CN112301001B - Staphylococcus aureus phage LSA2311 and application thereof - Google Patents
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Abstract
The invention discloses a staphylococcus aureus phage LSA2311 and application thereof, wherein the phage is of a wide spectrum type and can crack staphylococcus aureus and drug-resistant strains thereof, and is identified as myxococcaceae of the order of tailed phage with the preservation number of CCTCC NO: m2020562; phage LSA2311 is stable in titer at pH 6-11 and at a temperature of 40-50 ℃. According to the whole gene sequencing result of the phage, the phage has avirulence, antibiotic resistance and transduction related genes, and the safety of applying the phage to pathogenic bacteria in food and phage treatment is verified from genetic background. The phage provided by the invention can effectively control staphylococcus aureus in milk samples, and has the characteristics of high specificity, no residue and safety compared with antibiotics and chemical preservatives.
Description
Technical Field
The invention relates to the field of food safety, and in particular relates to staphylococcus aureus phage LSA2311 and application thereof.
Background
Staphylococcus aureus (s.aureus) is one of the most common food-borne pathogens, and is associated with health and safety issues in the food industry and medicine. As the staphylococcus aureus is widely distributed in nature, air, soil, water and tableware, and both human beings and animals have high bacteria carrying rate, the bacteria carrying rate of staphylococcus aureus in normal human groups can reach 30-80 percent. Staphylococcus aureus is one of important food-borne disease sources, and more food poisoning events caused by the staphylococcus aureus cause great influence on the field of food sanitation. Food safety events caused by staphylococcus aureus enterotoxin are reported in the united states about 185060 annually, with about 1750 hospitalized, already accounting for 33% of bacterial food poisoning, with losses of about $ 15 billion annually; in recent years, the food poisoning event caused by the disease annually in China is the 4 th bit of bacterial food poisoning. Therefore, the inhibition of the pollution of staphylococcus aureus is an urgent need in the field of food safety at home and abroad.
The monitoring and tracing of the pollution source of the food poisoning event show that: the pollution degree of various foods to staphylococcus aureus shows obvious difference, wherein milk, dairy products, fresh meat, quick-frozen products and the like are most easily polluted by the staphylococcus aureus. Especially, the milk is easy to be polluted by staphylococcus aureus due to the characteristic of balanced liquid nutritional ingredients.
For the prevention and treatment of staphylococcus aureus, antibiotic therapy is mainly adopted in the prior art, and the method has a certain effect on inhibiting staphylococcus aureus. However, as the antibiotics are used as chemical substances for treating bacterial infection, the situation of overdose and overrange use of antibiotics is common in China due to the fact that users lack necessary safety knowledge and manufacturers cannot use the antibiotics strictly according to standards in order to gain greater commercial benefits.
In recent years, the excessive dependence on antibiotic therapy has led to the emergence of resistant bacteria, most typically methicillin-resistant Staphylococcus aureus (MRSA) known as "superbacteria", which cause significant annual infections in humans. Therefore, many scientists are beginning to research to find a highly effective antibiotic substitute, and the bacteriophage as a therapeutic agent has the following significant advantages: the bacteriophage has strong specificity, only aims at specific pathogenic bacteria, and does not destroy the normal microecological balance; the bacteriophage has exponential multiplication capacity, and in general, the treatment by using the bacteriophage only needs to be carried out once through self multiplication and bacterium lysis to achieve the treatment effect; the side effect of the phage treatment is small, and even some patients with immunological defect are safe after taking the phage treatment, so that the application of the phage has wider prospect based on the advantages.
Candidate phages for use in therapy were mainly virulent phages belonging to the families Myoviridae (e.g., phage K or phiStau 2A) and Brevibacterium (phiSAP-2). These phages were isolated from various environments including dairy products, farms (mastitis-infected milk), and patients. The limitation of using phage therapy is the narrow host spectrum.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a staphylococcus aureus phage LSA2311 which is wide in spectrum and can crack staphylococcus aureus drug-resistant bacteria. The staphylococcus aureus phage LSA2311 is used as a phage with strong and effective lysis effect on staphylococcus aureus, and can effectively inhibit multiple strains of bacteria such as staphylococcus aureus ATCC25923, ATCC29213, 196, 6538 and the like.
Bacteriophage is an antibacterial agent that has been considered to have a good bactericidal effect and to be relatively safe in recent years. Bacteriophages (bacteriophages) are a generic term for viruses that infect microorganisms such as bacteria, fungi, actinomycetes, or spirochetes, and are called bacteriophages because they are partially responsible for the lysis of host bacteria. The bacteriophage is a virus for specifically cracking bacteria, has the advantages of fast self-proliferation and high safety, is widely distributed in the environment, is an important component of human microbiome, has the characteristics of high efficiency, high specificity, easy mass preparation and the like, is colorless and tasteless, and cannot influence the flavor of food.
At present, the bacteriophage products applied to food have been internationally, wherein the following are marked: in 2006, listShield was approved by the U.S. Food and Drug Administration (FDA) TM The phage product can be used as food additive for controlling Listeria monocytogenes pollution in instant food and poultry meat products. The phage therapy has the advantages of exponential proliferation, strong specificity, easy real-time update, small side effect, no residue, short period, low cost and the like, thereby having wide development prospect.
The invention separates a virulent phage with strong cracking ability, wide host range and high titer by taking staphylococcus aureus as a host bacterium, classifies and identifies the virulent phage by researching the physiological characteristics of the virulent phage, and provides a material basis for the application of the phage in food safety; in addition, the inhibiting effect of the bacteriophage on staphylococcus aureus in milk is researched.
The second purpose of the invention is to provide the application of the staphylococcus aureus phage LSA2311 in the bactericide for preventing staphylococcus aureus, in particular to the application in milk fresh-keeping. The sterilization mode used in the processing of drinking milk cannot completely meet the market demand. At present, the domestic common milk heat sterilization methods are pasteurization (62.8-65.6 ℃ for 30 min) and ultrahigh temperature instantaneous sterilization (135-147 ℃ for 4-0.5 s). In the former case, pasteurized milk, although it retains most of its flavor and nutrients, has a short shelf life and can be stored only at 4 ℃ for less than 10 days. In the latter case, although the shelf life is extended to 6 months, the milk suffers a certain loss of both its nutritional components and its flavor. The control of cow mastitis caused by staphylococcus aureus and other bacteria is also an important link in the dairy industry. Under the background, it is very important to develop a novel milk sterilization and preservation technology. The phage provided by the invention can effectively control staphylococcus aureus in milk samples, and has the characteristics of high specificity, no residue and safety compared with antibiotics and chemical preservatives.
In order to achieve the technical purpose, the invention adopts the following technical measures:
the invention provides a Staphylococcus aureus phage LSA2311, which has broad-spectrum property and can crack Staphylococcus aureus drug-resistant strains, and is identified as a cauda myophagae of the order of cauda phages, namely a Staphylococcus aureus phage LSA2311 and a Staphylococcus aureus bacteriophage LSA2311, wherein the preservation number is CCTCC NO: m2020562, the phage LSA2311 is preserved in China center for type culture Collection (CGMCC) at 29.9.2020, with the preservation address of Wuhan university, wuhan city, hubei province and the preservation number of: CCTCC M2020562, the preservation date is: 9/29 in 2020.
The staphylococcus aureus phage LSA2311 is a laboratory separated phage with stable titer at the pH of 6-11 and the temperature of 40-50 ℃, and the optimal complex number of infection is 0.01; it is a broad spectrum phage that can recognize staphylococcus aureus.
The invention also provides application of the staphylococcus aureus phage LSA2311 in preparation of a bactericide for preventing staphylococcus aureus, wherein the staphylococcus aureus is staphylococcus aureus standard strain (MSSA) ATCC25923.
The invention also provides an application of the staphylococcus aureus phage LSA2311 in milk preservation.
The method of the above application: adding a culture solution of staphylococcus aureus phage LSA2311 into milk, wherein the addition amount of the staphylococcus aureus phage LSA2311 is 1000-10000 PFU/mL.
An experiment of bacteriostatic effect of staphylococcus aureus bacteriophage LSA2311 in a milk sample comprises the following steps:
(1) Staphylococcus aureus ATCC25923 cultured in logarithmic phase was adjusted to a concentration of 1X 10 in PBS buffer 4 CFU/mL. Dividing the experiment into six groups, adding 100 μ L bacterial liquid into 9.8mL sterilized skimmed milk, placing three groups of milk samples in 25 deg.C incubator for 20min, and placing the other three groups of milk samples in 4 deg.C incubator for 20min to make the bacterial liquid fully adapt to the bacteriaThe environment.
(2) Of the three experimental groups at each temperature, two were experimental groups: 100 μ L of purified phage LSA2311 (titers 1X 10, respectively) was taken 8 PFU/mL、1×10 7 PFU/mL) is added into the milk added with the bacterial liquid, and the mixture is fully and evenly mixed; the other group was a control group: adding 100 μ L PBS buffer (pH 7.2-7.4) into the milk containing the bacteria liquid, and mixing well.
(3) Standing the two kinds of processed milk obtained in the step (2) in an incubator at 4 ℃ and 25 ℃, taking out the milk in 0, 1, 3, 6, 12 and 24 hours respectively, detecting the number of the staphylococcus aureus with enteronitis in the milk according to a GB-4789.10-2016 (national standard staphylococcus aureus detection method) plate counting method, and further calculating the bacteriostatic effect of the phage.
The invention has the beneficial effects that:
(1) The phage stock solution after solid enrichment has high titer, and in the invention, the titer of the phage LSA2311 is not less than 10 9 PFU/mL。
(2) Under different conditions, the bacteriostatic ability is strong. In the invention, the bacteriostatic action of phage LSA2311 on staphylococcus aureus under the conditions of MOI =10, MOI =1, MOI =0.1, MOI =0.01 and MOI =0.001 is compared, and the phage LSA2311 has a good bacteriostatic effect under different MOI conditions.
(3) The pH range is wider. The bacteriophage LSA2311 provided by the invention has pH stability (6-11) superior to that of staphylococcus aureus bacteriophage CCTCC No. M2017394 in patent 201810715693.0 and that of staphylococcus aureus bacteriophage CCTCC No. M2015142 in patent 201710048705.4 (5-9).
(4) The host spectrum is wide. The phage LSA2311 provided by the invention has a good cracking effect on 29 strains of staphylococcus aureus (including 3 strains of staphylococcus aureus drug-resistant strains).
(5) According to statistics, the application of the bacteriostatic agent to drug-resistant staphylococcus aureus is less at present, and the invention aims to provide a novel bacteriostatic agent capable of inhibiting the growth of staphylococcus aureus in a milk sample.
Drawings
FIG. 1 shows the plaque morphology of Staphylococcus aureus phage LSA2311 on double-layer agar plates;
FIG. 2 is an electron microscope observation image of Staphylococcus aureus phage LSA 2311;
FIG. 3 is the LSA2311 genome of Staphylococcus aureus phage and enzyme cutting electrophoresis picture;
in the figure, M:15000bp DNA marker;1: phage LSA2311 genome; 2: the phage LSA2311 genome is cut by Hind III enzyme; 3: the genome of the phage LSA2311 is cut by EcoRI enzyme;
FIG. 4 is a BLAST color scale bar of Staphylococcus aureus phage LSA 2311;
FIG. 5 is a graph showing the results of Staphylococcus aureus ATCC25923 lysis capacity of Staphylococcus aureus phage LSA2311 under different MOI values;
FIG. 6 is an adsorption curve of Staphylococcus aureus phage LSA 2311;
FIG. 7 is a one-step growth curve of Staphylococcus aureus phage LSA 2311;
FIG. 8 is a graph of the pH stability results of Staphylococcus aureus phage LSA 2311;
FIG. 9 is a graph of the results of the thermostability of Staphylococcus aureus phage LSA 2311;
FIG. 10 is a graph of the inhibitory effect of Staphylococcus aureus phage LSA2311 on Staphylococcus aureus in milk at low temperature 4 ℃ and room temperature 25 ℃;
in the figure, FIG. 10A is a bacteriostatic effect graph of phage LSA2311 at 4 ℃; FIG. 10B is a graph of the bacteriostatic effect of phage LSA2311 at 25 ℃.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
Example 1
The separation and screening method of staphylococcus aureus phage LSA2311 comprises the following steps:
(1) Sample collection
The sewage samples are respectively from a certain vegetable market in Wuhan city of Hubei province.
(2) Screening of Staphylococcus aureus phages
A10 mL sample of the contaminated water was taken and filtered through a 0.22 μm microporous filter. The culture medium is put into 50mL sterilized centrifuge tubes respectively, which are filled with 20mL sterilized LB broth culture medium (the components of the culture medium are 10.0g/L tryptone, 5.0g/L yeast extract and 10.0g/L, pH value is 7.3 +/-0.2; the use method of the culture medium is that 25.0g of the LB broth culture medium containing the components is weighed, heated, stirred and dissolved in 1000mL distilled water, and is respectively loaded into test tubes or other suitable containers, and is sterilized for 20min at 121 ℃ under high pressure, and 5mL of host bacterium liquid in logarithmic growth phase (cultured for 6 h-8 h) is additionally added. And carrying out shaking culture at 37 ℃ for 12-18 h to proliferate the phage, wherein the host bacterium is staphylococcus aureus ATCC25923. The culture solution was centrifuged at 8000r/min at 4 ℃ for 10min in a 50mL centrifuge tube, and the supernatant was filtered through a 0.22 μm filter. And (3) repeatedly enriching twice according to the method, namely adding a sterilized LB broth culture medium and adding a logarithmic phase host bacterium liquid for culturing for 6-8 h.
The sample application method is adopted for preliminary verification: the sample with obvious plaque is further subjected to gradient dilution by a double-layer plate method, and the plaque form is observed.
(3) Amplification culture and purification of bacteriophage
Relatively independent, large and smooth-edged plaques are picked from a double-layer plate for culturing phage stock solution, inoculated into 1mL of LB broth culture medium, and subjected to shaking culture at 37 ℃ and 200r/min for 12-18 h. Centrifuging at 8000r/min at 4 deg.C for 10min, filtering with 0.22 μm filter membrane for sterilization, and adding 200 μ L bacterial solution into the phage stock solution from high dilution to low dilution. And repeating the steps for 5 times to repeatedly purify the phage until a plaque with a uniform size is obtained, namely the purified phage, and numbering the bacterial strain as LSA2311. Measuring the titer of the separated phage by using a double-layer plate method; the phage LSA2311 was determined to have a titer of 3X 10 9 PFU/mL。
(4) Identification of strains
The head of the purified phage LSA2311 is of a regular dodecahedron structure, the diameter of the head is about 78nm, the phage contains a flexible tail, the length of the tail is about 85nm, and the diameter of the tail is about 10nm, and the phage is identified to be of the order of tailed phage, myoviridae.
(5) Preservation of phages
Short-term preservation can be realized by storing the filtered phage suspension in a refrigerator at 4 ℃; if the phage suspension is stored for a long time, sterilized glycerol (final concentration of 20%) is added into the phage suspension, and the phage suspension is stored in a refrigerator at-80 ℃.
The phage LSA2311 is preserved in China center for type culture collection (Wuhan university, wuhan city, hubei province) at 29 months 9 in 2020, with the preservation number of CCTCC NO: m2020562.
The plaque formed by phage LSA2311 is round and transparent, has a diameter of about 1mm (cultured for 12 h), has clear boundary without halo, has a head with a regular icosahedral structure, a head diameter of about 78nm, a phage with a contractile tail, a tail length of about 85nm and a tail diameter of about 10nm, and belongs to Myocapnoraceae.
Example 2: determination of phage LSA2311 host spectra
the above strains (29 strains of Staphylococcus aureus and 9 strains of other species) were each cultured to log phase. Taking 100 mu L of the logarithmic phase bacterial solution, when the temperature of upper agar (0.7 g of agar is added into 100mL of LB broth culture medium in the step (2) of the embodiment 1 and sterilized for 20min at 121 ℃) is reduced to 40 ℃, respectively taking 3mL of upper agar and the bacterial solution to be uniformly mixed, and then pouring 15mL of lower LB agar culture medium (the components of the culture medium are 10.0g/L of tryptone, 5.0g/L of yeast extract, 10.0g/L of sodium chloride and 10.0g/L, pH of agar have the value of 7.3 +/-0.2. The using method of the culture medium is that 35.0g of LB agar culture medium of the components are weighed, heated, stirred and dissolved in 1000mL of distilled water, subpackaged in a test tube or other suitable containers, and sterilized for 20min at 121 ℃) for later use; standing and air drying for about 10min, after the upper layer culture medium is solidified, respectively dripping 5 μ L of phage stock solution (the preparation method of phage stock solution is that staphylococcus aureus ATCC25923 is taken to be inoculated in 3mL of fresh LB broth culture medium, culture is carried out at 37 ℃ for about 6h, 100 μ L of the above-mentioned bacterial solution is taken to be added in 10mL of fresh LB broth culture medium, then 100 μ L of phage LSA2311 stored at 4 ℃ is respectively added, after mixing uniformly, culture is carried out in a shaking incubator at 37 ℃ for 12 h-18 h to proliferate the phage, 5mL of proliferation solution is taken to be put in a centrifuge tube, centrifuging is carried out at 8000r/min for 15min to remove bacterial debris, and the supernatant is filtered by a 0.22 μm filter membrane to obtain phage stock solution), and observing at night.
The results are shown in table 1, the phage can cleave a number of different staphylococcus aureus and exhibit a broad spectrum.
Due to the use of a large amount of broad-spectrum antibiotics, the number of staphylococcus aureus drug-resistant strains increases year by year, and food-borne diseases caused by multi-drug-resistant staphylococcus aureus seriously harm human health and life. The phage LSA2311 can effectively crack the drug-resistant strains, not only broadens the host spectrum of the phage, but also provides a new bactericide for killing the drug-resistant staphylococcus aureus.
TABLE 1 determination of phage LSA2311 host spectra
Table 1The host range of phage LSA2311
Note: "+ +" indicates clear and transparent plaques; "+" indicates plaque blur; "-" indicates no plaques; "+ +" Clear plane; "+" Opaque sequence; "-" No sequence.
Example 3: electron microscope observation of phage LSA2311
The phage suspension is ultracentrifuged for 1h at 40000r/min at 4 ℃ to precipitate phage particles by adopting a phosphotungstic acid negative staining method (Clokie and Kropinski 2009), the precipitate is resuspended by 0.1mol/L ammonium acetate, and 20 mu L of phage suspension and 20 mu L of phosphotungstic acid with the volume fraction of 2% and the pH =7 are respectively dripped on a sealing film. The copper mesh was gently taken, immersed in phage liquid for 10min and then excess liquid was removed by suction with filter paper. And then placing the copper mesh in phosphotungstic acid dye for dyeing for 10min, absorbing redundant liquid, naturally airing until the copper mesh is completely dried, observing the morphology of the phage on a transmission electron microscope by the prepared copper mesh, and measuring the size of the phage by using a software Digital Micrograph Demo 3.9.1.
As shown in FIG. 2, the purified phage LSA2311 had a regular icosahedral head structure with a head diameter of 78nm, a contractile tail-containing phage with a tail length of 85nm and a tail diameter of 10nm, and belongs to the Myoviridae family.
Example 4: extraction of phage genome and whole genome denovo sequencing
Adding 20 μ L of DNase I (1 mg/mL) and 20 μ L of RNase A (10 mg/mL) into 1mL phage LSA2311 stock solution, vortexing with a mini-vortexer for 2min, and incubating at 37 ℃ for 40min; 20 mu.L of 2mol/L ZnCl is added 2 Incubating at 37 deg.C for 7min, centrifuging, 10000r/min,1min; discarding the supernatant, adding 500. Mu.L TES buffer, blowing to clear and transparent state, no white particles, 65 deg.C, 15min (scattering), adding 10. Mu.L proteinase k (20 mg/mL), blowing with a gun head gently, turning upside down, incubating at 50 deg.C for 1h, and turning upside down every 10min, at which time the solution is clear. After incubation, cool and add 60. Mu.L of pre-cooled 3mol/L CH 3 COOK was brought up to 4 ℃ in advance, pH was adjusted to 5.2 with acetic acid), left on ice for 15min, centrifuged at 12000r/min,10min,4 ℃, the supernatant was taken, 600. Mu.L of phenol/chloroform/isoamyl alcohol (volume ratio: 25:24: 1) The mixture was gently inverted up and down repeatedly for 200 times, centrifuged at 12000r/min for 10min at room temperature, the supernatant was collected, 1 volume (about 600. Mu.L) of isopropanol was added to precipitate DNA at-20 ℃ and the mixture was inverted up and down to obtain floc, i.e., DNA, which was then left overnight. Then freezing and centrifuging, at 4 ℃,12000r/min for 10min, discarding the supernatant, adding 1mL of 70% (volume ratio) ethanol for washing once, blowing and sucking, centrifuging at 12000r/min for 10min, discarding the supernatant, centrifuging again for 1min, carefully sucking the residual ethanol by a white pipette tip according to the same direction, placing in an incubator at 37 ℃ for at least 40min, adding 20 mu L of TE for dissolving DNA at normal temperature, and standing for 30min. The sequencing work is completed by a sequencing company.
As a result of the whole gene sequencing of the phage LSA2311, the phage genome is double-stranded DNA, and the total length is 144592bp. The presumed virulence factors are screened by the virulence factor database, and the antibiotic resistance genes are screened by synthesizing the antibiotic resistance database, so that the phage LSA2311 is avirulent and resistant to antibiotics and transduces related genes.
Full database alignment by BLAST in NCBI failed to find completely similar sequences, as shown in FIG. 4, indicating that this phage is a novel phage.
Example 5: extraction and identification of staphylococcus aureus phage LSA2311 nucleic acid
(1) The phage suspension was ultracentrifuged at 40000r/min at 4 ℃ for 1h to precipitate phage particles.
(2) 20. Mu.L of DNase I (1 mg/mL) and 20. Mu.L of RNase A (10 mg/mL) were added and incubated at 37 ℃ for 40min with vortexing in a mini-vortexer.
(3) 20 μ L of 2mol/L ZnCl was added 2 Incubate at 37 ℃ for 7min. Centrifuging at 4 deg.C at 10000r/min for 1min; the supernatant was discarded.
(4) Add 500. Mu.L TES buffer, blow-suck 15min at 65 ℃ until clear and transparent, without white particles.
(5) Add 10. Mu.L proteinase k (20 mg/mL) and incubate at 50 ℃ for 1h, upside down every 10 min.
(6) After incubation, cool and add 60. Mu.L of pre-cooled 3mol/L CH 3 COOK (pH = 5.2), ice bath 15min.
(7) Centrifuging at 12000r/min,10min and 4 ℃. And taking the supernatant. Equal amounts of phenol chloroform isoamyl alcohol (25.
(8) Centrifuging at 12000r/min for 10min at normal temperature. The supernatant was collected, and 1-fold volume of isopropanol was used to precipitate DNA at-20 ℃ and left overnight.
(9) Centrifuging at 4 deg.C at 12000r/min for 10min, and discarding the supernatant. 1 wash with 1mL 70% ethanol. 12000r/min, centrifugation for 10min, discarding supernatant, and air-drying at 37 ℃ for 40min.
(10) The nucleic acid pellet was dissolved in TES buffer and stored at-20 ℃ until use.
(11) The nucleic acid was routinely digested with Hind III and EcoRI endonucleases and the digestion products were identified by 0.8% agarose gel electrophoresis.
As a result, as shown in FIG. 3, the phage LSA2311 nucleic acid was cleaved with HindIII endonuclease, and the genome size of the phage LSA2311 was estimated to be about 144592bp, based on the size of the cleaved fragment.
Example 6: optimal multiplicity of infection for phage LSA2311
Multiplicity of Infection (MOI) refers to the ratio of the number of phage to the number of host bacteria at the time of initial Infection. Mixing phage suspension and host bacterial liquid at 500 μ L each according to certain MOI value (0.001, 0.01, 0.1, 1, 10, 100, 1000), culturing at 37 deg.C for 3.5h, centrifuging at 8000r/min for 10min, and measuring phage titer of supernatants with different MOI values by double-layer plate method. The experiment was repeated 3 times, with 2 replicates each time.
The results of the optimal multiplicity of infection for phage LSA2311 are shown in Table 2. As can be seen from table 2, the phage titer reached the maximum at MOI =0.01 for phage LSA2311, i.e., the optimal multiplicity of infection of phage LSA2311 was 0.01, indicating that more phage could be propagated when the phage titer and the host bacterial count were infected at 0.01.
TABLE 2 optimal multiplicity of infection assay for phage LSA2311
Table2 MOI results for bacteriophage
Example 7: evaluation of phage LSA2311 ability to lyse bacteria
(1) A single colony of staphylococcus aureus ATCC25923 is picked and inoculated in 10mL LB broth medium, and cultured in a shaking table at the temperature of 37 ℃ at the speed of 150r/min for 8h. Then, the mixture was transferred to 10mL LB broth at a ratio of 1. The culture medium was counted by gradient dilution plating and was ready for use.
Number of bacteria (CFU/mL) = number of single colonies × dilution gradient × 10
( 2) Taking phage LSA2311 stock solution (the preparation method of the phage LSA2311 stock solution is as follows: staphylococcus aureus ATCC25923 is used as host bacteria, and phage LSA2311 is subjected to propagation culture for 12 to 18 hours by using LB broth culture medium; centrifuging the cultured mixed solution of the phage and the host bacteria at 8000r/min for 15min, removing the host bacteria from the supernatant with a 0.22 μm filter membrane to obtain pure phage solution, and measuring titer for use. )
Titer of phage (PFU/mL) = plaque number × dilution multiple × 10
(3) Taking staphylococcus aureus liquid in logarithmic phase, continuously diluting to 10 degrees of gradient in ten times 5 CFU/mL, taking the phage liquid, diluting to 10 in ten-fold gradient 8 PFU/mL、10 7 PFU/mL、10 6 PFU/mL、10 5 PFU/mL、10 4 PFU/mL、10 3 PFU/mL, MOI =1000, 100, 10, 1, 0.1, 0.01, 0.001 added to the corresponding experimental group, and 100. Mu.L 10 added 5 Uniformly mixing the CFU/mL staphylococcus aureus liquid; adding 100 μ L10 to positive control group 5 CFU/mL bacterial solution, 100 uL LB broth culture medium; negative control group 100. Mu.L 10 7 PFU/mL phage liquid, 100. Mu.L LB broth; setting parameters of a microplate reader, wherein lambda =600nm and T =37 ℃, preheating for 30min after starting up, and measuring OD (optical density) every 1h 600 A change in value. The cleavage curves were plotted using the software GraphPad Prism 6.
The phage shows certain lytic activity when infecting staphylococcus aureus at different MOIs. The light absorption value of the bacteria of the experimental group added with the phage is maintained at an initial value and does not have a growing trend, the growth of the bacteria can be completely inhibited, but the light absorption value is increased after 3h, which indicates that the host bacteria are not completely killed, the bacterial quantity is increased, and the experimental result shows that the phage LSA2311 has a strong cracking effect on the host bacteria, but cannot completely kill the host bacteria. The experimental group is also obviously lower than the positive control group, the lytic capacity of the phage added with different MOI values is slightly different, and the inhibition capacity to host bacteria is stronger when the MOI value is high. The result of the lysis curve provides experimental basis for the application experiment of the subsequent bacteriophage.
Example 8: adsorption experiments
Staphylococcus aureus LSA2311 was cultured to log phase. Phage liquid and staphylococcus aureus liquid were mixed at an MOI =0.01 ratio. Mixing in 5, 10, 15, 20, 25, 30min, respectively, adding 100 μ L each into 900 μ L SM buffer, and mixing. Immediately centrifuging at 13000g for 1min at 4 ℃, and adding 100. Mu.L of the supernatant to 900. Mu.L of SM buffer solution by suction and mixing for later use. The titer was determined on double-layer plates.
The experimental study of the adsorption of host bacteria by LSA2311 revealed that the abscissa represents the incubation time of the phage and the host bacteria together and the ordinate represents the percentage of phage adsorbed by the host bacteria, as shown in fig. 6. When LSA2311 and the host are incubated in a mixed state, most of the phage are adsorbed to the host after 10 min.
Example 9: determination of phage LSA2311 one-step growth curves
Mixing fresh phage liquid and host bacterial liquid at 500 μ L according to optimal MOI value (MOI = 0.01), incubating at 37 deg.C for 20min, centrifuging at 4 deg.C at 7000r/min for 2min, discarding supernatant as much as possible, washing with 1mL LB medium for 2 times, discarding supernatant, and resuspending with preheated 10mL LB medium. The resuspension was rapidly placed in a shaker at 37 ℃ for 160r/min shaking culture while timing, 300. Mu.L of sample was taken at 0min and every 10min, centrifuged at 4 ℃ at 7000r/min for 30s, and 100. Mu.L of supernatant was immediately extracted and diluted in 900. Mu.L of LB medium.
Sampling and selecting a proper dilution gradient at intervals of 10min according to the operation, and determining the titer of the phage.
The results are shown in FIG. 7, the latency of phage LSA2311 is about 10min; the cracking period is about 60min; the amount of cleavage was about 60PFU/cell.
Example 10: determination of the pH stability of the phage LSA2311
Adjusting pH of LB broth culture medium with HCl and NaOH, packaging 900 μ L of LB broth culture medium in sterile EP tube at pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, placing in 37 deg.C water bath, adding 100 μ L of phage solution (about 10 μ L) after temperature is stable 7 PFU/mL), water bath at 37 ℃ for 2h, and after the action time is over, properly diluting the sample according to the pre-experimental result and then determining the titer of the phage by adopting a double-layer plate method.
As shown in FIG. 8, the phage LSA2311 maintained high activity at pH 6-11 with little fluctuation in phage activity, and the phage titer decreased to a minimum value at pH 12 or more and remained unchanged.
Example 11: determination of the thermostability of phage LSA2311
Each 1mL of phage solution was diluted to 10 7 PFU/mL, and is divided into 21 mL sterile centrifuge tubes, each tube is 1mL, the EP tube is respectively placed in 30 ℃, 40 ℃,50 ℃, 60 ℃,70 ℃,80 ℃ constant temperature water bath kettle, respectively at 30min, 60min, from the EP tube to take 100 u L phage liquid, first cooling to room temperature, then using double agar plate method to determine the phage titer.
As shown in FIG. 9, the titer of the phage LSA2311 remained stable at 40-50 ℃ without significant change, and the titer began to decrease at 50-60 ℃, however, the presence of phage was not detected at 70 ℃ and 80 ℃ indicating that the phage was substantially inactivated. Therefore, the bacteriophage has stronger stability within 40-50 ℃.
Example 12: experiment of bacteriostatic effect of staphylococcus aureus by adding phage LSA2311 into milk at different MOI values (MOI =1000, MOI = 100) at low temperature of 4 ℃ and room temperature of 25 DEG C
The concentration of Staphylococcus aureus cultured to logarithmic phase was adjusted to 1X 10 with PBS buffer (pH7.2-7.4) 4 CFU/mL. The experiment was divided into six groups, 100. Mu.L of the bacterial solution was added to 9.8mL of sterilized skim milk, three groups of milk samples were placed in an incubator at 25 ℃ for 20min, and the other three groups of milk samples were placed in an incubator at 4 ℃ for 20min, allowing the bacterial solution to fully adapt to the environment.
Of the three experimental groups at each temperature, two were experimental groups: 100 μ L of purified phage LSA2311 solution (titers 1X 10, respectively) was taken 8 PFU/mL、1×10 7 PFU/mL) is added into the milk added with the bacterial liquid, and the mixture is fully and evenly mixed; the other group was a control group: 100 μ L of PBS buffer (pH 7.2-7.4) was added to the milk with the added bacterial liquid, and mixed well.
The two types of processed milk are respectively placed in incubators at 4 ℃ and 25 ℃, taken out at 0, 1, 3, 6, 12, 24 and 48 hours, and the number of staphylococcus aureus in the milk is detected according to a GB-4789.10-2016 (national standard staphylococcus aureus test method) plate counting method, so that the bacteriostatic effect of the phage is calculated.
As shown in FIG. 10, MOI =1000 at 25 ℃ (phage LSA2311 titer 1X 10 8 PFU/mL), the bacteria number is reduced by 4.3log CFU/mL compared with that of a control group after 6 hours in the milk added with the phage, and the sterilization efficiency reaches 50%;25 ℃ MOI =100 (phage LSA2311 titer 1X 10 7 PFU/mL), the bacteria number is reduced by 3.5log CFU/mL compared with the control group after 6 hours in the milk added with the phage, and the sterilization efficiency reaches 40.7 percent.
As shown in FIG. 10, 4 ℃ and MOI =1000 (phage LSA2311 titer 1X 10 8 PFU/mL), the bacterial count is reduced by 2.6log CFU/mL compared with the control group after 6 hours in the milk added with the phage, and the sterilization efficiency of the phage LSA2311 in the milk reaches 36.1 percent; 4 ℃ MOI =100 (phage LSA2311 titer 1X 10 7 PFU/mL), the bacteria number is reduced by 2.1log CFU/mL compared with the control group after 6 hours in the milk added with the phage, and the sterilization efficiency is 29.2 percent.
The calculation formula of the sterilization efficiency in the embodiment 12 is as follows: (amount of Staphylococcus aureus in control group-amount of Staphylococcus aureus in Experimental group) ÷ amount of Staphylococcus aureus in control group X100%.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (4)
1. Staphylococcus aureus bacteriophageStaphylococcus aureus bacteriophage) LSA2311, characterized by: the preservation number is CCTCC NO: m2020562.
2. Use of the staphylococcus aureus phage LSA2311 according to claim 1 for preparing a bactericide for staphylococcus aureus, wherein: the staphylococcus aureus is staphylococcus aureus standard strain ATCC25923.
3. Use of the staphylococcus aureus phage LSA2311 of claim 1 for milk refreshment.
4. Use according to claim 3, characterized in that: the application method comprises the following steps: adding a culture solution of staphylococcus aureus phage LSA2311 into milk, wherein the addition amount of the staphylococcus aureus phage LSA2311 is 1000-10000 PFU/mL.
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Inventor after: Wang Xiaohong Inventor after: Chen Muxiao Inventor after: Wang Jia Inventor after: Huang Chenxi Inventor after: Ding Yifeng Inventor after: Zhu Wenjuan Inventor after: Zhang Yu Inventor before: Wang Xiaohong Inventor before: Chen Muxiao Inventor before: Wang Jia Inventor before: Ding Yifeng Inventor before: Zhu Wenjuan Inventor before: Zhang Yu |