Phage aiming at various pathogenic bacteria of cow mastitis source and application of bacteriophage

Abstract

The invention belongs to the field of biotechnology dairy cow breeding bacterial disease prevention and control, and discloses a bacteriophage for various pathogenic bacteria of dairy cow mastitis sources and application. The preservation number of the bacteriophage is CCTCC (China Center For Type Culture Collection) NO: M 20241059. The bacteriophage GRNSAP05 has a good killing effect on clinical dairy cow mastitis source pathogenic bacteria separated across the country, including staphylococcus aureus, streptococcus agalactiae, streptococcus dysgalactiae, escherichia coli and / or pseudomonas aeruginosa and the like, and can be used for preventing and treating dairy cow bacterial diseases caused by the dairy cow mastitis source pathogenic bacteria. The acid and alkali resistance stability is high, the fermentation efficiency is extremely high, the cracking spectrum is wide, the production cost is greatly reduced, and the clinical application value is high.

Description

Technical Field

[0001] The invention belongs to the field of bacterial disease prevention and control in dairy cow breeding by biotechnology, and specifically relates to a bacteriophage targeting multiple pathogenic bacteria of dairy cow mastitis and its application. Background Art

[0002] Cow mastitis is a common disease in dairy farms, accounting for about 1 / 3 of the common production diseases in dairy farms. Since mastitis can seriously reduce the amount of milk, affect the milk quality, and even cause infertility in dairy cows, it has a serious impact on the production capacity of dairy cows. The main cause of bovine mastitis is various bacterial infections, among which Staphylococcus aureus, Escherichia coli and Streptococcus are the most common pathogens. Bacteria enter the mammary gland through the nipple opening, multiply rapidly in a suitable environment, and cause breast inflammation. Staphylococcus aureus has strong virulence and environmental adaptability, Escherichia coli often exists in adverse environments, and Streptococcus often exists in milk. According to statistics, the impact of mastitis on dairy cow production can lead to a 15% to 25% reduction in annual milk production per cow.

[0003] The treatment of mastitis mainly relies on antibiotics, and "treatment based on bacteria" is gradually being accepted. First, the type of pathogenic bacteria should be determined, and then antibiotics that are sensitive to pathogens should be screened out through in vitro drug sensitivity tests. The combined treatment of antibiotics involves multiple routes of administration, and breast injections are preferred, or multiple injections directly at the root of the breast can improve the clinical cure rate. Although antibiotics can treat mastitis in dairy cows, they have great shortcomings, especially long-term use, which can easily lead to the production of drug-resistant strains, making it impossible to exert the maximum efficacy of the drugs and increasing the difficulty of treating mastitis. Therefore, we should actively seek drugs that can replace antibiotics to eliminate strain resistance and reduce the difficulty of treating mastitis in dairy cows.

[0004] Bacteriophages are natural enemies of bacteria. They were discovered earlier than antibiotics and were used to treat bacterial diseases as soon as they were discovered. The discovery of antibiotics caused people to gradually ignore them. As bacterial resistance became more serious, phage therapy once again entered people's field of vision. However, bacteriophages are viral microorganisms and will be affected by the residual disinfectants in farms, thus affecting the effectiveness of their use. Therefore, screening out disinfectant-resistant bacteriophages has great clinical significance. The replacement of bacteriophages with disinfectants is more in line with the disinfection procedures of farms and prevents the generation of drug-resistant bacteria, which has a high clinical application value.

[0005] Mastitis is caused by a variety of pathogens. The bacteriophages reported so far only have inhibitory effects on a certain type of mastitis pathogen, and their scope of application and effectiveness are limited. Summary of the invention

[0006] The purpose of the present invention is to provide a bacteriophage against multiple pathogenic bacteria of dairy cow mastitis, and the preservation number of the bacteriophage is CCTCC NO: M20241059.

[0007] Another object of the present invention is to provide a bacteriophage GRNSAP 05 targeting multiple pathogenic bacteria of dairy cow mastitis for use in preparing a drug for treating or preventing diseases caused by infection with pathogenic bacteria of dairy cow mastitis.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] The applicant isolated a Staphylococcus aureus phage GRNSAP05 from piglet feces, which has a good killing effect on a variety of pathogens of clinical bovine mastitis isolated from all over the country, and can be used to prevent and treat bacterial diseases in cows caused by pathogens of bovine mastitis. The phage GRNSAP05 has a good killing effect on clinical bovine cocci isolated from all over the country, including Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli or Pseudomonas aeruginosa, etc., and can be used to prevent and treat bacterial diseases in cows caused by pathogens of bovine mastitis. It has strong acid and alkali stability, extremely high fermentation efficiency, and a wide lysis spectrum, which greatly reduces production costs and has high clinical application value.

[0010] The phage was sent to the China Center for Type Culture Collection for preservation on May 24, 2024, address: Wuhan University, Wuhan, China; the preservation number is CCTCC NO: M20241059, and the classification name is Staphylococcus aureus phageGRNSAP05.

[0011] A compound comprises a Staphylococcus aureus phage with a deposit number of CCTCC NO: M 20241059.

[0012] The use of the above-mentioned bacteriophage or complex in the preparation of a drug for treating or preventing bacterial infection, wherein the bacteria include Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. Equisimilis, Escherichia coli and / or Pseudomonas aeruginosa.

[0013] The use of the above-mentioned bacteriophage or compound in the preparation of a bacterial antibacterial agent, wherein the bacteria include Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and / or Pseudomonas aeruginosa.

[0014] The use of the above-mentioned bacteriophage or compound in non-therapeutic in vitro inhibition of bacteria includes Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and / or Pseudomonas aeruginosa.

[0015] Application of the bacteriophage or compound in preparing medicine for treating or preventing cow mastitis.

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

[0017] Bacteriophage GRNSAP05 has a good killing effect on a variety of pathogens of clinical bovine mastitis isolated from all over the country, including Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and / or Pseudomonas aeruginosa, etc. It can be used to prevent and treat bacterial diseases of dairy cows caused by bovine-derived cocci. It has strong acid and alkali stability, extremely high fermentation efficiency, and a wide lysis spectrum, which greatly reduces production costs and has high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : pH stability of Staphylococcus aureus phage GRNSAP05.

[0019] Figure 2 : Temperature stability of Staphylococcus aureus phage GRNSAP05. DETAILED DESCRIPTION

[0020] The present invention is further described below with reference to implementation cases, but the scope of the present invention is not limited to the scope of the embodiments. The technical solutions described in the present invention, unless otherwise specified, are all conventional technologies; the reagents or materials, unless otherwise specified, are all from commercial channels.

[0021] Embodiment 1:

[0022] Phage isolation and purification:

[0023] Take 10 portions of piglet manure from a pig farm in Shandong, add water, stir, and let stand overnight at room temperature. Take 10 mL of water sample from each portion, centrifuge at 6000 rpm for 10 min, and filter and sterilize the supernatant. Take 5 mL of the filtrate and mix with the same volume of 2×TSB liquid culture medium and 1 mL of fresh Staphylococcus aureus culture medium (10 7 cfu / mL) and cultured overnight in a shaker at 30°C and 200rpm to enrich phages. The enriched solution was centrifuged at 6000rpm for 10min, and the supernatant was sterilized with a 0.22μm microporous filter membrane to obtain a filtrate containing phages. The morphology of phage plaques was observed by double-layer plate method. After a single plaque grows, the double-layer plate experiment operation is repeated 3-5 times to obtain purified phages.

[0024] The phage was sent to the China Center for Type Culture Collection for preservation on May 24, 2024, address: Wuhan University, Wuhan, China; the preservation number is CCTCC NO: M 20241059, and the classification name is Staphylococcus aureusphage GRNSAP05.

[0025] Embodiment 2:

[0026] In vitro antibacterial performance of the polyvalent Staphylococcus aureus phage GRNSAP05 against bovine-derived cocci:

[0027] 20 strains of clinically isolated Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae were selected to prepare bacterial suspensions of each strain. 600 μL of bacterial suspension was mixed with TSB semi-solid culture medium and spread on the prepared NA plate to activate Staphylococcus aureus phage GRNSAP02, GRNSAP04, GRNSAP05, GRNSAP09, and GRNSAP12, and 5 μL of the above 5 phages were dripped on the plate. After natural air drying, culture at 37 ° C for 8-12 hours and observe the results. The experiment was repeated three times, and the results are shown in Table 1, indicating that GRNSAP05 is a multivalent phage that can kill cow-derived cocci from different regions, including Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae.

[0028] Table 1 Lysis of cocci from dairy cows by bacteriophages

[0029]

[0030]

[0031] Embodiment 3:

[0032] Determination of the titer of bacteriophage GRNSAP05 at different infection multiplicity:

[0033] The host bacteria Staphylococcus aureus SA202101 was activated on a plate and inoculated into a test tube containing 3ml TSB culture medium. The culture was shaken at 180rpm at 37℃ overnight to obtain a host bacterial suspension. 100μL of bacterial suspension was inoculated into 10ml TSB culture medium and shaken at 37℃ and 180rpm until the logarithmic growth phase. The phage and its corresponding host bacteria were diluted to a certain concentration and counted, mixed and cultured according to different infection multiplicity ratios (MOI = number of phages / number of bacteria), and TSB liquid culture medium was added to make the total volume of each tube the same. Shake and culture at 180rpm at 37℃ for 6h, and 5mL of culture medium was aseptically taken out for centrifugation at 5000rpm for 10min and the supernatant was collected to determine the phage titer. Each group was cultured in duplicate tubes and the average value was taken. The MOI that produced the highest phage titer was the optimal infection multiplicity. The experiment was repeated 3 times. The results are shown in Table 2. The titer of Staphylococcus aureus phage GRNSAP05 reached the highest level (3.2×10 11 PFU / mL).

[0034] Table 2: The titer of phage GRNSAP05 at different infection multiplicity

[0035]

[0036] Embodiment 4:

[0037] pH stability test of bacteriophage GRNSAP05:

[0038] Take 9 mL of TSB liquid culture medium and adjust it to different pH values ​​(2, 3, 4, 5, 6, 7, 8, 9, 10, 11), place it in a constant temperature water bath at 25°C, and add 1 mL of 1×10 10 PFU / mL Staphylococcus aureus phage pure culture solution was placed in a 25°C environment for 4 hours. After diluting some samples, the phage titer was determined by the double-layer plate method. Duplicate culture was performed at each point to obtain the average value. The experiment was repeated 3 times. The results are as follows Figure 1 As shown, it was shown that the phage GRN SAP05 was completely inactivated at pH = 2, and could maintain a high activity between pH 3-11.

[0039] Embodiment 5:

[0040] Temperature stability test of bacteriophage GRNSAP05:

[0041] Take 45mL TSB liquid culture medium into a sterile 50mL centrifuge tube and place it in a constant temperature water bath at the corresponding temperature. After the temperature is balanced, add 5mL of 1×10 10PFU / mL Staphylococcus aureus phage GRNSAP05 pure culture solution was exposed to 4℃, 25℃, 40℃, 50℃, 60℃, and 70℃ for 2h, 12h, and 24h. After the exposure time, the sample tube was taken out and immediately placed in an ice bath for cooling. After appropriate dilution, the phage titer was determined by the double-layer plate method. Duplicate culture was performed at each point to obtain the average value.

[0042] The results are as follows Figure 2 As shown, it shows that phage GRNSAP05 can survive stably below 50°C, loses its activity rapidly at 60°C and above, but is not completely inactivated even at 70°C for 24 hours.

[0043] Example 6:

[0044] The therapeutic effect of bacteriophage GRNSAP05 on mastitis in dairy cows:

[0045] In order to explore the effect of this bacteriophage in treating subclinical mastitis in dairy cows, this experimental example conducted a dairy cow feeding experiment. The specific methods and results are as follows:

[0046] Bacteria were isolated from dairy cows with late lactation who were suffering from latent mastitis, had similar parity, similar milk production, and were in the late lactation period. A total of 90 dairy cows with consistent health conditions and mixed infection with two or more of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, and Pseudomonas aeruginosa were selected and randomly divided into three groups, with 30 cows in each group, as shown in Table 3.

[0047] Set up group 1 blank group: feed the same brand, same specification, same batch of feed (blank feed), and control the same feeding conditions;

[0048] Group 2: After milking, 10 ml of 1×10 7 pfu / ml bacteriophage GRNSAP05, fed with feed of the same brand, specification, and batch (blank feed), and controlled with the same feeding conditions;

[0049] Group 3 control group: fed with feed of the same brand, specification and batch (blank feed) and controlled with the same feeding conditions, and injected 500,000 units of kanamycin through the teat canal after milking.

[0050] The cure rate is shown in Table 3 below. Bacteriophage GRNSAP05 is more effective than antibiotics in treating bovine mastitis and is safe and has no residue.

[0051] Table 3: Statistics on the therapeutic effect of mastitis in dairy cows

[0052]

Claims

1. An isolated Staphylococcus aureus phage ( Staphylococcus aureus The phage was deposited as GRNSAP05 and the deposit number of the phage is CCTCC NO: M 20241059.

2. A compound comprising a Staphylococcus aureus phage with a deposit number of CCTCC NO: M20241059.

3. Use of the bacteriophage according to claim 1 or the compound according to claim 2 in the preparation of a drug for treating or preventing bacterial infection, wherein the bacteria include Staphylococcus aureus ( Staphylococcus aureus )、Streptococcus agalactiae( Streptococcus agalactiae ), Streptococcus dysgalactiae ( Streptococcus dysgalactiae subsp. Equisimilis ), Escherichia coli ( Escherichia coli ) and / or Pseudomonas aeruginosa ( Pseudomonas aeruginosa ).

4. Use of the bacteriophage according to claim 1 or the compound according to claim 2 in the preparation of a bacterial antibacterial agent, wherein the bacteria include Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and / or Pseudomonas aeruginosa.

5. Use of the bacteriophage according to claim 1 or the compound according to claim 2 in non-therapeutic in vitro inhibition of bacteria, wherein the bacteria include Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and / or Pseudomonas aeruginosa.

6. Use of the bacteriophage according to claim 1 or the compound according to claim 2 in the preparation of a medicament for treating or preventing bovine mastitis.

Images