12 hours at MOI = 0.001, and its titer is as high as 1.1×10 12 PFU / mL, and the burst volume was 1060±51pfu / cell.

[0014] The bacteriophage can be used to prepare anti-infective drugs for Salmonella or Escherichia coli. Specifically, the drug can be used to treat or prevent diseases caused by Salmonella enteritidis infection in chicks.

[0015] Preferably, the drug is a phage preparation, and the phage reagent comprises an effective amount of the above-mentioned phage GRNPK and SM buffer.

[0016] Preferably, the phage preparation alone or in combination with other phages can be used for the control of Salmonella or Escherichia coli infection through oral administration, spraying, injection and the like.

[0017] (III) Beneficial effects

[0018] Compared with the prior art, the present invention provides a bacteriophage for lysing Salmonella and Escherichia coli and its application, which has the following beneficial effects:

[0019] 1. The Salmonella phage GRNPK is a Salmonella phage with a wide lysis spectrum that can lyse Escherichia coli across species. It has high titer, high burst volume, fast proliferation rate and good safety. The phage preparation can be used alone or in combination, and has strong lysis activity against Salmonella and Escherichia coli of various serotypes or various drug-resistant phenotypes. Most importantly, the phage can specifically kill bacteria in vitro and in vivo, and bacteria are not easy to develop resistance to the phage. The bactericidal effect is stable, indicating that the phage GRNPK can be used as an effective ingredient for a variety of products such as biocides, feed additives and therapeutic drugs, and is widely used. The phage of the present invention can provide an excellent phage source for the development of phage therapy, and has a good application development prospect, suitable for popularization and application;

[0020] 2. The Salmonella phage GRNPK has strong lysis activity against multiple serotypes of Salmonella and pathogenic Escherichia coli that are resistant to multiple antibiotics, indicating that the lysis spectrum of the phage is wide and can be used as a green and environmentally friendly product or means to prevent and treat Salmonella and Escherichia coli diseases in livestock and poultry. In vitro antibacterial tests showed that the phage can delay the development of bacterial resistance to it, and almost no phage-resistant bacteria were produced within 80 hours. The metabolism, safety and in vivo bactericidal tests of chicks have proved that the phage can enter the blood and intestines with a high titer, and is cleared from the body after 72 hours, indicating that the phage is safe, has no toxic side effects, and can effectively reduce the number of Enteritis Salmonella in the intestines of chicks and improve the intestinal health of chicks. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a transmission electron micrograph of the bacteriophage GRNPK of the present invention;

[0022] Figure 2 is a plaque image of bacteriophage GRNPK of the present invention;

[0023] Figure 3 is a one-step growth curve diagram of the bacteriophage GRNPK of the present invention;

[0024] Figure 4 It is the pH stability test result of the bacteriophage GRNPK of the present invention;

[0025] Figure 5It is the result of the thermal stability test of the bacteriophage GRNPK of the present invention;

[0026] Figure 6 This is the in vivo safety verification result of the bacteriophage GRNPK of the present invention;

[0027] Figure 7 It is the result of in vitro antibacterial test of bacteriophage GRNPK of the present invention;

[0028] Figure 8 The metabolic rules of the bacteriophage GRNPK of the present invention in the intestine and blood of chickens;

[0029] Fig. 9 This is the in vivo antibacterial test result of the bacteriophage GRNPK of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The culture medium formula involved in the present invention is as follows:

[0032] TSB liquid culture medium: Weigh 30 g of finished TSB, dissolve it in 1000 mL of distilled water, sterilize it with high-temperature and high-pressure steam at 121°C for 15 min, and cool it to room temperature for use.

[0033] TSB semi-solid agar medium: Weigh 30g of finished TSB and 10g of agar powder, dissolve in 1000mL of distilled water, sterilize at 121℃ high temperature and high pressure steam for 15min, and cool to 50-60℃ for use.

[0034] TSA solid culture medium: Weigh 40 g of finished TSA, dissolve it in 1000 mL of distilled water, sterilize it with high-temperature and high-pressure steam at 121°C for 15 min, cool it to 50-60°C, pour it into a sterile plate, and store it at 4°C after the agar is completely solidified.

[0035] 2×SM buffer (1000 mL): 200 mmol / L NaCl; 16 mmol / L MgSO4; 0.1 mol / L Tris-HCl, 0.02% gelatin, filter sterilize and store at 4°C.

[0036] SS agar medium: weigh 63.53 g of SS finished product, add 1000 mL of distilled water, heat and boil until completely dissolved, cool to 50-60°C, shake well, and pour into a sterile plate for later use.

[0037] The present invention provides a phage for lysing Salmonella and Escherichia coli. The phage was deposited in the China Type Culture Collection of Wuhan University, Wuhan City, Hubei Province, China on July 22, 2024, with a deposit number of CCTCC NO: M20241606, and the phage was named Salmonella phage GRNPK.

[0038] The Salmonella phage GRNPK of the present invention can infect a variety of pathogenic Salmonella and Escherichia coli at the same time, wherein the pathogenic Salmonella includes at least one of Salmonella pullorum, Salmonella fowltyphoid, Salmonella typhimurium, monophasic Salmonella typhimurium, Salmonella Arizona, Salmonella Enteritidis, Salmonella Derby, Salmonella Kentucky, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Salmonella Javana, Salmonella Indiana, Salmonella Rissen, Salmonella Potsdam, Salmonella Livingstone, Salmonella London, Salmonella Wertavreden, Salmonella Arizona, and Salmonella Brundenloops; and the pathogenic Escherichia coli includes at least one of enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), and extraintestinal pathogenic Escherichia coli (ExPEC).

[0039] The separation and purification method of Salmonella phage GRNPK of the present invention is as follows:

[0040] S1. Collect samples such as feces, sewage or chicken intestinal contents from poultry farms. If the sample is large insoluble particles, add 50 mL of sterile water, filter with gauze to remove larger solid particles, and then use filter paper to remove most of the insoluble impurities in the solution. Then centrifuge at 4°C 8000r / min for 10 minutes, collect the supernatant, filter and sterilize it with a 0.22μm microporous filter membrane, take 10 mL of the filtrate, add 30 mL of TSB liquid culture medium and mix well, and add 2.5 mL of OD 600nm Fresh bacterial suspensions of about 0.4-0.6 of Enteritidis Salmonella GXC200717, Fowl Typhoid Salmonella CVCC 1791, Enterohemorrhagic Escherichia coli ATCC 35150, and Enteropathogenic Escherichia coli ATCC 43887 were cultured at 37°C and 200 r / min for 12-16 hours to enrich phages;

[0041] S2. Centrifuge the culture solution at 8000r / min at 4℃ for 10min, take the supernatant and filter it through a 0.22μm microporous filter membrane and use the dot blot method to check whether there are phages that can lyse the above host bacteria in the sample. Specific steps: Take 100μL of the above host bacteria solution and mix it with 3mL of TSB semi-solid agar medium cooled to 50-60℃, pour it into the TSA solid plate prepared in advance, and prepare different double-layer plates. Add 5μL of filtrate to the solidified double-layer plate, and after the liquid dries, place the plate in a 37℃ constant temperature incubator for 12h, and observe whether a transparent lysis zone is formed on the plate. If a lysis zone is formed, it preliminarily indicates that the filtrate contains phages of the host strain;

[0042] S3. Use a pipette to pick up the lysis zone and add it to 2mL 2×SM buffer. After filtering and sterilizing with a 0.22μm microporous filter membrane, take 100μL of the filtrate and dilute it with 2×SM buffer in a 10-fold gradient to a suitable titer. Then take 100μL of the dilution and mix it with the corresponding 100μL host bacterial solution and 3mL TSB semi-solid agar medium cooled to 50-60℃, pour it into a TSA solid plate, and culture it at 37℃ for 12h. At this time, single plaques of different sizes or shapes may appear on the plate, and the phages in the lysis zone need to be purified;

[0043] S4. Phage purification: Use a pipette tip to pick up a single transparent, typical phage plaque and place it in 2 mL of 2×SM buffer. Mix by pipetting and sterilize, then filter through a 0.22 μm microporous filter to obtain the phage filtrate. Dilute 100 μL of the filtrate to an appropriate titer with 2×SM buffer, and obtain a single phage plaque by the double-layer plate method. Repeat the above steps 3-5 times until a single phage plaque of consistent size and morphology is formed on the plate, indicating that a single strain of Salmonella phage GRNPK has been purified.

[0044] The biological characteristics of the Salmonella phage GRNPK of the present invention were identified:

[0045] (1) Phage morphological characteristics

[0046] Phage particles were observed by transmission electron microscopy (TEM). Figure 1 As shown, the phage head capsid is icosahedral symmetrical, with a diameter of about 67±5nm; the tail is about 183±8nm long. Based on its morphology, the phage is classified as a long-tailed phage.

[0047] (2) Plaque morphology

[0048] like Figure 2 As shown, the Salmonella phage GRNPK produces regular, pinpoint-sized circular plaques with neat edges on the Salmonella lawn.

[0049] The lysis spectrum of the Salmonella phage GRNPK of the present invention was determined:

[0050] The lysis ability of GRNPK against 67 strains of Salmonella and 18 strains of Escherichia coli (all donated by Huazhong Agricultural University) was determined by the spot method. Single clones of Salmonella and Escherichia coli were picked and inoculated into 3 mL TSB, respectively, and cultured at 37°C, 200 r / min, until OD 600nm =1, take 100uL of bacterial solution and mix it with 3mL TSB semi-solid medium, pour it into TSA solid plate, wait for the upper agar to solidify, take 5uL of the titer of 10 9 The phage GRNPK lysis solution with a concentration of PFU / mL was spotted on the plate, and after drying naturally, the plate was incubated at 37°C for 12 hours to observe the lysis of the phage. The experiment was set up in triplicate.

[0051] The results are shown in Tables 1 and 2. The Salmonella phage GRNPK has a wide lysis spectrum and can lyse 65 of 67 Salmonella strains, including 19 different serotypes, and have antibiotic resistance, with a lysis rate of 97.0%; it can lyse 23 of 26 Escherichia coli strains, and these 18 Escherichia coli strains are classified as EPEC, ETEC, EHEC, and ExPEC according to their biological characteristics, and have antibiotic resistance, with a lysis rate of 88.5%, indicating that the Salmonella phage GRNPK has a wide lysis spectrum and can lyse multiple strains of Escherichia coli across species.

[0052] Table 1 Lysis effect of Salmonella phage GRNPK on Salmonella

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Table 2 The lysis effect of Salmonella phage GRNPK on Escherichia coli

[0060]

[0061]

[0062]

[0063]

[0064] Note: "-" indicates no lysis; "+" indicates that there are individual lysis spots or spots in the drip area; the lysis area is very turbid; "++" indicates slight lysis and turbid lysis area; "+++" indicates lysis, the lysis area is relatively clear, and only a few colonies grow; "++++" indicates complete lysis. TE: tetracycline, C: chloramphenicol, FON: florfenicol, SF: sulfisoxazole, S: streptomycin, K: kanamycin, CN: gentamicin, AMC: amoxicillin, AMP: ampicillin, FOX: cefoxitin, CRO: ceftriaxone, NA: nalidixic acid, CIP: ciprofloxacin.

[0065] Determination of the optimal MOI of the Salmonella phage GRNPK of the present invention:

[0066] The host bacteria Salmonella enteritidis GXC200717 was cultured to OD 600nm About 0.8-1.0, the number of live bacteria in the culture medium was determined by the plate count method, and the phage solution was diluted in multiples using SM buffer. According to MOI = 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 μL of phage solution was added to 100 μL of host bacterial solution, and supplemented to 10 mL with TSB medium. The mixed solution was cultured in a constant temperature shaker at 37°C and 200 r / min for 4 hours, and then centrifuged at 8000 r / min for 10 minutes to obtain the supernatant. The phage titer in the supernatant was determined by the double-layer agar plate method. The experiment was set up in 3 parallels, and the MOI that produced the highest phage titer was the best MOI.

[0067] The results are shown in Table 3. When the MOI was 0.001, the titer of Salmonella phage GRNPK was the highest, which was 1.1×10 12 PFU / mL, that is, the optimal MOI of Salmonella phage GRNPK is 0.001.

[0068] Table 3 Optimal MOI of Salmonella phage GRNPK

[0069]

[0070] Determination of the one-step growth curve of the Salmonella phage GRNPK of the present invention:

[0071] Phage GRNPK and GXC200717 cultured to the logarithmic phase were mixed at the optimal MOI, incubated in a constant temperature water bath at 37°C for 15 minutes, centrifuged at 6000r / min for 5 minutes, discarded the supernatant, and washed the precipitate twice with TSB medium to remove the unadsorbed phage. The precipitate was resuspended in 200μL TSB medium and supplemented to 10mL with TSB medium preheated to 37°C, cultured at 37°C and 200r / min for 160 minutes, and 100μL of culture solution was taken every 10 minutes or 20 minutes to determine the phage titer by the double-layer plate method. The parallel experiment was repeated 3 times. A one-step growth curve was drawn with the infection time as the horizontal axis and the logarithmic value of the phage titer as the vertical axis.

[0072] The results are as follows Figure 3 As shown, the incubation period and lysis period of Salmonella phage GRNPK infecting host bacteria are 40 minutes and 80 minutes respectively, and the burst volume is about 1060±51 pfu / cell (burst volume=phage titer at the end of the outbreak / host bacteria concentration at the beginning of infection).

[0073] The pH stability of the Salmonella phage GRNPK of the present invention was measured:

[0074] Adjust the pH of TSB medium with hydrochloric acid (HCl) and sodium hydroxide (NaOH) solution to a pH between 2 and 12. Take 900 μL TSB into a sterile EP tube and add 100 μL 10 11 PFU / mL of phage solution. Incubate EP in a constant temperature water bath at 37°C for 12 hours, and use the double-layer agar plate method to determine the phage titer. Repeat the experiment three times. Draw a graph with pH as the horizontal axis and the logarithm of phage titer as the vertical axis.

[0075] The results are as follows Figure 4 As shown, the titer of Salmonella phage GRNPK was incubated for 12 h in the pH range of 3-12, and was not much different from the initial titer, both within 10 9 PFU / mL; after incubation at pH 2 for 12 hours, the phage still maintained a high titer of about 10 7 PFU / mL or above, indicating that the phage has good tolerance to acidic and alkaline environments and can withstand strong acids and alkalis.

[0076] The thermal stability of the Salmonella phage GRNPK of the present invention was determined:

[0077] 1mL 10 9The phage solution of PFU / mL was divided into 3 sterile EP tubes. The EP tubes were placed at -20℃, 4℃, 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 60 minutes, and the phage titer was determined by double-layer agar plate method. The experiment was repeated three times. The temperature was used as the horizontal axis and the logarithm of the phage titer was used as the vertical axis to draw a graph.

[0078] The results are as follows Figure 5 As shown in the figure, the titer of Salmonella phage GRNPK in the experimental group remained stable at a temperature between -20℃ and 50℃, about 10 10 PFU / mL; after 60 min in a 60℃ water bath, the titer remained high, about 10 8 PFU / mL; After 60 min in 70℃ water bath, the titer dropped to 10 4 PFU / mL, and all phages died after being placed in a water bath at 80℃ for 60 min. The above results show that GRNPK has good thermal stability.

[0079] The Salmonella phage GRNPK of the present invention was sequenced:

[0080] Place 50mL of pure phage culture in a 100KD ultrafiltration centrifuge tube, centrifuge at 4℃6000r / min for 20min, concentrate the phage solution to about 1mL, add 10mL SM buffer to remove the culture medium, and then concentrate to about 1ml, twice in a row. Take 200μL of phage concentrate and use Viral DNA Kit (OMEGA) to extract phage DNA. Submit qualified DNA samples to a sequencing company for high-throughput sequencing, and determine the safety of the phage by analyzing the whole genome of Salmonella phage GRNPK.

[0081] Genome analysis showed that bacteriophage GRNPK belongs to the Demerecviridae family. The phage does not contain lysogeny-related genes such as integrase and repressor protein, and is a strictly virulent phage. It does not contain genes encoding virulence, toxins, and antibiotic resistance, indicating that there is no potential safety risk in the use of phage GRNPK in animal production.

[0082] The lysis test of Salmonella phage GRNPK on conditional pathogens and probiotics was carried out:

[0083] According to the method for determining the lysis spectrum of Salmonella phage GRNPK, 15 conditionally pathogenic bacteria and probiotics including non-pathogenic Escherichia coli, Proteus and Enterococcus were selected to test the lysis of Salmonella phage GRNPK on the above 15 intestinal commensal bacteria.

[0084] The results are shown in Table 4. The Salmonella phage GRNPK in this study did not lyse the 15 conditional pathogens and probiotics, indicating that the phage has strong host specificity.

[0085] Table 4 Evaluation of the lysis effect of Salmonella phage GRNPK on intestinal commensal bacteria

[0086]

[0087]

[0088] Note: “_” indicates no lysis; “+” indicates individual lysis spots in the drip area; “++” indicates slight lysis, with blurred lysis spots; “+++” indicates lysis, with clearer lysis spots; “++++” indicates complete lysis.

[0089] Safety assessment of Salmonella phage GRNPK:

[0090] The phage pure culture solution was concentrated in a 100KD ultrafiltration centrifuge tube and resuspended in SM buffer to a titer of 10 9 PFU / mL. Twenty 1-day-old Roman gray laying hens of similar weight purchased from Wuhan Lai De Ecological Agriculture Co., Ltd. were raised in a controlled environment with sufficient food and water. After 3 days of raising, the 20 chicks were randomly divided into 2 groups, with 10 chicks in each group. One group was orally administered with the above-prepared phage concentrate for 10 9 PFU / chicken; another group orally took an equal volume of SM buffer as a control. After oral administration for 3 consecutive days, the two groups of chicks were anesthetized and killed, and the chicks were dissected to examine the changes in organs such as the liver, stomach, intestines and the digestive tract mucosa.

[0091] The results are as follows Figure 6 The results showed that there was no significant difference in the liver, stomach, intestines and intestinal mucosa of the two groups of chicks, indicating that the application of 10 9 The phage preparations with PFU / mL did not have any adverse effects on their growth performance and immunity.

[0092] Determination of the in vitro bactericidal effect of Salmonella phage GRNPK:

[0093] According to the method of determining the lysis spectrum of Salmonella phage GRNPK, the time for Salmonella to develop phage resistance was studied by spot test. 9 10 μL of phage GRNPK lysate with a titer of 10 PFU / mL was spotted on a double-layer agar plate containing Salmonella enteritidis GXC200717. Other purified and concentrated phages with a titer of 10 9PFU / mL of Salmonella phage 1 (Siphoviridae), 3 (Autographiviridae), and 7 (Straboviridae) were used to evaluate the in vitro antibacterial effects of these Salmonella phages.

[0094] The results are as follows Figure 7 As shown in the figure, these four phages all produced nearly circular lysis zones at the inoculation site. The generation of resistant strains in the lysis zones was observed. Salmonella GXC200717 produced resistant mutants after 30 hours of phage 1, 36 hours of phage 3, and 80 hours of phage 7 and GRNPK. However, fewer resistant colonies grew in the lysis zone formed by phage GRNPK. This shows that phage GRNPK can efficiently lyse Salmonella Enteritidis in vitro and delay the generation of phage resistance in bacteria.

[0095] Determination of the metabolic pattern of Salmonella phage GRNPK in chicks:

[0096] 48 one-week-old chicks of similar weight were randomly divided into two groups, 24 in each group, and raised in a controlled environment with sufficient food and water. The chicks were raised normally for 3 days to relieve stress. Each chick was given 100 μL of 10 9 PFU / mL of GRNPK phage lysate. Four chicks from both groups were anesthetized and necropsied at 12h, 24h, 48h, 72h, 120h and 168h, and blood and cecal samples were obtained for phage quantification.

[0097] The results are as follows Figure 8 As shown in Figure 2, 12 hours after a single oral administration to chicks, phage GRNPK was able to enter the blood and had the highest titer, with an average titer of 10 4 PFU / mL, which is 3.5 orders of magnitude lower than the initial administration, and has a high blood entry rate; the average titer in the intestine is 10 6 PFU / mL, a decrease of 1.5 orders of magnitude compared with the initial administration. After 72 hours, phages were almost undetectable in the blood and intestines of chicks, and no animals died or had abnormal reactions during the test.

[0098] Determination of the in vivo bactericidal effect of Salmonella phage GRNPK:

[0099] Fifty-four one-week-old chicks of similar weight were randomly divided into three groups, with 18 chicks in each group, and kept in a controlled environment with adequate food and water. A single colony of Salmonella enteritidis GXC200717 was cultured in 3 mL TSB liquid medium until OD 600nm=1.5, centrifuge at 8000r / min for 1min, discard the supernatant, wash the bacterial pellet three times with 1mL of PBS and resuspend in 1mL of PBS buffer, adjust the OD 600nm =1.0. The phage preparation was obtained from Example 10. The chicks were raised normally for 3 days to relieve stress. Before the start of the experiment, two chicks were randomly anesthetized and dissected to check for Salmonella infection. On the 4th day, each group was treated as follows:

[0100] Model group: Each chicken was gavage-administered once with 100 μL of Salmonella Enteritidis GXC200717.

[0101] Treatment group: Each chick was gavaged once with 100 μL of Salmonella enteritidis GXC200717 and 100 μL of 10 9 PFU / mL of bacteriophage GRNPK.

[0102] Blank control group: Each chicken was gavage-administered once with 100 μL PBS.

[0103] During the experiment, no chicks died. At 12 h, 24 h and 36 h after treatment, 6 chicks in each group were anesthetized and dissected. The intestinal contents were separated and weighed, resuspended in 1 mL PBS buffer and homogenized. 100 μL of the homogenate was taken and the Salmonella was quantified using the selective medium SS agar.

[0104] The results are as follows Fig. 9 As shown in the figure, compared with the model group, the amount of Enteritidis Salmonella in the chicks in the treatment group decreased most significantly at 36 hours, with a decrease of about 10 4 times (from 10 6 CFU / g down to 10 2 CFU / g), indicating that the application of bacteriophage GRNPK in chickens can significantly reduce the number of Enteritidis Salmonella. 8 PFU of phage can effectively protect chicks without obvious side effects, indicating the feasibility of phage GRNPK as a medicinal antibacterial agent.

[0105] In summary, the Salmonella phage GRNPK of the present invention has the following good properties in terms of optimal MOI, one-step growth curve, pH stability, thermal stability, lysis spectrum, genome characteristics and bactericidal effect:

[0106] (1) The Salmonella phage GRNPK of the present invention can reach a titer of about 1.1×10 12 PFU / mL; the optimal infection multiplicity MOI is 0.001; the titer of Salmonella phage GRNPK is maintained at about 10 when incubated for 12 hours in the pH range of 3-12. 9PFU / mL, incubated at pH 2 for 12 h, the phage titer was still high, about 10 7 PFU / mL, indicating that the phage has a wide tolerance range to acid and alkali, and has good tolerance in relatively acidic and alkaline environments; incubated at -20℃-50℃ for 60min, its titer remains unchanged, about 10 10 PFU / mL, incubated at 60°C for 60 min, its titer is about 10 8 PFU / mL, indicating that the phage should be produced or used at a temperature of 30°C-50°C;

[0107] (2) The Salmonella phage GRNPK of the present invention has a genome length of 111280bp through whole genome sequencing. There are no genes with potential risks to the host, such as integrase, transposase, repressor protein, virulence gene, and drug resistance gene in the genome, and the safety is high; based on genome similarity analysis, the phage GRNPK belongs to the Demerecviridae family;

[0108] (3) The Salmonella phage GRNPK of the present invention has a wide host range, with a lysis rate of 97.0% for 67 strains of Salmonella and 88.5% for 26 strains of Escherichia coli;

[0109] (4) The Salmonella phage GRNPK of the present invention is a strictly virulent phage and has a strong lysis effect on Salmonella; it has a wide lysis spectrum and can lyse Escherichia coli across species, and can lyse various serotypes of Salmonella and different pathogenic Escherichia coli; it can specifically kill bacteria and the bactericidal effect is stable, and bacteria are not easy to develop resistance to the phage. The pure culture of the phage can survive stably at room temperature and the titer can remain stable at 4°C for up to 2 years. The present invention does not perform any genetic modification on the test phage. Therefore, the Salmonella phage GRNPK of the present invention can provide high-quality phage resources for the development of phage preparations and has good application development prospects. The Salmonella phage GRNPK of the present invention has a lysis rate of more than 95% for Salmonella and can lyse various pathogenic Escherichia coli across species. In addition, the phage has a good lysis effect on multidrug-resistant Salmonella and Escherichia coli;

[0110] (5) The Salmonella phage GRNPK of the present invention has broad application prospects. In the application of phage, those skilled in the art can prepare a therapeutic agent for preventing and treating diseases such as pullorum, fowl typhoid, fowl paratyphoid, gastroenteritis, pericarditis, perihepatitis, air sac inflammation, peritonitis, etc. caused by infection of Salmonella and Escherichia coli, or other diseases caused by mixed infection of Salmonella and Escherichia coli according to the present application;

[0111] (6) The Salmonella phage GRNPK of the present invention can be used in the preparation of drugs for inhibiting or killing Salmonella or Escherichia coli, wherein the Salmonella mainly includes 19 different serotypes of Salmonella such as Salmonella pullorum, Salmonella enteritidis, and Salmonella typhimurium, and the Escherichia coli mainly includes at least one of enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), and extraintestinal pathogenic Escherichia coli (ExPEC);

[0112] (7) The Salmonella phage GRNPK of the present invention can be used alone or in combination with other phages and antibiotics to prepare various products such as biocides, feed additives and therapeutic drugs, which can be used to prevent and control Salmonella and Escherichia coli pathogens in the environment, on the surface and in the body of animals, and in animal food.

[0113] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A bacteriophage for lysing Salmonella and Escherichia coli, characterized in that: The phage was deposited in the China Type Culture Collection of Wuhan University, Wuhan City, Hubei Province, China on July 22, 2024, with a deposit number of CCTCC NO: 0M20241606, and the phage was named Salmonella phage GRNPK.

2. Use of the bacteriophage according to claim 1 in the preparation of anti-infective drugs for Salmonella or Escherichia coli.

3. The use of the bacteriophage according to claim 2 in the preparation of anti-infective drugs for Salmonella or Escherichia coli, characterized in that: The medicine is used for preventing and treating diseases caused by Salmonella enteritidis infection in chicks.

4. The use of the bacteriophage according to claim 2 in the preparation of anti-infective drugs for Salmonella or Escherichia coli, characterized in that: The medicine is a phage preparation, and the phage reagent comprises an effective amount of the phage GRNPK as claimed in claim 1 and a SM buffer.

5. The use of the bacteriophage according to claim 4 in the preparation of anti-infective drugs for Salmonella or Escherichia coli, characterized in that: The bacteriophage preparation can be used alone or in combination with other bacteriophages to control salmonella or Escherichia coli infection through oral administration, spraying, injection and the like.

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