CN107099509B

CN107099509B - Aeromonas salmonicida phage, bactericidal composition containing same and application thereof

Info

Publication number: CN107099509B
Application number: CN201710250029.9A
Authority: CN
Inventors: 马迎飞; 陈玲; 袁盛建
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2017-04-17
Filing date: 2017-04-17
Publication date: 2020-08-25
Anticipated expiration: 2037-04-17
Also published as: CN107099509A

Abstract

The invention relates to Aeromonas salmonicida phage, and a bactericidal composition containing the phage and application thereof. The phage can rapidly inhibit the growth of Atlantic Aeromonas salmonicida in a short time, and inhibit the generation of phage resistance of pathogenic bacteria. In addition, the bactericidal composition of the bacteriophage is safe and effective, has strong specificity and low production cost, and makes up the defects of a single bacteriophage therapy in the control process of pathogenic bacteria.

Description

Aeromonas salmonicida phage, bactericidal composition containing same and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to an aeromonas salmonicida bacteriophage, a bactericidal composition containing the aeromonas salmonicida bacteriophage and application of the aeromonas salmonicida bacteriophage.

Background

Atlantic Aeromonas salmonicida (A), (B), (CAeromonas salmonicida) Gram-negative bacilli belonging to the genus Aeromonas under the phylum Proteobacteria are widely distributed in water environment, and are the main pathogenic bacteria causing the furunculosis and the ulcerative enteropathy of salmon and trout in the intestinal tracts of aquatic animals. Aeromonas salmonicida has a wide host range and can cause various infectious diseases inside and outside the intestinal tracts of people and animals under certain conditions. Under the condition of high-density culture, aeromonas salmonicida has high lethality and morbidity, and the pathogenic bacteria are considered to be one of the main causes of explosive death of various aquatic animals, so that the aeromonas salmonicida causes serious loss to salmonid and trout every year. In addition, the pathogenic bacteria can infect fishes in non-salmonidae, and bring huge economic loss to aquaculture.

Clinically, the pathogeny of the fish with diseases is not clearly known, so that the abuse of antibiotics is caused, the economic loss of farmers is caused, and the quality safety of aquatic products is influenced. Aiming at furunculosis and ulcerative enteropathy of salmon and trout in aquaculture, a main prevention and control mode is to use a large amount of antibiotics. Although the use of antibiotics is considered to be the most effective way to treat bacterial infections at present, the enormous pattern of antibiotic production and consumption also greatly contributes to the development of resistant bacteria and makes the resistance problem of pathogenic bacteria increasingly serious. It has been reported that a broad spectrum of drug-resistant Aeromonas salmonicida has been found in diseased animals with a range of resistance of up to 9 or more.

Bacteriophages are a class of viruses that infect bacteria and are widely present in the environment. The bacteriophage has extremely high host specificity, realizes self growth and reproduction by infecting a specific target host, finally leads to the lysis and death of host bacteria, and has no influence on other floras and human bodies. Phage therapy refers to a therapeutic means for treating diseases caused by pathogenic bacteria infection by killing pathogenic bacteria through specific lysis of phage. The theory of bacteriophage control of pathogenic bacteria has been proposed as soon as bacteriophage is discovered and successfully used in the case of treatment of bacterial infectious diseases. However, the use of bacteriophages in this respect has been impaired by the discovery of antibiotics, but the use of bacteriophages as antibacterial agents has been continued in eastern european countries.

In the last decade, due to the emergence of a large number of drug-resistant bacteria and even superbacteria caused by abuse of antibiotics, phage therapy for bacterial infectious diseases has returned to the human sight and has become increasingly highly appreciated. Currently, in most eastern European countries and America, bacteriophage has been widely used in the fields of environment, industry and agriculture, especially in the prevention and control of food-borne diseases during food processing and production. In addition, scientists are working on studying the mechanistic properties of phage infection of host bacteria by constructing models and trying to apply phage products to clinical studies for preventive control of epidemiology. As one of the important weapons against antibiotic resistance, bacteriophage has great potential for clinical application. However, as for the application of current single phage therapy, the potential disadvantages are gradually appeared in the application process, such as narrow cleavable host spectrum, high dosage required for cleaving a certain amount of host, resistance of host to evolve phage in a short time, etc.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In a first aspect of the invention, there is provided a bacteriophage which, in addition to specifically cleaving Aeromonas species such as Aeromonas salmonicida, has some cleaving properties with respect to Enterobacteriaceae such as Salmonella, Vibrio and Escherichia coli.

In one aspect, the invention relates to Aeromonas salmonicida bacteriophageAeromonas salmonicida phage) The preservation name is AS-ZJ, and the preservation number is AS follows: CCTCC NO: m2017091; the preservation time is as follows: 3, 6 months in 2017.

The phage are all preserved in China Center for Type Culture Collection (CCTCC) with the preservation addresses as follows: eight-way Lojia mountain in Wuchang region, Wuhan City, Hubei province, China center for type culture Collection, Wuhan university; the preservation time is as follows: 3, 6 months in 2017. The strains were detected as viable by the depository at 3, 16 and 2017.

The bacteriophage of the present invention can cleave not only Aeromonas species such as Aeromonas salmonicida, but also Enterobacteriaceae such as Salmonella, Vibrio and Escherichia coli.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a flowchart of phage selection in an example of the invention;

FIG. 2 is a plaque morphology of Aeromonas salmonicida phage;

FIG. 3 is an adsorption curve of Aeromonas salmonicida phage;

FIG. 4 is a one-step growth curve of Aeromonas salmonicida phage;

FIG. 5 is a graph of infection profiles of a host mixed with an individual Aeromonas salmonicida bacteriophage; a: MOI = 0.1; b: MOI = 1;

FIG. 6 is a graph of the infection profile of a host mixed with Aeromonas salmonicida phage cocktail.

Detailed Description

In a first aspect of the invention, there is provided a bacteriophage that is lytic to enterobacteriaceae such as Salmonella, Vibrio and Escherichia coli, in addition to specifically lysing Aeromonas salmonicida such as Aeromonas salmonicida.

In one aspect, the invention relates to Aeromonas salmonicida bacteriophageAeromonas salmonicidaphase), with the preservation name of AS-ZJ, preserved in China center for type culture Collection with the preservation number of: CCTCC NO: m2017091; the preservation time is as follows: 3, 6 months in 2017.

A bactericidal composition containing the bacteriophage as described above.

The invention establishes a feasible method for efficiently and quickly separating the bacteriophage of broad-spectrum drug-resistant Atlantic aeromonas salmonicida from the environment. In one embodiment, the infection capacity of a single bacteriophage is evaluated by knowing the characteristic of the single bacteriophage on an infected host, the bacteriophage cocktail is constructed by a certain mixing method, and finally the optimal cocktail composition is determined by specific indexes.

The phage bactericidal composition provided by the invention can quickly inhibit the growth of Atlantic aeromonas salmonicida in a short time and inhibit the generation of phage resistance of pathogenic bacteria. In addition, the phage cocktail is safe and effective, has strong specificity and low production cost, and makes up the defects of a single phage therapy in the control process of pathogenic bacteria.

The invention also relates to a bactericidal composition containing the bacteriophage and/or the mutant thereof, which is used for treating and/or preventing animal furunculosis or canker diseases such as fish.

The invention also relates to a bactericidal composition containing the bacteriophage and/or the mutant thereof, which is used for killing and/or preventing aeromonas salmonicida, salmonella, vibrio and escherichia coli.

Preferably, the bactericidal composition further comprises Aeromonas salmonicida phage AS-GZ with the following deposition number: CCTCC NO: m2017094;

aeromonas salmonicida phage AS-SW with the preservation number of CCTCC NO: m2017093;

aeromonas salmonicida phage AS-YJ with the preservation number of CCTCC NO: m2017095; and

the aeromonas salmonicida phage AS-SZW has the preservation number of CCTCC NO: one or more of M2017092;

the phages are all preserved in China center for type culture Collection for the following preservation time: 3, 6 months in 2017. The strains were detected as viable by the depository at 3, 16 and 2017.

In some embodiments of the invention, the active ingredient in the germicidal composition is predominantly Aeromonas salmonicida phage AS-ZJ.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phage AS-ZJ and AS-GZ.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-GZ and AS-YJ.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-GZ and AS-SW.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-GZ and AS-SZW.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-GZ, AS-SW, and AS-YJ.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-GZ, AS-SW, and AS-SZW.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-GZ, AS-YJ, and AS-SZW.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-GZ, AS-SW, AS-SZW, and AS-YJ.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ and AS-YJ.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phage AS-ZJ and AS-SW.

In some embodiments of the invention, the active ingredients in the bactericidal composition are mainly Aeromonas salmonicida phages AS-ZJ and AS-SZW.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-SW and AS-YJ.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-SW and AS-SZW.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-YJ and AS-SZW.

In some embodiments of the invention, the active ingredients in the germicidal composition are primarily Aeromonas salmonicida phages AS-ZJ, AS-SW, AS-YJ and AS-SZW.

In some embodiments of the invention, the germicidal composition further comprises one or more of a mutant of AS-GZ, a mutant of AS-SZW, a mutant of AS-YJ, a mutant of AS-ZJ, and a mutant of AS-SW;

preferably, the mutant sequence is at least 90% identical to the native sequence of the corresponding bacteriophage.

Since viruses are very susceptible to mutations during replication, it is preferred that mutants of the aforementioned phages are also within the scope of the present claims. Mutants of AS-GZ, AS-SZW, AS-YJ, AS-ZJ, AS-SW are at least 90% identical to the native sequence of the phage; and the mutant has substantially the same function of killing pathogenic bacteria as the original phage. More preferably, the mutants are 92%, 94%, 96%, 98% or 99% identical to the native sequence of the respective phage.

Mutants of phages AS-GZ, AS-SZW, AS-YJ, AS-ZJ, AS-SW may be point, deletion or addition mutations, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases being varied relative to the original phage sequence. It is not necessary for the skilled person to inventively work to select a mutant with a similar trait from the phages provided according to the invention.

As mentioned above, the phage may be present in a concentration high enough to induce lysis, and when compounded into a mixture, it is preferred that the bactericidal composition as described above contain greater than or equal to 10% of each Aeromonas salmonicida phage⁶PFU/mL；

Preferably, the bactericidal composition as described above, wherein the content of each Aeromonas salmonicida bacteriophage is 10⁶PFU/mL~10¹⁰PFU/mL, more preferably 10⁶PFU/mL~10⁹PFU/mL, more preferably 10⁶PFU/mL~10⁸PFU/mL, more preferably 10⁶PFU/mL~10⁷PFU/mL, 2 × 10⁶PFU/mL，3×10⁶PFU/mL，4×10⁶PFU/mL，5×10⁶PFU/mL，6×10⁶PFU/mL，7×10⁶PFU/mL，8×10⁶PFU/mL，9×10⁶PFU/mL，5×10⁷PFU/mL，5×10⁸PFU/mL，5×10⁹PFU/mL, etc.

Preferably, when AS-ZJ is mixed with one or more of AS-GZ, AS-SW, AS-YJ, AS-SZW, the mixture is mixed in such a way that the infection complex number is equal.

Preferably, the bactericidal composition as described above, further comprising an adjuvant;

the auxiliary material is one or more of SM buffer solution, sodium alginate, sucrose, maltodextrin and glucose;

the SM buffer is formulated by conventional methods, for example: NaCl 5.8g, MgSO₄·7H₂O2 g, 1mol/L Tris-HCl 50mL (pH 7.0), 5mL 2% gelatin, and purified water to make up to 1000 mL.

Preferably, the germicidal composition further comprises bacteriophages of specific pathogenic bacteria of different species of bacteria.

The bactericidal composition can be used as a virus preparation, and the used dosage form can be various common dosage forms, such as powder, aqua, lyophilized preparation, gel, cream, ointment and the like.

Preferably, the use of the bactericidal composition as described above for killing Aeromonas salmonicida, Salmonella, Vibrio and Escherichia coli; the use is therapeutic or non-therapeutic.

Preferably, the aeromonas salmonicida phage or the bactericidal composition is applied to preparing medicines for preventing and treating furunculosis or canker of animals;

preferably, for use as described above, the animal comprises: warm blooded animals and partially cold blooded animals;

preferably, for use as described above, the animal is a fish;

preferably, the fish is a salmonidae fish;

more preferably, the Salmonidae fish comprises salmon, camel salmon, chukari hucho, hucho taimen, hujia, brachymystax lenok, salvinia leucomao, anthocephalus pelargoni, north salmon, ussuriella pelargonium, catda whitefish, Atlantic salmon, Pacific salmon, silver salmon, rainbow trout, river trout, golden trout.

A method for preventing and treating furunculosis or canker of fish comprises adding the above bactericidal composition into fish feed as medicine, or spraying on fish body surface, or drenching or injecting, or dissolving the bactericidal composition in water and contacting with fish.

Preferably, the method as described above, the fish is a fish of the salmonidae family;

more preferably, the Salmonidae fish comprises salmon, camel salmon, chukarya, hucho taimen, hujia, brachymystax lenok, salvinia leucomao, anthocephalus pelargoni, north salmon, coreopsis pelargus, catda whitefish, Atlantic salmon, Pacific salmon, silver salmon, rainbow trout, river trout, golden trout.

In some embodiments, the invention aims at the problem that aquatic products, namely salmon and trout, generate furunculosis and ulcerative enteropathy due to Atlantic aeromonas salmonicida, on the basis of the occurrence of tolerance of the aeromonas salmonicida caused by abuse of antibiotics and the application defect of single phage, a pathogenic aeromonas salmonicida separated from muscles, livers and nephrosis foci of the Atlantic salmon with ulcerated skin is taken as a host, a double-layer plate method is adopted, 10 lytic phages are separated from a water environment, the infection capability of the pathogenic aeromonas salmonicida on the host is evaluated by knowing the infection characteristics of the single phage, and finally, a certain mixing method is adopted to construct and determine the optimal cocktail composition. The phage cocktail can rapidly inhibit the growth of Aeromonas salmonicida in a short time, and inhibit the generation of phage resistance of the pathogenic bacteria. The phage cocktail is safe and effective, has strong specificity and low production cost, and makes up the defects of a single phage therapy in the control process of pathogenic bacteria.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The invention discloses a research on a cocktail preparation for efficiently cracking Atlantic salmon aeromonas salmonicida phage. A broad-spectrum drug-resistant pathogenic salmon aeromonas salmonicida separated from muscles, livers and kidneys of skin ulcerated Atlantic salmon is taken AS a host, 10 fissile phages are separated from a water environment, the cracking diameters are all larger than 0.3mm, the optimal infection ratio MOI obtained by evaluating the cracking efficiency of the single phage is 0.1, and the single phages capable of being added with the phage cocktail are selected AS AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ by referring to indexes such AS an adsorption curve, a one-step growth curve, the highest titer and the like of the single phage. Through permutation and combination, a group of phage cocktail combinations capable of efficiently cracking Atlantic aeromonas salmonicida in short time are obtained according to the cracking amount and the cracking time of the host bacteria after the host bacteria and the phage are co-cultured. In addition, in order to further analyze the environmental tolerance analysis of the phage cocktail, the pH tolerance range of the two groups of phage cocktail combinations is determined to be 3-11, the phage cocktail still has certain activity at the temperature of 70 ℃, and the titer is slightly reduced to about 80% after the phage cocktail is cultured in seawater for 7 d. By adopting a conventional liquid mixing mode, the phage cocktail can quickly inhibit the growth of Atlantic aeromonas salmonicida, and the OD600 value of a growth curve in a range of 80h is below 0.05. The five phages AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ are all preserved in China center for type culture collection with the preservation date of 2017, 3 months and 6 days, and the preservation numbers are CCTCC NO: m2017092, CCTCC NO: m2017095, CCTCC NO: m2017091, CCTCC NO: m2017093, CCTCC NO: m2017094. The screening scheme of the phage of the present invention is shown in FIG. 1. Specifically, the pathogenic bacteria are detected by a diffusion method (K-B method) on the basis of the types of common antibiotics on the market, and the susceptibility test of 24 medicines such as chloramphenicol, florfenicol, enrofloxacin, tetracycline, cefotaxime, doxycycline, pipemidic acid, norfloxacin, ofloxacin, ceftizoxime, cefazolin, gentamicin, compound sulfamethoxazole, rifampin, vancomycin, erythromycin, streptomycin, neomycin, kanamycin, tobramycin, penicillin, neomycin, furazolidone, nitrofurantoin and the like is used to confirm that the pathogenic aeromonas salmonicida has strong drug resistance to the first 11 antibiotics.

The aeromonas salmonicida confirmed in the above steps is taken as a host, phage is separated from a seawater sample by adopting a double-layer plate method and combining sample characteristics, a transparent plaque is determined through multiple infections, a single plaque is picked for separation and purification, and finally the plaque is preserved in glycerol (20%) at the temperature of minus 80 ℃. The culture medium used in this separation and subsequent analyses was LB broth/agar medium, with 1.5% LB agar medium in the bottom layer and 0.7% agar medium in the upper layer of the double-layer plate.

Extracting the genome of each phage by using a phage genome extraction kit (Aidlab, DN 22), then performing whole genome sequencing by using a Miseq platform, splicing and annotating the sequence of the sequencing result, and determining that five phage strains, namely AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ, are salmon killing aeromonas phage after the sequence is compared by Blastn, wherein the similarity of the first 4 phage strains and the reported Aeromonas phage CC2 whole genome sequence is respectively 97%, 97%, 96% and 96%, and the similarity of the AS-GZ and the reported salmon killing aeromonas phage phiAS4 is 97%.

Evaluation of single phage morphology and microbiological levels: 1) and (3) morphology observation: after the single phage infects host bacteria, double-layer plates are paved, after the host bacteria are cultured for 12h at 30 ℃, the size of the original transparent plaque is measured by a vernier caliper, and the plaque shape is shown in figure 2. 2) Measurement of adsorption Capacity: taking MOI =0.005 mixed phage and host bacteria (Aeromonas salmonicida) in logarithmic phase, taking mixed culture solution according to preset time points (0, 1, 2, 3, 4, 5, 6, 10, 20 and 30 min), centrifuging 16000g, measuring free phage by adopting a double-layer plate method, and obtaining adsorption time of AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ by subtraction AS 5min, 10min, 10min, 5min and 20min respectively; the adsorption curve of the phage is shown in FIG. 3. 3) The determination of the phage one-step growth curve shows that the MOI is 0.01 (host OD600= 0.5), the host bacteria and the phage are incubated at 30 ℃ for 5min in advance, then centrifuged at 12000r/min for 30s, the clear liquid is removed, 5ml LB suspension is added, finally the culture is carried out at 30 ℃, samples are taken every 10min, and the total culture time is 90min (AS-SW is 120 min). Measuring the titer of the sample to obtain the cracking cycles of AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ of 80min, 40min, 100min and 60min respectively, and the cracking amounts of 145, 98, 86 and 135 respectively; the one-step growth curve of the phage is shown in FIG. 4. 4) And (3) determining a host spectrum, and evaluating the sensitivity of the phage to 40 selected indicator bacteria according to the characteristics of the formation of the transparent spots on the double-layer plate after infection culture (shown in table 1), so that the 5 phage not only can specifically lyse Aeromonas salmonicida under Aeromonas, but also has weak lysis on part of salmonella, vibrio and escherichia coli under enterobacteriaceae. In addition, the host spectrum range of the best phage cocktail AS-cocktail2-2,5 determined in the above experimental results is determined by a general method, and the obtained host spectrum is the superposition of the single-plant phage AS-YJ and AS-GZ.

TABLE 1 host spectra of 5 phage and optimal cocktail combinations provided by the invention

Note: "-" indicates no infectivity, "+" indicates infectivity, forming a transparent circle, but no transparent spot, "+ +" indicates strong infectivity, forming a transparent spot; "" denotes the host bacteria of the bacteriophage of the present patent content.

The combination method of the phage cocktail comprises the following steps: mixing the single-strain phage with the host bacteria at four multiplicity of infection (MOI =0.01, 0.1, 1 or 10), evaluating phage lysis efficiency according to growth curve (OD 600 value) measurement (FIG. 5) after the host bacteria are infected with the phage, determining the optimal MOI value, performing phage infection of the host bacteria according to the optimal MOI, mixing the preselected phage in a permutation combination manner, mixing the combinations according to 1:1, 1:1:1, 1:1:1:1 or 1:1:1:1:1, and then mixing and co-culturing the host bacteria and the phage in a logarithmic phase.

Determination of optimal phage cocktail: after the phage and the host bacteria are mixed and co-cultured, the change of the OD600 value of the culture solution is measured, the growth curve of the host bacteria is drawn (figure 6), and the optimal phage cocktail combination is determined; wherein the reference numerals 1, 2, 3, 4 and 5 in the figure represent five phage strains of AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ in sequence. The measurement time was 80 h. The best phage Cocktail combination was Cocktail2-2,5 (i.e., AS-YJ and AS-GZ), with a growth curve OD600 value of 0.01 or less over 80 h.

Evaluation of tolerance to optimal phage cocktail environmental factor: the pH, temperature and tolerance in seawater of the best cocktail combinations among the above experimental results were determined. Wherein the pH and temperature tolerance is determined as follows: a certain titer of phage (100 ul, 10)⁸PFU/mL) was added to a 1.5mL EP tube containing 900ul of liquid (LB liquid medium) at different pH (pH =2, 4, 6, 8, 10, 12; adjusted to the indicated pH with 0.1mol of HCl or NaOH) and temperature (4 ℃, 20 ℃, 37 ℃, 60 ℃, 80 ℃), after which samples are diluted by the appropriate fold and immediately plated in double plates to determine the titer. Wherein the determination time of pH is 3h, the temperature setting time is sampling every 15min, and the total determination time is 45 min. The determination of the tolerance in the seawater environment was consistent with the temperature determination, with a certain titer of phages (100 ul, 10 @)⁸PFU/mL) was added to a 1.5mL EP tube containing 900uL seawater (seawater samples taken from Shenzhen Bay, Shenzhen Dameisha, Huizhou Branth Bay and Zhuhai Port, centrifuged at 8000r/min for 10min, filtered at 0.22 μm), incubated at 20 deg.C, sampled every 1d, incubated for one week (7 d), assayed for phage activity, and assayed for phage activity. The obtained phage cocktail still has activity between pH 3-11, the optimum pH value is 8, certain cracking activity is still maintained at 60 ℃ for 30min, the activity is about 70%, the influence of the temperature of 37 ℃ and below on the activity is not great, the activity is inactivated at 80 ℃ for 30min, the cracking activity is lower than that of a detection line, the titer is not obviously reduced in seawater culture for 7d, and no difference exists among seawater samples.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Aeromonas salmonicida bacteriophage (Aeromonas salmonicidaphase), with the preservation name of AS-ZJ, preserved in China center for type culture Collection with the preservation number of: CCTCC NO: m2017091; the preservation time is as follows: 3, 6 months in 2017.

2. A bactericidal composition comprising the Aeromonas salmonicida bacteriophage of claim 1.

3. The bactericidal composition of claim 2, further comprising aeromonas salmonicida phage AS-GZ deposited under the following accession number: CCTCC NO: m2017094;

the phages are all preserved in China center for type culture Collection for the following preservation time: 3, 6 months in 2017.

4. The bactericidal composition of claim 3, wherein the bactericidal composition comprises the aeromonas salmonicida phage AS-YJ, the aeromonas salmonicida phage AS-ZJ, and the aeromonas salmonicida phage AS-GZ;

or, the bactericidal composition comprises the Aeromonas salmonicida phage AS-SZW, the Aeromonas salmonicida phage AS-YJ, the Aeromonas salmonicida phage AS-ZJ, and the Aeromonas salmonicida phage AS-GZ;

or, the bactericidal composition comprises the Aeromonas salmonicida phage AS-ZJ and the Aeromonas salmonicida phage AS-GZ.

5. The bactericidal composition according to any one of claims 2 to 4, further comprising one or more of phage mutants having a pathogen killing function similar to that of the corresponding phage.

6. The bactericidal composition of claim 5, wherein the mutant is at least 90% identical to a native sequence of a corresponding Aeromonas salmonicida bacteriophage.

7. The bactericidal composition of any one of claims 2 to 4, wherein each Aeromonas salmonicida bacteriophage is present in the bactericidal composition in an amount of 10 or more⁶PFU/mL。

8. The bactericidal composition of claim 7, wherein each Aeromonas salmonicida bacteriophage is present in the composition in an amount of 10⁶PFU/mL~10⁷PFU/mL。

9. The bactericidal composition according to any one of claims 2 to 4, wherein the bactericidal composition further comprises an auxiliary material.

10. The bactericidal composition of claim 9, wherein the excipient is one or more of SM buffer, sodium alginate, sucrose, maltodextrin, and glucose.

11. The germicidal composition of claim 9, further comprising a bacteriophage of a specific pathogen of a different species of bacteria.

12. The bactericidal composition according to any one of claims 2 to 4, wherein the bactericidal composition is in the form of one of powder, water, freeze-dried powder, gel, cream and ointment.

13. Use of an aeromonas salmonicida bacteriophage of claim 1, or the bactericidal composition of claim 2, in the manufacture of a medicament for killing and/or preventing aeromonas salmonicida.

14. Use of an aeromonas salmonicida bacteriophage of claim 1 or the bactericidal composition of claim 2 in the manufacture of a medicament for the treatment and/or prevention of salmonidae fish furunculosis or ulcer disease caused by aeromonas salmonicida.

15. The use according to claim 14, wherein the fish of the salmonidae family comprises salmon, camel salmon, chong hucho taimen, tiger jia, brachymystax lenok, salvinia leucomao, anthurium pelargonium, arctic salmon, ussuriella salmon, catda whitefish, Atlantic salmon, Pacific salmon, silver salmon, rainbow trout, river trout, golden trout.