KR101382893B1 - Bacteriophage of Pseudomonas aeruginosa and uses thereof - Google Patents

Abstract

The present application discloses novel bacteriophage, variants thereof, and progeny thereof, and uses thereof having infectivity against Pseudomonas aeruginosa. Bacteriophage according to the present invention is specifically infected with Pseudomonas aeruginosa, an opportunistic strain, has resistance to various antibiotics, and can effectively be used as an antibiotic by effectively removing the Pseudomonas strain which is a serious problem in the medical field. In addition, it may interfere with the formation of biofilm, and may also provide an antifouling effect on food hygiene and medical devices.

Description

Bacteriophage of Pseudomonas aeruginosa and uses approximately} Pseudomonas aeruginosa

The present application relates to bacteriophages and their applications.

The emergence of bacteria resistant to antibiotics used to eliminate pathogens is a major problem in the world and it is urgent to develop a substance that can replace antibiotics.

A bacteriophage is a bacterial virus that specifically infects a specific bacterium and kills the bacteria through a complex lysis process involving many proteins. The bacteriophage was highly specific for host bacteria, so it was highly evaluated as an antibiotic before the antibiotic was developed in earnest. However, the utility of the bacteriophage was reduced with the understanding of bacteriophage and the development of full-fledged antibiotics.

Recently, however, as a result of accumulation of research results on bacteriophage and the recent problem of antibiotic abuse, bacteriophage has been developed as an antibiotic for the treatment and prevention of infection.

Merril et al. (J INat Rev Drug Discov 2 (6): 489-197, 2005) for use of bacteriophage for human and livestock.

U.S. Patent No. 7674467 discloses a bacteriophage against Salmonella and its use.

U.S. Patent No. 7622293 discloses a bacteriophage against Pseudomonas and uses thereof.

Korean Patent No. 941892 discloses a bacteriophage against Salmonella gallinum and its use.

However, despite the various disclosures regarding the use of bacteriophages as antimicrobial agents and the like, there is still a need to develop new bacteriophage antigens and screened bacteriophages suitable for particular applications.

The present invention has been made to solve the above problems to provide a novel bacteriophage with a specificity for Pseudomonas aeruginosa and its use.

 In one embodiment the present application provides a bacteriophage having the RFLP pattern of FIG. 2 or the sequence of SEQ ID NO: 1 and having the ability to dissolve Pseudomonas aeruginosa. In one embodiment the bacteriophage according to the present application has a melting capacity for Pseudomonas aeruginosa ATCC 10145, ATCC 27853, ATCC 9027, ATCC 25619, ATCC 13388, or ATCC 15692.

The present application also provides a bacteriophage, which was deposited with the Korea Microorganism Conservation Center with accession number KFCC11533P, dated June 13, 2012.

The present invention also provides progeny bacteriophages exhibiting the same biological properties as the bacteriophages according to the present invention.

In another aspect, the present application provides a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa-induced diseases, including the bacteriophage or progeny thereof according to the present application.

In yet another aspect, the present invention provides antibiotics, feed additives, disinfectants, or biofilm formation inhibitors or inhibitors comprising the bacteriophage or its progeny according to the present invention.

In another aspect, the present invention provides a method of inhibiting or inhibiting biofilm formation comprising contacting the biofilm formation inhibitor or inhibitor according to the present invention with a bacteria.

Bacteriophage according to the present invention is specifically infected with Pseudomonas aeruginosa, an opportunistic strain, and resistant to various antibiotics, effectively removing the Pseudomonas strain, which is a serious problem in the medical field, to effectively remove human and livestock antibiotics. Of course, it can also be used to prevent the formation of biofilm (biofilm) can also provide an antifouling effect for food hygiene and medical devices and the like.

Brief Description of the Drawings Fig. 1 is an electron micrograph of a bacteriophage identified herein after negative staining with uranyl acetate. The scale bar is 100 nanometers.
Figure 2 is a restriction enzyme analysis of the bacteriophage DNA identified herein. Each lane: M1: lambda DNA marker (Hind III + EcoR I); M2: 1 kb DNA leather.
Figure 3 is a protein analysis of the bacteriophage identified in the circle. Lane 1: protein marker; Lane 2: Analysis of the phage according to the present invention.
Figure 4 is a result of analyzing the adsorption characteristics of the bacteriophage identified in the present application is the result of measuring pre-phage by time after infection with Pseudomonas eruginosa MOI 0.1.
5 is a one step growth curve of the bacteriophage identified herein. E: disappearance; L: latent period; B: Number of emissions.
FIG. 6 shows stability test results for temperature and pH of bacteriophage identified herein. (a) measuring the number of infectious bacteriophage particles after culturing the bacteriophage at different temperatures; (b) Infectious bacteriophage particle counts were measured after culturing bacteriophage at different pHs.
Figure 7 is the result of measuring the growth inhibition (a) and biofilm formation inhibition (b) effect on the bacteria of the bacteriophage identified herein.

Pseudomonas aeruginosa is an opportunistic infectious strain commonly found in the environment. It is a bacterium that causes inflammation and sepsis due to the main symptom of infection. Many varieties resistant to antibiotics are found, which makes it difficult to treat diseases, (biofilm) to cross infection, which is urgently needed.

In this context, a novel bacteriophage isolate that targets specific bacteria and can be selectively removed compared to antibiotics, has a lasting effect through replication in the host, and specifically acts on Pseudomonas aeruginosa without side effects. It was.

Thus, in one embodiment, the present application is directed to bacteriophages, variants, derivatives and progeny thereof having the ability to dissolve Pseudomonas aeruginosa having the sequence of SEQ ID NO: 1. In addition, the bacteriophage of the present application was deposited to the Korea Microorganism Conservation Center on June 13, 2012, accession number KFCC11533P.

Variants included herein have the same genetic and phenotypic characteristics as the bacteriophage according to the present application having a variation in the sequence of SEQ ID NO: 1 or a protein encoded thereby.

As used herein, the term “progeny” refers to the same biological properties as the bacteriophage of this invention from which it is derived, and is a copy of the bacteriophage according to the present invention produced via continuous passage culture or other known methods, the sequence of It is substantially identical to the sequence of No. 1, or substantially identical to the Restriction Fragment Length Polymorphism (RFLP) pattern of FIG. In the former case, "substantially the same" means, for example, at least 90%, in particular at least 95%, more in particular at least 99%, via sequence comparison programs or visual inspection, which are known to indicate similarities between the two sequences. It means to include a variant of the nucleic acid sequence with or without amino acid variation. In the latter case, "substantially the same" means intersubject variability according to criteria developed by Tenover et al. (Tenover, F.C. et al., (1995), J. Clin. Microbiol. 33: 2233-2239).

As used herein, the term " derivative " refers to a bacteriophage or a subunit or expression product constituting the bacteriophage according to the present invention, including, for example, a bacteriophage genome, all or part of a gene, a gene expression product, And the constituent products.

The bacteriophage of the present application specifically infects Pseudomonas aeruginosa. In one embodiment, Pseudomonas aeruginosa ATCC (American Type Culture Collection, USA, US Cell Line Bank) 15522, ATCC 10145, ATCC 27853, ATCC 9027, ATCC 25619, ATCC 13388, or ATCC 15692, but is not limited thereto.

In other embodiments, the bacteriophages, derivatives, variants or progeny thereof of the present invention may be used to control, inhibit or control the growth, division, or colonization of Pseudomonas aeruginosa, for example, treatment of processed or unprocessed food, Storage, various apparatuses, especially medical treatment of diseases, and antibiotics for the treatment of diseases.

In one aspect, the bacteriophage herein may be used to treat unprocessed food or foodstuffs for the prevention of food poisoning caused by Pseudomonas aeruginosa. In this case, the bacteriophage according to the present invention may be sprayed onto the subject food, the food material, or the equipment, or immersed in a solution containing the bacteriophage. The bacteriophage of the present invention may be used in a variety of foodstuffs, for example beef, especially ground beef, processed foods made from ground beef, such as hamburgers, various vegetables that grow on the feet of a nori, such as lettuce, spinach, , And foods that are susceptible to secondary infection by primary infected foodstuffs, but are not limited thereto.

The bacteriophages herein can also be used extensively in equipment used in the home, environmental, medical and industrial fields where control of biofilm formation by Pseudomonas aeruginosa is required. For example, various kinds of living tissues / tissues including epithelial cells, bones, teeth, and inner wall of vessels as well as objects such as equipment used for various food processing, food processing, various medical instruments, medical equipment, And various artificial implants applied to the living body.

The bacteriophage of the present application may be used in an amount effective to inhibit or inhibit the growth, division, colonization, control, inhibition and / or biofilm formation of Pseudomonas aeruginosa. The effective amount can be determined according to the conditions including the type and the area of the object to be treated, the degree of inhibition / control desired, the time of treatment, and the like, and those skilled in the art will appreciate the appropriate amount of the appropriate bacteriophage You will be able to select the concentration. For example, a concentration of about 1 × 10 ⁵ to 1 × 10 ¹⁵ PFU (Plaque Forming Unit) / ml, or about 10 ^-5 to 10 ⁵ MOI (multiplicity of infection) It can be processed over several days and it may be advantageous to treat it early. Those skilled in the art will be able to determine appropriate concentrations by reference to the literature on bacteriophage (Adams, MH (1959), Methods of study bacterial viruses. Bacteriophages. London, Interscience Publishers, Ltd. 443-519). For example, at a concentration of about 1 ml / 500 cm < ² > with respect to the surface to be treated.

Compositions comprising the bacteriophage of the present invention can be prepared in a variety of forms to meet specific needs in the home, industrial, medical, and environmental fields. For example, in liquid form, including spraying, spraying, spraying, spraying, spraying, spraying, spraying, spraying, spraying or spraying, in an amount effective to prevent, control, inhibit, and / or control the growth, colonization and division of Pseudomonas aeruginosa, And may contain additives such as a surfactant, a detergent, a disinfectant, a bactericide, an antibiotic, a fungicide, an acidity regulator, a dye, and a pigment, depending on the purpose. can do.

In one embodiment, in the case of a composition for controlling, controlling, inhibiting and / or preventing biofilm formation or inhibiting biofilm formation or inhibiting growth, division, colonization of Pseudomonas aeruginosa comprising the bacteriophage of this invention, An aqueous solution or suspension containing an effective amount of a bacteriophage. In other embodiments, it can be made in powder, coating, spray, dispense type, capsule / tablet form that can be dispensed into liquid. These compositions may further comprise a surfactant such as anionic, nonionic, amphoteric and biological surfactants and mixtures thereof.

Other embodiments may be made in the form of personal hygiene articles, for example contact lens disinfectants, soaps, shower gels, shampoos, or dental floss, toothpaste, dentifrices or gagging detergents for cleaning, cleaning, have.

In another embodiment it may be prepared in the form of a powder, coating, spray, wipe for cleaning, cleaning of medical equipment / apparatus / facilities.

In other embodiments, biofilm formation inhibitors or inhibitors comprising the bacteriophages herein may be used in industrial environments such as food processing, water treatment devices, and may be prepared in the form of additives, liquids, and coatings. When manufactured with additives, it can be used in water pipes, cooling towers, heat exchangers.

In another aspect, the present invention is also directed to a pharmaceutical composition comprising a bacteriophage according to the present invention. Since the bacteriophage according to the present invention specifically kills Pseudomonas aeruginosa, it can be usefully used for treatment, symptom relief, prevention and / or suppression of various diseases caused thereby. Examples of diseases include food poisoning caused by Pseudomonas aeruginosa, inflammation, and sepsis. But are not limited to, otitis media, cystic fibrosis, pneumonia, burns, fungal wound infections, and the like.

The pharmaceutically acceptable carriers to be included in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid But are not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention can be used as a method of applying or spraying on a diseased site, or may be administered through oral administration or parenteral administration. In the case of parenteral administration, intravenous administration, intraperitoneal administration, Intravenous, subcutaneous, or local administration.

The appropriate application, spray and dose of the pharmaceutical composition of the present invention may vary depending on factors such as formulation method, administration method, age, body weight, sex, disease symptom level, food, administration time, administration route, And responsiveness, and the ordinarily skilled physician or veterinarian can readily determine and prescribe dosages effective for the desired treatment. In general, the pharmaceutical composition of the present invention may be administered at a concentration of about 1 × 10 ⁵ to 1 × 10 ¹⁵ PFU / ml.

The pharmaceutical composition of the present invention may be formulated into pharmaceutically acceptable carriers and / or excipients according to a method which can be easily carried out by those skilled in the art, Or may be manufactured by penetrating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In another aspect, the invention also relates to an antibiotic comprising a bacteriophage as an active ingredient. In the present application, it is meant to include antiseptics, antiseptics, bactericides and antimicrobial agents. The antibiotic of the present invention can be used for prevention, treatment, alleviation and suppression of various diseases caused by Pseudomonas aeruginosa.

The bacteriophage of the present invention has a very high specificity to Pseudomonas aeruginosa as compared to conventional antibiotics. Therefore, it is highly valuable as an antibiotic without side effects because it can selectively remove the pathogen Pseudomonas without killing beneficial bacteria. Use of the bacteriophage of the present invention as an antibiotic does not induce tolerance or resistance to pathogens, unlike existing antibiotics, and thus can be used as a new antibiotic with a longer product life cycle than conventional antibiotics . That is, antibiotics containing the bacteriophage of the present invention as an active ingredient can fundamentally solve the problem of resistance, so that the product life cycle as antibiotics is expected to be longer.

In another aspect, the present invention also relates to a disinfecting agent comprising the bacteriophage of the present application as an active ingredient.

Disinfectants containing the bacteriophage of the present invention as an active ingredient are particularly valuable for food hygiene such as food poisoning prevention. It can be used as food disinfectant and food additive for the prevention of pollution of Pseudomonas in the food industry and can also be used in the field of animal husbandry. In addition, it can be used for spraying Pseudomonas bacteria in effluents from domestic sewage treatment plants.It can also be used as a disinfectant for cooking places, cooking facilities, sanitary facilities, medical facilities, public facilities such as daycare centers, and toys. Can be.

In another aspect, the present invention also provides a feed additive and drinking water comprising the bacteriophage of the present invention as an active ingredient. There are more antibiotics in livestock than humans, and the use of antibiotics in feed and livestock industry accounts for 54% of total antibiotic sales. In particular, administration of antibiotics for preventive purposes increases the possibility of producing resistant bacteria, and antibiotics remaining in livestock can be delivered to humans. If antibiotics are absorbed into human body through meat, they may cause antibiotic resistance and spread the disease and increase the probability of developing multidrug resistant bacteria. The antibiotics according to the present invention can be used as antibiotics for feed additives in place of conventional antibiotics.

The feed additives herein may be in the form of dry or liquid form preparations, and one or more enzyme preparations may be added. Enzyme preparations can be added either in dry or liquid form. Enzyme preparations include lipolytic enzymes such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, starches and glycogen ( amylase, an enzyme that hydrolyzes alpha-1,4-glycoside bonds in glycogen, phosphatase, an enzyme that hydrolyzes organic phosphate esters, Carboxymethylcellulase that breaks down cellulose, xylanase that breaks down xylose, and maltase that hydrolyzes maltose into two molecules of glucose. And sugar-producing enzymes such as invertase, which hydrolyzes saccharose to form a glucose-fructose mixture. It can be.

In addition, the feed additive comprising the bacteriophage of the present invention may be further supplemented with other non-pathogenic microorganisms. Examples of microorganisms that can be added include lactobacilli such as Bacillus subtilis capable of producing proteolytic enzymes, lipolytic enzymes and glycosyltransferases, lactobacilli having physiological activity and organic resolution at anaerobic conditions such as bovine, For example, Lactobacillus sp., Which increases the body weight of livestock and increases the milk yield of milk and increases the digestion rate of feed, such as Aspergillus oryzae, and Saccharomyces cerevisiae such as Aspergillus oryzae Yeast such as Saccharomyces cerevisiae may be used.

Feedstuffs such as peanuts, peas, beet pulp, pulp, grain by-products, animal-viscous powder, fish meal powder, etc., including various grains and soybean proteins, may be processed or processed.

In another aspect, the present invention relates to a method for preventing, inhibiting or inhibiting biofilm formation by Pseudomonas aeruginosa using a bacteriophage according to the present invention. The bacteriophage of the instant application can be treated on the surface where the bacteria can grow or propagate to prevent or inhibit the formation of the biofilm in advance. Contact with the bacteria includes treating the surface of the article with an anti-proliferative or inhibitory agent of the present invention. The biofilm formation inhibitor or inhibitor herein may be treated in an amount effective to inhibit the formation of the desired biofilm as mentioned above.

The surface refers to both the inner and outer surfaces, and includes both hard (solid) and flexible ones, and includes objects used in the home, industrial, environmental, and medical fields as well as living bodies. Solid surfaces include, but are not limited to, drain pipes, glazed ceramics, floors, glass, metal, wood, chrome, plastic, vinyl and cellular. The solid surface may also be an organism-derived tissue or organ such as skin, teeth, or the like. The solid surface may also be derived from a medical article. Medical articles include, but are not limited to, various medical devices, equipment, instruments, temporary or permanent artificial implants such as lenses, prosthetic valves, pacemakers, surgical pins, insertion conduits, catheters, and the like.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombinant techniques, unless otherwise indicated. A more detailed description of common techniques can also be found in Molecular Biotechnology: (Bernard et al., ASM press 1994); Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al., Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985).

Example

Example  Culture conditions of 1 strain

Pseudomonas aeruginosa (ATCC 27853) was used as a host and shaken at 37 ° C. in LB (Luria-Bertani) medium.

Example  2 Bacteriophage  Selection, RFLP  And sequencing

Water was collected from Songhak Reservoir located in Songhak-ri, Dado-myeon, Naju-si, Jeollanam-do, Korea, in order to screen phages infected with Pseudomonas eruginosa. The sampled water and Pseudomonas aeruginosa were shaken at 37 ° C. for 3 hours, and centrifuged at 500 rpm for 20 minutes to recover the supernatant. The supernatant was then filtered through a sterile membrane filter (0.45 μm). A double agar layer plaque assay was used for the filtered samples. In summary, the host bacteria and phage cultures were mixed with 0.1 MOI in 5 ml top agar and poured into agar plates and incubated at 37 ° C. for 24 hours to obtain plaques.

Subsequently, genome sequencing and RFLP analysis were performed from the bacteriophage. Pseudomonas aeruginosa was cultured in 200 ml of LB medium to OD ₆₀₀ = 0.5, and then infected with 10 ⁹ PFU / ml or 0.1 MOI of the filtered bacteriophage.

Thereafter, sodium chloride was added to a final concentration of 1 M and then placed at 4 ° C for 1 hour. After centrifugation at 11000xg for 10 minutes, PEG (polyethylene glycol 8000) was added to the precipitate at 10% (w / v) for 1 hour at 4 ° C. After centrifugation at 11000xg for 10 minutes, the supernatant was removed, and the precipitate was suspended in SM buffer (100 mM NaCl, 10 mM MgSO ₄ (heptahydrate), 50 mM Tris-HCl, pH 7.5). To this was added chloroform at a ratio of 1: 1, vortexed, and centrifuged at 3000xg for 15 minutes to obtain supernatant. 3 ml of 40% glycerol was added to a polycarbonate test tube, followed by 4 ml of 5% glycerol. The prepared phage sample was added thereto and centrifuged at 15000 × g for 1 hour at 4 ° C. The supernatant was then removed and the precipitate was resuspended in SM buffer. Bacteriophage genomic DNA was isolated using phage DNA isolation kit (Norgen biotek corp.) According to manufacturer's instructions.

For the analysis of restriction length polymorphism (RFLP), the isolated genome was digested with BamH I, Hind III, and EcoR V restriction enzymes, followed by electrophoresis with 0.8% agarose gel. The results are described in FIG.

The nucleotide sequence of the thus isolated total genome was analyzed. Sequence analysis was performed by combining shot sequence analysis with shotgun cloning (Macrogen, Roche 454 Next Generation Genome Analyzer).

As a result of BLAST search, a sequence comparison program for the obtained sequence, it was confirmed that the sequence was a new sequence unknown to the existing sequence (the comparison result is not shown). Genomic analysis confirmed that the total length of 72,321 bp linear double helix DNA (see SEQ ID NO: 1), it was named PA26.

Example  3 Analysis of virus type using transmission electron microscope

To analyze the morphology of the bacteriophage obtained in Example 2, centrifugation using a cesium chloride (CsCl) density gradient method (density 1.15 g / ml, 1.45 g / ml, 1.50 g / ml and 1.70 g / ml) The bacteriophage was purified through separation (38,000 rpm, 22 hours, 4 ° C) and then dropped on formvar carbon film (200 mesh copper grids), followed by negative staining with 2% Uranyl acetate The shape was photographed through a transmission electron microscope (LIBRA 120 Energy-Filtering Transmission Electron Microscope (Carl Zeiss)).

The results are shown in Fig. The isolated bacteriophages were considered to belong to the Myobiridae and the bacteriophages with icosahedral heads with a diameter of about 110 nm and long tails of 160 nm.

Example 4 Bacteriophage  Structural protein analysis

Sample buffer solution (SDS 10% w / v, 10 mM DTT, glycerol 20% v / v, 0.2M tris-HCl, pH 6.8, bromophenol blue 0.05% w / v) in 10 ¹⁰ PFU of bacteriophage isolated in Example 2 After mixing 4 μl, it was boiled for 3 minutes, loaded onto a 12% SDS-PAGE gel, and electrophoresed, and the separated bands were visualized by staining with Coomassie blue G-250. The results are described in FIG. As a result of analysis, PA26 was composed of 14 proteins. The molecular weight ranged from 15 to 130 kDa, and the most important protein was 65 kDa.

Example  5 Bacteriophage  Growth and adsorption characterization

Pseudomonas aeruginosa was cultured to OD ₆₀₀ = 0.5 and then infected with the bacteriophage isolated in Example 2 of 0.1 MOI. Thereafter, while stirring and incubating at 37 ° C, samples were taken at 0, 5, 10, and 20 minutes, respectively, and the number of bacteriophage was confirmed using the double agar layer plaque assay described in Example 1.

The results are described in FIG. Adsorption was the first step for bacteriophage to infect the host, less than 10% when the number of prephages was more than 5 minutes and less than 1% after 10 minutes. Therefore, it can be seen that rapid adsorption of 90% or more occurs within 5 minutes.

One-step growth assays were incubated with Pseudomonas aeruginosa up to OD ₆₀₀ = 0.5, followed by infection with bacteriophage isolated in Example 2 of 0.1 MOI. After allowing the bacteriophage adsorption to occur at 37 ° C for 5 minutes, the pre-phage was removed by centrifugation at 13000 rpm for 5 minutes. The precipitate was then suspended in 20 ml LB medium and incubated with stirring at 37 ° C. Subsequently, two samples were taken at 5 minute intervals during incubation, one of which was treated with chloroform (1% v / v) to measure phage titer, and the other was not treated with chloroform, and the phage concentration ( phage titer) was measured. Phage concentration was performed using the double agar layer plaque assay described in Example 1. [

One-step growth analysis confirmed the eclipse period and burst size of PA26. The results are described in FIG. The disappearance of PA-26 was 10 minutes and the incubation time was 15 minutes, and the discharge number was 252.

Example  6 Bacteriophage  Temperature and pH  Stability analysis

Different pH buffers (2M sodium acetate pH3, 4, 5, and 6; 2M Tris-Cl pH7, 8, 9; 2M glycine pH 10 and 11) were prepared. The buffer and phages of 4 × 10 ⁶ PFU / ml were mixed at a ratio of 1: 1 to analyze stability for temperature and pH.

For stability analysis on temperature, the phage diluted with SM buffer as described above was incubated for 1 hour at each temperature (4 ° C, 37 ° C, 45 ° C, 55 ° C, 65 ° C, 80 ° C). Subsequent plaque analysis (RIZVI S and MORA PT. 1963. Nature 200: 1324-1325) confirmed the stability of PA26 at different temperatures. The results are described in Figure 6 (a). PA26 showed about 100% stability at 4-37 ° C. Stability decreased by about 30% at 45 ° C and decreased by more than 99% above 65 ° C.

The stability of phage to pH in each buffer was analyzed. The result is in FIG. 6 (b). PA26 was found to be the most stable at pH 7, and was found to be more than 90% stable at pH 6 and pH 8. At other pHs, the stability of the phage was reduced by more than 40%, and no phage was observed at pH 3.

Considering that the growth temperature of Pseudomonas aeruginosa is 4-45 ° C., the optimum temperature is 37 ° C., and the optimum pH is pH 7, the phages of the present application are stable under these conditions.

Example  7 On a bacteriophage  By Pseudomonas Eruginosa  Growth inhibition and Biofilm  Formation Inhibition Assay

The inhibitory effect of biofilm formation by bacteria was basically performed in the same manner as described in Knezevic and Petrovic paper (Knezevic and Petrovic, 2008, J Microbiol Methods. 74 (2-3): 114-8.). In summary, 96 well plates were inoculated with Pseudomonas aeruginosa at 1 × 10 ⁶ CFU / ml. PA26 phages were then infected with various 10 ⁰ , 10 ⁻² and 10 ⁻⁴ MOIs. Phage was not treated for control wells. The plates were then incubated at 37 ° C. and 120 rpm for 18 hours. Thereafter, the suspended cells were removed, and each well was washed twice with PBS (phosphate-buffered saline), followed by air drying. Then fixed for 15 minutes with 40% methanol, the plate was air dried and stained with 0.4% crystal violet solution for 15 minutes. Subsequently, the mixture was washed with running water, air dried, and incubated for 20 minutes after adding 33% acetic acid. The degree of biofilm formation was measured at 595 nm using a 96 well plate reader (ELISA reader, BioRad). Three experiments were performed independently to use the mean value.

In order to confirm the growth inhibition of bacteria, as described above, after infection with Pseudomonas aeruginosa with PA26 of each MOI, OD was measured by time. The results are described in FIG. As shown in FIG. 7. PA26 has been shown to effectively inhibit Pseudomonas aeruginosa specifically its growth and biofilm formation. As shown in FIG. 7, it was shown that the MOI 0.01 or more inhibits the biofilm formation of 95% or more.

Pseudomonas aeruginosa ATCC 15522, ATCC 10145, ATCC 9027, ATCC 25619, ATCC 13388, and ATCC 15692 were also performed as described above. The results are shown in Table 1. PA26 shows a broad host range capable of infecting all Pseudomonas strains except ATCC 15522.

Pseudomonas Eruginosa Infection degree ATCC 15522 - ATCC 10145 + ATCC 27853 + ATCC 9027 + ATCC 25619 + ATCC 13388 + ATCC 15692 +

Attach an electronic file to a sequence list

Claims (10)

A bacteriophage having a restriction enzyme fragment length polymorphism (RFLP) pattern of FIG. 2 or a sequence of SEQ ID NO: 1, wherein the bacteriophage has a melting ability against Pseudomonas aeruginosa.
The method of claim 1, wherein the Pseudomonas aeruginosa US Cell Line Bank (ATCC) 10145, US Cell Line Bank (ATCC) 27853, US Cell Line Bank (ATCC) 9027, US Cell Line Bank (ATCC) 25619, US Cell Line Bank (ATCC) 13388 Or bacteriophage, which is the American Cell Line Bank (ATCC) 15692.
Progeny of the bacteriophage according to claim 1, which exhibits the same biological properties as the bacteriophage.
The bacteriophage according to claim 1, wherein the bacteriophage has been deposited with the Korea Microorganism Conservation Center under the accession number KFCC11533P as of June 13, 2012.
delete An antibiotic comprising the bacteriophage according to any one of claims 1 to 4.
Feed additive comprising a bacteriophage according to any one of claims 1 to 4.
Disinfectant comprising a bacteriophage according to any one of claims 1 to 4.
An agent for preventing or inhibiting biofilm formation comprising the bacteriophage according to any one of claims 1 to 4.
10. A method for inhibiting or inhibiting biofilm formation, comprising contacting a biofilm formation preventing or inhibitor according to claim 9 with Pseudomonas aeruginosa.

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