Salmonella phage, microbial preparation and application of salmonella phage

Abstract

The invention belongs to the technical field of microbiology and agricultural application thereof, and particularly relates to a salmonella bacteriophage, a microbial preparation and application of the salmonella bacteriophage. The salmonella bacteriophage disclosed by the invention belongs to a long-tail bacteriophage, has relatively good killing and destroying capability on host bacteria drug-resistant salmonella typhimurium SB56, also can have different degrees of splitting action on other 16 clinical drug-resistant salmonella typhimurium, is relatively wide in antibacterial spectrum, and has the potential of being applied to drugs for preventing and treating salmonella typhimurium; the salmonella bacteriophage pSty.S56. XXXSZ disclosed by the invention is relatively short in latency period and relatively strong in environmental tolerance, and can achieve a synergistic effect with antibiotics, so that the antibacterial ability is relatively lasting, and the dosage of avidin is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology and its agricultural application, and specifically relates to a Salmonella phage, a microbial preparation and application thereof. Background Art

[0002] Salmonella is a common foodborne Gram-negative pathogen. At present, more than 2,500 serotypes of Salmonella enterica have been identified, accounting for about 99% of pathogenic strains of Salmonella. Salmonella mainly causes salmonellosis through oral infection of contaminated food and water sources. It can live in the intestines of humans and animals and even cause systemic infection, manifested as diarrhea, fever and other symptoms. There are an estimated 1.3 billion cases of gastroenteritis caused by Salmonella each year, of which about 3 million people die. The prevention and treatment of Salmonella mainly relies on antibiotics. However, with the overuse of antibiotics, the drug resistance of Salmonella continues to increase, which significantly reduces the effectiveness of traditional antibiotic treatment and leads to an increase in the failure rate of clinical treatment. Secondly, drug-resistant strains not only affect individual health, but may also be widely spread through food, environment and other channels, further increasing the pressure on the public health system and increasing the complexity and cost of infection control.

[0003] Bacteriophages are viruses that can selectively infect specific bacteria. They are widely found in water, soil, hospitals, communities, the human body, and even all places where bacteria exist. Phages can be divided into two categories according to their morphology: tailed phages and tailless phages. Tailed phages account for 96% of known phages. Phages are usually composed of proteins and internal genetic material, which can be either DNA or RNA. As one of the alternatives to antibiotics, phages have the advantages of wide sources, simple separation, low cost, high stability, and strong specificity. Phage therapy has entered the clinical trial stage in some countries, and some patients have shown good efficacy. There are successful cases in the fields of skin infections, respiratory infections, and digestive system-related diseases. In short, phage therapy, as an emerging treatment method, has important clinical significance and development potential.

[0004] The existing Salmonella phages screened from the natural environment are not rich enough in species resources, and there are still defects in dealing with host resistance evolution and environmental stability. Therefore, screening and discovering new Salmonella phages plays an important role in enriching related species resources and improving the prevention and control of Salmonella infections. Summary of the invention

[0005] In order to solve the problems existing in the prior art, one of the objects of the present invention is to provide a Salmonella phage, which is a long-tailed phage with excellent environmental stability and a broad antibacterial spectrum.

[0006] The second object of the present invention is to provide a microbial preparation comprising the Salmonella phage provided by the present invention, which can be used as a medicine, feed additive, cleaning agent and / or disinfectant, and has a high efficiency and lasting ability to disinfect and destroy Salmonella.

[0007] The third object of the present invention is to provide an application of a microbial preparation. The Salmonella phage microbial preparation provided by the present invention can be used in combination / compound with antibiotics to exert a synergistic effect and have a more lasting antibacterial ability.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A Salmonella phage is named pSty.S56.XXXSZ, and its deposit number is CCTCC No: M20242172.

[0010] A microbial preparation comprising the above-mentioned Salmonella phage.

[0011] In a specific embodiment of the present invention, the content of Salmonella phage in the above-mentioned microbial preparation is ≥10 1 PFU / mL.

[0012] The specific embodiments of the present invention verify that the use of the Salmonella phage of the present invention alone can produce an antibacterial effect, and the use in combination with antibiotics can produce a more lasting antibacterial effect, and can reduce the dosage of antibiotics. As an example, the microbial agent of the present invention also contains antibiotics, and the content of antibiotics is less than 60% of the recommended antibacterial content of a single antibiotic; preferably, the content of antibiotics is less than 30% to 60% of the recommended antibacterial content of a single antibiotic; as an example, the antibiotics can be selected from ciprofloxacin and ceftriaxone sodium.

[0013] Optionally, the antibiotin is ciprofloxacin and the content of Salmonella phage in the microbial preparation is 10 1 ~10 7 PFU / mL, ciprofloxacin content was 2-8 ng / mL;

[0014] Optionally, the antibiotin is ceftriaxone sodium, and the content of Salmonella phage in the microbial preparation is 10 1 ~10 4 PFU / mL, ceftriaxone sodium is 0.01~0.2μg / mL.

[0015] As an example, the microbial preparation can be used alone or in combination with antibiotics to prevent and / or treat salmonella contamination and / or infection. Specifically, the microbial preparation can be used to prepare a drug for preventing and / or treating salmonella infection, or to prepare a cleaning agent or disinfectant for preventing and treating salmonella contamination, or to prepare a feed additive.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The Salmonella phage pSty.S56.XXXSZ of the present invention belongs to a long-tailed phage and is a new type of Salmonella phage;

[0018] (2) The Salmonella phage pSty.S56.XXXSZ of the present invention has good disinfecting and destruction ability against the host bacteria resistant Salmonella typhimurium SB56, and can also have different degrees of lysis effect on other 16 clinical resistant Salmonella typhimurium strains, with a wide antibacterial spectrum, and has the potential to be used as a drug for the prevention and treatment of Salmonella typhimurium;

[0019] (3) The incubation period of the Salmonella phage pSty.S56.XXXSZ of the present invention is relatively short, only 20 minutes, and the lysis amount is about 173 PFU / mL. It can be seen that it can quickly kill the host bacteria and has a strong lysis ability, and has the potential to be developed into an antibacterial preparation;

[0020] (4) The Salmonella phage pSty.S56.XXXSZ of the present invention has strong environmental tolerance and is still active when exposed to water bath conditions at 80°C for 40 minutes. Its activity remains stable within the pH range of 5 to 11.

[0021] (5) The Salmonella phage pSty.S56.XXXS of the present invention can synergize with antibiotics to achieve more lasting antibacterial ability and reduce the amount of antibiotics used. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 This is a plaque picture of the Salmonella phage pSty.S56.XXXSZ of the present invention;

[0024] Figure 2 It is a transmission electron microscope image of the Salmonella phage pSty.S56.XXXSZ of the present invention;

[0025] Figure 3 It is the best infection multiplicity diagram of the Salmonella phage pSty.S56.XXXSZ of the present invention;

[0026] Figure 4It is a one-step growth curve diagram of the Salmonella phage pSty.S56.XXXSZ of the present invention;

[0027] Figure 5 Schematic diagram of the effect of temperature on the activity of the Salmonella phage pSty.S56.XXXSZ of the present invention;

[0028] Figure 6 Schematic diagram showing the effect of pH on the activity of the Salmonella phage pSty.S56.XXXSZ of the present invention;

[0029] Figure 7 Schematic diagram of the sterilization of the Salmonella phage pSty.S56.XXXSZ in the culture medium;

[0030] Figure 8 Schematic diagram of the combined antibacterial effect of the Salmonella phage pSty.S56.XXXSZ and ciprofloxacin of the present invention;

[0031] Fig. 9 It is a schematic diagram of the combined antibacterial effect of the Salmonella phage pSty.S56.XXXSZ and ceftriaxone sodium of the present invention. DETAILED DESCRIPTION

[0032] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0033] The strains, reagents and culture media involved in the embodiment are:

[0034] The host bacteria for the screening of Salmonella phages, Salmonella typhimurium SB56, and the Salmonella strains used for the host spectrum analysis of the screened Salmonella phages are all clinical drug-resistant strains, and their direct source is the strains preserved in the laboratory.

[0035] 0.7% LB semisolid medium (400 mL): weigh 4.0 g of peptone, 2.0 g of yeast powder, 4.0 g of NaCl, and 2.8 g of agar powder, add ddH2O to 400 mL, and sterilize under high pressure at 121°C for 20 min.

[0036] LB solid medium (400 mL): Weigh 4.0 g of peptone, 2.0 g of yeast powder, 4.0 g of NaCl, and 6.0 g of agar powder, add 400 mL of ddH2O, sterilize at 121°C for 20 min, cool to 50°C, pour the plate, cool and solidify, and then invert for later use.

[0037] SM buffer, PEG8000, NaOH, HCl, NaCl, ciprofloxacin powder, and ceftriaxone sodium powder were all commercially available.

[0038] Example 1 Isolation and Purification of Salmonella Phage pSty.S56.XXXSZ

[0039] 1) The sample was collected from sewage in Xinxiang City, Henan Province. The sewage sample was centrifuged at 4°C and 8000rpm / min for 15min, and the supernatant was filtered with a 0.22μm filter membrane. Take 600μL of the filtrate, add 100μL of the host bacterial solution, and then add 5mL of LB culture medium, place it in a constant temperature oscillator and culture it for 12h. The next day, centrifuge the above culture at 8000rpm / min for 10min, and filter the supernatant with a 0.22μm filter membrane to sterilize and obtain the filtrate containing bacteriophages;

[0040] 2) Take the host bacteria SB56 and streak it on LB solid medium. After culturing overnight, pick a single clone and inoculate it into 5 mL LB broth medium. After shaking culture at 37°C for 12 hours, use it as the host bacterial liquid for later use;

[0041] 3) Mix 100 μL of the above-mentioned reserved host bacterial solution with 8 mL of LB semi-solid medium at about 50°C in a 15 mL sterile centrifuge tube, pour it on the solid medium, and after it is dried, divide the plate into three areas. Take 10 μL of the above-mentioned filtrate containing phages and drop it in two areas of the plate, and add 10 μL of PBS to the other area as a blank control. After it is naturally dried, invert it in a 37°C incubator and culture it for 8 hours, and observe whether there are plaques formed in the area where the phages are added. If plaques are formed, it proves that phages exist;

[0042] 4) If plaques are formed, dilute the filtrate containing phages 10 times in succession using LB liquid medium. -4 ~10 -7 100μL of the diluent was mixed with 100μL of the host bacterial solution, and then allowed to stand for 20 minutes. 8mL of 0.7% LB semi-solid medium at about 50°C was added, and then mixed and poured onto the LB solid medium plate. After culturing at 37°C for 12 hours, the growth of plaques was observed. A single transparent plaque of uniform size was picked up and placed in a 1.5mL centrifuge tube containing LB broth medium, and vortexed to mix. 100μL of the above solution was diluted 10 times again, and 100μL of the diluent was cultured with the same volume of host bacterial solution using the double-layer plate method. Repeat this process for about 3-5 times to obtain phages with uniform plaque size, which were stored at 4°C for later use.

[0043] Plaque results Figure 1 As shown, the phage can form clear plaques with clear and regular edges in the solid LB culture medium plate.

[0044] Example 2 Amplification and Concentration of Salmonella Phage pSty.S56.XXXSZ

[0045] 1) Take 100 μL of the phage storage solution prepared in Example 1 and 100 μL of the host bacterial solution prepared in Example 1 and place them in a centrifuge tube for 15 min, add 5 mL of LB liquid culture medium, culture at 37° C. for 12 h, centrifuge at 4° C. and 8000 rpm for 10 min, take the supernatant, filter with a 0.22 μm filter membrane, and the filtrate is the phage stock solution;

[0046] 2) PEG concentration: Add NaCl to the phage stock solution to a final concentration of 1 mol / L and place on ice for 2 h. Centrifuge at 4°C, 10,000 rpm / min for 20 min. Take the supernatant and add PEG8000 at a final concentration of 10%, place on ice for 16 h, and then centrifuge at 4°C, 12,000 rpm / min for 15 min. Carefully discard the supernatant, invert for 5 min to allow the liquid to drain, add 500 μL SM buffer to resuspend the precipitate, and obtain the purified and concentrated phage stock solution.

[0047] Example 3 Transmission electron microscopic observation of Salmonella phage pSty.S56.XXXSZ

[0048] Example 2 Electron microscopic observation of the concentrated phage stock solution: 10 μL of the concentrated phage stock solution of Example 2 was dropped on a copper sheet, and precipitated naturally for 2 to 3 minutes. The excess liquid was absorbed with filter paper, and 10 μL of 2% phosphotungstic acid (PTA, 2% w / v) was dropped for staining. After drying at room temperature, the phage stock solution was observed using a transmission electron microscope. The observation results are as follows: Figure 2 As shown: The phage pSty.S56.XXXSZ is a long-tailed phage with a regular icosahedral structure in the head, a head diameter of approximately 59.3±0.47nm, and a tail length of approximately 123±0.82nm.

[0049] The phage was named pSty.S56.XXXSZ and deposited in China Center for Type Culture Collection with the deposit number: CCTCC No: M 20242172.

[0050] Example 4 Determination of the Optimal Multiplicity of Infection of Salmonella Phage pSty.S56.XXXSZ

[0051] The host bacterial solution SB56 prepared in Example 1 was cultured to the logarithmic phase, and the phage pSty.S56.XXXSZ with MOI of 100, 10, 1, 0.1, 0.01, and 0.001 were added respectively, and co-cultured at 37°C for 6 hours. The culture solution was centrifuged at 8000 r / min for 10 minutes, and the supernatant was filtered through a 0.22 um filter membrane. The titer of the phage at different MOIs was determined by the double-layer plate method, and the optimal MOI of the phage was determined. The test results are shown in FIG. Figure 3 As shown, the optimal infection multiplicity of phage pSty.S56.XXXSZ is 0.1.

[0052] Example 5 Determination of one-step growth curve of Salmonella phage pSty.S56.XXXSZ

[0053] The host bacterial liquid SB56 reserved in Example 1 was mixed with the phage stock solution preserved in Example 1 (the infection multiplicity was about 0.1), incubated at 37°C for 15 minutes, and then centrifuged at 7000rpm / min for 5 minutes. The supernatant was discarded, and the precipitate was resuspended with 5mL LB broth medium. The precipitate was quickly placed in a 37°C shaker for shaking culture. Starting from 0min, 200μL of the culture was taken every 10min, and the bacteria were removed by centrifugation at 4°C and 10000rpm / min for 2min. The supernatant was taken and diluted 10 times to the appropriate concentration, and the phage titer was determined by the double-layer plate method. A one-step growth curve was drawn with the sampling time as the horizontal axis and the logarithm of the phage titer as the vertical axis. The results of the one-step growth curve are shown in FIG. Figure 4 As shown: The incubation period of Salmonella phage pSty.S56.XXXSZ infecting the host bacteria is 20 minutes, and it enters the stable period after the 90th minute.

[0054] Example 6 Temperature and pH tolerance test of Salmonella phage pSty.S56.XXXSZ

[0055] The phages prepared in Example 1 were placed in an environment at 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C, and samples were taken every 20 minutes to 60 minutes. The phages stored in Example 1 were diluted 10 times and then the titer was determined by the double-layer plate method. The test results are as follows: Figure 5 As shown: the phage can still maintain a high titer at 30℃, 40℃, and 50℃. When it is exposed to 60℃ water bath conditions for 60 minutes, the phage titer begins to decrease. When it is exposed to 80℃ water bath conditions for 60 minutes, the phage is inactivated.

[0056] Take 12 sterile 1.5mL centrifuge tubes, add 100μL of the phages preserved in Example 1 and 900μL of LB broth medium with pH values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively, incubate at 37°C for 1h, and use the double-layer plate method to determine the titer of the phages after treatment. The test results are as follows: Figure 6 As shown: Salmonella phage pSty.S56.XXXSZ is stable in an environment with a pH value of 5 to 11. When the pH is lower than 5 or higher than 11, the phage is completely inactivated.

[0057] Example 7 Host spectrum analysis of Salmonella phage pSty.S56.XXXSZ

[0058] The experiment selected 37 clinical drug-resistant bacteria preserved in the laboratory as host bacteria (numbered as Table 1), and analyzed the host spectrum of Salmonella phage pSty.S56.XXXSZ. The specific operation is as follows: Take 100 μL of the logarithmic growth period culture solution of 37 strains of bacteria in a 15mL sterile centrifuge tube, add 8mL LB semi-solid medium at about 50°C, mix by inversion, pour it on the LB medium plate and wait for it to solidify, take 10 μL of the Salmonella phage pSty.S56.XXXSZ phage stock solution preserved in Example 1 and drop it on the surface of the strain plate, and use PBS as a negative control. After drying, invert and culture at 37°C for 12h. If plaques are produced, it is recorded as "+", otherwise it is recorded as "-". The results are shown in Table 1: Salmonella phage pSty.S56.XXXSZ can lyse the host Salmonella SB56, and can also lyse other 16 clinical drug-resistant Salmonella.

[0059] Table 1

[0060]

[0061]

[0062] Example 8 Bactericidal effect of Salmonella phage pSty.S56.XXXSZ in culture medium

[0063] The host bacterial solution SB56 prepared in Example 1 was adjusted to OD using LB broth medium. 600 to 0.6 (the number of viable bacteria is about 1×10 8 CFU / mL). The phage stock solution pSty.S56.XXXSZ preserved in Example 1 was mixed with the host bacterial solution in liquid LB medium at a multiplicity of infection of 100, 10, 1, 0.1, 0.01, and 0.001, respectively, and cultured in a 37°C constant temperature shaker. A control group was set up, in which the phage solution was replaced with an equal amount of sterile PBS. The absorbance at 600nm of the culture was measured every 1h, with time as the horizontal axis and OD as the horizontal axis. 600 The value is taken as the vertical axis, and the in vitro inhibition curve is drawn.

[0064] The results of the bacteriophage killing experiment are as follows Figure 7 As shown, the five phages with different MOIs all had an antibacterial effect on bacteria. In a short period of time, the antibacterial effect was best when the phage MOI was 100.

[0065] Example 9 Antibacterial effect of Salmonella phage pSty.S56.XXXSZ combined with ciprofloxacin and ceftriaxone sodium

[0066] The host bacterial solution SB56 prepared in Example 1 was adjusted to OD using LB broth medium. 600 to 0.6 (the number of viable bacteria is about 1×108 CFU / mL) and keep on ice until use.

[0067] Take a sterile 96-well plate and add 50 μL of ice bath culture to each well in the clean bench (except the 11th column). Add 50 μL of 1×10 9 PFU / mL of phage stock solution, 10-fold concentration gradient dilution of phage stock solution was added to the second row, the concentration decreased from top to bottom, and 50μL LB broth was added to the last row; each column of wells was diluted with 2-fold concentration gradient of antibiotic stock solution, the concentration decreased from left to right, antibiotics were added to the first column, 50μL LB broth was added to the 10th column, 150μL LB broth was added to the 11th column as a negative control, and 50μL bacterial solution and 100μL LB were added to the 12th column as a positive control.

[0068] After the above system configuration is completed, a combination of different concentrations of phages and different concentrations of antibiotics is obtained in the form of a chessboard. The last row is the antibacterial drug alone (the third well is the antibiotic mic value), and the tenth column is the antibacterial phage alone. The 96-well plate was incubated at 37°C for 12 hours, the absorbance at 600nm was measured by an ELISA instrument, and a heat map of bacterial growth was drawn (the color changes from green to red, indicating that there are more bacteria in the well).

[0069] Ciprofloxacin and ceftriaxone sodium were selected as antibacterial drugs respectively. The antibacterial performance of phage pSty.S56.XXXSZ combined with ciprofloxacin and ceftriaxone sodium was verified according to the above scheme. The results were as follows: Figure 8 and Fig. 9 As shown in the figure, the minimum inhibitory concentration of ciprofloxacin alone is 8 ng / mL; when the phage pSty.S56.XXXSZ and ciprofloxacin are combined for antibacterial activity, when the ciprofloxacin concentration is 2-8 ng / mL, the phage titer is 10 1 ~10 7 PFU / mL, the bacteria were completely eliminated.

[0070] The minimum inhibitory concentration of ceftriaxone sodium alone was 0.26 μg / mL. When the phage pSty.S56.XXXSZ was combined with ceftriaxone sodium for inhibition, the minimum inhibitory concentration of ceftriaxone sodium alone was 0.26 μg / mL. 1 ~10 4 PFU / mL, and when the concentration of ceftriaxone sodium was 0.01-0.2 μg / mL, the combined antibacterial effect was the best, and the combined antibacterial effect was reduced by 30%-60% compared with the single antibacterial effect.

[0071] This experiment proves that the combined use of bacteriophage with ciprofloxacin and ceftriaxone sodium can not only enhance the bactericidal effect, but also reduce the dosage of antibiotics, thus playing a certain role as an alternative to antibiotics.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Salmonella phage, characterized in that It is named as pSty.S56.XXXSZ, and its deposit number is CCTCCNo:M20242172.

2. A microbial preparation, characterized in that: The method comprises the Salmonella phage according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of Salmonella phage is ≥10 1 PFU / mL.

4. The microbial preparation according to claim 2 or 3, characterized in that Also includes antibiotics.

5. The microbial preparation according to claim 4, characterized in that The content of the antibiotic is less than 60% of the recommended bacteriostatic content of a single antibiotic.

6. The microbial preparation according to claim 5, characterized in that The content of the antibiotic is 30% to 60% of the recommended antibacterial content of a single antibiotic.

7. The microbial preparation according to claim 6, characterized in that The antibiotic is selected from ciprofloxacin and ceftriaxone sodium; Optionally, the antibiotin is ciprofloxacin and the content of Salmonella phage in the microbial preparation is 10 1 ~10 7 PFU / mL, ciprofloxacin content was 2-8 ng / mL; Optionally, the antibiotin is ceftriaxone sodium, and the content of Salmonella phage in the microbial preparation is 10 1 ~10 4 PFU / mL, and the content of ceftriaxone sodium was 0.01-0.2 μg / mL.

8. A use of the microbial preparation according to claims 2 to 7, characterized in that: The invention can be used for preparing medicines for preventing and / or treating salmonella infection, or for preparing cleaning agents or disinfectants for preventing and treating salmonella contamination, or for preparing feed additives.

9. A use of the microbial preparation as claimed in claim 2 or 3, characterized in that: For use in combination with antibiotics to prevent and / or treat Salmonella contamination and / or infection.

10. The use according to claim 8 or 9, characterized in that: The Salmonella is selected from Salmonella typhimurium.

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