CN115851617B - Coliphage LHE83 and application thereof - Google Patents
Coliphage LHE83 and application thereof Download PDFInfo
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- CN115851617B CN115851617B CN202211265553.0A CN202211265553A CN115851617B CN 115851617 B CN115851617 B CN 115851617B CN 202211265553 A CN202211265553 A CN 202211265553A CN 115851617 B CN115851617 B CN 115851617B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses an escherichia coli bacteriophage LHE83 and application thereof, wherein the preservation number of the escherichia coli bacteriophage LHE83 is CCTCC NO: m20221473, storage time is 2022, 9 and 22 days. The coliphage LHE83 provided by the invention is a new phage separated from the natural world, and has the characteristics of wide host spectrum, high cracking activity and good physical and chemical factor tolerance; the natural enemy of the bacteria can specifically lyse pathogenic escherichia coli without destroying normal flora constitution, is not influenced by bacterial drug resistance, has no problems of drug residue and the like, and has high use safety; in addition, the coliphage LHE83 provided by the invention has remarkable antibacterial effect when being used together with low-dose antibiotics, and has good application prospect in the aspect of preventing and treating colibacillosis.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to an escherichia coli bacteriophage LHE83 and application thereof.
Background
Coli is widely used as a conditional pathogen in a breeding environment, and once the animal is infected, enteritis, peritonitis, septicemia and the like can occur, so that the weight gain of the animal body is obviously affected, the death rate of animal groups is rapidly increased, and huge loss of breeding industry is caused. At present, antibiotics are mainly used for preventing and controlling colibacillosis in farms, but various side effects exist in antibiotic therapy, such as damage to animal intestinal microbial flora structures, drug residues and the like, and meanwhile, the large-scale use of antibiotics accelerates the generation and diffusion of serious drug resistance and multi-drug resistance escherichia coli and the like, so that the difficulty in preventing and controlling colibacillosis is increased, the healthy development of the breeding industry is seriously influenced, and the national policy implementation requirements on resistance reduction, replacement and no resistance strategy of the breeding industry are seriously met, so that the development of novel antibiotic substitutes for preventing and controlling drug resistance bacterial diseases which occur increasingly frequently is urgently needed.
Phage as a virus of a specific infectious bacterium has shown great potential in controlling bacterial diseases. The phage lysis bacteria mechanism is different from antibiotics, so that the phage lysis bacteria mechanism is not influenced by bacterial drug resistance, has high species specificity on the lysed bacteria, can kill target bacteria efficiently and specifically under the condition of not damaging normal flora structure, has no side effect on animal organisms, has no drug residue problem, has wide phage sources and low production cost, and has many successful reports on clinical drug-resistant bacteria prevention and control. As a natural enemy of bacteria, theoretically, any pathogenic bacteria have phage corresponding to the bacteria, but the types and the numbers of the phage reported at present are only the iceberg, and a large number of phage resources have not been developed yet. The developed phage with wide cracking spectrum, strong cracking performance, high titer and good physical and chemical factor tolerance has wide application potential in clinic, meets the national requirements of the national policies of resistance reduction, resistance replacement and no resistance strategy, and therefore, the development of phage preparations with the characteristics has great significance for the healthy and rapid development of the aquaculture.
Disclosure of Invention
The invention mainly aims at providing an escherichia coli bacteriophage LHE83 and application thereof, and aims to provide an escherichia coli bacteriophage which has wide cracking spectrum, strong cracking performance and good physical and chemical factor tolerance.
In order to achieve the aim, the invention utilizes environmental samples collected from chicken farm in De-Zhou, shandong, to screen, separate and purify the environmental samples to obtain the coliphage LHE83. The coliphage LHE83 obtained by separation and purification is preserved in China center for type culture collection (CCTCC for short), and the preservation address is: the classification of the Chinese university of Wuhan is named as coliphage (Bacteriophage) LHE83 (hereinafter, also simply referred to as coliphage LHE 83), and the preservation number is CCTCC NO: m20221473, storage time is 2022, 9 and 22 days.
Furthermore, the invention also provides application of the coliphage LHE83 in preparing biological agents for preventing and treating diseases or pollution caused by pathogenic escherichia coli.
Alternatively, the pathogenic E.coli includes chicken-derived E.coli.
Optionally, the chicken-derived escherichia coli comprises a virulent escherichia coli.
Optionally, the chicken-derived escherichia coli comprises at least one of O8 type, O55 type, O78 type, O111 type, O114 type, O119 type and O125 type escherichia coli.
Still further, the present invention also proposes a biological agent for controlling diseases or pollution caused by pathogenic E.coli, comprising the E.coli phage LHE83 as described above.
Optionally, the biological agent is a feed additive, a drinking water additive, an environmental disinfectant or an environmental cleaner.
Optionally, the feed additive is for addition to chicken feed or duck feed.
Optionally, the environmental cleaner is a liquid, a lyophilized powder or a tablet; and/or the number of the groups of groups,
the environment disinfectant is liquid, freeze-dried powder or tablet.
Optionally, the biological agent is used in combination with an antibiotic for controlling diseases or pollution caused by pathogenic E.coli.
The coliphage LHE83 provided by the invention is a new phage separated from the natural world, and has the characteristics of wide host spectrum, high cracking activity and good physical and chemical factor tolerance; the natural enemy of the bacteria can specifically lyse pathogenic escherichia coli without destroying normal flora constitution, is not influenced by bacterial drug resistance, has no problems of drug residue and the like, and has high use safety; in addition, the coliphage LHE83 provided by the invention has remarkable antibacterial effect when being used together with low-dose antibiotics, and has good application prospect in the aspect of preventing and treating colibacillosis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a transmission electron microscope image of an E.coli phage LHE83 provided by the invention;
FIG. 2 is a photograph of the plaque morphology of the E.coli phage LHE83 provided by the present invention;
FIG. 3 is a graph showing the pH stability of the E.coli phage LHE83 provided by the present invention;
FIG. 4 is a graph showing the results of a thermal stability assay for E.coli phage LHE83 provided by the present invention;
FIG. 5 is a graph showing the results of ultraviolet stability measurements of the E.coli phage LHE83 provided by the present invention;
FIG. 6 is a graph showing the results of one-step growth curve measurement of the E.coli phage LHE83 provided by the invention;
fig. 7 is a graph showing the measurement results of an in vitro combined antibiotic bacteriostasis test of the coliphage LHE83.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides an escherichia coli phage LHE83, wherein the escherichia coli phage LHE83 is obtained by screening, separating and purifying an environmental sample collected from a chicken farm in Texas of Shandong province, and is preserved in China center for type culture collection (CCTCC for short), and the preservation address is: the classification of the Chinese university of Wuhan is named as coliphage (Bacteriophage) LHE83 (hereinafter, also simply referred to as coliphage LHE 83), and the preservation number is CCTCC NO: m20221473, storage time is 2022, 9 and 22 days.
Specifically, the coliphage LHE83 provided by the invention is separated by the following method:
(1) Test materials:
environmental samples, collected from chicken farm in texas, shandong; the phage host bacteria for screening are 20 pathogenic escherichia coli clinical isolates which are stored in the laboratory.
(2) The test method comprises the following steps:
20 clinical Escherichia coli isolates are used as host bacteria, and phage separation is carried out on fecal samples collected from farms. Taking 20g of fecal sample, putting the fecal sample into a 50mL centrifuge tube, adding the fecal sample into 30mL of LB liquid medium, soaking for 4 hours at 37 ℃, centrifuging at 4000rpm for 30 minutes, taking 20mL of supernatant into a new centrifuge tube, adding 30mL of escherichia coli 20 strain mixed bacterial liquid, mixing uniformly, putting into a constant temperature incubator at 37 ℃ for culturing for 4 hours, centrifuging at 4000rpm for 30 minutes, taking 5mL of supernatant, filtering the supernatant by a 0.22 mu m water filter, marking the filtrate, and storing the filtrate in a refrigerator at 4 ℃ for later use.
Phage isolation was performed using the spot plate method and the double-layer plate method. Mixing 3 μl of the prepared filtrate with 7 μl of the bacterial liquid, sucking 7 μl of the agar plate, marking, culturing at 37deg.C for 4 hr, observing plaque (shown in figure 2), recording bacterial liquid and filtrate corresponding to the plaque, taking the recorded bacterial liquid as host bacteria, and purifying the recorded corresponding filtrate by double-layer plate method.
The phage thus isolated were purified by the single spot method. After single plaque is taken out and added with normal saline, after incubation is carried out for 1h at 37 ℃, supernatant is taken out and filtered by a 0.22 mu L filter after centrifugation at 12000rpm for 5min, phage filtrate is taken out and added with an equal volume of host bacteria, and the culture is carried out by a double-layer flat plate method. The process is repeated for 3 times to obtain purified phage with uniform plaque morphology, glycerol with the final concentration of 30% is added, and the phage is preserved in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for standby, namely the coliphage LHE83.
The coliphage LHE83 can form transparent plaques on a solid medium, the edges are clear and regular, and the diameter is 1-1.5 mm; under a transmission electron microscope, the coliphage has an obvious regular polyhedron head structure and a telescopic tail structure, the head diameter is about 110nm, and the tail length is about 80-120 nm, and belongs to myotail phage.
The biological performance of the E.coli phage LHE83 was determined as follows:
a. according to a lysis spectrum measurement test, the LHE83 of the escherichia coli bacteriophage can lyse 19 strains in 20 pathogenic escherichia coli, and the lysis rate is 95%;
b. as can be seen from the potency assay and the optimal multiplicity of infection assay, the titer of the coliphage LHE83 is 3.02X10 9 pfu/mL, optimal multiplicity of infection is 0.01;
c. the pH, heat and ultraviolet stability tests show that the coliphage LHE83 has stable performance under the condition of pH4-10, stable activity and certain resistance to ultraviolet when the temperature is below 50 ℃;
d. the incubation period of the coliphage LHE83 is about 10min, the outbreak period is about 170min and the outbreak amount is about 22 as determined by a one-step growth curve;
e. as determined by phage-antibiotic in vitro combined bacteriostasis test, the concentration is 10 7 The pfu/mL coliphage LHE83 can effectively inhibit host bacteria by single useIs used cooperatively with spectinomycin (2 mug/mL) with sub-MIC concentration, and has remarkable antibacterial effect;
in summary, the invention provides a newly discovered coliphage LHE83, which has the characteristics of wide host spectrum, good physical and chemical factor tolerance, short hidden period, high cracking performance, good safety and the like, and is a novel product and means for preventing and treating colibacillosis; the coliphage LHE83 can inhibit the growth of pathogenic coliform bacteria in the environment; the coliphage LHE83 can be used together with antibiotics to effectively kill pathogenic colibacillus in the environment, so that the coliphage LHE83 can be used together with commercial antibiotics, the antibacterial effect of the coliphage LHE is further improved, the remarkable synergistic antibacterial effect can be achieved at the low-concentration antibiotic level, the consumption of the antibiotics is reduced, and the harm caused by the large consumption of the antibiotics is avoided.
The invention also provides application of the coliphage LHE83 in preparing a biological agent for preventing and treating diseases or pollution caused by pathogenic escherichia coli. The coliphage LHE83 can effectively inhibit the growth of pathogenic colibacillus in the environment, and the purified coliphage LHE83 can be used as an effective component in a biological agent for preventing and treating diseases caused by pathogenic colibacillus, can be singly applied to prepare a corresponding biological agent, can also be compounded with other phages to prepare a compound biological agent, and has remarkable antibacterial effect.
In some embodiments of the invention, the pathogenic E.coli comprises chicken-derived E.coli.
Further, the chicken-origin escherichia coli includes a virulent escherichia coli.
Still further, the chicken-derived E.coli includes at least one of O8 type, O55 type, O78 type, O111 type, O114 type, O119 type and O125 type E.coli.
In addition, the invention also provides a biological agent for preventing and treating diseases or pollution caused by pathogenic escherichia coli, and the biological agent comprises the escherichia coli bacteriophage LHE83. The purified coliphage LHE83 is used as an active ingredient of a biological agent, so that diseases caused by pathogenic colibacillus can be effectively prevented and treated, and the proliferation of pathogenic colibacillus in the environment can be inhibited.
Specifically, the active ingredient of the biological agent may include only the purified coliphage LHE83, or may include both the coliphage LHE83 and other phages with antibacterial effect, which are all within the scope of the present invention. In addition, in some embodiments of the invention, the biological agent can be used in combination with commercial antibiotics when being used for preventing and treating diseases or infections caused by pathogenic escherichia coli, has excellent antibacterial effect, can achieve remarkable synergistic antibacterial effect at low-concentration antibiotic level, is beneficial to reducing the consumption of antibiotics and avoids the harm caused by large consumption of antibiotics. Further, in some preferred embodiments of the invention, the antibiotic is preferably spectinomycin, which in combination with a sub-MIC concentration (2. Mu.g/mL) of spectinomycin produces a pronounced bacteriostatic effect.
The biological agent may be used in a variety of forms, and in some embodiments of the invention, the biological agent is a feed additive, a potable water additive, an environmental disinfectant, or an environmental cleaner.
In some embodiments of the invention, the biological agent may be added as an additive to animal feed and/or drinking water for controlling pathogenic E.coli contamination during cultivation. Further, in some embodiments of the invention, the feed additive is for addition to chicken feed or duck feed.
In other embodiments of the invention, the biological agent may be used as an environmental cleaner or an environmental disinfectant to clean the environment during application to prevent and treat pathogenic E.coli contamination during cultivation. Further, in some embodiments of the invention, the environmental cleaner is a liquid, a lyophilized powder, or a tablet; and/or the environmental disinfectant is liquid, freeze-dried powder or tablet. When the environment cleaning agent or environment disinfectant is used, the environment cleaning agent or environment disinfectant can be used for disinfection or cleaning of the culture environment after being diluted.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1: separation and preparation of coliphage LHE83
(1) Test materials:
fecal samples collected from chicken farm in texas, shandong; the total 20 strains of pathogenic escherichia coli of phage host bacteria for screening are stored in the laboratory.
(2) The test method comprises the following steps:
20 clinical Escherichia coli isolates are used as host bacteria, and phage separation is carried out on fecal samples collected from farms. Taking 20g of fecal sample, putting the fecal sample into a 50mL centrifuge tube, adding the fecal sample into 30mL of LB liquid medium, soaking for 4 hours at 37 ℃, centrifuging at 4000rpm for 30 minutes, taking 20mL of supernatant into a new centrifuge tube, adding 30mL of escherichia coli 20 strain mixed bacterial liquid, mixing uniformly, putting into a constant temperature incubator at 37 ℃ for culturing for 4 hours, centrifuging at 4000rpm for 30 minutes, taking 5mL of supernatant, filtering the supernatant by a 0.22 mu m water filter, marking the filtrate, and storing the filtrate in a refrigerator at 4 ℃ for later use.
Phage isolation was performed using the spot plate method and the double-layer plate method. Mixing 3 μl of the prepared filtrate with 7 μl of the bacterial liquid, sucking 7 μl of the agar plate, making a mark, culturing at 37deg.C for 4 hr, observing plaque (shown in figure 2), recording bacterial liquid and filtrate corresponding to the plaque, taking the recorded bacterial liquid as host bacteria, and separating phage from the recorded corresponding filtrate by double-layer plate method.
Example 2: purification and mass proliferation of E.coli phage LHE83
(1) Purification of E.coli phage LHE83
The phage initially isolated in example 1 above were purified by the single spot method. After single plaque is taken out and added with normal saline, after incubation is carried out for 1h at 37 ℃, supernatant is taken out and filtered by a 0.22 mu L filter after centrifugation at 12000rpm for 5min, phage filtrate is taken out and added with an equal volume of host bacteria, and the culture is carried out by a double-layer flat plate method. The process is repeated for 3 times to obtain purified phage with uniform plaque morphology, glycerol with the final concentration of 30% is added, and the phage is stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for standby.
(2) Purified proliferation of E.coli phage LHE83
Adding 1mL of phage stock solution and 1mL of freshly cultured host bacteria E.coli-E82 bacterial solution into 100mL of LB liquid medium, shake culturing for 2h at 37 ℃, adding chloroform with the volume ratio of 5%, continuing shake culturing for 30min, centrifuging at 12000rpm for 2min, and collecting supernatant to obtain a large amount of multiplication solution of phage, thus obtaining the coliphage LHE83.
Example 3: transmission electron microscope morphological observation of E.coli phage LHE83
Taking 10 mu L of purified coliphage LHE83 proliferation liquid drop on a copper net, standing for about 15min, and sucking redundant liquid by using filter paper. mu.L of 2% phosphotungstic acid (PTA) was added dropwise to the copper mesh and stained for 5min, the excess dye was removed by filter paper, dried and observed by a transmission electron microscope.
As shown in FIG. 1, the transmission electron microscope result of the coliphage LHE83 shows that the coliphage LHE83 has obvious regular polyhedron head structure and telescopic tail structure, the head diameter is about 110nm, the tail length is about 80-120 nm, and the coliphage LHE83 belongs to myotail phage.
Example 4: detection of cleavage spectrum of E.coli phage LHE83
The test selects 20 pathogenic E.coli clinical isolates (all isolated from the pathogenic chickens) and the cleavage profile of E.coli phage LHE83 was determined by double-layer agar plate method. The specific operation is as follows: 50. Mu.L of 20 E.coli proliferation solution and 50. Mu.L of phage proliferation solution (3.02X10) 9 pfu/mL) was added to 3mL of a heated (about 40 ℃) upper nutrient agar medium, immediately after mixing, poured onto a normal nutrient agar solid plate, cultured upside down in a 37℃incubator for 4 hours, and the results were observed and recorded as shown in Table 1.
The results in Table 1 show that the 20 E.coli strains of different serotypes selected in the test grew well on LB solid medium plates to form a lawn. Plaques with smooth edges and uniform sizes appear on the plates of 19 E.coli, which indicates that the isolated E.coli phage LHE83 can lyse 19 strains of 20 E.coli with a lysis rate of 95%.
TABLE 1 results of determination of the cleavage spectra of E.coli phage LHE83
Example 5: potency determination of E.coli phage LHE83
Sequentially diluting the multiplication liquid of the coliphage LHE83 by 10 times, taking 100 mu L of multiplication liquid at each dilution, respectively and uniformly mixing with the equal volume of host bacteria E.coli-E82 bacterial liquid, and carrying out plaque counting by a double-layer flat plate method, wherein each dilution is 3 in parallel. Phage titers were calculated from the number of plaques, and the titers of E.coli phage LHE83 were measured to be 3.02X10 9 pfu/mL。
Example 6: determination of optimal multiplicity of infection for E.coli phage LHE83
E.coli phage LHE83 proliferation liquid and host bacteria E.coli-E82 bacterial liquid cultured to logarithmic phase are taken for counting. Mixing uniformly according to the ratio of the infection complex numbers of 0.001, 0.01, 0.1, 1, 10, 100 and 1000, adding 5mL of LB broth, shaking culture at 37 ℃ for 4h, centrifuging at 12000rpm for 20min, filtering with a 0.22 mu m filter, sterilizing to obtain phage proliferation liquid, measuring phage titer by a double-layer flat plate method, performing three parallel operation on each group, calculating the optimal infection complex number according to the measurement result of the phage titer, and recording the result as shown in Table 2.
The results in Table 2 show that the titer of the E.coli phage LHE83 reaches a maximum of 3.02X10 when the multiplicity of infection is 0.01 9 pfu/mL. Thus the optimal multiplicity of infection of the E.coli phage LHE83 is 0.01.
TABLE 2 determination of optimal multiplicity of infection for E.coli phage LHE83
Example 7: determination of the pH stability of the E.coli bacteriophage LHE83
Titers of 500. Mu.L were 10 9 The pfu/mL phage proliferation liquid is respectively added with 4.5mL of LB liquid culture medium with different pH values (3, 4, 5, 6, 7, 8, 9, 10, 11 and 12), and is placed in the condition of 37 ℃ for shake culture, sampling is respectively carried out for 1h, 2h and 3h, ten-fold specific dilution is carried out, 100 mu L of each gradient dilution is respectively and evenly mixed with an equal volume of E.coli-E82 bacterial liquid, and phage titer is measured by a double-layer flat plate method, wherein each group is parallel. The phage pH stability curve was plotted according to the measurement results, and the results are shown in FIG. 3.
The results in FIG. 3 show that the E.coli phage LHE83 is relatively stable over the pH range of 4-10 when pH<4 or pH>At 10, the activity of the E.coli phage LHE83 rapidly decreased with increasing pH. When the pH is reduced to 3, the phage acts for 1-3 hours, and the titer is only 10 4 pfu/mL; when the pH is raised to 12, the LHE83 titer of the coliphage is 10 after 1h of action 4 pfu/mL; after 2h of action, the phage were completely inactivated. Thus, the E.coli phage LHE83 is stable at pH4 to 10.
Example 8: thermal stability assay of E.coli phage LHE83
500 mu L of the mixture is taken to have the concentration of 10 8 The pfu/mL coliphage LHE83 proliferation liquid is incubated in water bath at 40 deg.c, 50 deg.c, 60 deg.c, 70 deg.c and 80 deg.c for 20min, 40min and 60min to sample 200 μL, ten times the dilution successively, 100 μL of the dilution is mixed with equal volume of E.coll-E82 bacteria liquid, and the phage titer is measured in double-layer plate process. The thermal stability of phage was plotted against the statistics and the results are shown in FIG. 4.
The results in FIG. 4 show that the E.coli phage LHE83 has a substantially constant titer at 40℃and 50 ℃; strips at 60 DEG CUnder the condition of the part, the titer of the coliphage LHE83 is reduced by two orders of magnitude for 20min, and the titer of the phage is reduced to 10 when the coliphage is acted for 60min 5 pfu/mL; when the temperature is 70 ℃, the phage activity rapidly decreases with the extension of the action time; phage were rapidly inactivated at 80 ℃. Thus, the E.coli phage LHE83 activity is more stable when the temperature is below 60 ℃.
Example 9: ultraviolet stability assay for E.coli phage LHE83
Taking 4mL of the sample with the potency of 10 9 The pfu/mL phage LHE83 was placed in a dish and continuously irradiated by a UV lamp (power: 30W) for 40 cm. 200 mu L of phage multiplication liquid is taken every ten minutes, diluted by 10 times, 100 mu L of each gradient dilution liquid is respectively and uniformly mixed with E.coli-E82 bacterial liquid with equal volume, phage titer is measured by a double-layer flat plate method, continuous measurement is carried out for 60 minutes, and each group is provided with two parallel groups. The phage ultraviolet stability curve was plotted according to the measurement results, and the results are shown in FIG. 5.
The results in FIG. 5 show that the phage activity starts to decrease after a continuous irradiation of the E.coli phage LHE83 for 10min under UV light. Phage titers decreased by about an order of magnitude when irradiated with ultraviolet light for 10 min. When irradiated continuously with ultraviolet light for 40min, the titer of phage decreased by about two orders of magnitude. When the phage was continuously irradiated for 60min, the titer of phage was reduced to 10 5 pfu/mL. Indicating that the coliphage LHE83 has a certain resistance to ultraviolet rays.
Example 10: one-step growth curve of E.coli phage LHE83
The concentration is 10 according to the optimal infection complex ratio 6 pfu/mL of E.coli phage LHE83 proliferation solution and concentration of 10 8 The cfu/mL of the overnight cultured host bacteria E.coli-E82 bacteria solution were 200. Mu.L each, mixed well, incubated at 37℃for 5min, centrifuged at 13000rpm for 30s, washed twice with physiological saline, and the supernatant was discarded. The pellet was resuspended in 7mL of LB liquid medium and shake cultured at 37 ℃. Respectively taking 200 μL of proliferation solution at different time points (from zero time, every 10min in the first 1h, every 20min in the second 2h, and every 30min in the third 3 h), centrifuging at 16000rpm for 1min, taking supernatant, and double-layer plate methodPhage titers were measured, three replicates were set per group, one-step growth curves were drawn based on the measurement results, and the incubation period, outbreak period, and outbreak amount of phage were calculated, and the results are shown in fig. 6.
The results in FIG. 6 show that the incubation period for the E.coli phage LHE83 is about 10min, the outbreak period is about 170min, and the outbreak is about 22.
Example 11: phage in vitro bacteriostasis test
Under aseptic condition, 200 mu L of the composition is taken to have a concentration of about 10 according to the optimal infection complex ratio 7 phage LHE83 in pfu/mL and 200. Mu.L concentration of about 10 8 cfu/mL of host bacteria E.coli-E82 bacterial liquid is cultured in 5mL of LB broth at 37 ℃ in a shaking way, 200 mu L of mixed culture is taken every 2 hours from the zero moment, ten times ratio dilution is carried out, colony counting is carried out by a pouring method, and the in vitro antibacterial capacity of LHE83 is analyzed through the change condition of bacterial quantity, and the result is shown in figure 7.
The results in FIG. 7 show that the concentration is 10 7 The pfu/mL coliphage LHE83 can effectively inhibit bacterial growth within 12 hours.
Example 12: phage-antibiotic combined bacteriostasis test
The spectinomycin solution having a concentration of 256. Mu.g/mL was sequentially diluted 2-fold. Spectinomycin solutions of different concentrations (0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 8. Mu.g/mL, 16. Mu.g/mL, 32. Mu.g/mL, 64. Mu.g/mL, 128. Mu.g/mL, 256. Mu.g/mL) were separated from the solutions of different concentrations (10) 3 pfu/mL、10 4 pfu/mL、10 5 pfu/mL、10 6 pfu/mL、10 7 pfu/mL、10 8 pfu/mL、10 9 pfu/mL) of each 100 mu L of the E.coli phage LHE83 proliferation liquid was added to a 96-well plate and mixed well, and then added at a concentration of 10 8 cfu/mL of host bacterium E.coli-E82 bacterial liquid 100 mu L, static culture at 37 ℃ for 12 hours, and OD measurement every 30 minutes 630 Values, a phage-antibiotic combined antibacterial effect curve is drawn according to the measurement results, and the results are shown in figure 7.
The results in FIG. 7 show that the concentration is 10 7 The pfu/mL coliphage LHE83 can effectively inhibit the growth of bacteria, and can generate obvious effect when being used together with spectinomycin with sub-MIC concentration (2 mu g/mL)Is effective in inhibiting bacteria.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. An escherichia coli Bacteriophage LHE83, wherein the escherichia coli Bacteriophage (Bacteriophage) LHE83 has a preservation number of CCTCC NO: m20221473, storage time is 2022, 9 and 22 days.
2. Use of the coliphage LHE83 of claim 1 in the preparation of a biological agent for the control of diseases or pollution caused by pathogenic escherichia coli of chicken origin.
3. Use of the coliphage LHE83 in accordance with claim 2 for the preparation of a biological agent for controlling diseases or pollution caused by chicken-derived pathogenic e.
4. Use of the bacteriophage LHE83 according to claim 3 for the preparation of a biological agent for controlling diseases or pollution caused by chicken-derived pathogenic e.coli, comprising at least one of e.coli of O8, O55, O78, O111, O114, O119 and O125.
5. A biological agent for controlling diseases or pollution caused by pathogenic escherichia coli of chicken origin, comprising the escherichia coli bacteriophage LHE83 of claim 1.
6. The biologic of claim 5, wherein the biologic is a feed additive, a potable water additive, an environmental disinfectant, or an environmental cleaner.
7. The biologic of claim 6 wherein the feed additive is for addition to chicken feed or duck feed.
8. The biologic of claim 6 wherein said environmental cleaner is a liquid, lyophilized powder or tablet; and/or the number of the groups of groups,
the environment disinfectant is liquid, freeze-dried powder or tablet.
9. The biological agent according to claim 5, wherein the biological agent is used in combination with an antibiotic for controlling diseases or pollution caused by pathogenic escherichia coli of chicken origin.
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| WO2016003307A1 (en) * | 2014-07-02 | 2016-01-07 | Limited Liability Company "Bphage" | Bacteriophage strains, compositions and related methods |
| CN110129283A (en) * | 2019-05-24 | 2019-08-16 | 青岛诺安百特生物技术有限公司 | A kind of short-tail coliphage and its application |
| CN110607284A (en) * | 2019-10-23 | 2019-12-24 | 青岛农业大学 | Escherichia coli phage vB _ EcoM _ swi3 and application thereof |
| CN112280749A (en) * | 2020-10-16 | 2021-01-29 | 青岛农业大学 | Escherichia coli phage vB _ EcoM _ swi3 and application thereof |
| CN113583972A (en) * | 2021-08-05 | 2021-11-02 | 瑞科盟(青岛)生物工程有限公司 | Escherichia coli bacteriophage capable of reducing antibiotic resistance and application thereof |
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| KR101591783B1 (en) * | 2014-04-15 | 2016-02-04 | 씨제이제일제당(주) | Novel bacteriophage and composition comprising the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016003307A1 (en) * | 2014-07-02 | 2016-01-07 | Limited Liability Company "Bphage" | Bacteriophage strains, compositions and related methods |
| CN110129283A (en) * | 2019-05-24 | 2019-08-16 | 青岛诺安百特生物技术有限公司 | A kind of short-tail coliphage and its application |
| CN110607284A (en) * | 2019-10-23 | 2019-12-24 | 青岛农业大学 | Escherichia coli phage vB _ EcoM _ swi3 and application thereof |
| CN112280749A (en) * | 2020-10-16 | 2021-01-29 | 青岛农业大学 | Escherichia coli phage vB _ EcoM _ swi3 and application thereof |
| CN113583972A (en) * | 2021-08-05 | 2021-11-02 | 瑞科盟(青岛)生物工程有限公司 | Escherichia coli bacteriophage capable of reducing antibiotic resistance and application thereof |
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