CN113881641B - Escherichia coli phage EP01 and application thereof - Google Patents
Escherichia coli phage EP01 and application thereof Download PDFInfo
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- CN113881641B CN113881641B CN202111216498.1A CN202111216498A CN113881641B CN 113881641 B CN113881641 B CN 113881641B CN 202111216498 A CN202111216498 A CN 202111216498A CN 113881641 B CN113881641 B CN 113881641B
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Abstract
The invention discloses an escherichia coli bacteriophage EP01, and the escherichia coli (Escherichia coli phage) EP01 has a preservation number of CCTCC M20211159. The escherichia coli bacteriophage EP01 has short incubation period, can quickly kill host bacteria in a culture medium, has small toxic and side effects, high safety and wide temperature and acid-base tolerance range, and has good killing, prevention and control effects on environments, foods and the like polluted by salmonella and pathogenic escherichia coli.
Description
Technical Field
The invention relates to the technical field of coliphage, in particular to an coliphage EP01 and application thereof.
Background
Salmonella belongs to the enterobacteriaceae, and gram-negative enterobacteria is a common food-borne pathogenic bacterium. Nearly one thousand strains have been found to be classified into basic groups of bacteria, A, B, C, D, E, etc., based on their antigenic components. Among them, there are mainly paratyphoid A bacteria of group A, paratyphoid B bacteria and typhimurium bacteria of group B, paratyphoid C bacteria of group C and cholera bacillus of pig, paratyphoid B bacteria of group D and enteritis bacillus, etc. related to human diseases. Most of the diseases of human beings caused by typhoid bacillus, paratyphoid bacillus and paratyphoid bacillus can only cause diseases of animals such as domestic animals, mice, birds and the like, but sometimes, the diseases can pollute food of human beings to cause food poisoning.
Escherichia coli is a medium-sized bacillus, is in a short rod shape, has the size of 1-3 um multiplied by 0.5-0.7 um, has flagella and no spores, can form capsules by some strains, is gram-negative, is aerobic or facultative anaerobic, has active biochemical reaction, is easy to proliferate on common culture, has strong adaptability, is common bacteria in intestinal tracts of people and animals, and can be divided into two categories of pathogenicity and non-pathogenicity. The antigen components of the escherichia coli are complex and can be divided into a thallus antigen (O), a flagella antigen (H) and a surface antigen (K), and the escherichia coli can be divided into more than two hundred types according to the thallus antigens, wherein some escherichia coli with special serotypes have pathogenicity to human beings and animals, especially to infants and young animals, and often cause severe diarrhea and septicemia. Coli is more resistant to heat than other enterobacteria and still has some bacteria surviving after heating at 60 ℃ for a short period of time. Coli can survive in water in nature for weeks to months and longer in lower temperature feces. Colibacillosis can occur throughout the year, and is frequent in rainy, stuffy and humid seasons.
Phage (phage) is a generic term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, and spirochetes, and is called a phage because some of them cause lysis of host bacteria. Phage is a virus that is characterized by a specific bacterium as the host, and as with other viruses, phage is simply a mass of genetic material surrounded by a protein coat, most phage also have a "tail" that is used to inject genetic material into the host.
Chinese application CN112680423a discloses a broad-spectrum strong-lytic escherichia coli bacteriophage which can lyse four bacteria simultaneously, named EC35P1, and the bacteriophage can lyse shigella, salmonella and enterobacter cloacae in addition to having a better lysis effect on escherichia coli, and can realize large-scale industrial production. Korean patent KR102003786B1 discloses a new escherichia coli-specific phage EcoH7 and a method for preparing the same. The phage EcoH7 has very high specificity to the escherichia coli, and can solve the problems of drug resistance of the escherichia coli to antibiotics and residue of the escherichia coli in foods. Chinese application CN108103031A discloses a broad-spectrum bacteriophage such as Escherichia coli phage NTHP01 for aquaculture, which is prepared by fermenting genetically engineered Escherichia coli DH5 alpha as host bacteria, and the phage has better lysis effect on aeromonas bacteria, can lyse other bacteria (common bacteria such as Edwardsiella tarda, vibrio parahaemolyticus, vibrio alginolyticus, vibrio cholerae, kwangsi bacteria, citrobacter freundii, enterobacteria and the like) and also has lysis effect (the broad spectrum is not disclosed).
The research on coliphage mainly focuses on the aspect of intestinal hemorrhagic phage lytic property, and the research reports on coliphage bacterial and application aspects of bacterial lysis of other genera are not so much. Therefore, it is very necessary to find a novel coliphage isolate which can kill various bacteria at the same time, and has high stability, good safety and high potency.
Disclosure of Invention
Aiming at the technical problems, the invention provides an ultra-broad-spectrum multivalent escherichia coli bacteriophage capable of cracking 31 escherichia coli and 1 salmonella enteritidis, and aims to provide a novel prevention and treatment means for environmental and food pollution and the like caused by escherichia coli and salmonella.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
the escherichia coli phage EP01, the escherichia coli (Escherichia coli phage) EP01 has a preservation number of CCTCC M20211159, and the preservation date is as follows: 2021, 9, 13, deposit address: the university of Wuhan China center for type culture collection of Wuhan in Wuhan district of Hubei province.
Use of coliphage (Escherichia coli phage) EP01 as described above for the manufacture of a medicament for the prevention and treatment of diseases of escherichia coli infection.
A phage composition comprising escherichia coli phage (Escherichia coli phage) EP01 as described above.
A phage pharmaceutical formulation whose active ingredient comprises e.coli phage (Escherichia coli phage) EP01 as described above or a phage composition as described above.
The phage pharmaceutical preparation also comprises a pharmaceutically acceptable carrier, and the dosage form of the phage pharmaceutical preparation is solution, powder, gel, granule or freeze-drying agent.
A water disinfectant comprising, as an active ingredient, escherichia coli bacteriophage (Escherichia coli phage) EP01 as described above or said bacteriophage composition; other active ingredients for the inhibition or elimination of viruses, bacteria in the environment are also included.
Wherein, the disinfectant can disinfect the breeding environment, the breeding apparatus and the feed with colibacillus by spraying and soaking.
The escherichia coli phage (Escherichia coli phage) EP01 has a good disinfection effect on host escherichia coli GXEC-N01 separated from pig manure in a pig farm, has a strong cracking effect on salmonella enteritidis separated from piglets (1 strain) and escherichia coli separated from piglets (16 strains), dogs and cats (1 strain), birds (5 strains), humans (1 strain) and environment (8 strains), has a wide antibacterial spectrum, and can be widely applied to the fields of food, environment and feed disinfection of salmonella and escherichia coli.
Compared with the prior art, the invention has the following beneficial effects:
(1) The escherichia coli phage EP01 is a newly discovered phage, has a better cracking effect on host escherichia coli GXEC-N01 separated from piglet excrement swabs, has a very strong cracking effect on salmonella enteritidis separated from piglets (1 strain) and escherichia coli separated from piglets (16 strains), dogs and cats (1 strain), birds (5 strains), humans (1 strain) and environment (8 strains), has a wider antibacterial spectrum, and can be applied to preparing products for preventing and treating pathogenic escherichia coli, salmonella and other pathogenic bacteria;
(2) The escherichia coli bacteriophage EP01 has short incubation period, can quickly kill host bacteria in a culture medium, has small toxic and side effects, high safety and wide temperature and acid-base tolerance range, and has good killing, prevention and control effects on environments, foods and the like polluted by salmonella and pathogenic escherichia coli.
Description of preservation information
The coliphage EP01 is preserved in the China center for type culture collection of university of Wuhan, gmelin, hubei province at 9 and 13 days of 2021, and the preservation number is CCTCC M20211159.
Drawings
FIG. 1 is a photograph of an E.coli bacteriophage EP01 plaque according to the present invention.
FIG. 2 is a transmission electron microscope image of the E.coli phage EP01 of the present invention.
FIG. 3 is a graph showing the optimal multiplicity of infection of E.coli phage EP01 of the present invention.
FIG. 4 is a graph showing the one-step growth of E.coli phage EP01 of the present invention.
FIG. 5 is a schematic diagram showing the effect of temperature on the activity of the E.coli phage EP01 of the invention.
FIG. 6 is a schematic representation of the effect of pH on the activity of the E.coli phage EP01 of the invention.
FIG. 7 is a schematic diagram of the sterilization of E.coli phage EP01 of the present invention in liquid medium.
FIG. 8 is a schematic diagram of the sterilization of the E.coli phage EP01 of the invention in fresh milk.
Detailed Description
The following detailed description, in conjunction with the accompanying drawings, describes in detail, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were commercially available unless otherwise specified.
The host bacterium used in the experiment is an escherichia coli clinical isolate GXEC-N01 which is separated from the excrement of a piglet in a pig farm in Guangxi nan Ning, and is transacted with the escherichia coli phage EP01 of the invention to be preserved in China center for type culture collection, eight-way university of Wuhan in Wuhan district of Hubei province, and the preservation number is CCTCC M20211159.
LB (Luria broth) liquid Medium (1L): peptone 10g, yeast powder 5g, naCl10g, add ddH 2 O to 1L, adjusting the pH to 7.0, and autoclaving at 121 ℃ for 20 min.
0.6% LB semisolid medium (1L): peptone 10g, yeast powder 5g, naCl10g, agar powder 6g, add ddH 2 O to 1L, adjusting the pH to 7.0, and autoclaving at 121 ℃ for 20 min.
1.2% LB solid Medium (1L): peptone 10g, yeast powder 5g, naCl10g, agar 12g, ddH is added 2 O to 1L, regulating the pH to 7.0, sterilizing at 121 ℃ for 20min under high pressure, cooling to 50 ℃, pouring the flat plate, cooling and solidifying, and inverting for later use.
Eosin metablue solid medium (1L): 5.445g of eosin and bluish agar powder and adding ddH 2 O to 1L, adjusting the pH to 7.0, and autoclaving at 121 ℃ for 20 min.
SM buffer (1L): 6.055g of Tris-HCI (pH 7.5) was weighed to a volume of 100ml and 5.800g NaCl,2.000g MgSO was added 4 After that, ddH is added 2 O is fixed to volume to 1L.
1mol/L sterile CaCl 2 Solution (1L): weigh Ca Cl with balance 2 111g of solid is poured into a beaker, added with water for dissolution, the solution is poured into a 1L volumetric flask and is rinsed for 3 times by distilled water, the rinse solution is poured into the volumetric flask together, the volume is fixed, and the beaker is sterilized under high pressure for standby.
DNase I, RNase A, PEG8000, phosphotungstic acid (PTA, 2% w/v) are commercially available.
Example 1
Isolation of E.coli phage EP01
The sample is collected from sewage in a pig farm septic tank in Guangxi province, the sample is centrifuged for 10min at 4 ℃ and 12000rpm, the supernatant is centrifuged for 3 times again, and the final supernatant is filtered by a filter membrane of 0.45 μm and a filter membrane of 0.22 μm respectively; taking 5mL of filtrate, adding 0.1mL of host bacteria preserved in China center for type culture collection, adding 5mL of 2 XLB liquid culture medium, placing the mixture at 37 ℃ for culturing for 14-16 hours, culturing for 10min after the next day at 4 ℃ and 12000rpm centrifugal center, and filtering and sterilizing the supernatant by using a 0.22 mu m filter membrane to obtain stock solution containing phage, namely phage suspension.
And (3) streaking host bacteria preserved in the China center for type culture collection, inoculating the streaked host bacteria to an eosin blue solid culture medium, culturing overnight, picking up a monoclonal and inoculating the monoclonal bacteria to 5mL LB (Luria broth) liquid culture medium, and performing shaking culture at 37 ℃ for 8 hours to serve as a host bacteria culture for later use.
Dividing 1.2% LB solid culture medium into 2 areas, sucking the above standby host bacteria culture 0.1mL and 3mL of 0.6% LB semisolid, uniformly mixing, spreading on 1.2% LB solid culture medium, air drying, taking 10 μl of phage suspension, naturally air drying, placing in a 37 ℃ incubator for culture, observing whether plaque is formed in the dropwise adding phage area, and if plaque is formed, proving that phage exists.
Another 0.1ml of the phage suspension was serially diluted 10-fold and taken out 10-fold -2 、10 -4 、10 -6 0.1ml of diluent is added with 0.1ml of host bacteria preserved in China center for type culture collection, and the mixture is kept stand for 15min, 3.5ml of 0.6% semisolid LB culture medium with the temperature of about 45 ℃ is added, and the mixture is evenly spread on 1.2% LB solid culture medium prepared in advance, and plaque growth is observed after culturing for 8h at 37 ℃; single transparent halo-free plaque with uniform size and neat edges is picked up and placed in an EP tube containing 0.1mL of host bacteria culture and LB liquid medium, and co-cultured overnight at 37 ℃; the following day, the co-culture was centrifuged, and the filtrate was diluted 10-fold with SM buffer and double with 0.1ml host bacteriaThe layer is repeated for about 10 times, and phage with uniform plaque size can be obtained and stored at 4 ℃ for standby.
As a result of examining the above-mentioned standby phage by a double-layer plate method, as shown in FIG. 1, the phage can form a needlepoint transparent plaque in an agar medium, and the phage has no halo around, and has clear and regular edges, which is a typical lytic phage.
Example 2
Amplification and purification of E.coli phage EP01
Taking 0.1ml of phage for standby in example 1 and 0.1ml of host bacteria culture for standby in example 1, acting for 15min in a test tube, adding 10ml of LB liquid medium, culturing for 6h at 37 ℃, centrifuging for 20min at 12000rpm, taking the supernatant, filtering with a 0.22 mu m filter membrane, and obtaining the filtrate as phage lysate.
PEG purification: DNase I and RNase A are added into phage lysate to a final concentration of 1 mug/ml, incubated for 30min at 37 ℃, naCl ice bath with a final concentration of 1M is added (i.e. sodium chloride is added to make the final concentration of sodium chloride in the mixed solution 1M), centrifugation is carried out at 4 ℃ and 12000rpm for 10min, PEG8000 with a final concentration of 10% is taken out from the supernatant, overnight at 4 ℃ and 12000rpm for 10min, the supernatant is discarded, inverted for 5min, redundant water is removed as much as possible, SM buffer solution is added into the rest solid matters for heavy suspension, chloroform with an equal volume is added and shaking is carried out for 30s, and centrifugation is carried out at 5000rpm for 15min to separate an organic phase and a hydrophilic phase, and a hydrophilic phase containing phage particles is recovered, thus obtaining purified phage suspension.
Phage titers were detected by double-layer plate method: diluting the purified phage suspension by 10 times gradient, mixing 0.1ml phage diluent and 0.1ml host bacteria liquid, spreading double-layer agar plates, culturing at 37deg.C for about 6-10 hr, counting plaques on each agar plate, selecting plates with plaques about 30-300, calculating phage initial concentration according to dilution multiple to obtain phage titer, wherein phage titer (PFU/ml) =dilution multiple×number of plaques×10, and phage titer is 1.3X10 9 PFU/ml。
Example 3
Transmission electron microscope observation of coliphage EP01
Performing electron microscope observation on the phage suspension purified in the example 2, dripping the phage suspension purified in the example 2 on a copper sheet, naturally precipitating for 5-10 min, sucking redundant liquid by using filter paper, dripping 2% phosphotungstic acid (PTA, 2% w/v) for dyeing, drying at room temperature, and observing by using a transmission electron microscope; as a result of observation, as shown in FIG. 2 (100 kV), the phage has a head with a regular icosahedron, a head diameter of about 45nm and a tail of 150nm, and belongs to the myotail virus family (Myoviridae) and is designated as EP01 according to the eighth report of the International Commission for viral Classification-International Classification of viruses (ICTV) published 2015.
Example 4
Analysis of E.coli phage EP01 host profile
The phage titer for use in example 1 was adjusted to 10 9 PFU/ml was used for analysis of the host profile of phages with 66 strains of bacteria isolated from different animal sources, as follows: taking 0.1ml of 66 bacterial overnight culture, adding 3ml of 0.6% LB semisolid culture medium at about 45 ℃, uniformly spreading on 1.2% LB solid culture medium which is prepared in advance, and then equally dividing each plate into two areas, wherein 10 mu L of each area is taken and the titer is adjusted to 10 9 PFU/ml of the standby phage is dripped on the surface, physiological saline is dripped in the other area to serve as a control, the liquid drops are inverted at 37 ℃ for culturing for 12 hours after being dried, and the observed result is marked as "+" if plaque is generated, otherwise, the observed result is marked as "-"; the results are shown in Table 1: the coliphage EP01 can lyse host bacteria GXEC-N01 and also can lyse other 1 strain of salmonella of swine origin and 31 strains of colibacillus separated from different animal sources.
TABLE 1 host profile information for E.coli phage EP01
Note that: mainly aiming at OK1, OK2, H6 and O157 serum (italics and bold are host bacteria).
Example 5
Determination of optimal multiplicity of infection of E.coli phage EP01 (multiplicity of infection is the ratio of the number of phages at the initial stage of infection to the number of host bacteria)
The host bacterium culture prepared in example 1 was used and the concentration was adjusted to 1X 10 9 CFU/mL was added to the phage used in example 1 and the host bacterial culture used in example 1 in a ratio of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, respectively, and LB (Luria broth) liquid medium was added to make the total volume of the culture system the same. Stationary culturing at 37℃for 5 hours, centrifuging at 12000rpm for 10 minutes, collecting supernatant, diluting to an appropriate concentration, and measuring titer by a double-layer plate method, and the optimal multiplicity of infection of E.coli phage EP01 is 0.001 as shown in FIG. 3.
Example 6
Determination of one-step growth curve of coliphage EP01
The host bacterial cultures prepared in example 1 were mixed with excess phage prepared in example 1 (MOI >10, ensuring adsorption of all bacteria to phage), centrifuged at 12000rpm for 1min after incubation at 37℃for 15min, the supernatant (unadsorbed phage) was discarded, the pellet was washed 1 time with LB liquid medium, resuspended in 10ml of pre-warmed LB liquid medium, rapidly placed in a shaking table at 37℃for shaking culture, 120. Mu.l of culture was taken every 10min, after centrifugation at 4℃for 2min, the supernatant was diluted to the appropriate concentration (appropriate concentration, i.e.concentration of 30-300 plaques formed on plates), the phage titer was determined by bilayer assay, 130min, total sampling was performed 14 times, the log of phage titer was plotted on the ordinate with the sampling time as abscissa, and the latency, burst period, burst size of phage were plotted on the one-step growth curve. The results of the one-step growth curve are shown in FIG. 4, and the incubation period of the infected host bacteria is short (< 10 min), the burst period is 80min, and the burst amount is about 130.
Example 7
Temperature and pH tolerance test of coliphage EP01
Taking 10 sterile EP tubes, adding 0.5ml of phage prepared in example 1, respectively, allowing to act at 30deg.C, 40deg.C, 50deg.C, 60deg.C, 70deg.C, 80deg.C for 30min and 60min, immediately cooling in water bath, and measuring phage titer; the detection results are shown in fig. 5: the phage can withstand high temperature of 60 ℃, the titer is basically stable within 60min, the phage titer is obviously reduced or even inactivated with time when the temperature is higher than 60 ℃, and the phage is completely inactivated when the temperature reaches 80 ℃.
Taking 11 parts of 0.1ml of phage prepared in example 1, respectively placing in SM buffer solutions (0.9 ml) with pH of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, allowing the phage to act at 37 ℃ for 1-2h, and then measuring the titer of the phage after reaction by a bilayer method; the detection results are shown in fig. 6: the titer of the coliphage EP01 is less changed in the environment with the pH value of 5-10, and the activity is basically unchanged; when the environmental pH is >10 or pH <5, the titer of phage decreases sharply with the enhancement of acid and alkali; when the pH is >12 or <3, the phage titer is 0, and the phage is in an inactivated state, namely, the optimum pH of the phage is 5-10.
Example 8
Sterilization effect of coliphage EP01 in liquid culture medium
The host strain culture is prepared by diluting Salmonella GXSM-N02 and Escherichia coli GXEC-N01 (O114: K90 (B90)), GXEC-N07 (O157: H7), GXEC-N11 (O142: K86 (B)) of different serotypes to 1×10 9 CFU/ml, taking 30 sterile test tubes, adding 3ml of LB liquid medium into a control group, respectively adding 1.5ml of the host bacteria culture and 1.5ml of the phage for standby in example 1 with different concentrations into an experimental group according to moi=0.001 (optimal multiplicity of infection) and moi=1, placing the test group into a shaking table (180 rpm) at 37 ℃ for continuous shaking, and repeating each group for 3 times; after 5h, the OD of the co-culture solution of the host bacteria and the phage was measured by a spectrophotometer 600nm Phagocytosis ofAs shown in FIG. 7, the results of the bacterial sterilization test are shown in FIG. 7, wherein only the host bacteria in the positive control group, namely the culture solution, have no phage, and the OD is within 5h 600nm Significantly rise and remain at a higher level; the salmonella viable count of the phage treatment group was slowly increased within 2 hours and then significantly decreased after 3-5 hours of treatment. When moi=0.001, phage EP01 was added to different e.coli culture solutions, all e.coli content increased slowly over 1h, and after 2-5 h, stable levels were reached. When moi=1, the number of live escherichia coli bacteria in all tested is slightly increased within 1h, and the phage EP01 gradually decreases to the initial level after 2-5 h of action. Furthermore, the antibacterial effect of phage EP01 showed MOI dependence, with MOI of 1 being better than that of MOI of 0.001. In conclusion, the coliphage EP01 has a good application prospect in preventing and treating salmonella and pollution of escherichia coli with different sources and serotypes.
Example 9
Sterilization effect experiment of coliphage EP01 in fresh milk
Fresh skimmed milk is obtained from market, and host strain culture salmonella GXSM-N02 and different serotypes of Escherichia coli GXEC-N07 (O157: H7), GXEC-N01 (O114: K90 (B90)), GXEC-N11 (O142: K86 (B)) are diluted to 1×10 respectively 8 CFU/ml, 100. Mu.L was inoculated into 5ml milk. Control group was added with 100. Mu.L SM buffer, experimental group was added with 100. Mu.L of the above host bacteria culture and 100. Mu.L of phage for use in example 1 at different concentrations according to MOI=0.001 (optimal multiplicity of infection) and MOI=1, and incubated at 4℃and 28℃for 24 hours, respectively. At incubation times 2, 4, 6,24h, 500 μl each was taken and serially diluted with PBS buffer and the bacterial count was determined by plate counting. Each group was replicated 3 times (since phage titers corresponding to salmonella GXSM-N02 could not be determined, the experimental group could only be set with two phage-treated groups at different temperatures, whereas the host bacteria established 4 phage-treated groups for the experimental group of e.coli, including 2 phage-treated groups at different MOI and 2 phage-treated groups at different temperatures). The result of the sterilization experiment of phage in milk is shown in FIG. 8, and the positive control group, namely, milk, is not added with only host bacteriaPhage were introduced and bacterial numbers increased significantly over 24h with prolonged incubation time and maintained at a higher level. When moi=0.001 and the temperature is 4 ℃, the phage-treated group e.coli O157:h7 viable count is significantly reduced at 2,24 hours. When moi=1, e.coli O157: H7 viable count was significantly reduced at 2,6,24H (fig. 8A). The phage treatment group with moi=1 showed stronger antibacterial effect than phage treatment group with moi=0.001 at 28 ℃ (fig. 8B). When moi=0.001, the number of viable bacteria of e.coll O114: K90 (B90) was significantly reduced in the EP01 phage-treated group at 6,24 hours compared to the positive control group at a temperature of 4 ℃. When moi=1, the viable count was significantly reduced in the EP01 phage-treated group at 4, 6,24h (fig. 8C). At 28℃the E.coli O114:K90 (B90) viable count was significantly reduced in phage treatment groups after 4, 6,24h incubation at MOI of 0.001 and 1 (FIG. 8D). The addition of phage EP01 at MOI of 0.001 and 1 significantly reduced the viable count of E.coli O142:K86 (B) at 4, 6,24h at 28℃culture (FIG. 8F). When moi=1, the phage-treated group exhibited the best bactericidal effect at 4h. When the antibacterial effect of moi=0.001 was higher than moi=1, the phage-treated groups of two different MOIs began to show a significant difference after 4h incubation (fig. 8F). At each time point of 4 ℃ and 28 ℃, the number of live salmonella bacteria in the phage EP01 treated group was significantly reduced compared to the positive control group (fig. 8G). As can be seen from the above, the coliphage EP01 of the invention has great potential in controlling the pollution of common food-borne pathogenic bacteria such as salmonella, escherichia coli O157H 7, O114K 90 (B90), O142K 86 (B) and the like in fresh milk.
The escherichia coli bacteriophage EP01 has good tolerance at the pH value of 5-10 and the temperature of 30-60 ℃, the incubation period is short (20 min), and the burst period is long (80 min). In liquid culture medium and fresh milk, phage EP01 has remarkable reduction effect on the number of living bacteria of tested salmonella, escherichia coli O157: H7, escherichia coli O114: K90 (B90) and escherichia coli O142: K86 (B), and shows the potential of preventing and controlling the food-borne pathogens.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (7)
1. ColiphageEscherichia coli phage) EP01, characterized in that the coliphageEscherichia coli phage) EP01 has a preservation number of CCTCC NO. M20211159.
2. An escherichia coli bacteriophage according to claim 1Escherichia coli phage) Use of EP01 in the manufacture of a medicament for the prevention and treatment of diseases caused by escherichia coli infection.
3. A phage composition characterized in that: comprising the coliphage of claim 1Escherichia coli phage）EP01。
4. A phage pharmaceutical formulation characterized in that: the effective components of the bacteriophage comprise the coliphage as set forth in claim 1Escherichia coli phage) EP01 or phage composition according to claim 3.
5. The phage pharmaceutical formulation of claim 4, wherein: the phage pharmaceutical preparation also comprises a pharmaceutically acceptable carrier, and the dosage form of the phage pharmaceutical preparation is solution, powder, gel, granule or freeze-drying agent.
6. A water disinfectant, characterized in that: the effective components of the bacillus coli phage comprise the coliphage as claimed in claim 1Escherichia coli phage) EP01 or the phage composition of claim 3; wherein it also comprisesOther active ingredients for viral and bacterial inhibition or elimination in the environment.
7. The water disinfectant as set forth in claim 6, wherein: the disinfectant disinfects escherichia coli in the culture environment, the raising apparatus and the feed in a spraying and soaking mode.
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| CN110438091B (en) * | 2019-07-12 | 2020-12-22 | 广西大学 | Novel Klebsiella pneumoniae phage and application thereof |
| CN112280749B (en) * | 2020-10-16 | 2022-03-11 | 青岛农业大学 | Escherichia coli phage vB _ EcoM _ swi3 and application thereof |
| CN112680423B (en) * | 2020-12-29 | 2022-07-08 | 菲吉乐科(南京)生物科技有限公司 | Wide-spectrum escherichia coli bacteriophage capable of simultaneously cracking four bacteria and composition, kit and application thereof |
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| WO2002014468A2 (en) * | 2000-08-15 | 2002-02-21 | Phage Biotechnology Corporation | Phage-dependent superproduction of biologically active protein and peptides |
| WO2019051603A1 (en) * | 2017-09-15 | 2019-03-21 | Syntbiolab Inc. | Bacteriophage composition and method of preventing bacterial infections in livestock |
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