Aeromonas sobria phage with wide splitting property and application of aeromonas sobria phage

Abstract

The invention discloses a wide-splitting-property aeromonas sobria bacteriophage and application thereof, and relates to the technical field of biological medicines. The invention relates to a wide splitting property aeromonas sobria bacteriophage, which is named as RKP-AE24005, is preserved in the China General Microbiological Culture Collection Center (CGMCC), and has a preservation number of CGMCC No.45948. The wide splitting property aeromonas sobria bacteriophage can be used for preparing an aeromonas sobria vaccine. The bacteriophage RKP-AE24005 is successfully separated by taking aeromonas sobria as a host, and experiments prove that when the bacteriophage RKP-AE24005 is used for cracking 100 different aeromonas sobria strains, 92 strains can be cracked, and the cracking rate is 92%; and the cracking property is relatively wide.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a broad-lytic temperate Aeromonas phage and its applications. Background Art

[0002] Aeromonas belongs to Gammaproteobacteria, Aeromonadales, Aeromonadaceae, and Aeromonas. There are more than 30 species and subspecies under Aeromonas, such as Aeromonas hydrophila, Aeromonas caviae, Aeromonas salmonicida, Aeromonas media, Aeromonas fonticola, Aeromonas veronii, Aeromonas anguillarum, Aeromonas sobria, Aeromonas punctata, etc.

[0003] Aeromonas sobria is a Gram-negative, non-fermenting bacterium belonging to the genus Aeromonas. Aeromonas sobria widely exists in water environments and can be transmitted through water bodies, feeds, or direct contact. It can infect the skin, gills, and internal organs of aquatic animals. After infection, aquatic animals may show symptoms such as ulcers, bleeding, and fin damage, and may even die in severe cases. It can cause serious harm to aquatic animals, especially fish. The infection routes of fish include wound infection, digestive tract infection, and direct contact infection. After infecting fish, Aeromonas sobria can cause various diseases, such as septicemia, ascites, and erythroderma. In addition, Aeromonas sobria may also cause septicemia, resulting in economic losses to the aquaculture industry.

[0004] Currently, antibiotics are mainly used to control Aeromonas sobria in aquaculture. Due to the improper use of antibiotics, the phenomenon of bacterial drug resistance is becoming more and more common. It is necessary to find antibiotic alternatives to control Aeromonas sobria, reduce the use of antibiotics, and protect human health and safety.

[0005] Phages are a type of virus that can infect and kill bacteria. Phages attach to the surface of bacteria, inject their genetic material, and use the bacterial cell mechanism to replicate themselves, ultimately causing the bacteria to lyse and release new phages. Phages have a high degree of host specificity in the process of inhibiting bacteria, can precisely target specific bacteria, and reduce the interference with normal flora; moreover, they have a fast reproduction rate and can rapidly increase after infecting bacteria, effectively controlling the number of bacteria; at the same time, phage therapy usually does not produce drug resistance because they act on bacteria through different mechanisms, reducing the risk of drug resistance development compared with traditional antibiotics; in addition, phage therapy has low toxicity and causes less damage to host cells. Therefore, phages are an effective alternative to antibiotic treatment for bacterial infections in aquatic animals.

[0006] Related studies have found that bacteriophages infecting Aeromonas sobria cannot infect all species of Aeromonas sobria, but only some species. The host range of bacteriophages is usually affected by the following factors:

[0007] (1) Host cell surface receptors: Bacteriophages rely on specific molecules on the bacterial surface as receptors to enter the cell; different strains of Aeromonas sobria may have different surface receptors or different receptor expression levels, so not all strains of Aeromonas sobria can be hosts for the same bacteriophage.

[0008] (2) Genetic differences: Different strains of Aeromonas sobria may have genetic differences. Some strains may lack the receptors or specific molecular mechanisms required by certain bacteriophages, resulting in the inability of the bacteriophages to infect them.

[0009] (3) Specificity of bacteriophages: Most bacteriophages have high host specificity. They usually can only infect bacteria that match their receptor structure; therefore, even among bacteria of the same species, there may be differences between different strains, resulting in some strains being able to be infected while others cannot.

[0010] Therefore, how to solve the above technical problems and develop an Aeromonas sobria bacteriophage with broad lysis ability against a majority of Aeromonas sobria is a technical problem currently faced by those of ordinary skill in the art.

[0011] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0012] In view of the above technical problems, embodiments of the present invention provide a broad-lysis Aeromonas sobria bacteriophage and its application to solve the problems raised in the above background art.

[0013] A broad-lysis Aeromonas sobria bacteriophage, named RKP-AE24005, was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on June 14, 2024, with the deposit number CGMCC No. 45948.

[0014] Preferably, the gene sequence of the broad-lysis Aeromonas sobria bacteriophage is as shown in SEQ ID NO.1.

[0015] It should be emphasized that when the phage RKP-AE24005 was deposited, the proposed taxonomic name was: Aeromonas sobria phage; however, the taxonomic name of Aeromonas sobria phage only indicates that the phage RKP-AE24005 in the present invention can lyse Aeromonas sobria, which does not mean that phages capable of lysing Aeromonas sobria belong to the same phage.

[0016] Currently, when phages are taxonomically named, due to the limitations of traditional naming, they are generally named after the host; however, whether phages are of the same kind cannot be judged solely by the host, and there are several reasons and factors to consider as follows:

[0017] (1) Different phages may have similar or cross-infecting host ranges, but are essentially different phages with genomic differences. For example, even if multiple phages can use Escherichia coli as a host, their genomes may be completely different.

[0018] (2) Phenotypic differences. Different phages, even if they can infect the same host, may have different phenotypic characteristics (such as morphological characteristics, infection efficiency, lysis pattern, lysis curve, drug resistance, etc.). Different phenotypes may indicate differences in their genomes or evolutionary paths.

[0019] A phage product comprising the broad-lysing Aeromonas sobria phage as described above.

[0020] A phage pharmaceutical preparation, the active ingredient of which comprises the broad-lysing Aeromonas sobria phage as described above.

[0021] Preferably, the phage pharmaceutical preparation further comprises other antibacterial or bactericidal active ingredients; the dosage form of the phage pharmaceutical preparation is one of powder, solution, emulsion, gel, granule or lyophilized product.

[0022] Preferably, the titer of the phage in the phage pharmaceutical preparation is 10 11 PFU / mL or more.

[0023] A water environment disinfectant, the active ingredient of which comprises the broad-lysing Aeromonas sobria phage as described above.

[0024] A biological antibacterial agent for treating fresh or frozen aquatic products, the active ingredient of which comprises the broad-lysing Aeromonas sobria phage as described above.

[0025] An application of the broad-lysing Aeromonas sobria phage as described above in aquaculture.

[0026] Preferably, the broad-lytic Aeromonas sobria phage is used to prevent fish from being infected with Aeromonas sobria or to treat fish infected with Aeromonas sobria.

[0027] The broad-lytic Aeromonas sobria phage and its application provided by the embodiment of the present invention have the following beneficial effects:

[0028] (1) In the present invention, a phage RKP-AE24005 was successfully isolated using Aeromonas sobria as the host. Through experiments, it was confirmed that phage RKP-AE24005 lysed 92 out of 100 different Aeromonas sobria strains, with a lysis rate of 92%; it has broad lysis ability.

[0029] (2) Under the condition of 30 passages of phage RKP-AE24005, a relatively high titer can still be maintained, indicating that this phage has strong genetic stability, and high stability is of great significance for production and transportation.

[0030] (3) The phage obtained in the present invention can be used for the prevention and treatment of diseases caused by Aeromonas sobria, and has extremely high safety characteristics. Description of the Drawings

[0031] Figure 1 This is the colony morphology of Aeromonas sobria on a BHI plate in the present invention;

[0032] Figure 2 This is the plaque of the phage on the host bacterium in the present invention;

[0033] Figure 3 This is the electron microscope observation photo of the phage in the present invention;

[0034] Figure 4 This is the result of the optimal multiplicity of infection of the phage in the present invention;

[0035] Figure 5 This is the result of the one-step growth curve of the phage in the present invention;

[0036] Figure 6 This is the result of the optimal growth temperature of the phage in the present invention;

[0037] Figure 7 This is the result of the pH stability of the phage in the present invention;

[0038] Figure 8 This is the result of the genetic stability experiment of the phage in the present invention. Detailed Embodiments

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the protection scope of the present invention.

[0040] In view of the above technical problems, the embodiments of the present invention provide a broad-lytic Aeromonas sobria phage and its application to solve the problems raised in the above background technology.

[0041] Example 1: Isolation and identification of Aeromonas sobria

[0042] Samples were collected from a Qingdao aquaculture farm. The samples were streaked and inoculated on BHI solid medium and cultured in a constant temperature incubator at 37°C for 24 h. The colony morphology and color on the plate were observed. Different colonies were picked, streaked and inoculated on BHI medium, and subcultured and purified 3-5 times Figure 1 as shown, until colonies with uniform morphology were obtained; single colonies were picked and streaked and inoculated in BHI agar medium, and colonies were picked, and then identified by 16S rRNA gene sequencing technology. It was identified as Aeromonas sobria; after the identification was completed, the bacterial lawn was scraped and placed in 30% glycerol broth and stored at -80°C.

[0043] Example 2: Isolation and purification of phage

[0044] (1) Treatment of aquaculture water samples: Sludge water samples were taken from the farm. 5 g was weighed and added to 10 mL of SM buffer and soaked overnight. Then the overnight leachate was centrifuged at 10000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter. The filtered solution was stored at 4°C for later use;

[0045] (2) Phage enrichment: 0.1 mL of Aeromonas sobria suspension and 1 mL of the filtered solution were added to 5 mL of LB broth, cultured overnight at 37°C on a shaker, and then centrifuged at 10000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter, and the filtered solution was reserved for use.

[0046] (3) Phage isolation: The double-plate method was used for phage isolation. 0.1 mL of Aeromonas sobria suspension was mixed with 0.6% LB soft agar and spread on an LB solid plate. After the soft agar solidified, 0.1 mL of the filtered solution was spotted on the plate, left to stand until the mixture was absorbed, and then placed in a 37°C incubator for 8 h. The transparent phage plaque was picked with an inoculation loop and placed in 2 mL of SM buffer and stored at 4°C overnight to allow the phage to be completely released, thus obtaining the phage.

[0047] (4) Phage purification: The double-plate method was used for phage isolation. 0.1 mL of the filtered mixed bacterial suspension and 0.2 mL of the host Aeromonas sobria bacterial suspension were mixed evenly, added to the soft agar cooled to about 50 °C and mixed evenly, then spread on a double plate and placed in an incubator at 37 °C for 8 h. A single transparent plaque with a smooth edge was picked and placed in 1 mL of SM buffer and stored overnight at 4 °C. 0.1 mL of the overnight-preserved leachate and 0.1 mL of the bacterial suspension were mixed, added to the soft agar cooled to about 50 °C and mixed evenly, spread on a double plate, and cultured in an incubator at 37 °C for 4 - 6 h. Purification was carried out according to this step until the phage plaques were of uniform size and had smooth edges, and the purified phage was obtained.

[0048] (5) Phage preparation: 0.2 mL of the Aeromonas sobria bacterial suspension and 1 mL of the purified phage suspension were added to 50 mL of LB broth, cultured overnight on a shaker at 37 °C, then centrifuged at 10000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter, and the phage filtrate was reserved for use.

[0049] (6) Phage preservation method: The phage suspension was mixed with 60% glycerol at a ratio of 1:1 and stored at -80 °C, named RKP-AE24005.

[0050] Example 3: Electron microscopy observation of phage

[0051] 20 μL of the liquid containing phage crude particles was dropped on a copper grid, allowed to precipitate naturally for 15 min, and the excess liquid was blotted from the side with filter paper. A drop of 2% phosphotungstic acid (PTA) was added to the copper grid to stain the phage for 10 min, then the staining solution was blotted from the side with filter paper. After the sample was dried, the morphology of the phage was observed with an electron microscope.

[0052] Figure 3 As shown: The phage has a polyhedral icosahedral head that encapsulates nucleic acid, with a diameter of about 94 nm, and a tail about 100 nm long. The neck connects the head and the tail. According to the Ninth Report of the International Committee on Taxonomy of Viruses, this phage is classified into the order Caudovirales, family Myoviridae.

[0053] Example 4: Whole-genome sequencing

[0054] Phage whole-genome sequencing and analysis: The Illumina TruSeq TM Nano DNA Sample PrepKit method was used to construct a library; the specific steps were as follows:

[0055] (1) Construct a library starting from 1 μg of phage genomic DNA;

[0056] (2) Covaris M220 ultrasonic disrupts DNA to 300 - 500 bp;

[0057] (3) Fill in the 3' end, add A, and ligate the index adapter (TruSeq TM Nano DNA Sample Prep Kit);

[0058] (4) Enrich the library and perform 8 cycles of PCR amplification;

[0059] (5) Recover the target band using 2% agarose gel (Certified Low Range Ultra Agarose);

[0060] (6) Quantify using TBS380 (Picogreen) and mix and load onto the machine according to the data ratio;

[0061] (7) Perform bridge PCR amplification on the cBot solid support to generate clusters;

[0062] (8) Use the Illumina Hiseq sequencing platform for 2×150 bp sequencing.

[0063] Analysis of the phage genome: The whole genome of phage RKP - AE24005 is 171,224 bp in size, containing 257 genes. The average length of the genes is 662 bp, and the G + C% content is 43.3%. Through prediction and analysis, the genome of phage RKP - AE24005 contains 257 open reading frames (ORFs), with an average length of 207 bp. It contains 105 known coding functional proteins, which are divided into 3 parts according to protein function: 38 structural proteins, 67 functional proteins, and 6 other proteins. Among them, ORF11, ORF48, and ORF183 are its lyases, as well as 146 hypothetical proteins.

[0064] Example 5: Determination of phage titer

[0065] Take 100 μL of the host bacterial liquid cultured to the exponential phase, mix it well in a 5 mL centrifuge tube, place it on the LB semi-solid agar, and completely cover it on a 20 mL LB solid agar plate (9 cm in diameter). After the semi-solid agar dries, respectively pipette 100 μL of the purified phage concentrate onto the plate. Spread one phage on 3 plates and incubate overnight at 37°C. Pick the phage plaques into 20 mL of SM buffer, place it at 4°C. The next day, take the supernatant, filter it through a 0.22 μm filter membrane. Take 100 μL of the filtrate and dilute it 10-fold serially with sterile water. Then, mix 100 μL of the bacterial liquid and the phage liquid each in a double plate, and incubate the plate overnight in a 37°C incubator. The phage titer of phage RKP-AE24005 was determined by the above method. Select countable plates, count the phage plaques, and calculate the phage titer (PFU / mL) as the number of plaques × dilution factor / sample volume (mL). Experimental results and analysis: After measurement, the phage titer can reach 10 11 PFU / mL.

[0066] Example 6: Determination of the optimal multiplicity of infection (MOI) of phage

[0067] MOI refers to the ratio of the number of phages to the number of host bacteria at the initial infection. Adjust the concentration of the host bacterial liquid cultured to the exponential phase to 10 8 CFU / mL. Set MOIs to 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 respectively. Add the phage liquid with the determined titer to the bacterial liquid according to the ratio. Mix 500 μL of the bacterial liquid and the phage liquid each, and incubate with shaking at 37°C and 200 r / min for 8 h. Centrifuge the mixed culture at 10000 r / min for 10 min and measure the phage titer. The multiplicity of infection with the highest titer is the optimal multiplicity of infection. The optimal multiplicity of infection of phage RKP-AE24005 was determined by the above method.

[0068] The results are as Figure 4 shown. When the multiplicity of infection of the phage is 0.1, the phage titer is the highest. Therefore, the optimal multiplicity of infection of phage RKP-AE24005 is 0.1, and the corresponding highest titer is 10 11 PFU / mL.

[0069] Example 7: One-step growth curve of phage

[0070] Mix 500 μL of the phage solution and 500 μL of the host bacteria solution in the ratio required for the optimal multiplicity of infection, and incubate at 37 °C for 15 min. Centrifuge at 10,000 r / min for 1 min, and wash the precipitate three times with LB liquid. Add 10 mL of pre-warmed LB liquid medium at 37 °C, mix well, and immediately place it in a shaker at 37 °C for shaking culture. Take samples every 10 min to measure the phage titer. Do three parallels, and take the average of the results. Use the infection time as the abscissa and the logarithm of the phage titer in the infection system as the ordinate to plot a one-step growth curve, obtain the latent period and burst period of phage RKP-AE24005, and calculate the burst size. Burst size = total number of phages at the end of the phage burst / total number of bacteria at the initial stage of the phage.

[0071] Results of the determination of the one-step growth curve of phage ( Figure 5 ) showed that within 100 min after the phage infected the host bacteria, the number of phages did not increase, and this period was called the latent period; from 100 to 250 min after infecting the host bacteria, the number of phages increased rapidly, and this period was the burst period of the phage, that is, the burst period of the phage was about 150 min, and the burst size was 10 4 pfu mL -1 .

[0072] Example 8: Determination of the optimal growth temperature of phage

[0073] Dilute the phage stock solution by ten-fold gradient to a suitable concentration, take 100 μL and mix it with an equal volume of the bacteria solution, pour double-layer plates, and incubate at 15 °C, 20 °C, 25 °C, 30 °C, 37 °C, 40 °C, 45 °C and 50 °C for 4 hours, and detect the phage titer. Use the temperature as the abscissa and the logarithm of the phage titer as the ordinate to plot the growth temperature curve of the phage respectively. Figure 6 As shown, for phage RKP-AE24005, the titer was the highest at 30 °C, and its optimal growth temperature was 30 °C.

[0074] Example 9: Determination of the pH stability of phage

[0075] Adjust the pH values of the LB liquid medium to 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. Take 100 μL of each and mix it with an equal volume of the phage stock solution. After incubating at 37 °C for 2 h, use the double-layer plate method to measure the titer, and set three replicates for each pH. Use the pH as the abscissa and the logarithm of the phage titer as the ordinate to plot the pH stability curve of the phage respectively.

[0076] The results of the phage pH stability determination are shown in 7; its titer was the highest at pH = 7, and its titer could maintain a relatively high level of 10 8 -10 11 pfu·mL-1 within the range of pH 5 - 10.

[0077] Example 10: Genetic Experiment on the Stability of Bacteriophage

[0078] The bacteriophage was serially passaged 30 times. An equal amount of bacteriophage and host bacteria was added to the culture medium for each generation, and the culture time for each generation was 6 h. Then, double plates were spread. After the bacteriophage was serially passaged 29 times, its titer was measured. The results are as Figure 8 shown.

[0079] Figure 8 shown. During the passage process of the bacteriophage, the titer was generally stable, indicating that the biological genetic stability of the bacteriophage was good, which was an excellent choice for the production process.

[0080] Example 11: Analysis of the Lysis Spectrum of Aeromonas sobria Bacteriophage

[0081] The double-layer plate dotting method was used to measure the lysis effect of bacteriophage RKP-AE24005 on 100 strains of Aeromonas sobria preserved in the strain library. Different Aeromonas sobria bacterial solutions were prepared. 200 μL of the bacterial solution to be tested cultured for 6 h was added to 0.6% LB semi-solid medium and mixed evenly, then poured into a petri dish and left to solidify. 20 μL of the bacteriophage suspension was dropped onto the soft agar petri dish and cultured at 37°C for about 6 - 8 h, and the appearance of plaques was observed. The results are shown in Table 1.

[0082] As can be seen from Table 1, bacteriophage RKP-AE24005 could lyse 92 out of 100 different Aeromonas sobria strains, and the lysis rate was 92%, showing a relatively wide lysis rate.

[0083] Table 1 Lysis Spectrum of Aeromonas sobria Bacteriophage

[0084]

[0085]

[0086] Example 12: Experiments on the Prevention and Treatment of Fish Infections by Aeromonas sobria Bacteriophage

[0087] Preventive experiment: To evaluate the role of bacteriophage in preventing Aeromonas sobria infection. Healthy zebrafish were divided into two groups; prevention group: intraperitoneally injected with bacteriophage (10 11 PFU / fish) 24 hours before infection. Control group: injected with an equal amount of normal saline 24 hours before infection. Both groups were inoculated with the same dose of Aeromonas sobria (10 mL). The clinical symptoms, survival rate, and histopathological changes within 7 days after infection were observed. The experimental results are shown in Table 2 below.

[0088] Table 2 Results of the Preventive Experiment

[0089]

[0090] Therapeutic experiments: To comprehensively evaluate the therapeutic effect of phages, the following experiments were also conducted. Different infection dose experiments: Test the harm of different concentrations of Aeromonas sobria (such as 10 5 CFU / mL, 10 7 CFU / mL, 10 9 CFU / mL) to fish. Different phage dose experiments: Test the influence of different phage concentrations (such as 10 8 PFU / fish, 10 11 PFU / fish, 10 13 PFU / fish) on the therapeutic effect. The experimental results are shown in Table 3-4 below.

[0091] Table 3 Results of different infection dose experiments

[0092]

[0093]

[0094] Table 4 Results of different phage dose experiments

[0095]

[0096] Results: The results of the preventive experiment show that the application of phages 24 hours before infection can significantly reduce the infection rate, and the survival rate is increased to 90%. The different infection dose experiments show that phages can still effectively control the infection at a high infection dose (10 9 CFU / mL), and the survival rate reaches 80%. The different phage dose experiments show that the higher the phage dose, the more significant the therapeutic effect (the survival rate reaches 95% at 10 11 PFU / fish). In the fish infection model, after the experimental group of fish was treated with phages, the clinical symptoms were alleviated and the survival rate was increased. The results of statistical analysis show that there is a significant difference between the phage treatment group and the control group, and phage treatment has a positive therapeutic effect on Aeromonas sobria infection in fish.

[0097] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A broad-lytic Aeromonas sobria phage, characterized in that, It is named RKP-AE24005 and is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 45948.

2. The Aeromonas sobria phage with broad lysis property according to claim 1, characterized in that, Its gene sequence is shown in SEQ ID NO.

1.

3. A phage product, characterized in that, Comprising the broad-lytic temperate Aeromonas phage as described in claim 1 or 2.

4. A phage pharmaceutical preparation, characterized in that, Its active ingredient comprises the broad-lytic temperate Aeromonas phage as described in claim 1 or 2.

5. The phage pharmaceutical preparation according to claim 4, characterized in that, The phage pharmaceutical preparation further comprises other antibacterial or bactericidal active ingredients; the dosage form of the phage pharmaceutical preparation is one of powder, solution, emulsion, gel, granule or freeze-dried preparation.

6. The phage pharmaceutical preparation according to claim 4, characterized in that, The titer of the phage in the phage pharmaceutical preparation is 10 11 PFU / mL or higher.

7. A water environment disinfectant, characterized in that, The active ingredient of the water environment disinfectant comprises the broad-lytic temperate Aeromonas phage as described in claim 1 or 2.

8. A biological bacteriostatic agent for processing fresh or frozen aquatic products, characterized in that, The active ingredient of the biological bacteriostatic agent comprises the broad-lytic temperate Aeromonas phage as described in claim 1 or 2.

9. Use of the broad-lytic temperate Aeromonas phage as described in claim 1 or 2 in aquaculture.

10. Use of the Aeromonas sobria phage with broad lysis ability according to claim 9 in aquaculture, characterized in that, The broad-lytic temperate Aeromonas phage is used for preventing fish from being infected with Aeromonas or treating fish infected with Aeromonas.

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