KR101174809B1 - A bacteriophage killing Staphylococcus aureus - Google Patents

Abstract

The present invention relates to a bacteriophage that kills Staphylococcus aureus, more specifically, bacteriophage of Siphoviridae, which has a killing ability against Staphylococcus aureus and has a full-length genome represented by SEQ ID NO: 1. (Bacteriophage) relates to SAP26.
According to the present invention, the bacteriophage SAP26 can be used to effectively kill or reduce Staphylococcus aureus having a biofilm formation ability. Bacteriophage, in particular, is a bacterium-based virus that is known to not work at all in humans and animals, so it can be applied to the pharmaceutical industry, food industry, biotechnology, etc. Effective killing of target material.

Description

Bacteriophage killing Staphylococcus aureus

The present invention relates to a bacteriophage that kills Staphylococcus aureus, more specifically, bacteriophage of Siphoviridae, which has a killing ability against Staphylococcus aureus and has a full-length genome represented by SEQ ID NO: 1. (Bacteriophage) relates to SAP26.

Staphylococcus aureus is a representative bacterium that causes infection in hospitals and is a common flora that is easily found in normal people and the environment. The bacterium infects through wounds, immunocompromised patients, and medical devices contaminated with surgical treatments, and is resistant to antimicrobial treatments and causes purulent disease. Due to the characteristics of the bacteria, they adhere to the surface of medical devices or non-living organisms and survive for a long time, so they are difficult to remove completely with general detergents or alcohols. This is called biofilm formation of bacteria and is more difficult to treat during biofilm formation and shows higher resistance to antimicrobial agents. When infected, the biofilm resists the body's immune system, and the resistance to antimicrobial substances increases by 500 times to 1000 times that of the suspended state, so antimicrobial treatment alone is not expected to be effective. In addition, the emergence and proliferation of antimicrobial resistant bacteria is so severe that super bacteria that are resistant to almost all types of antimicrobial treatments that have been developed and used up to now have emerged, and development of alternative treatments other than antimicrobial agents is urgently and urgently needed. .

Bacteriophage is a bacterium that specifically infects bacteria and causes lysis, and has been studied for the treatment of various bacterial diseases since its first discovery in 1915.To date, phage founder Felix d'Herelle was the first to treat phage. Used for the purpose.

Research using bacteriophages has been continuously conducted in Georgia, Poland and the Soviet Union since the past, and has been used to treat pathogenic microorganisms. In 1994, the bacteriophage treated with E. coli infected with a diaphragmatic tumor recurred after gastrectomy was treated cleanly. In 1995, 92 patients were clinically recovered by treating bacteriophage in 46 patients with acute chronic urinary inflammatory disease, and 84% completely eliminated pathogenic microorganisms. In 1999, Klebsiella pueumoniae , a bacterium that causes meningitis in newborns, failed to be treated with antibiotics. In 2001, 20 patients with refractory tumors were treated with oral bacteriophage three times a day for 2 to 9 weeks. In 2001, 54 patients treated with phage in the burn area 1.5 to 2 times the number of infected bacteria to protect against Staphylococcus, Streptococcus and Proteus . In 2002, the bacteriophage and biodegradable mixtures were treated with wounds and tumors, recovering 67 of 96 patients.

Recently, as pharmacokinetic studies on bacteriophage, which have a wide range of effects on species selectivity, and on the transmission of bactericidal effects, their applicability is gradually increasing. Research is just the beginning.

World-renowned journals Nature review and Nature biotechnology report on the treatment of bacteriophage and the methods of screening and treatment of the bacteriophage for treatment.In addition, the method of inhibiting the growth of infectious bacteria using the products of genomic genes is introduced. It was.

In 2007, the U.S. Food and Drug Administration (FDA) approved a phase 1 clinical trial using phage to treat bacterial infections of diabetic foot necrosis, which opened the way for treatment not only for infectious diseases in animals but also for humans. . In 2008, the FDA approved a method to reduce Salmonella contamination during chicken processing using Salmonella specific bacteriophages. In addition to developing phage itself for the treatment of infectious diseases, it is necessary to search and apply bactericidal factor of phage. As it is expected to have a great influence on the economic effect of infectious diseases and the improvement of health medical technology, it is required to improve related technologies and research base through national research support.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, confirming that bacteriophage SAP26 isolated from clinical isolates of multi-drug resistant Staphylococcus aureus exhibits specific killing ability against Staphylococcus aureus. The invention was completed.

Therefore, the main object of the present invention is to provide a bacteriophage SAP26 having excellent killing ability against Staphylococcus aureus.

Another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of Staphylococcus aureus-induced diseases containing the bacteriophage SAP26 as an active ingredient.

It is another object of the present invention to provide an antibiotic and a disinfectant comprising the bacteriophage SAP26.

According to one aspect of the invention, the present invention provides a bacteriophage SAP26 of Siphoviridae, which has a killing ability against Staphylococcus aureus, and has a full-length genome represented by SEQ ID NO: 1. do.

In the present invention, the bacteriophage SAP26 was isolated and identified for the first time in the present invention, the sequence of the genome was also first revealed by the present inventors. By separating and sequencing the full-length genome of the isolated bacteriophage SAP26, the present inventors confirmed that the phage genome is about 41 kbp double-stranded DNA having 63 protein codes and an open reading frame (ORF) (Table 2). Reference). In addition, the bacteriophage SAP26 isolated from the present invention was confirmed to have excellent killing ability in both methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus (Table 1 and Figure 4).

In the present invention, the bacteriophage is characterized in that the accession number KCTC 11665BP. We selected new bacteriophages that have the ability to kill Staphylococcus aureus from clinical isolates of multi-drug resistant Staphylococcus aureus, and deposited them on March 22, 2010 to the Gene Bank of Korea Biotechnology Research Institute. It was.

In the present invention, the Staphylococcus aureus is characterized in that the methicillin-resistant Staphylococcus aureus or methicillin-sensitive Staphylococcus aureus. In particular, it showed high killing ability against methicillin resistant Staphylococcus aureus formed biofilm (Fig. 4b). In addition, the combination with a low concentration of antimicrobial agents showed excellent antimicrobial activity (Fig. 4d). In other words, the treatment of Staphylococcus aureus on which the bacterium was formed with the antibacterial agent alone (FIG. 4c) had low killing ability of Staphylococcus aureus, but when treated with the bacteriophage of the invention (FIG. 4d), Staphylococcus aureus was formed. The bacteriophage of the present invention was directly acted on to obtain a synergy effect. Therefore, by applying the bacteriophage SAP26 of the present invention to the medical industry, food industry, biotechnology industry, etc., it is possible to effectively kill the harmful staphylococcus aureus at the target place or the target substance without the problem of antimicrobial resistance.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating diseases caused by Staphylococcus aureus having a biofilm-forming ability comprising the bacteriophage SAP26 as an active ingredient.

The bacteriophage included in the composition of the present invention is less resistant to the emergence of resistant bacteria than general antimicrobial agents, and has a selective toxic effect only on bacteria but does not act on eukaryotic cells. Can be.

The term "treatment" as used herein refers to the prevention of diseases caused by Staphylococcus aureus; Suppression of diseases caused by Staphylococcus aureus; And alleviation of diseases caused by Staphylococcus aureus.

As described above, the bacteriophages included in the composition of the present invention can kill Staphylococcus aureus and effectively remove the biofilm formed therefrom, and thus various diseases caused by Staphylococcus aureus and chronicized due to biofilm formation, such as mastitis and dermatitis. , Sepsis, purulent disease, food poisoning, pneumonia, osteomyelitis, impetigo, bacteremia, endocarditis, and enteritis. The composition for this purpose may further comprise a component that has already demonstrated antimicrobial activity against Staphylococcus aureus in order to obtain the effect of further treatment.

Ingredients that have proven antimicrobial activity against Staphylococcus aureus that can be applied to the present invention include methicillin, oxacillin, vancomycin, and the like, but various antibiotics may be applied.

In addition, the composition of the present invention may further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no.

In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition of the present invention can be used as a method of spraying or spraying on a diseased site, and can also be administered by oral or parenteral administration. In the case of parenteral administration, intravenous administration, intraperitoneal administration, muscle Administration may be by intra-, subcutaneous or topical administration.

Suitable application, spraying, and dosage of the pharmaceutical compositions of the present invention may include factors such as formulation method, mode of administration, age, weight, sex, degree of disease symptom, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient. The physician, who is usually skilled in the art, can readily determine and prescribe a dosage effective for the desired treatment. In general, the pharmaceutical compositions of the present invention comprise 1 × 10 ³ to 1 × 10 ¹⁰ pfu / ml of bacteriophage.

The pharmaceutical compositions of the present invention are prepared in unit dose form by being formulated with pharmaceutically acceptable carriers and / or excipients according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating 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.

According to another aspect of the present invention, the present invention provides a cosmetic antibacterial agent and a pharmaceutical antibiotic comprising the bacteriophage SAP26 as an active ingredient.

Staphylococcus aureus is a harmful bacterium that is frequently found in cosmetics along with Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Cosmetics contain oil and water as the main ingredients, and glycerin or sorbitol, which is a carbon source of microorganisms, and amino acid derivatives and proteins, which are nitrogen sources, are easily blended, so it is easy to penetrate microorganisms such as bacteria. In addition, since the use period is very long compared to food, the risk of contamination by microorganisms can be said to be much greater. Addition of antimicrobial agent is essential for long-term protection of cosmetics from discoloration or odor caused by microbial contamination.

Synthetic preservatives such as parabens, which are most widely used in cosmetics, have recently been highlighted, and it is recognized that synthetic preservative-containing cosmetics may be unexpectedly dangerous. The American Academy of Dermatology has also put synthetic preservatives on the list of the second leading sources of skin allergy. In addition, there is a concern that the use of artificial preservatives for children's products may be more dangerous since the use of preservative-containing products from childhood can increase the duration and amount of preservatives compared to adults. Development of antiseptics is urgently needed.

The bacteriophage of the present invention has the advantage of having excellent killing ability of Staphylococcus aureus and elimination of biofilm formed by Staphylococcus aureus compared to conventional antibiotics. When the bacteriophage of the present invention is used as an antibiotic, it has a significant advantage that it does not induce resistance or resistance of Staphylococcus aureus, unlike conventional antibiotics, and thus has a longer life cycle than conventional antibiotics. It can be provided as a novel antibiotic. While most antibiotics face increasing resistance, the range of use is inevitably decreased. On the other hand, antibiotics containing the bacteriophage of the present invention as an active ingredient can fundamentally solve the problem of antibiotic resistance. It is expected.

Therefore, the antibiotic containing bacteriophage of the present invention which kills Staphylococcus aureus as an active ingredient can be usefully used as an antibiotic having excellent antibacterial, bactericidal and antiseptic effects. As used herein, the term "antibiotic" refers to preservatives, fungicides and antimicrobials.

According to another aspect of the invention, the present invention provides a disinfectant comprising the bacteriophage as an active ingredient.

Disinfectants containing the bacteriophage of the present invention having an effective killing ability against Staphylococcus aureus as an active ingredient can be usefully used as a disinfectant for hospitals and health care to prevent hospital infections, and also as a general household disinfectant, food and cooking place And it can be usefully used as a disinfectant of equipment, a livestock disinfectant of the livestock industry.

In addition, the bacteriophage used in the disinfectant of the present invention is very useful as a disinfectant for removing staphylococcus aureus, which is present in a general environment or a medical device because it is naturally decomposed and can be prepared in liquid form. The cost of producing bacteriophages is also much more economical than the development and production of antimicrobial agents.

As described above, according to the present invention, effectively killing Staphylococcus aureus is very important for disease treatment and prevention. In order to treat diseases caused by Staphylococcus aureus or to kill causative bacteria, antimicrobial agents are administered to humans or livestock, and the causative bacteria may be reduced by physicochemical therapy on food materials or the environment. However, the current epidemic of resistant bacteria due to antimicrobial abuse is a major hazard to public health, and physicochemical therapy has the disadvantages of high environmental pollution and economic cost. Bacteriophage SAP26 of the present invention shows excellent killing ability against Staphylococcus aureus having a biofilm formation ability. Therefore, SAP26, which kills Staphylococcus aureus, which causes various diseases, is highly valued as a food and eco-friendly alternative antibacterial agent as well as the health industry.

Figure 1 shows phage plaques generated in methicillin resistant Staphylococcus aureus (MRSA) strain.
Figure 2 is a photograph of hemolysis of the MRSA strain Staphylococcus aureus bacteriophage SAP26 by direct drip method.
3 is an electron micrograph of the bacteriophage SAP26.
4 is a result of comparing the removal effect of Staphylococcus aureus bacterium according to the treatment of bacteriophage SAP26, fifampicin, and a combination thereof with respect to MRSA Staphylococcus aureus MRSA. a: untreated control biofilm, b: biofilm treated with bacteriophage SAP26, c: biofilm treated with rifampicin, d: biofilm treated with bacteriophage SAP26 and rifampicin simultaneously.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Production and Separation of SAP26 Phage

Inoculated with domestic clinical isolates methicillin resistant Staphylococcus aureus (MRSA) in LB (Luria Bertani) liquid medium and treated with mitomycin C at a concentration of 1 μg / ml in exponential growth conditions And incubated overnight at 37 ° C. The bacteria were removed by centrifugation at 4000 g for 10 minutes and filtered using a 0.45 μm filter. About 50 μl of chloroform was added and stored at 4 ° C. This solution was used to spread the Staphylococcus aureus to LB solid medium, dropping it directly onto a dried bacterial lawn, incubated at 37 ° C. overnight, and then confirming bacterial hemolysis the next day (direct point method). Bacteriophage was present on the chromosomes of the isolates and was isolated by artificially treating mitomycin. Plaque (plaque) test for the pure separation of the bacteriophage, a single plaque was separated and cultured again with Staphylococcus aureus to amplify the bacteriophage was repeated three times in a row was pure separation (see Figure 1). The inventors of the present invention separated the pure bacteriophage into the Gene Bank (KCTC) of the Korea Biotechnology Research Institute (KCTC 11665BP) and named it 'bacteriophage SAP26'. In order to harvest a large amount of bacteriophage, purely separated phages were mixed with host bacteria in the exponential phase and mixed with agar melted to a final 0.7% agar, poured into LB solid medium, dried and incubated overnight at 37 ° C. Next, centrifugation and filtration were performed. This was repeated until the desired plaforming unit (PFU) was obtained.

Example 2. Host specificity and killing ability of Staphylococcus aureus of SAP26

A total of 171 Staphylococcus aureus strains were used, including 61 strains of MSSA (methicillin susceptible Staphylococcus aures ) and 110 strains of MRSA (methicillin resistant Staphylococcus aures ) isolated from patients with atopic dermatitis and wounds using the purely isolated phage solution obtained in Example 1. Direct dropping was performed on Table 1 (FIG. 2). As a result, as shown in Table 1 below, SAP26 phage killed 93.4% of MSSA and 99.1% of MRSA.

[Table 1]

Example 3 Morphology Observation of SAP26 Using Electron Microscopy

Scanning electron microscope was used to observe the shape of the phage. Approximately 10 ¹² pfu of purely isolated phage was applied to a copper plate coated with carbon, then stained with 2% uranyl acetate and observed using a scanning electron microscope (Philips EM 300).

Observation results In FIG. 3, an icosahedral head and one long tail were observed and determined to belong to Siphoviridae through morphological classification. The head size was about 50 nm and the tail length was 120-200 nm.

Example 4 Genetic Properties of SAP26

Phage genomes were purified using Lambda midikit (Qiagen). The separated phage genome was entrusted to Genotech Co., Ltd. to determine the whole gene sequence. Total sequencing was performed by shotgun method, phosphide library construction, and automated sequencing (ABI PRISM 377). In the sequencing results, open coding frames (ORFs) were analyzed using Sequin software version 9.5. The results are shown in Table 2 below.

As a result, the phage genome was double-stranded DNA, approximately 41 kbp in size (SEQ ID NO: 1), and composed of 63 ORFs. As a result of BLAST search of NCBI for the entire sequence, the genome of SAP26 phage showed the closest to SAP26 with 75% Query Coverage of phiNM1 and phiETA2, respectively. These results indicate that SAP26 is a new phage.

TABLE 2-1

Table 2-2

Example 5.Staphylococcus aureus killing effect and biofilm removal effect test by single treatment with SAP26 or simultaneous treatment with SAP26 and antimicrobial agent

MRSA strain S. aureus D43-a strain was used to test the ability of Staphylococcus aureus to kill SAP26. The D43-a strain was incubated overnight by shaking at 37 ° C. using a BHI liquid medium, and the culture medium adjusted to OD ₆₀₀ = 1 was diluted with 100-fold BHI and 100 μl was divided into 96-well plates. The bacterium at this time is about 5 × 10 ⁶ cfu / well. And left overnight culture at 37 ℃ to form a bacterial film for 24 hours. After 24 hours, the culture medium and the floating bacteria were removed and 100 μl of SAP26 phage was added to each well to give 5 × 10 ⁸ pfu / well. For bacterial membrane treatment only for antimicrobials, rifampicin was used to prepare a concentration of 0.6 μg / ml, 10 times higher than the MIC concentration of the D43-a strain, and then 100 μl was added to each bacterial membrane-formed well. For simultaneous treatment of rifampicin and phage, SAP26 phage 5 × 10 ⁸ pfu / well and rifampicin were added at a concentration of 0.6 μg / ml to a total volume of 100 μl and added to wells formed with bacterial membranes. The phages, antimicrobial agents and phage-antibacterial treatments were added and cultured at 37 ° C. for 24 hours, and the state of bacteria was observed using a scanning electron microscopy.

Referring to FIG. 4, compared to the untreated biofilm (FIG. 4 a), the SAP26-treated biofilm (FIG. 4 b) retained a considerable amount of biofilm removed more than rifampicin-treated (FIG. 4 c). The bacteria present in the form are also unhealthy. On the other hand, the biofilm treated with SAP26 phage and rifampicin simultaneously (Fig. 4d) showed better removal of biofilm than antibacterial or phage alone treatment. Based on these results, SAP26 phage can be effectively removed by killing MRSA Staphylococcus aureus with antimicrobial agents.

Korea Biotechnology Research Institute KCTC11665BP 20100311

Attach an electronic file to a sequence list

Claims (6)

Bacteriophage SAP26 of Siphoviridae, which has a killing ability against Staphylococcus aureus and has a full-length genome represented by SEQ ID NO: 1.
2. The bacteriophage SAP according to claim 1, wherein the bacteriophage is a bacteriophage of Accession No. KCTC 11665BP.
The bacteriophage SAP according to claim 1, wherein the Staphylococcus aureus is methicillin resistant Staphylococcus aureus or methicillin-sensitive Staphylococcus aureus.
delete An antibiotic comprising the bacteriophage SAP26 of any one of claims 1 to 3.
A disinfectant comprising the bacteriophage SAP26 of any one of claims 1 to 3.

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