CN107119025A - Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application - Google Patents
Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application Download PDFInfo
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Abstract
Bactericidal composition the present invention relates to aeromonas salmonicida bacteriophage and comprising the bacteriophage and application.The bacteriophage can quickly suppress the growth of Atlantic Ocean aeromonas salmonicida in a short time, and suppress the generation of the pathogen phage resistance.In addition, the bactericidal composition of the bacteriophage is safely, effectively, and high specificity, production cost is low, and the deficiency of single phagotherapy is compensate in the control process of pathogenic bacteria.
Description
Technical field
The present invention relates to biological technical field, in particular to aeromonas salmonicida bacteriophage, the sterilization comprising it
Composition and its application.
Background technology
Atlantic Ocean aeromonas salmonicida (Aeromonas salmonicida) belongs to Aeromonas under Proteobacteria
Gram negative bacillus, is distributed extremely wide in water environment, is to cause Salmons especially in the enteron aisle of aquatic animal
Occur furunculosis and the Main Pathogenic Bacteria of routed enteropathy.The host range of aeromonas salmonicida is wide, in some cases, can trigger
The multi-infection disease allowed inside and outside humans and animals enteron aisle.Under conditions of high-density breeding, because aeromonas salmonicida has
Very high lethal and the incidence of disease, such pathogenic bacteria are considered as to cause the explosive main causes of death of a variety of aquatic animals
One of, every year Salmons are caused with extremely serious loss.In addition, such pathogenic bacteria can infect non-salmon fishes, feedwater
Production cultivation brings huge economic loss.
Clinically because the cause of disease to the fish that falls ill does not recognize clearly, cause the abuse of antibiotic, both cause raiser
Economic loss, also have impact on the quality safety of aquatic products.The furunculosis and routed intestines occurred for Salmons in aquaculture
Disease, the main prevention of use and control mode are a large amount of antibiotic that come into operation.Although the use of antibiotic being considered as currently to control
Bacterium infection most effective way is treated, but huge antibiotics production and consumption patterns have also greatly encouraged drug-fast bacteria simultaneously
Formed, and make it that the drug resistance problems of pathogenic bacteria are increasingly serious.Report, has found one plant in the animal body for suffer from disease at present
Wide spectrum drug resistance aeromonas salmonicida, up to more than 9 kinds of its drug resistance scope.
Bacteriophage is the virus of a class energy bacterial infection, is widely present in the environment.Bacteriophage has high host special
The opposite sex, self growth and breeding are realized by infecting specific targeting host, and ultimately result in the cracking death of Host Strains, and right
Other floras and human body are without influence.Phagotherapy refers to kill pathogen to treat cause of disease using the single-minded cracking performance of bacteriophage
The treatment means of disease caused by bacterium infection.The theory of pathogenic bacteria is controlled using bacteriophage, after bacteriophage is found soon
It is suggested, and is used successfully in the treatment case of bacterial infection disease.However, due to the discovery of antibiotic, weakening
The application of bacteriophage in this respect, but be continued for so far as the application of antiseptic in East European countries bacteriophage.
Nearly ten years, because abuse of antibiotics causes a large amount of drug-fast bacterias even appearance of superbacteria, directed toward bacteria infection
The phagotherapy of property disease returns the sight of people again, and is gradually highly valued.Currently, in most of Eastern Europe
Country and the U.S., bacteriophage are widely used to during field, the especially food processing and productions such as environment, industry and agricultural
Prevention and control to food origin disease.In addition, current scientists from all over the world are directed to by building scale-model investigation Phage Infection place
The mechanism characteristic of main bacterium, and attempt bacteriophage products application in the clinically research of prevention and control epidemiology.As
One of important weapon of antibiotic resistance is tackled, the clinical practice of bacteriophage has a high potential.But with regard to its current single phagocytosis physical exercise therapy
For the application of method, it engenders some potential shortcomings, such as cleavable host range narrow range in application process, cracking
The dosage that a certain amount of host needs is high, and resistance of bacteriophage etc. is evolved in host's short time.
In view of this, it is special to propose the present invention.
The content of the invention
The first aspect of the present invention is provided in addition to selectivity cracking Aeromonas such as aeromonas salmonicida, also right
Enterobacteriaceae such as Salmonella, vibrios and Escherichia coli have the bacteriophage of certain cracking performance.
One aspect of the present invention is related to aeromonas salmonicida bacteriophage (Aeromonas salmonicida phage), preservation
Entitled AS-SW, is preserved in China typical culture collection center, and deposit number is： CCTCC M 2017093；The preservation time
For：On March 6th, 2017.
The bacteriophage is preserved in China typical culture collection center (CCTCC), and preservation address is：Wuhan City, Hubei Province
City Wuchang District Bayi Road Luo Jia Shan, Wuhan University's China typical culture collection center；The preservation time is：On March 6th, 2017.
Through collection survival strains are detected as on March 11st, 2017.
The bacteriophage of the present invention can not only crack Aeromonas such as aeromonas salmonicida, and can also crack intestines bar
Cordycepps such as Salmonella, vibrios and Escherichia coli.
Brief description of the drawings
, below will be to tool in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in body embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing be some embodiments of the present invention, for those of ordinary skill in the art, do not paying creative work
Under the premise of, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 is the phage selection flow chart in the embodiment of the present invention；
Fig. 2 is the plaque morphology of aeromonas salmonicida bacteriophage；
Fig. 3 is the adsorption curve of aeromonas salmonicida bacteriophage；
Fig. 4 is the one step growth curve of aeromonas salmonicida bacteriophage；
Fig. 5 is the infection curve map that host mixes with individual plant aeromonas salmonicida bacteriophage；A：MOI=0.1；B：MOI=
1；
Fig. 6 is the infection curve map that host mixes with aeromonas salmonicida bacteriophage cocktail.
Aeromonas salmonicida bacteriophage AS-YJ (Aeromonas salmonicida phage AS-YJ) provided by the present invention,
Preserving number is CCTCC NO:M 2017095；
Aeromonas salmonicida bacteriophage AS-GZ (Aeromonas salmonicida phage AS-GZ) provided by the present invention,
Preserving number is CCTCC NO:M 2017094；
Aeromonas salmonicida bacteriophage AS-SW (Aeromonas salmonicida phage AS-SW) provided by the present invention,
Preserving number is CCTCC NO:M 2017093；
Aeromonas salmonicida bacteriophage AS-SZW (Aeromonas salmonicida phage AS- provided by the present invention
SZW), preserving number is CCTCC NO:M 2017092；
Aeromonas salmonicida bacteriophage AS-ZJ (Aeromonas salmonicida phage AS-ZJ) provided by the present invention,
Preserving number is CCTCC NO:M 2017091；
The preservation address of above-mentioned bacterial strains is：Wuhan City, Hubei Province Wuchang District Bayi Road Luo Jia Shan, Wuhan University's Chinese Typical Representative training
Support thing collection；The preservation time is：On March 6th, 2017.Survival is detected as on March 16th, 2017 through collection
Bacterial strain.
Embodiment
The first aspect of the present invention is provided in addition to selectivity cracking aeromonas salmonicida category such as aeromonas salmonicida,
Also to the bacteriophage of enterobacteriaceae such as Salmonella, vibrios and E. coli lytic.
One aspect of the present invention is related to aeromonas salmonicida bacteriophage (Aeromonas salmonicida phage), preservation
Entitled AS-SW, is preserved in China typical culture collection center, and deposit number is： CCTCC M 2017093；The preservation time
For：On March 6th, 2017.
Bactericidal composition containing bacteriophage as described above.
The present invention establishes a kind of feasible method, and efficiently quickly salmon gas is killed in the separating broad spectrum resistance Atlantic Ocean from environment
The bacteriophage of monad.In one embodiment, by recognizing single bacteriophage for infecting the characteristic of host, assess single
The infection ability of one bacteriophage, bacteriophage cocktail is built using certain mixed method, is determined most finally by specific indexes
Good cocktail composition, the bacteriophage cocktail had both had the selectivity feature of bacteriophage, single phagotherapy is compensate for again
The deficiency of Host Strains resistance is easily caused, application prospect is extensive.
The bacteriophage bactericidal composition that the present invention is provided can quickly suppress Atlantic Ocean aeromonas salmonicida in a short time
Growth, and suppress the generation of the pathogen phage resistance.In addition, the bacteriophage cocktail is safely, effectively, and high specificity,
Production cost is low, and the deficiency of single phagotherapy is compensate in the control process of pathogenic bacteria.
The invention further relates to a kind of bactericidal composition containing bacteriophage as described above and/or its mutant, for treating
And/or prevention animal such as fish furunculosis or canker.
The invention further relates to a kind of bactericidal composition containing bacteriophage as described above and/or its mutant, for killing
And/or prevention aeromonas salmonicida, Salmonella, vibrios and Escherichia coli.
It is preferred that, the bactericidal composition also includes aeromonas salmonicida bacteriophage AS-GZ, and preserving number is：CCTCC M
2017094；
Aeromonas salmonicida bacteriophage AS-YJ, preserving number is CCTCC M 2017095；
Aeromonas salmonicida bacteriophage AS-ZJ, preserving number is CCTCC M 2017091；And
Aeromonas salmonicida bacteriophage AS-SZW, preserving number is the one or more in CCTCC M 2017092；
Above-mentioned bacteriophage is preserved in China typical culture collection center, and the preservation time is：On March 6th, 2017.Through
Collection was detected as survival strains on March 11st, 2017.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW and AS-GZ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-GZ and AS-ZJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-GZ and AS-YJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-GZ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-GZ, AS-YJ and AS-ZJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-GZ, AS-YJ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-GZ, AS-ZJ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-GZ, AS-YJ, AS-SZW and AS-ZJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW and AS-ZJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW and AS-YJ.
In certain embodiments of the present invention, the active ingredient in the composition is mainly that aeromonas salmonicida is bitten
Thalline AS-SW and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-YJ and AS-ZJ.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-YJ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-ZJ and AS-SZW.
In certain embodiments of the present invention, the active ingredient in the bactericidal composition is mainly to kill salmon gas unit cell
Bacterium bacteriophage AS-SW, AS-YJ, AS-ZJ and AS-SZW.
In certain embodiments of the present invention, the bactericidal composition is also including AS-GZ mutant, AS-SZW
One or more in mutant, AS-YJ mutant, AS-ZJ mutant and AS-SW mutant
It is preferred that, the mutant sequence at least 90% is identical with the native sequences of corresponding bacteriophage.
Because virus is very easy to undergo mutation in a replication process, thus preferred, the mutant of above-mentioned bacteriophage
In the range of the application is claimed.AS-GZ, AS-SZW, AS-YJ, AS-ZJ, AS-SW mutant at least 90% and institute
The native sequences for stating bacteriophage are identical；And the mutant has and roughly the same with initial bacteriophage to kill pathogen
Function.It is furthermore preferred that mutant has the native sequences of 92%, 94%, 96%, 98% or 99% and each self-corresponding bacteriophage
It is identical.
Bacteriophage AS-GZ, AS-SZW, AS-YJ, AS-ZJ, AS-SW mutant can be point mutation, deletion mutation or add
Plus mutation, relative to initial bacteriophage sequences, there is 1,2,3,4,5,6,7,8,9,10 or more
Many bases can change.To those skilled in the art, the phage selection provided according to the present invention goes out and its character
Similar mutant simultaneously would not require any inventive effort.
As described above, bacteriophage can exist to induce bacteriolysis with sufficiently high concentration, when mixture resulting mixture
When, it is preferred that the content of every kind of aeromonas salmonicida bacteriophage in bactericidal composition as described above, the composition >=
106PFU/mL；
It is preferred that, bactericidal composition as described above, the content of every kind of aeromonas salmonicida bacteriophage in the composition
For 106PFU/mL~1010PFU/mL, more preferably 106PFU/mL~109PFU/mL, more preferably 106PFU/mL~
108PFU/mL, more preferably 106PFU/mL~107PFU/mL, can also take 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 × 109The intermediate values such as PFU/mL.
It is preferred that, as AS-SW and AS-GZ, AS-YJ, AS-ZJ when one kind in AS-SZW or many plants carry out mixture, are pressed
The mode of the ratio such as infection multiplicity is mixed.
It is preferred that, bactericidal composition as described above, the bactericidal composition also includes auxiliary material；
The auxiliary material is the one or more in SM buffer solutions, sodium alginate, sucrose, maltodextrin, glucose；
The compound method of SM buffer solutions is conventional method, for example：NaCl 5.8g, MgSO4·7H2O 2g, 1mol/L's
TrisHCl 50mL (pH=7.0), 5mL 2%gelatin, add pure water and complement to 1000mL.
It is preferred that, the bacteriophage of the bactericidal composition also specific pathogen bacterium including variety classes bacterium.
Above-mentioned bactericidal composition can be used as virus formulation, formulation used can be various common dosage forms, such as pulvis,
Aqua, freeze-dried, gel, creme, paste etc..
It is preferred that, bactericidal composition as described above is killing aeromonas salmonicida, Salmonella, vibrios and large intestine
Application in bacillus；The application is therapeutical uses or non-therapeutic use.
It is preferred that, aeromonas salmonicida bacteriophage as described above or bactericidal composition are being prepared for preventing and treating animal furuncle
Application in the medicine of sore disease or canker；
It is preferred that, application as described above, the animal includes：The cold-blooded property animal of warm-blooded animal and part
It is preferred that, application as described above, the animal is fish；
It is preferred that, application as described above, the fish are salmon fishes；
It is furthermore preferred that application as described above, the salmon fishes include cherry salmon, humpbacked salmon, big
Fiber crops breathe out white in fish, Ishikawa taimen, taimen, brave Jiayu, fine-scaled graphite, hickie torgoch, Salvelinus malma, northern salmon, Usu
Salmon, card reach whitefish, Atlantic salmon, Pacific Ocean salmon, silverside, rainbow trout, grayling, golden trout.
A kind of method for preventing and treating fish furunculosis or canker, bactericidal composition as described above is added as medicine
Spray, or gavaged to fish into fish meal, or to fish body surface, or give fish injection, or the bactericidal composition is dissolved in
Contacted again with fish in water.
It is preferred that, method as described above, the fish are salmon fishes；
It is furthermore preferred that method as described above, the salmon fishes include cherry salmon, humpbacked salmon, big
Fiber crops breathe out white in fish, Ishikawa taimen, taimen, brave Jiayu, fine-scaled graphite, hickie torgoch, Salvelinus malma, northern salmon, Usu
Salmon, card reach whitefish, Atlantic salmon, Pacific Ocean salmon, silverside, rainbow trout, grayling, golden trout.
In some embodiments, the present invention is directed to causes aquatic products Salmons to be sent out by Atlantic Ocean aeromonas salmonicida
The problem of raw furunculosis and routed enteropathy, occur and single bacteriophage in aeromonas salmonicida drug resistance caused by abuse of antibiotics
It is pathogenic with one plant isolated from the Atlantic salmon muscle of exanthemv, liver, nephrosis stove on the basis of applied defect
Aeromonas salmonicida is host, using double-layer agar technique, isolated 10 plants of lytic phages from water environment, by recognizing
The infectivity of single bacteriophage is known, so that its infection ability to Host Strains is assessed, finally using certain mixed method structure
Build and determine optimal cocktail composition.The bacteriophage cocktail can quickly suppress the Atlantic Ocean and kill salmon gas unit cell in a short time
The growth of bacterium, and suppress the generation of the pathogen phage resistance.The bacteriophage cocktail safely, effectively, and high specificity,
Production cost is low, and the deficiency of single phagotherapy is compensate in the control process of pathogenic bacteria.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the present invention.It is unreceipted in embodiment
Actual conditions person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm of instrument
Person, is the conventional products that can be obtained by commercially available purchase.
It is used to efficiently crack the research of Atlantic Ocean aeromonas salmonicida bacteriophage cocktail agent the invention discloses one kind.
Using from the Atlantic salmon muscle of exanthemv, liver, kidney separation one plant of wide spectrum resistance pathogenic aeromonas salmonicida as place
Main, isolated 10 plants of fragility bacteriophages from water environment, cracking diameter is all higher than 0.3mm, with individual phage lysis efficiency
It is 0.1 that evaluation, which obtains optimal infection proportion MOI, with reference to the adsorption curve of individual phage, one step growth curve and highest potency
The individual phage that can add bacteriophage cocktail Deng index screening is AS-SZW, AS-YJ, AS-ZJ, AS-SW, AS-GZ.It is logical
Permutation and combination is crossed, the burst size and pyrolysis time of Host Strains obtain one group in short-term as foundation after being trained altogether using Host Strains and bacteriophage
The bacteriophage cocktail thereof of interior efficiently cracking Atlantic Ocean aeromonas salmonicida.In addition, further to analyze the bacteriophage chicken
The environmental resistance analysis of tail wine, it is determined that the pH tolerances scope of two groups of bacteriophage cocktail thereofs is 3~11 more than,
Still there is certain activity at a temperature of 70 DEG C, and 7d potency is cultivated in seawater and slightly have reduction, about 80%.Using conventional liq
The mode of mixing, the bacteriophage cocktail can quickly suppress the growth of Atlantic Ocean aeromonas salmonicida, be grown in the range of 80h
Curve OD600 values are less than 0.05.The above five plants of bacteriophages AS-SZW, AS-YJ, AS-ZJ, AS-SW, during AS-GZ is preserved in
State's Type Tissue Collection, preservation date on March 6th, 2017, deposit number be respectively CCTCC M 2017092,
CCTCC M 2017095、CCTCC M 2017091、CCTCC M 2017093、CCTCC M 2017094.Bacteriophage of the present invention
Screening process figure it is as shown in Figure 1.Specifically, the present invention is based on common antibiotics species on the market, using diffusion method
(K-B methods) detects the pathogenic bacteria to chloramphenicol, Florfenicol, Enrofloxacin, tetracycline, CTX, Doxycycline, pyrrole piperazine
Acid, Norfloxacin, Ofloxacin, Ceftizoxime, cephazoline, gentamicin, SMZco, rifampin, vancomycin,
Erythromycin, streptomysin, neomycin, card receive 24 kinds of mycin, TOB, penicillin, neomycin, furazolidone, furantoin etc.
The drug sensitive test of medicine, confirms that the pathogenic aeromonas salmonicida has very strong drug resistance to preceding 11 kinds of antibiotic.
More than walk in the aeromonas salmonicida that confirms be host, using double-layer agar technique and combine sample characteristic from seawater
Bacteriophage is separated in sample, speculum is determined by many subinfections, the single plaque of picking is isolated and purified, most finally -80
DEG C glycerine (20%) is preserved.This is separated and subsequent analysis used medium is LB meat soups/agar medium, double-layer plate
In, bottom is 1.5%LB agar mediums, and upper strata is 0.7% agar medium.
The genome of each bacteriophage is extracted using phage genome extraction agent box (Aidlab, DN22), it is rear to use
Miseq platforms carry out genome sequencing, splicing and the annotation of sequence are carried out to sequencing result, after sequence is compared through Blastn
Determine that five plants of bacteriophages of AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ are aeromonas salmonicida bacteriophage, wherein preceding 4
Strain bacteriophage is respectively 97%, 97%, 96% with the Aeromanas phage CC2 whole genome sequence similitudes reported,
96%, AS-GZ and the aeromonas salmonicida bacteriophage phiAS4 reported similitude are 97%.
The evaluation of the single morphology of phages and microbiology level：1) morphologic observation：Single phage-infect Host Strains
Afterwards, spread after double-layer plate, 30 DEG C of culture 12h, determine original speculum size with slide measure, obtain bacteriophage AS-SZW,
AS-YJ, AS-ZJ, AS-SW, AS-GZ size are respectively 0.83mm, 0.48mm, 0.5mm, 0.5mm, 1mm.Plaque morphology
As shown in Figure 2.2) measure of adsorption capacity：(salmon gas list is killed with the MOI=0.005 Host Strains for mixing bacteriophage and logarithmic phase
Born of the same parents bacterium), mixed-culture medium is taken according to time point (0,1,2,3,4,5,6,10,20,30min) is pre-seted, after 16000g centrifugations,
Free state bacteriophage is determined using double-layer agar technique, AS-SZW is obtained according to subtraction, AS-YJ, AS-ZJ, AS-SW, AS-GZ inhales
The attached time is respectively 5min, 10min, 10min, 5min, 20min；The adsorption curve of bacteriophage is as shown in Figure 3.3) bacteriophage
The measure of one step growth curve, MOI is 0.01 (host OD600=0.5), and Host Strains at advance 30 DEG C of bacteriophage with being incubated
5min, then 12000r/min centrifugations 30s, removes clear liquid, adds 5ml LB and suspends, is cultivated at last 30 DEG C, every 10min
Sampling, co-cultures 90min (AS-SW is 120min).The potency of determination sample, obtains AS-SZW, AS-YJ, AS-ZJ, AS-SW,
AS-GZ lytic cycle is respectively 80min, 40min, 40min, 100min and 60min, burst size is respectively 145,98,86,
86 and 135；The one step growth curve of bacteriophage is as shown in Figure 4.4) measure of host range, and according to double-deck flat after infection culture
The feature of speculum formation on plate, evaluates sensitiveness (table 1 shown in) of the bacteriophage to 40 plants of selected indicator bacterias, obtains the above 5
Outside aeromonas salmonicida under strain bacteriophage decapacitation selectivity cracking Aeromonas, to the part Salmonella under enterobacteriaceae
Bacillus, vibrios and Escherichia coli have faint cracking performance.Determined in addition, being determined using universal method in above-mentioned experimental result
Optimal bacteriophage cocktail AS-cocktail2-2,5 host's spectral limit, obtain host range for individual phage AS-YJ and
AS-GZ superposition.
5 plants of bacteriophages and the host range of optimal cocktail thereof that the present invention of table 1 is provided
Note："-" represents that, without infectivity, "+" represents infectious, forms transparent circle, but does not form speculum, "+
+ " strong infectivity is indicated, speculum can be formed；" * " is expressed as the Host Strains of this patent content bacteriophage
The combined method of bacteriophage cocktail：With four kinds of infection multiplicities (MOI=0.01,0.1,1 or 10) mixing individual plant bite
Thalline and Host Strains, growth curve (Fig. 5) measure (OD600 values) the evaluation bacteriophage after bacteriophage is infected according to Host Strains and is split
Efficiency is solved, it is determined that optimal MOI values, and according to the phage-infect of optimal MOI progress Host Strains, will be pre- with permutation and combination method
Select bacteriophage to be mixed, respectively combine according to 1:1,1:1:1,1:1:1:1 or 1:1:1:1:After 1 is mixed, by logarithmic phase
Host Strains carry out mixing co-cultivation with bacteriophage.
The determination of optimal bacteriophage cocktail：Bacteriophage is mixed with Host Strains after co-cultivation, by determining nutrient solution
The change of OD600 values, draws the growth curve (Fig. 6) of Host Strains, it is determined that optimal bacteriophage cocktail thereof；Wherein figure acceptance of the bid
Numbers 1,2,3,4,5 represent five plants of bacteriophages of AS-SZW, AS-YJ, AS-ZJ, AS-SW and AS-GZ successively.A length of 80h during measure.
Optimal bacteriophage cocktail thereof is obtained for Cocktail2-2,5 (i.e. AS-YJ and AS-GZ), it grows song in the range of 80h
Line OD600 values are less than 0.01
The evaluation of optimal bacteriophage cocktail envirment factor tolerance：Determine optimal cocktail group in above-mentioned experimental result
The pH of conjunction, temperature and tolerance in the seawater.Wherein pH, temperature tolerance are determined as：The bacteriophage of a certain amount of potency
(100ul, 108PFU/mL in the EP pipes for) being added to the 1.5mL of liquid containing 900ul (LB fluid nutrient mediums), in different pH (pH
=2,4,6,8,10,12；Adjusted with 0.1mol HCl or NaOH to specified pH) and temperature (4 DEG C, 20 DEG C, 37 DEG C, 60 DEG C,
80 DEG C) under cultivate, rear sampling dilution suitable multiple simultaneously spreads double-layer plate and determines potency at once.Wherein pH minute is 3h,
The temperature setting time is, every 15min samplings, 45min to be determined altogether.Tolerance in briny environment is determined and temperature measuring one
Cause, bacteriophage (100ul, 10 of a certain amount of potency8PFU/mL) be added to seawater containing 900uL (seawater sample be derived from Bay in Shenzhen,
Shenzhen great Mei is husky, Huizhou Xun Liaowan and Zhuhai Port, 8000r/min centrifugations 10min, 0.22 μm of filtering) 1.5mL EP pipes
In, in being cultivated at 20 DEG C, every 1d samplings, one week (7d) is cultivated, bacteriophage activity is determined, bacteriophage activity is determined.It is somebody's turn to do
Bacteriophage cocktail is still active between pH 3~11, and its optimum pH is 8, and 30min still keeps one at a temperature of 60 DEG C
Fixed lytic activity, about 70%, to its activity influence less, 30min is inactivated at 80 DEG C for 37 DEG C and temperature below, and it is cracked
Activity is less than detection line, and the potency in seawater in culture 7d is without significantly reducing, and indifference between each seawater sample.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；
Although the present invention is described in detail with reference to foregoing embodiments, it will be understood by those within the art that：
It can still modify to the technical scheme described in foregoing embodiments, or to which part or whole technologies
Feature carries out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from each implementation of the present invention
The scope of example technical scheme.
Claims (10)
1. aeromonas salmonicida bacteriophage (Aeromonas salmonicida phage), the entitled AS-SW of preservation, in being preserved in
State's Type Tissue Collection, deposit number is：CCTCC M 2017093；The preservation time is：On March 6th, 2017.
2. the bactericidal composition containing the aeromonas salmonicida bacteriophage described in claim 1.
3. bactericidal composition according to claim 2, it is characterised in that the bactericidal composition also includes killing salmon gas unit cell
Bacterium bacteriophage AS-GZ, preserving number is：CCTCC M 2017094；
Aeromonas salmonicida bacteriophage AS-YJ, preserving number is CCTCC M 2017095；
Aeromonas salmonicida bacteriophage AS-ZJ, preserving number is CCTCC M 2017091；And
Aeromonas salmonicida bacteriophage AS-SZW, preserving number is the one or more in CCTCC M 2017092；
Above-mentioned bacteriophage is preserved in China typical culture collection center, and the preservation time is：On March 6th, 2017.
4. bactericidal composition according to claim 3, it is characterised in that the bactericidal composition includes the Bactericidal composite
Thing includes the aeromonas salmonicida bacteriophage AS-SW, the aeromonas salmonicida bacteriophage AS-ZJ and described kills salmon gas list
Born of the same parents bacterium bacteriophage AS-GZ；
Or, the bactericidal composition includes the aeromonas salmonicida bacteriophage AS-SW and the aeromonas salmonicida phagocytosis
Body AS-GZ.
5. the bactericidal composition according to any one of claim 2~4, it is characterised in that also wrapped in the bactericidal composition
Include the one or more in each bacteriophage mutants.
It is preferred that, the mutant at least 90% is identical with the native sequences of corresponding aeromonas salmonicida bacteriophage.
6. the bactericidal composition according to any one of claim 2~4, it is characterised in that every kind of in the bactericidal composition
Content >=10 of aeromonas salmonicida bacteriophage6PFU/mL；
It is preferred that, the content of every kind of aeromonas salmonicida bacteriophage is 10 in the composition6PFU/mL~107PFU/mL。
7. the bactericidal composition according to any one of claim 2~4, it is characterised in that the bactericidal composition also includes
Auxiliary material；
It is preferred that, the auxiliary material is the one or more in SM buffer solutions, sodium alginate, sucrose, maltodextrin, glucose；
It is preferred that, the bacteriophage of the bactericidal composition also specific pathogen bacterium including variety classes bacterium.
8. the bactericidal composition according to any one of claim 2~4, it is characterised in that the formulation of the bactericidal composition
For pulvis, aqua, freeze-dried, gel, creme, paste.
9. the bactericidal composition described in aeromonas salmonicida bacteriophage or any one of claim 2~8 described in claim 1
Application in killing and/or preventing aeromonas salmonicida, Salmonella, vibrios and Escherichia coli.
10. the bactericidal composition described in aeromonas salmonicida bacteriophage or any one of claim 2~8 described in claim 1
Preparing the application in being used to treat and/or prevent animal such as fish furunculosis or the medicine of canker；
It is preferred that, the fish are salmon fishes；
It is furthermore preferred that the salmon fishes include cherry salmon, humpbacked salmon, chum salmon, Ishikawa taimen, wise man
Sieve fish, brave Jiayu, fine-scaled graphite, hickie torgoch, Salvelinus malma, northern salmon, Coregonus ussuriensis, card reach whitefish, Atlantic salmon, too
Flat ocean salmon, silverside, rainbow trout, grayling, golden trout.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022529A (en) * | 2017-04-17 | 2017-08-08 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107099509A (en) * | 2017-04-17 | 2017-08-29 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107119024A (en) * | 2017-04-17 | 2017-09-01 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107129972A (en) * | 2017-04-17 | 2017-09-05 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022529A (en) * | 2017-04-17 | 2017-08-08 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107099509A (en) * | 2017-04-17 | 2017-08-29 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107119024A (en) * | 2017-04-17 | 2017-09-01 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107129972A (en) * | 2017-04-17 | 2017-09-05 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
-
2017
- 2017-04-17 CN CN201710250600.7A patent/CN107119025B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022529A (en) * | 2017-04-17 | 2017-08-08 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107099509A (en) * | 2017-04-17 | 2017-08-29 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107119024A (en) * | 2017-04-17 | 2017-09-01 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107129972A (en) * | 2017-04-17 | 2017-09-05 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
Non-Patent Citations (2)
| Title |
|---|
| S. PEREIRA ET AL: "Potential of phage cocktails in the inactivation of Enterobacter cloacae—An in vitro study in a buffer solution", 《VIRUS RESEARCH》 * |
| SUSANA MERINO ET AL: "Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila", 《FEMS MICROBIOLOGY LETTERS》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022529A (en) * | 2017-04-17 | 2017-08-08 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107099509A (en) * | 2017-04-17 | 2017-08-29 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107119024A (en) * | 2017-04-17 | 2017-09-01 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107129972A (en) * | 2017-04-17 | 2017-09-05 | 深圳先进技术研究院 | Aeromonas salmonicida bacteriophage, the bactericidal composition comprising it and its application |
| CN107099509B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
| CN107129972B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
| CN107119024B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
| CN107022529B (en) * | 2017-04-17 | 2020-08-25 | 深圳先进技术研究院 | Aeromonas salmonicida phage, bactericidal composition containing same and application thereof |
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