CN114703150B - Environment-tolerant aeromonas hydrophila phage ZPAH34 and application thereof - Google Patents
Environment-tolerant aeromonas hydrophila phage ZPAH34 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to an environment-tolerant aeromonas hydrophila phage ZPAH34 and application thereof, wherein the preservation number of the aeromonas hydrophila phage is CCTCC NO: m2022141. The aeromonas hydrophila phage is separated from natural environment, has the characteristics of high specificity, strong cracking capacity, good temperature and pH tolerance and the like, and can have strong inhibition and removal effects on a biofilm formed by aeromonas hydrophila. The phage is used as a means for controlling bacteria, and can reduce the number of live aeromonas hydrophila in lettuce and fish fillets. Meanwhile, the phage can effectively prevent and treat the infection caused by aeromonas hydrophila on zebra fish, and has wide application prospect in aquaculture. The research provides scientific basis for phage application research and theoretical reference for the development of biological prevention and control strategies of aeromonas hydrophila.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an environment-tolerant aeromonas hydrophila bacteriophage ZPAH34 and application thereof.
Background
Aeromonas hydrophila (Aeromonas hydrophila) belongs to the family Aeromonas, genus Aeromonas, and is a gram-negative Brevibacterium. It is widely used in river, pond, sewage, sea water, etc. and can infect various freshwater fishes to result in bacterial septicemia. It is reported that bacterial septicemia caused by aeromonas hydrophila is most serious in megalobrama amblycephala cultivation, the morbidity is high, the duration is long, the death rate is over 10 percent, and meanwhile, a large amount of fish intercropping death is caused, so that huge economic loss is caused for cultivation. Aeromonas hydrophila also has the characteristic of wide host range, and can infect various animals such as fishes, amphibians, reptiles, mammals and the like. It is reported that aeromonas hydrophila can be parasitic in human intestinal tracts, and can cause diseases such as septicemia, necrotizing fasciitis, acute gastroenteritis and the like of human beings and animals in severe cases.
Phage is a virus that specifically infects bacteria and lyses them. In recent years, bacterial resistance has become a global concern due to the massive emergence of drug-resistant strains. Phage therapy is again of interest to the scientific community. With the gradual deepening of the research on phage, the phage therapy is found to have obvious advantages compared with the antibiotic therapy for bacterial infection, and because the phage has strong host specificity, the phage only can kill one or a class of specific pathogenic bacteria, so that the microbial community of the organism is not influenced, and the phage therapy has higher safety and effectiveness; the phage can be used alone, can be made into phage mixed cocktail therapy, and can be combined with antibiotics or other bactericidal medicines; meanwhile, the phage is easy to obtain from the environment and cannot cause environmental pollution. Thus providing more possibilities for the application of phage in different fields.
Disclosure of Invention
The invention screens aeromonas hydrophila phage ZPAH34 from natural environment. The phage can specifically lyse aeromonas hydrophila, has strong environmental tolerance and stronger bactericidal activity, and the preservation number is CCTCC NO: m2022141.
It is another object of the present invention to provide the use of phage ZPAH34.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the applicant screens out a phage from the southern lake of the mountain area of the martial arts in the city of hubei, which was sent to the China center for type culture Collection for storage on the 2 nd month 21 of 2022, and is named by classification: aeromonas hydrophila phage (Aeromonas hydrophila phage) ZPAH34 with preservation number of CCTCC NO: m2022141, address: university of martial arts in chinese.
The aeromonas hydrophila phage ZPAH34 (Aeromonas hydrophila phage ZPAH 34) is a lytic phage, and is observed and displayed by a transmission electron microscope: a head having a short tail and an icosahedron structure with a diameter of 50 + -3 nm, the head and tail being separated by a neck. Phage strains formed clear and transparent plaques on double-layered plates, with halos around them, with regular edges and a diameter of about 1mm. The phage has a specific sequence shown in SEQ ID NO. 1.
The invention also includes the application of the phage ZPAH34, including the application of the phage ZPAH34 in food preservative or in preparing medicine for treating or preventing aeromonas hydrophila infection.
Compared with the prior art, the invention has the following advantages:
1 Aeromonas hydrophila phage ZPAH34 (Aeromonas hydrophila phage ZPAH 34) when cultured for 4h under MOI=0.001 condition, the phage titer reached a maximum of 4×10 8 PFU/mL, the optimal multiplicity of infection is 0.001.
2. Has good acid-base tolerance, stable phage titer in the interval of pH 4-11 of Aeromonas hydrophila phage ZPAH34 (Aeromonas hydrophila phage ZPAH) and is kept at 10 8 PFU/mL or more. Phage ZPAH34 has better acid-base resistance.
3. Has a certain temperature tolerance, and the titer of the aeromonas hydrophila phage ZPAH34 (Aeromonas hydrophila phage ZPAH) is stabilized at 10 between 30 ℃ and 50 DEG C 7 PFU/mL or more.
4. The adsorption speed is high, the highest adsorption speed of the aeromonas hydrophila phage ZPAH34 (Aeromonas hydrophila phage ZPAH 34) in 5min reaches 81.75%, and 94.45% of phages are adsorbed to host bacteria in 15min.
5. The incubation period is short, and the propagation is easy. The one-step growth curve of aeromonas hydrophila phage ZPAH34 (Aeromonashydrophilaphage ZPAH 34) shows that the phage has a short incubation period of 20min and a high platform-stage titer of 10 9 PFU/mL or more.
6. The food preservative can be directly used for food preservation, and the aeromonas hydrophila bacteriophage ZPAH34 (Aeromonas hydrophila phage ZPAH) can effectively inhibit food deterioration caused by aeromonas hydrophila in the food storage aspects of lettuce, fish meat and the like, and can properly prolong the food preservation time by adopting a soaking or spraying mode.
7. Can be used for preparing medicines for treating diseases, and aeromonas hydrophila bacteriophage ZPAH34 (Aeromonas hydrophila phage ZPAH) can effectively prevent and treat diseases caused by aeromonas hydrophila and improve the survival rate of zebra fish.
Drawings
FIG. 1 is a plaque morphology map of the Aeromonas hydrophila phage ZPAH34.
Fig. 2 is a schematic structural diagram of the aeromonas hydrophila phage ZPAH34 under transmission electron microscope observation.
FIG. 3 is a graph of the pH tolerance of the Aeromonas hydrophila phage ZPAH34.
FIG. 4 is a graph showing the temperature tolerance of the Aeromonas hydrophila phage ZPAH34.
FIG. 5 is a graph of the adsorption rate of the Aeromonas hydrophila phage ZPAH34.
FIG. 6 is a graph showing one-step growth of the Aeromonas hydrophila phage ZPAH34.
FIG. 7 is a graph showing clearance of the host biofilm by the aeromonas hydrophila phage ZPAH 34;
a is a graph of host biofilm removal on a 96-well plate; b is a graph of host biofilm removal on a 12-well plate.
FIG. 8 is a graph showing the bactericidal effect of the aeromonas hydrophila phage ZPAH34 on lettuce;
a is a graph of the sterilization effect of phage ZPAH34 on lettuce at 4 ℃; b is a graph showing the sterilization effect of phage ZPAH34 on lettuce at 25 ℃.
FIG. 9 is a graph showing the bactericidal effect of the aeromonas hydrophila phage ZPAH34 on fish;
a is a graph of the sterilization effect of phage ZPAH34 on fish at 4 ℃; b is a graph showing the sterilization effect of phage ZPAH34 on fish meat at 25 ℃.
FIG. 10 is a diagram showing a test of prevention and treatment protection of the zebra fish by the aeromonas hydrophila phage ZPAH 34;
a is a survival rate diagram of zebra fish in a prevention test; b is a survival chart of zebra fish in the treatment test.
Detailed Description
Those skilled in the art can refer to the study of the present application. It should be noted that any minor changes and substitutions to the present invention are intended to be included within the scope of the present invention. The experimental specific conditions not specified in the invention are usually carried out according to conventional conditions; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified. Aeromonas hydrophila ZYAH75 used in the present invention is disclosed in GenBank Accession No NZ _CP 016990.
Example 1:
isolation, purification and potency determination of phages
The water sample of the aeromonas hydrophila phage ZPAH34 is collected in the southern lake of the mountain area of Wuhan, hubei province, the water sample is filtered by filter paper, the water sample is centrifuged for 10min at 8000r/min at 4 ℃, the supernatant is carefully sucked by a syringe and filtered by a filter membrane of 0.22 mu m, host bacteria and other impurities are removed, and the obtained phage stock solution is stored at 4 ℃.
(1) Phage separation and purification: preparing a plate of LA solid culture medium, diluting phage stock solution by 10 times gradient, and finally diluting to 10 -6 . 100 mu L of diluted phage solution and 100 mu L of host bacteria solution are taken by a double-layer plate method and placed in a 5mL centrifuge tube, 3.8mL of 0.7% LB semisolid culture medium is added, the mixture is quickly turned over up and down and mixed evenly, poured onto a solid plate, and cultured for 12 hours at 28 ℃. Clear and transparent plaques on a double-layer flat plate are obtained, single plaques are selected and added into 10mL of LB liquid medium, 100 mu L of aeromonas hydrophila liquid is added at the same time, shake cultivation is carried out for 12h at 28 ℃, the mixed liquid after cultivation is centrifuged for 10min at 10000r/min/min, the supernatant is sucked and filtered by a filter membrane with the thickness of 0.22 mu m, purified phage is obtained, and the steps are repeated four times to obtain plaques with uniform size. As a result, as shown in FIG. 1, phage ZPAH34 formed clear and transparent plaques on double-layered plates with halos around them, with regular edges and a diameter of about 1mm.
(2) The titers were determined using the double-layer plate method. Phage titer (PFU/mL) refers to the number of phage per milliliter of liquid. The phage stock was diluted 10-fold, and the diluted solution was incubated at 28℃for 12 hours by a double-plate method, and plaques on the plates were counted. The concentration calculation formula is: phage titer = number of plaques on double-layer agar plates x dilution x 10.
The phage were sent to China center for type culture Collection, and were named after the year 2022, month 21: aeromonas hydrophila phage (Aeromonas hydrophila phage) ZPAH34 with preservation number of CCTCC NO: m2022141, address: university of martial arts in chinese.
Example 2:
electron microscope observation of phage ZPAH34
Firstly, performing expansion culture on phage, preparing a 250mL conical flask, adding 100mL LB liquid culture medium and 200 mu L host bacteria liquid, performing shake culture at 28 ℃ for 3 hours, picking upper agar containing plaque on a double-layer flat plate into the conical flask by using a sterile inoculating loop, and performing shake culture at 28 ℃ for 4 hours. After the completion of the culture, the mixture was centrifuged at 10000r/min for 10min, and the supernatant was collected and filtered with a 0.22 μm filter membrane. Ultracentrifugation of the filtrate for 2h at 30000r/min under vacuum, discarding supernatant, adding 500 μl ammonium acetate solution, repeatedly gently blowing the centrifugal column, transferring the solution into 1.5mL centrifugal tube to obtain extract with titer of not less than 10 10 PFU/mL phage enrichment. Preparing a copper mesh, washing the copper mesh with sterile water for several times, immersing the copper mesh in the enriched phage stock solution, adsorbing for 5min, sucking redundant liquid with filter paper, dripping 2% phosphotungstic acid on the copper mesh, dyeing for 1min, drying, and observing under a 100kV transmission electron microscope. The length measurement is performed using image processing software Digital Micrograph Demo 3.9.1. As a result, as shown in FIG. 2, the aeromonas hydrophila phage ZPAH34 has a short tail and a head of regular polyhedron structure with a diameter of 50.+ -.3 nm, the head and tail being separated by a neck.
Example 3: determination of optimal multiplicity of infection for phage ZPAH34
Culturing host strain Aeromonas hydrophila ZYAH75 (NZ_CP 016990) overnight, and adjusting OD 600 About 0.8, the concentration of the bacterial liquid is 10 8 CFU/mL, diluting bacterial liquid to 10 times 5 CFU/mL. According to the infection complex, 1000, 100, 10, 1, 0.1, 0.01, 0.001 are added with 100 mu L of phage solution (10) 8 -10 2 PFU/mL) and 100. Mu.L of the host bacterial liquid in 800. Mu.L of LB medium. Vortex mixing, and shake culturing the mixed solution at 28deg.C for 4 hr. After the culture is finished, centrifuging at 4 ℃ and 8000r/min for 10min, taking supernatant, diluting different concentration gradients according to ten times ratio, and measuring the titer of phage by adopting a double-layer agar plate method. The ratio of phage titers at the highest is the optimal multiplicity of phage infection. The results are shown in Table 1, which show that when MOI=0.001, the titer of phage ZPAH34 is a maximum of 4×10 8 PFU/mL, the optimal multiplicity of infection for phage ZPAH34 is 0.001。
TABLE 1 determination of optimal multiplicity of infection for phage ZPAH34
Example 4: phage ZPAH34pH tolerance assay
The pH value of the LB liquid medium is regulated to 2-13. 100. Mu.L of phage stock was added to 900. Mu.L of LB liquid medium at different pH values (initial phage concentration 10) 8 PFU/mL), culturing at 28℃for 1h, and then determining the titer of phage under each pH culture by a double-layer agar culture method, and repeating the experiment three times.
As shown in FIG. 3, the titer of phage ZPAH34 was reduced to 0PFU/mL at pH 2, 3, 12 and 13, and the phage titer was stable at pH 4 to 6 under acidic conditions and maintained at 10 8 PFU/mL or more, phage titer can reach 10 under alkaline condition at pH 7-11 9 PFU/mL。
The titer of phage AhyVDH1 in the paper is stable at pH 5-10, and the survival rate is reduced to 10% when pH=4, 11 [1] . The results show that the phage ZPAH34 has wide acid-base tolerance range and high potency.
Example 5: phage ZPAH34 temperature tolerance assay
The experiments were divided into 6 groups, each group was charged with 500. Mu.L of phage stock solution in a sterile centrifuge tube (initial phage concentration was 10) 8 PFU/mL), then placing the centrifuge tube with phage at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃,80 ℃ respectively, sampling at two time points of 30min and 60min, measuring the titer of phage at each temperature by adopting a double-layer agar culture method, and repeating the experiment three times.
The results are shown in FIG. 4, where the temperature is stable between 30℃and 50 ℃. The aging at 60 ℃ and 70 ℃ is reduced by 10 respectively 3 PFU/mL. Phage ZPAH34 retains good activity at temperatures between 30-50 ℃. In published articles, the survival rate of phage CC2 was reduced to 30% after treatment at 40 ℃ [2] In the published patent, phagocytesThe titer of the body PZY-Ah after treatment at 70 ℃ is 0PFU/mL [3] Therefore, the phage ZPAH34 has better temperature tolerance.
Example 6: phage ZPAH34 adsorption rate assay
According to the ratio corresponding to the optimal infection complex number (MOI=0.001), 5mL of host bacterium aeromonas hydrophila ZYAH75 (NZ_CP 016990) bacterial liquid and 5mL of phage liquid are added into a 50mL centrifuge tube, and the mixed liquid is placed in a shaking table at 28 ℃ for shake culture. The concentration of free phage was determined every 5min starting at 0min, 300. Mu.L of liquid was aspirated at each time point, centrifuged at 8000r/min for 30s, and the supernatant was aspirated for gradient dilution. Depending on the nature of the phage, a different and appropriate dilution gradient was selected at each time point and the titers of free phage in the supernatant were determined by the double-layer plate method.
As a result, as shown in FIG. 5, the maximum adsorption rate of phage ZPAH34 reached 81.75% in 5min and 94.45% of phage was adsorbed to host bacteria in 15min. According to the prior publication of the applicant, compared with phage ZPAH7 with high adsorption rate, the adsorption rate of the ZPAH7 is 79% at 5min [4] The bacteriophage ZPAH34 can reach higher adsorption rate than ZPAH7 in 5min, and has obvious difference from the bacteriophage ZPAH34, and the bacteriophage ZPAH34 can invade host bacteria in a large amount in a short time.
Example 7: phage ZPAH34pH one-step growth curve assay
According to the ratio corresponding to the optimal multiplicity of infection (MOI=0.001), 500. Mu.L of phage and 500. Mu.L of Aeromonas hydrophila ZYAH75 (NZ_CP 016990) bacterial liquid were added, and incubated at 28℃for 15min. The mixture was centrifuged at 8000r/min for 5min, the phage removed by removal was discarded, 1mL of LB liquid medium was added to resuspend the pellet, the procedure was repeated twice, and 9mL of LB liquid medium was added. And (3) placing the mixed solution at 28 ℃ for shake culture. Starting from 0min, sampling every 5min, sampling every 10min after 20min, sucking 600 μl of the mixed solution at each time point, centrifuging at 8000r/min for 30s, and determining phage titer in the supernatant by double-layer plate method. The experiment was repeated three times.
The results are shown in FIG. 6, showing a one-step growth curve for phage ZPAH34 with a masking period of 10min, a incubation period of 20min, and a growth period of 90min.
Example 8: phage genome extraction and sequencing
(1) 1mL of phage solution was taken, 3. Mu.L of DNase I and RNaseA were added thereto, and incubated overnight at 28℃and then inactivated at 80℃for 15min.
(2) To the system, 24. Mu.L of 0.5M EDTA solution, 1.5. Mu.L of 20mg/mL proteinase K, 30. Mu.L of 10% SDS solution were added, and incubated at 56℃for 1 hour.
(3) An equal volume of Tris-equilibrated phenol solution was added, gently shaken for 1min, centrifuged at 12000r/min for 10min, and the upper aqueous phase was transferred to a new EP tube.
(4) An equal volume of DNA extract (phenol-chloroform-isoamyl alcohol 25:24:1) was added, gently shaken for 1min, centrifuged at 12000r/min for 10min, and the upper aqueous phase was transferred to a new EP tube.
(5) Adding equal volume of chloroform, mixing thoroughly, centrifuging at 10000r/min for 5min, and transferring the upper water phase into new EP tube. (repeating this step 2 times)
(6) 400. Mu.L of isopropanol was added thereto, and the mixture was left at-20℃for 1 hour or more. Centrifugation was performed at 13000r/min for 20min at 4℃and the supernatant was slowly decanted.
(7) Adding 1mL of 75% ethanol (precooled at-20deg.C), standing, centrifuging at 12000r/min for 10min, discarding supernatant, standing at room temperature, and completely volatilizing ethanol.
(8) The precipitate was dissolved by adding 30. Mu.LTE buffer, and the obtained phage DNA was stored at-20 ℃.
Whole genome sequencing work was performed at Illumina company and phages were sequenced in high throughput using a Miseq sequencer. Sequencing results showed that the phage ZPAH34 genome was 234,540 bp in full length, with a total of 234 Open Reading Frames (ORFs), 2 trnas, of which 111 ORFs predicted function, 47% of all ORFs, 132 ORFs predicted hypothetical proteins, 53% of all ORFs. The phage ZPAH34 is determined to be free of virulence genes, and does not cause potential health risks to animals or human bodies in practical application. According to the classification of viruses in International Committee on Taxonomy of Viruses (ICTV), the main capsid protein amino acid sequences of representative phages of all genera in the myotail phage family, all aeromonas phages in the myotail phage family, and the statistical phages with homology to phage ZPAH34 were downloaded in the NCBI database. And (3) performing multiple sequence comparison on all amino acid sequences by using MEGA software, selecting neighborhood connection to construct a phylogenetic tree, and finally drawing the phylogenetic tree by using RAxML (v 8.2.4) software. The result shows that phage ZPAH34 belongs to a new phage genus of the family myophagidae and can be named ZPAH34. The phage has a specific sequence shown in SEQ ID NO. 1.
Example 9: phage to host biofilm clearance map
The method comprises the steps of culturing a host biofilm by adopting two modes of a 12-hole plate and a 96-hole plate, and determining the cleaning effect of phage on the biofilm by using two methods of crystal violet staining and viable bacteria counting.
96-well plates: 180 mu L of LB culture medium and 20 mu L of aeromonas hydrophila ZYAH75 bacterial liquid in logarithmic growth phase are added into each hole, and the mixture is placed into a constant temperature incubator at 28 ℃ for culturing for 48 hours and 72 hours, and two time groups are set. Control groups were added with 20 μl PBS instead of phage. Each group was replicated three times. The clearance condition of phage on the biofilm is determined by using crystal violet staining, and the specific method comprises the following steps: each well was washed 3 times with 200 μl sterile PBS to remove planktonic bacteria and media. 200. Mu.L of methanol was added to each well and the mixture was fixed for 30min, and the methanol was sucked out and dried in an oven at 28 ℃. 1% crystal violet 200uL per well was added for 20min and then the dye was aspirated and gently washed three times with PBS. The 96-well plate was placed in an oven for drying. 200. Mu.L of 33% glacial acetic acid was added to each well and incubated on a constant temperature culture shaker at 30℃for 30min to substantially release crystal violet from the biofilm. OD determination with an ELISA apparatus 600 Values.
12 orifice plate: the climbing slices were placed in the bottom of a 12-well plate, and 2.7mLLB medium, 300uL of Aeromonas hydrophila in logarithmic growth phase was added. Culturing at 28deg.C for 48 hr and 72 hr. The clearance of the phage to the biofilm was determined by viable bacteria count. 3mL of PBS is added into each hole for three times, the climbing sheet is transferred into a clean 12-hole plate, 2mL of PBS is added into each position in the climbing sheet for full blowing for 10 times, ten-fold gradient dilution is carried out, 100 mu L of PBS is respectively coated on an LB solid plate, the plate is placed in a constant temperature incubator at 28 ℃ for overnight culture, counting is carried out, and the experiment is repeated three times.
As a result, as shown in FIG. 7, the clearance of phage ZPAH34 to host bacteria, aeromonas hydrophila, was 54% in 48 hours in the environment, resulting in a 1.4log reduction in the microbial load of the biofilm formed on the 12-well plate slide.
Example 10: bactericidal activity of bacteriophage on lettuce
Lettuce leaves were cut into 1cm x 1cm pieces. Soaking in 75% ethanol for 1min, sterilizing, and washing the leaf with sterile water. Then transferred to a sterile petri dish in a flow-through air cabinet at room temperature and air dried. Finally, ultraviolet irradiation is carried out for 1h. Soaking lettuce slices to a concentration of 10 6 CFU/mL of Aeromonas hydrophila solution was used for 10min. And waiting for the leaves to air dry at room temperature to allow bacteria to adhere to the leaves. Soaking lettuce treated with bacteria at titer of 10 7 PFU/mL (moi=10) and 10 8 PFU/mL (moi=100) phage ZPAH34, for 10min. Transfer to a clean petri dish and air dry for 30min. Culturing in an incubator at 4 ℃ and 25 ℃. The bacterial load on lettuce samples was measured at time points 0, 1, 3, 6, 9 and 12 hours after phage addition. The control group used the same volume of PBS solution instead of phage solution. Each group of three are parallel. And (3) detecting bacterial load: the lettuce samples are transferred to a 5mL EP tube added with 1mL PBS buffer solution, ground by a sterilization grinding rod, then mixed by vortex, centrifuged for 10min at 3000 Xg, and the supernatant is discarded to collect the precipitated bacteria, wherein the step is to prevent the influence of phage on the samples on the bacteria, re-suspend the bacterial precipitate by 1mL PBS, and the obtained bacterial suspension is subjected to continuous dilution plating to count the bacterial amount. As shown in fig. 8, when the aeromonas hydrophila phage ZPAH34 is treated for 12 hours at the temperature of 4 ℃, the aeromonas hydrophila bacterial amount is reduced by 3.4log10, and the phage ZPAH34 has good inhibition capability on host aeromonas hydrophila under the treatment of different MOI concentration conditions, so that the fresh-keeping effect of food can be prolonged.
Example 11: bactericidal activity of bacteriophage on fish
Cutting fish into slices of 2cm×1cm×0.5cm, soaking in 75% ethanol at room temperature for 5min for sterilizing, and flushing with sterile waterWashing for 10 times, and air drying for 30min to remove water. 10mL of the mixture was placed in a dish at a concentration of 10 6 CFU/mL of aeromonas hydrophila broth, and immersing the fish meat in the culture dish for 5min. And waits for air drying at room temperature. Each fish fillet sample was soaked at a titer of 10 7 PFU/mL (moi=10) and 10 8 PFU/mL (moi=100) phage ZPAH34, for 10min. Air-drying for 30min. Culturing in incubator at 4deg.C and 25deg.C respectively. Bacterial load on fish fillet samples was measured at time points 0, 1, 3, 6, 9 and 12 hours after phage addition. The control group used the same volume of PBS solution instead of phage solution. Each group of three are parallel. And (3) detecting bacterial load: the lettuce samples were transferred to a 5mL EP tube with 1mL PBS buffer added, ground with a sterile grinding rod and vortexed for mixing, centrifuged at 3000 Xg for 10min, the supernatant was discarded to collect the precipitated bacteria, the bacterial pellet was resuspended with 1mL PBS, the resulting bacterial suspension was plated by serial dilution, and the bacterial count was counted. As a result, as shown in fig. 9, when aeromonas hydrophila phage ZPAH34 is treated for 12 hours at the temperature of 4 ℃ at the MOI=100, the bacterial load is reduced by 3.3log10; the bacterial load is reduced by 2.3log10 when the bacterial load is treated for 3 hours at the temperature of 25 ℃. The phage ZPAH34 has a certain sterilization effect on host bacteria aeromonas hydrophila in a short time after being treated under different MOI concentration conditions, and can be suitable for enhancing the freshness of food on a meat product production chain.
Example 11: prevention and treatment effect of bacteriophage on infection of zebra fish with aeromonas hydrophila
The aeromonas hydrophila infection zebra fish model adopts a scratch soaking infection method. The method can more truly simulate the conditions that some fish body surfaces are scratched and infected by bacteria in aquaculture. Test fish (zebra fish) were randomized into three groups of 90 total fish: preventive, therapeutic and control groups. Phage concentrations of 4 MOIs were selected for treatment and prophylaxis groups. Ten per group. Firstly, scraping a small amount of scales from the dorsal fin of the zebra fish by a surgical knife to form a micro wound, and firstly, treating the scratched zebra fish by phage soaking in a prevention group, and then, carrying out toxicity attack treatment. The treatment group is to soak the infected and scratched zebra fish with aeromonas hydrophila and then treat the zebra fish with bacteriophage. The specific scheme is as follows:
(1) Control group: after the zebra fish was wounded, the zebra fish was soaked with 10LD50 Aeromonas hydrophila AH75 (NZ_CP 016990) for 20min, and then soaked with PBS for 20min.
(2) Treatment group: after the zebra fish is wounded, 10LD is used 50 Aeromonas hydrophila AH75 is used for soaking the zebra fish for 20min, and bacteriophage is used for soaking the zebra fish for 20min after one hour.
(3) Preventive group: after the zebra fish is wounded, the zebra fish is soaked in phage for 20min, and 10LD is used after one hour 50 Aeromonas hydrophila AH75 soaked zebra fish for 20min and transferred to clean water.
Daily observations recorded the death of zebra fish, and data from all groups were plotted and the results are shown in figure 10, with 100% survival rate at moi=1 in phage prophylaxis test. Under the condition of phage prevention and treatment, the protection rate of the zebra fish can be improved.
TABLE 2 prevention of infection of Zebra fish with Aeromonas hydrophila ZYAH75 by bacteriophage ZPAH34
TABLE 3 therapeutic Effect of phage ZPAH34 on infection of Zebra fish with Aeromonas hydrophila ZYAH75
Survival (%) = number of surviving zebra fish/number of original zebra fish x 100.
Reference is made to:
[1]Cheng Y,Gao D,Xia Y,Wang Z,Bai M,Luo K,et al.Characterization of Novel Bacteriophage AhyVDH1 and Its Lytic Activity Against Aeromonas hydrophila.Curr Microbiol. 2021；78(1):329-37.
[2] shen Chanjuan the biological properties of Aeromonas hydrophila phage and genomics research [ D ] Nanjing university of agriculture, 2013.
[3] Zhang Lei an aeromonas hydrophila bacteriophage and its use in China, 202110789459.4[ P. 2021.09.28 ].
[4]Islam MS,Yang X,Euler CW,Han X,Liu J,Hossen MI,et al.Application of a novel phage ZPAH7 for controlling multidrug-resistant Aeromonas hydrophila on lettuce and reducing biofilms.Food Control.2021；122.
Sequence listing
<110> university of agriculture in China
<120> environmental tolerant aeromonas hydrophila phage ZPAH34 and uses
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 255
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ttattcttca agacctaaac gacttgatac ttcagccact acagtaactt ggagtgcttt 60
agtgccacca ttggttaaat taatattaat tgaaactttg tctgatactc catttgtagg 120
agtgatcgca ttgggatcaa taccgtacgt tccatatttg gcactaaaat ctaatgcgac 180
agtatttcca acgacacgat acatacccgt tactttaaaa caagatgtat tgttactagc 240
aggatctctt gtcat 255
Claims (3)
1. Isolated aeromonas hydrophila bacteriophageAeromonas hydrophilaphage), the preservation number of the phage is CCTCC NO: m2022141.
2. Use of the bacteriophage of claim 1 for the preparation of a food preservative.
3. Use of the bacteriophage of claim 1 for the manufacture of a medicament for treating or preventing infection by aeromonas hydrophila.
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