Aeromonas veronii bacteriophage pAEv1818 and its application

Abstract

This application relates to the field of microbial technology, and discloses a Aeromonas veronii bacteriophage pAEv1818 and its application, specifically relating to a phage isolate that can specifically lyse Aeromonas veronii ( Vibrio parahaemolyticus ), and the application of this phage in bacteriostatic agents for crucian carp septicemia, thereby improving the survival rate of crucian carp infected by pathogenic bacteria, and being able to increase the total weight gain of crucian carp and enhance the enterprise benefits. This application strategy is green and pollution-free, can protect the aquaculture environment, and reduce the usage amount of antibiotics from the source. This phage belongs to the family Siphoviridae, and the strain preservation number is CGMCC No. 20295. The Aeromonas veronii bacteriophage provided by the present invention can be applied to bacteriostatic agents for preventing bacterial diseases of crucian carp, and can effectively improve the survival rate of crucian carp.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an Aeromonas veronii phage pAEv1818 and its application. Background Art

[0002] Crucian carp ( Crucian carp ) is a common variety of freshwater cultured fish and belongs to the genus Carassius. The annual loss of freshwater cultured fish due to bacterial diseases is high. Research has proven that Aeromonas septicemia is the most important bacterial infectious disease affecting the crucian carp aquaculture industry, and for a long time, researchers have believed that Aeromonas hydrophila ( Aeromonas hydrophila ) is the main pathogen of this disease. The latest research results of the Aquatic Animal Feed Innovation Team of the Feed Research Institute of the Chinese Academy of Agricultural Sciences show that Aeromonas veronii ( Aeromonas veronii ) is more virulent than Aeromonas hydrophila and is the main pathogenic bacterium in the crucian carp aquaculture industry. The relevant research results were published as a cover article in Environmental Microbiology. At the same time, Aeromonas veronii is also a recognized conditional pathogenic bacterium that is common to humans, livestock, and aquatic animals, and can cause epidermal ulcers, enteritis, and hemorrhagic septicemia in target animals.

[0003] Usually, to solve the problem of Aeromonas septicemia in the crucian carp aquaculture industry, vaccines or a large amount of antibiotics are used. However, the attenuated vaccines or antibiotic drugs for this disease have been used for many years, and the outbreak of Aeromonas septicemia and the resulting economic losses have not decreased. At the same time, the excessive use of antibiotics has caused new environmental problems and exacerbated the harm of viral diseases in crucian carp, seriously hindering the sustainable development of the crucian carp aquaculture industry. Therefore, a new antibacterial method is urgently needed. So far, the mainstream alternative method in the international research field is to use phage therapy, which has been involved in many fields, such as agriculture, animal husbandry, aquaculture, food safety assurance, and even human skin burn treatment. Phages are a general term for viruses that infect bacteria and fungi. They can specifically recognize and lyse host pathogenic bacteria. Currently, the research on using phage therapy to control bacterial diseases has become a new hot spot and trend and has gradually been recognized by authoritative institutions.

[0004] Compared with traditional antibiotics, the outstanding advantages of phage therapy are as follows: (1) Specifically lysing target pathogenic microorganisms without destroying the normal flora in the water environment. Traditional antibiotic therapy not only kills pathogenic bacteria but also destroys the normal microbial community in the water body, causing microecological imbalance and leading to opportunistic infections; (2) Phages are host-dependent and only act at the site of bacterial infection, disappearing with the clearance of the host bacteria and not remaining in the animal body; (3) Phages can self-replicate and proliferate, and their infection occurs in the water medium; (4) Safe and non-toxic side effects. Phages are composed of proteins and nucleic acids, and the end products after degradation are amino acids and nucleic acid fragments, which are safe for humans and animals.

[0005] Through the implementation of the action to reduce the use of aquaculture drugs in various places, the total amount of veterinary drugs used by aquaculture enterprises participating in the action has decreased by more than 5% on average year-on-year, and the use of antibiotic veterinary drugs has decreased by more than 20% on average; zero drug addition to feed has been achieved, and actively exploring the use of veterinary antibacterial drug alternatives is encouraged, gradually reducing the varieties and usage amounts of growth-promoting veterinary antibacterial drugs, and improving the level of healthy aquaculture. Several application modes of phages as drugs for preventing and treating bacterial diseases in aquaculture have been recorded, including feeding by mixing in feed, intramuscular or intraperitoneal administration, anal intubation, and immersion or direct release into the culture system. Although each application mode has its own advantages and disadvantages, it mainly depends on the nature of the bacterial pathogen. Currently, the phages and related patents on their applications that can be consulted mainly involve Vibrio (ZL201410108994) and Bacillus phages (ZL201410235510), and there is no report in the category of Aeromonas. Therefore, this invention application is the first of its kind. Summary of the Invention

[0006] In order to prevent bacterial septicemia caused by Aeromonas veronii during the breeding process of crucian carp and reduce the mortality rate caused by this disease, based on the pond breeding mode of fry crucian carp stocking and the outbreak characteristics of Aeromonas, the present invention provides a preparation of Aeromonas veronii phage that can be used for immersion or directly sprayed into the pond when and after crucian carp are stocked, and at the same time provides an application strategy for this phage.

[0007] To achieve the above invention purpose, the present invention adopts the following technical solutions:

[0008] An Aeromonas veronii phage pAEv1818, which is isolated with Aeromonas veronii AEv1810 as the host. This phage was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 10, 2020, with the deposit number CGMCC No.20295, and the taxonomic name is Aeromonas veronii phage; Address of the General Microbiological Center of the China Committee for Culture Collection of Microorganisms: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101.

[0009] Another object of the present application is to provide the use of Aeromonas veronii bacteriophage pAEv1818 with the preservation number of CGMCC No. 20295 in the preparation of an inhibitor against Aeromonas veronii.

[0010] Another object of the present application is to provide the use of Aeromonas veronii bacteriophage pAEv1818 with the preservation number of CGMCC No. 20295 in the preparation of a medicament for preventing diseases caused by Aeromonas veronii.

[0011] Further, the disease caused by Aeromonas veronii is septicemia.

[0012] Another object of the present application is to provide the use of Aeromonas veronii bacteriophage pAEv1818 with the preservation number of CGMCC No. 20295 in killing Aeromonas veronii in the spatial environment.

[0013] Further, the spatial environment includes water bodies, ground, sludge, feces, litter and feed.

[0014] Further, the specific steps of the use of Aeromonas veronii bacteriophage pAEv1818 in killing Aeromonas veronii in the spatial environment are as follows: when juvenile crucian carp (10 - 15 g) are stocked in large water bodies, the crucian carp are immersed in the suspension of the bacteriophage, and the application amount is ≥ 10 8 PFU / L of water body.

[0015] Further, the specific steps of the use of Aeromonas veronii bacteriophage pAEv1818 in killing Aeromonas veronii in the spatial environment are as follows: the suspension of the bacteriophage is diluted and then sprayed on the aquaculture pond, sprayed once every 10 days, continuously for 3 times, and the application amount each time is ≥ 10 5 PFU / M 3 of aquaculture water body.

[0016] The present application also discloses an antibacterial agent, which comprises Aeromonas veronii bacteriophage pAEv1818 with the preservation number of CGMCC No. 20295.

[0017] The tail of the Aeromonas veronii bacteriophage of the present application is 96 ± 1.1 nm long, and the head diameter is 35 ± 1.2 nm. It has a long and narrow tail structure and belongs to the family Siphoviridae. The one-step growth curve shows that the latent period of this bacteriophage infecting the host is 40 min, the lysis period is 80 min, and the burst size is 190 PFU / cell, indicating strong lytic ability.

[0018] Beneficial effects:

[0019] The Vibrio aerogenes phage pAEv1818 provided by the present invention and its applications have the following advantages compared with the prior art:

[0020] 1. Vibrio aerogenes infection is a serious bacterial disease in the process of crucian carp farming. By applying the lytic phage of the present invention in the form of immersion or spraying, the abundance of Vibrio aerogenes can be reduced, the infection of Vibrio aerogenes during the farming process can be effectively prevented, the survival rate of juvenile crucian carp can be improved, and economic losses can be reduced;

[0021] 2. By using the phage of the present invention to control the abundance of Vibrio aerogenes, it can replace antibiotics to a certain extent in preventing bacterial infections, reduce the usage amount of antibiotics in the process of sea cucumber farming, reduce the emergence probability of drug-resistant strains, improve the effect of antibiotic treatment for other diseases, and safeguard the production safety of the aquaculture industry and the ecological environment safety from the source;

[0022] 3. The phage preparation of the present invention can replace the use of traditional chemical drugs and antibiotics, and has the characteristics of being pollution-free, residue-free, cheap, highly efficient in antibacterial, etc. It is a natural bacteriostatic agent with broad application prospects and great potential. Description of the Drawings

[0023] Figure 1 These are the plaque and electron microscope pictures of the Vibrio aerogenes phage pAEv1818 of the present application. The left picture is the plaque of the Vibrio aerogenes phage pAEv1818, and the right picture is the electron microscope picture of the Vibrio aerogenes phage pAEv1818;

[0024] Figure 2 This is the one-step growth curve of the Vibrio aerogenes phage pAEv1818 of the present application;

[0025] Figure 3 This is the in vitro bacteriostatic curve of the Vibrio aerogenes phage pAEv1818 of the present application;

[0026] Figure 4 This is the picture of bacterial septicemia of crucian carp of the present application;

[0027] Figure 5 This is the picture of the effect of the Vibrio aerogenes phage pAEv1818 of the present application on the survival rate of crucian carp. Detailed Embodiments

[0028] Based on the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0029] Example 1

[0030] Screening and Purification of Aeromonas veronii Bacteriophage pAEv1818

[0031] (I)Water Sample Collection and Treatment

[0032] Take 200 mL of wastewater samples from a crucian carp breeding base in Yancheng and the sewage outlet of a certain aquatic product market respectively. After mixing, perform pretreatment on the water sample: add CaCl2 and MgCl2 to make their final concentrations 1 mmol / L, and act for 10 min.

[0033] (II)Enrichment of Bacteriophages in Water Samples

[0034] Centrifuge the pretreated water sample above at 10000 g for 5 min, and collect the supernatant; filter the supernatant through a 0.22 μm filter membrane to remove bacteria, impurities and other components, and obtain the original bacteriophage solution; take 10 mL of the filtrate and add it to 50 mL of bacterial liquid in the logarithmic growth phase (5 h after inoculation, when the final concentration of bacteriophages reaches 10 6 -10 7 CFU / mL, that is, the logarithmic growth phase), and culture overnight at 28℃ for 12 - 18 h. The purpose is to amplify the number of bacteriophages, observe and record; collect the bacterial liquid, centrifuge at 10000 g for 5 min, collect the supernatant, and then filter through a membrane (0.22 μm) to obtain the proliferated bacteriophage supernatant.

[0035] (III)Screening of Aeromonas veronii Bacteriophage pAEv1818

[0036] Adopt the double-layer plate method for the identification of bacteriophages. The lower layer is a 2216E solid medium with 1.5% agar, placed at 4℃ for standby, and placed at 28℃ for 30 min before use; the upper layer is a 2216E medium with 0.7% agar. Heat and dissolve the upper layer medium, place it in a water bath at 50℃ for standby. Take 8 10 mL centrifuge tubes, add 1 mL of host bacterial liquid to each tube, and aspirate the bacteriophage suspension for gradient dilution to 10 -6 . Take 10 μL of each of the 7 different concentration gradients of bacteriophage dilutions and add them to 10 mL centrifuge tubes. Set a control group, which only adds 1 mL of host bacterial liquid and 10 μL of 2216E medium. Act at 28℃ for 10 min, add 5 mL of 2216E medium containing 0.5% agar, mix well, and immediately add it to the upper layer. After solidification, incubate upside down for 24 h and observe whether there are plaques formed. If clear plaques are formed on the plate, it indicates that there are lytic bacteriophages against this host bacterium in the filtrate.

[0037] (IV)Purification of Aeromonas veronii Bacteriophage pAEv1818

[0038] The sizes and shapes of the initially isolated plaques were inconsistent. The isolated phages were further purified to form plaques with consistent sizes and morphologies on the plate. Use a sterile 200 μL pipette tip to pick one plaque each with significantly different sizes and morphologies, and add them separately to centrifuge tubes containing 1 mL of sterile PBS. Vortex for 1 min and place at 4°C for 4 h to fully release the phages into the PBS. Centrifuge at 4°C and 10,000 g for 5 min, collect the supernatant, filter through a membrane (0.22 μm), perform gradient dilution on the phage filtrate, and conduct double-layer culture. Repeat the above operations 3 times until the morphologies and sizes of the plaques that appear are completely consistent, thus obtaining purified phages. The purified phages were observed under a transmission electron microscope, and the electron micrographs of the phages are shown in the appendix Figure 1 。

[0039] (5) Enrichment and amplification of Aeromonas veronii phage pAEv1818

[0040] Liquid proliferation method was used for proliferation. The specific method is as follows: Add the phage solution to the host bacteria solution cultured for 12 h, then culture in a shaker at 28°C for 12 h. Centrifuge the mixture at 4°C and 10,000 g for 5 min to remove bacterial debris. Filter the supernatant through a 0.22 μm filter membrane to obtain a high-titer phage enrichment solution.

[0041] Example 2

[0042] One-step growth curve determination of Aeromonas veronii phage pAEv1818

[0043] The multiplicity of infection (MOI) of phage refers to the ratio of the number of phages to the number of host bacteria added at the initial infection. Add phages with an MOI of 0.1 to the host bacteria solution, mix well, let stand at 28°C for 15 min for adsorption; centrifuge at 4°C and 11,000 rpm for 10 min, discard the supernatant, resuspend the precipitate with LB liquid medium, repeat the above centrifugation operation to remove all unadsorbed phages. Resuspend the precipitate in 100 mL of LB liquid medium, and record the time as T0 = 0 at this time; culture at 28°C and 120 rpm, and take samples every 10 min to measure the phage titer.

[0044] The results showed that the latent period of this phage infecting the host was 40 min, the lysis period was 80 min, and the burst size was 190 PFU / cell, indicating strong lytic ability. The results are shown in Figure 2 。

[0045] Example 3

[0046] In vitro antibacterial curve determination of Aeromonas veronii phage pAEv1818

[0047] When detecting the in vitro lysis efficiency of phages in this example, the MOI value was set to 10, that is, the phage concentration was 10 times higher than the concentration of the bacterial suspension in the logarithmic phase, so as to increase the differences in the antibacterial curves between different treatment groups. The specific operations are as follows: The host bacteria were cultured to 10 8 CFU / mL, centrifuged at 4°C and 8000 × g for 5 min, and the supernatant was discarded. The precipitate was resuspended with an equal volume of LB medium; 100 μL of the resuspended solution was added to each well of a 96-well cell culture plate. At the same time, doxycycline sarafloxacin hydrochloride (10 mg / L) was used as a positive control, and three parallels were set for each treatment group (n = 3); 100 μL of the host bacteria suspension was set in the blank control group; the grouped cell culture plates were uniformly left standing and placed in an enzyme-linked immunosorbent assay (ELISA) reader for culture, cultured with shaking at 28°C, and the OD 600 value was detected with an ELISA reader every 1 h, the data were recorded, and graphs were plotted and analyzed using PrismGraphpad 5.0 data analysis software. The results are shown in Figure 3 , and phages can effectively inhibit the growth of Aeromonas veronii in vitro.

[0048] Example 4

[0049] Analysis of the application effect of Aeromonas veronii phage pAEv1818 in preventing and controlling diseases of crucian carp caused by Aeromonas veronii (laboratory level)

[0050] 120 healthy crucian carp purchased from a crucian carp farm in Yancheng were randomly divided into three groups, with 10 in each group and 4 replicates were set. The infection method was immersion treatment, and 30 L of water was placed in each culture tank.

[0051] The first group: negative control group, Aeromonas veronii was added to a final concentration of 10 5 CFU / mL;

[0052] The second group: positive control group, Aeromonas veronii was added to a final concentration of 10 5 CFU / mL, and after 3 h, sarafloxacin hydrochloride was added to a final concentration of 10 mg / L;

[0053] The third group: phage experimental group, Aeromonas veronii was added to a final concentration of 10 5 CFU / mL, and after 3 h, phages were added to a final concentration of 10 6 CFU / mL (MOI = 10).

[0054] The survival status of crucian carp in each group was observed, and the survival rate was statistically analyzed. The results of diseased crucian carp in the control group are shown in the appendix Figure 4 as shown. The intestine was swollen and congested, and the gills were bleeding. The results showed that although the survival rate of crucian carp in the phage treatment group was significantly lower than that in the antibiotic treatment group ( p <0.05), but compared with the negative control group, phages could effectively increase the survival rate of infected crucian carp (p <0.05), see attachment Figure 5 .

[0055] Example 5

[0056] Purify the phage lysate by the NaCl-PEG method (10 10 PFU / ml), expand the culture in a 50 L fermenter, filter through a plate filter and a 0.22 μm filter membrane, and collect the filtrate in a 30 L plastic bucket (10 8 PFU / ml). During the seedling release period (May - June), when stocking in large water bodies, immerse the crucian carp in the suspension of the phage, with a dosage of ≥10 8 PFU / L of water body, or dilute the phage suspension and spray it on the culture pond, spray once every 10 days, continuously for 3 times, with a dosage of ≥10 5 PFU / M 3 culture water body. The specific implementation details are as follows:

[0057] Experimental site: A certain agricultural development company in Yancheng

[0058] Time: May 16, 2021 - June 20, 2021

[0059] Crucian carp specifications: Healthy crucian carp with an average weight of 12 ± 1.6 g

[0060] Grouping of culture ponds: 1 control pond and 1 experimental pond, each covering an area of 5 mu

[0061] Product of Aeromonas veronii phage pAEv1818: Immerse the fry in the phage suspension each time when releasing the fry.

[0062] Cultivation conditions: Control the water temperature at 20 - 28 °C, pH is 7.3 - 7.6; During the experiment, the culture pond is aerated for 24 hours and fed once a day.

[0063] Experimental sampling: After 30 days of cultivation, randomly select 30 crucian carp from the control pond and the experimental pond respectively to calculate the weight gain and feeding rate, and investigate the growth indicators of the crucian carp.

[0064] The preliminary application results of the phage suspension are statistically shown in Table 1 below.

[0065] Table 1 Changes in some growth performance indicators of crucian carp after phage treatment

[0066]

[0067] It can be seen from Table 1 that immersing in the phage suspension of the present invention can effectively improve the growth performance of crucian carp. Among them, the total weight gain rate of the experimental pond exceeds that of the control pond by 14 percentage points after one month of cultivation, and the individual feeding rate is also significantly improved.

Claims

1. Aeromonas veronii bacteriophage pAEv1818, characterized in that, The Aeromonas veronii phage pAEv1818 is deposited in the China General Microbiological Culture Collection Center, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is September 10, 2020, and the deposit number is CGMCC No. 20295.

2. Application of the Aeromonas veronii phage pAEv1818 with the deposit number CGMCC No. 20295 in the preparation of an Aeromonas veronii inhibitor.

3. Application of the Aeromonas veronii phage pAEv1818 with the deposit number CGMCC No. 20295 in the preparation of a drug for preventing Aeromonas veronii-induced septicemia in crucian carp.

4. Application of the Aeromonas veronii phage pAEv1818 with the deposit number CGMCC No. 20295 in killing Aeromonas veronii in the space environment.

5. The application according to claim 4, wherein: The space environment includes water bodies, ground, silt, feces, litter, and feed.

6. The application according to claim 4, wherein The specific steps are as follows: When releasing 10 - 15 g of juvenile crucian carp in a large water body, immerse the crucian carp in the suspension of the phage, and the application amount is ≥ 10 8 PFU / L of water body.

7. The application according to claim 4, wherein The specific steps are as follows: Dilute the phage suspension and spray it on the aquaculture pond, spray once every 10 days, conduct it continuously for 3 times, and the application amount each time ≥ 10 5 PFU / M 3 aquaculture water body.

8. An antibacterial agent, characterized in that: It includes the Aeromonas veronii phage pAEv1818 with the deposit number CGMCC No. 20295.

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