Staphylococcus bacteriophage and application thereof

Abstract

The invention discloses a staphylococcus bacteriophage which is Staphyloccus pseudodintermedius Phage vBPTSHC01, the preservation number of the staphylococcus bacteriophage vBPTSHC01 is CCTCC (China Center For Type Culture Collection) NO: M 20252005, and application of the staphylococcus bacteriophage in preparation of a medicine for preventing and treating diseases infected by staphylococcus. The staphylococcus bacteriophage can split 29 swine staphylococcus aureus strains and 4 staphylococcus pseudointermedius strains, can continuously inhibit bacteria for a long time, is high in stability and good in safety, can be easily prepared into preparations and the like, and has very good killing, preventing and controlling effects on environments, foods and the like of staphylococcus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacteriophages, in particular to a staphylococcus bacteriophage and application thereof. BACKGROUND

[0002] Staphylococcus is a kind of gram-positive bacteria, not strictly anaerobic. It widely exists in sewage, soil, food and animal and human skin and mucosa (such as nasal cavity, throat) is one of the most common normal flora members, and belongs to conditional pathogenic bacteria. The pathogenicity of Staphylococcus aureus highly depends on various toxins secreted by it, which can be divided into invasive enzymes, cytolysin toxins and superantigen toxins according to the mechanism of action.

[0003] The abuse of antibiotics to treat human and animal infections has led to the rise of pathogen and symbiotic bacteria drug resistance. As a virus that specifically lyses bacteria, bacteriophages are being developed again for the treatment of various bacterial infections to reduce or even replace the use of antibiotics. The post-antibiotic era is coming, and we are currently facing the danger of having no antibiotics available, and there is an urgent need to research new antibacterial preparations. Bacteriophages are specific bacteria-lysing viruses with great application prospects. However, there are few studies on staphylococcus bacteriophages. Since Staphylococcus aureus and Staphylococcus pseudintermedius have various toxin types, and each toxin type has different hosts, it is urgent to expand the staphylococcus bacteriophage library, especially bacteriophages with strong lytic effect. SUMMARY

[0004] To solve the above technical problems, the present application provides a staphylococcus bacteriophage, which has strong bacteriostatic effect and can lyse 29 strains of porcine Staphylococcus aureus and 4 strains of Staphylococcus pseudintermedius. The present application provides a new treatment for staphylococcus infection and a new disinfection method for staphylococcus pollution of the environment and food.

[0005] To achieve the above purpose, the technical scheme provided by the present application is as follows:

[0006] A staphylococcus bacteriophage is Staphylococcus pseudintermedius Phage vB_PTS_HC01, which was preserved in China Center for Type Culture Collection on September 11, 2025, with the preservation number CCTCC NO: M 20252005, and the address of preservation is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The staphylococcus bacteriophage as described above is used in the preparation of a medicine for preventing and treating staphylococcus infection diseases.

[0008] A bacteriophage pharmaceutical preparation, wherein the effective component comprises the staphylococcus bacteriophage as described above.

[0009] The bacteriophage pharmaceutical preparation further comprises a pharmaceutically acceptable carrier, and the dosage form is a solution, a powder, a gel, a granule or a lyophilized agent.

[0010] A biological bacteriostatic agent for aquatic product disinfection, comprising the staphylococcus bacteriophage as described above, wherein the biological bacteriostatic agent is used by soaking or spraying the surface of the aquatic product to inhibit the proliferation of staphylococcus during the processing or preservation of the product.

[0011] The staphylococcus bacteriophage has a strong killing effect on the host bacteria Staphylococcus pseudintermedius isolated from clinical companion animals, and has great application prospects for preparing a new biological agent for preventing and treating staphylococcus.

[0012] Compared with the prior art, the staphylococcus bacteriophage has the following beneficial effects:

[0013] The staphylococcus bacteriophage can lyse 29 strains of Staphylococcus aureus from pigs and 4 strains of Staphylococcus pseudintermedius, has a long-lasting bacteriostatic effect, high stability, good safety, and is easy to be made into a preparation, and has a good killing and prevention effect on staphylococcus in the environment and food.

[0014] Deposit information

[0015] The staphylococcus bacteriophage Staphylococcus pseudintermedius Phage vB_PTS_HC01 was preserved in the China Center for Type Culture Collection on September 11, 2025, and the preservation number is CCTCC NO: M 20252005. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a plaque picture of the staphylococcus bacteriophage vB_PTS_HC01 of the present application.

[0017] Figure 2 is a transmission electron microscope picture of the staphylococcus bacteriophage vB_PTS_HC01 of the present application.

[0018] Figure 3 is an optimal infection multiple figure of the staphylococcus bacteriophage vB_PTS_HC01 of the present application.

[0019] Figure 4 is a one-step growth curve figure of the staphylococcus bacteriophage vB_PTS_HC01 of the present application.

[0020] Figure 5Figure 1 is a schematic diagram of the effect of pH on the activity of the staphylococcus phage vB_PTS_HC01 of the present application.

[0021] Figure 6 Figure 2 is a schematic diagram of the effect of temperature on the activity of the staphylococcus phage vB_PTS_HC01 of the present application.

[0022] Figure 7 Figure 3 is a schematic diagram of the bacteriostatic curve of the staphylococcus phage vB_PTS_HC01 of the present application.

[0023] Figure 8 Figure 4 is an effect diagram of the biofilm removal ability of the staphylococcus phage vB_PTS_HC01 of the present application on strong biofilm-forming staphylococcus. DETAILED DESCRIPTION

[0024] The specific embodiments will be described in detail below with reference to the accompanying drawings, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. The raw materials and reagents used in the examples are commercially available unless otherwise specified. The TSA solid medium used in the examples was purchased from Qingdao Haibo Biological Company, and the PBS buffer was commercially available.

[0025] The staphylococcus phage Staphylococcus pseudintermedius Phage vB_PTS_HC01 of the present application has completed whole genome sequencing work by Shengong Bioengineering (Shanghai) Co., Ltd., and the sequence has been uploaded to the GenBank database with a submission ID number of 3007841.

[0026] The host bacteria used in the experiment were Staphylococcus pseudintermedius clinical strains HCP21, which were isolated from an animal hospital in Hechi, Guangxi, and were deposited together with the staphylococcus phage in the China Center for Type Culture Collection.

[0027] TSB liquid medium (100 mL): casein (tryptic hydrolysis) 1.5 g, yeast powder 0.5 g, glucose 0.5 g, sodium thioethanolate 0.05 g, L-cystine 0.05 g, resazurin 0.0001 g, NaCl 2.5 g, agar powder 0.075 g, add ddH2O to 1 L, adjust pH to 7.0, 121℃, 20 min high pressure sterilization.

[0028] TSB semi-solid medium (100 mL): protein peptone 0.5 g, tryptone 1.5 g, yeast extract 0.5 g, NaCl 0.5 g, agar powder 0.6 g, add ddH2O to 100 mL, 121℃, 20 min high pressure sterilization.

[0029] TSA solid medium (100 mL): Proteose peptone 0.5 g, Tryptone 1.5 g, Yeast extract 0.5 g, NaCl 0.5 g, Agar powder 1.2 g, add ddH2O to 100 mL, 121℃, 20 min high pressure sterilization, cool to 50℃, pour plate, cool and solidify, then invert and store.

[0030] SM buffer (1 L): weigh 6.055 g Tris-HCI (pH 7.5) to 100 ml, add 5.800 g NaCl, 2.000 g MgSO4, then add ddH2O to 1 L.

[0031] 1 mol / L sterile CaCl2solution (1 L): weigh CaCl2solid 111 g with a balance, pour into a beaker and dissolve with water, pour the solution into a 1 L volumetric flask and rinse the beaker with distilled water 3 times, pour the rinse into the volumetric flask, then make up the volume, sterilize with high pressure and store.

[0032] DNase I, RNase A, PEG8000, phosphotungstic acid (PTA, 2% w / v) are commercially available.

[0033] Example 1

[0034] Isolation and purification of staphylococcus phage vB_PTS_HC01

[0035] The sample was taken from sewage in a septic tank of a pet hospital in Hechi, Guangxi. The sewage was centrifuged at 12000 rpm for 10 min at 4℃, and the supernatant was centrifuged again 3 times. The final supernatant was filtered with 0.45 μm and 0.22 μm filters. 5 mL of the filtrate was taken, and host bacteria HCP21 isolated from skin swabs of clinical cases from a pet hospital in Hechi, Guangxi were added. The host bacteria were mixed with 60% glycerol at a ratio of 1:1 and stored at -40℃. 0.1 mL of the mixture was added to 5 mL of TSB liquid medium and incubated at 37℃ for 14-16 h. The next day, the culture obtained after incubation for 14-16 h was centrifuged at 4℃ and 12000 rpm for 10 min. The supernatant was filtered with a 0.22 μm filter to remove bacteria, and the phage-containing stock solution, i.e., phage suspension, was obtained.

[0036] The host bacteria HCP21 were streaked on TSA solid medium and incubated overnight. A single colony was picked and inoculated in 5 mL of TSB liquid medium, which was incubated at 37℃ for 8 h to obtain the phage vB_PTS_HC01 culture, which was stored for later use.

[0037] The 0.1 mL and 4 mL TSB semi-solid culture of the above-mentioned standby phage vB_PTS_HC01 were mixed evenly and then plated on TSA solid culture medium. After air-drying, 10 μL of the above-mentioned phage suspension was dropped on one of the regions, and after natural air-drying, the region was placed in culture at 37°C. The region where the phage was added was observed for the presence of plaque formation. If plaque formation was observed, it was proved that the phage existed.

[0038] Another 0.1 mL of the above-mentioned phage suspension was taken and diluted by 10 times successively, i.e. 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -2 , 10 -4 , 10 -6 dilutions, respectively. 0.1 mL of each dilution was added to 0.1 mL of host bacteria, and after standing for 15 min, 4 mL of TSB semi-solid culture medium at about 50°C was added, and then the mixture was evenly plated on the previously prepared TSA solid culture medium. After aerobic culture at 37°C for 8 h, the growth of phage plaques was observed. A single transparent plaque without halo, uniform in size and with neat edges was picked and placed in an EP tube containing 0.1 mL of the above-mentioned standby phage vB_PTS_HC01 culture and TSB liquid medium, and then the tube was cultured overnight at 37°C. The next day, the co-culture was centrifuged and filtered, and the filtrate was diluted by 10 times with SM buffer and then double-layered with 0.1 mL of host bacteria. This process was repeated for about 4 times, and then a phage plaque with uniform size was obtained. The phage plaque was stored at 4°C and was named as vB_PTS_HC01.

[0039] The above-mentioned phage stored at 4°C was detected by double-layer plating method, and the results are shown in Table 1. Figure 1 The phage can form a needle-shaped transparent plaque in agar culture medium, and the plaque has no halo and clear edges, which is a typical lytic phage.

[0040] Example 2

[0041] Purification and amplification of staphylococcus phage

[0042] 0.1 mL of the phage stored at 4°C in Example 1 and 0.1 mL of the phage vB_PTS_HC01 culture prepared in Example 1 were mixed in a test tube and allowed to react for 15 min. Then, 10 mL of TSB liquid medium was added, and the mixture was cultured at 37°C for 10 h. The mixture was centrifuged at 12000 rpm for 20 min at 4°C, and the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was the phage stock solution.

[0043] PEG purification: DNase I, RNase A were added to the phage stock solution to a final concentration of 1 μg / ml, incubated at 37°C for 30 min, 1 M NaCl was added to the final concentration of 1 M and the mixture was incubated in ice bath for 1 h, centrifuged at 4°C, 12000 rpm for 10 min, the supernatant was collected and 10% PEG8000 was added to the final concentration of 10%, and the mixture was incubated at 4°C overnight, then centrifuged at 4°C, 12000 rpm for 10 min, the supernatant was discarded, and the residual water was removed as much as possible by inverting the tube for 5 min, then the remaining solid material was resuspended with SM buffer, an equal volume of chloroform was added and the mixture was gently shaken for 30 s, then the mixture was centrifuged at 4°C, 5000 rpm for 15 min to separate the organic phase and the hydrophilic phase, and the hydrophilic phase containing phage particles was recovered to obtain the purified phage stock solution.

[0044] Double-layer plate method for detecting phage titer: the purified phage stock solution was diluted by 10-fold gradient, i.e. 10 -1 , 10 -2 , 10 -3 , 10 -4 , 0.1 mL of each gradient of phage diluent was mixed with 0.1 mL of host bacteria, 4 mL of TSB semi-solid medium was added, and the mixture was evenly spread on the pre-prepared TSB solid medium and incubated at 37°C for about 6-10 h, the phage plaques were counted for each TSB solid medium, and the plates with about 30-300 plaques were selected, and the initial concentration of the phage calculated according to the dilution factor was the titer of the phage, the titer of the phage (PFU / ml) = dilution factor x plaque number x 10, and the titer of the phage was 6.0 x 10 6 PFU / ml.

[0045] Example 3

[0046] Transmission electron microscopy observation of staphylococcus phage

[0047] Example 2 The purified phage suspension was observed by electron microscopy, the purified phage suspension of Example 2 was dropped on a copper sheet, naturally precipitated for 5-10 min, the excess liquid was absorbed with filter paper, a drop of 2% phosphotungstic acid (PTA, 2% w / v) was dropped for staining, and the copper sheet was dried at room temperature and observed by transmission electron microscopy; the observation results are shown in Figure 2 Fig. 2, the phage has a head with a regular icosahedron, the head diameter is about 101 nm, the tail is 173 nm, according to the “Virus Classification - Eighth Report of the International Committee on Taxonomy of Viruses” published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, the phage belongs to the Myoviridae of the Caudovirales, and is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 20252005.

[0048] Example 4

[0049] Determination of optimal multiplicity of infection (MOI) of Staphylococcus phage vB_PTS_HC01 (MOI is the ratio of the number of phages to the number of host bacteria at the initial stage of infection)

[0050] Take the phage vB_PTS_HC01 culture prepared in Example 1, adjust the concentration to 1 x 10 6 CFU / mL, add the phage prepared in Example 1 for preservation at 4°C at a ratio of 100, 10, 1, 0.1, 0.01, respectively, add TSB liquid medium to make the total volume of the culture system the same, and incubate at 37°C for 5 h, centrifuge at 10,000 rpm for 10 min, collect the supernatant, dilute to an appropriate concentration, and determine the titer by the double-layer method. The results are shown in Table 3, and the optimal MOI of Staphylococcus phage vB_PTS_HC01 is 10. Figure 3

[0051] Example 5

[0052] Host spectrum analysis of Staphylococcus phage vB_PTS_HC01

[0053] Adjust the titer of the phage prepared in Example 1 for preservation at 4°C to 1 x 10 6 PFU / ml, and analyze the host spectrum of the phage using Staphylococcus isolated from different animals (the host spectrum information of Staphylococcus phage vB_PTS_HC01 is shown in Table 1), as follows: add 0.1 ml of 40 bacterial overnight cultures to 4 ml of TSB semi-solid medium at about 50°C, evenly spread on the TSB solid medium prepared in advance, then divide each plate into two regions, add 10 μL of the phage prepared in Example 1 for preservation at 4°C with a titer of 1 x 10 8 PFU / ml to one region, and add TSB without inoculating bacteria to the other region as a control, invert and incubate at 37°C for 12 h after the droplets are dried, and observe the results. If plaques are produced, record as “+”, otherwise as “-”. The results are shown in Table 2: Staphylococcus phage vB_PTS_HC01 can lyse 29 strains of Staphylococcus aureus from pigs and 4 strains of Staphylococcus pseudintermedius.

[0054] Table 1. Host spectrum information of Staphylococcus phage vB_PTS_HC01

[0055] Number Strain name Strain species Strain source Animal source 1 23YZ1 Staphylococcus aureus Jiangsu Nanjing Pig 2 23YZ2 Staphylococcus aureus Jiangsu Nanjing Pig 3 23YZ3 Staphylococcus aureus Jiangsu Nanjing Pig 4 23YZ4 Staphylococcus aureus Jiangsu Nanjing Pig 5 23YZ5 Staphylococcus aureus Jiangsu Nanjing Pig 6 23YZ9 Staphylococcus aureus Jiangsu Nanjing Pig 7 23YZ7 Staphylococcus aureus Jiangsu Nanjing Pig 8 23JC1 Staphylococcus aureus Jiangsu Nanjing Pig 9 23JC2 Staphylococcus aureus Jiangsu Nanjing Pig 10 23JC3 Staphylococcus aureus Jiangsu Nanjing Pig 11 23JC4 Staphylococcus aureus Jiangsu Nanjing Pig 12 23JC5 Staphylococcus aureus Jiangsu Nanjing Pig 13 23JC6 Staphylococcus aureus Jiangsu Nanjing Pig 14 23RZ1 Staphylococcus aureus Jiangsu Nanjing Pig 15 23RZ2 Staphylococcus aureus Jiangsu Nanjing Pig 16 23RZ3 Staphylococcus aureus Jiangsu Nanjing Pig 17 23RZ5 Staphylococcus aureus Jiangsu Nanjing Pig 18 23RZ6 Staphylococcus aureus Jiangsu Nanjing Pig 19 23RZ7 Staphylococcus aureus Jiangsu Nanjing Pig 20 23RZ8 Staphylococcus aureus Jiangsu Nanjing Pig 21 23RZ9 Staphylococcus aureus Jiangsu Nanjing Pig 22 23RZ10 Staphylococcus aureus Jiangsu Nanjing Pig 23 23RZ11 Staphylococcus aureus Jiangsu Nanjing Pig 24 23RZ12 Staphylococcus aureus Jiangsu Nanjing Pig 25 23RZ13 Staphylococcus aureus Jiangsu Nanjing Pig 26 23RZ14 Staphylococcus aureus Jiangsu Nanjing Pig 27 23RZ15 Staphylococcus aureus Jiangsu Nanjing Pig 28 23RZ16 Staphylococcus aureus Jiangsu Nanjing Pig 29 23RZ17 Staphylococcus aureus Jiangsu Nanjing Pig 30 23RZ18 Staphylococcus aureus Jiangsu Nanjing Pig 31 26001 Staphylococcus aureus Jiangsu Nanjing Pig 32 SH1 Staphylococcus aureus Jiangsu Nanjing Pig 33 SH2 Staphylococcus aureus Jiangsu Nanjing Pig 34 HCP4 Staphylococcus pseudintermedius Guangxi Hechi Dog 35 HCP6 Staphylococcus pseudintermedius Guangxi Hechi Cat 36 HCP7 Staphylococcus pseudintermedius Guangxi Hechi Cat 37 HCP9 Staphylococcus pseudintermedius Guangxi Hechi Cat 38 HCP19 Staphylococcus pseudintermedius Guangxi Hechi Cat 39 HCP21 Staphylococcus pseudintermedius Guangxi Hechi Dog 40 HCP24 Staphylococcus pseudintermedius Guangxi Hechi Cat 41 HCP25 Staphylococcus pseudintermedius Guangxi Hechi Dog

[0056] Table 2. Spotting results of Staphylococcus phage vB_PTS_HC01

[0057] Strain name Animal source Spotting result 23YZ1 Pig + 23YZ2 Pig + 23YZ3 Pig + 23YZ4 Pig + 23YZ5 Pig + 23YZ9 Pig + 23YZ7 Pig - 23JC1 Pig + 23JC2 Pig - 23JC3 Pig - 23JC4 Pig + 23JC5 Pig + 23JC6 Pig - 23RZ1 Pig + 23RZ2 Pig + 23RZ3 Pig + 23RZ5 Pig + 23RZ6 Pig + 23RZ7 Pig + 23RZ8 Pig + 23RZ9 Pig + 23RZ10 Pig + 23RZ11 Pig + 23RZ12 Pig - 23RZ13 Pig + 23RZ14 Pig + 23RZ15 Pig + 23RZ16 Pig + 23RZ17 Pig + 23RZ18 Pig + 26001 Pig + SH1 Pig + HCP4 Dog - HCP6 Cat - HCP7 Cat + HCP9 Cat + HCP19 Cat + HCP21 Dog + HCP24 Cat + HCP25 Dog + ​

[0058] +: spotted, positive; - : not spotted, negative

[0059] Example 6

[0060] Determination of one-step growth curve of staphylococcus phage vB_PTS_HC01

[0061] Mix the phage vB_PTS_HC01 culture prepared in Example 1 with excess of the phage prepared in Example 1 (MOI > 10, to ensure all bacteria are adsorbed with phage), incubate at 37℃ for 15 min, centrifuge at 12000 rpm for 1 min, discard the supernatant (unadsorbed phage), add 25 mL TSB liquid medium to dilute, incubate at 37℃. From 0 min, take 100 μl culture every 10 min, centrifuge at 10000 rpm for 2 min at 4℃ to remove bacteria, take the supernatant and dilute to appropriate concentration (appropriate concentration is the concentration that can form 30-300 plaques on the plate), determine the phage titer by double-layer method, measure for 120 min, a total of 14 times of sampling, take the sampling time as the abscissa, the logarithm of the phage titer as the ordinate, draw the one-step growth curve to obtain the latent period, burst period and burst size of the phage. The one-step growth curve result is shown in Figure 4 , the latent period of the phage is 30 min, and the burst size is about 20 PFU / CFU.

[0062] Example 7

[0063] Temperature and pH tolerance experiment of staphylococcus phage vB_PTS_HC01

[0064] Take 10 sterile EP tubes, add 0.5 ml of the phage prepared in Example 1 stored at 4℃ to each, respectively, and act at 4℃, 25℃, 37℃, 45℃, 50℃, 60℃, 70℃ for 30 min and 60 min, respectively, immediately cool in a water bath after the action time, and then determine the phage titer; the detection result is shown in Figure 5 : the phage can tolerate 45℃ temperature, and the titer is basically stable within 30 min, and the phage titer decreases obviously with time when the temperature is greater than 45℃.

[0065] Take 6 portions of 0.1 ml of the phage prepared in Example 1 stored at 4℃, and put them into SM buffer (0.9 ml) with pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, respectively, act at 37℃ for 1-2 h, and then determine the titer of the reacted phage by double-layer method; the detection result is shown in Figure 6As shown: Staphylococcus phage vB_PTS_HC01 titer changes little in the environment with pH value of 6-11, and the activity is basically unchanged; when the environment pH > 11 or pH < 6, the titer of the phage decreases sharply with the increase of acidity or alkalinity; when pH = 5 or 12, the titer of the phage is 0, and all inactivated, so the optimum pH of the phage is 6-11.

[0066] Example 8

[0067] Staphylococcus phage vB_PTS_HC01 in vitro inhibition curve

[0068] Dilute the host bacteria HCP21 isolated in Example 1 to 1×10 6 CFU / mL, add 3ml TSB liquid medium to the control group, and mix 100µL of the phage vB_PTS_HC01 culture with 100µL of the phage at MOI = 100, 10, 1, 0, 0.1, 0.01 in the 96-well plate, and place it in 37℃ culture, with 3 repeats for each group; measure the OD 450 value every 1h with spectrophotometer. The results of the phage sterilization experiment are shown in Figure 7 , MOI = 0, i.e. only host bacteria in the culture medium without phage interference, the OD 450 starts to rise obviously after 5h, and enters the logarithmic growth phase; compared with this, the OD 450 of the different MOI groups maintains at a very low level (OD 450 <0.25) before 13h, and the bacteria are almost completely killed, indicating that the phage with low titer can also well inhibit the growth of bacteria and the antibacterial time is very long.

[0069] Example 9

[0070] Staphylococcus phage vB_PTS_HC01 inhibits the ability of biofilm formation

[0071] The host bacteria with strong biofilm formation ability are cultured in TSB liquid medium to logarithmic phase, and then the logarithmic phase bacterial liquid is mixed with TSB liquid medium in a ratio of 1:1 (100 μL+100 μL), and cultured for 36 h. The biofilm is formed and attached to the plate hole, simulating the attachment scene of bacterial biofilm in medical devices as a positive group. The experimental groups are also different phage infection multiplicity (MOI=100, MOI=10, MOI=1, MOI=0.1, MOI=0.01) added to the plate hole with bacterial liquid and TSB liquid medium, each plate hole is repeated three times, as a biofilm removal experimental group. After 36 h, the plate hole is washed with sterile PBS buffer for 3 times, and the floating bacterial biofilm is washed clean. Then methanol is used to fix the biofilm attached to the plate hole for 15 min, and after removing the methanol, the 96-well plate is dried at room temperature. Then 1% crystal violet solution is used for biofilm staining for 5 min, and after removing the staining solution, the excess crystal violet dye is washed clean with PBS buffer and dried naturally at room temperature. Finally, 33% acetic acid is added to the plate hole to dissolve the stained attached biofilm, and the reflection wavelength is measured at OD=630 nm. The experimental results show that the phage has strong biofilm removal ability, and can treat the biofilm attached by the bacteria with strong biofilm formation ability under different infection multiplicity conditions. From the above, it can be seen that the staphylococcus phage vB_PTS_HC01 has great potential in controlling the biological pollution caused by staphylococcus.

[0072] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only and is not intended to limit the application in any way. Many alternatives, modifications, and variations will be apparent to those skilled in the art upon reading this description. The scope of the application is intended to be defined only by the claims appended hereto and equivalents thereof.

Claims

1. A bacteriophage of Staphylococcus, characterized in that, The staphylococcus phage is Staphylococcus pseudintermedius Phage vB_PTS_HC01, and the preservation number is CCTCC NO: M 20252005.

2. The staphylococcus phage according to claim 1 is used for preparing a medicine for preventing and treating staphylococcus infection.

3. A bacteriophage pharmaceutical formulation, characterized in that: The effective component comprises the staphylococcus phage according to claim 1.

4. The bacteriophage pharmaceutical preparation of claim 3, wherein: The medicine further comprises a pharmaceutically acceptable carrier, and the dosage form is a solution, a powder, a gel, a granule or a freeze-dried agent.

5. A biological bacteriostatic agent for disinfecting aquatic products, characterized in that: The method for using the staphylococcus phage according to claim 1 comprises: soaking or spraying the surface of aquatic products to inhibit the proliferation of staphylococcus in the processing or preservation process of the products.

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