Wide-splitting-spectrum salmonella typhimurium bacteriophage, bacteriophage composition thereof and application of bacteriophage composition

Abstract

The invention discloses a wide-splitting-spectrum salmonella typhimurium bacteriophage, a bacteriophage composition and application thereof, the bacteriophage is named as salmonella bacteriophage PC817 which is preserved in China General Microbiological Culture Collection Center on August 16, 2024, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.9687. The invention further discloses a preparation method of the wide-splitting-spectrum salmonella typhimurium bacteriophage. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection The bacteriophage PC817 is a salmonella typhimurium bacteriophage with a wide lysis spectrum, has the characteristic of high lysis rate for different types of salmonella, and can be used for preparing drugs for treating or preventing salmonellosis, environment disinfectants, feed additives, drinking water additives, food preservatives, detection kits and the like. While salmonella typhimurium infection is solved, the problems of antibiotic residues and pathogen drug resistance caused by use of antibiotics are avoided. The bacteriophage and the composition thereof can provide a more effective scheme for prevention and control of pigeon salmonellosis.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Salmonella typhimurium phage, a phage composition thereof, and their applications. Background Art

[0002] Salmonella, as a common foodborne pathogen, is widely distributed in nature. It not only infects livestock and poultry as the source of infection and spreads among animals through horizontal and vertical transmission, but also can infect humans through food and other routes, causing foodborne diseases. Some studies have shown that 20% of poultry products in the world are contaminated with Salmonella, which can exist in the animal and human environment for a long time by forming biofilms, or attach to the surfaces of carriers such as drinking water equipment, medical equipment, and food processing. The formation of biofilms greatly improves the survival ability of Salmonella in nature. Approximately 80% of chronic infections are related to the formation of biofilms. According to statistics, 70% - 80% of bacterial food poisoning incidents in China are caused by Salmonella, and about 90% of the foods in which Salmonella poisoning occurs are livestock products such as meat, eggs, and milk. Slaughterhouses are also recognized as the best storage places for Salmonella.

[0003] Salmonella Typhimurium is a Gram-negative bacterium mainly parasitic in the intestine. As a common Salmonella and an important zoonotic pathogen, its infection incidence ranks first among Salmonella infections, and its detection rate is relatively high in foodborne diseases and food poisoning cases in China. Salmonella Typhimurium was first discovered in 1885. American veterinary experts Salmon and Smith isolated the first bacterium other than typhoid bacillus from cholera-infected pigs and named it Salmonella Typhimurium. Salmonella Typhimurium is the main serotype causing salmonellosis. This bacterium is mainly transmitted through food or water. Infecting poultry will cause clinical manifestations such as high fever, abdominal pain, and diarrhea. If not treated in time, it will weaken the immunity of animals, lead to the occurrence of intestinal diseases, and pose a serious threat to the healthy development of the poultry industry. Therefore, preventing and controlling Salmonella Typhimurium infection is of great importance.

[0004] In recent years, after the improvement of the material level, the demand for pigeon meat by people has been increasing year by year. Some studies have shown that the characteristics of pigeon meat with high protein and low fat at 12 months of age can better meet the nutritional needs of consumers. In order to meet the increasing demand for pigeon meat, the breeding scale of pigeon farms has been continuously expanded. In some pigeon farms, the environmental sanitation and feeding management levels have not been improved, and bacterial diseases are likely to occur. Pigeon salmonellosis is mainly caused by Salmonella Typhimurium, also known as pigeon paratyphoid, which can occur throughout the year. Not only in intensive pigeon farms, but also the isolation rate of Salmonella-resistant strains in wild pigeons is increasing. It can be seen that pigeons infected with Salmonella are very common.

[0005] To avoid the adverse effects of bacterial drug resistance on the aquaculture industry and public health, relevant laws were promulgated in China in 2020 to prohibit or restrict the use of antibiotics, and appropriate alternatives were actively sought to promote animal intestinal health, which has become an urgent problem to be solved in the poultry industry. In the context of antibiotic bans, phages have received extensive attention due to their antibacterial effects. Phages have the characteristics of obligate parasitism and strong specificity. They only destroy the corresponding host bacteria, have no effect on other bacterial populations, and die with the clearance of the host bacteria. When phages infect host bacteria, the depolymerases and lysins encoded by themselves can degrade polysaccharides and peptidoglycans, destroy the extracellular polymeric structure, and lyse biofilms.

[0006] However, there is currently no phage with high lysis performance specifically targeting Salmonella typhimurium from pigeons. Existing phage products are difficult to be effectively used for preventing and treating pigeon paratyphoid caused by Salmonella typhimurium from pigeons. Therefore, the existing technology needs to be further improved. Summary of the Invention

[0007] To solve the above problems, the present invention provides a Salmonella typhimurium phage PC817, its phage composition and its applications. The above phage PC817 is a virulent broad-spectrum lysing Salmonella typhimurium phage, which can effectively prevent and treat salmonellosis and can be used to prepare drugs for treating or preventing salmonellosis, environmental disinfectants, feed additives, drinking water additives, food preservatives and detection kits, etc. While solving Salmonella typhimurium infections, it avoids the problems of antibiotic residues and pathogen drug resistance caused by the use of antibiotics.

[0008] To solve the above problems, the technical solutions of the present invention are specifically as follows:

[0009] In the first aspect, the present application provides a Salmonella typhimurium phage, which is named Salmonella phage PC817, and its deposit number is CGMCC No. 46169. This phage was deposited on August 16, 2024 at the China General Microbiological Culture Collection Center, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Observed by electron microscopy, this phage PC817 has a polyhedral head structure and a non-contractile tail. The head is 60-64 nm wide and 68-72 nm long, and the tail is about 132-138 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of this phage PC817 in the present application conforms to the characteristics of the family Siphoviridae and belongs to the Siphoviridae.

[0011] In this application, the phage PC817 includes mutant strains with a homology higher than 98% or 99% after point mutation, deletion mutation, or addition mutation and maintaining substantially the same bactericidal activity. Since phages are very prone to mutation during replication, the mutants of the above phages are also within the scope of protection claimed in this application. The sequence of phage PC817 can be obtained by sequencing through well-known methods based on the biological material preserved according to the present invention. For those skilled in the art, it does not require creative labor to screen mutants with extremely similar traits from the phages provided by the present invention.

[0012] The lysis rate of the Salmonella typhimurium phage PC817 against pigeon-derived Salmonella is 93.87%. The pigeon-derived Salmonella includes Salmonella typhimurium, Salmonella paratyphi A, Salmonella newport, and Salmonella kisangani. Among them, the lysis rate of this phage against pigeon-derived Salmonella typhimurium is 100%, the lysis rate against chicken-derived Salmonella is 84.61%, the lysis rate against duck-derived Salmonella is 75%, and the lysis rate against food-derived Salmonella is 80%. Thus, it can be seen that the Salmonella typhimurium phage PC817 has a broad lysis spectrum and the highest lysis rate against pigeon-derived Salmonella typhimurium.

[0013] In a second aspect, this application also provides a phage composition, which includes the aforementioned Salmonella typhimurium phage and other phages. In practical applications, in order to further broaden the lysis spectrum of phage preparations, give full play to the differences in the lysis spectra of different phages, and make complementary advantages, the above Salmonella typhimurium phage PC817 and other phages can be used in combination.

[0014] Optionally, in the phage composition, the other phages include: one or both of phage PC204 (specifically see the publication number CN115786279A) and phage SP8 (specifically see the publication number CN116286671A); among them, the preservation number of phage PC204 is: CGMCC No. 22368; the preservation number of phage SP8 is: CGMCC No. 45256. By using phages in combination, the lysis spectrum is broadened to kill different types of Salmonella in the farm environment for better prevention and treatment of Salmonella diseases in poultry and pigeons.

[0015] Phage PC204 is preserved in the China General Microbiological Culture Collection Center, with the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is April 12, 2021, and the preservation number is No. 22368. For specific information, see the patent with the publication number CN115786279A.

[0016] Phage SP8, with the deposit number of No. 45256, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 12, 2022. For specific information, please refer to the patent with the publication number CN116286671A.

[0017] In addition, the above-mentioned phage PC817 can also be combined with other different types of phages (which inhibit different pathogenic bacteria causing the same type of disease) for the prevention and treatment of the same type of disease, such as acute septicemia caused by mixed infection of different pathogenic bacteria.

[0018] In a third aspect, the present application also provides the use of the aforementioned Salmonella typhimurium phage or the aforementioned phage composition in the preparation of drugs, feed additives, environmental disinfectants, raw meat disinfectants, food preservatives, and detection kits for preventing and treating diseases caused by Salmonella infection. The prevention and treatment include prevention and treatment. The term "prevention" herein refers to all actions including inhibiting or delaying the disease by administering the composition. The term "treatment" herein refers to all actions including improving or ameliorating the disease by administering the composition.

[0019] Preferably, the Salmonella is Salmonella typhimurium of pigeon origin. The diseases caused by the infection of Salmonella of pigeon origin are: pigeon salmonellosis, also known as pigeon paratyphoid. According to its symptoms and lesions, it can be divided into four types: intestinal type, joint type, visceral type, and nervous type.

[0020] In a fourth aspect, the present application also provides a phage pharmaceutical preparation, the active ingredient of which includes the aforementioned Salmonella typhimurium phage or the aforementioned phage composition; preferably, the phage pharmaceutical preparation further includes other antibacterial or bactericidal active ingredients.

[0021] Optionally, the form of the phage pharmaceutical preparation is an oral dosage form, an external dosage form, or a parenteral dosage form. The application method of the phage pharmaceutical preparation is: adding the phage or its composition as a therapeutic drug to drinking water or feed, or administering it to pigeons by gavage, subcutaneous injection, or intramuscular injection. By the above methods, salmonellosis can be prevented and treated, and the survival rate can be improved, etc.

[0022] Optionally, the phage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not eliminate the biological activity and properties of the administered active ingredient. In order to formulate the pharmaceutical composition into a liquid preparation, the pharmaceutically acceptable carrier must be suitable for sterility and biocompatibility. Examples include saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, glucose solution, maltodextrin solution, glycerol, ethanol, various culture media, etc. They can be used alone or in any combination thereof. As needed, other conventional additives can be added, such as antioxidants, buffers, bacteriostatic agents, etc. When combined with diluents, dispersants, surfactants, binders, and / or lubricants, the composition of the present invention can also be prepared into injection and oral dosage forms (e.g., aqueous solutions, suspensions, and emulsions, pills, capsules, granules) and other intermediate dosage forms, such as lyophilized products.

[0023] In a fifth aspect, the present application also provides a feed additive or drinking water additive, the active ingredient of which comprises the aforementioned Salmonella typhimurium phage or the aforementioned phage composition. Preferably, the concentration of the phage is 10 7 PFU / mL or more.

[0024] By adding the above-mentioned drinking water additive or feed additive to water or mixing it with feed, pigeons are fed, thereby disinfecting and sterilizing the drinking water and feed in the pigeon farm. The acid resistance of the phage allows it to survive in the gastric acid environment, effectively preventing and treating Salmonella disease.

[0025] Optionally, the drinking water additive or feed additive further comprises other active ingredients for inhibiting or eliminating bacteria in water; the drinking water additive and feed additive are in the form of liquid dosage form, powder dosage form, or solid dosage form, but are not limited to the above three dosage forms.

[0026] In a sixth aspect, the present application also provides an environmental disinfectant, the active ingredient of which comprises the aforementioned Salmonella typhimurium phage or the aforementioned phage composition. Preferably, the concentration of the phage is 10 8 PFU / mL or more.

[0027] Optionally, the environmental disinfectant further comprises other active ingredients for inhibiting or eliminating bacteria in the pigeon breeding farm environment; preferably, the environments where the environmental disinfectant can be applied include feed, water, and the breeding environment, and the breeding environment includes feeding tools such as pigeon houses, pigeon cages, waterers, feed troughs, feces, and bedding.

[0028] In a seventh aspect, the present application further provides the use of the environmental disinfectant in the disinfection of the pigeon farm environment. The application method is as follows: The disinfectant is used in pigeon slaughterhouses, pigeon product processing workshops and utensils, and pigeon breeding environments to prevent the contamination of Salmonella from pigeons in the environment. The breeding environment includes feeding tools such as pigeon coops, pigeon cages, waterers, and feed troughs, as well as feces and bedding. The application method includes, but is not limited to, disinfecting and decontaminating the water distribution system, aquaculture facilities, feeding utensils or other environmental surfaces of the pigeon farm in the form of liquid immersion, spraying, combined use with a water-containing carrier, etc., and disinfecting and preserving the feed. This disinfectant can be used to replace antibiotics or traditional disinfection products, and it will not cause harm to humans and poultry.

[0029] In an eighth aspect, the present application further provides a raw meat disinfectant or a raw meat preservative, which comprises the aforementioned Salmonella typhimurium phage or the aforementioned phage composition.

[0030] In a ninth aspect, the present application further provides the use of the above raw meat disinfectant or raw meat preservative. The application method is as follows: The raw meat disinfectant can be used to kill Salmonella by spraying or soaking the surface of raw meat after processing in the slaughterhouse.

[0031] In a tenth aspect, the present application further provides a phage preparation, which is a spray-dried powder. The preparation method is as follows: Using skim milk powder and lactose as protectants, adding the phage PC817 proliferation solution and mixing evenly, then performing spray drying, and finally collecting the spray-dried particles, which is the phage preparation.

[0032] Among them, the content of skim milk powder is 8-12% (mass / volume percentage), and the content of lactose is 0.5-2.5% (mass / volume percentage); preferably, the content of skim milk powder is 10%.

[0033] The present invention has the following beneficial effects:

[0034] 1. The present invention provides a virulent Salmonella typhimurium phage PC817 with a broad lysis spectrum, which has good lysis effects on different types and serotypes of Salmonella, can effectively prevent and control Salmonella diseases, has a specific lysis advantage for Salmonella typhimurium from pigeons, and has a more prominent effect on pigeon paratyphoid.

[0035] 2. Based on the above biological characteristics, the phage can be used for the prevention and control of Salmonella diseases in pigeon farms, and has the effects of reducing the mortality rate of pigeon flocks and maintaining the freshness of meat quality. The phage can be used as an active ingredient of disinfectants, feed and water additives, food preservatives, etc., and while solving Salmonella infections, it avoids the problems of antibiotic residues and pathogen drug resistance caused by the use of antibiotics.

[0036] 3. Bacteriophage PC817 has strong stability in an acidic environment and can adapt to a certain range of strong acid environments. The above-mentioned bacteriophage is obtained from nature and is easy to be industrially produced. The drugs or disinfectants prepared from the above-mentioned bacteriophage can not only reduce costs but also have the advantages of being green and environmentally friendly. Description of the Drawings

[0037] Figure 1 It is a plaque picture of Salmonella typhimurium bacteriophage PC817;

[0038] Figure 2 It is an electron microscope picture of Salmonella typhimurium bacteriophage PC817;

[0039] Figure 3 It is the pH value stability test result of Salmonella typhimurium bacteriophage PC817;

[0040] Figure 4 It is the temperature stability test result of Salmonella typhimurium bacteriophage PC817;

[0041] Figure 5 It is the one-step growth curve of Salmonella typhimurium bacteriophage PC817;

[0042] Figure 6 It is the influence of Salmonella typhimurium bacteriophage PC817 on the TVB-N value of meat. Detailed Implementation Modes

[0043] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0044] Example 1 Isolation and Purification of Salmonella typhimurium Bacteriophage PC817

[0045] 1. Resuscitation and Cultivation of Host Bacteria

[0046] Select 18 strains of Salmonella typhimurium preserved in this laboratory, use a sterilized inoculation loop to dip the cryopreservation solution and streak it on an SS medium for resuscitation, and culture it in an incubator at 37°C for 18 - 24 h to obtain single colonies; pick the single colonies and inoculate them into 5 mL of NB broth, and culture them with shaking at 37°C and 170 rpm / min for 16 h to obtain a fresh Salmonella typhimurium bacterial solution.

[0047] 2. Isolation of Bacteriophages

[0048] Take appropriate amounts of samples such as pigeon feathers and fecal water from Guangdong region and place them in a sample bottle. Add an appropriate amount of broth medium and 18 strains of Salmonella typhimurium bacterial liquid. Put the mixed solution in a shaker at 37°C and 170 rpm / min for 12 h, centrifuge at 11000 rpm for 5 min, and then filter through a 0.22-μm sterile microporous filter membrane to obtain a phage proliferation solution. Dilute the phage stock solution by 10-fold serial dilution. Take appropriate gradient phage dilution solutions and mix them with 18 strains of Salmonella typhimurium one by one at a ratio of 1:1 evenly. After incubating at 37°C for 5 min, pipette 200 μL of the mixed solution into the upper agar (agar concentration is 0.7%), mix well and quickly pour it onto the lower agar (agar concentration is 1.5%) petri dish. Shake well and leave it flat until the medium solidifies. Place it in an incubator at 37°C and incubate it upside down for 4 - 6 h to obtain a double-layer plate with phage plaques formed.

[0049] 3. Purification of Bacteriophages

[0050] Pick a single phage plaque from the double-layer agar medium with phage plaques formed and place it in 1 mL of NB broth. Culture it in a constant-temperature shaker at 37°C and 170 rpm for about 30 min to obtain a phage leaching solution. Take the phage leaching solution and mix it evenly with the proliferation solution of the corresponding plaque-forming Salmonella typhimurium (referred to as the host bacterium later) at a ratio of 1:1 (incubate at 37°C for 5 min). Pipette 200 μL, mix it evenly in the upper agar and quickly pour it onto the lower agar petri dish. Shake well and leave it flat until the medium solidifies. Place it in an incubator at 37°C and incubate it upside down for 4 - 6 h to obtain a double-layer plate with phage plaques formed again. Use a sterilized forceps to pick a single phage plaque from the double-layer medium with plaques formed and place it in 1 mL of NB broth. Culture it in a constant-temperature shaker at 37°C and 170 rpm for about 30 min to obtain a phage leaching solution. Repeat the above steps 3 times to obtain a purified phage leaching solution.

[0051] 4. Phage Proliferation and Titer Determination

[0052] Take equal amounts of the purified phage leaching solution and the host bacterium proliferation solution in 5 mL of liquid NB medium. Culture it in a shaker at 37°C and 170 rpm until the liquid becomes clear. Centrifuge the clear liquid at 11000 rpm for 10 min, take the supernatant, filter it through a 0.22-μm sterile microporous filter membrane to obtain a phage proliferation solution, and determine the titer of the newly isolated phage by the double-layer plate method.

[0053] 5. Experimental Results and Analysis

[0054] According to the above experimental method, 10 Salmonella typhimurium phages were screened from 20 Salmonella typhimurium isolates, numbered PC810 - PC819 respectively. All 10 Salmonella typhimurium phages formed clear plaques on the double - layer agar medium plate, without a halo around them, with clear edges, and the diameter was about 0.5 mm - 1.2 mm. The titers of these ten phages were between 3.1×10 9 PFU / mL and 7.2×10 10 PFU / mL (the results are shown in Table 1). Among them, the phage PC817 had the highest titer, and it was used as the experimental object for various experiments in the following examples.

[0055] Table 1 Titers of 10 newly isolated Salmonella typhimurium phages (PFU / mL)

[0056] Phage name Titer Phage name Titer PC810 <![CDATA[3.4×10 9 > PC815 <![CDATA[7.4×10 9 > PC811 <![CDATA[3.1×10 9 > PC816 <![CDATA[3.8×10 9 <!-- 5 -->]]> PC812 <![CDATA[4.6×10 10 > PC817 <![CDATA[7.3×10 10 > PC813 <![CDATA[7.2×10 9 > PC818 <![CDATA[4.6×10 10 > PC814 <![CDATA[3.7×10 9 > PC819 <![CDATA[8.2×10 9 >

[0057] Example 2 Morphological observation and identification of Salmonella typhimurium phage PC817

[0058] 1. Copper grid preparation and electron microscopy observation

[0059] Take 20 μL of the phage sample and drop it on a copper grid with a carbon - coated film. Wait for it to precipitate naturally for 15 min. After slightly blotting it with filter paper, stain it with 2% (W / V) phosphotungstic acid (PTA) for 1 - 2 min. Slightly blot it with filter paper again, and after drying, observe and take pictures under a transmission electron microscope.

[0060] 2. Identification results

[0061] As Figure 2 shown, phage PC817 has a polyhedral head structure and a non - contractile tail. The head is 60 - 64 nm wide and 68 - 72 nm long, and the tail is about 132 - 138 nm long. According to the classification method of the International Committee on Taxonomy of Viruses (ICTV), the morphology of the phage in this application conforms to the characteristics of the Siphoviridae family and belongs to the Siphovirus.

[0062] Example 3 Determination of the lysis spectrum of Salmonella typhimurium phage PC817

[0063] 1. Experimental method:

[0064] Select 80 pathogenic Salmonella strains with different sources and serotypes isolated from different regions and preserved in the laboratory. Determine the lysis rate of PC817 against these 80 Salmonella strains by the double - layer plate method. The detailed strain information and lysis spectrum information are shown in Table 2 below. At the same time, the lysis rate of another existing Salmonella typhimurium phage PC204 was also measured synchronously. The specific information of this phage can be found in the patent with the publication number CN 115786279 A.

[0065] 2. Experimental Results and Analysis

[0066] It can be seen from the experimental results of the lysis spectrum in Table 2 that:

[0067] (1) Salmonella typhimurium phage PC817 can lyse 71 out of 80 Salmonella strains of different types and serotypes, with a lysis rate as high as 88.75%. The lysis rate of this phage against 25 Salmonella typhimurium strains among the 80 Salmonella strains is 96%, and the lysis rate against 23 Salmonella typhimurium strains from pigeons is 100%. This result shows that phage PC817 has a higher lysis rate against Salmonella typhimurium in pigeons and has a specific lysis advantage over it.

[0068] Among 49 Salmonella strains from pigeons, including Salmonella typhimurium, Salmonella paratyphi A, Salmonella newport, and Salmonella kisangani, 46 strains can be lysed, and the lysis rate is 93.88%.

[0069] The phage PC817 of this application has a high lysis effect on both Salmonella enteritidis and Salmonella gallinarum. Among them, the lysis rate against 13 Salmonella strains from chickens is 84.61%, and the lysis rate against 8 Salmonella strains from ducks is 75%. The lysis rate of this phage against 10 foodborne Salmonella strains is 80%. This result shows that this phage can also be used to effectively prevent the colonization of Salmonella in food.

[0070] (2) Phage PC204 can lyse 67 out of 80 Salmonella strains of different types and serotypes, with a lysis rate as high as 83.75%; it can lyse 22 out of 25 Salmonella typhimurium strains, with a lysis rate of 88%, and can lyse 20 out of 23 Salmonella typhimurium strains from pigeons, with a lysis rate of 87% against Salmonella typhimurium strains from pigeons.

[0071] It can be seen that the lysis rate of phage PC817 of this application against various Salmonella strains is higher than that of phage PC204. It can be seen that phage PC817 has a good lysis effect and broad-spectrum property against Salmonella. In addition, the lysis effect of phage PC817 of this application against Salmonella typhimurium strains from pigeons is also significantly better than that of phage PC204.

[0072] In order to improve the use effect of the phage product, phage PC817 can be combined with phage PC204 and SP8 used in the previous patent to improve the lysis spectrum of the phage product.

[0073] Table 2 Lysis Spectrum Results of Salmonella Phage PC817 Against 80 Salmonella Strains from Different Sources

[0074]

[0075]

[0076]

[0077]

[0078] Example 4 Lysis Test of Salmonella typhimurium Phage PC817 against Non-host Bacteria

[0079] 1. Experimental Method

[0080] Select 10 strains of Escherichia coli, 5 strains of Staphylococcus, 5 strains of Proteus and 5 strains of Clostridium perfringens, a total of 25 different types of non-host bacteria, and determine the lysis spectrum of Salmonella typhimurium phage PC817 according to the method for determining the lysis spectrum in Example 4.

[0081] 2. Experimental Results and Analysis

[0082] No clear lytic plaques were found in the double-layer plates of Salmonella typhimurium phage PC817 and the above 25 strains of non-host bacteria, indicating that the phage could not recognize these 10 strains of Escherichia coli, 5 strains of Staphylococcus, 5 strains of Proteus and 5 strains of Clostridium perfringens. This shows that the tested phage PC817 has extremely strong host specificity and has no damaging effect on the microbial community, and its specificity can be used to prepare a detection kit.

[0083] Example 5 pH Tolerance of Salmonella typhimurium Phage

[0084] 1. Experimental Method

[0085] Add 4.5 mL of NB broth with different pH values (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) to sterile test tubes, three for each pH value. Then place the test tubes in a water bath at 37 °C. After the temperature stabilizes, add 500 μL of 5×10 10 pfu / mL phage proliferation solution to each tube, mix well, and incubate in a 37 °C water bath for 1 h, 2 h, and 3 h. After the incubation, immediately add an appropriate amount of 1 mol / L HCl or NaOH to the mixture to adjust the pH value of the mixture to about 7, perform 10-fold serial dilutions, and take appropriate dilution gradients to determine the titer. Set 2 replicates for each test tube with a specific pH value. Plot the phage pH stability curve with the pH value as the abscissa and the logarithm of the phage titer as the ordinate.

[0086] 2. Experimental Results and Analysis

[0087] From Figure 3 it can be seen that within the pH range of 5.0 - 9.0, the titer of phage PC817 remains at 10 9 pfu / mL and its activity is stable. When acting at pH 2.0 for 2 h, the titer is at 105 pfu / mL, it can resist a certain strong acid environment and still retain a certain activity after being treated at pH 1.0 for 1 h; after being treated at pH 11 for 3 h, the titer of this phage is at 10 7 pfu / mL, and when treated at pH 13 for 3 h, the titer drops to 10 2 pfu / mL. Therefore, phage PC817 has strong stability in an acidic environment and can adapt to a certain range of strong acid environments.

[0088] Example 6 Temperature tolerance of Salmonella typhimurium phage

[0089] (1) Experimental method

[0090] 5×10 10 PFU / mL of phage PC817 proliferation solution was incubated at 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, and 90 °C respectively, with three parallel samples at each temperature. After incubating for 20 min, 40 min, and 60 min, immediately cool the samples in an ice bath after the treatment, and then detect the phage titer at different temperatures by the double-layer plate method. Plot the thermal stability curve of phage PC817 with temperature as the abscissa and the logarithm of phage titer as the ordinate.

[0091] (2) Experimental results and analysis

[0092] The results are as Figure 4 It can be seen that phage PC817 still maintains its original activity after being treated at 60 °C for 60 min; when treated at 70 °C for 40 min, the phage still contains 10 8 PFU / mL of active phage. When treated at 80 °C for 20 min, the phage titer is about 2.21×10 6 PFU / mL. When treated at 90 °C for 20 min, phage PC817 still has a certain activity, and the titer is about 1.28×10 4 PFU / mL. Thus, it can be seen that Salmonella phage PC817 has good temperature stability and can adapt to a relatively high temperature environment.

[0093] Example 7 One-step growth curve of Salmonella typhimurium phage PC817

[0094] 1. Experimental method

[0095] Mix 1 mL of the phage proliferation solution with a multiplicity of infection of 10 and 1 mL of the fresh proliferation solution of the host bacteria thoroughly (start timing at this point), incubate at 37 °C for 5 min, centrifuge at 13000 rpm for 30 s, aspirate the supernatant as much as possible with a micropipette, wash once with 5 mL of NB broth (centrifuge at 13000 rpm for 30 s), and discard the supernatant. Suspend the precipitate with pre-warmed NB broth (total volume is 5 mL) and mix thoroughly, quickly place it in a shaker at 37 °C and shake-culture at 170 rpm. Take out 150 μL at 0 h and every 5 min, centrifuge at 10000 rpm for 1 min,

[0096] Perform 10-fold serial dilutions with NB broth and then use the double-layer plate method to measure the phage titer. Do 3 parallels and take the average value. Use the infection time as the abscissa and the phage titer in the infection system as the ordinate to plot a one-step growth curve, obtain the latent period and burst period of the phage, and calculate the burst size.

[0097] Burst size = total number of phages at the end of the phage burst / total number of bacteria at the beginning of the burst

[0098] 2. Experimental results and analysis

[0099] From Figure 5 the results, it can be seen that after phage PC817 infects the host bacteria, the lysis cycle duration of the phage is about 75 min, the latent period is about 10 min, and the burst period of the phage is about 60 min; after 75 min, the phage number is basically unchanged and enters the stationary phase. At this time, the titer can reach 10 10 pfu / mL, and the burst size of phage PC817 is 121.

[0100] Example 8 Determination of the Optimal Multiplicity of Infection (MOI) of Salmonella typhimurium Phage PC817 for Salmonella 1. Experimental method

[0101] Pick a single Salmonella colony and inoculate it into 5 ml of NB broth medium, culture at 37 °C and 170 rpm for 12 - 16 h to obtain a bacterial solution. Determine the bacterial concentration by the pour plate method, and adjust the bacterial solution concentration to 1×10 9 cfu / mL, 1×10 8 cfu / mL... 1×10 5 cfu / mL according to the measurement results. Adjust the phage concentration obtained from the separation in Example 1 to 1×10 5 ~1×10 7PFU / mL. Add phages and bacterial suspensions to NB medium according to the ratio of phage number to bacterial number in Table 3, and proliferate them in a shaker at 37 °C and 170 rpm until the liquid becomes clear, and record the proliferation time. Take an appropriate amount of the clear liquid, centrifuge it at 11000 rpm for 10 min, take the supernatant, filter it through a 0.22-μm sterile microporous membrane, and determine the phage titer in the filtrate by the double-layer plate method. The MOI (phage number / bacterial number) with the highest phage titer is the optimal multiplicity of infection of the phage.

[0102] 2. Experimental results

[0103] Table 3 shows that when the MOI is 0.01:1, the titer of PC817 reaches the highest, 1.2×10 11 PFU / mL. At this time, the initial input amount of phages is small, and the highest reproduction yield can be achieved, which is beneficial to large-scale industrial production.

[0104] Table 3 Titers of Salmonella phage PC817 at different multiplicities of infection

[0105]

[0106] Example 9 Environmental disinfection test of Salmonella typhimurium phage PC817

[0107] 1. Experimental method

[0108] In a certain pigeon hatchery, a large number of Salmonella were detected, and the bactericidal effects of chemical disinfectants and others were not obvious. In this experiment, phage PC817 was designed to be used for spray disinfection of the hatching workshop.

[0109] Among them, the preparation method of the phage disinfectant is as follows: Mix the phage liquid and the host bacteria in a 1:1 ratio in NB broth medium, culture at 37 °C and 170 rpm for 4 - 6 h until the liquid becomes clear and bacterial debris can be seen. Take the clear liquid and filter to remove the bacterial debris residue, and determine the phage titer to be 1×10 10 pfu / ml, dilute the phage liquid 100 times to prepare a phage disinfectant with a titer of 1×10 8 pfu / mL, seal it and store it at 2 - 8 °C for standby.

[0110] Adopt the air disinfection method, and the specific operation is as follows:

[0111] Two hatching workshops of the same size were selected to collect air samples before and after disinfection in the hatching workshops by the natural sedimentation method. Five points, including the central point and four points at the corners of the wall, were used as test points. The sampling points were 0.5 m above the ground, and the four points at the corners were 1 m away from the wall. XLD agar plates with a diameter of 9 cm were placed at each point. Before disinfection, 2 XLD agar plates were used at each sampling point. The lids of the culture dishes were opened and sampled for 10 min. Disinfection was carried out using the fog line provided by the pigeon farm, and two disinfectants were used respectively for disinfection, namely phage preparation disinfection (10 mL / m 3 , phage titer 1×10 8 pfu / mL) and benzalkonium bromide disinfection (benzalkonium bromide diluted 1:25). After 30 min of disinfection, 2 more XLD agar plates were placed at each of the five sampling points. The lids of the culture dishes were opened and sampled for 10 min. The sampled culture dishes before and after disinfection were placed in a constant temperature incubator at 37 °C and cultured for 12 - 24 h, and the cultured bacteria were counted.

[0112] The total number of colonies C was counted according to the Orr formula: C = 50000N / AT, where C is the total number of colonies per cubic meter (CFU / m3); N is the number of colonies per dish; A is the area of the culture dish (cm2); T is the sampling time (min).

[0113] 2. Experimental results and analysis

[0114] As shown in Table 4, after disinfection with benzalkonium bromide disinfectant, the extinction rate of Salmonella in the air of the hatching workshop was 53.19%; after disinfection with phage PC817, the extinction rate of Salmonella in the air of the hatching workshop was 88.31%. It can be seen that the environmental disinfection effect of phage PC817 is significantly better than that of the existing chemical disinfectant benzalkonium bromide, and it is safer to use.

[0115] Therefore, the above results show that phage PC817 has excellent disinfection and killing effects on Salmonella in the hatching workshop, can significantly reduce Salmonella in the environment, and can be promoted and applied as a new type of biological environmental disinfectant.

[0116] Table 4 Salmonella colony counts before and after disinfection with phage PC817 and benzalkonium bromide disinfectants

[0117]

[0118] Example 10 Treatment test of phage as a drinking water additive for pigeon salmonellosis

[0119] 1. Experimental method

[0120] Forty diseased pigeons infected with Salmonella were randomly divided into 2 groups, with 20 pigeons in each group, namely the control group and the experimental group. The experimental group drank water containing phage (10 8Drinking water containing phages at a concentration of [[ID=]], and the control group drank drinking water without phages. Medication was administered continuously for 1 week, followed by 1 week of observation, and then the mortality rate of diseased pigeons was counted.

[0121] 2. Experimental results and analysis

[0122] As can be seen from the results in Table 5 below, the mortality rate of diseased pigeons in the experimental group was 10% lower than that in the control group. This indicates that the above phages can be used as a biological additive in drinking water to prevent and control pigeon salmonellosis. It also has a preventive and control effect on pigeon salmonellosis when used as a feed additive.

[0123] Table 5 Death results of diseased pigeons

[0124]

[0125] Experiment on the preventive and control effect of the freeze-dried powder of phage PC817 as a feed additive on salmonellosis in a pigeon farm in Example 11 1. Experimental method

[0126] Select a laying pigeon farm in the south with a relatively high positive rate of salmonellosis antibodies, and select two pigeon houses with 1200 pigeons each as the experimental group and the control group respectively.

[0127] (1) Preparation of phage spray-dried powder: Using skim milk powder and lactose as protectants, the composition of the protectants is 8 - 12% by weight of skim milk powder and 0.5% - 2.5% by weight of lactose. After adding the phage PC817 proliferation solution and mixing evenly, it is dried using a Swiss type nano spray dryer, and the process parameters are: inlet temperature (120 °C), air flow rate (120 L / min), spray rate (100%), nozzle cap size (4.0 μm). After spray drying for about 1 h, the spray-dried particles are collected. Finally, the phage PC817 is made into a spray-dried powder dosage form with a concentration of 1×10 9 PFU / g.

[0128] (2) Treatment of the experimental group: The prepared phage PC817 powder was added to the feed at a rate of 500 g / ton and used starting from the squab stage. Treatment of the control group: Phages were not added to the feed. Other immunization and medication procedures were carried out according to the farm procedures. (3) Serum samples of 28-day-old and 49-day-old laying pigeons from the experimental group and the control group were collected respectively and subjected to a serum plate agglutination test with positive serum of Salmonella typhimurium.

[0129] 2. Experimental results and analysis

[0130] (1) The results are shown in Table 6. For 28-day-old laying pigeons, the positive rate of Salmonella typhimurium antibody in the control group was 17.5%, while that in the phage group was 0.34%, a decrease of 17.16%. For 49-day-old laying pigeons, the positive rate of Salmonella typhimurium antibody in the control group was 29.17%, and that in the phage group was 1.5%, a decrease of 27.67%. At the same time, comparing the positive rate of antibody and the mortality rate of PC204 in the previous period, the results showed that for 28-day-old laying pigeons, the positive rate of Salmonella typhimurium antibody decreased by 16.1%; for 49-day-old laying pigeons, the positive rate of Salmonella typhimurium antibody decreased by 27.5%. These results indicate that by adding Salmonella typhimurium phage PC817 to the feed for a long time, the positive rate of Salmonella typhimurium antibody in the pigeon farm can be significantly reduced, and long-term use can even achieve the effect of purifying Salmonella in the breeding farm.

[0131] (2) Through statistical analysis of the mortality rate of 49-day-old pigeons in the phage group and the control group, it can be seen that the mortality rate of pigeons treated in the phage group decreased by 16.17% compared with the control group, and the mortality rate of PC204 pigeons decreased by 14.5% compared with the control group. This shows that long-term addition of Salmonella typhimurium phage PC817 to the feed can reduce the mortality rate of pigeons, has a good prevention and control effect on pigeon-derived Salmonella, and its effect is better than that of phage PC204 in the previous patent.

[0132] Table 6 Statistical results of positive detection of Salmonella typhimurium antibody and mortality rate

[0133]

[0134] Example 12 Safety test of phage

[0135] 1. Experimental method

[0136] Select 40 one-day-old pigeon chicks, divide them into a phage group and a control group. The phage group orally takes 0.2 mL of PC817 1×10 10 PFU phage, and raise and observe for 7 days. The control group orally takes the same dose of sterile normal saline. Perform autopsy to observe the lesions of the heart, liver, spleen, lungs, kidneys, brain, and intestines, and observe the mental state, food intake, etc. during the feeding process.

[0137] 2. Experimental results and analysis

[0138] During the entire administration period, no diseases or toxic symptoms were observed in the phage group and the control group, and the mental state and food intake were normal. After detailed clinical autopsy observation, whether it was the phage group or the control group, the main organs and intestines of the animals were normal. This shows that phage PC817 has high safety and has no adverse effects on the animal body.

[0139] Example 13 Preservation or disinfection effect of phage on meat quality

[0140] 1. Experimental method:

[0141] Prepare 300 g of sterile fresh pigeon meat, equally divide it into 6 portions, and then evenly divide it into two groups: an experimental group and a control group, with 3 portions in each group. Prepare a high-concentration phage suspension, and then dilute it with normal saline to an appropriate working concentration (about 10 6 pfu / mL). Take 250 μL of the diluted phage and spray it onto the surface of the pigeon meat in the experimental group, and spray an equal amount of sterile water onto the control group. Place the treated samples in sterile sealable bags and store them at 4°C. Samples are taken at 3 d, 7 d, 10 d, and 14 d of storage, and the total volatile basic nitrogen (TVB-N) value is determined according to the standard of "Determination of total volatile basic nitrogen in foods - GB 5009.228-2016". Each experimental group has 3 replicates and is repeated 3 times.

[0142] 2. Experimental results:

[0143] The results are as Figure 6 shown. The TVB-N in the control group reached the spoilage threshold (15 mg / 100 g) after 3 d of storage at 4°C, while the experimental group reached the spoilage threshold after 7 d of storage. These results indicate that phage PC817 inhibits the total volatile basic nitrogen in pigeon meat products and plays a very important role in preserving the quality of pigeon meat.

[0144] Example 14 Whole-genome analysis of Salmonella typhimurium phage PC817

[0145] Extract the genome of phage PC817, perform whole-genome sequencing and sequence analysis, and the results are as follows:

[0146] (1) The full length of the genome is 85,988 bp, the G + C content is 38.95%, and the contents of bases C, G, A, and T are 20.53%, 18.42%, 29.30%, and 31.74% respectively. The online annotation result of the whole genome by RAST shows that this genome contains 122 open reading frames (ORFs). Among these 122 open reading frames (ORFs), 7 structural proteins are found, mainly including the structural and packaging proteins of the phage (tail protein, binding protein, phage fiber protein, large terminase subunit, etc.), phage-like proteins, regulatory proteins, and other functional proteins. At the same time, among the 122 ORFs, analysis by the software tRNAscan-SE shows that this genome does not contain tRNA genes. Analysis by the online tool CGE server shows that this genome does not contain drug resistance genes and virulence genes. Analysis by PHASTER shows that this genome does not contain lysogeny-related genes.

[0147] (2) In the genome of the phage: The sequence of the highly conserved large terminase subunit protein gene is shown as Sequence 1 in the sequence listing.

[0148] Table 7 Gene Sequence Information Table

[0149] type Start stop function Sequence 1 Peg23 18550 20151 terminase large subunit

[0150] It can be understood that for those of ordinary skill in the art, equivalent substitutions or changes can be made according to the technical solution of the present invention and the inventive concept of the present invention, and all such changes or substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A broad lytic spectrum Salmonella typhimurium phage, characterized in that: It was named as Salmonella typhimurium phage PC817, and its deposit number is CGMCC No.46169.

2. A bacteriophage composition, characterized in that: The invention comprises the Salmonella typhimurium phage described in claim 1 and other phages.

3. The bacteriophage composition according to claim 2, characterized in that The other bacteriophages include: one or both of bacteriophage PC204 and bacteriophage SP8; wherein the deposit number of bacteriophage PC204 is: CGMCC No. 22368; the deposit number of bacteriophage SP8 is: CGMCC No. 45256.

4. Use of the Salmonella typhimurium phage according to claim 1 or the phage composition according to claim 2 in the preparation of drugs, feed additives, environmental disinfectants, raw meat disinfectants, food preservatives and detection kits for preventing and treating diseases caused by Salmonella infection; preferably, the Salmonella is pigeon-derived Salmonella typhimurium.

5. A phage pharmaceutical preparation, the active ingredient of which comprises the Salmonella typhimurium phage according to claim 1 or the phage composition according to claim 2; preferably, the phage pharmaceutical preparation further comprises other antibacterial or bactericidal active ingredients.

6. The bacteriophage pharmaceutical preparation according to claim 5, characterized in that The bacteriophage drug preparation is a spray-dried powder, which is prepared by the following method: skimmed milk powder and lactose are used as protective agents, the bacteriophage PC817 proliferation liquid as claimed in claim 1 is added, mixed evenly, spray-dried, and finally the spray-dried particles are collected to obtain the bacteriophage preparation.

7. A feed additive or drinking water additive, characterized in that: The method comprises the Salmonella typhimurium phage according to claim 1 or the phage composition according to claim 2.

8. An environmental disinfectant, characterized in that The active ingredient comprises the Salmonella typhimurium phage according to claim 1 or the phage composition according to claim 2.

9. The application of the environmental disinfectant as claimed in claim 7 in the environmental disinfection of a pigeon farm, characterized in that: The environmental disinfectant can disinfect the aquaculture environment and feeding equipment from salmonella by spraying or soaking, and the aquaculture environment includes feed troughs, floors, walls, feces and bedding.

10. A raw meat disinfectant or raw meat preservative, characterized in that: The method comprises the Salmonella typhimurium phage according to claim 1 or the phage composition according to claim 2.

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