KR101461096B1 - Biofilm formation inhibitors comprising streptomyces sp. or kribbella sp. and a method using thereof - Google Patents

Abstract

The present invention provides a composition for preventing or inhibiting biofilm formation by Staphylococcus aureus containing Streptomyces sp. BFI 250, Krivela sp. BFI 1562 or a supernatant thereof as an active ingredient. The present invention provides a method for preventing or inhibiting biofilm formation through contact with a bacterial preparation according to the present invention and is usefully used as a pharmaceutical composition, a disinfecting composition, a medical cleaning composition, and an environmental purification composition .

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, comprising an agent for preventing or inhibiting biofilm formation by bacteria and a step of treating the surface or administering the agent to a subject.

In its natural state, germs coexist with diverse species forming a cluster with other bacteria, competing 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 biofilm in order to find non-toxic compounds that inhibit and degrade biofilms without the development of bacterial drug resistance.

In some parts of the bacterium that are infected, there is a mucus, a colony of bacteria surrounded by a polymer matrix. A mucus-producing bacterial complex formed by this bacterium is called a biofilm, biofilm or biofilm (J Bacteriol 176: 2137-2142, 1994). The biofilm encompasses the bacterial colonization of the extracellular matrix (mucosal surface) composed of polymer matrix (polysaccharide and polypeptide). The biofilm is a composite of bacterial colonies, a solid biological surface, and an outer membrane, a non-biological surface. Therefore, the biofilm in the present specification means the entire film including the outer membrane and the bacterial colonies contained therein. Biofilm is a concept proposed by Professor Costerton, director of the Center for Biofilm Engineering, Montana State University, USA, in the late 1970s. It is based on the idea that a viscous substance secreted by bacteria, such as a bacteria on a solid surface, secretes a viscous substance such as polysaccharide. Refers to an environment in which germs coexist. Biofilms are commonly found in nature, such as mucus found in rocks and ponds. The biofilm is a small city made of bacteria in which bacteria communicate with each other and defend against the outside world. This makes biofilm possible to survive under various environmental stresses including antibiotics.

These biofilms are often found in relation to bacterial infectious diseases in addition to the natural environment. It may be formed in a person's organs, in the form of plaque in the teeth, or in industrial equipment or medical implants. Because of this, the biofilm has been of interest to scholars studying periodontal disease, pneumonia associated with cystic fibrosis, and earache in the middle ear. In the 2002 report, the US National Institutes of Health estimated that up to 80% of bacterial populations are spreading pathogens through biofilm formation. Separately, antibiotics that have beneficial effects on planktonic bacteria also tend to lose efficacy when bacteria form biofilms (Trends Microbiol 9: 34-39, 2001). When bacteria form a biofilm, they can not pass through the outer membrane of the biofilm, making the host immune system ineffective and increasing the resistance of bacteria to antibiotics to about 1,000 times (Antimicrob Agents Chemother. 47: 3407-3414, 2003). The cause of increased resistance to antibiotics due to biofilm formation has not yet been clarified.

The first is the ecological change of microorganisms. When the biofilm is formed, the binding force between the bacteria is strengthened, and as a result, bacterial colonization does not spread well and bacterial growth is also lowered. This weakens the dependence on exchange with the environment, slows down metabolism, and results in a lower susceptibility 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 with antibiotics, thus interfering with the spread of antibiotics by binding with antibiotics. In other words, antibiotics do not transmit to individual bacteria, and antibiotics are difficult to exert their effects. 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. Once the biofilm is formed, bacteria that do not have resistance in the biofilm tend to acquire resistance gene related genes through horizontal gene transfer from surrounding resistant bacteria and become resistant bacteria. That is, when a biofilm is formed at the site of infection, it can be regarded as an antibiotic resistant state. For these reasons, the formation of a biofilm makes antibiotics, which are widely used in the treatment of infectious diseases, difficult to treat and, consequently, the therapeutic effect of antibiotics is weakened. For this reason, the formation of a biofilm implies a chronic bacterial infection. In this case, as described above, the susceptibility of bacteria to antibiotics is low, so it is hardly effective using antibiotics. To overcome them, simply over-prescribing antibiotics increases the antibiotic resistance of bacteria. In other words, bacterial infections with biofilms are simply treated with antibiotics, which means that they are no longer effective treatments. Especially, the infection caused by the bacteria forming the biofilm becomes more serious due to the multi-drug resistance bacteria which are resistant to various antibiotics. Therefore, in order to prevent the disabling of the antibiotic treatment due to the formation of the biofilm, a new antibiotic that can remove the biofilm may be developed even if the biofilm is formed, or the outer membrane of the biofilm may be destroyed The ingredients should be used with existing antibiotics.

Enterohemorrhagic E. coli O157: H7 is the most common human pathogen causing hemorrhagic enteritis worldwide. Notable outbreaks occurred in the United States in 1982 and 2006, and its 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 isolated globally from soil, sea, and organic matter (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). Because intestinal E. coli O157: H7 is found in actinomycetes dominant soil, intestinal E. coli O157: H7 is more likely to encounter actinomycetes (McNeil and Brown 1994).

Staphylococcus aureus is an important human pathogen capable of acquiring resistance to antibiotics. Staphylococcus aureus is a causative organism of global human infection (Lowy 1998). One of the important mechanisms of antibiotic resistance in Staphylococcus aureus is biofilm formation. It is therefore important to understand how Staphylococcus aureus controls the biofilm in order to find non-toxic compounds that inhibit and degrade the biofilm without the bacteria developing drug resistance.

Staphylococcus aureus is a gram-positive bacterium that is a pathogenic microorganism that causes pneumonia, abscess formation, various purulent infections, and sepsis. The average resistance rate to the antibiotic methicillin tested in Korea is 73%, which is the highest level in the world. This means that Staphylococcus aureus, which does not die even with the use of antibiotics, accounts for 73%, which is a very serious pathogen. In addition, Staphylococcus aureus has many species that can form a biofilm, and when a biofilm is formed, it is impossible to penetrate the drug, and thus, it exhibits a chronic infection pattern. Because of this, biofilm formation by Staphylococcus aureus results in chronic infections (FEMS Microbiology Letters 252: 89-96, 2005). The treatment of diseases caused by biofilm-forming Staphylococcus aureus 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. Therefore, the biofilm treatment formed by Staphylococcus aureus requires a new material approach rather than a method dependent on conventional antibiotics. There have been several attempts to treat the biofilm formed by Staphylococcus aureus, but no effective method was found. Nevertheless, a somewhat effective attempt has been made to utilize resource lysostaphin, which has been reported to be able to be used to remove biofilms formed by Staphylococcus aureus (Antimicrob Agents Chemother 47: 3407-3414, 2003). Resource Tapin, an antimicrobial enzyme obtained from Staphylococcus aureus, is an enzyme capable of destroying the cell membrane of Staphylococcus aureus. It specifically cleaves pentaglycine cross bridges found in the peptidoglycan structure of Staphylococcus aureus as glycylglycine endopeptidase. Therefore, it can be said that the resource tapin has the potential to develop an extremely potent anti-Staphylococcal agent against Staphylococcus aureus. However, even though Resource Tapin has excellent efficacy, it can not be said to be perfect. There is considerable 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 that resource tapin will be effective in all Staphylococcus aureus because the staphylococcus aureus have different lysostaphin sensitivities. In addition, another problem is that there is a Staphylococcus aureus that is resistant to resource tampering. Variants that have acquired resistance to these resource tags are termed resource protamine-resistant strains of Staphylococcus aureus (Antimicrob Agents Chemother 51: 475-482, 2007). Although the mechanism of their resistance to resource tapin is not yet known, one mechanism reported is as follows. A mutation in the FemA gene results in the expression of an inactive FemA protein (nonfunctional FemA protein) resulting in monoglycine cross bridges in the peptidoglycan structure, disabling the action of the resourcetapine (J Bacteriol 188: 6288-6297 , 2006). In an attempt to overcome the disadvantages of this resource tapin, the use of other enzymes, such as lysozyme, or resource taps, with antibiotics such as methicillin, oxacillin, vancomycin, Research is 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 substances are used together, biofilms composed of strains resistant to resource-toxin resistance, such as Staphylococcus aureus or resource-tapkin resistant strains, are also unable to remove effective biofilms. In the end, it will be necessary to develop new materials that can complement the disadvantages of resource tapping. Substances that meet these objectives may be used alone or in combination with resource taps or conventional antibiotics. Especially, it is more desirable to be able to exhibit the antimicrobial effect by a mechanism different from the resource tapin 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, 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 these biofilms and the information of these antifibrotic compounds are not known, and the strain of the present invention having antibacterial 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 "preventing" and "inhibiting" refer to all actions that delay the formation of the biofilm or decompose the biofilm formed 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, the nucleotide sequence of the 16S rRNA gene was determined in order to identify a strain that inhibits biofilm formation most significantly. 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 conventional antibiotics or other proven substances, the composition of the present invention inhibits or degrades the biofilm, so that existing antibiotics or other substances proved to be efficacious can exert their effects according to their initial purpose . In the medical therapeutic use of the composition in the present invention, the effective dose of the composition will ordinarily be readily determined and prescribed by a skilled practitioner. The specific dose will depend on the age or weight of the animal, including the human being treated, or the age and weight of the animal and the clinical symptoms and method of administration. The appropriate application, spraying, spraying, and dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, disease symptom, food, administration time, administration route, And the like, and the ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment.

The composition of the present invention may be administered orally or parenterally. In the case of parenteral administration, the composition may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously or locally. 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 contain lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. The composition of the present invention may be prepared in unit dose form by being formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. It may be manufactured by inserting it into a multi-capacity 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. The use of the compositions of the present invention in the treatment of bacterial 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 contain a component that has been proven to have antibacterial activity against the bacteria to obtain additional effects, and may further include ingredients for ordinary disinfection, medical cleaning, and environmental cleaning compositions. In addition, the composition of the present invention may include, but is not limited to, a method of spraying the composition of the present invention to a part to prevent biofilm formation, that is, a method of spraying on artificial joints, a catheter surface, an endoscope, or a wound. In addition, the composition of the present invention may include a method of applying, spraying or spraying onto a biofilm formation 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.

Nearly all bacteria can proliferate in close contact with the surface, forming a monolayer, forming aggregates, or forming biofilms. A biofilm is a three-dimensional structure composed of an extracellular polymeric matrix secreted by microorganisms and proliferating bacteria, and can be formed on almost all kinds of solid surfaces including living tissues. Such biofilms are commonly found in living, medical and industrial environments that are often encountered around them. In the case of pathogenic bacteria that can live in the living body, the biofilm can be used for various tissues / organs including the host's epithelial cells, bones, teeth, and blood vessel walls, various artificial implants (catheter, implant) Can be formed. When a biofilm is formed, bacteria are resistant to harsh environments as well as have strong resistance to antibiotics and immune cells. Therefore, they are very difficult to remove, cause chronic inflammatory diseases, and cause microbiologically induced corrosion do. Therefore, control of biofilm formation is very important in preventing / treating infections caused by bacterial corrosion or by pathogenic bacteria.

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 composition of the present invention on a surface, the surface may be coated with any one selected from the group consisting of a water pipe, glazed ceramic, posileine, glass, metal, wood, chrome, plastic, Or more combinations.

The biofilm by bacteria is also found in most medical implanted artificial surfaces such as catheters, heart valves, shunts, and prosthetic devices (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 general, it is very important to prevent biofilm formation because the only way to remove biofilm is surgical removal.

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

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of 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 Korean soils at the initial stage were cultured for 7 days at 28 ℃ in 10g glucose, 1g yeast extract (Difco), 2g peptone (Difco), 1g beef extract (Difco) and 15g agar Lt; RTI ID = 0.0 &gt; (Bennett &apos; 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. Aureus  The highest for Antifibrotic membrane  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.
delete A composition for preventing or inhibiting biofilm formation by Staphylococcus sp. Containing Streptomyces sp. BFI 250 strain deposited with deposit number KCTC 12190BP or a culture thereof as an active ingredient.
The composition for preventing or inhibiting biofilm formation according to claim 3, wherein the Staphylococcus sp. Is Staphylococcus aureus .
Streptomyces sp. BFI 250 deposited with the deposit number KCTC 12190BP or a Staphylococcus sp. Containing the culture medium as an active ingredient for disinfection or disinfection for biofilm formation or inhibition &Lt; / RTI &gt;
A method for preventing or inhibiting biofilm formation by bacteria comprising contacting a strain of Streptomyces sp. BFI 250 deposited with in vitro accession number KCTC 12190BP or a culture thereof with Staphylococcus sp. .
7. The method according to claim 6, wherein the Staphylococcus sp . Is Staphylococcus aureus .
[Claim 6] The method according to claim 6, wherein the contact with the genus Staphylococcus is characterized in that Streptomyces sp. BFI 250 strain deposited with the deposit number KCTC 12190BP or a culture thereof is treated on the surface or administered to an individual A method of preventing or inhibiting biofilm formation.
9. The method according to claim 8, wherein the surface is a combination of one or more selected from the group consisting of a surface of a medical article, a drain pipe, a glazed ceramic, a flocculant, a glass, a metal, a wood, A method for preventing or inhibiting the formation of a biofilm 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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