Staphylococcus phage capable of preventing and treating poultry staphylococcal disease and its application

Abstract

The present invention provides a staphylococcal phage for preventing and treating poultry staphylococcal disease, including staphylococcal phage GSA-P12. The applicant screened out a Staphylococcus aureus phage from a water sample near a chicken farm in Hubei Province. The phage was sent to the China Center for Type Culture Collection for preservation on May 26, 2022. The preservation address is: Wuhan University, Wuhan, China, with a preservation number of CCTCCNO: M2022725 and a classification name of Staphylococcus aureus phage GSA-P12. The staphylococcal phage GSA-P12 is used for the prevention and control of poultry staphylococcal disease and can be used for large-scale industrial fermentation production. It has a good therapeutic effect on poultry arthritis-type staphylococcal disease, can effectively reduce joint infections caused by poultry staphylococci, and improve the survival rate, thereby providing a safe, non-toxic and non-side-effect biological prevention and control measure that can be fermented and produced on a large scale for the treatment of staphylococcal disease.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a staphylococcal phage capable of preventing and treating poultry staphylococcal disease and an application thereof. Background Art

[0002] Poultry staphylococcal disease is an acute or chronic infectious disease mainly caused by Staphylococcus aureus. It is particularly harmful to chickens and is an infectious disease that can easily cause the death of chickens. Clinically, it often manifests in various types, such as arthritis, tenosynovitis, foot pad swelling, omphalitis and staphylococcal septicemia, causing great losses to the poultry industry. Staphylococcal disease in chickens is a susceptible infectious disease, so it is necessary to strengthen the research and prevention of staphylococcal disease in chickens, strengthen its attention, find prevention and control measures and methods, and avoid staphylococcal disease in chickens from causing greater harm to breeding, thereby causing greater economic and property losses.

[0003] In recent years, bacteriophages have been widely used to prevent and treat bacterial infections and have achieved excellent results. Their high efficiency and specificity in killing bacteria have given them great advantages in clinical use. Many studies have shown that staphylococcal phages have good application prospects in preventing and treating staphylococcal infections in poultry.

[0004] Application CN 102417896 A discloses a bacteriophage of Staphylococcus aureus and its application. The bacteriophage is directly sprayed on the surface of solid food or mixed in bacteria-containing food to inhibit Staphylococcus aureus in the food.

[0005] Most of the existing Staphylococcus aureus phages are used in the food field or for the treatment of mastitis in dairy cows, but there are no reports on their use in controlling staphylococcal disease in poultry farming. The invention proposes a staphylococcal phage GSA-P12 that can prevent and treat poultry staphylococcal disease, providing a new biological control measure for the prevention and treatment of poultry staphylococcal disease. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a Staphylococcus aureus phage GSA-P12, the deposit number of which is CCTCC NO: M 2022725. Another object of the present invention is to provide the use of Staphylococcus aureus phage in the preparation of a biological drug for preventing and treating arthritis-type staphylococcal disease.

[0007] In order to achieve the above-mentioned purpose, the present invention is realized through the following technical scheme: a staphylococcal phage that can prevent and treat poultry staphylococcal disease, including staphylococcal phage GSA-P12. The applicant screened out a Staphylococcus aureus phage from a water sample near a chicken farm in Hubei Province. The phage was sent to the China Center for Type Culture Collection for preservation on May 26, 2022. The preservation address is: Wuhan University, Wuhan, China, the preservation number: CCTCC NO: M 2022725, and the classification name: Staphylococcus aureus phage (staphylococcus aureus phage) GSA-P12.

[0008] Furthermore, the Staphylococcus phage GSA-P12 has the morphological characteristics of conventional Staphylococcus aureus phage and has high fermentation activity. When the final concentration of phage inoculation in the fermentation system is 3.2×10 7 PFU / mL, the final concentration of host bacteria is 2.9×10 8 CFU / mL, after fermentation at 28℃ for 12h, the titer of the fermentation broth can reach 3.7x10 11 PFU / ml.

[0009] Beneficial effects of the present invention:

[0010] 1. The staphylococcal phage GSA-P12 for preventing and treating poultry staphylococcal disease not only has high fermentation efficiency and can be used for large-scale industrial fermentation production, but also has a good therapeutic effect on poultry arthritis-type staphylococcal disease, can effectively reduce joint infections caused by poultry staphylococci, and improve survival rates.

[0011] 2. The staphylococcal phage capable of preventing and treating poultry staphylococcal disease and its application provide a safe, non-toxic and non-side-effect biological control measure capable of large-scale fermentation production for the treatment of staphylococcal disease. DETAILED DESCRIPTION

[0012] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0013] Example 1: Isolation and purification of Staphylococcus aureus bacteriophage GSA-P12

[0014] Eight water samples were collected from a chicken farm in Hubei Province where Staphylococcus aureus was found. Each sample was 30 mL. The water sample was centrifuged at 5000 rpm for 10 min and 15 mL of the supernatant was filtered and sterilized. 10 mL of the filtrate was mixed with 10 mL of 2×TSB liquid culture medium and 1 mL of Staphylococcus aureus culture medium in the logarithmic phase (10 8cfu / mL) and cultured overnight at 180rpm at 37°C to enrich phages. Centrifuge the sample enrichment solution at 5000rpm for 10min, take the supernatant and sterilize it through a 0.22μm microporous filter membrane to obtain a filtrate containing phages. Take 1mL of the filtrate and mix it evenly with 500uL of its host Staphylococcus aureus bacterial solution, let it stand for 15min to allow it to fully bind to the receptors on the bacterial surface. Add 7mL of TSB semi-solid agar medium cooled to 50°C to the above mixture, mix well and immediately spread it on the solidified TSA plate. After the agar solidifies, invert and culture it at 37°C for 8-12h to observe the growth of phage plaques. On the double-layer plate where plaques are formed, pick up large and transparent plaques with a sterile pipette tip, desorb them in 1mL SM solution by shaking, and sterilize them through a 0.22μm microporous filter to obtain a phage filtrate. Inoculate the filtrate in 5mL TSB liquid culture medium, add 100uL of host Staphylococcus aureus activated overnight and mix well, culture overnight at 37℃ and 180rpm, centrifuge at 5000rpm for 10min, take the supernatant and pass it through a 0.22μm microporous filter, and observe the plaque morphology of the filtrate using the double-layer plate method. Repeat the operation 3-5 times to obtain plaques of uniform shape and size.

[0015] Staphylococcus aureus phage GSA-P12 was isolated from a water sample from a chicken farm in Hubei. The phage produces a single circular plaque on the Staphylococcus aureus lawn, with a clear center and a diameter of about 1 mm. The phage was sent to the China Center for Type Culture Collection on May 26, 2022, and the classification name is: Staphylococcus aureus phage (staphylococcus aureus phage) GSA-P12, address: Wuhan University, Wuhan, China, and the collection number is CCTCC NO: M 2022725.

[0016] Example 2: Determination of the optimal fermentation conditions for Staphylococcus aureus phage GSA-P12

[0017] Pick a single colony of the host Staphylococcus aureus and inoculate it into a test tube containing 3 ml TSB culture medium. Cultivate it at 180 rpm in a shaker at 37 ° C for 12 hours to obtain a host bacterial suspension. Transfer the bacterial suspension to 10 ml TSB culture medium at a ratio of 1:100 and culture it at 180 rpm at 37 ° C until the early logarithmic period. Dilute the phage GSA-P12 and its host Staphylococcus aureus to a certain concentration and count them, mix and culture them according to different ratios, and add TSB liquid culture medium to make the total volume of each tube the same. Prepare four copies for each concentration ratio and culture them at 180 rpm for 12 hours at 25 ° C, 28 ° C, 32 ° C and 37 ° C respectively. After the culture is completed, centrifuge the fermentation broth at 6000g for 10 minutes and collect the supernatant. After the supernatant is filtered through a 0.22 μm microporous filter, use the double-layer plate method to count. Duplicate tube culture is performed at each point to take the average value, and the condition that produces the highest phage titer is the optimal fermentation condition. The experiment was repeated 3 times.

[0018] The results are shown in Table 1. When the concentration of bacteriophage GSA-P12 was 3.2×10 7 PFU / mL, host bacteria concentration is 2.9×10 8 CFU / mL, when the fermentation temperature was 28°C, the fermentation titer of bacteriophage GSA-P12 reached a maximum of 3.7x10 11 PFU / mL.

[0019] Table 1 The titer of staphylococcal phages at different infection multiplicities

[0020]

[0021] Example 3: Temperature stability test of Staphylococcus phage GSA-P12

[0022] Take several sterile 15mL centrifuge tubes, add 9mL TSB to each tube and place it in a constant temperature water bath at the corresponding temperature. After the temperature is balanced, add 1mL of pure phage culture solution to each tube and act at 4℃, 25℃, 37℃, 45℃, 55℃, 65℃, 75℃, and 85℃ for 1h, 2h, 12h, 24h, 48h, and 72h, respectively. After the action time is over, take out the sample tube and immediately cool it in a room temperature water bath. After appropriate dilution, use the double-layer plate method to determine the phage titer. Duplicate culture was performed at each point to obtain the average value, and the experiment was repeated 3 times.

[0023] The results are shown in Table 2. The activity of Staphylococcus phage GSA-P12 remains basically unchanged at a temperature below 45°C, and still has a high titer after being placed in a 55°C water bath for 24 hours. It has good stability at 4-45°C and can be stored for a long time.

[0024] Table 2 The titers of Staphylococcus phages after storage at different temperatures for different periods of time (initial titer: 3.5x10 9 PFU / mL)

[0025]

[0026] Example 4: Experiment on the lysis range of Staphylococcus aureus by Staphylococcus phage GSA-P12

[0027] The titer is 5.6x10 9 PFU / mL Staphylococcus phage GSA-P12 stock solution, 10 times diluted to 10 -4 , take the original solution and 10 -3 and 10 -4 The lysis spectrum of phage was determined by the spotting method at three concentrations.

[0028] 36 chicken-derived Staphylococcus aureus monoclonals, 12 slow Staphylococcus monoclonals, and 5 chromogenic Staphylococcus monoclonals isolated from clinical sites were selected and inoculated into centrifuge tubes containing 3 mL TSB, and cultured at 37 °C 180 rpm for 12 h to obtain bacterial suspensions of each strain. 1 mL of bacterial suspension was mixed with TSB semi-solid medium and spread on the prepared TSA plate, and 5 uL of phage culture solution and dilution solution were dripped on the plate. After natural air drying, cultured at 37 °C for 12-16 h, and the results were observed. The experiment was repeated three times.

[0029] The results are shown in Table 3. The phage stock solution can lyse 32 of 36 chicken-derived Staphylococcus aureus, the diluent can lyse 29 of 36 chicken-derived Staphylococcus aureus (lysis rate is 80.6%), 12 slow Staphylococci can lyse 6 (lysis rate is 50%), and 5 chromogenic Staphylococci can lyse 2 (lysis rate is 40%). In addition, combined with the lysis effects of the stock solution and the diluent, it can be inferred that the phage may contain phage lytic enzymes that can inhibit or kill bacteria. Through the lysis spectrum measurement, it was found that the phage has a wide lysis spectrum and has great application potential in preventing and treating infections caused by Staphylococci.

[0030] Table 3 Results of lysis spectrum determination of Staphylococcus phage GSA-P12

[0031]

[0032]

[0033]

[0034] “-” indicates no lysis; “+” indicates lysis with blurred lysis spots; “++” indicates lysis with clear lysis spots.

[0035] Example 5: Fermentation preparation of bacteriophage GSA-P12:

[0036] Pick a single colony of host Staphylococcus aureus, inoculate it into a test tube containing 3ml TSB culture medium, shake and culture it at 180rpm in a shaker at 37℃ for 12h to obtain a host bacterial suspension. The bacterial suspension was transferred to 500ml TSB culture medium at a ratio of 1:100, shaken and cultured at 37℃ and 180rpm to the early logarithmic stage and the concentration of the bacterial suspension was determined. The fermentation system was 6L, and the fermentation medium was TSB medium. Flame inoculation was used to inoculate, and phages and host bacteria were inoculated into the fermenter under the best fermentation conditions. Sterile air was introduced during the fermentation process, and 3‰ defoaming agent was added. The fermentation preparation time was 12h. After the fermentation was completed, all the mixed solutions of phage and host bacteria were taken out from the sampling port and connected to a sterile container, centrifuged at 6000rpm for 15min, and the supernatant was filtered into a sterile filter device through a vacuum pump to obtain phage fermentation liquid and stored at 4℃.

[0037] As shown in Table 4, after fermentation at 28°C for 12 h, the titer of bacteriophage GSA-P12 can reach 5.6x10 11 PFU / ml. Therefore, it is feasible to use the fermentation method for large-scale industrial preparation of bacteriophages, which provides a feasible method for the industrial production and promotion and application of the bacteriophages.

[0038] Table 4 Fermentation dynamics of Staphylococcus aureus phage GSA-P12

[0039] Final concentration of phage Final concentration of inoculated host bacteria Fermentation temperature Fermentation broth titer <![CDATA[6.1×10 7 PFU / mL]]> <![CDATA[5.5×10 7 CFU / mL]]> 28℃ <![CDATA[5.6×10 11 PFU / mL]]>

[0040] Example 6: Effect of bacteriophage GSA-P12 on the prevention and treatment of Staphylococcus aureus arthritis in chickens

[0041] Sixty 3-day-old broiler chickens were randomly divided into 3 groups (blank group, challenge group and treatment group), with 20 chickens in each group. After 3 days of pre-feeding, the leg joints of the treatment group were given 10 8 cfu / kg of Staphylococcus aureus and 10 9 pfu / kg phage, the challenge group was given 10 8 cfu / kg of Staphylococcus aureus, and the blank group was given an equal amount of PBS buffer. The treatment group was given phage treatment for three consecutive days from the day of challenge. All chickens were autopsied after 3 days of observation after the treatment, and the results are shown in Table 5.

[0042] The results in Table 5 show that Staphylococcus aureus phage GSA-P12 can effectively prevent and treat poultry staphylococcal disease, reduce the Staphylococcus carrying rate in infected poultry joints, and improve the survival rate.

[0043] Table 5 The therapeutic effect of bacteriophage on staphylococcal disease

[0044] Grouping Quantity (pcs) Swelling rate Carrier rate Survival rate Blank group 20 0 0 100％ Treatment Group 20 0 5％ 100％ Anti-virus group 20 85％ 100％ 65％

[0045] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention.

[0046] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A staphylococcal phage for preventing and treating staphylococcal disease in poultry, characterized in that: Including Staphylococcus phage GSA-P12, which was sent to the China Center for Type Culture Collection for preservation on May 26, 2022. The preservation address is: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M 2022725, classification name: Staphylococcus aureus phage (staphylococcus aureus phage) GSA-P12.

2. The use of the staphylococcal phage according to claim 1, characterized in that: The poultry oral biological drug is prepared using Staphylococcus aureus phage with the deposit number CCTCCNO: M 2022725 as the active ingredient or the only active ingredient, and is used for the prevention and control of poultry staphylococcal disease.

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