KR101329639B1 - Novel bacteriophage asp-1 and its use for preventing proliferation of aeromonas salmonicida - Google Patents

Abstract

The present invention relates to an isolated and identified novel phage and a composition containing the same as an active ingredient for suppressing proliferation and inducing apoptosis of Aeromonas salmonicida. The phage is variously used as a composition for preventing or treating Aeromonas salmonicida infectious diseases, antibiotics, a disinfectant, and a feed additive for fish and crustaceans.

Description

Novel bacteriophage ASP-1 and its use for preventing proliferation of Aeromonas salmonicida}

The present invention is newly isolated and identified new phage, Aeromonas Salmonici comprising the same as an active ingredient ( Aeromonas salmonicida ) A composition for inhibiting or killing proliferation, Aeromonas salmonisida , for preventing or treating infectious diseases, antibiotics, disinfectants, and feed additives for fish and shellfish.

Aeromexico belonging to monada seae (Aeromonadaceae), non-motile an O Monad (non -motile aeromonad) to the, ah Monastir Monitor Let us live with the (Aeromonas salmonicida) is bacterial sepsis in a wide variety of species (septicemia) and It causes furunculosis, the most important economic loss in agriculture worldwide (Wiklund T, Dalsgaard I (1998) Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species: a review. Dis Aquat Organ 32: 49-69). Until recently, antibiotic-resistant Aeromonas Salmonis, including Korea Strain infections are the biggest problem in agriculture (Kim JH, Hwang SY, Son JS, Han JE, Jun JW, Shin SP, Choresca CH, Choi YJ, Park YH, Park SC (2011) Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea. J Vet Sci 12: 41-48). Oxytetracycline, the most commonly used in fish farming, has already been reported to be resistant to aeromonas salmonicida in 1951, but is currently being used in a variety of ways. In addition, the development of resistant bacteria due to antibiotic abuse of Aeromonas salmonisida Are constantly being reported. R-plasmid endogenous antibiotic resistant bacteria have also been reported. In addition, the occurrence of resistance to second-generation antibiotics of the 4-Quinolones-Fluroquinolones family represented by Enrofloxacin and Sarofloxacin has also been reported. Therefore, an alternative control method for this is urgently needed.

In theory, as an alternative approach to controlling infectious bacterial diseases, phage can be used to treat pathogenic bacteria. Indeed, phage has been used as a therapeutic or prophylactic for some fish and crustacean pathogens (Karunasagar I, Shivu MM, Girisha SK, Krohne G, Karunasagar I (2007) Biocontrol of pathogens in shrimp hatcheries using bacteriophages. Aquaculture 268: 288- 292; Kim JH, Gomez DK, Nakai T, Park SC (2010) Isolation and identification of bacteriophages infecting ayu Plecoglossus altivelis altivelis specific Flavobacterium psychrophilum . Vet Microbiol 140: 109-115; Munro J, Oakey J, Bromage E, Owens L (2003) Experimental bacteriophage-mediated virulence in strains of Vibrio harveyi . Dis Aquat Org 54: 187-194; Nakai T, Park SC (2002) Bacteriophage therapy of infectious diseases in aquaculture. Res Microbiol 153: 13-18; Vinod MG, Shivu MM, Umesha KR, Rajeeva BC, Krohne G, Karunasagar I, Karunasagar I (2006) Isolation of Vibrio harveyi bacteriophage with a potential for biocontrol of luminous vibriosis in hatchery environments. Aquaculture 255: 117-124). Similarly, Aeromonas Salmoncida Phage HER110 (Imbeault S, Parent S, Lagace M, Uhland CF, Blais JF (2006) Using bacteriophages to prevent furunculosis caused by Aeromonas salmonicida in farmed brook trout. J Aquat Anim Health 18: 203-214) and phages O, R and B (Verner-Jeffreys DW, Algoet M, Pond MJ, Virdee HK, Bagwell NJ, Roberts EG (2007) Furunculosis in Atlantic salmon ( Salmo salar L.) is not readily controllable by bacteriophage therapy.Aquaculture 270: 475-484).

However, to date, many aeromonadia infectious phages have been isolated and characterized, but according to the VIIIth ICTV report (http://www.ictvdb.org/ Ictv / index.htm), most of them are myobiri. P1, P2 and T4-like phages belonging to Myoviridae . Moreover, recent studies on aeromonas phages have focused on toxic T4-like phages. There are relatively few studies on other aeromonas phages belonging to myobiride but not classified as T4-like phages, except for aeromonas phage φO18P. Infectious against Aeromonas salmonicida as well as infecting Aeromonas media ( A. media ) has not been reported.

Therefore, the problem to be solved by the present invention is to isolate a new phage having lytic activity to aeromonas Salmonida, a composition for inhibiting or killing aeromonas Salmonidada growth, including aeromonas Salmonida The present invention provides a composition for preventing or treating a infectious disease, an antibiotic, a disinfectant, and a feed additive for fish and shellfish.

In one aspect, the invention relates to phages having specific killing ability against Aeromonas salmonicida .

Specifically, ah buy as Pseudomonas spp. Let us live monitor Monitor Let (Aeromonas salmonicida subsp. Salmonicida), Oh, live with Pseudomonas spp Arc Monitor Let our jeneseu (Aeromonas on salmonicida subsp . Monastir-year-old achromogenes) Oh, and let us monitor the subspecies Massonnat woosida (Aeromonas salmonicida subsp . masoucida ) has a specific kill ability,

In the morphological Mai ohbiri to (Myoviridae) and (family) kawoodo Bilal less (Caudovirales) neck (order) form type (morphotype) A1, and

RNA polymerase, DNA polymerase, large subunit terminase, tail fiber, muramidase, head portal protein and The access number of the nucleotide sequence of one hypothetical protein gene is JF342689, JF342683, JF342686, JF342690, JF342687, JF342688 and JF342684, respectively.

The phage is characterized in that the phage of the accession number KCTC 12127BP.

The present inventors collected sediment samples collected from the rainbow trout culture farm in Korea, separated and identified phages having the characteristics described above with the ability to kill against Aeromonas salmonicida ( Aeromonas salmonicida ), ASP It was named -1 and deposited on February 3, 2012 with accession number KCTC 12127BP to BRC.

The inventors took a sample from a rainbow trout culture farm, and from this sample, aeromonas Salmonida subspecies Salmonida ( Aeromonas salmonicida subsp . Salmonicida ) Isolate phage (hereinafter referred to as ASP-1) that lysate it as a host, and ASP-1 is Aeromonas salmonicida subsp . achromogenes , Aeromonas Let us live monitor subspecies Massonnat woosida (Aeromonas salmonicida subsp. masoucida) Oh, and as Pseudomonas spp. Let us live live monitor monitor Let (Aeromonas in salmonicida subsp . It was confirmed that the plaque was formed on salmonicida ) and had bacteriolytic activity. In addition, it was confirmed that the lysed aeromonas salmonisid subspecies Salmonida includes multidrug resistant strains (AS09, 10, 11, 12, 13, 14 and 15) including antibiotic resistance. In particular, Aeromonas Salmonida subspecies Salmonida da AS09 ( Aeromonas salmonicida subsp . salmonicida Highest EOP value for AS09) (see Table 1).

Further, it was found for these ASP-1 as belonging to a result of observation of the morphological through an electron microscope, Mai ohbiri to (Myoviridae) and (family) kawoodo Bilal less (Caudovirales) neck (order) form type (morphotype) A1.

In addition, analysis of the genome of ASP-1 revealed that the DNA was composed of double-stranded DNA (dsDNA) and had a genome size of about 48 kb, indicating that four of the phage-associated proteins (RNA polymerase, DNA polymerase, large subunit terminase, tail fiber) Analysis of three partial open reading frame (ORF) sequences and three complete ORF sequences (muramidase, head portal protein, hypothetical protein) revealed that the access number of each nucleotide sequence was JF342689, JF342683, JF342686, JF342690, and JF342687, respectively. , JF342688 and JF342684. In addition, the entire ASP-1 genome showed no apparent homology with other phages, and ASP-1 was found to be a novel phage.

In another embodiment, the present invention is the Aeromonas Salmonida comprising the phage or culture thereof as an active ingredient ( Aeromonas salmonicida ) relates to a composition for inhibiting or killing growth.

In another embodiment, the present invention is a phage or culture thereof, or Aeromonas Salmonida comprising the same as an active ingredient ( Aeromonas salmonicida ) relates to antibiotics, disinfectants and feed additives for fish and shellfish, including compositions for inhibiting or killing growth.

Aeromonas Salmonici according to the present invention ( Aeromonas salmonicida ) Proliferation suppressing or killing composition, antibiotics, disinfectants, and feed additives have a very high specificity for Aeromonas salmonicida compared to the existing ones, so that the fungus can kill only certain pathogens without killing them. There is no side effect, it can kill the multidrug-resistant pathogens not only the effect is excellent, but also does not induce the resistance or resistance of the pathogen, the product life is longer than the conventional one.

Phage ASP-1 according to the present invention can be cultured in large quantities by conventional phage culture methods. As the culture medium, a medium composed of carbon source, nitrogen source, vitamins and minerals can be used. For example, NB (nutrient broth), TSB (tryptic soy broth) liquid medium, or TSA (tryptic soy agar) solid medium can be used. The culture can be carried out under ordinary culture conditions. Centrifugation or filtration may be performed to remove the culture medium in the culture and recover only the concentrated cells, and this step may be performed according to the needs of those skilled in the art. The concentrated microbial cells can be preserved by freezing or freeze-drying according to a conventional method so as not to lose their activity.

Preferably, the Oh with live Trichomonas monitor Let (Aeromonas salmonicida) is O to Pseudomonas sub-species live live monitor Let Let monitor (in the Aeromonas salmonicida subsp . salmonicida ), Aeromonas Salmonida subspecies ( Aeromonas) salmonicida subsp . Monastir-year-old achromogenes) Oh, and let us monitor the subspecies Massonnat woosida (Aeromonas salmonicida subsp . masoucida ) is one or more selected from the group consisting of. More preferably, the aeromonas Salmonida subspecies Salmonida is aeromonas Salmonida subspecies Salmonida AS01, AS02, AS04, AS05, AS06, AS07, AS08, AS09, AS10, AS11, AS12, AS13, AS14, AS15 or ATCC 33658, wherein the Aeromonas Salmonida subspecies Acromogenes is Aeromonas Salmonida subspecies Acromogenes AS03 and the Aeromonas Salmonida subspecies Masocidae subs. Eromonas Salmonida subspecies Masourida ATCC 27013.

Also preferably, the Aeromonas salmonicida is a multidrug resistant strain.

Aeromonas Salmonida ( Aeromonas) salmonicida ) Proliferation inhibiting or killing compositions, antibiotics, disinfectants and feed additives for fish and shellfish may further comprise an acceptable carrier, and may be formulated with the carrier. The acceptable carrier refers to a carrier that does not irritate the organism and does not interfere with its biological activity and properties. For the purpose of stable formulation suitable for actual packaging, it may be prepared in the form of a wetting agent, a granular wetting agent, a liquid wettable powder, a liquid agent, a water-soluble agent, a water-soluble granule or an encapsulating agent using a surfactant or an extender. Examples of the surfactant include alkyl (C ₈ -C ₁₂ ) aryl sulfonate, dialkyl (C ₃ -C ₆ ) aryl sulfonate, dialkyl (C ₈ -C ₁₂ ) sulfosuccinate, lignin sulfonate, naphthalene Sulfonate condensates, naphthalene sulfonate formalin condensates, alkyl (C ₈ -C ₁₂ ) naphthalene sulfonate formalin condensates, sodium salts of sulfonate compounds such as polyoxyethylene alkyl (C ₈ -C ₁₂ ) phenylsulfonate, or calcium salts, alkyl (C ₈ ~ C ₁₂₎ sulfates, alkyl (C ₈ ~ C ₁₂₎ aryl sulfates, polyoxyethylene alkyl (C ₈ ~ C ₁₂₎ sulfates, polyoxyethylene alkyl (C ₈ ~ C ₁₂₎ phenyl sulphate , Sodium salts or calcium salts of succinate compounds such as polyoxyalkylene succinates, sodium benzoates, anionic surfactants such as alkylcarboxylates, polyoxyethylene alkyl (C ₁₂ ~ C ₁₈₎ ethers, polyoxyethylene al (C ₈ ~ C ₁₂₎ phenyl ether, polyoxyethylene alkyl (C ₈ ~ C ₁₂₎ phenyl polymer and ethylene oxide propylene oxide from the non-ionic surfactants, polycarboxylate, a group consisting of Triton 100 and Tween 80, such as a copolymer It will be readily understood by one of ordinary skill in the art that one or more selected may be used in combination and other surfactants may be used. Extenders include bentonite, talc, clay, kaolin, calcium carbonate, silica sand, pumice, diatomaceous earth, acidic clay, zeolite, pearlite, white carbon, ammonium sulfate, urea, glucose, dextrin, soy flour, rice, wheat It may be easily understood by those skilled in the art that ocher, glucose and starch, water, and the like may be used alone or in combination of two or more thereof, and other extenders may be used. will be.

The term "antibiotic" is meant to include preservatives, fungicides, and antibacterial agents.

The feed additive may be added to the feed for fish or sensory foods.

Alternatively, ASP-1 may be prepared separately in the form of feed additives and not mixed with the feed. Alternatively, it may be prepared by adding a culture thereof.

In another embodiment, the present invention is aeromonas Salmonida comprising a phage or a culture thereof as an active ingredient ( Aeromonas salmonicida) and infectious disease prevention or treatment composition or use it for a live ah Monastir Monitor Let (Aeromonas salmonicida ) relates to a method for preventing or treating an infectious disease.

As described above, the phage according to the present invention has a specific killing ability against Aeromonas salmonicida , and therefore, Aeromonas Salmonisi. salmonicida ) is effective in the prevention or treatment of various diseases caused by infection.

Preferably, the Aeromonas Salmonida ( Aeromonas salmonicida ) Infectious diseases include, but are not limited to, bacterial septicemia or furunculosis.

The 'prophylaxis or treatment' includes the prevention, inhibition, and alleviation of Aeromonas salmonicida infectious diseases.

The composition of the present invention may be administered to an animal in the form of a pharmaceutical preparation, or mixed with feed or water and fed to the animal.

The administration route of the composition of the present invention may be administered through various routes of oral or parenteral administration as long as it can reach the target tissues. Specifically, oral administration, rectal administration, topical administration, intravenous injection, intraperitoneal injection, intramuscular injection, Transdermal, non-intranasal, inhalation, and the like.

A prophylactic or therapeutic method of the present invention includes administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to one of ordinary skill in the art that a suitable total daily dose may be determined by a physician within the scope of good medical judgment. The specific therapeutically effective amount for a particular subject will depend upon the type and extent of response to be achieved, the age, body weight, general health, sex and diet of the subject, the time of administration, the route of administration and the fraction of the route of administration, It is preferable to apply differently depending on various factors including drugs used or co-used and similar factors well known in the medical field.

The newly isolated phage according to the present invention Aeromonas Salmonida ( Aeromonas salmonicida ) It has a proliferation inhibitory or killing effect, and in particular, it has a proliferation inhibitory or killing ability against antibiotic-resistant aeromonas salmonicida , and therefore, aeromonas such as chorus or sepsis. Salmonida Effective in preventing or treating infectious diseases, it can be used not only as a feed but also as an antibiotic or disinfectant. Therefore, it is expected to contribute to the competitiveness of the aquaculture industry and the improvement of public health.

FIG. 1 shows electron staining of negatively stained ASP-1 virions. Arrows A and B represent neck and contracted tail, respectively.
Figure 2 shows a one-step growth pattern of phage ASP-1 in the E. monarch Salmonida subspecies Salmonida AS01 ( A. salmonicida subsp. Salmonicida AS01). The results show mean ± standard deviation, and latency and burst size values were obtained from three replicates.
FIG. 3 shows the pulsed-field gel electrophoretogram of the degradation products by ASP-1 gDNA and 12 restriction endonucleases. The M line represents the low range PFG marker (New England BioLab). Line 1 shows phage gDNA without degradation. Lines 2-13 show phage gDNA degradation patterns generated by SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI, respectively (New England Biolab). The genome size of ASP-1 was predicted to be about 48 kb from the NcoI distinguishing fragment.
Figure 4 shows the protein profile by ASP-1 virion SDS-PAGE profile and liquid chromatography-mass spectrometry (LC-MS / MS) analysis.
Figure 5 shows a Pseudomonas live monitor let (A. salmonicida) strain lytic activity of ASP-1 for the three kinds in the Ah. Oh live Trichomonas spp live monitor let to let the monitor (A. salmonicida subsp salmonicida.) AS01 pre-IX Four nensyeol culture (Fig. 5a), to the live monitor ah Pseudomonas spp let our jeneseu arc (A. salmonicida subsp . achromogenes) free of AS03 - Extreme Four nensyeol culture (Fig. 5b), and ah live in Monastir Monitor Let Massonnat woosida subspecies (A. salmonicida in subsp . masoucida ) The pre-exponential culture of ATCC 27013 (FIG. 5C) was co-cultured with ASP-1 with muliplicity of infection (MOI) 0, 0.1, 1 and 10, respectively. The results showed mean ± standard deviation in three tests.
Figure 6 shows the therapeutic effect of a rainbow trout ah Monastir Monitor Let us live (A. salmonicida) ASP-1 on phage infection.
7 is jeolchang increase ah let to monitor the live Trichomonas generated (A. salmonicida) infection showing a group of rainbow trout (a) and jeolchang authentication is not affected area is treated with ASP-1 treated group without generating rainbow trout (b) will be.

≪ Materials and methods >

Bacterial strain

The experiment includes 17 A. salmonicida species from three different subspecies and 20 species of the genus Aeromonas (10 A. hydrophila , 5 A. sorbia , and Five different A. media ) and eleven different strains belonging to different genera were used (see Table 1). Bacterial strains were cultured in triptic soy broth (TSB), or sub-cultured all Aeromonas strains in a 20 ° C. tryptic soy agar and the remaining bacteria belonging to other genera. Strains were sub-cultured in tryptic soy agar at 37 ° C. All strains were stored in 10% glycerol at -80 ° C until use.

[12]: Kim JH, Hwang SY, Son JS, Han JE, Jun JW, Shin SP, Choresca CH, Choi YJ, Park YH, Park SC (2011) Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea. J Vet Sci 12: 41-48

[7]: Han JE, Kim JH, Choresca CH, Shin SP, Jun JW, Chai JY, Han SY, Park SC (2011) First description of the qnrS -like ( qnrS5 ) gene and analysis of quinolone resistance-determining regions in motile Aeromonas spp. from diseased fish and water. Res Microbiol. doi: 10.1016 / j.resmic.2011.09.001.

Phage isolation and host range determination

The double-layered method commonly used for phage separation and the number of plaque-forming units (PFUs) thereof (AdamsM (1959) Bacteriophages. Interscience Publishers, New York) Was used. AS01 was used as a host bacterial strain for phage separation, which was previously identified as an antibiotic-resistant Aeromonas Salmonida subspecies ( Aeromonas salmonicida subsp . Salmonicida ) strain. The plaque morphology was observed after 18-24 hours of incubation. Bacterial cultures in exponential phage were inoculated with sediment samples or water obtained from rainbow trout culture farms in Korea. The mixture was incubated at 20 ° C. for 36 hours, centrifuged at 10,000 × g for 20 minutes and then filtered through a 0.45 μm filter. Pure phage strains were obtained through three series of single-plaque separations and named ASP-1. The filtered phage lysate was precipitated with 10% (wt / vol) polyethylene glycol 8000 in 1M NaCl at 4 ° C for 12 hours and recovered by centrifugation at 10,000 x g for 10 minutes at 4 ° C. Purified phage was subjected to CsCl step gradient ultracentrifugation (density gradient: 1.15, 1.45, 1.50 and 1.70 g / ml; 250,000 × g; 22h; 4 ° C.) and SM buffer (10-mM NaCl, 50-mMTris [pH7. 5] and 10-mM MgSO ₄ ) and stored at 4 ° C. until use.

The host range of ASP-1 was determined by dropping 10 μl of diluted phage suspension (3.3 × 10 ⁷ PFU / ml) onto bi-layered agar plates inoculated with all 37 Aeromonas genus. All Aeromonas genus strains were then incubated at 20 ° C. and other bacterial strains at 37 ° C. for 20 hours and checked for plaque formation. Whether or not phage was able to form plaques in each strain was measured by plating efficiency (EOP). Diluted phage suspension (3.5 × 10 ⁴ PFU / ml) was analyzed using a double-layered agar method for each phage-tolerant bacterial strain and found to show clean plaques and the number of plaques was incubated for 24 hours. After the decision was made. PFU and Aeromonas Salmonida subspecies obtained from each phage-tolerant strain salmonicida subsp . salmonicida ) The ratio of PFU obtained in AS01 was calculated to obtain the EOP value.

Electron microscope

Purified phage samples were loaded onto a copper grid, then negatively stained with 2% uranyl acetate and dried. The phage was observed at a voltage of 80 kV using Zeiss TEM EM902 (Zeiss). Phage size was calculated by at least 10 measurements.

One-step growth

10 μl of purified phage suspension (9.3 × 10 ⁹ PFU / ml) was added to 10 ml host bacterial strain AS01 as an inoculum (OD ₆₀₀ : 0.2) in the initial exponential phage state in TSB. Absorbed for 5 minutes and then centrifuged at 10,000 x g for 1 minute. After removing the supernatant, pellets containing phage-infected bacterial cells were suspended in 20 ml of clean TSB and incubated at 20 ° C. with shaking at 250 rpm. Some samples were taken at 10 minute intervals for 120 minutes, to immediately determine titration in the aliquots.

Phage Genome Analysis

Purified phage genomic DNA (gDNA) was obtained from Son JS et al ,. (2010) Complete genome sequence of a newly isolated lytic bacteriophage, EFAP-1 of Enterococcus faecalis, and antibacterial activity of its endoly sin EFAL-1.J Appl Microbiol 108: 1769-1779, and described in the Guidance. Nuclease treatment was performed using DNase I (20U / μl), RNase A (5U / μl) and Mung bean nuclease (20U / μl) (Takara). In addition, sizing and restriction enzyme analysis of phage gDNA was performed by Uchiyama J et al., (2008) Iso-lation and characterization of a novel Enterococcus faecalis bacteriophage EF24C as a therapeutic candidate. FEMS Microbiol Lett 278: 200-206 was performed via pulsed-feild gel electrophoresis according to a slightly modified method. Briefly, 500 μl of phage suspension was mixed with 2% (wt / vol) NuSieve GTG agarose (FMC BioProducts), poured into a plug mold and solidified. The flocs were punched through the mold into a small amount of digestion buffer (500 mM EDTA, 10 mM Tris [pH 8.0], 1% SDS [wt / vol] and 1 mg / ml proteinase K) and incubated overnight at 50 ° C. . Decompose the digestion buffer, wash the sample three times with TE buffer, then 10 U of SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI (New England Biolabs) ) At 37 ° C for 1 hour each. The flocs were washed three times with TE buffer, placed in wells of 1.2% Pulsed Field Certified agarose (Bio-Rad) in 0.5X TBE and covered with molten 0.5% NuSieve GTG agarose. The samples were electrophoresed using the CHEF-DR III System (Bio-Rad) at 6 V / cm with pulse ramps for 5 to 15 seconds at 14 ° C for 16 hours in 0.5X TBE buffer.

Phage genome sequencing is performed by Macrogen Inc. It was performed through (Korea). Phage gDNA was sheared with nebulizer (Invitrogen) and blunt-ended repair. DNA fragments of the desired size (2 to 3 kb) were blunt-enterated into the pCR4 blunt-TOPO vector (Invitrogen) and inserted into E. coli DH10B. Some genomic sequences were obtained through sequencing using primer walking. Potential ORFs were predicted using GeneMark.hmm, and gene sequence homology was investigated using the NCBI BlastP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Phage Genome Analysis

Phage ghosts are described by Konopa G, Taylor K (1979) Coliphage λ ghosts obtained by osmotic shock or LiCl treatment are devoid of J- and H-gene products. Prepared as described in J Gen Virol 43: 729-733. Purified phage suspension (8.4 × 10 ¹¹ PFU / ml) was re-concentrated via ultra-centrifugation at 100,000 × g at 4 ° C. for 30 minutes. They were re-suspended in 10 M LiCl, heated at 46 ° C. for 20 minutes and then diluted 10-fold with 50 mM Tris / HCl (pH 8.0) in 100 mM NaCl and 5 mM MgCl ₂ and 37 ° C. Treated with DNase I (20U / μl) for 2 hours at. Prepared phage ghosts were analyzed via standard Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using SDS Ready-Gel (4-20% polyacrylamide gradient; Bio-Rad), and PlusOne ™ Silver Staining Kit, Protein Staining was performed using (GE Healthcare). Protein bands were extracted from the gel, digested with trypsin, and then confirmed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a proteomic platform at the National Instrumentation Center for Environmental Management (NICEM) at Seoul National University. . All MS / MS data were examined using ProteinPilot ^™ 3.0 Software (Applied Biosystems) from the GenBank non-redundant protein database.

Host cell lysis test ( Host cell lysis test )

Phage-sensitive Aeromonas salmonicida salmonicida ) , AS01 ( A. salmonicida subsp . salmonicida ), AS03 ( A. salmonicida subsp . achromogenes ) and ATCC 27013 ( A. salmonicida subsp . masoucida ) was used to assess the lytic activity of ASP-1. Aeromonas Salmonida in purified phage and three early-exponential stages salmonicida ) strains (OD ₆₀₀ : 0.1) were co-cultured in 10 ml of clean TSB to values of multiplicity of infection (MOI): 0, 0.1, 1 and 10. The product was co-cultured at 20 ° C with shaking at 250 rpm. Bacteria inoculated in phage free (MOI: 0) TSB were used as controls. Absorbance (OD ₆₀₀ ) was determined after inoculation, 0, 3, 6, 12 and 24 h, respectively.

Aeromonas Let's Salmon Aeromonas salmonicida) on  Artificially infected In experiment  About ASP -1 efficacy test of phage

Rainbow trout (Oncorhynchus mykiss, 4-5 weeks old, average body length: 15.5 cm, average body weight: 24.71 g) was used for Aeromonas salmonicida. Aeromonas salmonisida, used for artificial infection, used an AS09 strain from which a type III secretion system (TTSS) gene was detected. Infection was used intramuscularly and ASP-1 was administered in the same way. The experimental group consisted of 4 groups that were not treated after bacterial infection (20), those treated with phage after bacterial infection (20), and inoculated with phage only (20), and those who inoculated only PBS (20). Configured. The AS09 strain used for bacterial infection was artificially infected after dilution in PBS at a concentration of 2.97 × 10 ³ CFU / fish. ASP-1 used for phage treatment was inoculated at a concentration of 1.9 × 10 ⁸ CFU / fish. Each experimental group observed the prognosis for 14 days in a water bath of 18 ℃.

<Result>

Aeromonas  scrap paper( Aeromonas phage ) ASP -1 form, host range and one step growth

Phage, named ASP-1, was isolated from the sediment samples of the rainbow trout culture farm, and the A. salmonicida subsp . salmonicida ) plaques of approximately 2 mm were formed in the AS01 strain. Phage was classified, according to the Ackermann classification, Mai ohbiri to (Myoviridae) in the form surface and the (family) kawoodo Bilal less (Caudovirales) neck (order) form type (morphotype) A1 (an icosahedral head and long contractile sheathed tails) ( 1). Tail length and width were 123 ± 18 nm (mean ± SD) (n = 10) and 16 ± 2 nm (n = 10), respectively, and the head diameter was 53 ± 7 nm (n = 10).

To assess the host range of ASP-1, a variety of kinetic Aeromonas genus as well as the Aeromonas salmonisid strain were tested. Of the 14 antibiotic-resistant aeromonas salmonisid subspecies, nine strains formed perfectly clean plaques in the bi-layered agar and the other five strains formed cloudy plaques. Therefore, it was determined that ASP-1 could infect all 14 Aeromonas Salmonida subspecies Salmonida. Aeromonas Salmonida S. Salmonida subsp . salmonicida ) ATCC 33658, Aeromonas Salmonida subspecies A. salmonicida subsp . achromogenes) Monastir Monitor to live in. Oh, and let us AS03 Massonnat woosida subspecies (A. salmonicida subsp . masoucida ) ATCC 27013 also showed clear plaques, and the phage was judged to be susceptible to these strains. There were various EOP values per Aeromonas salmonicida, and the highest EOP was detected in the multidrug resistant AS09 strain (Table 1). However, ASP-1 was not able to lyse 11 other bacterial species or the motility Aeromonas genus used in the study. In addition, one step growth of ASP-1 was tested to assess the growth pattern and number of progeny phage released through the lysate of AS01, the indicator host strain. The incubation period and mean burst size were estimated to be approximately 40 minutes and 116.7 PFU / cell, respectively (FIG. 2).

Aeromonas  scrap paper( Aeromonas phage ) ASP -1 of Genomic  And Proteomics  characteristic

In general, myribide phage is known to have a double helix (ds) DNA genome. Likewise, the gDNA of ASP-1 was completely degraded by DNase I but not completely by RNase A or Mung bean nuclease; Therefore, it was assumed to be ds DNA. In addition, gDNA was degraded by Sau3AI, MspI, NotI, NcoI, HpaII, and its size was assessed as a separate fragment of NcoI at approximately 48 kb (FIG. 3). For genomic analysis of ASP-1, a preliminary phage genome database was constructed via random shotgun sequencing. We describe four partial ORF sequences of phage-related proteins (RNA polymerase, DNA polymerase, large subunit terminase, tail fiber) and three complete ORF sequences (muramidase, head portal protein, hypothetical protein) in the Genbank database. Could be found through search. Amino acid size, homology and E-values are shown in Table 2. The predicted RNA polymerase, DNA polymerase, large subunit terminase and one hypothetical protein of ASP-1 showed similarities to those of enterobacterial phage φEcoM-GJ1 [GenBank accession number NC_010106], and the expected tail fiber ) And putative muramidase protein showed similarity with Aeromonas phage φO18P [GenBank accession number NC — 009542] classified as P2-like Myoviridae phage.

To analyze the structural proteins of ASP-1, purified phage ghosts were analyzed by SDS-PAGE and LC-MS / MS. It was separated into 14 or more distinct protein bands, having a molecular weight of 8 to 140 kDa, and nine major protein bands were subjected to LC-MS / MS analysis for peptide sequencing. From these results, the seven partial peptide sequences of the structural protein (tail sheath (140 and 11 kDa), head morphogenesis (52 kDa), wac fibritin neck whiskers (48 kDa), major capsid (30 kDa) and prohead core (15 kDa) )) Could be obtained (FIG. 4).

Aeromonas In Salmoncida  About Aeromonas  scrap paper ASP - 1 of  Host Cell Lysis Test

The bactericidal activity of ASP-1 was investigated in early-exponential stages of aeromonas salmonisid subspecies Salmonida (AS01), aeromonas salmonisid subspecies Acromogenes (AS03) and aeromonas sal Monyida subspecies Masoucida (ATCC 27013) cultures were tested (FIG. 5). When the cultures of those three strains were not infected with ASP-1 (MOI: 0), OD ₆₀₀ values continued to increase during incubation. In contrast, bacterial growth by the AS01 and ATCC 27013 strains was inhibited at MOI 0.1, 1 and 10 until 24 hours after ASP-1 infection. However, bacterial growth by the AS03 strain was inhibited only in MOI 10, and OD ₆₀₀ values in MOI 0.1 and 1 groups began to increase at 3 and 6 hours after phage infection, respectively, at almost 1.5 and 0.5 at 24 hours. Reached. Additionally, the presence of viable phage-resistant Aeromonas salmonisida was determined by plating lysates of all phage-inoculated MOI groups (MOI: 0.1, 1 and 10), and phage-resistant colonies only Only MOI 0.1 and 1 groups of AS03 strain were recovered.

Aeromonas In Salmoncida  Artificially infected In experiment  About ASP -Efficacy test results of -1 phage

All patients who were inoculated with aeromonas salmonisida died completely within 4 days, and delayed mortality and reduced mortality were observed in the group treated with ASP-1, and the group inoculated with only phage and PBS only. No mortality was observed for 14 days (FIG. 6).

In addition, as a result of comparing the symptoms between the experimental group, no symptoms of chorus was found in the case of infected fish undergoing ASP-1 treatment (FIG. 7).

Nucleotide Assertion  number( Nucleotide accession numbers )

RNA polymerase, DNA polymerase, large subunit terminase, tail fiber, muramidase of Aeromonas phage ASP-1 Nucleotide sequence data of muramidase, head portal protein, and one hypothetical protein gene are stored in the Genbank database with the assay numbers JF342689, JF342683, JF342686, JF342690, JF342687, JF342688, and JF342684, respectively. Deposited.

BRC KCTC12127BP 20120203

Claims (10)

To remain on to have a specific killing capability for Pseudomonas live monitor Let (Aeromonas salmonicida), morphologically Mai ohbiri to (Myoviridae) and (family) kawoodo Bilal less (Caudovirales) neck in the (order) form type (morphotype) A1 and,
RNA polymerase, DNA polymerase, large subunit terminase, tail fiber, muramidase, head portal protein and The access number of the nucleotide sequence of one hypothetical protein gene is JF342689, JF342683, JF342686, JF342690, JF342687, JF342688 and JF342684, respectively. The phage according to claim 1, wherein the phage is a phage of accession number KCTC 12127BP. Aeromonas Salmonida comprising the phage of claim 1 or 2 or a culture thereof as an active ingredient ( Aeromonas salmonicida ) composition for inhibiting or killing growth. 4. The method of claim 3 wherein the Oh with live Trichomonas monitor Let (Aeromonas salmonicida) is O to Pseudomonas sub-species live live monitor Let Let monitor (in the Aeromonas salmonicida subsp . salmonicida), in the Oh Pseudomonas live monitor let subspecies arc our jeneseu (Aeromonas salmonicida subsp. achromogenes) and remain on as Pseudomonas live monitor let subspecies Massonnat woosida (Aeromonas salmonicida subsp., characterized in that at least one selected from the group consisting of masoucida) Composition. The composition of claim 3, wherein the Aeromonas salmonicida is a multidrug resistant strain. Aeromonas Salmonida comprising the phage of claim 1 or 2 or a culture thereof as an active ingredient ( Aeromonas salmonicida ) A composition for preventing or treating infectious diseases. 7. The composition of claim 6, wherein said Aeromonas Salmonida infectious disease is bacterial septicemia or furunculosis. An antibiotic comprising the phage of claim 1 or 2 or a culture thereof as an active ingredient. A disinfectant comprising the phage of claim 1 or 2 or a culture thereof as an active ingredient. A feed additive for fish or shellfish comprising the phage of claim 1 or 2 or a culture thereof as an active ingredient.

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