Phage for splitting bovine-derived salmonella and application thereof
By developing the phage Seoulvirus GX2401 that lyses Salmonella bovine, the prevention and treatment problems of Salmonella bovine infection have been solved, and efficient killing and safe application of a variety of salmonella bacteria has been achieved.
Patent Information
- Application Number
- CN202411684108.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art is difficult to effectively prevent and treat Salmonella infections from bovine origin, especially when antibiotic use is limited.
A phage Seoulvirus GX2401, which lyses Salmonella bovine, was developed and provided with spray or soaking agents for the prevention and treatment of Salmonella bovine infection. The phage is obtained by specific culture methods, including the action with the host bacteria, DNase and RNaseA treatment, PEG8000 purification and other steps.
Seoulvirus GX2401 has a wide host spectrum, can quickly kill a variety of salmonella, and proliferate in a short time, tolerate temperature and acid-base environment changes, is easy to make drugs, is safe, and is suitable for preventing and treating cattle sources. Salmonella infection.
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Figure CN119432774A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a bacteriophage for lysing bovine salmonella and application thereof. Background Art
[0002] Salmonellosis in cattle (Pulomm Disea) is an acute or chronic disease of cattle caused by any one or more of the Salmonella genus. Cattle of all breeds, genders, and ages can be infected, among which calves are the most susceptible, and the highest incidence rate is in calves of one month old. Sick cattle and carrier cattle are the main sources of infection of the disease. Salmonella in cattle can survive in the lymphatic tissue and gallbladder of cattle for a long time, and be excreted from the body through feces, urine, milk, aborted fetuses, afterbirth, lochia, etc., polluting the surrounding environment, and entering the body of healthy cattle through the digestive tract, causing infection. It can also be transmitted through mating and artificial insemination.
[0003] The problem of abuse of antibiotics in the aquaculture industry is still difficult to solve completely, and the continuous emergence of drug-resistant strains has attracted the attention of the whole society. Bacteriophage (phage) is a virus that infects bacteria and relies on its bacterial host to replicate. It consists of a genome composed of single-stranded or double-stranded DNA or RNA and an outer protein capsid, which does not harm animal cells. In recent years, the Ministry of Agriculture and Rural Affairs has banned the addition of antibiotics to feed. As a natural antibacterial agent, bacteriophage targets and kills harmful bacteria. It is a natural bactericidal substance. Phage has great potential in treating or preventing diseases due to its efficient and specific bactericidal effect.
[0004] According to Announcement No. 194 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, starting from July 1, 2020, feed production enterprises shall stop producing commercial feed containing growth-promoting drug feed additives (except traditional Chinese medicine). Banning antibiotics in feed is an inevitable trend to produce green and safe food and reduce the risk of environmental drug-resistant bacteria. In order to reduce the use of antibiotics in the breeding process, it is necessary to develop new antibiotic reduction and replacement technologies. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a new option for preventing and treating bovine salmonella infection.
[0006] The technical solution of the invention is a bacteriophage Seoulvirus GX2401 for lysing bovine Salmonella, and its preservation number is CCTCC NO: M20241156.
[0007] The present invention also provides the use of the bacteriophage Seoulvirus GX2401 in preparing a preparation for preventing and treating bovine Salmonella;
[0008] Furthermore, the preparation is a spray or a soak.
[0009] The present invention also provides a method for culturing bacteriophage Seoulvirus GX2401, comprising the following steps:
[0010] S1. Take the phage and host bacteria and react for 15 minutes, add LB liquid medium, culture at 37℃ for 6 hours, centrifuge, take the supernatant, filter, and the filtrate is the phage lysate;
[0011] S2. Add DNase and RNaseA to the phage lysate, incubate, add NaCl and place in an ice bath, centrifuge, take the supernatant and add PEG8000, incubate at 4°C overnight, centrifuge, discard the supernatant, add SM buffer to resuspend, add an equal volume of chloroform and gently shake for 30 seconds, centrifuge to separate the organic phase and the hydrophilic phase, recover the hydrophilic phase containing the phage particles, and obtain a purified bacterial suspension.
[0012] The volume ratio of bacteriophage to host bacteria is 1:1;
[0013] Further, in step S1, the centrifugation conditions are 4°C, 12000 rpm, 20 min;
[0014] Particularly, in step S1, the filtration is performed using a 0.22 μm filter membrane.
[0015] Wherein, in step S2, DNase and RNaseA are added to the phage lysate to a final concentration of 1 μg / mL each;
[0016] Specifically, in step S2, incubate for 30 min;
[0017] Particularly, in step S2, NaCl was added and ice bathed for 1 h;
[0018] Wherein, in step S2, the first two centrifugation conditions are 4°C and 12000 rpm for 10 min;
[0019] Particularly, in step S2, centrifugation is performed at 4°C and 5000 rpm for 15 min to separate the organic phase and the hydrophilic phase;
[0020] Wherein, in step S2, the final concentration after adding PEG8000 is 10%.
[0021] Beneficial effects of the present invention: The present invention discloses a bacteriophage SeoulvirusGX2401 that lyses bovine Salmonella. The bacteriophage has a wide host spectrum and can lyse 6 different Salmonella. It has a wide antibacterial spectrum and can be used to prepare drugs for preventing and treating diseases caused by bovine Salmonella infection. The bovine Salmonella bacteriophage Seoulvirus GX2401 can quickly kill bovine Salmonella and can proliferate in large quantities in a short period of time. It has a wide tolerance range for temperature, acid and alkali, and is easy to make into a spray and soaking agent. It does not carry drug resistance and virulence genes, has small toxic and side effects, is highly safe, and has no toxic effects on animals. When used alone or in combination with other agents, it has a good inhibitory or killing effect on the cattle farm environment contaminated by Salmonella.
[0022] The bacteriophage Seoulvirus GX2401 for lysing bovine Salmonella of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on June 6, 2024, with the deposit address: Wuhan University, Wuhan, China, Postal Code 430072; the deposit number is CCTCC NO: M20241156. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 , plaque image of bovine Salmonella phage Seoulvirus GX2401.
[0024] Figure 2 , the optimal multiplicity of infection (MOI) of bovine Salmonella phage Seoulvirus GX2401.
[0025] Figure 3 , growth curve of bovine Salmonella phage Seoulvirus GX2401.
[0026] Figure 4 , temperature tolerance chart of bovine Salmonella phage Seoulvirus GX2401.
[0027] Figure 5 , pH tolerance chart of bovine Salmonella phage Seoulvirus GX2401.
[0028] Figure 6 , environmental disinfection effect diagram of bovine Salmonella bacteriophage Seoulvirus GX2401. DETAILED DESCRIPTION
[0029] The technical solutions in the embodiments of the present application will be described in detail below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present application are within the scope of protection of the present application.
[0030] The host bacteria GXSE-A11 used in the examples is a clinical strain of Salmonella isolated from a cattle farm in Nanning, Guangxi. The host bacteria and the Salmonella phage Seoulvirus GX2401 are co-deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, with the deposit number being CCTCCM20241156.
[0031] LB (Luriabroth) liquid medium (1 L): peptone 10 g, yeast powder 5 g, NaCl 10 g, add ddH20 to 1 L, adjust pH to 7.0, and sterilize by high pressure at 121°C for 20 min.
[0032] 0.6% LB semisolid medium (1 L): peptone 10 g, yeast powder 5 g, NaCL 10 g, agar powder 6 g, add ddH20 to 1 L, adjust pH to 7.0, and sterilize by high pressure at 121°C for 20 min.
[0033] 1.2% LB solid medium (1L: peptone 10g, yeast powder 5g, NaCl 10g, agar powder 12g, add ddH2O to 1L, adjust pH to 7.0, sterilize at 121℃, 20min, cool to 50℃, pour the plate, cool and solidify, and then invert for use.
[0034] SM buffer (1 L): weigh 6.055 g, dilute to 100 ml with Tris-HCl (pH 7.5), add 5.8 g NaCL and 2.0 g MgS04, and add ddH2O to dilute to 1 L.
[0035] 1mol / L sterile CaCl2 solution (1L): Weigh 111g of CaCl2 solid, pour into a beaker and add water to dissolve, pour the dissolved solution into a 1L volumetric flask and rinse the beaker with distilled water for 3 times, pour the rinse solution into the volumetric flask, then make up to 1L, sterilize at 121℃ for 20min, and cool for use.
[0036] SS medium, DNase I, RNase A, PEG8000, and phosphotungstic acid (PTA, 2% w / v) were commercially available.
[0037] Example 1 Isolation of bovine Salmonella phage Seoulvirus GX2401
[0038] Wastewater was collected from a septic tank in a cattle farm in Guangxi. The sample was centrifuged at 4°C and 12000rpm for 10 minutes. The supernatant was centrifuged three times again, and the supernatant was filtered with 0.45μm and 0.22μm filter membranes respectively. 5mL of the filtrate was taken, 0.1mL of the host bacteria preserved in the China Center for Type Culture Collection was added, and 5mL of 2×LB liquid culture medium was added, and the culture was placed at 37°C for 14-16h. The next day, the culture obtained after the above culture for 14-16h was centrifuged at 4°C and 12000rpm for 10 minutes, and the supernatant was filtered with a 0.22μm filter membrane for sterilization to obtain the original solution containing bacteriophages, that is, the bacteriophage suspension.
[0039] The host bacteria preserved in the China Center for Type Culture Collection were streaked on SS medium and cultured overnight. A single clone was picked out and inoculated into 5 mL LB (Luriabroth) liquid medium. After shaking culture at 37°C for 8 h, it was used as the host bacteria culture for later use.
[0040] Divide the 1.2% LB solid culture medium into two areas, take 0.1mL of the above-mentioned spare host bacteria culture and 3ml of 0.6% LB semi-solid, mix them evenly and spread them on the 1.2% LB solid culture medium. After it is dried, take 10μL of the above-mentioned phage suspension and drop it on one of the areas. After it is naturally dried, place it in a 37℃ incubator for incubation, and observe whether there are plaques formed in the phage-dropped area. If plaques are formed, it proves the presence of phages.
[0041] Take another 0.1 mL portion of the above phage suspension and dilute it 10 times continuously. Take out 0.1 mL of 10-2, 10-4, and 10-6 dilutions respectively, add 0.1 mL of the host bacteria preserved in the China Center for Type Culture Collection, let it stand for 15 minutes, add 3.5 mL of 0.6% semi-solid LB culture medium at about 45°C, evenly spread it on the pre-prepared 1.2% LB solid culture medium, and observe the growth of plaques after culturing at 37°C for 8 hours; pick a single transparent plaque with no halo, uniform size, and neat edges and put it into an EP tube containing 0.1 mL of host bacteria culture and LB liquid culture medium, and co-culture at 37°C overnight; on the next day, take the co-culture and centrifuge and filter it, dilute the filtrate 10 times with SM buffer, and make a double layer with 0.1 mL of host bacteria, repeat this for about 5-10 times, and you can get phages with uniform plaque size, store at 4°C for later use.
[0042] The above-mentioned spare bacteria were tested by double-layer plate method, and the results were as follows Figure 1 As shown, the phage can form translucent pinpoint plaques in agar culture medium, with no halo around them, clear and regular edges, and a diameter of about 0.5 mm, which is a typical lytic phage.
[0043] Example 2 Amplification and purification of bovine Salmonella phage Seoulvirus GX2401
[0044] Take 0.1 mL of the reserved phage in Example 1 and 0.1 mL of the reserved host bacteria culture in Example 1 in a test tube and act for 15 min, add 10 ml of LB liquid culture medium, culture at 37° C. for 6 h, centrifuge at 4° C. and 12000 rpm for 20 min, take the supernatant, filter with a 0.22 μm filter membrane, and the filtrate is the phage lysate.
[0045] PEG purification: Add DNase I and RNase A to the phage lysate to a final concentration of 1 μg / mL, incubate at 37°C for 30 min, add NaCl to a final concentration of 1 M and bathe on ice for 1 h (i.e., add sodium chloride to make the final concentration of sodium chloride in the mixed solution 1 M), centrifuge at 4°C and 12000 rpm for 10 min, take the supernatant and add PEG8000 with a final concentration of 10%, incubate at 4°C overnight, then centrifuge at 4°C and 12000 rpm for 10 min, discard the supernatant, invert for 5 min, remove excess water as much as possible, then add SM buffer to the remaining solid material to resuspend, add an equal volume of chloroform and gently shake for 30 s, centrifuge at 4°C and 5000 rpm for 15 min to separate the organic phase and the hydrophilic phase, recover the hydrophilic phase containing phage particles, and obtain a purified bacterial suspension.
[0046] The double-layer plate method was used to detect the phage titer: the purified phage suspension was diluted 10 times in a gradient, 0.1 mL of the phage dilution solution in each gradient was taken and mixed with 0.1 mL of the host bacterial solution, and then spread on a double-layer agar plate. The plate was cultured at 37°C for about 6-10 h, and the plaques were counted on each agar plate. The plate with about 30-300 plaques was selected, and the phage titer was obtained by calculating the initial phage concentration according to the dilution multiple. The phage titer (PFU / mL) = dilution multiple × number of plaques × 10, and the phage titer was 2.3 × 10 9 PFU / ml.
[0047] Example 3 Determination of the optimal multiplicity of infection of bovine Salmonella phage Seoulvirus GX2401 (the multiplicity of infection is the ratio of the number of phages to the number of host bacteria at the initial stage of infection)
[0048] Take the host bacterial culture prepared in Example 1 and adjust the concentration to 1×10 9 CFU / mL, the reserved bacteria in Example 1 and the reserved host bacteria culture in Example 1 were added at a ratio of 1000, 100, 10, 1, 0.1, 0.01, and 0.001, respectively, and LB liquid culture medium was added to make the total volume of the culture system the same, static culture was carried out at 37°C for 5h, centrifuged at 10000rpm for 10min, the supernatant was collected and diluted to an appropriate concentration, and the titer was determined by the double-layer method. The results are as follows Figure 2As shown, the optimal infection multiplicity of bovine Salmonella phage Seoulvirus GX2401 is 0.01.
[0049] Example 4 One-step growth curve determination of bovine Salmonella phage Seoulvirus GX2401
[0050] The host bacteria culture prepared in Example 1 was mixed with an excess of the prepared phages in Example 1 (MOI>10 to ensure that all bacteria and phages are adsorbed), incubated at 37°C for 15 minutes, centrifuged at 12000 rpm for 1 minute, and the supernatant (unadsorbed phages) was discarded. The precipitate (mutually adsorbed bacteria and phage particles) was washed once with LB liquid culture medium, and the precipitate was resuspended with 10 ml of preheated LB liquid culture medium, and quickly placed in a 37°C shaker for shaking culture. Starting from 0 min, 120 μL of the culture was taken every 10 min, and the bacteria were centrifuged at 4°C and 10000 rpm for 2 min to remove the bacteria. The supernatant was taken and diluted to an appropriate concentration (an appropriate concentration is a concentration that forms 30-300 plaques on the plate), and the bacterial titer was determined by the double-layer method. The measurement was performed for 180 minutes, and a total of 18 samples were taken. The sampling time was used as the horizontal axis and the logarithm of the phage titer was used as the vertical axis to draw a one-step growth curve to obtain the incubation period, outbreak period, and outbreak volume of the phage. The one-step growth curve results are shown as follows: Figure 3 As shown in the figure, the incubation period of the host bacteria is short (<20min) and the outbreak period is long (>60min).
[0051] Example 5 Temperature and pH tolerance test of bovine Salmonella phage Seoulvirus GX2401
[0052] Take 10 sterile EP tubes, add 0.5 mL of the reserved bacteria in Example 1 to each tube, and act at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 60 min, immediately cool in a water bath after the action time, and then measure the titer of the phage; the test results are as follows: Figure 4 As shown: the phage can withstand a high temperature of 60°C, and the titer is basically stable within 90 minutes. When the temperature is above 60°C, the phage titer decreases significantly over time. In a 70°C environment, the titer begins to decrease to an extremely low level, and the phage is rapidly inactivated in an 80°C environment.
[0053] Eleven portions of 0.1 mL of the phage prepared in Example 1 were placed in SM buffer (0.9 mL) at pH 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, and reacted at 37° C. for 1 h. The titer of the phage after the reaction was then determined by the double-layer method. The test results are shown in FIG. Figure 5As shown: the titer of the bovine Salmonella phage Seoulvirus GX2401 changes little in an environment with a pH value of 4 to 11, and the activity remains basically unchanged; when the environmental pH is > 11 or pH < 4, the titer of the phage decreases sharply with the increase of acidity and alkalinity; when pH is > 12 or pH < 2, the phage titer is 0 and is completely inactivated. Therefore, it can be seen that the optimal pH of the phage is 4 to 11.
[0054] Example 6 Host spectrum analysis of bovine Salmonella phage Seoulvirus GX2401
[0055] The titer of the phage used in Example 1 was adjusted to 10 9 PFU / m1 was used for standby, and the host spectrum of 20 Salmonella phages isolated from various parts of Guangxi was analyzed. The specific operation was as follows: 0.1 mL of the overnight culture of 20 Salmonella strains was taken, 3 mL of 0.6% LB semi-solid medium at about 45°C was added, and evenly spread on the pre-prepared solid 1.2% LB solid medium, and then each plate was divided into two areas, and 10 mL of the titer was adjusted to 10 in one area. 9 PFU / mL of the above-mentioned reserved phage was dripped on the surface, and physiological saline was dripped on another area as a control. After the droplet dried, it was inverted and cultured at 37℃ for 12 hours. The results were observed. If plaques were produced, it was recorded as "+", otherwise it was "-". The results are shown in Table 1: The bovine Salmonella phage Seoulvirus GX2401 can lyse 6 different types of Salmonella.
[0056] Table 1 Host spectrum of bovine Salmonella phage Seoulvirus GX2401
[0057]
[0058]
[0059] Example 7 Safety Experiment of Bovine Salmonella Phage Seoulvirus GX2401
[0060] Twenty 7-day-old mice, half male and half female, were randomly divided into two groups, 10 mice in each group (5 females and 5 males). One group was intraperitoneally injected with 10 9 PFU / mL / 0.25mL / piece (the concentration of the phage prepared in Example 1 was adjusted to 10 9 PFU / ml), the control group was injected with an equal volume of PBS intraperitoneally, and the mice were observed for 14 consecutive days. The mice were killed by bleeding and the changes in the internal organs, digestive tract and fissure membrane were observed. The results showed that this amount of phage had no effect on the daily behavior of the mice, and no abnormalities were found in the tissues and organs during anatomical examination.
[0061] Example 8 Environmental Disinfection Effect of Bovine Salmonella Phage Seoulvirus GX2401
[0062] The cattle farm was used as the test site, and the host bacterial culture prepared in Example 1 was diluted to 1×10 4 CFU / mL and spray the wine evenly on the ground (mL / m 2 ), and then adjust the concentration of the phage prepared in Example 1 to 10 9 PFU / mL, the obtained concentration was 10 9 PFU / ml phage spraying on the ground (mL / m 2 ), every h, the number of ground host bacteria was detected by plate counting method, and the results were as follows Figure 6 Shown: After 1 hour, the number of Salmonella on the ground dropped to 10 3 After 2 hours, the number of Escherichia coli on the ground dropped to 10 CFU, and after 3 hours, almost no Salmonella was detected on the ground, indicating that the bovine Salmonella phage Seoulvirus GX2401 of the present invention can effectively kill Salmonella on the ground in the breeding environment.
Claims
1. A bacteriophage Seoulvirus GX2401 for lysing bovine Salmonella, with a deposit number of CCTCC NO: M20241156.
2. Use of the bacteriophage Seoulvirus GX2401 according to claim 1 in the preparation of a preparation for preventing and treating bovine Salmonella; the preparation is a spray or a soak.
3. The method for culturing bacteriophage Seoulvirus GX2401 according to claim 1, characterized in that: The steps include: S1. Take the phage and host bacteria and react for 15 minutes, add LB liquid medium, culture at 37℃ for 6 hours, centrifuge, take the supernatant, filter, and the filtrate is the phage lysate; S2. Add DNase and RNaseA to the phage lysate, incubate, add NaCl and place in an ice bath, centrifuge, take the supernatant and add PEG8000, incubate at 4°C overnight, centrifuge, discard the supernatant, add SM buffer to resuspend, add an equal volume of chloroform and gently shake for 30 seconds, centrifuge to separate the organic phase and the hydrophilic phase, recover the hydrophilic phase containing the phage particles, and obtain a purified bacterial suspension.
4. The culture method according to claim 3, characterized in that: In step S1, the volume ratio of the bacteriophage to the host bacteria is 1:
1.
5. The culture method according to claim 3, characterized in that: In step S1, the centrifugation conditions are 4°C and 12000 rpm for 20 min.
6. The culture method according to claim 3, characterized in that: In step S1, the filtration is performed using a 0.22 μm filter membrane.
7. The culture method according to claim 3, characterized in that: In step S2, DNase and RNaseA are added to the phage lysate to a final concentration of 1 μg / mL each.
8. The culture method according to claim 3, characterized in that: In step S2, the incubation time is 30 min, and NaCl is added and ice bathed for 1 h.
9. The culture method according to claim 3, characterized in that: In step S2, the first two centrifugation conditions are 4°C, 12000 rpm for 10 min; in step S2, 4°C, 5000 rpm for 15 min to separate the organic phase and the hydrophilic phase.
10. The culture method according to claim 3, characterized in that: in, In step S2, PEG8000 is added to a final concentration of 10%.