KR100949389B1 - Preparation containing bacteriophage KCTC 11120BP to inhibit or kill bacteria - Google Patents

Abstract

The present invention relates to a bacteriophage KCTC 11120BP-containing bacterial growth inhibitory or killing agent, the bacteriophage KCTC 11120BP exhibits the effect of reducing the pathogenicity of the host even in a soluble state, effectively inhibiting the growth of a wide range of hosts And killable features.

Bacteriophage, Death, Bacteria, Tolerance

Description

Preparation containing bacteriophage KCTC 11120BP to inhibit or kill bacteria}

The present invention relates to an agent for killing bacteria, and more particularly, to bacteriophages that reduce pathogenicity of a host even in a soluble state.

After the discovery of plant and animal viruses, a virus that infects bacteria (bacteria) was known, and several years later, in 1915, Twart observed lytic phenomena of staphylococci. In 1917, the herler at the Pasteur Institute observed this in the bacterium and found that it was caused by a virus. Since then, the virus that has been infected with bacteria has been called "bacteriophage" in the sense of "Phage," or simply "phage."

The size of the phage is about 1/10 of the bacteria and its shape can only be observed by electron microscopy. However, when phage lysates bacteria, transparent spots (plaques) are formed on bacterial colonies that have grown on agar medium.

About 30 minutes after the bacterial infection, the phages are made and 100 to 200 new phages are made and break out of the bacterial membrane. Bacteria grow in two divisions, and the rapid one consists of two individuals in about 20 minutes, four individuals in 40 minutes, and eight individuals in 60 minutes, but in the case of phage, more than 100 new phages are generated in about 30 minutes. Phage is much more prevalent in speed. If a particular bacterium grows indefinitely once every 20 minutes, a bacterium starting from one cell takes only 36 hours to weigh like the earth, so phage can be considered as an important candidate to suppress the growth of bacteria. have.

Phage are classified into lytic phages and lysogenic phages. Viral phage is defined as repeating the lysis cycle only and temperate phage is repeated only the lysis cycle. Because weak phage inserts phage genetic information into the host bacterium's genome, the phage disappears but the host bacteria are not destroyed. This condition continues as long as the host bacterium is in good environmental conditions. If the host bacterium is deteriorated, the phage gene in the host bacterium gene is made into phage particles, which are then converted into a lysis cycle to lyse the host bacterium and out of the host cell. To escape.

From 1910 to 1930, when phages were found, phages were not resolved scientifically at that time, including host specificity, endotoxins and exotoxins of host bacteria, penicillin by flaming in 1929 and streptomycin by Worksman in 1943. The discovery of representative chemotherapeutic agents has limited their use.

Chemotherapeutic agents have been widely used because they are effective against multiple infections (with a wide range of antimicrobial agents) and have no problem with resistant bacteria at the time of first application, and phage therapy, which had no comparative advantage, has almost disappeared. Since then, in the 1950s, phage therapy has been declared invalid by the WHO and has only been maintained in the Eastern bloc and the Soviet Union.

The reason why phage law was declared invalid at the time was as follows. First, as discussed above, the host range of phage is very narrow, so even if the same species is different, the sensitivity of phage varies depending on the state. Because there was a problem to prepare a lot of landlords.

Secondly, in the case of phages that show very distinct lytic action, oral administration of strong gastric acid is inevitable, and when applied to systemic infections, phages that enter the body are removed by the reticuloendothelial system. to be.

Third, because phage resistant bacteria are present in the same bacterial population.

However, the idea of phage therapy changed in the 1980s. In 1982, British researchers, Smith and Huggins, used mice to test meningitis treatment models. When the bacteria were injected into the subcutaneous muscle of the mouse and the other subcutaneous was injected with the bacterium specific lysate phage, the control group that did not inject phage survived 100% when the phage was injected (experimental group). It was found to be superior to the effects of antibiotics (such as streptomycin). Even more surprising is that phage was recovered from the brain both when the bacteria were injected directly into the brain and when the phage was injected into the brain, which means that the injected phage moved to the brain tissue through the bloodstream. It means that it can cross the blood-brain barrier.

Recently, reports of successful treatment and treatment of various diseases such as E. coli diarrhea in calves, various diseases of fish, vancomycin-resistant enterococci sepsis, Mycobacterium tuberculosis, Chicken paratyphoid and E. coli O157: H7 have been reported. In addition to using phage directly, the development of antibiotics specific to anthrax, pneumococci, etc. has been made using lysin, which acts to specifically destroy the cell wall of host bacteria. It is time to find new phages.

Accordingly, an object of the present invention is to provide a new bacteriophage that can induce the death of bacteria.

In order to achieve the above object, the present invention provides a bacteriophage inhibiting or killing agent, characterized in that it comprises KCTC 11120BP as an active ingredient.

In the present invention, bacteriophages having proliferation inhibitory or killing ability against both Salmonella and Escherichia coli were isolated from Cheonggyecheon water in Seoul, and deposited with KCTC (Korea Research Institute of Bioscience and Biotechnology) and given accession number KCTC 11120BP. This phage belongs to the complex Siphoviridae because it has a long tail and no contractile sheath, and can be classified as a type 1 virus according to the Baltimor classification.

Bacteriophage KCTC 11120BP of the present invention can be widely dissolved in Salmonella and Escherichia coli, and it has been confirmed that it has sensitivity to Salmonella enteritidis isolated from human intestines and Salmonella typhimurium isolated from animals.

Meanwhile, the bacteriophage KCTC 11120BP of the present invention, unlike other bacteriophages, is characterized by lowering the pathogenicity of cells in a lysogenic state. It was confirmed that the bacteriophage infected with the bacteriophage KCTC 11120BP of the present invention was significantly inferior to epithelial cells compared to the control bacteria that were not lysed.

The bacteriophage of the present invention exerts an effect of reducing the pathogenicity of the host even in a soluble state, and can effectively inhibit and kill its growth for a wide range of hosts beyond the use of limited bacteriophages caused by the host specificity of the bacteriophage. There is a characteristic.

Bacterial phage having bacteriostatic or killing ability of the present invention can be used in place of antibiotics in animal husbandry, fish farming, etc., thereby reducing the possibility of antibiotic resistant bacteria through reducing antibiotic use and increasing environmental safety.

Hereinafter, the content of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples.

Example  1: Bacteriophage Separation

Bacteriophage for Salmonella and E. coli were isolated from Cheonggyecheon water in Seoul.

Cheonggyecheon water was filtered with a 0.45 μm membrane filter that passed bacteria but bacteriophage. The filtrate was added with LB broth composed of 10% NaCl, 5% yeast extract , and 10% tryptone, and bacteria ( Salmonella t yphimurium ), Escherichia coli ) and mixed with the culture.

The sample was incubated at 37 ° C. for 12 hours and the supernatant was filtered through a 0.45 μm membrane filter by centrifugation at 8000 rpm. A pipette of 0.1 ml of the filtrate and 0.1 ml of bacteria (Salmonella or E. coli) cultured for 12 hours or more were extracted with a pipette and mixed. The mixture was mixed with agar (0.7%) medium, poured into a plate containing 1.5% agar medium, and then incubated overnight at 37 ° C.

When the desired bacteriophage appeared on the plate, independent and transparent plaques were observed. The plaque region was separated using a platinum loop and then placed in a new bacterial culture, which observed clearing of the culture.

Thereafter, the culture solution was centrifuged, and only the supernatant was taken out and filtered through a 0.45 μm membrane filter to remove all bacteria remaining in the lysate, thereby recovering a filtrate containing only bacteriophage.

The bacteriophage solution thus obtained was diluted and injected on agar medium, and then pure bacteriophage was obtained by re-separation of the plaque three times.

Example  2: shape and classification of acquired phage

Phages infecting both Salmonella and Escherichia coli were selected from the bacteriophages obtained in Example 1, which were deposited with KCTC (Korea Research Institute of Bioscience and Biotechnology) and given accession number KCTC 11120BP. Electron micrographs of the phage were analyzed, and FIG. 1 shows the results.

The electron microscope was an Energy-Filtering Transmission Electron Microscope. Bacteriophage samples to be observed were placed on a carbon coated copper grid and negatively stained with 2% liquid uranyl acetate. Stained samples were observed using an energy filtered transmission electron microscope (LIBRA 120, Carl Zeiss) at a voltage of 80 kV.

The observed bacteriophage belongs to the complex Siphoviridae because of its long tail and no contractile sheath, and according to the Baltimor classification, Could be classified.

Example  3: Host Susceptibility to Isolated Bacteriophage susceptibility ) decision

Host susceptibility was determined through serial dilution dropping method. After mixing the culture broth with 0.7% agar medium for 2 hours, the mixture was poured into a plate and dried at 37 ° C. for 15 minutes, and the diluted phages were dropped by dilution factor, dried at room temperature, and allowed to stand at 37 ° C. for 10 hours. . If the strain is susceptible to phage, it shows a clear area, or plaque, on the plate, proving that it is the result of complete lysis of bacterial cells. If the sensitivity is weak, cloudy areas are formed or independent bold spots appear.

Table 1 below shows the results of examining the susceptibility to Salmonella and E. coli of bacteriophage KCTC 11120BP of the present invention.

Determination of Host Susceptibility of Bacteriophage KCTC 11120BP (+ count indicates degree of sensitivity) host scrap paper Host (Salmonella) scrap paper Host (Salmonella) scrap paper LT2     Salmonella ++ S.E 1 ++ S.T 1 - SL1344 ++ S.E 2 ++ S.T 2 ++ 14028 s ++ S.E 3 ++ S.T 3 ++ S. Dublin ++ S.E 4 ++ S.T 4 ++ UK1 ++ S.E 5 ++ S.T 5 ++ KCTC 1925 +++ S.E 6 ++ S.T 6 ++ NCTC 12023 ++ S.E 7 ++ S.T 7 ++ ST 4/74 ++ S.E 8 ++ S.T 8 - S. enteritidis ++ S.E 9 ++ S.T 9 ++ DH5α    Escherichia coli +++ S.E 10 +++ S.T 10 ++ MC4100 +++ JM109 +++ JM109 (DE3) +++ MG1655 +++ BL21 (DE3) -

As a result of the experiment, it was confirmed that the two strains can be widely dissolved, Salmonella enteritidis (SE 1-10) isolated from human intestine and Salmonella typhimurium (ST 1-10) isolated from animals also bacteriophage Susceptibility was confirmed by KCTC 11120BP.

Example 4 Determining Factors Susceptible to Salmonella and Escherichia Coli

Partial sequencing of the bacteriophage KCTC 11120BP revealed a phage protein that binds to the host cell. 2 shows the nucleotide sequence of this protein. This protein is attached to the BtuB protein of the host cell, and the mutation of the base sequence of the protein in the host cell to prevent the expression of the protein, and to determine what changes in the sensitivity of the phage through this example.

Figure 3 shows the results, the host cells mutated BtuB protein was significantly less susceptible than the control group. In Figure 3 ▲ is a wild type SL1344, ■ is SL1344 mutated FhuA protein, □ is SL1344 mutated BtuB protein.

Example  5: After infecting Salmonella with bacteriophage Yongwon Province  Salmonella Isolation

A suspension of 0.1 ml of Salmonella cultured for 12 hours or more was extracted with a pipette, mixed in agar (0.7%) medium, and poured into a plate containing 1.5% agar medium. The agar was hardened for about 10 minutes, the bacteriophage solution of the present invention was diluted by one-tenth to make ten samples, and 5 μl each was dropped onto the agar medium (FIG. 4). This plate was then incubated at 37 ° C. for 24 hours.

After the incubation, colonies generated in plaques were separated using a platinum loop, which was then further cultured in the medium. It was confirmed by the polymerase chain reaction whether the cells were lysogenic cells, and even when passaged more than 10 times, the cells were stable enough to maintain the soluble state.

Example  6: Yongwon Province  Reduced pathogenicity of infected salmonella

In order to confirm the pathogenicity of Salmonella infected with lysogenicity, we conducted animal cell invasion assay and survival assay of Salmonella model, which is a common experiment to measure Salmonella's pathogenicity. If shown, can determine whether the pathogenicity is reduced.

First, ICE-6 cells, which are small intestinal epithelial cells, and RAW264.7 cells, which are macrophages, were cultured in DMEM (Dulbecco's modified Eagle's medium 'Gilbco-BRL) medium to which 10% petal bovine serum was added. Culture conditions were 37 ℃, 5% CO ₂ , each cell was passaged at intervals of about 3 to 4 days. Salmonella were then incubated to a mid-log phase of OD ₆₀₀ = 0.6 and then inoculated at 10 bacteria / animal cells to animal cells cultured in 12-well tissue culture plates. The bacteria were infected for 1 hour and washed 3-4 times with phosphate-buffered saline (PBS). Afterwards, the cells were incubated in DMEM medium containing gentamicin to remove Salmonella from outside the animal cells, and the cells were lysed with PBS containing 1% triton X-100 (Sigma Co.). Cultured in LB plate at the appropriate dilution ratio. Thereafter, the invasion ratio was determined by comparing the number of colonies grown with the number of Salmonella seeded at the initial inoculation step.

In the case of Survival assay, after the gentamicin treatment, the medium was exchanged with fresh DMEM and the animal cells were lysed at each time to investigate the decrease of intracellular bacteria.

The experiments were performed by mutating two bacterial tail genes (fliC, fljB), which are known to affect the reduction of pathogenicity, as a control of each experiment.

As a result of experiments with the above parent cells and lysogenic cells, it was confirmed that infiltrating and viability of epithelial cells was significantly decreased in the case of lysogenic cells (FIG. 5). 5A and 5B, 'SL1344' is a Salmonella wild-type strain, 'lysogen' is a lysogenic strain, 'fliC' and 'fljB' are strains mutated the tail gene of Salmonella. 5a is the number of bacteria in the epithelial cells (cfu, colony forming unit) 3 hours after the strain is infected with the intestinal epithelial cells of the animal, 5b is 6 after infecting 4 strains to the macrophages of the animal 6 This is a graph counting the number of bacteria (cfu, colony forming unit) in macrophages after 18 hours. At this time, 6, 18 on the 5b bar graph means time.

The above results demonstrate the effect of reducing the pathogenicity of bacteria even when phage is lysogenic to the bacteria, which is a feature of the present invention.

Example  7: Production of a preparation containing the bacteriophage of the present invention

Bacteriophage KCTC 11120BP of the present invention was mixed in a Salmonella culture cultured for 12 hours, and incubated at 37 ° C. for 3 hours. After clear culture (dissolution) appeared, the samples were kept cold until the next day. In order to remove unlyzed bacterial cells, the lysate was filtered through a 0.45 μm membrane filter, which gave a bacteriophage solution.

Sterile cultured phage lysates show relatively long lytic activity. If kept cold, they will survive for many years and in some cases for more than ten years.

The bacteriophage solution thus recovered was used for the preparation of the phage drug.

Phage drugs are mixtures comprising bacteriophage solution. After neutralization of this mixture as a drug (e.g. using Gel Alumini phosphorici, soda or VIchy water), 10 ml at 30 minutes before meals three times daily It can be administered orally by the amount.

When this mixed solution is applied directly to the wound, it can be performed three times over 24 hours. At this time, the wound should not be cleaned with a disinfectant, as this may lead to inactivation of the phage. If cleaning is required, the wound may be washed with sterile medium or 0.9% NaCl physiological solution.

Meanwhile, if necessary, the phage therapy of the present invention may be applied in connection with antibiotic therapy.

1 is an electron micrograph of the bacteriophage isolated from the present invention.

Figure 2 is a gene sequence of a protein that affects the broadening of the host bacteriophage of the present invention.

Figure 3 is a graph analyzing the factors affecting the bacteriophage of the present invention to broaden the range of the host. (▲ is SL1344 of wild type, ■ is SL1344 mutated FhuA protein, □ is SL1344 mutated BtuB protein)

Figure 4 is an explanatory diagram showing a process for selecting Salmonella lysogenically infected by the bacteriophage of the present invention.

Figures 5a and 5b is a graph showing that the infiltration capacity to epithelial cells and viability in macrophages is significantly lower than the parent cells in the case of lysogenic cells.

Claims (2)

Salmonella growth inhibition or killing agent comprising bacteriophage KCTC 11120BP as an active ingredient E. coli growth inhibiting or killing agent comprising bacteriophage KCTC 11120BP as an active ingredient

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