CN117737002A - Broad-spectrum phage with high cracking rate and capable of cross-species cracking and application thereof - Google Patents
Broad-spectrum phage with high cracking rate and capable of cross-species cracking and application thereof Download PDFInfo
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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 belongs to the technical field of biology, and particularly relates to a broad-spectrum phage with high cracking rate and cross-species cracking and application thereof. A broad-spectrum bacteriophage with high cracking rate and cross-species cracking function is colibacillus bacteriophageEscherichia coli phageThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC) for 4 months and 20 days in 2022, and the preservation number is CGMCC NO.45087. The broad-spectrum bacteriophage which has high cracking rate and can be subjected to cross-species cracking has strong cracking effect on escherichia coli and salmonella, can effectively prevent and control the spread of the escherichia coli and the salmonella, has higher cracking rate on the escherichia coli and the salmonella, has a wide cracking spectrum and high antibacterial rate, can inhibit the growth of bacteria for a long time, has certain tolerance to temperature, and is favorable for industrial production and preparation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a broad-spectrum phage with high cracking rate and cross-species cracking and application thereof.
Background
Phage is a bacterial virus, has high abundance in nature, and can exist in places where bacteria exist. Phage of intestinal bacteria are often contained in human and animal excretions or contaminated well water, river water. In the soil, phages of soil bacteria can be found. Phages have strict host specificity, i.e. in most cases one phage is directed against only one bacterium and only colonizes the susceptible host organism. Moreover, the phage has biological activity, so that the phage is sensitive to temperature and easy to inactivate at high temperature. The host specificity and thermal sensitivity of phage cause difficulties in their industrial production and use.
Disclosure of Invention
The invention aims at overcoming the defects of the existing phage and providing a broad-spectrum phage with high-temperature-resistant high-cracking rate capable of cross-species cracking, which can crack escherichia coli and salmonella simultaneously and shows high-temperature tolerance, thus providing a new material basis for the industrial application of the phage.
In order to achieve the above purpose, the invention adopts the technical means that: a broad-spectrum bacteriophage with high cracking rate and cross-species cracking function is colibacillus bacteriophageEscherichia coli phageThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC) for 4 months and 20 days in 2022, and the preservation number is CGMCC NO.45087.
Preferably, the phage is capable of lysing E.coli and Salmonella.
Preferably, the phage has an optimal multiplicity of infection of E.coli of 1:100.
Preferably, the phage is capable of maintaining lytic activity at 80 ℃.
Preferably, the present invention also provides the use of said phage, which phage can be used to prepare a bactericidal composition.
Preferably, the invention also provides a bactericidal composition, wherein the bactericidal composition comprises the bacteriophage.
The broad-spectrum phage with high cracking rate and cross-species cracking provided by the invention has strong cracking effect on escherichia coli and salmonella, can effectively prevent and control the generation and transmission of escherichia coli and salmonella, has higher temperature tolerance, and is favorable for industrialized production and preparation. The phage can be used as bactericide to effectively and reliably treat livestock and poultry diseases caused by escherichia coli and salmonella, and improve the breeding environment.
Drawings
FIG. 1 shows plaques of phage RDP-EC-20055 in an example of the invention;
FIG. 2 shows the morphology of phage RDP-EC-20055 under transmission electron microscope in the examples of the present invention;
FIG. 3 is a graph showing the effect of phage RDP-EC-20055 on E.coli BE-20054 growth curve in an embodiment of the present invention;
FIG. 4 is a thermal stability assay of phage RDP-EC-20055 in an example of the invention;
FIG. 5 is a graph showing the acid-base stability of phage RDP-EC-20055 in an example of the invention;
FIG. 6 is a phage RDP-EC-20055 titer stability assay in examples of the invention.
Detailed Description
In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The invention separates a strain of escherichia coli BE-20054 from viscera of a dead chicken in a certain farm of a eastern Weifang, determines pathogenicity of escherichia coli BE-20054 through experiments, takes escherichia coli BE-20054 as a host, separates a virulent phage from the environment, and determines the genotype of phage RDP-EC-20055 through a whole genome sequence.
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.
Example 1 isolation and purification of pathogenic bacteria and phages:
(1) Isolation and identification of pathogenic bacteria:
sampling from the sick broiler chickens by adopting a sterile operation method, streaking on a Maiconk and SS culture medium, culturing for 18-24 hours at 37 ℃, and then presenting round pink colonies with neat edges on a Maiconk agar plate, wherein the diameters are about 1-2 mm; then picking a typical colony, continuing streaking and purifying for 3 times, then picking a single colony, inoculating the single colony into 5mL of LB liquid medium, and carrying out shaking culture for 8 hours at 37 ℃ and 200rpm to obtain a uniform and turbid bacterial suspension. Then the strain is identified by 16sRNA molecules, and is determined to BE escherichia coli, and one strain is named as escherichia coli BE-20054.
(2) Isolation of phage RDP-EC-20055: the fecal sample for the test of the present invention was collected from a farm of a Weifang in Shandong in 2020 and used as a phage-isolated sample.
10g of excrement is taken, soaked in 20 ml of sterile physiological saline for 5min, centrifuged at 10000rpm for 5min, and filtered and sterilized by a 0.22 micron microporous filter membrane after removing larger impurities and most bacteria. 3 ml of the filtrate and 3 ml of the host bacterium suspension were taken and added together to 20 ml of the autoclaved LB broth, followed by overnight culture in a 37℃incubator. After culturing, 5ml is taken, centrifugated at 10000rpm for 5 minutes, filtered and sterilized by a 0.22 micron microporous filter, and the filtrate is the stock solution which is intended to contain phage. Then, a double plate method is used to identify whether phage exists, if so, the phage is indicated, otherwise, no phage is isolated and a new screen is needed.
(3) Purification of phage RDP-EC-20055:
the initially isolated plaques are often of inconsistent size, morphology and therefore require further purification. And (3) selecting single independent, uniform-shape, clear and transparent plaques on a double-layer plate with plaques, immersing the plaques in 1 milliliter of physiological saline for 10 minutes, taking clear liquid, filtering the clear liquid by a 0.22 micrometer microporous filter, properly diluting filtrate (the single plaques are when the double-layer plate is paved), paving the double-layer plate with host bacterial suspension, repeating the steps for 3-4 times, and obtaining the purified phage when the sizes, the shapes and the definition of the plaques on the double-layer plate are consistent.
(4) Titer determination of phage RDP-EC-20055:
spreading the purified phage RDP-EC-20055 on a double-layer plate, taking single plaque, adding into LB broth, adding host bacterial suspension, proliferating phage RDP-EC-20055, centrifuging proliferation liquid at 10000rpm for 5min, filtering with 0.22 μm microporous filter, and 10 times gradient diluting the filtrate to 10 times 7 The phage titer was determined by taking 0.1 ml of phage dilutions of the last 3 dilutions and 0.1 ml of host bacterial suspension, and plating double-layered plates. Phage titer (pfu/mL) =number of plaques × dilution fold ≡0.1.
As a result, the phage RDP-EC-20055 showed consistent plaque morphology, size and clarity, and typical lytic properties, as shown in FIG. 1. The titer of the phage RDP-EC-20055 can reach 10 9 pfu/mL or more.
Example 2 morphological observations of phage RDP-EC-20055:
the phosphotungstic acid negative dyeing method is used: phage solution (titer 10) was dropped onto paraffin plates 10 pfu/m L) 100 μl, placing the copper mesh film side on phage droplets, taking off after 10min, and naturally drying in air for 2-3 min. And then, a drop of 2% phosphotungstic acid (PTA) aqueous solution is dripped on the copper wire for dyeing, the copper wire is taken down after 10min, dried in the air for 10-15 min, observed by an electron microscope, and a clear phage image is selected for photographing. As can be seen from the electron micrograph shown in FIG. 2, the phage RDP-EC-20055 has a tail with a head size of 30nm×100nm and a tail length of about 100 nm.
Example 3 genome sequencing and biological preservation of phage RDP-EC-20055:
after enrichment culture of individual phages, the strips were grown at 4 ℃Centrifuging 8000g under the piece for 15 minutes, adding 10% PEG8000 and 0.5M NaCl, standing overnight, adding equal amount of chloroform, mixing, standing, layering, centrifuging for 10 minutes under 5000g, removing chloroform layer and PEG layer, adding restriction endonuclease for digestion treatment, suspending phage under the condition of gradient density cesium chloride, dialyzing for 3 times by using TM buffer solution for 30 minutes each time at the later stage, and finally reserving a part of dialyzed phage for electron microscopy, and carrying out whole genome sequencing by a part of China megagene biological sequencing company. Sequencing results show that the genome of the phage has the total length of 53067bp, as shown in SEQ ID: 1. It was named Escherichia coli phageEscherichia coli phageAnd is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.45087 at 20 months of 2022. The preservation unit addresses are: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Example 4 action diagram of phage RDP-EC-20055 on E.coli BE-20054 growth curve:
phage were added to the bacterial suspension, resulting in a change in the OD600 value due to the lysis of the host by the phage, thereby resulting in a lysis curve of the phage against the host. As a control, a suspension of host bacteria (E.coli BE-20054) cultured for 6 hours was incubated at 2%80℃for 30min, 3 parallel controls were made, and the results were averaged.
As a result, as shown in FIG. 3, the OD600 of the host to which no phage was added gradually increased as the host proliferated, the logarithmic phase ended after 3.5 hours, whereas the OD600 of the host to which phage was added increased in the first 80 minutes when phage was in the adsorption and infection phase to the host, and the OD600 began to decrease after 80 minutes when phage was lysed, but gradually fluctuated with the lapse of time.
The phage RDP-EC-20055 is added into the host bacterium BE-20054 bacterial suspension, and a certain cracking effect appears after 80 minutes of action, compared with a control group, the OD600 of a host without phage is obviously increased, which shows that the phage has stronger cracking capability to the host and can continuously crack the host bacterium. As the phage is in the adsorption and infection stage of host bacteria, only a small part of phage begins to lyse the host, so that the amount of the lysed host is far smaller than the growth amount of the host, and the OD600 still keeps a certain ascending trend within the first 80min, and the number of the lysed host is larger than the proliferation speed of the host after 80min along with the exponential increase of the number of phage, so that the OD600 value begins to gradually decrease, and the mixed bacterial liquid reaches the clearest. After 510min of continuous action, the mixed bacterial liquid returns to turbidity, the original sensitive bacteria are cracked by phage, and one flora replaced by mutant tolerant bacteria is alternated. Therefore, in order to obtain phage with high titer, the mixed bacterial liquid should be controlled within the time range from the highest definition to the beginning of the turbidity returning phenomenon, so as to avoid the influence on phage titer.
Example 5 determination of the thermal stability of phage RDP-EC-20055:
the phage stock solution was packed into EP tubes, incubated at 40℃at 50℃at 60℃at 70℃at 80℃for 60min, diluted with 10-fold ratio of physiological saline, and counted in double plates. As can be seen from FIG. 4, the phage activity was lower and lower with increasing temperature. Although the phage was inactivated by incubation at 80℃for 60min, the phage was allowed to act at 80℃for 10min and the titer was maintained at 10 5 pfu/mL proves that the phage has a certain tolerance to instantaneous high temperature, and is beneficial to the production of industrial powder.
Example 6 determination of acid-base stability of phage RDP-EC-20055:
regulating pH of physiological saline with diluted hydrochloric acid and diluted NaOH solution, preparing buffer solutions with pH value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, diluting the diluted solution at 37deg.C in water bath for 1 hr, diluting with physiological saline 10 times, and spreading on double plates. Inverted culturing at 37deg.C for 4-6 hr, and counting. As can be seen from FIG. 5, phage RDP-EC-20055 maintained good activity at pH 3-11 with small potency change.
Example 7 phage RDP-EC-20055 bacteriostasis test:
under aseptic conditions, 1mL of sample and 1mL of host bacterial liquid (1×10) 5 CFU/mL), incubation at 37℃for 15 min, mixing wellThen diluted to 10 with normal saline -1 -10 -3 Each gradient was plated onto LB agar plates at 100. Mu.L, incubated at 37℃for 6 hours, and repeated twice. Simultaneously taking 1mL of physiological saline and 1mL of host bacteria liquid (1×10) 5 CFU/mL) as a blank, the above procedure was repeated. Plates with a colony count of 30-300 were selected for counting. This test was repeated 3 times and the average was taken.
Phage inhibition = (1-number of colonies of treatment group/number of colonies of control group) ×100%. The result shows that the bacteriostasis rate of the phage RDP-EC-20055 reaches 99%, has a better cracking effect on hosts, and is suitable for being used in the cultivation process.
Example 8 determination of phage RDP-EC-20055 cleavage spectrum:
the bacteriophage RDP-EC-20055 is subjected to a lysis spectrum experiment by selecting escherichia coli, salmonella, klebsiella pneumoniae and staphylococcus aureus, and the experiment shows that the bacteriophage has cross-species lysis capability and has lysis capability on the escherichia coli and the salmonella. 120 strains of escherichia coli (30 strains of swine source, 30 strains of chicken source, 30 strains of duck source, 30 strains of sea cucumber source and 30 strains of culture environment) and 100 strains of salmonella (30 strains of chicken source, 30 strains of duck source, 30 strains of pig source, 30 strains of shrimp source and 30 strains of environment source) are selected for carrying out a lysis spectrum experiment, and experimental results show that the phage can lyse escherichia coli which is easily separated from animal source and environment source, the lysis rate is 90%, and salmonella which is easily separated from animal source and environment source is all lysed, and the lysis rate is 95%. It was further found that the bacterial serotypes have an effect on the lysis profile, and that the selected E.coli and Salmonella were subjected to serotype analysis, and that the selected E.coli had 9 serotypes, the Salmonella had 21 serotypes, 6 of which were lysed by phage RDP-EC-20055, and 14 of which were lysed by 21 serotypes of Salmonella. The phage has a wide range of lysis to both E.coli and Salmonella. The final experimental results are shown in tables 1 and 2.
TABLE 1 results of the determination of the cleavage spectra of phage RDP-EC-20055
| Bacteria and method for producing same | Animal origin lytic property | Environmental Source cleavage Property |
| Coli bacterium | + | + |
| Salmonella bacteria | + | + |
| Klebsiella pneumoniae | - | - |
| Staphylococcus aureus | - | - |
| Clostridium perfringens | - | - |
TABLE 2 determination of the results of the lytic spectra of phage RDP-EC-20055 on different serotypes of E.coli and Salmonella
| Salmonella serotype | Cleavage Property | Coli serotype | Cleavage Property |
| Salmonella enteritidis | + | O18 | + |
| Salmonella pullorum | + | O2 | + |
| Salmonella typhimurium | + | O45 | + |
| Salmonella typhi | - | O145 | - |
| Salmonella paratyphi B | - | O1 | + |
| Salmonella enterica | - | O78 | - |
| Salmonella paratyphi A | - | O3 | + |
| Salmonella indiana | + | O4 | + |
| Salmonella zhang Wei | + | O5 | - |
| Salmonella in famous and ancient houses | - | ||
| Salmonella rison | + | ||
| Lei Gensen Salmonella | + | ||
| Salmonella mountain fudberg | + | ||
| Salmonella neotame | + | ||
| Salmonella anatipestifer | + | ||
| Cloth Long Si Salmonella burica | - | ||
| Salmonella cotibus | + | ||
| Shang Boxun Salmonella | + | ||
| Kentucky gateBacteria of the genus Shewanella | + | ||
| Salmonella moelleri Ban Daka | - | ||
| Salmonella stanli | + |
Note that: "+" represents cleavable; "-" means non-cleavable.
Example 9 stability experiments of phage RDP-EC-20055:
taking the initial potency as 8.9 x 10 9 The liquid phage of (2) was dispensed into sterile 1.5mL EP tubes, and a total of 90 were dispensed in 1mL each tube. The cells were randomly divided into 3 groups, with no group 30. The phage were stored at 4℃and 20℃and 37℃respectively, and the change in the phage titer was monitored periodically. Experimental results show that the phage RDP-EC-20055 has stable titer, is suitable for long-term placement, and has longer product validity period after industrial production. The phage titer remained essentially unchanged when placed at 4℃for 12 months, at which temperature phage were better stored. At 20℃and 37℃the titer of the phage was slightly decreased by about 1 order of magnitude when left for 12 months. The specific experimental results are shown in fig. 6.
Example 10 phage RDP-EC-20055 optimal multiplicity of infection experiments:
according to the infection complex in Table 3Ratio of numbers phage RDP-EC-20055 proliferation fluid and host were added to LB broth and the total volume of the culture system was ensured to be the same. After shaking culture at 200rpm at 37℃for 4-6 hours, centrifugation was carried out at 12000r/min for 5min at room temperature, and the titer was measured by taking the supernatant and spreading on a double plate. As shown in Table 3, the optimal infection complex of phage RDP-EC-20055 to host bacteria is 1:100, and phage titer can reach 10 10 pfu/mL。
TABLE 3 Complex infection results of phage RDP-EC-20055 provided by the invention on host bacteria
| Phage: host bacterium | Valency of |
| 10:1 | 8.9*10 8 |
| 1:1 | 3.6*10 9 |
| 1:10 | 4.8*10 9 |
| 1:100 | 5.3*10 10 |
| 1:1000 | 7.6*10 9 |
| 1：10000 | 6.2*10 9 |
Claims (6)
1. A broad-spectrum phage with high cracking rate and cross-species cracking is characterized in that: the phage is Escherichia coli phageEscherichia coli phageThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC) for 4 months and 20 days in 2022, and the preservation number is CGMCC NO.45087.
2. The high-lysis-rate cross-species-cleavable broad-spectrum phage of claim 1 wherein: the phage can lyse E.coli and Salmonella.
3. The high-lysis-rate cross-species-cleavable broad-spectrum phage of claim 2 wherein: the optimal infection complex of the phage to the escherichia coli is 1:100.
4. The high-lysis-rate cross-species-cleavable broad-spectrum phage of claim 1 wherein: the phage was able to maintain lytic activity at 80 ℃.
5. Use of a broad spectrum of high rate cross-species cleavable phages according to claim 1, characterized in that: the phage are used to prepare a bactericidal composition.
6. A bactericidal composition characterized in that: the bactericidal composition contains the phage of any one of claims 1-4.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310839152.XA CN117737002A (en) | 2023-07-10 | 2023-07-10 | Broad-spectrum phage with high cracking rate and capable of cross-species cracking and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310839152.XA CN117737002A (en) | 2023-07-10 | 2023-07-10 | Broad-spectrum phage with high cracking rate and capable of cross-species cracking and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN117737002A true CN117737002A (en) | 2024-03-22 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202310839152.XA Pending CN117737002A (en) | 2023-07-10 | 2023-07-10 | Broad-spectrum phage with high cracking rate and capable of cross-species cracking and application thereof |
Country Status (1)
| Country | Link |
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| CN (1) | CN117737002A (en) |
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2023
- 2023-07-10 CN CN202310839152.XA patent/CN117737002A/en active Pending
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