KR20130128166A - Biofilm formation inhibitors comprising streptomyces sp. or kribbella sp. and a method using thereof - Google Patents

Abstract

The present invention provides a preventive or inhibitory composition of biofilm formation caused by Staphylococcus aureus containing Streptomyces sp., Kribbella sp., or a supernatant thereof as an active ingredient. The present invention provides a method for preventing or inhibiting biofilm formation through a contact of a formulation or a composition according to the present invention to bacteria. The composition is used as a pharmaceutical composition, a composition for disinfection, a medical washing composition, and a composition for environmental purification.

Description

TECHNICAL FIELD The present invention relates to a biofilm formation preventing or inhibiting agent including Streptomyces spp. Or Chrysomel spp., And a method of preventing or inhibiting biofilm formation using the same. OR KRIBBELLA SP. AND A METHOD USING THEREOF}

The present invention relates to a method for preventing or inhibiting biofilm formation by bacteria and a method for preventing or inhibiting biofilm formation comprising treating the surface or administering the same to a subject.

In nature, bacteria co-exist in different species, flocking to other bacteria, and compete for resources and space. Many bacteria form biofilms that are sticky microbial communities that attach to surfaces using polysaccharides or proteins. The biofilm is a complex, well-organized population that is resistant to common antibiotics, host immune and external stresses and contributes to bacterial resistance to chronic infections (Costerton et al. al . 1999; Hoffman et al . 2005). Therefore, it is important to understand how bacteria control their biofilms in order to find nontoxic compounds that inhibit and degrade biofilms without developing bacterial drug resistance.

In some places infected with bacteria, mucus, which is a colony of bacteria wrapped in a polymer substrate, is sometimes present. The bacterial complex of mucus formed by this bacterium is called a biofilm, biofilm or biofilm (J Bacteriol 176: 2137-2142, 1994). In the biofilm, an extracellular matrix (mucosal surface) composed of a polymer matrix (polysaccharide and polypeptide mainly) surrounds the colony. In other words, a biofilm is a complex composed of a bacterial colony that is a solid biological surface and an outer membrane that is a non-biological surface. Therefore, the biofilm of the present specification means the whole including the outer membrane and the bacterial colonies contained therein. Biofilm is a concept proposed in the late 1970s by Professor Costerton, Director of Biofilm Engineering Center, Montana State University, USA. The environment in which bacteria coexist. Biofilms are commonly found in nature, such as mucus found in rocks or ponds. Biofilms are small cities made of bacteria, in which bacteria communicate with each other and defend against the outside world. This allows the biofilms to survive bacteria under various environmental stresses, including antibiotics.

Such biofilms are often found in connection with bacterial infectious diseases in addition to the natural environment. It can form on human organs, appear in the form of plaques on teeth, or on industrial equipment or medical implants. Because of this, biofilms have been of interest to researchers studying pneumonia accompanied by periodontal disease, cystic fibrosis, and earaches in the middle ear. The National Institutes of Health estimated in 2002 that up to 80 percent of the bacterial population spread pathogens through the formation of such biofilms. Antibiotics, which are effective against separately planktonic bacteria, tend to lose their efficacy when bacteria form biofilms (Trends Microbiol 9: 34-39, 2001). When bacteria form a biofilm, antibodies cannot penetrate the outer membrane of the biofilm, rendering the host immune system incapacitated, and increasing the resistance of the bacteria to antibiotics by up to 1,000 times (Antimicrob Agents). Chemother 47: 3407-3414, 2003). The cause of the increase in resistance to antibiotics due to the formation of such biofilms is not yet precisely explained.

The first is the ecological change of microorganisms. In the biofilm formed state, the binding force between the bacteria is strengthened, and as a result, the bacterial colonies do not spread well, and thus bacterial growth is also reduced. This weakens our dependence on exchange with the environment, slows our metabolism and consequently decreases our sensitivity to antibiotics. The second is the physical properties of the outer membrane composed of mucopolysaccharides. The mucopolysaccharides that form the outer membrane have electrical properties and tend to bind to antibiotics, which prevents them from spreading. In other words, antibiotics are not easily transmitted to individual bacteria, and antibiotics are difficult to exert their efficacy. The third is presumably the production of inhibitors that are associated with the general antibiotic resistance acquisition mechanism. The most commonly known inhibitors of antibiotic efficacy are beta-lactamases produced by Pseudomonas spp. When the biofilm is formed, non-resistant bacteria present in the biofilm tend to become resistant bacteria by obtaining resistance-related genes through horizontal gene transfer from the surrounding resistant bacteria. In other words, if the biofilm is formed in the infected area, it can be said that the antibiotic resistance state. For these reasons, the formation of biofilms makes it difficult for antibiotics, which have been widely used to treat infections, and as a result, the therapeutic effects of antibiotics are weakened. For this reason, biofilm formation means entering a chronic bacterial infection state. In this case, as described above, the bacteria are less susceptible to antibiotics, and the use of antibiotics is almost ineffective. To overcome this, simply overdose antibiotics to increase the antibiotic resistance of the bacteria. In other words, biofilm-forming bacterial infections simply mean that treatment with antibiotics is no longer effective. In particular, infections caused by bacteria forming the biofilm are often caused by multi-drug resistance bacteria that are resistant to various antibiotics. Therefore, in order to prevent the antibiotic treatment from being formed due to the formation of such biofilms, new antibiotics can be developed to remove the biofilm even if the biofilm is formed, or it can destroy the outer membrane of the biofilm so that the existing antibiotic can be effective. Ingredients should be used in conjunction with existing antibiotics.

Enterohemorrhagic E. coli O157: H7 is the most common human pathogen causing hemorrhagic enteritis worldwide. Popular occurrences occurred in the United States in 1982 and 2006, and their chimerical strain occurred in Europe in 2011. However, there is no effective treatment for intestinal E. coli O157: H7 infection (Tarr et al. 2005) and their serotypes form a strong biofilm on surfaces of various materials such as leaves, stainless steel, glass and polystyrene.

Several non-toxic antifibrotic compounds against intestinal E. coli O157: H7 have been identified, for example exopolysaccharides isolated from Lactobacillus acidophilus (Kim et < RTI ID = 0.0 > al . 2009), limonoids isolated from grapes (Vikram et al . 2010a), plant flavonoids, naringenin, kaempferol, quercetin and apigene (Vikram et al . 2010b) reduce intestinal E. coli O157: H7 biofilm formation .

Actinomycetes , including Streptomyces , are widespread in the environment; They can be separated from soil, sea and organic material worldwide (McNeil and Brown 1994). The family of Streptomyces is an abundant resource of bioactive compounds, important antibiotics, enzymes, enzyme inhibitors, and pharmacologically active substances (Chun et al . 1997; Labeda et al . 1997; Xu et al . 2004). E. coli O157: H7 is found in the soil where actinomycetes dominate, so E. coli O157: H7 is likely to encounter actinomycetes (McNeil and Brown 1994).

Staphylococcus aureus is an important human pathogen that can acquire antibiotic resistance. Staphylococcus aureus is the causative agent of the development of world-class human infections (Lowy 1998). One of the important mechanisms of antibiotic resistance in Staphylococcus aureus is biofilm formation. Therefore, it is important to understand how Staphylococcus aureus controls biofilms in order to find nontoxic compounds that inhibit and degrade biofilms without bacteria developing drug resistance.

Staphylococcus aureus is a Gram positive bacterium that is a pathogenic microorganism that causes purulent, abscess formation, various purulent infections, and sepsis. The resistance rate to antibiotic methicillin, which is investigated in Korea, averages 73%, which is the world's highest harmful pathogen. This means that 73% of Staphylococcus aureus, which does not die even with antibiotics, is a very serious pathogen. In addition, Staphylococcus aureus has many species that can form a biofilm, and when the biofilm is formed, it is impossible to penetrate the drug, thereby showing a chronic infection pattern. For this reason, the formation of biofilms by Staphylococcus aureus leads to chronic infections (FEMS Microbiology Letters 252: 89-96, 2005). The treatment of diseases caused by Staphylococcus aureus with biofilms is particularly difficult compared to the treatment of diseases caused by biofilms formed by other bacteria. Because of the difficulties of drug delivery due to the biofilm even when drugs are used for the treatment of diseases, the antibiotic-based treatment is not effective because of the nature of Staphylococcus aureus, which has a large number of resistant strains even if the drug is delivered. Thus, biofilm treatment formed by Staphylococcus aureus requires an approach by a new substance rather than an antibiotic-dependent one. Several attempts have been made with regard to the treatment of biofilms formed by Staphylococcus aureus, but no clear effective method has been found. A rather effective attempt, however, has been reported to use lysostaphin, which can be used to remove biofilms formed by Staphylococcus aureus (Antimicrob Agents Chemother 47: 3407-3414, 2003). Lysostaphin is an antibacterial enzyme obtained from Staphylococcus aureus and is an enzyme capable of destroying the cell membrane of Staphylococcus aureus. It specifically cuts the pentaglycine cross bridges found in the peptidoglycan structure of Staphylococcus aureus as glycylglycine endopeptidase. Thus, lysostaphin has potential for development as an extremely potent anti-Staphylococcal agent for Staphylococcus aureus. However, although lysostapine has excellent efficacy, it is not perfect. There is a significant presence of lysostaphin-resistant strain in Staphylococcus aureus (J Clin Microbiol 11: 724-727, 1980; Antimicrob Agents Chemother 47: 3407-3414, 2003). In other words, it is difficult to expect Lysostaphin to be effective in all Staphylococcus aureus because Staphylococcus aureus has different lysostaphin sensitivity. In addition, another problem is the emergence of Staphylococcus aureus, which has become resistant to Rixotaffin. Such strains that have acquired resistance to lysostaphin are called lysostaphin-resistant Staphylococcus aureus variants (Antimicrob Agents Chemother 51: 475-482, 2007). The mechanism of obtaining resistance to lysostaphin is not yet known exactly, but one mechanism reported is as follows. Mutations in the FemA gene result in the expression of an inactive FemA protein, resulting in monoglycine cross bridges in the peptidoglycan structure that neutralizes the action of lysostaphin (J Bacteriol 188: 6288-6297). , 2006). In an attempt to overcome these disadvantages of lysostaphin, the use of lysostim and other enzymes such as lysozyme or antibiotics such as methicillin, oxacillin, and vancomycin together Studies are active (Antimicrob Agents Chemother 21: 631-535, 1982; J Antimicrob Chemother 59: 759-762, 2007; Folia Microbiol (Praha) 51: 381-386, 2006). However, even if these materials are used together, biofilms consisting of Staphylococcus aureus with low susceptibility to susceptibility or susceptibility to susceptibility may not be effective. In the end, it is necessary to develop new materials that can compensate for the drawbacks of lysostaphin. Substances that meet these objectives may be used alone or in combination with resource taps or conventional antibiotics. In particular, if the antimicrobial effect can be achieved by a mechanism different from lysostaphin or other antibiotics.

Various mechanisms and environmental conditions involved in biofilm formation and degradation of Staphylococcus aureus include quorum sensing, protease, DNase, cis-2-decenoic acid, and D-. Amino acids, phenol-soluble polypeptides and pH changes (Boles and Horswill 2011). In particular, proteases in Staphylococcus aureus have been identified by the agr quorum sensing system (Vuong et al . 2000; Boles and Horswill 2008) to inhibit their biofilm formation and break down the biofilm. Serine protease in S. epidermidis inhibits biofilm formation of Staphylococcus epidermis (Iwawse et < RTI ID = 0.0 > al . 2010).

Recently, actinomycetes have been reported to produce compounds that inhibit the biofilm of some pathogenic bacteria. For example, Streptomyces albus extract, identified by screening for 88 marine Actinomycetes , Vibrio harvey ) (You et al. 2007). However, the mechanism of reducing their biofilm and the information of these anti-biofilm compounds are not known, and the strain in the present invention having anti-biofilm activity by bacteria is not known.

Accordingly, the present inventors screened 458 actinomycetes isolates to identify non-toxic biofilm inhibitors for bacteria. Streptomyces sp. BFI 250, and Klebella sp. BFI 1562 inhibited the biofilm formation of Staphylococcus, thereby completing the present invention.

An object of the present invention is to provide a novel genus Streptomyces belongs to the actinomycetes (Actinomycetes) having a biofilm formed prevented or reduced activity of the bacteria (Streptomyces sp.) BFI 250 strain (KCTC 12190BP).

Another object of the present invention is to provide a novel Cri Bella in (Kribbella sp.) BFI 1562 strain (KCTC 12189BP) belonging to the actinomycetes (Actinomycetes) having a biofilm formed prevented or reduced activity of the bacteria.

Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically effective amount of Streptomyces sp. BFI 250, Kribbella The present invention also provides a pharmaceutical composition for preventing or inhibiting biofilm formation by Staphylococcus sp. containing Staphylococcus sp. containing at least one selected from the group consisting of BFI 1562 or a supernatant thereof.

Another object of the present invention is to provide a biofilm of Staphylococcus sp. Containing Streptomyces sp. BFI 250, Kribbella sp. BFI 1562 or a culture thereof as an active ingredient, And to provide a composition for disinfection, medical cleaning, and environmental purification for preventing or inhibiting the formation of a microorganism.

Another object of the present invention is to provide a method for preventing or inhibiting biofilm formation, comprising the step of contacting the biofilm formation preventing or inhibiting composition with a genus Staphylococcus.

In order to achieve the above object the present invention provides a novel genus Streptomyces (Streptomyces sp.) BFI 250 strain (KCTC 12190BP) belonging to prevent biofilm formation or actinomycetes (Actinomycetes) having an inhibitory activity of the bacteria.

In another aspect, the present invention provides a novel Cri Bella in (Kribbella sp.) BFI 1562 strain (KCTC 12189BP) belonging to the actinomycetes (Actinomycetes) having a biofilm formed prevented or reduced activity of the bacteria.

The present invention also relates to a method for preventing biofilm formation by Staphylococcus sp. Containing Streptomyces sp. BFI 250, Kribbella sp. BFI 1562 or a culture thereof as an active ingredient Or inhibiting the proliferation of cancer cells.

The present invention also relates to a method for preventing biofilm formation by Staphylococcus sp. Containing Streptomyces sp. BFI 250, Kribbella sp. BFI 1562 or a culture thereof as an active ingredient Or disinfecting detergent, medical cleaning, and environmental cleaning composition.

The present invention also provides a method for preventing or inhibiting biofilm formation comprising contacting the biofilm formation preventing or inhibiting composition with a genus Staphylococcus.

Streptomyces sp. BFI 250 and Kribella sp. BFI 1562 according to the present invention have an effect of inhibiting biofilm formation of Staphylococcus sp. And decomposing biofilms already formed.

Figure 1 is a graph showing the distribution of Staphylococcus aureus biofilm formation on the actinomycetes library: the numbers above each bar graph represent the number of actinomycetes and the biofilm formation (%) on the X axis represents the relative biofilm formation (100 X Biofilm formation in actinomycetes culture supernatant / biofilm formation of untreated group).
Figure 2 is a genus Streptomyces (Streptomyces sp.) BFI 250 strain and Cri Bella in (Kribbella sp.) BFI 1562 strains are S. Aureus (S. aureus) is a graph showing to reduce the biofilm formation: two S. S. aureus (ATCC 25923 and ATCC 6538) strains were used (A and B) and S. Cell growth of S. aureus (ATCC 25923) was measured by culturing actinomycetes culture supernatant (1%) at 37 ° C and 250 rpm (C); Proteinase K was added at the beginning of culture for biofilm analysis (D).
FIG. 3 is a graph showing the effect of dispersing the formed Staphylococcus aureus biofilm in Streptomyces sp. BFI 250 strain and Krivela sp. BFI 1562 strain: ATCC 25923 (A) and ATCC 6538 (B) After incubation, the culture supernatant of Actinomycetes was added to the Staphylococcus aureus culture in 96-well plates and incubated for 17 hours; The total biofilm formation was measured at OD ₅₇₀ , stray cell growth of S. aureus was measured at OD ₆₂₀ , Pro K was 1 unit (ug / ml), BFI 250 and BFI 1562 were the supernatants of actinomycetes AN090250 and AN091562 %, v / v).
FIG. 4 is a photograph showing the proteolytic activity of Streptomyces sp. BFI 250 and Krivela sp. BFI 1562. The culture supernatant (10 L) was treated with milk agar plate and reacted at 37 ° C for 24 hours; GSS medium was used as a negative control, and protinase K was used as a positive control; Pro K 0.1 and Pro K 0.01 mean 0.1 and 0.01 μg / mL proteinase K in a 10 μL dose; BFI 250 and BFI 1562 represent actinomycetes AN090250 and AN091562, respectively.

Hereinafter, terms used in the present invention will be described.

As used herein, the terms "prevention" and "inhibition" refer to any action that delays the formation of the biofilm or degrades the biofilm produced by administration of the composition of the present invention.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method.

As used herein, the term "individual" means any animal such as a human, a monkey, a dog, a goat, a pig or a mouse having a disease in which biofilm formation by bacteria is inhibited by administration of the composition of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Streptomyces sp. BFI 250 strain (Deposit No. KCTC 12190BP) belonging to Actinomycetes having an activity of preventing or inhibiting biofilm formation by bacteria.

The present invention also provides a novel Kribbella sp. BFI 1562 strain (Deposit No. KCTC 12189BP) belonging to Actinomycetes having an activity of preventing or inhibiting biofilm formation by bacteria.

In one embodiment of the present invention, actinomycetes library screening was performed to find actinomycetes having Staphylococcus aureus biofilm inhibiting activity. Of the 458 actinomycetes culture supernatants tested, two supernatants showed biofilm inhibitory activity against the strongest Staphylococcus aureus.

In one embodiment of the present invention, 16S rRNA gene sequences were determined to identify strains that significantly inhibit biofilm formation. As a result, strains having the biofilm inhibiting activity against the strongest Staphylococcus aureus were named Streptomyces sp. BFI 250 and Krivela sp. BFI 1562.

The present invention also provides a pharmaceutical composition for inhibiting or inhibiting biofilm formation by Staphylococcus sp. Containing Streptomyces sp. BFI 250, Krivela sp. BFI 1562 or a culture thereof as an active ingredient.

The Staphylococcus spp . Can be, but is not limited to, S. aureus , S. simiae , S. auricularis , S. canosus ( S. carnosus , S. condimenti , S. massiliensis , S. piscifermentans , S. simulans , S. capitis , S. capitis , S. caprae , S. epidermidis , S. saccharolyticus , S. devriesei , S. aureus , S. haemolyticus , S. hominis , S. chromogenes , S. felis , S. delphini , S. aureus , S. hyicus , S. intermedius , S. lutrae , S. microti , S. muscae , , S. pseudointermedius , S. rostri , S. gallinarum , And preferably S. aureus .

In one embodiment of the present invention, in the crystal violet biofilm assay, the supernatant of Streptomyces sp. BFI 250, Krivela sp. BFI 1562 significantly reduced biofilm formation by Staphylococcus aureus.

In one embodiment of the present invention, the growth rate of the Streptomyces sp. BFI 250 and the Klebella sp. BFI 1562 in the LB medium were measured to confirm the toxicity against growth of Staphylococcus aureus. As a result, It did not inhibit the growth of Aureus. Therefore, the biofilm inhibition of Staphylococcus aureus against the supernatant of the Streptomyces sp. BFI 250 and Kribella sp. BFI 1562 of the present invention is due not only to their antibiotic activity but also to their antifungal activity.

Therefore, the composition containing the Streptomyces sp. BFI 250, the Klebella sp. BFI 1562 or the culture medium thereof as an active ingredient according to the present invention does not affect the cell growth of Staphylococcus sp., And thus the probability of developing antibiotic resistance And inhibits biofilm formation. Therefore, it can be usefully used in pharmaceutical compositions for preventing or inhibiting biofilm formation due to Staphylococcus sp.

In the administration of the composition of the present invention to a subject, the composition of the present invention can effectively remove the biofilm formed by the genus Staphylococcus, as described above. Therefore, the biofilm formation due to Staphylococcus spp. And can be usefully used for treatment of diseases.

Compositions for this purpose may further comprise ingredients that have been proven to have antimicrobial activity against the genus Staphylococcus to obtain the effect of additional treatment. When used in combination with other existing antibiotics or other proven substances, the composition of the present invention inhibits or decomposes the biofilm so that the existing antibiotics or other proven substances can exert their efficacy for the initial purpose. . For medical therapeutic uses of the composition in the present invention, the effective dose of the composition is usually readily determined and prescribed by a skilled physician. The particular dose will depend on the age or weight of the animal, including the human being to be treated, the weight and clinical symptoms and method of administration. Suitable application, spraying, spraying and dosage of the pharmaceutical compositions of the present invention may be formulated with the method of formulation, mode of administration, age, weight, sex, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response of the patient. Depending on the same factors, usually skilled practitioners can easily determine and prescribe a dosage effective for the desired treatment.

The composition of the present invention may be administered by oral or parenteral administration, and may be administered by parenteral administration using intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration or topical administration. The pharmaceutically acceptable carriers to be contained in the composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no. The composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. The compositions of the present invention may be prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. It may also be prepared by incorporation 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 extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers. The use of the compositions of the present invention in the treatment of bacterial related diseases provides additional advantages over conventional antibiotic based treatments.

In addition, the present invention can be provided as a composition for disinfection, medical cleaning, or environmental purification containing Streptomyces sp. BFI 250, Krivela sp. BFI 1562 or a culture thereof as an active ingredient.

The composition containing the culture medium of Streptomyces genus BFI 250 and Krivela genus BFI 1562 as an active ingredient of the present invention inhibits the biofilm formation by Staphylococcus sp., And therefore the composition for sterilization, medical cleaning or environmental purification And can be usefully used in compositions. The composition for this purpose may further include a component that has proven antimicrobial activity against bacteria in order to obtain additional effects, and may further include a component that is included in the composition for conventional disinfection, medical cleaning, environmental purification. In addition, the composition of the present invention may include, but is not limited to, spraying the composition of the present invention in the form of a spray on a part to prevent biofilm formation, that is, artificial joint, catheter surface, endoscope or wound. The composition of the present invention may also include a method of applying, spraying or spraying to a biofilm forming site.

The present invention also provides a method for preventing or inhibiting biofilm formation, comprising the step of contacting Streptomyces sp. BFI 250, Krivela sp. BFI 1562 or a culture thereof with Staphylococcus sp.

Almost all bacteria can grow in close contact with the surface, in which case they form a monolayer, aggregates, or biofilms. Biofilm is a three-dimensional structure composed of extracellular polymeric matrix and microorganisms secreted by microorganisms, and can be formed on almost all kinds of solid surfaces including biological tissues. Such biofilms are commonly found in biological, medical and industrial environments that are commonly encountered in the environment. In the case of pathogenic bacteria that can live in a living body, biofilms are formed on various tissues / organs including host epithelial cells, bones, teeth, and inner walls of blood vessels, various artificial implants, and various medical instruments / equipments / facilities. Can be formed. When the biofilm is formed, bacteria can withstand harsh environments and have strong resistance to antibiotics and immune cells, making it very difficult to remove, causing chronic inflammatory diseases, and causing microbiologically induced corrosion in objects. do. Therefore, in the prevention of corrosion by bacteria or the prevention / treatment of infection by pathogenic bacteria, control of biofilm formation is very important.

Thus, contact with the Staphylococcus genus of the present invention may be by treating the surface or administering the biofilm formation preventing or inhibiting composition to the subject, although not exclusively.

In addition, said Staphylococcus spp. Is preferably Staphylococcus aureus.

In the method of treating the surface of the composition of the present invention, the surface is not limited to any one selected from the group consisting of drainage pipe, glazed ceramic, posulin, glass, metal, wood, chromium, plastic, vinyl or pomica or And combinations thereof.

Bacterial biofilms are also found in most implanted artificial surfaces, such as catheters, heart valves, shunts and prosthetic devices in the medical field (New Microbiol). 22: 337-341, 1999; J Med Microbiol 50: 582-587, 2001; Infections Associated with Indwelling Medical Devices, pp. 55-88, 2000, ASM, Washington, DC). Thus, implantable medical devices are preferably coated with an antibacterial agent. In the case of artificial implants (implant) is the only way to remove the biofilm is formed surgically because it is very important to prevent the formation of the biofilm.

Thus, in the method of treating the surface of the composition of the present invention, the surface may be, but is not limited to, the surface of a tissue or organ derived from an organism, or a medical article.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, and the present invention is not limited to the following examples.

Streptomyces sp. Streptomyces sp .) BFI  250 and Creebella ( Kribbella sp .) BFI  Of the 1562 strain Staphylococcus  Aureus ( Staphylococcus aureus ) Biofilm  Inhibitory Activity Assay

< Example  1> Preparation of strain

All experiments were performed at 37 ° C and were performed using Luria-Bertani medium (Sambrook et al., 1989) with S. aureus (ATCC 25923), S. aureus (ATCC 6538) # 11862). Actinomycetal library obtained from soil actinomycetes obtained from the Microbiological Resource Center in Korea Biotechnology Research Institute (Daejeon, Korea) was used for screening biofilm inhibitors. Actinomycetes isolated from soil in Korea initially contained 10 g glucose, 1 g yeast extract (Difco), 2 g peptone (Difco), 1 g beef extract (Difco) and 15 g agar (7 g) per liter (pH 7.0) for 7 days at 28 ° C. Incubation was carried out in Bennett's agar plates. All actinomycetes were cultivated in GSS medium (pH 7.2, 10 g water-soluble starch, 20 g glucose, 25 g soy flour, 1 g beef extract, 4 g yeast extract, 2 g sodium chloride, 0.25 g K ₂ HPO ₄ and 2 g CaCO ₃ ). The actinomycetes supernatant was stored at 4 ° C until used.

< Example  2> Culture of bacteria

A single colony of S. aureus was inoculated into LB (25 ml) in a 250 ml flask and cultured at 37 ° C and 250 rpm. The overnight culture was re-inoculated at 1: 100 in fresh medium. Growth was observed at OD ₆₀₀ values converted to dry cell weights (OD _{600 of} 1 = 240 mg / l). Each experiment was performed with at least two independent cultures.

< Example  3> S. Aureus Biofilm  Screening of Actinomycetes Library to Suppress Formation

To identify the antifungal membrane compounds against S. aureus (ATCC 25923), supernatants from 458 soil actinomycetes were screened. Antibody screening demonstrated a diverse ability to control the biofilm formation of S. aureus (Fig. 1). A static biofilm formation assay was performed on a 96-well polystyrene plate (SPL Bioscience, Korea) (Pratt and Kolter 1998). Briefly, cells were incubated at 37 ° C without shaking for 24 hours at an OD ₆₀₀ of 0.05. The total biofilm count was measured at 570 nm using crystal violet. For a dispersion assay S. aureus was initially incubated in 96 well plates at 37 [deg.] C for 7 or 14 hours without shaking. The supernatant of actinomycetes was added and the culture was further cultured for 7 or 17 hours before biofilm assay. The supernatant of actinomycetes was filtered through a 0.45 탆 filter to remove bacteria before incubation in S. aureus. Each data point was averaged over at least three independent cultures.

As a result, 77 actinomycetes reduced the biofilm formation of S. aureus by more than 80%, while 16 actinomycetes increased biofilm formation by more than 50% and screened more than 3 times. Therefore, the supernatant of soil actinomycetes mainly inhibited biofilm formation of S. aureus.

< Example  4> active type Actinomycosis  Sympathy

10S actinomycetes with the highest antifungal activity were identified using 16S rRNA gene sequences. A list of various actinomycetes was obtained using BLAST searching for the GenBank database (Table 1). Nine strains were Streptomyces genus and one was Kribbella genus. The supernatants of the two most active Streptomyces strains (AN090250 and AN091562) were selected and named Streptomyces sp. BFI 250 and Kribbella sp. BFI 1562, On May 2, KCTC 12190BP, KCTC 12189BP was deposited with KCTC at Korea Research Institute of Bioscience and Biotechnology. Streptomyces sp. BFI 250 is shown in SEQ ID NO: 1, and Kribbella sp. BFI 1562 is shown in SEQ ID NO: 2.

S. In Aureus  The highest for Antibiotics  Ten active Actinomycetes ID Strains Sequence identity (%) Biofilm formation
reduction (%) Relative protease activity AN090185 Streptomyces sp. HB096 100 82 ± 8 ++ AN090230 Streptomyces hebeiensis NBRC 101006 100 95 ± 4 +++ AN090250 Streptomyces halstedii 98 95 ± 1 +++ AN090282 Streptomyces laceyi 99 92 ± 6 + AN090374 Streptomyces turgidiscabies strain HBUM174876 99 92 ± 8 + AN090410 Streptomyces griseoruber 99 92 ± 8 + AN090414 Streptomyces akiyoshiensis strain HBUM174525 99 87 ± 5 + AN090423 Streptomyces sp. DA10202 99 83 ± 4 ++ AN090426 Streptomyces lanatus 99 93 ± 7 + AN091562 Kribbella hippodromi 99 94 ± 4 +++

< Example  5> floating S. Aureus  Without lowering cell growth, low concentrations of Actinomycetes  The supernatant is S. Aureus Biofilm  Inhibition of Formation Activity Analysis

To analyze the inhibitory activity of S. aureus biofilm formation, a static biofilm formation assay was performed on a 96-well polystyrene plate (SPL Bioscience, Korea) (Pratt and Kolter 1998). Briefly, cells were incubated at 37 ° C without shaking for 24 hours at an OD ₆₀₀ of 0.05. Total biofilm activity was measured at 570 nm using crystal violet. Each data point was averaged over at least three independent cultures.

As a result, the supernatants of Streptomyces sp. BFI 250 and Krivela sp. BFI 1562 inhibited the biofilm formation of two S. aureus strains (ATCC 25923 and ATCC 6538) in an apparent dose-dependent manner (Fig. 2A, 2B, 2E ). Very small concentrations (0.01%) of Streptomyces sp. BFI 250 and Krivela sp. BFI 1562 also inhibited biofilm formation of S. aureus (ATCC 25923) by more than 80% (Figs. 2A, 2B). However, the supernatants of Streptomyces sp. BFI 250 and Krivela sp. BFI 1562 were not toxic to S. aureus cells (Fig. 2C). These results indicate that the biofilm inhibition of S. aureus to actinomycetes is due not only to their antibiotic activity but also to their antifungal activity.

< Example  6> Actinomycetes  Identify the active ingredient in the supernatant

Solvent extraction was performed using a solvent such as methanol, hexane, ethyl acetate, ethyl ether or chloroform in order to identify the active ingredient of the actinomycetism supernatant that inhibits the biofilm of S. aureus. However, none of the solvent extracts exhibited anti - biofilm activity, while the aqueous residue after solvent extraction had antifungal activity. Also, heat treatment of the supernatant at 100 &lt; 0 &gt; C for 10 min removed the antifibrotic activity of the complete supernatant (Fig. 2A, 2B). These results indicate that the antifibrotic active ingredient in the supernatant is a protein or a peptide.

Since protease activity plays an important role in the S. aureus biofilm (Boles and Horswill 2011), the addition of proteinase K (0.1 μg ml ^-1 ) apparently results in biofilm formation of S. aureus at least 80% (Fig. 2D). Therefore, the protease activity assay was performed on milk agar plates. Protein hydrolysis activity was measured using skim milk agar plates containing 5 g of non-fat milk powder and 0.5 g of bacto-agar (Difco) in 50 ml of distilled water (Quiblier et al. 2011). To measure extracellular protease activity, the supernatant of actinomycetes was added to the pores in milk / agar plates and incubated at 37 ° C for 24 hours. GSS medium (10)) and protease K (Sigma-Aldrich) were used as negative and positive control, respectively. Proteolysis was observed using growth inhibition rings surrounded by actinomycetes supernatant.

Obviously, as the positive control proteinase K showed high protease activity, the supernatant of Streptomyces sp. BFI 250 and Krivela sp. BFI 1562 showed a high protease activity (transparent circular while decomposing milk) (Fig. 4). The amounts of protease in the Streptomyces sp. BFI 250 and Kribella sp. BFI 1562 supernatants were 0.1 μg protease K ml ^-1, respectively. In addition, all ten actinomycetes showed protease activity to different degrees, while the 16 biofilm-forming actinomycetes (Fig. 1) showed no protease activity. Therefore, the culture supernatant of actinomycetes forms a large amount of extracellular protease that inhibits biofilm formation of S. aureus. As a result, extracellular protease was found to be a major antifungal active ingredient in the actinomycetes supernatant.

< Example  7> Actinomycetes  S. of the supernatant. Aureus Biofilm  Separation activity assay

Like biofilm formation, the dispersion of biofilms produced is important for biofilm control. Therefore, biofilm dispersion activity was investigated. For a dispersion assay S. aureus was initially incubated in 96 well plates at 37 [deg.] C for 7 or 14 hours without shaking. The supernatant of actinomycetes was added and the culture was further cultured for 7 or 17 hours before biofilm assay. The supernatant of actinomycetes was filtered through a 0.45 탆 filter to remove bacteria before incubation in S. aureus.

As a result, when treated with Proteinase K, the culture supernatant of actinomycetes significantly inhibited the biofilm previously present in S. aureus and decomposed in a dose-dependent manner (Fig. 3). Specifically, the supernatant at 0.01% (v / v) of Streptomyces sp. BFI 250 and Krivela sp. BFI 1562 separated more than 80% of the generated S. aureus biofilm for 17 hours. Similarly, after adding the supernatant of Streptomyces sp. BFI 250, the short dispersion time (7 hours instead of 17 hours in FIG. 3) and the long incubation time (14 hours instead of 7 hours) before adding the actinomycete supernatant solution Almost the same results were obtained. Therefore, the supernatant of actinomycetes inhibited and dispersed the biofilm of S. aureus.

Korea Biotechnology Research Institute KCTC12190BP 20120502 Korea Biotechnology Research Institute KCTC12189BP 20120502

<110> Korea Research Institute of Bioscience and Biotechnology <120> BIOFILM FORMATION INHIBITORS COMPRISING STREPTOMYCES SP. BFI250          OR KRIBBELLA SP. BFI1562 AND A METHOD USING THEREOF <130> 12p-04-37 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1446 <212> DNA <213> Streptomyces sp. BFI 250 <400> 1 ggcaatanca tgcagtcgac gatgaagccc ttcggggtgg attagtggcg aacgggtgag 60 taacacttgg gcaatctgcc cttcactctg ggacaagccc tggaaacggg gtctaatacc 120 ggataacact ctgtcccgca tgggacgggg ttaaaagctc cggcggtgaa ggatgagccc 180 gt; tgagagggcg accggccaca ctgggactga gacacggccc agactcctac gggaggcagc 300 agtggggaat attgcacaat gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac 360 ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg aaagtgacgg tacctgcaga 420 agaagcgccg gctaactacg tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc 480 cggaattatt gggcgtaaag agctcgtagg cggcttgtca cgtcggatgt gaaagcccgg 540 ggcttaaccc cgggtctgca ttcgatacgg gctagctaga gtgtggtagg ggagatcgga 600 attcctggtg tagcggtgaa atgcgcagat atcaggagga acaccggtgg cgaaggcgga 660 tctctgggcc attactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac 720 cctggtagtc cacgccgtaa acgttgggaa ctaggtgttg gcgacattcc acgtcgttcg 780 gtgccgcagc taacgcatta agttccccgc ctggggagta cggccgcaag gctaaaactc 840 aaaggaattg acgggggccc gcacaagcag cggagcatgt ggcttaattc gacgcaacgc 900 gaagaacctt accaaggctt gacatatacc ggaaagcatc agagatggtg ccccccttgt 960 ggtcggtata caggtggtgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 1020 tcccgcaacg agcgcaaccc ttgttctgtg ttgccagcat gcccttcggg gtgatgggga 1080 ctcacaggag actgccgggg tcaactcgga ggaaggtggg gacgacgtca agtcatcatg 1140 ccccttatgt cttgggctgc acacgtgcta caatggccgg tacaatgagc tgcgatgccg 1200 cgaggcggag cgaatctcaa aaagccggtc tcagttcgga ttggggtctg caactcgacc 1260 ccatgaagtc ggagttgcta gtaatcgcag atcagcattg ctgcggtgaa tacgttcccg 1320 ggccttgtac acaccgcccg tcacgtcacg aaagtcggta acacccgaag ccggtggccc 1380 aaccccttgt gggagggagc tgtcgaaggt gggactggcg attgggacga agtcgtaaaa 1440 ggggaa 1446 <210> 2 <211> 1427 <212> DNA <213> Kribbella sp. BFI 1562 <400> 2 tcgagcggta aggccccttc gggggtacac gagcggcgaa cgggtgagta acacgtgagc 60 aacctaccct caactttggg ataagcctcg gaaacggggt ctaataccga atatcatctc 120 ctgcttcatg gtggggggtt gaaagttctg gcggttgggg atgggctcgc ggcctatcag 180 cttgttggtg gggtaatggc ctaccaaggc gtcgacgggt agccggcctg agagggcgac 240 cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat 300 tgcgcaatgg acgaaagtct gacgcagcaa cgccgcgtga gggatgacgg ccttcgggtt 360 gtaaacctct ttcagcaggg acgaagcgag agtgacggta cctgcagaag aaggaccggc 420 caactacgtg ccagcagccg cggtaatacg tagggtccga gcgttgtccg gaattattgg 480 gcgtaaaggg ctcgtaggcg gttcgtcacg tcgggagtga aaactcgggg cttaaccccg 540 agcctgcttc cgatacgggc agactagagg taggcagggg agagcggaac tcctggtgta 600 gcggtggaat gcgcagatat caggaagaac accggtggcg aaggcggctc tctgggcctt 660 acctgacgct gaggagcgaa agcgtgggta gcgaacagga ttagataccc tggtagtcca 720 cgccgtaaac gttgggcgct aggtgtgggg gacattccac gtcctccgtg ccgcagctaa 780 cgcattaagc gccccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg 840 ggggcccgca caagcggcgg agcatgcgga ttaattcgat gcaacgcgaa gaaccttacc 900 tgggtttgac atatagggaa atctcccaga gatggggggt ccgtaagggt cctatacagg 960 tggtgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1020 caaccctcgt cctatgttgc cagcacgtta tggtggggac tcataggaga ctgccggggt 1080 caactcggag gaaggtgggg atgacgtcaa gtcatcatgc cccttatgtc cagggcttca 1140 cgcatgctac aatggccggt acaaagggct gcgaagctgt aaggtggagc gaatcccaaa 1200 aagccggtct cagttcggat tggggtctgc aactcgaccc catgaagtcg gagtcgctag 1260 taatcgcaga tcagcaacgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1320 cacgtcatga aagtcggcaa cacccgaagc cggtggccta acccttgtgg agggagccgt 1380 cgaaggtggg gctggcgatt aggacgaagt cgtaacaagg taaccaa 1427

Claims (10)

A Streptomyces sp. BFI 250 strain deposited with Accession No. KCTC 12190BP, which has a biofilm formation inhibitory or inhibitory activity by bacteria.
Kribbella sp. BFI 1562 strain deposited with Accession No. KCTC 12189BP, which has a biofilm formation prevention or inhibitory activity by bacteria.
The genus Streptomyces (Streptomyces deposited with Accession No. KCTC 12190BP sp.) BFI 250 strain, accession number KCTC the Cri Bella deposited in a 12189BP (Kribbella sp.) BFI 1562 strain or star containing these as an active ingredient of the culture Philo Rhodococcus genus (Staphylococcus Pharmaceutical composition for preventing or inhibiting biofilm formation by sp.).
The pharmaceutical composition for preventing or inhibiting biofilm formation according to claim 3, wherein the Staphylococcus sp. Is Staphylococcus aureus .
The genus Streptomyces (Streptomyces deposited with Accession No. KCTC 12190BP sp.) BFI 250 strain, accession number KCTC the Cri Bella deposited in a 12189BP (Kribbella sp.) BFI 1562 strain or star containing these as an active ingredient of the culture Philo Rhodococcus genus (Staphylococcus Disinfection, medical cleaning or environmental purification composition for preventing or inhibiting biofilm formation by sp.).
The genus Streptomyces (Streptomyces deposited with Accession No. KCTC 12190BP sp.) Method of preventing or inhibiting biofilm formation by bacteria comprising contacting BFI 250 strain, Kribbella sp. BFI 1562 strain deposited with Accession No. KCTC 12189BP or a culture thereof with Staphylococcus spp. .
7. The method according to claim 6, wherein the Staphylococcus sp . Is Staphylococcus aureus .
7. The method of claim 6 wherein the star contact with the pillow Paracoccus genus is the genus Streptomyces (Streptomyces deposited with Accession No. KCTC 12190BP sp.) Method of preventing or inhibiting biofilm formation, characterized in that the BFI 250 strain, Kribbella sp. BFI 1562 strain deposited with Accession No. KCTC 12189BP, or a culture thereof are treated on a surface or administered to a subject.
The method of claim 8, wherein the surface is one or more combinations selected from the group consisting of a surface of a medical article, a drain pipe, a glazed ceramic, posulin, glass, metal, wood, chromium, plastic, vinyl or pomica. A method of preventing or inhibiting biofilm formation by bacteria.
9. The method for preventing or inhibiting formation of biofilm according to claim 8, wherein the surface is a tissue or an organism derived from an organism.

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