CN116751753A - Edwardsiella tarda phage with high lytic property and composition and application thereof - Google Patents
Edwardsiella tarda phage with high lytic property and composition and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a high-schizolysis Edwardsiella tarda bacteriophage and a composition and application thereof. Edwardsiella tarda phage with high lytic property, which is Edwardsiella tarda phage PKP-ET-2022001, latin literature nameEdwardsiella tarda phageThe strain is preserved in China general microbiological culture Collection center (CGMCC) at the 8 th month of 2022 and the 31 th day, and the preservation number is CGMCC NO.45264; the host is Edwardsiella tarda of marine origin. The phage provided by the invention has higher cracking property on the Edwardsiella tarda from the ocean and the Edwardsiella tarda from the fresh water, and has wider cracking spectrum, thus having important significance and application prospect on preventing and treating the Edwardsiellosis in the aquaculture industry.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a high-schizolysis Edwardsiella tarda bacteriophage and a composition and application thereof.
Background
Edwardsiella tardaEdwardsiella tarda) Is a short-rod gram-negative pathogen, and can cause systemic hemorrhagic septicemia of various light and marine fishes. Is also an important zoonotic primordium, which is the only member of Edwardsiella that infects humans. During the course of the infection process, the process,E.tardacan be planted in the important organs of a host in an intracellular parasitic mode to cause the ulceration of the epidermis, abscess and intestinal tissue of the host to spoil, and finally erupt to form Edwardsiellosis. At present, fish infection exists all over the worldE.tardaCausing serious economic losses in the aquaculture industry.
In recent years, the scale of aquaculture has been increasedE.tardaBecome a bacterial pathogen which seriously threatens light and marine fishes, and cause huge economic loss for the aquaculture industry. The bacterium causes various digestive tract diseases of economic fish species, and hemorrhagic septicemia caused by infection is considered as a typical condition of Edwardsiellosis. In addition, in the case of the optical fiber,E.tardathe geographical location of the disease is widely varied.
There are cases reported in Bohai sea, yellow sea, east coastal farms, and even inland farms such as freshwater fish and soft-shelled turtle.E.tarda Also has the characteristic of facultative intracellular parasitic, is a pathogenic bacterium causing zoonosis of people and fish, and is directly taken as a medicineThe hypochondrium is healthy. Human infections caused by raw food fish products or body wounds to dateE.tardaCases show a growing trend year by year and can induce a variety of complications.
Currently, in aquaculture, antibiotics or other related chemicals are commonly used for treatment of Edwardsiella tarda disease in aquatic animals. Such as gentamicin, ampicillin, neomycin, streptomycin, sulfonamides, etc. However, it is known that the use of drugs such as antibiotics can naturally achieve a very obvious curative effect in a short period of time, but has the great disadvantage of causing pathogenic drug resistance, and meanwhile, the use of antibiotics can also generate great damage to other non-pathogenic microorganisms or probiotic bacteria in the culture environment, thereby breaking the competition and balance of the microbial bacteria in the culture environment.
Phages are viruses that specifically infect bacteria and replicate within bacterial cells. They are the most abundant organisms on earth, most commonly playing an important role in microorganisms, physiology, evolution and therapy. In recent years, the use of bacteriophages for the treatment of chronic bacterial infections in humans and animals has been the case of many successes. Phages have higher effectiveness and safety than antibiotics. Compared with antibiotics, the phage has high specificity, and the advantage of the action mechanism is that the phage only lyses target strains and does not cause the damage of symbiotic intestinal flora. Meanwhile, the phage has the advantages of self replication, small dosage required by treatment, no toxicity, short research and development period, low production cost and the like. Phage therapy has very broad and attractive prospects as an alternative means for eliminating pathogenic bacteria, and deserves deep exploration by researchers.
Phage is derived from the environment, is specially invaded by bacteria as a type of bacteria-dependent virus, and is a candidate of a novel antibacterial preparation in the face of frequent phenomena of antibiotic residues and bacterial drug resistance. Because of the characteristics of self efficient replication, specific pathogen targeting, no influence on normal flora, no generation of resistance and the like, the drug has been reported to be used successfully abroad, and has great advantages in the aspect of treating multi-drug resistant bacteria.
Although phage application is promising, it is not neglected that phages often have a narrow host spectrum, one phage usually only lyses one bacterium, or only certain serotypes of the same bacterium. Some phages are also selective for the source of the host, e.g., some phages are lytic to a host of terrestrial origin and not to a host of marine origin. The reason for this is that phages have strict specificity for the host pair due to environmental impact on the host.
Disclosure of Invention
The invention aims to provide a high-schizolysis Edwardsiella tarda bacteriophage, a composition and application thereof, and aims to solve the problems of ineffective medicine application and the like caused by the infection of the Edwardsiella tarda in the aquaculture industry.
In order to achieve the above purpose, the present invention provides the following technical solutions: a high-lytic Edwardsiella tarda phage, which is Edwardsiella tarda phage PKP-ET-2022001, is named Latin literatureEdwardsiella tarda phageThe strain is preserved in China general microbiological culture Collection center (CGMCC) at the 8 th month of 2022 and the 31 th day, and the preservation number is CGMCC NO.45264; the host is Edwardsiella tarda of marine origin.
The invention further provides application of the phage in preparing medicines for resisting Edwardsiellosis, aquatic feed additives or environment disinfectants for aquafarms.
The invention further provides compositions comprising said phage.
Preferably, the composition is an Edwardsiellosis-resistant drug, an aquatic feed additive, an aquatic feed or an environmental disinfectant for an aquaculture farm.
The phage provided by the invention has higher cracking property on the Edwardsiella tarda from the ocean and the Edwardsiella tarda from the fresh water, and has wider cracking spectrum, thus having important significance and application prospect on preventing and treating the Edwardsiellosis in the aquaculture industry.
Drawings
FIG. 1 shows the colony morphology of Edwardsiella tarda ET-2022001 on TSA blood agar plates;
FIG. 2 shows the plaque of the phage PKP-ET-2022001 against a host bacterium;
FIG. 3 is a photograph of phage PKP-ET-2022001 observed by electron microscopy;
FIG. 4 shows the effect of the phage PKP-ET-2022001 on the lysis assay of host bacteria;
FIG. 5 is a stable genetic experiment of phage PKP-ET-2022001.
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.
1. Isolation and identification of host Edwardsiella tarda:
(1) Sampling from a Weifang fish farm, streaking and inoculating the samples on a TSA blood agar medium, placing the samples in a constant temperature incubator at 37 ℃ for culturing for 18-24 hours, and observing the morphology and the color of colonies on a flat plate. Picking out colony with smooth, moist and semitransparent surface and narrow beta-type hemolytic ring;
(2) Streaking was inoculated on TSA blood agar medium, and subcultured and purified 3-5 times until colonies of uniform morphology were obtained, as shown in FIG. 1. Single colony is picked up, streaked and inoculated in LB agar medium, colony is picked up, and then identification is carried out by 16S rRNA gene sequencing technology; after the identification, the scraped lawn was placed in 30% glycerol broth and stored at-80℃and the strain was designated ET-2022001. The strain ET-2022001 is confirmed to be Edwardsiella tarda by 16S rRNA gene sequencingEdwardsiella tarda。
2. Isolation of phages:
(1) And (3) treating a culture water sample: taking a sludge water sample from a farm, weighing 5g, adding the sludge water sample into 10mL of SM buffer solution for soaking overnight, centrifuging the overnight leachate at 10000rpm for 5min, taking supernatant, filtering the supernatant by a 0.22 mu m filter, and preserving the filtrate at 4 ℃ for later use;
(2) Phage enrichment: adding 0.1mL of Edwardsiella tarda suspension and 1mL of filtrate into 5mLLB broth, shaking at 37deg.C overnight, centrifuging at 10000rpm for 10min, and filtering the supernatant with 0.22 μm filter;
(3) Phage isolation: separating phage by double-plate method, mixing 0.1mL of Edwardsiella tarda suspension with 0.6% LB soft agar, spreading on LB solid plate, standing to absorb the mixed solution after soft agar is solidified, culturing in 37 deg.C incubator for 6-8 hr, collecting transparent plaque with inoculating loop, placing in 2mL SM buffer solution, preserving at 4deg.C overnight, and releasing phage completely to obtain phage, as shown in figure 2;
(4) Phage purification: separating phage by double-plate method, mixing mixed bacterial suspension filtrate 0.1mL and host Edwardsiella tarda bacterial suspension 0.2mL, adding soft agar cooled to about 50deg.C, mixing, spreading double-plate, culturing in 37 deg.C incubator for 6-8 hr, picking out smooth-edged single transparent spot in 1mLSM buffer solution, and storing at 4deg.C overnight. Mixing 0.1mL of leaching solution stored overnight with 0.1mL of bacterial suspension, adding soft agar cooled to about 50 ℃ for uniform mixing, paving double plates, culturing for 4-6 hours in a 37 ℃ incubator, and purifying until plaques are uniform in size and smooth in edge, thus obtaining purified phage;
(5) Phage preparation: adding 0.2mL of Edwardsiella tarda suspension and 1mL of purified phage suspension into 50mLLB broth, shaking at 37 ℃ for overnight, centrifuging at 10000rpm for 10min, filtering the supernatant with 0.22 μm filter, and collecting phage filtrate;
(6) Preservation of phage: phage suspension 1:1 was mixed with 60% glycerol and stored at-80 ℃. The phage is named Edwardsiella tarda phageEdwardsiella tarda phageThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC) at the 8 th month of 2022 and the 31 th day, and the preservation number is CGMCC NO.45264, and the preservation unit address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
3. Electron microscopy of phage PKP-ET-2022001:
taking 20 mu L of liquid containing crude phage particles, dripping the liquid on a copper mesh, naturally precipitating the liquid for 15 min, sucking redundant liquid from the side by using filter paper, adding a drop of 2% phosphotungstic acid (PTA) on the copper mesh to dye phage for 10min, sucking the dyed liquid from the side by using filter paper, and observing the phage form by using an electron microscope after a sample is dried. As shown in FIG. 3, phage PKP-ET-2022001 has a polyhedral, stereosymmetrical head, surrounding nucleic acid, about 107nm in diameter, and a tail about 208nm in length, the neck connecting the head and tail. According to the ninth report of the International taxonomic organization of viruses, the phage were classified as having the order Myoviridae.
4. Whole genome sequencing and analysis of phage PKP-ET-2022001:
constructing a library by using an Illumina TruSeq ™ Nano DNA Sample Prep Kit method; the method comprises the following specific steps:
library construction with an initial amount of 1. Mu.g of phage genomic DNA;
the Covaris M220 breaks the DNA to 300-500bp by ultrasound;
filling up the 3' end, adding A, and connecting an index joint (TruSeq ™ Nano DNA Sample Prep Kit);
library enrichment, PCR amplification for 8 cycles;
recovering the target band (Certified Low Range Ultra Agarose) from the 2% agarose gel;
quantitative TBS380 (PicoGreen), mixing and loading according to the data proportion;
bridge PCR amplification is carried out on the cBat solid carrier to generate clusters;
the Illumina Hiseq sequencing platform was subjected to 2X 150bp sequencing. Sequencing results show that the total length of the phage PKP-ET-2022001 genome is 42232bp, as shown in a sequence table SEQ ID: 1.
5. Cleavage effect test of phage PKP-ET-2022001 on host bacterium ET-2022001:
the host bacterial suspension cultured for 6 hours was inoculated into 100mLLB liquid medium at a ratio of 1:100, and cultured at 37℃for 2 hours (OD about 0.15, about 5.0X10) 8 cfu/mL, or adjusting bacterial liquid by using LB liquid culture mediumTo OD of 0.15) the bacterial suspensions were aliquoted into 6 sterile conical flasks, to 3 of which 0.2mL (about 2.5 x 10) of phage suspension was added at optimal multiplicity of infection ((MOI 0.1 and 1 respectively) 9 pfu/mL, filtered through a 0.22 μm membrane) with no phage added and only an equivalent amount of LB added as controls. After 0h, 2h, 4h, 6h, 8h and 10h, respectively, 0.2ml of detection OD600 values were taken and the changes in OD600 after phage addition were compared. Phage were added to the bacterial fluid, resulting in a change in the OD600 value due to the lysis of the host by the phage, thereby obtaining a phage lysis profile for the host. As shown in FIG. 4, the OD600 of the bacterial liquid added with the phage PKP-ET-2022001 is reduced compared with that of the control group, and particularly the lysis effect on bacteria of the system phage with MOI of 1 is obvious. Therefore, the phage has remarkable cracking effect on host bacteria Edwardsiella tarda ET-2022001, and the growth of the Edwardsiella tarda can be effectively inhibited by different action concentrations.
6. Stability genetic experiments with phage PKP-ET-2022001:
phage were serially passaged 30 times, equal amounts of phage and host bacteria were added to each culture medium, each culture time was 6h, then double plates were spread, phage were serially passaged 29 generations, and their titers were determined. As shown in FIG. 5, the phage PKP-ET-2022001 has stable titer as a whole in the passage process, which indicates that the phage has good biological genetic stability and is an excellent choice in the production process.
7. Analysis of the lytic lineage of phage PKP-ET-2022001:
the splitting effect of the bacteriophage PKP-ET-2022001 on 19 Edwardsiella tarda strains stored in a strain library is determined by adopting a double-layer flat plate method spot plate method, wherein 10 strains are of marine origin and 9 strains are of fresh water origin. Preparing different Edwardsiella tarda bacterial solutions, adding 200 mu L of bacterial solution to be detected which is cultured for 6 hours into 0.6% LB semisolid culture medium, uniformly mixing, pouring into a plate, standing and solidifying. mu.L phage suspension was added dropwise to a soft agar plate, and incubated at 37℃for about 6-8 hours, and the presence or absence of plaque was observed. As can be seen from Table 1, the phage PKP-ET-2022001 of the invention lyses up to 95% of Edwardsiella tarda strains of different origins. Thus, the phage PKP-ET-2022001 has good lysis effect on both fresh water and marine-derived Edwardsiella tarda strains.
TABLE 1 cleavage Profile of Edwardsiella tarda phage PKP-ET-2022001
The invention discovers and separates the Edwardsiella tarda of the aquatic product, and takes the Edwardsiella tarda as a host to separate and obtain phage PKP-ET-2022001, which has strong cracking effect on the Edwardsiella tarda in the aquaculture environment, and provides a good phage source for the control of the Edwardsiella tarda in the aquaculture environment for the industrialized production of phage. Under the condition that the phage is passaged for 30 times, the titer of the phage can still be kept at a higher level, which shows that the phage has stronger genetic stability, and the high stability has very important significance for production and transportation. The phage obtained by the invention can be used for preventing and treating diseases caused by Edwardsiella tarda, and has the characteristic of extremely high safety. The biological characteristics of the phage obtained by separation are researched, so that theoretical guiding significance is provided for the next step of developing new antibacterial drugs and biological control agents.
Claims (4)
1. A high-schizolysis Edwardsiella tarda phage is characterized in that: the phage is Edwardsiella tarda phage PKP-ET-2022001, latin literature nameEdwardsiella tarda phageThe strain is preserved in China general microbiological culture Collection center (CGMCC) at the 8 th month of 2022 and the 31 th day, and the preservation number is CGMCC NO.45264; the host is Edwardsiella tarda of marine origin.
2. The use of the high lytic Edwardsiella tarda phage of claim 1, characterized in that: the phage can be used for preparing medicine for resisting Edwardsiellosis, aquatic feed additive or environment disinfectant for aquaculture farm.
3. A composition characterized by: a phage comprising the Edwardsiella tarda having high lytic property according to claim 1.
4. A composition according to claim 3, characterized in that: the composition is an Edwardsiellosis-resistant drug, an aquatic feed additive, an aquatic feed or an environmental disinfectant for an aquatic farm.
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