CN112725287A - Strong-lytic staphylococcus aureus phage RDP-SR-20001 and application thereof - Google Patents

Abstract

The invention discloses a strong lytic staphylococcus aureus bacteriophage RDP-SR-20001 and application thereof in pathogenic staphylococcus aureus infection of poultry. The method comprises the steps of screening a staphylococcus aureus BS-20001 strain from diseased broiler tissues in Qingdao and Jimo broiler farms by a double-layer plate method, and then separating staphylococcus aureus bacteriophage RDP-SR-20001 from the sewage of the farms by using a staphylococcus aureus BS-20001 host, wherein the preservation number is CGMCC No. 21409. The pathogenic staphylococcus aureus BS-20001 in the poultry breeding environment of the staphylococcus aureus bacteriophage RDP-SR-20001 provided by the invention has strong lytic effect, and provides a bacteriophage source for the industrial production of the bacteriophage for preventing and treating the pathogenic staphylococcus aureus in the poultry breeding environment.

Description

Strong-lytic staphylococcus aureus phage RDP-SR-20001 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a strong lytic staphylococcus aureus bacteriophage RDP-SR-20001 and application thereof.

Background

Staphylococcus aureus (Staphylococcus aureus) is an important pathogenic bacterium in human beings, belongs to the genus Staphylococcus (Staphylococcus), is called as a "mesophilic bacterium", is a representative of gram-positive bacteria, and can cause many serious infections. Staphylococcus aureus is ubiquitous in nature and is found in air, water, dust and human and animal excreta. Thus, there are many opportunities for food to become contaminated therewith. Staphylococcus aureus is pathogenic to both animals and humans. Wherein air pollution is the major route of transmission in animal populations. Rabbits are sensitive to staphylococcus aureus and can cause rabbit septicemia when infected through skin surface lesions. Infection of mice with staphylococcus aureus can bow a skin soybean-sized spot or septicemia. Staphylococcus aureus infection of poultry such as broiler chicken can cause arthritis, tendonitis, septicemia, peritonitis, etc. Staphylococci always exist in places such as farms, hospitals and communities, and if the prevention and treatment are not good, large-area infection can be caused, so that not only is serious loss caused to the breeding industry, but also the life safety of human beings can be endangered.

The traditional treatment mode of staphylococcus aureus is antibiotic treatment, but along with the use and abuse of a large amount of antibiotics in the breeding industry, the drug resistance of staphylococcus aureus to antibiotics is gradually enhanced, so that the treatment of infection caused by the staphylococcus aureus is more and more difficult, especially in recent years, "super bacteria" capable of resisting methicillin is separated clinically, so that the prevention and treatment of staphylococcus aureus are more and more difficult, and therefore, a novel drug is urgently needed to replace antibiotics for treatment.

Bacteriophages (phages) are a generic term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochetes, and are called phages because they partially cause lysis of host bacteria. The phage exists widely in nature, and corresponding phage may exist in places where bacteria grow. About 10 on earth is predicted³¹The phage has been classified and identified and reported to be close to 5100 phage. These abundant phage resources provide valuable materials for the development and utilization of phage.

Compared with antibiotics, the bacteriophage has the characteristics of strong specificity, difficult antibody generation, safety, no drug residue, strong proliferation and the like, and can safely, efficiently and quickly kill specific pathogenic bacteria. Therefore, screening and applying suitable phage is an effective way to prevent and treat staphylococcus aureus infection.

The invention provides a safe and reliable strong lytic staphylococcus aureus bacteriophage for treating staphylococcus aureus, which is used for solving the problems of bacterial drug resistance and antibiotic abuse in order of comprehensive banning of corresponding countries.

Disclosure of Invention

The invention aims to provide a strong lytic staphylococcus aureus bacteriophage RDP-SR-20001, and aims to solve the difficulties that poultry in a farm are not treated timely due to acute disease caused by infection of staphylococcus aureus and drug taking is ineffective due to drug resistance of pathogenic bacteria.

In order to achieve the purpose, the invention adopts the following technical scheme:

a strong lytic staphylococcus aureus bacteriophage RDP-SR-20001, which is observed by a transmission electron microscope, has a diameter of 1-2mm, is round and transparent, has no halo, has a head part with a regular polyhedron structure, a diameter of about 41nm, and no tail part, and is a capsphage family; the total length of the genome is 17085bp, and the genome has no virulence genes and lysogenic genes; the phage is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 17 days in 2020, and the preservation addresses are as follows: the number of the collection is CGMCC No.21409, No. 3 of Xilu No. 1 of Beijing Chaoyang district.

The bacteriophage is a virulent bacteriophage, has strong lytic property to pseudomonas aeruginosa, has a lysis rate of 95 percent, and provides a bacteriophage source for industrialized, large-scale and programmed production of the bacteriophage and treatment of poultry breeding animal bacterial diseases caused by staphylococcus aureus.

Further, the bacteriophage RDP-SR-20001 is treated at 50 ℃ for 1h, and the titer is still kept at 10⁹pfu/ml or more.

Further, the bacteriophage RDP-SR-20001 is cultured at 37 deg.C for 60min at pH5.0-9.0, and the titer is still maintained at 10¹⁰pfu/mL or more, completely losing the ability to infect a host at pH 2.0; at pH 12.0, the titer of the phage remained at 10³pfu/mL, phage RDP-SR-20001 has acid intolerance and alkalophilicity.

The bacteriophage can be used for industrial production, can be specifically amplified by host bacteria, and can be used as a biological disinfectant to be applied to poultry farms, animal husbandry, food, medicines, medical instrument factories, hospitals and other environment disinfectants for human activities.

The bacteriophage is added into poultry feed as a feed additive, has obvious inhibiting and killing effects on staphylococcus aureus in poultry bodies, and can effectively prevent the occurrence of poultry staphylococcus aureus.

The invention also provides a bactericidal composition for preventing and treating staphylococcus aureus, and the effective component of the bactericidal composition comprises staphylococcus aureus bacteriophage RDP-SR-20001.

The invention also provides a feed for preventing and/or treating diseases caused by pseudomonas aeruginosa, and the effective component of the feed comprises the staphylococcus aureus bacteriophage RDP-SR-20001.

The invention also provides a cleaning agent or a disinfectant, the effective component of which comprises the staphylococcus aureus bacteriophage RDP-SR-20001.

The invention has the beneficial effects that:

(1) the invention obtains a staphylococcus aureus BS-20001 from diseased broiler tissues of a Qingdao, i.e. Memo-broiler farm by screening, and obtains a staphylococcus aureus bacteriophage RDP-SR-20001 from the sewage of the farm by taking the staphylococcus aureus BS-20001 as a host through separation, wherein the bacteriophage RDP-SR-20001 has strong cracking effect on pathogenic staphylococcus aureus BS-20001 in a poultry breeding environment, and provides a bacteriophage source for industrial production of the bacteriophage for preventing and treating the pathogenic staphylococcus aureus BS-20001 in the poultry breeding environment;

(2) the staphylococcus aureus bacteriophage RDP-SR-20001 provided by the invention can maintain better activity in the range of pH5.0-9.0, the bacteriophage titer is not obviously changed, and the bacteriophage titer is highest when the pH is 9.0; the titer of the phage is obviously reduced along with the reduction of the pH value, and the capability of infecting the host is completely lost when the pH value is 2.0; but has better stability in alkaline environment, and the titer of the phage is still kept at 10 when the pH is 12.0³pfu/mL or so; has acid and alkalophilicity;

(3) the staphylococcus aureus bacteriophage RDP-SR-20001 can be used for industrial production and can be specifically amplified by host bacteria staphylococcus aureus BS-20001; the bacteriophage can also be used as a safe biological disinfectant for environmental disinfection and animal body surface disinfection, thereby treating animal farm pollution; the bacteriophage can also be used as a medicine for preventing and treating diseases caused by staphylococcus aureus.

Drawings

The above and other features, properties and advantages of the present invention will become more apparent from the following description of the embodiments with reference to the accompanying drawings:

FIG. 1 is a schematic diagram of the separation and purification of Staphylococcus aureus BS-20001 according to example 1 of the present invention;

FIG. 2 is a schematic representation of the phage plaques obtained in example 2 of the present invention.

FIG. 3 is an electron micrograph of Staphylococcus aureus bacteriophage RDP-SR-20001;

FIG. 4 is a graph of one step growth of Staphylococcus aureus bacteriophage RDP-SR-20001 of the present invention;

FIG. 5 is a schematic diagram of the acid-base tolerance test of Staphylococcus aureus bacteriophage RDP-SR-20001 according to the present invention;

FIG. 6 is a schematic diagram of the heat stability experiment of Staphylococcus aureus bacteriophage RDP-SR-20001 of the present invention;

Detailed Description

The invention is further illustrated by the following figures and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The staphylococcus aureus BS-20001 is obtained by screening diseased broiler tissues in a Qingdao, Yimo and broiler farm, pathogenicity and drug resistance of the BS-20001 are determined through experiments, and a staphylococcus aureus bacteriophage RDP-SR-20001 is obtained by separating sewage of the chicken farm by taking the BS-20001 as a host; and the genotype of RDP-SR-20001 is determined by the whole genome sequence.

Example 1 isolation and identification of pathogenic Staphylococcus aureus BS-20001

Sampling from a diseased farm, taking a joint exudate, a liver and a spleen of a diseased chicken through aseptic operation, streaking on a BHI culture medium, culturing at 37 ℃ for 24-48 h, carrying out gram staining and microscopic observation on bacteria growing on a plate, carrying out plate streaking purification on bacterial colonies with gram staining as positive cocci again, repeating the steps for 3 times until bacterial colonies with uniform size and shape are grown, then selecting a single bacterial colony to inoculate in 5mL LB broth, and carrying out shaking culture at 37 ℃ and 200rpm for 10h to obtain a uniform and turbid bacterial suspension. And determining the strain to be pathogenic staphylococcus aureus by biochemical identification and 16sRNA molecular identification, and storing one strain of the pathogenic staphylococcus aureus under the name of BS-20001 (shown in figure 1) in a refrigerator at the temperature of-80 ℃.

Example 2 isolation and characterization of Staphylococcus aureus phage RDP-SR-20001

(1) Sewage treatment: collecting sewage from a farm, primarily filtering the collected sewage sample by using a plurality of layers of gauze, then placing the filtrate at 4 ℃ for 10000 Xg centrifugation for 15min, filtering the supernatant by using a disposable filter with the pore diameter of 0.22 mu m, and placing the filtrate at 4 ℃ for later use;

(2) preparing a mixed bacterial suspension: 0.2mL of the bacterial suspension and 1mL of the filtrate were added to 5mL of LB broth, incubated overnight at 37 ℃ with shaking at 200rpm, and then centrifuged at 10000rpm for 5min, and the supernatant was filtered through a 0.22 μm filter to prepare a filtrate.

(3) Phage separation: separating phage by using a double-plate method, uniformly mixing 0.1mL of mixed bacteria suspension filtrate with 0.8978 mL of staphylococcus aureus BS-200010.2 mL, carrying out water bath at 37 ℃ for 10min, then paving double plates, putting the double plates in an incubator at 37 ℃ for culturing for 6-8h, and observing that transparent spots are formed on the plates; and (3) picking the transparent spots, fully and uniformly mixing the transparent spots in 1mL of SM buffer solution, standing the mixture at 4 ℃ for 6 hours, then diluting the phage soaking solution in a multiple ratio, and repeating the operation of the double-layer plate. This procedure was followed 3 times to obtain a phage, which was designated as RDP-SR-20001. As can be seen from FIG. 2, the plaques formed by the phage are uniform in shape and size, round and transparent, clear in edge, and about 1-2mm in diameter.

(4) The purified phage multiplication fluid was mixed with glycerol (30%) and stored in liquid nitrogen.

(5) Measuring the titer of the phage: centrifuging bacteriophage proliferation solution at 10000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter to obtain filtrate, diluting the filtrate with SM buffer solution by 10 times, and diluting to 10 times⁸Taking 0.1mL of each of the diluent and the bacterial suspension of the last 3 dilutions, preparing a double-layer flat plate, inverting after condensation, and culturing at constant temperature of 37 ℃. Phage titer (pfu/mL) is plaque number × dilution multiple × 10; the result shows that the titer of the phage can reach 10¹¹pfu/mL or more.

EXAMPLE 3 Electron microscopy of phages

The phage is treated by adopting a phosphotungstic acid negative staining method: dropping 100 μ L of purified and multiplied phage on a paraffin plate, placing the surface of a copper net with a membrane on a phage droplet, taking the copper net off the droplet after 10min, placing the copper net on clean filter paper, drying for 3min, adding a drop of 2% phosphotungstic acid (PTA) on the copper net to dye the phage for 10min, then sucking the dyeing solution from the side by using the filter paper, and observing the form of the phage by using an electron microscope after the sample is dried.

As can be seen from FIG. 3, the head of the bacteriophage RDP-SR-20001 is a regular polyhedron structure, the diameter is about 41nm, and no tail is present; according to the ninth report of the organization of viral taxonomy of International taxonomy of viruses, the form of the phage conforms to the characteristics of the Capsioviridae family.

Example 4 phage genome sequencing

After enrichment culture of a single phage, centrifuging for 15 minutes at 8000r/min at 4 ℃, adding 10% PEG8000 and 0.5M NaCl, standing overnight, adding equal amount of chloroform, mixing uniformly, standing for layering, centrifuging for 10 minutes at 5000r/min, removing a chloroform layer and a PEG layer, adding restriction endonuclease for digestion treatment, suspending the phage under the condition of gradient density cesium chloride, dialyzing for 3 times at the later stage by TM buffer solution, and performing whole genome sequencing by Chihuada gene biosequencing company for 30 minutes each time. Sequencing results show that the whole length of the phage genome is 17085bp, and the phage genome does not have virulence genes and lysogenic genes.

Example 5 optimal multiplicity of infection assay

Optimal multiplicity of infection (MOI) assay: 1 in terms of multiplicity of infection, respectively: 1. 1: 10. 10: 1. 100, and (2) a step of: 1. 1000: 1, adding the phage and BS-20001 into a nutrient broth culture medium, carrying out shaking culture at 37 ℃ for 10h, carrying out 12000 Xg centrifugation at 4 ℃ for 15min, filtering the supernatant by using a disposable filter with the pore diameter of 0.22 mu m, and measuring the titer of the phage at the moment, wherein the corresponding multiplicity of infection when the obtained phage titer is the highest is the optimal multiplicity of infection of the phage. The results are shown in Table 1.

TABLE 1 optimal multiplicity of infection for bacteriophage RDP-SR-20001

As can be seen from the data in Table 1, when the infection number is 1:100, the growth medium is relatively clear and the titer is the highest after 10 hours of culture, indicating that the optimal infection number of RDP-SR-20001 is 1: 100.

Example 6 determination of phage growth curves

Taking staphylococcus aureus BS-20001 in logarithmic growth phase, inoculating the staphylococcus aureus BS-20001 and phage RDP-SR-20001 according to the optimal complex number of infection, carrying out water bath for 10min at 37 ℃, then centrifuging for 5min at 12000r/min at normal temperature, removing supernatant, removing free phage not adsorbed on a host, and washing twice by using LB broth culture medium. The pellet was resuspended in LB broth at 37 ℃ and quickly placed on a shaker at 37 ℃ and 200r/min for shaking culture and timing. Sampling and counting every 5min for the first 30min, counting every 10min for 30-200min, drawing a growth curve by taking time as a horizontal coordinate and a bacteriophage titer logarithm value as a vertical coordinate, obtaining the incubation period and the lysis period of the bacteriophage, and calculating the average lysis amount. Wherein the average lysis amount is the phage titer at the end of outbreak/the host bacteria concentration at the initial stage of infection.

As can be seen from FIG. 4, the titer did not change significantly within 15min after the phage infected the host bacteria, indicating that the incubation period was about 15min, and the titer of the phage increased significantly within 20-90min after infection, then became stable, indicating that the phage lysis period was about 70 min. By calculation, the average lysis amount of the phage is 108, and the phage shows extremely strong lysis and replication capacity.

Example 7 acid-base resistance test

Adjusting pH of normal saline with dilute hydrochloric acid and dilute NaOH solution to obtain buffer solution with pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and diluting bacteriophage with SM buffer solution to 1 x 10¹⁰pfu/mL, the dilution was incubated at 37 ℃ for 1h in a water bath, diluted 10-fold with physiological saline and plated on double plates. Counting after inverted culture at 37 ℃ for 4-6 h.

Referring to FIG. 5, it can be seen that the phage RDP-SR-20001 maintained good activity at pH5.0-9.0 after cultivation under different pH conditions, the phage titer did not change significantly, and the phage titer was highest at pH 9.0. The titer of the phage decreased significantly with decreasing pH, and completely lost the ability to infect the host at pH 2.0. But has better stability in alkaline environment, and the titer of the phage is still kept at 10 when the pH is 12.0³pfu/mL, which shows that the bacteriophage RDP-SR-20001 has acid and alkalophilicity; the phage grows optimally in a neutral environment.

Example 8 thermal stability experiment

The phage stock solution is subpackaged into EP tubes, incubated for 30min and 60min at 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ respectively, then diluted by 10 times of SM buffer solution, and spread on a double plate to determine the titer.

As can be seen from FIG. 6, the activity of the phage remained stable within 30 ℃ to 50 ℃, the activity of the phage was reduced by about half at 60 ℃ and 70 ℃ and was substantially inactivated at 80 ℃ and higher temperatures. Therefore, the bacteriophage RDP-SR-20001 has poor high temperature tolerance and is suitable for living in the environment of 50 ℃ and below.

Example 9 cracking Rate experiments

Under the aseptic condition, respectively taking 1mL bacteriophage RDP-SR-20001 and 1mL host bacterium BS-20001 bacterial liquid, incubating for 15 minutes at 37 ℃, mixing uniformly, diluting with SM buffer solution to10^-1-10^-3Each gradient was plated at 100. mu.L onto a plate and incubated at 37 ℃ for 24 hours, with each gradient repeated twice. Simultaneously taking 1mL of SM buffer solution and 1mL of host bacterium BS-20001 bacterium solution as blank controls, and repeating the steps. Plates with 30-300 colonies were picked for enumeration. This test was repeated 3 times, and the average value was taken to calculate the phage lysis rate. The phage lysis rate was (1-number of treated colonies/number of control colonies) × 100%. Through calculation, the cracking rate of RDP-SR-20001 reaches 95%, and the microbial composite has a good cracking effect on a host and is suitable for being used in a culture process.

Example 10 fragmentation Spectrum experiments

30 staphylococcus (including 17 staphylococcus aureus, 7 staphylococcus epidermidis and 6 staphylococcus saprophyticus), 10 salmonella, 5 escherichia coli, 1 vibrio alginolyticus, 1 vibrio harveyi and 3 vibrio parahaemolyticus which are identified in a laboratory are selected to carry out a lysis spectrum experiment. The phages were reacted with different species of standard strains. As shown in table 2.

TABLE 2

As can be seen from Table 2, the bacteriophage RDP-SR-20001 can not crack Escherichia coli, Salmonella, Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus, but can crack staphylococcus, which shows that the bacteriophage RDP-SR-20001 has specificity only to staphylococcus. In 17 strains of staphylococcus aureus participating in detection, the phage can form plaques on plates of 15 strains of staphylococcus aureus, 3 strains of staphylococcus epidermidis and 4 strains of staphylococcus saprophyticus, so that the phage can crack 15 strains of staphylococcus aureus (chicken source, cattle source, pig source and duck source including one MRSA strain) and can crack staphylococcus saprophyticus and staphylococcus epidermidis and the like. This experiment shows that the bacteriophage RDP-SR-20001 has a broad host spectrum and can crack part of drug-resistant strains separated from meat products.

It should be noted that the specific embodiments are merely representative examples of the present invention, and it is obvious that the technical solution of the present invention is not limited to the above-mentioned examples, and many variations are possible. Those skilled in the art, having the benefit of this disclosure and the benefit of this written description, will appreciate that other embodiments can be devised which do not depart from the specific details disclosed herein.

Sequence listing

<110> Ray union (Qingdao) bioengineering Co., Ltd

<120> strong lytic staphylococcus aureus bacteriophage RDP-SR-20001 and application thereof

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<170> SIPOSequenceListing 1.0

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cacccatact caaatgaaga ggttatgttg tcaaaagtag tactcaatgg tttatatggc 5040

atacctgcgt tacgttcaca ttttaattta tttcgtttag atgaaaataa cgaattgtat 5100

aacatcatta acggttataa aaacacagaa cgtaatattt tattttctac atttgtaaca 5160

tcacgttcat tgtataactt attagtacca ttccaatatt taacagaaag tgaaattgat 5220

gataatttta tttattgtga tactgatagt ttgtatatga aatccgttgt aaagccctta 5280

ttgaacccca gtttattcga ccctatatca ttaggcaaat gggatattga aaacgaacag 5340

atagataaga tgtttgtact caatcataaa aaatatgcat atgtaacaaa tggaaagatt 5400

aaaatagcct cagctggtat accgaaaaac gcctttgata caagcgtcga ttttgaaacc 5460

tttgtacgtg aacaattctt tgacggtgcc attattgaaa acaataaaag tatctataat 5520

gctcaaggta caatatcgat atatccgtct aaaactgaaa ttgtatgcgg taatgtatat 5580

gatgaatatt ttactgatga acttaatatg aaacgtgaat ttatcttaaa agacgctaga 5640

gaaaattttg accatagtca atttgatgat attctttata ttgaaagcga cattggttcg 5700

ttctcactta atgacttatt tccagttgta cgttcagtac ataataaatc aaatttaaat 5760

atattaaaaa atcaacatga tgacatcaaa aaggcaactg ttaaataaca gttgcctttt 5820

tcttttgaga taacatgaaa aatgtgtacg aaaattgatt atgttttgta ttttatttac 5880

tagcattact agcatgtatt tattatatca tagttaatta agcaatacca ctgaagaata 5940

caatattatc accagcgtta tgaggtacac cattaatgag tgtatacaat acaacccttg 6000

acggagcaac gtatggtgga acattatagt ttgctactaa gaatgaacca tcgtcaaaca 6060

cagcaacaac tacacccgtg tgaccgatac catatatgct tgcttgtaag tatggtggtt 6120

tacttgaaaa gccataaccg acagtagggt tgtgcgttgt tttagcgcct aactttttat 6180

aaacatacca cacacgttga ccgtttgtta tttgtccatc gtcggtaggt tgtcttttac 6240

catgtagttg tgacatatac gcccatgtta attctgtaca ttgtcctgca ttacccgttt 6300

gaggaaatat gttacctggt ttgtataaat attctttttt gaataaaggt acaccaattg 6360

cttttttata tttttctggt aactgtgcat acgtccagtt accaccaatg acacgaccac 6420

ttttaccatt tggtttgact gatttaccac taattggttt atggtctccg tcatcatcag 6480

tagggtttga actactaccc ccactatcta cttgcacgct atcaatcagt ttttttaatg 6540

aatcgagtag cccaatcgtc attttaatat gatatgtgtt gttaaatgtt ttttgtaatg 6600

taaaataatc attactaaaa aatttgtcac tacctatact gtgtacatcc cattgtaatg 6660

cgtcttgtac tttttttaat aattcttgca tggcttgttt tgctaaagcg agcagtgaac 6720

taccactgtc accactacta ccactgtcag acgaatcact aggtgaacca cctttaccgt 6780

ctaatttacc accccacgca agaatagtat tcgcaccgtc taaaaatgga ttaccatagt 6840

tttgtacttt attatatgac gctttcaaac ctaggggata atatgccgcc caagtagctg 6900

ctgctgttaa tggaatatac gcacgtccga ttgtacctgc tttcatattt ttagcaaaat 6960

ctgcattacc ttttctttgt acgtcttgag gtacaaagtg aacgatgtta cctgcgtcat 7020

accaagacgg ttgtcctgct tgttttgatt gtgatactaa ttttcttgca atgaatttag 7080

cgtctgttaa atagtcacct cgtgcagatg tatggttcaa ccaacctaaa ccagcgctat 7140

acccctcatt tttttcatat acagcaaaaa gagtaggcga cacacctatc tcttttactg 7200

cttttaatac ttgtctgatt ttactttcat taccaccaag ccatacatta aagcgtccat 7260

acccttttac tttagggact aactggtcta tcgttaaacc gaaatcatca ttaatatacg 7320

aatgcgtaaa tttatctatc ttctcttggt cgttcattat tatcactctt ttcagaatcg 7380

tttttaatta ctcttaattt atctttaatt tgttctggca ctaatacgtc catttctgca 7440

caattttcta cgatagataa gccctcattc gcaatataat agaaaatcgt aatcatgagt 7500

aagccacctt ttaattgtaa aatctggtca atgatgttcg ctaaaatgat aatacagaat 7560

atcaataatt ttttagcaaa acctttcatt gatttttttg accatagatt attattttta 7620

atggattttg aaatacctgt aataatatca acaaacatta atacaaataa aaaatatagt 7680

aattttaaat cgcctgcata tataaacatg tgaaacgctt ctgaatctgt aaacctaaat 7740

tttacttcat tcattttaca caccctctct aaatttgtta tttaatggat tttgtaacat 7800

agggttacct gaaccatcat tatgccaaaa tctcacacca gattctaaaa tagcttttaa 7860

ttgttccatt aacatagggt caatgtcacg tatggtatac gtaccagtac attttaaata 7920

gttgcaaaca gtcatactgt taatcggttc aataaatgaa ttatagtcat ttacttcaaa 7980

accaaataac atataatatt tttgtaagaa tgtaatttct ttgggagatg gaacactaat 8040

cttcatcgtt aaaccattaa tactatttgc aatttggaag gcgtttccca tttctgattc 8100

tgttactgat ggcggttgta atgctaaatc tttatattct gcttgttgtt gtttatagaa 8160

attatattct tcattaaact taccaaataa tgcagttgga cttaaattac ttgctacact 8220

tactgcgtca taaaaacgtg attttgggtc actaccattt aatacattat ctatacgact 8280

tgtgattaat tgactttctg cattacgctg tctattagct tgctgtgatt gacctaaaat 8340

accgttattg attaaaattg gtacttgtgc aaaactatta aatgttatat ttgtatttaa 8400

gaatgaacct gtatcaatta atatatcttt atttttagca agtatcggtc tatcgttttc 8460

agcactgtta taatcaactg gatatactcg cacttcatta tgataaccaa tgatggattt 8520

tgtacgtaac ttaacacctg ttttttgtga aattttacca gcgtcaagta acattgtgtt 8580

accgttccag tcataaaatt caatcgtcat gtactcatta cgtatcatat gtttaaactc 8640

gtctttttta gacaacatca tctcttgtag tttagtaaaa cttaatgata aatcgtttaa 8700

actccattct tttgattttc caccctgttt taatgtcttt aatccagtaa ttttttcact 8760

tgtcttaacg tcctctaaat cttttgtatt aataaaatct ttaggtaaca tttgaacctt 8820

ttgaaagttt tgtgtaatcc atggatatgc actcatttta tccataaagt taataaagtc 8880

accatattcc ataacgtata agttgactgg tgatgtgata ttgtcatata tcgtaccttt 8940

agacgtatct aagtttggct ctttttttgt accaaatttc tttgataaat cagcgcttga 9000

ctggaataac actaaatttt ccaaatactg ttgcatttgg ttatacacat agtttttatt 9060

agatactttt aacacatcat cattgttacg taacattggt aacatatagt tatacgtgcg 9120

ttttgataag tgttgacgtt caatattaac gtttgagagt tgctctaata cattaccttg 9180

cgtgtacgtc ataatagtat caatcacaaa atatatttta accactacat cattcacgta 9240

ttctatttga ttcacaaaag cgtaatagcg tctgtcctca aaatctgata aaaacgtcat 9300

atagttaatt ccttgtgcgt catgccactg catatctaca ttaatttcca ttctatcgcg 9360

tataaaatta tacggttgtt tggaatagtc taacgattta aaatgacgac catttaaaaa 9420

ataatcatca cgttcttgat tactattaaa atgaattgta ttttgataat ctgtaaacgg 9480

tgtgttatag aaaaacttaa aattcgttaa ttttctcata ttttcctcct aatataaaaa 9540

tagtcgtata aattatttat acgactatta tatcattttt attcaatgat ttgtgtatct 9600

ataacaaaac gtttatctcc atttgttaag tcactatctt gataagatga tgtaacaaaa 9660

tgtaattcgt tattaaagtt taaatacatt cttgtattta tcatttttgt atcaatggca 9720

cattgcgtat aatgatgtgt tgattttaaa tttgcattaa tactgcctaa ttttatatca 9780

ccatttttct tgattccttt aattacacct cttaattgaa tcgtgcgtat attattaatt 9840

gtaattatac gatattgcgg tgcaggataa ccactattac tatcacttgc aataatacca 9900

ctttctaatg taatatcttg ccaccctgta tcattcacag ttgttttatt ttcattaatt 9960

gtatttaaaa tttctatttt atcattagtt attgtagctg ttaaattgtt aatactttgg 10020

gtattattac ctacactttc ttttgtagag ataatatctt gtttattttt ttcaatatct 10080

gcttcatttt ttgtgttttt atcatctaat aaaagaattg cagattcatg attacttagt 10140

ttatttgtat gttctgattg aacatctgat aaatttttta tttttttatc ttgttgtaca 10200

ttatcttctt taatattaat aatgtctgta gcgttttgag aaatattatt tttatttgta 10260

gcgatatcat ttttattttt attaatgtct tttgtgttcg tattaatttt acttaataat 10320

tcatctttaa aggttaactt ataataatcc tcatcacgtc ttatataaat gttaccgtcc 10380

tttgtagtaa ttaagtcatt tgcttctact aaattatcat ttaatttata tacagagtca 10440

atgttgcgca aacttcttac aattctatca gccattgttt acacctctta tttatatcgt 10500

ttccaactaa attcaaagaa aaatcctaaa atacccatta tgagaacacc ccccaaggta 10560

caccaatact atatgcatta cctgtttttc cattccattg tctaactggt aaataataac 10620

gagttccttg ccagttataa ccaatccaaa ctaacccatc tgataaacaa acttcgtcat 10680

atggtgtata tccatttggt tggaaccaat atccattagg ttcacttaat ttaggactac 10740

ctacacgtgc aaatattggt aaaaatccac atgtaaatgt tgcattttca tttctgtaat 10800

atgttccgta ttggttttgt ttccaatttc caaatgtatt tacttttgat gtttctaata 10860

cattactatt actagcagaa aatttaggtc taataaagtg tgttacaccg tcataataat 10920

gtgttcttat tgttgctttt tcccaaccgt caaacccacc acctaaccag ttttgctcta 10980

aacatgtata ataatcaaga tttccacttg ttacacattg gatatgtcca tattgagaat 11040

ttgtgtatac tccaacatca cctaattgag gtttaaagct cggtgtattt tcatacaccg 11100

ttgctaaacc tttaaagtca ttattaattg cgtctttggc attaccccac atacgcactt 11160

taccgtcagt aatataatag atataagcga cagctaagtc catacattga aaaccatatg 11220

caccatcaaa gtcaacacca acaccttcat gtttatatat ccaatctttc gcttgttgtt 11280

gtgatttcat ttataacact cctatttttt atgttttgct acccattcat attcacgatg 11340

tttcgtatca ctgtttacat tactgaaaaa ctctttatat tctgatatat tagcctctaa 11400

tgtttgtctt acttcactaa ctgcgttacc acttgacaca cgtaaccatg caccaacacg 11460

ttttatttct tccggtgcgt ctttgaataa ttccatttgg ttacctgtaa tgtaatattc 11520

tccgggtgtt gtaacgtaag ctatccaatt attatattga cttgcttcta aatattcttg 11580

atattgtgcg tctgttttga ttgttgtcca taaaccataa tcccatttta atgtaaatac 11640

atcgagcgtc ataccacgca tgattttaac catttttcga ccagtagaaa aacgcgttaa 11700

ttcttgtact gtacctaatg tttgtgttgt agggtataca ttgatgaaac aaccagcgtc 11760

tataattttt ttactaccat tcattggcat atttttaagt ttactagctg taccaccgtc 11820

aatataatag aaccctgctt gtgttaagtc atttaaatcg tcaatatgat ctggaataga 11880

taacgcacga ccatcgtctt ttgttaattt atagttttgt gaacctctcg cacgtaatgc 11940

ttcaaagtgt tcatattctc caagttggaa gaaaccatat aaattattga atcgttttcc 12000

accgccgcca ttagtcatag caagtaataa cgatttacgc tttgttttag gattggtata 12060

aatacaaata ccctcaggct ctttaaagtt atcacgtggg aaattaattc cgtcttgata 12120

tgataattta aacggataat cataaatctt ttgacctgtt gttaatgaat atttaccgat 12180

ttgaacgtgt gaattaactg aactgttacc acttaaccag tataaatcat ctccgtcaac 12240

ggctatccct tgcatccaac gattatcatt gttttctgaa ttatcaatag tcatttcttt 12300

ttctacatta tcaatgtgat tttttacgtc agctcttgaa cgcacctgta ttgtcccgtc 12360

accgaatctt aaaacgattt tgtcatttgc ttcatcaatt aacggtgtaa acgtatgttt 12420

gtttaaaagt gactgtggtg tataatcggt taaccctttt gcttcttcta aatctaatac 12480

atagttatct ttataagcca cttgtaacag ttttgcaaca ccatcgtgat gtaaccatat 12540

tttcatttct ccattagatt gacgttctaa tccaattgtt gtaccatgac caccctgtac 12600

aatacgcata cttgaaatta agtcaccact aggcgttaat ttattaatcc aaaatccctc 12660

aggtgtttgt gagtcggatt gtgttgagta catttgatta gtttctttat caatattaat 12720

agattggttc acagcgttac gaataccgcc aaagcccatt acaaacttag gttcaagctc 12780

atttaattca aaaccattaa caaaacggtc aatgtcttta attaagtctt tcacttctgc 12840

tttaaaatca ttcatttgtt tcatttctgc aactttaaat aatgcaaatg cagatgtaag 12900

accagcacta tatttagtaa attcatcatg aataatttta tctattgtac cgtcatttaa 12960

ccaacctcta aataagtctt tagcttgttc tgggaatgct ttcattaatt catcccagtt 13020

tctgaaacgt ttttttaact catcgtcata gtcccaaata cgatgtgcta atacttcaat 13080

gagctttgat aatcttgaaa tataatcata atatgatttt gaattggtat tataatctgc 13140

tctatcatcg taaaacggtg tataacgttc tctcgtttta tatatttcgt ctaagaatgg 13200

acgaatgtca tcaaaatatt taaaatcgtt ttcattatat gccattattt aaccacctct 13260

accaaatttg taaaaaacat tttttatcaa attcatttaa aattttcttt cttaaatcgt 13320

atactttatc aatattatca attaaatact gttttgaaaa ttgtgtacct ttcgcattac 13380

ctttttgatt ttgattacgt tttgcgtttt gattactttc gttacttgat ttattcacag 13440

ttttaccgtt atcaatcgtg ttattgtcag caaattgtaa cgttgtttta tcaacatcaa 13500

tgttaacttc gctttgtggt aatgacgcat aagcatttct gtttgctgtc ataccagttg 13560

aattgtctaa agatgttgta ttttgatttg aagtttcgtc agtgttgctt gttgtatctt 13620

cgttatgttc tgtgaagcct tgcgattgta gatatttttc tacttcactt gatgaataaa 13680

ccacatttaa ataatcctca tgtgtgatac atacagtaat aacttgcata ccaaacgctt 13740

caactgtttg tctgttgatt tctctatcta aaaagtgtat tgtaaatgat tttttgaaaa 13800

gtaagtcaga taactcatct ttaagtttaa aacctttaaa cactttttca ttaatgattg 13860

ataaaacgtc tttgtcaaac ttcaacattt tttgcatgaa ttgaaattca tcatcataaa 13920

acgttaattt atcattattt acaaattcat taaaaccttt tttaattaat tctgatttaa 13980

taaaatcaaa taaagtcatt gtatatctag ccattgtatt cactactttc atcattagac 14040

aatgtatcta tcattgtgat ttctgaagta acttcatcgt cgtaatacgg tttaatatct 14100

aaaccataac gttttgataa gaatgtaatc ggttcacgac cttttaaata aatattacta 14160

tttgatgtgg taaaacctcg gttgcttttt gcctcttcgt cagatacacc actttcttta 14220

tcaacagcta aagagttaat acctaaatag ttacttaatt cactaatttt attttggtat 14280

tctcttttca tctcagttag tgctggaatc acactattac tggttaaatc tatgatatca 14340

tcatcggcgt taaacatagg tgacatttta acaaatggtg caccgttgta tatttccgat 14400

acaagttgat taactgactc atcattaatg tctgatttaa atattttgct aaattttgct 14460

tgcataatta atgaaaatcg agataaaaca acctcagata attcatcagt ataatgttct 14520

ataatttcta tatcactatt atattgaatt ggtttatttt gcatgacaac aaagttgcca 14580

ctcatacagt tatcatatat tttatgaatc tgtaagcatt catctggtat taaatagtct 14640

gggacaatga aataaatatc atcttttgtt aatcgttttt gaaattggaa gttaaagttt 14700

gatgaaaaat ttggtgcttg attaaagtaa gtattattta cgtaaccaag aatcataatt 14760

tgttcatttc ttgctttgcc aaccactaca ttgatgtttt gtcttaatgc tgattctaat 14820

tgaataaaat ctataccaac cgtatcacga ttggtatagt tgatgagtag tggtaaaaat 14880

tccaaataac gattaaacat aagacgttta aatctgttgc gatgttcaac aactcttttg 14940

ttgatttctt ttgataattc aacgtttaaa cctcttttat cgttgttcat atttacgctc 15000

cttttattct gttgcttctt cctctggttt tggtgttaca tcttggtcag taattaatat 15060

tttattaaag aatggactaa tagccttgaa tgaataataa tgaatccagt gtgtgacctc 15120

atcaaattca ccattataga atggttgttt taacatacct ttggtataac gtttgtattt 15180

aattgcatta atatctaaaa taaatgcata taaatctgat tttggtttaa tttcttcaat 15240

gttaccagta aactctttaa gtttagaaac atcataagta aatactgcac caactggaat 15300

tgtgtcaccc atttgcgact gataatcacc gtaagcacgt aaaaagttaa tagattcttg 15360

gttttgtaac ttaaattctt ttgttacttt aaacacacca cctaaatcgt caaaactgat 15420

aacgtggtct gtgaagtcaa taccagcaac ttggaacgta ttcgcaattt ttgtatctaa 15480

aagataagat tttaaagagt cagtcgttaa aataacaata tcttttaatt ttgatacagt 15540

tgtatattgt ccaattgcac caccagaagc acgatgaact tcattgtatt tagcgctgtt 15600

gttttgtaag tttaaaattg cttcaaatac tttgcttgct aaatcttctt tagatgttgt 15660

tttacgtaca tttgtatcag ataattgatt caatgaataa tcaactaaca ttgcacgcat 15720

ttctttttct tctaatacat taatatcaga aattttcttt ttatatacac ctaaagcata 15780

attagttgcg tctgctaatg tttggaaatt gaaacgtgta tcattattgt ttaatgtgaa 15840

tttttgtttc ttaacaatac cactaccata taacttagta gccatacgtg gatagttacg 15900

ttttaacatt aactcttcgt ttttagataa atccatgttg attggaacag tatccataat 15960

cacatattct tcactatatt gaccaataaa gtcttgttct ttagctaacc aattaaaacg 16020

attacctaaa gcgatatcga ttaataaagt ctcattaatc ttagggaata aatatttatt 16080

tacaaatgtt tcaaacattg tatttgaatt atcccatttg tctccaaatg tccaagattt 16140

tgaataatca tgattaaaat cttgcaatgc tgattttgcg gattttgcaa ctaaaagggc 16200

agtttcattt ttagtactcg cttgtgccat aatttattat tcctcctcta cgtctccgct 16260

aaaagactgt tttgaaagtg agtgaatttg tacaccgtac tcatcctcac ttttatttac 16320

atcgattgac atattttcat ttaattctgt tcgtttgttt aatcttgaat cttcatatga 16380

tgttcccatc atagaacgca tgttgttacc ttcatacata atattttcct cctaatctaa 16440

atctaacttg tcaactaatt cttcatctga atagtctttg tcttctttgt ctgcgtttgt 16500

tacatctggt tgtgtttgtt gtggttgttg aatttgtgat gaaacaaaag tagtcatttg 16560

ttgctctaat gatgtaatac gttgttctaa tacaacaggg tcgtattttg aactatcttc 16620

atctgttata gtaggttcta atttattctc attttctact tcaattgttt ctactgtttt 16680

atcttcagta ggttcttcag ttggttcttc agttagttct tcagttggtt cttcagttgg 16740

ttcttcagtt ttttcattgt cgtctggttt tacgatttca tcaaattctg tcattgtgac 16800

acctccaaaa tattttataa ctaattatat catagaatat ttaaataagt aaattaaatt 16860

tatttaaaag cgtgaacata gttttgaata aaagtcaata gatacataaa ttttgtattt 16920

gatgaatatt taataggttg gataagttgg aaaagttgtt gcacagtatt tataagttta 16980

gtttagaaat gataagttaa tttataagtt tttgatttta taatcgttta ttttaatcgg 17040

tggggaggtg taaatttaca taaaaaaatc acatatacta ttt 17083

Claims (10)

1. A strong lytic staphylococcus aureus bacteriophage RDP-SR-20001 is characterized in that the bacteriophage plaque has the diameter of 1-2mm, is round and transparent and has no halo when being observed through an electron microscope, the head of the bacteriophage plaque is of a regular polyhedron structure, the diameter of the head is about 41nm, the tail of the bacteriophage is not, and the bacteriophage is of Capricoraceae; the total length of the genome is 17085bp, and the genome has no virulence gene and lysogen gene and has a preservation number of CGMCC No. 21409.

2. The staphylococcus aureus bacteriophage RDP-SR-20001 according to claim 1, wherein the bacteriophage is a virulent bacteriophage, which has strong lytic activity against pseudomonas aeruginosa, and the lysis rate is up to 95%, thereby providing a bacteriophage source for industrialized, large-scale, programmed production of bacteriophage and treatment of avian breeding animal bacterial diseases caused by staphylococcus aureus.

3. Staphylococcus aureus bacteriophage RDP-SR-20001 as in claim 1, wherein the bacteriophage RDP-SR-20001 has a potency of 10 after 1h treatment at 50 ℃⁹pfu/ml or more.

4. Staphylococcus aureus bacteriophage RDP-SR-20001 as claimed in claim 1, wherein the bacteriophage RDP-SR-20001 is cultured at 37 ℃ for 60min at pH ranging from 5.0 to 9.0 and the titer is still maintained at 10¹⁰pfu/mL or more, completely losing the ability to infect a host at pH 2.0; at pH 12.0, the titer of the phage remained at 10³pfu/mL, phage RDP-SR-20001 has a pH value of alkalophilicity.

5. Use of the staphylococcus aureus bacteriophage RDP-SR-20001 according to claim 1 in the manufacture of a medicament for the prevention and or treatment of a disease caused by staphylococcus aureus.

6. Use of the staphylococcus aureus bacteriophage RDP-SR-20001 according to claim 1 for the preparation of a food additive for the prevention and or treatment of diseases caused by staphylococcus aureus.

7. Use of the staphylococcus aureus bacteriophage RDP-SR-20001 according to claim 1 for preparing a feed for preventing and or treating diseases caused by staphylococcus aureus.

8. The effective component of the bactericidal composition for preventing and treating staphylococcus aureus comprises staphylococcus aureus bacteriophage RDP-SR-20001.

9. The active ingredient of the feed for preventing and/or treating diseases caused by pseudomonas aeruginosa comprises the staphylococcus aureus bacteriophage RDP-SR-20001.

10. A detergent or disinfectant, the active ingredient of which comprises Staphylococcus aureus bacteriophage RDP-SR-20001 as set forth in claim 1.

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