A Salmonella phage SPYS_1 and its applications

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a Salmonella phage SPYS_1 and its application. The present invention provides a Salmonella phage SPYS_1, which is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M2024984. The Salmonella phage SPYS_1 of the present invention has strong tolerance to temperature and pH, adapts to various environments, proliferates rapidly, is suitable for large-scale production, and can effectively kill Salmonella, and can be used for the prevention or treatment of Salmonella infection in animal breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Salmonella phage SPYS_1 and its application. Background Art

[0002] Salmonella is a Gram-negative, intracellular parasitic intestinal bacterium and one of the main foodborne pathogenic bacteria. It can not only cause acute, chronic or latent infections in livestock, poultry and other animals, but also lead to food poisoning in humans through food contamination, posing a great threat to humans. Currently, the most commonly used method for preventing and controlling Salmonella is the use of antibiotics, but the use of antibiotics is prone to residues, and long-term use will also cause Salmonella to develop drug resistance. Summary of the Invention

[0003] The purpose of the present invention is to provide a Salmonella phage SPYS_1 and its application. The Salmonella phage SPYS_1 of the present invention can lyse Salmonella, effectively kill Salmonella, and be used for the prevention or treatment of Salmonella infection in animal breeding.

[0004] The present invention provides a Salmonella phage SPYS_1, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2024984.

[0005] The present invention also provides a bactericide or disinfectant for Salmonella, and the active ingredient of the bactericide or disinfectant contains the Salmonella phage SPYS_1 described in the above scheme.

[0006] Preferably, the concentration of Salmonella phage SPYS_1 in the bactericide or disinfectant ≥ 1×10 7 PFU / mL 。

[0007] Preferably, the Salmonella includes Salmonella typhimurium and / or Salmonella choleraesuis.

[0008] The present invention also provides the application of the Salmonella phage SPYS_1 described in the above scheme or the bactericide or disinfectant described in the above scheme in the preparation of a drug for preventing and / or treating diseases caused by Salmonella.

[0009] The present invention also provides the application of the Salmonella phage SPYS_1 described in the above scheme or the bactericide or disinfectant described in the above scheme in the preparation of a Salmonella bactericidal product.

[0010] Preferably, the Salmonella bactericidal product is used for the prevention or treatment of Salmonella infection in animal breeding.

[0011] Preferably, the animal breeding includes one or more of cattle breeding, chicken breeding and pig breeding.

[0012] The present invention also provides a method for lysing Salmonella for non-disease treatment purposes, by spraying the Salmonella phage SPYS_1 described in the above scheme or the bactericide or disinfectant described in the above scheme.

[0013] The present invention provides a strain of Salmonella phage SPYS_1, which is preserved in the China Center for Type Culture Collection, with the preservation number of CCTCC NO: M2024984. The Salmonella phage SPYS_1 of the present invention has strong tolerance to temperature and pH, adapts to various environments, has rapid proliferation, is suitable for large-scale production, and can effectively kill Salmonella, and can be used for the prevention or treatment of Salmonella infection in animal breeding.

[0014] The Salmonella phage SPYS_1 of the present invention has strong specificity and will only specifically infect and lyse the host bacteria (Salmonella); at the same time, the phage has the ability of self-proliferation and evolution, and one administration can achieve the effect of multiple administrations; the phage stays in the body for a short time and has rapid metabolism. The Salmonella phage SPYS_1 described in the present invention can specifically kill Salmonella, has high safety, and is a perfect antibiotic substitute. Moreover, the Salmonella phage SPYS_1 of the present invention can be used for the prevention or treatment of Salmonella infection in animal breeding, is convenient to use, greatly reduces the occurrence and spread of diseases caused by Salmonella, and has good application prospects.

[0015] Biological deposit description

[0016] Salmonella phage SPYS_1 was deposited in the China Center for Type Culture Collection (CCTCC) on May 17, 2024. The deposit address is: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M2024984. Description of the drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is the transmission electron microscope image of phage SPYS_1 in Example 2;

[0019] Figure 2 It is the one-step growth curve of phage SPYS_1 in Example 2;

[0020] Figure 3 It is the graph of the pH tolerance of phage SPYS_1 in Example 3;

[0021] Figure 4 It is the graph of the temperature tolerance of phage SPYS_1 in Example 3;

[0022] Figure 5 It is the bacteriostatic curve of phage SPYS_1 against Salmonella typhimurium ATCC14028 in Example 4;

[0023] Figure 6 It is the whole gene spectrum of phage SPYS_1 in Example 5;

[0024] Figure 7 It is the graph of the biological control result of phage SPYS_1 against Salmonella typhimurium ATCC14028 in milk in Example 7. Detailed implementation mode

[0025] The present invention provides a Salmonella phage SPYS_1, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M2024984.

[0026] In the present invention, the Salmonella phage SPYS_1 is isolated from the sewage collected from the Hanchengqiao Convenience Market at the intersection of Hanchengqiao Road and Yueyahe West Road, Jinnan District, Tianjin.

[0027] The Salmonella phage SPYS_1 of the present invention is a long-tailed phage with an icosahedral head, a diameter of 58.30 ± 5.04 nm, a long and non-retractable tail, a length of 133.15 ± 8.65 nm, and a width of 11.37 ± 2.63 nm. The latent period of this phage is 15 - 30 min, the lysis period is 5 - 15 min, and the burst size is 119 PFU / cell; the Salmonella phage SPYS_1 can maintain stable activity between pH 3 - 12 and between 4°C - 70°C, and it is a virulent phage that can adapt to various environments. The full-length genome of the Salmonella phage SPYS_1 of the present invention is 45220 bp, with a total of 82 open reading frames, 39 of which are annotated as hypothetical proteins, and the remaining 43 ORFs encode functional proteins; among the 43 encoded functional proteins, there are endolysin that can lyse the bacterial peptidoglycan layer and holin that can form pores in the cell membrane.

[0028] The present invention also provides a bactericide or disinfectant for Salmonella, and the active ingredient of the bactericide or disinfectant comprises the Salmonella phage SPYS_1 described in the above solution. The concentration of the Salmonella phage SPYS_1 in the bactericide or disinfectant of the present invention is preferably ≥ 1×10 7 PFU / mL 。 The phage SPYS_1 of the present invention has strong tolerance to temperature and pH, can adapt to various environments, has a wide host spectrum, and can play a bactericidal role against various Salmonella.

[0029] In the present invention, the Salmonella preferably includes Salmonella typhimurium and / or Salmonella choleraesuis; more preferably, it is one or more of Salmonella typhimurium ATCC14028, Salmonella typhimurium CMCC50115, Salmonella typhimurium CMCC50220, Salmonella typhimurium ATCC25241, and Salmonella choleraesuis CVCC3383.

[0030] The present invention also provides the application of the Salmonella phage SPYS_1 described in the above solution or the bactericide or disinfectant described in the above solution in the preparation of a drug for preventing and / or treating diseases caused by Salmonella.

[0031] The present invention also provides the application of the Salmonella phage SPYS_1 described in the above solution or the bactericide or disinfectant described in the above solution in the preparation of a Salmonella bactericidal product.

[0032] In the present invention, the Salmonella bactericidal product is preferably used for the prevention or treatment of Salmonella infection in animal breeding. The animal breeding of the present invention preferably includes one or several of cattle breeding, chicken breeding, and pig breeding.

[0033] The present invention also provides a method for lysing Salmonella for non-disease treatment purposes, by spraying the Salmonella phage SPYS_1 described in the above solution or the bactericide or disinfectant described in the above solution. In the present invention, the spraying dosage is preferably ≥ 1×10 6 PFU / cm 2 .

[0034] In order to further illustrate the present invention, the following describes in detail a Salmonella phage SPYS_1 and its application provided by the present invention with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] Screening, purification and enrichment of phage

[0037] ① Screening of phage

[0038] (1) Collect 5000 g of sewage from the local market (Hanchengqiao Convenience Vegetable Market at the intersection of Hanchengqiao Road and Yueyahe West Road, Jinnan District, Tianjin), centrifuge at 4 °C for 10 min at 5000 g. After centrifugation, discard the precipitate and obtain the first supernatant filtrate by passing the supernatant through a 0.22 μm filter membrane.

[0039] (2) Mix 10 mL of 2×LB liquid medium with 10 mL of the first supernatant filtrate, and inoculate 20 μL of an overnight bacterial solution (OD 600 = 0.9) of Salmonella Typhimurium ATCC14028. Incubate at 37 °C and 160 rpm for 12 h to preliminarily enrich for phages that may be present in the sewage.

[0040] (3) After centrifuging the cultured mixture at 5000 g for 20 min at 4 °C, pass the supernatant through a 0.22 μm filter membrane to obtain the second supernatant filtrate.

[0041] (4) Mix 100 μL of an overnight bacterial solution (OD 600 = 0.9) of Salmonella Typhimurium ATCC14028 with 100 μL of the second supernatant filtrate, and then culture using the double-layer plate method to detect whether plaques are formed on the plate.

[0042] ② Purification of phages

[0043] (1) After obtaining plaques, use a sterile pipette tip to pick the phage plaques and transfer them to 1 mL of SM Buffer.

[0044] (2) Let the phages obtained in the above step stand at 4 °C for 24 h, and then obtain the plaques on the plate by the double-layer plate method.

[0045] (3) Repeat the above method at least 3 times until plaques of uniform size and shape are obtained.

[0046] ③ Enrichment of phages

[0047] (1) After three purifications, if plaques of consistent size, shape, etc. appear on the plate, indicating successful purification, proceed to the next step, enrichment of phages.

[0048] (2) Gradient dilute the purified phages, and then use the double-layer plate method to have about 10 4 plaques on the plate.

[0049] (3) Add 5 mL of SM buffer to the plate and let it stand for 2 h.

[0050] (4) Chop the upper agar, mix it with SM buffer, transfer the mixed liquid to a centrifuge tube, centrifuge at 3000 rpm and 4 °C for 15 min, and then filter the supernatant through a 0.22-μm filter membrane.

[0051] (5) Use the double-layer plate method to determine the titer of the filtered phage.

[0052] Example 2

[0053] Characterization test of phage

[0054] ① Electron microscopy observation

[0055] Drop 20 μL of the phage sample obtained in Example 1 (phage concentration is 1.2×10 12 PFU / mL) onto a copper grid, allow it to adsorb naturally for 10 min, then absorb the excess liquid droplets with a filter paper strip and air dry. Then pipette 20 μL of 2% phosphotungstic acid negative staining solution and drop it onto the copper grid, and let it stand for 5 min. Finally, use a filter paper strip to absorb the excess liquid droplets, air dry under an incandescent lamp, and observe and take pictures under a transmission microscope. The results are shown in Figure 1 .

[0056] According to Figure 1 , it can be seen that the screened phage belongs to the family Siphoviridae and is named SPYS_1. The head of the phage SPYS_1 is icosahedral, with a diameter of 58.30 ± 5.04 nm. The tail is slender and non-retractable, with a length of 133.15 ± 8.65 nm and a width of 11.37 ± 2.63 nm.

[0057] ② One-step growth curve

[0058] (1) Add 1 mL of the overnight culture of Salmonella typhimurium ATCC14028 to 50 mL of LB solution and culture it to the logarithmic phase (OD 600 = 0.3), then add phage SPYS_1 with an MOI of 0.1 and culture it at 37 °C and 160 rpm for 3 h.

[0059] (2) Take two samples at the time points of 0, 5, 15, 30, 45, 60, 90, 120, 150, and 180 min. One sample is directly measured for concentration by titration, denoted as the chloroform-free treatment group, and the other sample is added with chloroform at a volume ratio of chloroform: sample = 1:200, allowed to stand for at least 15 min, and then measured for concentration by titration, denoted as the chloroform treatment group.

[0060] (3) Repeat the experiment three times and draw a one-step growth curve based on the phage concentration measurement results. The results are shown in Table 1 and Figure 2 as follows.

[0061] Table 1 Concentration measurement results of the one-step growth curve of phage SPYS_1

[0062]

[0063]

[0064] According to Figure 2 it can be seen that phage SPYS_1 can complete two lysis cycles within 90 min, the adsorption time is 5 min, and the adsorption rate is 87%. The first lysis cycle takes a total of 30 min, the latent period is 15 - 30 min, the lysis period is 5 - 15 min, and the burst size is 119 PFU / cell. The second lysis cycle occurs at 30 - 90 min, the latent period is 15 - 30 min, and the burst size is 100 PFU / cell.

[0065] Example 3

[0066] Phage stability test

[0067] ① pH stability

[0068] Add 100 μL of the phage SPYS_1 sample to 900 μL of SM buffer solutions with different pH values (2 - 13) respectively, and let it stand at 4°C for 24 h. Subsequently, determine the phage concentration by titration, repeat three times, and plot the effect of pH on the titer of phage SPYS_1. The results are shown in Table 2 and Figure 3 as follows.

[0069] Table 2 Tolerance results of phage SPYS_1 to pH

[0070] pH Phage titer (log10PFU / mL) 2 - 3 9.038 4 8.994 5 9.136 6 9.160 7 9.053 8 9.231 9 9.141 10 8.994 11 9.499 12 8.952 13 -

[0071] According to Figure 3 it can be seen that phage SPYS_1 is relatively stable in the pH range of 3 - 12.

[0072] ② Thermal stability

[0073] Place 1 mL of the phage SPYS_1 sample at different temperatures (4°C, 37°C, 50°C, 60°C, 70°C, and 80°C) for 1 h respectively. Subsequently, determine the phage concentration by titration, repeat three times, and plot the effect of temperature on the titer of phage SPYS_1. The results are shown in Table 3 and Figure 4 .

[0074] Table 3 Tolerance results of phage SPYS_1 to temperature

[0075] Temperature (°C) Phage titer (log10PFU / mL) 4 9.033 37 9.017 50 9.012 60 9.145 70 8.981 80 -

[0076] According to Figure 4It can be seen that phage SPYS_1 is relatively stable within the temperature range of 4 to 70 °C.

[0077] Example 4

[0078] Bacteriostatic curve of phage SPYS_1

[0079] Phage SPYS_1 at different doses (MOI = 0.1, 1, and 10) was added to Salmonella typhimurium ATCC14028 in the logarithmic phase, with SM buffer as a control, and cultured at 37 °C and 160 rpm. The absorbance (OD 600nm ) was measured every 1 h. The experiment was repeated three times, and a bacteriostatic curve was plotted based on the absorbance changes. The results are shown in Table 4 and Figure 5 .

[0080] Table 4 Bacteriostatic effect of phage SPYS_1 (OD 600nm )

[0081]

[0082]

[0083] According to Figure 5 it can be seen that phage SPYS_1 at different doses (MOI = 0.1, 1, or 10) can reduce the absorbance of the host bacteria suspension to less than 0.3 within 6 h.

[0084] Example 5

[0085] Purification and sequencing of the genome of phage SPYS_1

[0086] The specific steps are as follows:

[0087] (1) The genome of phage SPYS_1 was purified and extracted using a λ phage genomic DNA rapid extraction kit (Beijing Zhuangmeng International Biotechnology Co., Ltd.) to obtain the DNA of phage SPYS_1.

[0088] (2) The DNA of phage SPYS_1 was sequenced by Illumina, and the results were filtered using Soapnuke (v2.0.5). Subsequently, the influence of the host bacteria on the sequencing was removed by BWA (v0.7.17), and finally, the sequencing results were assembled by Megahit (v1.1.2).

[0089] (3) RAST (https: / / rast.nmpdr.org / ) and Uniprot ( https: / / www.uniprot.org / ) were used to annotate the open reading frames of the phage genome, and in Proksee (https: / / proksee.ca / ) Draw a genetic map, and the results are shown in Figure 6 .

[0090] According to Figure 6 , it can be known that the full length of the genome of phage SPYS_1 is 45,220 bp, and there are 82 open reading frames in total. Among them, 39 are annotated as hypothetical proteins, and the remaining 43 ORFs encode functional proteins; among the 43 functional proteins encoded, there are endolysin that can lyse the bacterial peptidoglycan layer and holin that can form holes in the cell membrane.

[0091] Example 6

[0092] Determination of the host spectrum of phage SPYS_1

[0093] Salmonella typhimurium ATCC14028 used in this example is stored in this laboratory (the research group of Wang Shuo at Nankai University), and other Salmonella strains are donated by the research group of Xu Yongping at Dalian University of Technology.

[0094] The Salmonella to be tested was cultured overnight for 18 h to obtain the overnight bacterial solution of the Salmonella to be tested. 100 μL of the overnight bacterial solution of the Salmonella to be tested was added to the LB medium containing 0.7% agarose respectively, mixed well and cooled to 55 °C, and then quickly spread on the LB plate containing 1.5% lower agar to make a double-layer plate. After the upper agar cooled, 5 μL of the phage SPYS_1 lysate was titrated onto the surface of the upper agar respectively. If a clear plaque appeared, it was judged as positive (+), and if there was no plaque, it was recorded as (-). The concentration of the phage SPYS_1 lysate was 1×10 9 PFU / mL. The detection results are shown in Table 5.

[0095] Table 5 Results of the determination of the host spectrum of phage SPYS_1

[0096] Salmonella strains Phage SPYS_1 Salmonella Typhimurium ATCC14028 + Salmonella Typhimurium CMCC50115 + Salmonella Typhimurium CMCC50220 + Salmonella Typhimurium CICC21484 - Salmonella Typhimurium ATCC25241 + Salmonella Paratyphi B CICC10437 - Salmonella Paratyphi A CICC21501 - Salmonella Typhi CICC10871 - Salmonella Typhi CVCC3378 - Salmonella Gallinarum CICC21510 - Salmonella Choleraesuis CVCC3383 + Salmonella Choleraesuis CVCC503 - Salmonella Choleraesuis CVCC79102 -

[0097] According to Table 5, it can be known that phage SPYS_1 can lyse 5 Salmonella strains, namely Salmonella typhimurium ATCC14028, Salmonella typhimurium CMCC50115, Salmonella typhimurium CMCC50220, Salmonella typhimurium ATCC25241, and Salmonella choleraesuis CVCC3383. This strain of phage has a relatively broad host range and is a broad-spectrum phage.

[0098] Example 7

[0099] The pasteurized fresh milk was divided into an experimental group and a control group.

[0100] Control group: Take 19.96 mL of milk, add 20 μL of Salmonella typhimurium ATCC14028 and 20 μL of SM buffer. The concentration of Salmonella typhimurium ATCC14028 is 1×10 8 CFU / mL.

[0101] Experimental group: Take 19.96 mL of milk, add 20 μL of Salmonella typhimurium ATCC14028 and 20 μL of phage SPYS_1 lysate. The concentration of Salmonella typhimurium ATCC14028 is 1×10 8 CFU / mL, the MOI of phage SPYS_1 is 0.1, and the concentration of phage SPYS_1 lysate is 1×10 7 PFU / mL.

[0102] Cultivate the experimental group and the control group respectively at 37 °C and 160 rpm; Take 500 μL of the culture at 0 h, 2 h, 4 h, and 6 h of cultivation, centrifuge at 5000 g for 5 min to obtain cell pellets; Take the cell pellets and resuspend them with PBS, measure the viable cell count, with 3 biological replicates in each group. The measurement results are shown in Table 6 and Figure 7 .

[0103] Table 6 Antibacterial assay results of phage SPYS_1

[0104]

[0105] According to Figure 7 it can be seen that the phage SPYS_1 of the present invention can significantly kill Salmonella typhimurium ATCC14028 in milk.

[0106] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bactericide or disinfectant for Salmonella, characterized in that: The active ingredient of the bactericide or disinfectant comprises Salmonella phage SPYS_1, and the Salmonella phage SPYS_1 is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M2024984.

2. The bactericide or disinfectant according to claim 1, characterized in that The concentration of Salmonella phage SPYS_1 in the bactericide or disinfectant is ≥1×10 7 PFU / mL.

3. Use of the bactericide or disinfectant according to claim 1 or 2 in the preparation of a medicament for preventing and / or treating diseases caused by Salmonella; the Salmonella includes Salmonella typhimurium and / or Salmonella choleraesuis.

4. Use of the bactericide or disinfectant according to claim 1 or 2 in the preparation of a Salmonella sterilization product; the Salmonella includes Salmonella typhimurium and / or Salmonella choleraesuis.

5. The use according to claim 4, characterized in that: The salmonella sterilization product is used for preventing or treating salmonella infection in animal breeding.

6. The use according to claim 5, characterized in that: The animal breeding includes one or more of cattle breeding, chicken breeding and pig breeding.

7. A method for lysing Salmonella for non-disease treatment purposes, characterized in that: Spraying the bactericide or disinfectant according to claim 1 or 2; the Salmonella includes Salmonella typhimurium and / or Salmonella choleraesuis.

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