CN115747171A - Aeromonas veronii phage pAEv1818 and application thereof - Google Patents

Abstract

The application relates to the technical field of microorganisms, discloses an aeromonas veronii phage pAEv1818 and application thereof, and particularly relates to an aeromonas veronii (a strain capable of specifically cracking aeromonas veronii)Vibrio parahaemolyticus) The phage isolate of (2), and theThe application of the phage in the crucian bacterial septicemia bacteriostatic agent improves the survival rate of crucian infected by pathogenic bacteria, can improve the total weight gain of crucian, and increases enterprise benefits. The application strategy is green and pollution-free, can protect the aquaculture environment, and reduces the use amount of antibiotics from the source. The bacteriophage belongs to the long-tail bacteriophage family, and the strain preservation number is CGMCC No.20295. The Aeromonas veronii phage provided by the invention can be applied to a crucian bacterial disease prevention bacteriostatic agent, and can effectively improve the survival rate of crucian.

Description

Aeromonas veronii phage pAEv1818 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to an aeromonas veronii phage pAEv1818 and application thereof.

Background

Crucian carp (A)Carassius auratus) Is a common species of freshwater fish, and belongs to the genus Carassius. The annual loss of freshwater fish due to bacterial diseases is high. Studies have shown that aeromonas septicemia is the most predominant bacterial infectious disease affecting the crucian breeding industry, and researchers have long thought that aeromonas hydrophila (A)Aeromonas hydrophila) Is the main etiological agent of the disease. The latest research results of aquatic animal feed innovation team of feed institute of agricultural academy of sciences show that Aeromonas veronii: (Aeromonas veronii) The toxicity of the strain is stronger than that of aeromonas hydrophila, the strain is a main pathogenic bacterium in the crucian breeding industry, and related research results are published on Environmental Microbiology as cover articles. Meanwhile, the aeromonas veronii is also a well-known conditioned pathogen which is commonly suffered by people, livestock and aquatic animals and can cause epidermal ulcer, enteritis and hemorrhagic septicemia of target animals.

Generally, in order to solve the problem of bacterial septicemia in the crucian breeding industry, a vaccine or a large amount of antibiotics are used, however, attenuated vaccines or antibiotic medicines for the disease have been used for many years, and the outbreak of aeromonas septicemia and the economic loss caused by the aeromonas septicemia are not reduced. Meanwhile, the excessive use of antibiotics causes new environmental problems, aggravates the virus disease damage of the crucian, and seriously inhibits the sustainable development of the crucian breeding industry, so a novel antibacterial mode is urgently needed. To date, the mainstream alternative in the international research field is to use phage therapy, which has been applied to many fields such as agriculture, animal husbandry, aquaculture, food safety guarantee, and even human skin burn treatment. Bacteriophage is a generic name of viruses infecting bacteria and fungi, which can specifically identify and lyse host pathogenic bacteria, and the current research on applying bacteriophage therapy to control bacterial diseases has become a new hot spot and trend and is gradually approved by authorities.

The outstanding advantages of phage therapy over traditional antibiotics are: (1) Target pathogenic microorganisms are specifically cracked, and normal flora in water environment cannot be damaged. The traditional antibiotic treatment destroys the normal microbial community in the water body while killing pathogenic bacteria, causes micro-ecological imbalance and causes opportunistic infection; (2) The phage is host-dependent, only acts on the infected part of the bacteria, disappears along with the removal of host bacteria, and cannot remain in the animal body; (3) The phage can be replicated and proliferated, and the infection of the phage occurs in an aqueous medium; (4) The bacteriophage consists of protein and nucleic acid, and the degraded end product is amino acid and nucleic acid segment, and is safe to human and animal.

By implementing the drug decrement action of aquaculture in various places, the total amount of veterinary drugs used by enterprises participating in action culture is averagely reduced by more than 5 percent in the same ratio, and the total amount of antibiotic veterinary drugs used by enterprises is averagely reduced by more than 20 percent; realizes zero drug addition of the feed, encourages active exploration and use of veterinary antibacterial drug substitutes, gradually reduces the use variety and the use amount of the growth-promoting veterinary antibacterial drug, and improves the healthy breeding level. Several modes of application of bacteriophages as agents for the prevention and treatment of bacterial diseases in aquaculture have been described, including feeding with dressing, intramuscular or intraperitoneal administration, anal intubation, and immersion or direct release in culture systems. Although each mode of application has advantages and disadvantages, it depends primarily on the nature of the bacterial pathogen. The related patents of the phage and the application class thereof which can be found at present mainly relate to the aspects of vibrio (ZL 201410108994) and bacillus phage (ZL 201410235510), but are not reported in the category of aeromonas, so the application of the invention is the first time.

Disclosure of Invention

In order to prevent bacterial septicemia caused by Aeromonas veronii in the crucian breeding process and reduce the mortality caused by the disease, the invention provides an Aeromonas veronii phage preparation which can be used for immersion bath or directly sprayed on a pond during and after the crucian breeding based on the pond breeding mode of the crucian breeding in the seedling stage and the outbreak characteristics of the Aeromonas veronii, and also provides an application strategy of the phage.

In order to realize the purpose, the invention adopts the following technical scheme:

an Aeromonas veronii phage pAEv1818 is obtained by separating an Aeromonas veronii AEv1810 serving as a host, and is preserved in the China general microbiological culture Collection center (CGMCC) in 9, 10 and 2020, with the preservation number of CGMCC No.20295 and is named as the Aeromonas veronii phage in a classified manner; china general microbiological culture Collection center address: west road No. 1, north chen, chaoyang district, beijing, zip code: 100101.

another object of the present application is the use of the Aeromonas veronii phage pAEv1818 with the accession number CGMCC No.20295 in the preparation of an inhibitor of Aeromonas veronii.

Another objective of the application is to use the Aeromonas veronii phage pAEv1818 with the collection number of CGMCC No.20295 in preparing a medicament for preventing diseases caused by Aeromonas veronii.

Further, the disease caused by Aeromonas veronii is sepsis.

Another objective of the application is to provide the application of the Aeromonas veronii phage pAEv1818 with the preservation number of CGMCC No.20295 in killing Aeromonas veronii in a space environment.

Further, the space environment comprises water, ground, sludge, excrement, bedding and feed.

Further, the application of the Aeromonas veronii phage pAEv1818 in killing Aeromonas veronii in a space environment specifically comprises the following steps: carassius auratus in seedling stage(10-15 g) when the large water body is put in a stocking, the crucian is soaked in the phage suspension, and the application amount is more than or equal to 10 ⁸ PFU/L water body.

Further, the application of the Aeromonas veronii phage pAEv1818 in killing Aeromonas veronii in a space environment specifically comprises the following steps: diluting the phage suspension, spraying the phage suspension to a culture pond for 3 times continuously at intervals of 1 time and 10 days or more, wherein the application amount of each time is more than or equal to 10 ⁵ PFU/M ³ And (5) cultivating a water body.

The application also discloses a bacteriostatic agent which comprises Aeromonas veronii phage pAEv1818 with the preservation number of CGMCC No.20295.

The Aeromonas veronii phage has a tail length of 96 +/-1.1 nm and a head diameter of 35 +/-1.2 nm, has a long and narrow tail structure, and belongs to the family of long-tail phage (Siphoviridae); the one-step growth curve shows that the incubation period of the phage infected host is 40 min, the lysis period is 80 min, the lysis amount is 190 PFU/cell, and the lysis is strong.

Has the advantages that:

compared with the prior art, the aeromonas veronii phage pAEv1818 provided by the invention and the application thereof have the following advantages:

1. the aeromonas veronii infection is a serious bacterial disease in the crucian breeding process, and by using the lytic bacteriophage of the invention and applying the bacteriophage in a bath or splashing way, the abundance of the aeromonas veronii can be reduced, the infection of the aeromonas veronii in the breeding process can be effectively prevented, the survival rate of the crucian in the seedling stage can be improved, and the economic loss can be reduced;

2. the bacteriophage is used for controlling the abundance of Aeromonas veronii, can replace antibiotics to a certain extent to prevent and treat bacterial infection, can reduce the use amount of the antibiotics in the stichopus japonicus culture process, reduce the occurrence probability of drug-resistant strains, improve the effect of the antibiotics on treating other diseases, and maintain the production safety and ecological environment safety of the culture industry from the source;

3. the phage preparation can replace the use of traditional chemical drugs and antibiotics, has the characteristics of no pollution, no residue, low price, high-efficiency antibacterial property and the like, and is a natural bacteriostatic agent with wide application prospect and huge potential.

Drawings

FIG. 1 is a plaque and an electron microscope picture of the Aeromonas veronii phage pAEv1818, wherein the left picture is the plaque of the Aeromonas veronii phage pAEv1818, and the right picture is the electron microscope picture of the Aeromonas veronii phage pAEv 1818;

FIG. 2 is a graph of one step growth of the Aeromonas veronii phage pAEv1818 of the present application;

FIG. 3 is a graph of in vitro inhibition of Aeromonas veronii phage pAEv1818 of the present application;

fig. 4 is a diagram of crucian bacterial septicemia of the present application;

FIG. 5 is a graph showing the effect of Aeromonas veronii phage pAEv1818 on crucian survival rate.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1

Screening and purification of Aeromonas veronii phage pAEv1818

Water sample collection and treatment

Taking 200mL of wastewater samples from a certain crucian breeding base in a salt city and a sewage outlet of a certain aquaculture market, mixing the wastewater samples, and then pretreating the water samples: adding CaCl ₂ 、MgCl ₂ The final concentration was 1 mmol/L and the reaction was allowed to proceed for 10 min.

(II) enrichment of bacteriophages in water sample

Centrifuging the pretreated water sample for 5min by 10000 g, and collecting the supernatant; filtering the supernatant with 0.22 μm filter membrane, removing thallus and impurities to obtain bacteriophage stock solution; 10 mL filtrate was added to the medium in the logarithmic growth phase (5 h after inoculation, final phage concentration of 10 ⁶ -10 ⁷ CFU/mL, namely logarithmic growth phase), in the 50 mL bacterial solution, culturing 12-18 h at 28 ℃ overnight so as to amplify the quantity of the phage, observing and recording; collecting bacterial liquid, centrifuging for 5min at 10000 g, and collectingCollecting the supernatant, and filtering with a filter membrane (0.22 μm) to obtain the supernatant of the proliferated phage.

Screening of Aeromonas (tri) Vickers phage pAEv1818

Identifying bacteriophage by double-layer plate method, placing 2216E solid culture medium with lower layer of 1.5% agar at 4 deg.C for use, and placing at 28 deg.C for 30 min before use; the upper layer is 0.7% agar 2216E culture medium, the upper layer culture medium is heated and dissolved, and is placed in a water bath kettle at 50 ℃ for standby, 810 mL centrifuge tubes are taken, 1 mL host bacterium liquid is respectively added, and the bacteriophage suspension is sucked to carry out gradient dilution to 10 ^-6 Taking out 10 mu L of 7 phage dilutions with different concentration gradients, adding the dilutions into a 10 mL centrifuge tube, arranging a control group, adding only 1 mL host bacterial liquid and 10 mu L2216E culture medium, acting at 28 ℃ for 10 min, adding 5 mL 2216E culture medium containing 0.5% agar, mixing uniformly, immediately adding the mixture into the upper layer, and after solidification, carrying out inverted culture on 24 h to observe whether plaques are formed. If a plaque forms on the plate, this indicates the presence of lytic phage in the filtrate against the host bacterium.

Purification of Aeromonas (tetra) Vickers phage pAEv1818

The size and shape of the plaque are not consistent, and the phage is further purified to form the plaque with consistent size and shape on the plate. Picking up each of the plaques with obvious difference in shape and size by using a sterile 200 mu L gun head, respectively adding the plaques into a centrifuge tube of 1 mL sterile PBS, carrying out vortex oscillation for 1 min, placing at 4 ℃, and 4 h to fully release the phage into the PBS; centrifuging at 4 deg.C and 10000 g for 5min, collecting supernatant, passing through membrane (0.22 μm), performing gradient dilution on phage filtrate, performing double-layer culture, and repeating the above operation for 3 times until the plaque shape and size are completely consistent to obtain purified phage. The purified phage was observed by transmission electron microscopy, and the photograph of the phage electron microscopy is shown in figure 1.

Enrichment and amplification of Aeromonas veronii phage pAEv1818

Proliferation is carried out by liquid proliferation method. The specific method comprises the following steps: adding phage liquid into host bacterial liquid cultured in 12 h, culturing in 12 h in 28 deg.C shaking table, centrifuging the mixed liquid at 4 deg.C and 10000 g for 5min, removing bacterial debris, and filtering the supernatant with 0.22 μm filter membrane to obtain high titer phage enrichment liquid.

Example 2

Determination of Aeromonas veronii phage pAEv1818 one-step growth Curve

The multiplicity of infection (MOI) of a bacteriophage refers to the ratio of the number of phage and host bacteria added at the time of initial infection. Adding phage with multiplicity of infection of 0.1 into host bacterial liquid, mixing, standing at 28 deg.C, and adsorbing for 15 min; centrifuging at 4 deg.C 11000 rpm for 10 min, discarding supernatant, re-suspending the precipitate with LB liquid culture medium, and repeating the above centrifugation operation to remove unadsorbed phage. The pellet was resuspended in 100 mL LB broth, at which time T was timed ₀ =0; the culture was carried out at 28 ℃ and 120 rpm, and samples were taken every 10 min to determine the titer of the phage.

The result shows that the incubation period of the phage infection host is 40 min, the lysis period is 80 min, the lysis amount is 190 PFU/cell, and the lysis is strong, and the result is shown in figure 2.

Example 3

Determination of in vitro inhibition curve of Aeromonas veronii phage pAEv1818

In the embodiment, when the in-vitro phage lysis efficiency is detected, the MOI value is set to 10, that is, the concentration of the phage is 10 times higher than that of log-phase bacteria liquid, so that the difference of the bacteriostatic curves among different treatment groups is improved. The specific operation is as follows: culturing the host bacterium to 10 ⁸ CFU/mL，4℃、8000 × gCentrifuging for 5min, discarding the supernatant, and resuspending the precipitate with an equal volume of LB medium; adding 100 mu L of resuspension into a 96-well cell culture plate, taking doxycycline sarafloxacin hydrochloride (10 mg/L) as a positive control, and setting three non-treatment groups in parallel (n = 3); setting 100 mu L of host bacterium suspension liquid in a blank control group; uniformly standing the grouped cell culture plates, culturing in a microplate reader, performing shake culture at 28 ℃, and detecting OD (optical density) by the microplate reader every 1h ₆₀₀ Data were recorded and plotted using Prism graphic 5.0 data analysis software, and the results are shown in FIG. 3, where the phage was effective in inhibiting the growth of Aeromonas veronii in vitro.

Example 4

Application effect analysis (laboratory level) for preventing and controlling crucian diseases caused by Aeromonas veronii phage pAEv1818

Taking 120 tails of healthy crucian carps purchased from a certain crucian breeding factory in the salt city, randomly dividing the healthy crucian carps into three groups, setting 10 carps in each group for 4 times, treating by soaking bath in a poison counteracting mode, and placing 30L of water in a non-breeding box.

A first group: negative control group, adding Aeromonas veronii to final concentration of 10 ⁵ CFU/mL；

Second group: positive control group, aeromonas veronii was added to a final concentration of 10 ⁵ CFU/mL,3 h, adding sarafloxacin hydrochloride to a final concentration of 10 mg/L;

third group: phage panel, aeromonas veronii added to a final concentration of 10 ⁵ CFU/mL,3 h, was added to phage to a final concentration of 10 ⁶ CFU/mL（MOI=10）。

Observing the survival state of each group of crucian, counting the survival rate, and showing the results of the diseased crucian in the control group as shown in figure 4, intestinal swelling and congestion, and gill bleeding. The results showed that although the survival rate of the phage-treated crucian carp was significantly lower than that of the antibiotic-treated crucian carp group: (p <0.05 But phage was effective in increasing the survival rate of infected crucian carp relative to the negative control group (p <0.05 See fig. 5).

Example 5

Phage lysate (10) purified by NaCl-PEG method ¹⁰ PFU/ml), 50L fermenter scale up, plate filter and 0.22 μm filter membrane filtration, collecting filtrate in 30L plastic bucket (10) ⁸ PFU/ml). During the stocking period (5-6 months), when the large water body stocking is carried out, the crucian is bathed in the phage suspension, and the application amount is more than or equal to 10 ⁸ PFU/L water body, or spraying the phage suspension liquid to the culture pond after dilution, spraying for 1 time every 10 days, continuously spraying for 3 times, wherein the application amount is more than or equal to 10 each time ⁵ PFU/M ³ And (5) cultivating a water body. The specific implementation details are as follows:

an experimental field: salt city certain agriculture development limited

Time: 2021.5.16-2021.6.20

The crucian carp specification is as follows: healthy crucian carp with average weight of 12 +/-1.6 g

Grouping culture ponds: the control pool and the experimental pool are 1 pool each and occupy 5 mu of land

Product of aeromonas veronii phage pAEv 1818: and (3) carrying out immersion bath by using the phage suspension every time the fry is put.

The culture conditions are as follows: controlling the water temperature at 20-28 deg.C and pH at 7.3-7.6; in the experimental process, the culture pond is aerated by adopting 24 h, and the feed is fed once a day.

Sampling in an experiment: and after 30 days of culture, randomly taking 30 crucian carps in a control pool and an experimental pool, calculating the weight gain and the ingestion rate, and inspecting the growth indexes of the crucian carps.

The results of the initial application of the phage suspension are shown in Table 1 below,

TABLE 1 index change of growth performance of part of crucian after phage treatment

As can be seen from Table 1, the growth performance of crucian carps can be effectively improved by soaking the phage suspension liquid, wherein the total weight gain rate of the crucian carps cultured for one month exceeds 14 percent of the total weight gain rate of a control pool, and the individual ingestion rate is also obviously improved.

Claims (9)

1. An aeromonas veronii phage pAEv1818 characterized by: the preservation number is CGMCC No.20295.

2. Application of Aeromonas veronii phage pAEv1818 with the preservation number of CGMCC No.20295 in preparing Aeromonas veronii inhibitor.

3. Application of Aeromonas veronii phage pAEv1818 with the preservation number of CGMCC No.20295 in preparing a medicament for preventing diseases caused by Aeromonas veronii.

4. Use according to claim 3, characterized in that: the disease caused by Aeromonas veronii is septicemia.

5. The application of the Aeromonas veronii phage pAEv1818 with the preservation number of CGMCC No.20295 in killing Aeromonas veronii in a space environment.

6. Use according to claim 5, characterized in that: the space environment comprises water, the ground, sludge, excrement, padding and feed.

7. A bacteriostatic agent is characterized in that: comprises Aeromonas veronii phage pAEv1818 with the preservation number of CGMCC No.20295.

8. The application of claim 5, comprising the following specific steps: when crucian (10-15 g) in the seedling stage is cultured in large water body, the crucian is bathed in the phage suspension with the application amount of more than or equal to 10 ⁸ PFU/L water body.

9. The application of claim 5, comprising the following steps: diluting the phage suspension, spraying the phage suspension to a culture pond for 3 times continuously at intervals of 1 time and 10 days or more, wherein the application amount of each time is more than or equal to 10 ⁵ PFU/M ³ And (5) cultivating a water body.

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