A Salmonella phage SPYS_2 and its application

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to a Salmonella phage SPYS_2 and its application. The present invention provides a Salmonella phage SPYS_2, which is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M2024985. The Salmonella phage SPYS_2 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_2 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 contaminated food, posing a great threat to humans. Currently, the most common method for preventing and controlling such bacteria is the use of antibiotics, but the use of antibiotics is prone to residues, and long-term use will also make Salmonella resistant. Summary of the Invention

[0003] The object of the present invention is to provide a Salmonella phage SPYS_2 and its application. The Salmonella phage SPYS_2 of the present invention can lyse Salmonella, effectively kill Salmonella, and is used for the prevention or treatment of Salmonella infection in animal breeding.

[0004] The present invention provides a Salmonella phage SPYS_2, which is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC NO: M2024985.

[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_2 described in the above scheme.

[0006] Preferably, the concentration of Salmonella phage SPYS_2 in the bactericide or disinfectant is ≥1×10 7 PFU / mL.

[0007] Preferably, the Salmonella includes one or more of Salmonella typhimurium, Salmonella choleraesuis, Salmonella typhi and Salmonella paratyphi B.

[0008] The present invention also provides the application of the Salmonella phage SPYS_2 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 a method for lysing Salmonella for non-disease treatment purposes, by spraying the Salmonella phage SPYS_2 described in the above scheme or the bactericide or disinfectant described in the above scheme.

[0010] The present invention provides a Salmonella phage SPYS_2, which is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M2024985. The Salmonella phage SPYS_2 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.

[0011] The Salmonella phage SPYS_2 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 a fast metabolism. The Salmonella phage SPYS_2 described in the present invention can specifically kill Salmonella, has high safety, and is a perfect alternative to antibiotics. Moreover, the Salmonella phage SPYS_2 of the present invention can be used for the prevention or treatment of Salmonella infection in animal breeding, is easy to use, greatly reduces the occurrence and spread of diseases caused by Salmonella, and has good application prospects.

[0012] Biological deposit description

[0013] Salmonella phage SPYS_2 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: M2024985. Description of the drawings

[0014] 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 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.

[0015] Figure 1 It is the transmission electron microscope image of phage SPYS_2 in Example 2;

[0016] Figure 2 It is the one-step growth curve of phage SPYS_2 in Example 2;

[0017] Figure 3 It is the tolerance graph of phage SPYS_2 to pH in Example 3;

[0018] Figure 4 It is the tolerance graph of phage SPYS_2 to temperature in Example 3;

[0019] Figure 5 It is the bacteriostatic curve of phage SPYS_2 against Salmonella typhimurium ATCC14028 in Example 4;

[0020] Figure 6 It is the whole gene spectrum of phage SPYS_2 in Example 5;

[0021] Figure 7 It is the biological control result diagram of phage SPYS_2 against Salmonella typhimurium ATCC14028 in chicken in Example 7. Detailed implementation manners

[0022] The present invention provides a Salmonella phage SPYS_2, which is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M2024985.

[0023] In the present invention, the Salmonella phage SPYS_2 is isolated from the sewage collected from the Hanchengqiao Convenience Vegetable Market at the intersection of Hanchengqiao Road and Yueyahe West Road, Jinnan District, Tianjin.

[0024] The Salmonella phage SPYS_2 of the present invention is a self-replicating short-tailed phage with an icosahedral head having a diameter of 54.32 ± 5.37 nm, a short and non-contractile tail with a length of 11.67 ± 3.46 nm and a width of 10.07 ± 3.99 nm. The latent period of this phage is 15 - 30 min, the lysis period is 5 - 15 min, and the burst size is 1516.7 PFU / cell; the Salmonella phage SPYS_2 can maintain stable activity between pH 3 - 11 and temperature 4°C - 50°C, and it is a virulent phage that can adapt to various environments. The genome of the Salmonella phage SPYS_2 of the present invention is 40176 bp in full length, with a total of 49 open reading frames, 14 of which are annotated as hypothetical proteins, and the remaining 35 ORFs encode functional proteins. Among the 35 encoded functional proteins, there are endolysin that can lyse the bacterial peptidoglycan layer and holin that can form holes in the cell membrane.

[0025] 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_2 described in the above solution. The concentration of the Salmonella phage SPYS_2 in the bactericide or disinfectant of the present invention is preferably ≥1×10 7 PFU / mL.

[0026] The phage SPYS_2 of the present invention has strong tolerance to temperature and pH, can adapt to various environments, has a broad host spectrum, and can play a bactericidal role against various Salmonella bacteria.

[0027] The Salmonella bacteria described in the present invention preferably include one or more of Salmonella typhimurium, Salmonella choleraesuis, Salmonella typhi, and Salmonella paratyphi B; more preferably one or more of Salmonella typhimurium ATCC14028, Salmonella typhimurium CMCC50115, Salmonella typhimurium CMCC50220, Salmonella typhimurium ATCC25241, Salmonella paratyphi B CICC10437, Salmonella typhi CICC10871, and Salmonella choleraesuis CVCC3383.

[0028] The present invention also provides the use of the Salmonella phage SPYS_2 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 bacteria.

[0029] The present invention also provides the use of the Salmonella phage SPYS_2 described in the above solution or the bactericide or disinfectant described in the above solution in the preparation of a Salmonella bactericidal product.

[0030] The Salmonella bactericidal product described in the present invention is preferably used for the prevention or treatment of Salmonella infection in animal breeding. The animal breeding described in the present invention preferably includes one or several of cattle breeding, chicken breeding, and pig breeding.

[0031] The present invention also provides a method for lysing Salmonella bacteria for non-disease treatment purposes, by spraying the Salmonella phage SPYS_2 described in the above solution or the bactericide or disinfectant described in the above solution. In the present invention, the dosage of the spraying is preferably ≥1×10 6 PFU / cm 2 。

[0032] To further illustrate the present invention, the following describes in detail a Salmonella phage SPYS_2 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.

[0033] Example 1

[0034] Screening, purification, and enrichment of phages

[0035] ① Screening of phages

[0036] (1) The sewage collected from the local market (Hanchengqiao Convenience Vegetable Market at the intersection of Hanchengqiao Road and Yueyahe West Road, Jinnan District, Tianjin), 5000 g, was centrifuged at 4 °C for 10 min, and the precipitate was discarded. The supernatant was passed through a 0.22 μm filter membrane to obtain the first filtered supernatant.

[0037] (2) 10 mL of 2×LB liquid medium was mixed with 10 mL of the first filtered supernatant, and 20 μL of an overnight bacterial solution (OD 600 = 0.9) of Salmonella Typhimurium ATCC14028 was inoculated. It was cultured at 37 °C and 160 rpm for 12 h to preliminarily enrich the phages that might exist in the sewage.

[0038] (3) After the cultured mixture was centrifuged at 5000 g and 4 °C for 20 min, the supernatant was passed through a 0.22 μm filter membrane to obtain the second filtered supernatant.

[0039] (4) 100 μL of an overnight bacterial solution (OD 600 = 0.9) of Salmonella Typhimurium ATCC14028 was mixed with 100 μL of the second filtered supernatant, and then the double-layer plate method was used for culture to detect whether plaques were produced on the plate.

[0040] ② Purification of phages

[0041] (1) After obtaining the plaques, a sterile pipette tip was used to pick the phage plaques and transfer them to 1 mL of SM Buffer.

[0042] (2) The phages obtained in the above steps were left standing at 4 °C for 24 h, and then the double-layer plate method was used to obtain the plaques on the plate.

[0043] (3) The above method was repeated at least 3 times until plaques of uniform size and shape were obtained.

[0044] ③ Enrichment of phages

[0045] (1) After three purifications, plaques of consistent size, shape, etc. appeared on the plate, indicating successful purification, and then the next step, the enrichment of phages, was carried out.

[0046] (2) The purified phages were serially diluted and then the double-layer plate method was used to have about 10 4 plaques on the plate.

[0047] (3) 5 mL of SM buffer was added to the plate and left standing for 2 h.

[0048] (4) Chop the upper agar and 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.

[0049] (5) Use the double-layer plate method to determine the titer of the filtered phage.

[0050] Example 2

[0051] Characterization test of phage

[0052] ① Electron microscopy observation

[0053] Drop 20 μL of the phage sample obtained in Example 1 (phage concentration is 8×10 10 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 as Figure 1 shown.

[0054] According to Figure 1 , it can be known that the screened phage belongs to the self-replicating short-tailed phage, named SPYS_2. The head of the phage SPYS_2 is icosahedral, with a diameter of 54.32 ± 5.37 nm, the tail is short and non-contractile, with a length of 11.67 ± 3.46 nm and a width of 10.07 ± 3.99 nm.

[0055] ② One-step growth curve

[0056] (1) Add 1 mL of the overnight culture of Salmonella typhimurium ATCC14028 to 50 mL of LB solution, and culture it until the logarithmic phase (OD 600 = 0.3), then add phage SPYS_2 with an MOI of 0.1 and culture it at 37 °C and 160 rpm for 3 h.

[0057] (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, recorded 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, recorded as the chloroform treatment group.

[0058] (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 shown.

[0059] Table 1 Concentration measurement results of the one-step growth curve of phage SPYS_2

[0060]

[0061]

[0062] According to Figure 2 it can be seen that the one-step lysis cycle of phage SPYS_2 is 60 min, the adsorption time is 5 min, the adsorption rate is 41%, the latent period is 15 - 30 min, the lysis period is 5 - 15 min, and the burst size is 1516.7 PFU / cell.

[0063] Example 3

[0064] Phage stability test

[0065] ① pH stability

[0066] Add 100 μL of the phage SPYS_2 sample to 900 μL of SM buffer solutions with different pH values (pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13), 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_2. The results are shown in Table 2 and Figure 3 as follows.

[0067] Table 2 pH tolerance results of phage SPYS_2

[0068] pH Phage titer (log10PFU / mL) 2 - 3 7.537 4 8.221 5 8.380 6 8.327 7 8.354 8 8.153 9 8.020 10 8.269 11 8.333 12 - 13 -

[0069] According to Figure 3 it can be seen that phage SPYS_2 is relatively stable in the pH range of 3 - 11.

[0070] ② Thermal stability

[0071] Place 1 mL of the phage SPYS_2 sample at different temperatures (4°C, 37°C, 50°C, 60°C, 70°C, and 80°C) for 1 h, then determine the phage concentration by titration, repeat three times, and plot the effect of temperature on the titer of phage SPYS_2. The results are shown in Table 3 and Figure 4 as follows.

[0072] Table 3 Temperature tolerance results of phage SPYS_2

[0073]

[0074]

[0075] According to Figure 4As shown, phage SPYS_2 is relatively stable within the temperature range of 4°C to 50°C, and its activity only begins to decline after 60°C.

[0076] Example 4

[0077] Bacteriostatic curve of phage SPYS_2

[0078] Phage SPYS_2 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 the bacteriostatic curve was plotted based on the absorbance changes. The results are shown in Table 4 and Figure 5 .

[0079] Table 4 Bacteriostatic effect of phage SPYS_2 (OD 600nm )

[0080] Time (h) ATCC14028 MOI = 0.1 MOI = 1 MOI = 10 0 0.337 0.377 0.378 0.378 1 0.564 0.490 0.530 0.551 2 0.579 0.393 0.429 0.583 3 0.615 0.153 0.221 0.635 4 0.668 0.132 0.210 0.565 5 0.741 0.198 0.259 0.459 6 0.798 0.293 0.344 0.468 7 0.836 0.381 0.415 0.512 8 0.856 0.434 0.465 0.539 9 0.860 0.500 0.533 0.546 10 0.861 0.543 0.579 0.572 11 0.869 0.575 0.607 0.584 12 0.867 0.591 0.624 0.588

[0081] According to Figure 5 , phage SPYS_2 at a low dose (MOI = 0.1) can reduce the absorbance of the host bacteria broth below 0.3 within 6 h, while a medium dose (MOI = 1) can inhibit the absorbance of the host bacteria below 0.3 within 5 h.

[0082] Example 5

[0083] Purification and sequencing of the genome of phage SPYS_2

[0084] The specific steps are as follows:

[0085] (1) The genome of phage SPYS_2 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_2.

[0086] (2) The DNA of phage SPYS_2 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).

[0087] (3) Annotate the open reading frames of the phage genome using RAST (https: / / rast.nmpdr.org / ) and Uniprot (https: / / www.uniprot.org / ) respectively, and draw a gene map on Proksee (https: / / proksee.ca / ). The results are shown in Figure 6 .

[0088] According to Figure 6 , the genome of the phage SPYS_2 is 40,176 bp in length and contains 49 open reading frames. Among them, 14 are annotated as hypothetical proteins, and the remaining 35 ORFs encode functional proteins. Among the 35 functional proteins, there are endolysin that can lyse the bacterial peptidoglycan layer and holin that can form pores in the cell membrane.

[0089] Example 6

[0090] Determination of the host spectrum of phage SPYS_2

[0091] Salmonella typhimurium ATCC14028 used in this example is stored in our laboratory (the research group of Wang Shuo at Nankai University), and other Salmonella strains were donated by the research group of Xu Yongping at Dalian University of Technology.

[0092] Cultivate the Salmonella to be tested overnight for 18 h to obtain the overnight bacterial liquid of the Salmonella to be tested. Add 100 μL of the overnight bacterial liquid of the Salmonella to be tested to the LB medium containing 0.7% agarose respectively, mix well and cool to 55 °C, and then quickly spread it on the LB plate containing 1.5% lower agar to make a double-layer plate. After the upper agar cools, titrate 5 μL of the phage SPYS_2 lysate onto the surface of the upper agar respectively. If clear plaques appear, it is judged as positive (+), and if no plaques appear, it is recorded as (-). The concentration of the phage SPYS_2 lysate is 1×10 9 PFU / mL. The detection results are shown in Table 5.

[0093] Table 5 Results of the determination of the host spectrum of phage SPYS_2

[0094]

[0095]

[0096] According to Table 5, the phage SPYS_2 can lyse 7 strains of Salmonella, namely Salmonella typhimurium ATCC14028, Salmonella typhimurium CMCC50115, Salmonella typhimurium CMCC50220, Salmonella typhimurium ATCC25241, Salmonella paratyphi B CICC10437, Salmonella typhi CICC10871, and Salmonella choleraesuis CVCC3383. This phage has a relatively broad host range and is a broad-spectrum phage.

[0097] Example 7

[0098] Cut the raw chicken into small pieces of 1 cm×1 cm×1 cm, disinfect the surface with 75% alcohol, and irradiate with ultraviolet light for 1 h. Turn the chicken over after 30 min of irradiation. After the irradiation is completed, randomly select one side of each piece of chicken and add 100 μL of Salmonella typhimurium ATCC14028 in the way of 10 μL×10 times. Air dry for 15 min on the super clean bench and randomly divide them into an experimental group and a control group. The concentration of the Salmonella typhimurium ATCC14028 is 1×10 8 CFU / mL.

[0099] Control group: Add 100 μL of SM buffer to the same position of the chicken with Salmonella typhimurium ATCC14028 in the way of 10 μL×10 times.

[0100] Experimental group: Add 100 μL of the phage SPYS_2 lysate to the same position of the chicken with Salmonella typhimurium ATCC14028 in the way of 10 μL×10 times. The MOI of the phage SPYS_2 is 0.1, and the concentration of the phage SPYS_2 lysate is 1×10 7 PFU / mL.

[0101] Place the experimental group and the control group in sterile petri dishes and culture at 37°C. At 0 h, 2 h, 4 h, and 6 h of culture, take one piece of chicken and put it into a sterile centrifuge tube containing PBS, shake vigorously for 1 min to mix evenly, and measure the number of viable bacteria in the PBS. Each experiment is repeated 3 times. The measurement results are shown in Table 6 and Figure 7 .

[0102] Table 6 Bacteriostatic assay results of phage SPYS_2

[0103]

[0104] According to Figure 7 it can be seen that the phage SPYS_2 of the present invention can effectively kill Salmonella typhimurium ATCC14028 in chicken.

[0105] 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 these embodiments 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 contains Salmonella phage SPYS_2, and the Salmonella phage SPYS_2 is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M2024985.

2. The fungicide or disinfectant according to claim 1, characterized in that, The concentration of Salmonella phage SPYS_2 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 drug for preventing and / or treating diseases caused by Salmonella.

4. The application according to claim 3, characterized in that The Salmonella includes one or more of Salmonella typhimurium, Salmonella choleraesuis, Salmonella typhi, and Salmonella paratyphi B.

5. A method for lysing Salmonella for non-therapeutic purposes, characterized in that, Spray the bactericide or disinfectant according to claim 1 or 2.

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