Patent Application
20100203019
The present invention relates to a novel Podoviridae bacteriophage having killing activity specific to Staphylococcus aureus.
Bacteriophage is a kind of virus-like microorganism infecting bacteria and generally called ‘phage’ in short. Bacteriophage is an organism having a simple structure wherein a central genetic material composed of nucleic acid is covered by a protein envelope. The nucleic acid is single stranded or double stranded DNA or RNA. To survive, bacteriophage needs a host bacterium and every bacterium has a specific partner bacteriophage. When bacteriophage invades into a host bacterium, it multiplicates itself and then induces expressions of enzymes involved in the decomposition of cell wall of the host bacterium. The enzymes destroy cell wall by attacking the peptidoglycan layer which is responsible for rigidity and mechanical strength of cell wall.
Bacteriophage was first found by Twort, an English bacteriologist, in 1915 during his research on the phenomenon that micrococcus colony is decomposed turning transparent by something. And in 1917, a French bacteriologist d'Herelle found out that there was something that decomposes Shigella disentriae in filtrate of feces of a patient with dysentery, and he continued to study to identify the material, leading to the finding of bacteriophage which means “eating bacteria”. Since then, bacteriophages against Shigella dysenteriae, Salmonella typhi, and Vibrio cholerae were further identified. Since penicillin was found by Flemming in 1950, antibiotics have been widely used and the study on bacteriophage continued only in some East European countries and it became out of concern in many other countries. However, since 2000, multidrug-resistant pathogenic bacteria resulted from over-use and/or mis-use of antibiotics have been frequently reported. Because of potential as an alternative for the conventional antibiotics, bacteriophage became in the spotlight again and the studies on bacteriophage are actively undergoing led by advanced countries.
Even though antibiotics (or antibacterial agents) are still major therapeutic agents for the treatment of various infectious diseases, it has been a serious problem since 1980s that the excessive use of such antibiotics generates numbers of multi-drug resistant strains. In 1986, Staphylococcus aureus having resistance against vancomycin, which is so called ‘the drug of last resort’, and other multi-drug resistant strains were found, giving a great shock to those in medical field. Vancomycin resistant enterococci (VRE) were first reported in France in 1986 and first separated in USA in 1988. Since then, the cases of VRE infection have been increased every year with high frequency, everywhere including Europe, USA, Singapore, Japan, Australia, Korea, etc, making the VRE as a causal agent of nosocomial infections. In Korea, VRE was first isolated in 1992. As for Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus (VRSA) was first found in the early 1990s and was first found in Korea in June, 1996.
Therefore, it is an urgent request to develop a novel antibiotic to treat the infectious diseases caused by bacteria resistant against conventional antibiotics and further to lead national health and medical techniques. Again, it is urgently required to develop an alternative antibiotic to solve the problems of multi-drug resistant bacteria along with the abuse or misuse of the conventional antibiotics and the bio-accumulation of antibiotics. To solve the problem of such resistant bacteria, an alternative antibiotic has to be developed by a completely and fundamentally different method.
In the previous study, the present inventors isolated a Myoviridae bacteriophage capable of killing specifically Staphylococcus aureus, and deposited the bacteriophage at Korean Agricultural Culture Collection, National Institute of Agricultural Biotechnology on Jun. 14, 2006 (Accession No: KACC 97001P) and at Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology on Jul. 18, 2007 (Accession No: KCTC 11153BP). The related matters have been applied for a patent (Korean Patent Application No. 2006-55461). Based on the experiences of the present inventors, it can be said that each bacteriophage works on different strains of Staphylococcus aureus, precisely every bacteriophage demonstrates bacteriolytic activity against different strains of Staphylococcus aureus and shows different spectrums by different mechanisms. Therefore, it is preferred to obtain as diverse bacteriophages as possible. Identifying additional bacteriophage facilitates establishing a method to cope with larger spectrum of Staphylococcus aureus and bacteriophage cocktail containing several bacteriophages is expected to have broadened bacteriolytic activity, compared with single kind of bacteriophage.
Thus, the present inventors first isolated Staphylococcus aureus and separated bacteriophage that is able to kill selectively the isolated Staphylococcus aureus from the natural system. Then, the inventors investigated the morphological and genetic characteristics of the separated bacteriophage to provide the gene sequence of this genome to distinguish it from other bacterophages. The present inventors finally completed this invention by confirming that the Staphylococcus aureus specific bacteriophage isolated by the inventors can be effectively used for the prevention and treatment of the disease caused by Staphylococcus aureus.
It is an object of the present invention to provide a novel bacteriophage that is able to selectively kill Staphylococcus aureus, the causing agent of infectious disease in animals including human.
It is another object of the present invention to provide a pharmaceutical composition for the prevention and treatment for the disease caused by Staphylococcus aureus containing the bacteriophage as an active ingredient.
It is a further object of the present invention to provide an antibiotic containing the bacteriophage as an active ingredient.
It is also an object of the present invention to provide a disinfectant containing the bacteriophage as an active ingredient.
The present invention provides a bacteriophage belonging to φ29-like virus genus, Podoviridae family, which has killing activity specific to Staphylococcus aureus.
The bacteriophage of the present invention has the genome comprising the nucleotide sequence represented by SEQ. ID. NO: 1
Staphylococcus aureus is a causing agent of skin infection and food poisoning. It is a very dangerous pathogenic bacterium having strong resistance against methicillin as high as 73% at average, which is the top level among all the antibiotic resistant bacteria in the world. That means 73% of Staphylococcus aureus cannot be killed by the antibiotics, which means this bacterium is highly antibiotic resistant.
The present inventors have been studied to kill Staphylococcus aureus selectively. The inventors isolated Staphylococcus aureus from pathogen and a novel Podoviridae bacteriophage that is able to kill the isolated Staphylococcus aureus selectively. This novel bacteriophage having killing activity specific to Staphylococcus aureus, separated by the inventors, was named ‘SAP-2’ and deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on Jul. 18, 2007 (Accession No: KCTC 11154BP).
The present invention provides a nucleic acid sequence of the genome of bacteriophage SAP-2 selected by the present inventors.
In this invention, the term ‘nucleic acid’ indicates DNA (gDNA and cDNA) and RNA in a large sense. Nucleotide, the basic unit of nucleic acid, can be not only natural nucleotide but also its analogue having modified sugar or base (Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman and Peyman, Chemical Reviews, 90:543-584(1990)).
The present invention also provides a pharmaceutical composition for the prevention and treatment of a disease caused by Staphylococcus aureus containing the bacteriophage SAP-2 as an active ingredient.
The bacteriophage included in the pharmaceutical composition of the present invention can kill Staphylococcus aureus specifically, so that it is very effective in the treatment of various diseases caused by Staphylococcus aureus.
Staphylococcus aureus is the number one reason to cause infectious mastitis in cattle. Staphylococcus aureus is found in 90% of the total dairy cows in USA and 10% of the total dairy cows are presumably infected by this pathogenic bacterium. Staphylococcus aureus is a causing agent of acute dermatitis in human, and this acute dermatitis can be suddenly developed into sepsis taking a patient's life. Staphylococcus aureus is also a causing agent of pyogenic disease, sweat odor and food poisoning.
Thus, the pharmaceutical composition of the present invention can be used for the treatment of various diseases caused by Staphylococcus aureus such as mastitis, acute dermatitis, sepsis, pyogenic disease, food poisoning, pneumonia, osteomyelitis, impetigo, bacteremia, endocarditis and enteritis. According to a preferred embodiment of the present invention, every day spray of the bacteriophage of the invention on the lesion of dairy cow having mastitis could significantly reduce the symptoms of mastitis, suggesting that the bacteriophage of the invention is effective in the treatment of mastitis.
The term ‘treatment’ herein indicates (i) the prevention of the disease caused by Staphylococcus aureus; (ii) the inhibition of the disease caused by Staphylococcus aureus; and (iii) the relief of the disease caused by Staphylococcus aureus.
The pharmaceutical composition of the present invention can additionally include a pharmaceutically acceptable carrier, which is exemplified by 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 and mineral oil, but not always limited thereto. The pharmaceutical composition of the present invention can also include a lubricant, a wetting agent, a sweetener, a flavor, an emulsifier, a suspending agent, and a preservative, in addition to the above ingredients.
The pharmaceutical composition of the present invention can be applied or sprayed on the lesion, and administered orally or parenterally (for example, intravenous, intramuscular, hypodermic, local or peritoneal injection).
The effective dosage of the pharmaceutical composition of the present invention varies from the formulation, administration pathway, age, weight, gender, severity of a disease, diet, administration frequency and pathway, excretion and sensitivity. In general, the dosage can be determined by an experienced doctor with consideration of the goal of the treatment or preventive effect. In this invention, the pharmaceutical composition contains bacteriophage at the concentration of 1×103-1×1010 pfu/ml, and more preferably 1×106-1×109 pfu/ml.
The pharmaceutical composition of the present invention can be formulated as a unit dose medicine or as a medicine in multidose vehicle by mixing with a pharmaceutically acceptable carrier and/or excipient by the method well known to those in the art. The pharmaceutical formulation can be selected from a group consisting of ointments, solutions, suspensions or emulsions, extracts, powders, granules, tablets or capsules and additionally includes a dispersing agent or a stabilizing agent.
In another preferred embodiment of the present invention, the present invention provides an antibiotic containing the bacteriophage SAP-2 as an active ingredient.
The term ‘antibiotic’ is used herein as a general term for antiseptics, bactericidal agents and antibacterial agents.
Staphylococcus aureus is frequently found in cosmetics along with Bacillus subtilis, E. coli and Pseudomonas aeruginosa. Cosmetics use oil or water as a major ingredient, to which glycerin and sorbitol, which are carbon sources of a microorganism, and amino acid derivatives and a protein which are nitrogen sources of a microorganism, are added, suggesting that there are enough nutrition and ingredients to attract microorganisms including bacteria. In addition, the term of use of the cosmetics is comparatively long, indicating that it is in high risk of contamination by a microorganism. To prevent color changes or odor changes caused by the contamination of a microorganism, an antibacterial agent is necessarily added to cosmetics for a long shelf-life.
A synthetic antiseptic such as parabens is widely used as an additive for cosmetics, but it is potentially dangerous. Particularly, since its accumulation in breast cancer cells was detected, it has been recognized that the accumulation of such synthetic antiseptic via cosmetics might be very harmful. The American Academy of Dermatology's Committee acclaimed the synthetic antiseptic as the number two allergen causing skin trouble. Recently what worries us is that cosmetics for children also include such artificial synthetic antiseptic, suggesting that children are exposed on such harmful antiseptic longer and much, raising the risk seriously. Therefore, it is sincerely requested to develop a natural antiseptic.
The bacteriophage SAP-2 of the present invention is characterized by its high specificity to Staphylococcus aureus, compared with other conventional antibiotics. That is, the bacteriophage SAP-2 can selectively kill Staphylococcus aureus only without killing useful bacteria, suggesting that it is a highly valuable antibiotic that has less side effects. The bacteriophage-based antibiotics, unlike the conventional antibiotics, do not induce resistance so that their life cycles are comparatively long. Most conventional antibiotics are gradually limited in use because of the increasing resistance. On the other hand, the antibiotic containing the bacteriophage SAP-2 of the present invention as an active ingredient can solve the problem of the antibiotic-resistance and thus has longer life cycling. Therefore, the antibiotic containing the bacteriophage SAP-2 of the present invention as an active ingredient that is able to kill Staphylococcus aureus selectively can be effectively used as a novel antibiotic with excellent antibacterial, bactericidal and antiseptic effects. The term ‘antibiotic’ is used herein as a general term for antiseptics, bactericidal agents and antibacterial agents.
In another preferred embodiment of the present invention, the invention provides a disinfectant containing the bacteriophage SAP-2 as an active ingredient.
The distribution of bacteria separated from nosocomial infection has been changed over the time. According to NNIS (National Nosocomial Infection Surveillance System), USA, Gram-positive bacteria particularly Staphylococcus aureus has been increasing in number among those separated bacteria since late 1980s, and this phenomenon is consistent with that in Korea. According to a report made in Korea in 1985, the dominant distribution is E. coli, and Pseudomonas aeruginosa, coagulase negative Staphylococcus and Staphylococcus aureus follows in that order. But, the separation of Staphylococcus aureus is increasing gradually. Korean Society for Nosocomial Infection Control (KSNIC) reported in 1996 that Staphylococcus aureus took 17.2% of total isolated pathogenic microorganisms and Pseudomonas aeruginosa (13.8%) and E. coli (12.3%) followed. And, 78.8% of the total Staphylococcus aureus separated were confirmed to have resistance against antibiotics.
Based on the above finding, the disinfectant containing the bacteriophage SAP-2 of the present invention that is able to kill specifically Staphylococcus aureus can be effectively used as a disinfectant specifically for hospitals and public health. It is also available as a general life disinfectant, a food and kitchen disinfectant, and a stall disinfectant.
As explained hereinbefore, the present invention provides a Podoviridae bacteriophage that is able to kill specifically Staphylococcus aureus, a pathogenic microorganism. The bacteriophage of the present invention can be effectively used in a variety of fields as a preventive/therapeutic agent for infectious disease caused by Staphylococcus aureus, as an antibiotic, as a antibacterial agent for cosmetics, as a natural antiseptic, and as a multi-purpose disinfectant.
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Isolation of Staphylococcus aureus and Bacteriophage Having Killing Activity Specific to Staphylococcus aureus
<1-1> Isolation of Staphylococcus aureus
Bacteriophage generally lives together with bacteria in natural system. To separate the bacteriophage specifically infecting Staphylococcus aureus, samples were collected from everywhere where the inventors expected Staphylococcus aureus lives. To investigate the samples where Staphylococcus aureus really exists, the Baird-Parker agar medium, a Staphylococcus aureus selection medium, was used.
Particularly, the present inventors selected dairy cow mastitis as a target disease to isolate Staphylococcus aureus, the target microorganism. Mastitis is one of the most representative diseases caused by Staphylococcus aureus. A sample was taken from milk of a dairy cow with mastitis and Staphylococcus aureus was isolated therefrom using the Baird-Parker agar medium, a Staphylococcus aureus selection medium. The isolated Staphylococcus aureus was identified as Staphylococcus aureus by biochemical analysis including Gram staining method, catalase test and analysis with Vitek of bioMeriuex. The results are shown in Table 1.
Staphylococcus aureus
<1-2> Isolation of the Staphylococcus aureus Specific Bacteriophage
To isolate the Staphylococcus aureus specific bacteriophage, samples expected to contain the bacteriophage were cultured together with Staphylococcus aureus. The culture broth was centrifuged, filtered and then cultured again with Staphylococcus aureus, the bait for the isolation of the bacteriophage, and then lysis of Staphylococcus aureus was investigated by plaque assay.
Particularly, to isolate the bacteriophage having killing activity specific to Staphylococcus aureus, samples were collected from soil and straw in a cowshed and sewage where the bacteriophage was expected to be. The samples were shaking-cultured with Staphylococcus aureus at 37° C. for 3-4 hours. Centrifugation was performed for 20 minutes at 8,000 rpm to obtain supernatant. The supernatant was filtered with a 0.45 μm filter. The Staphylococcus aureus specific bacteriophage was detected from the filtrate by plaque assay. The method used for the assay is shown in the schematic diagram of
Nucleotide sequence of the obtained bacteriophage SAP-2 was analyzed. First, the genome of the bacteriophage SAP-2 was extracted by the conventional method, and genetic characteristics thereof were investigated. Particularly, 50 ml of Staphylococcus aureus suspension (OD600=1) and 1 ml of filtered bacteriophage suspension (1×108 pfu/ml) were added into 200 ml of TSB (Tryptic Soy Broth) medium (casein digest, 17 g/l; soybean digest, 3 g/l; dextrose, 2.5 g/l; NaCl, 5 g/l; dipotassium phosphate, 2.5 g/l) in a 1 l flask, followed by shaking-culture at 37° C. for 3-4 hours. Then, lysis of Staphylococcus aureus was observed. After confirming lysis, the culture broth was filtered with a 0.45 μm filter. To eliminate DNA and RNA of Staphylococcus aureus remaining in the filtered culture broth, DNase and RNase (200 U each) were added to 10 ml of the filtered culture broth, which stood at 37° C. for 30 minutes. To inactivate the enzymes (DNase and RNase) therein, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto, which stood for 10 minutes. Next, to destroy outer wall of bacteriophage, 100 μl of proteinase K (20 mg/ml) and 500 μl of 10% sodium dodecyl sulfate (SDS) were added thereto, followed by incubation at 65° C. for 1 hour. After one hour incubation, 10 ml of phenol:chloroform:isoamylalcohol mixture (25:24:1) was added thereto and mixed well. The mixture was centrifuged at 18,000 rpm to separate layers. Upper layer was recovered, to which two times the volume of 100% cold alcohol was added, followed by extraction of pure genome. To investigate whether the genome extracted from bacteriophage was DNA or RNA, DNase I (10 U/μl) and RNase A (10 μg/μl) were added respectively, followed by incubation at 37° C. for 1 hour. The genome was also treated with mung bean nuclease (45 U/μl) for 15 minutes at room temperature to determine whether it was a single stranded DNA or a double-stranded DNA, in case it would be confirmed to be DNA. Electrophoresis was performed with those treated samples using 0.8% agarose gel and fragmentation pattern by each enzyme was investigated. As a result, the obtained genome was sensitive to DNase I (
The genome extracted from the isolated bacteriophage was a genomic DNA (gDNA). To analyze the gene sequence of the gDNA, the genome was treated with several restriction enzymes and fragmentation patterns by these enzymes were observed (
Particularly, DNA fragments were obtained by treating the gDNA of bacteriophage SAP-2 with Nde I according to the conventional method. Vector fragments were also prepared by treating the modified pGEM T-easy vector (Promega) with Nde I. The pGEM T-easy vector was the vector designed for TA-cloning. So, the vector could not be used as it was. Instead, T-overhang of the end of the vector was eliminated by the conventional method known to those in the art and then blunt-ended ligation was carried out, resulting in a circular modified vector. The DNA fragments and the vector fragments derived from the modified vector were ligated using T4 ligase. The resultant recombinant vector having the DNA fragment of bacteriophage SAP-2 was introduced into E coli Top10F′ via electroporation, a kind of electro-transformation. The transformant transformed with the vector was selected on the agar plate medium containing ampicillin supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) and isopropyl β-D-1-thiogalacto-pyranoside (IPTG) by using Blue-White colony selection. The selected single colony was inoculated into the medium containing ampicillin, followed by shaking-culture for overnight. Plasmids were extracted from the culture cells above using a plasmid purification kit (iNtRON). The extracted plasmids were electrophoresed using 0.8% agarose gel to confirm the size. Based on the size, recombinant plasmids were selected.
The numbers of selected plasmids were 3 in total and thus the numbers of clones obtained were also 3. The clones were cultured again and plasmids were extracted from the culture cells by the same manner as described above and the nucleotide sequences of the extracted plasmids were analyzed. Direct nucleotide sequencing of the gDNA of bacteriophage SAP-2 was also performed. Sequences of primers used herein are shown in Table 2.
The nucleotide sequence of the total genome of the bacteriophage SAP-2 obtained by the above two methods was represented by SEQ. ID. NO: 1. The total number of nucleotides forming the genome of bacteriophage SAP-2 was 17938.
100 μl of bacteriophage SAP-2 suspension (1×108 pfu/ml) prepared in Example 1 was added into 9 ml of nutrient broth (beef extract 3 g/l, peptone 5 g/l). A control medium was prepared without the addition of the bacteriophage suspension.
Staphylococcus aureus suspension was added into each medium at a starting optical density at 600 nm (OD600) of 0.5, followed by investigation of the growth of Staphylococcus aureus. As shown in table 3, in the medium not treated with the bacteriophage suspension, Staphylococcus aureus was growing so well (30 minutes later: OD600=0.8). On the other hand, in the medium treated with the bacteriophage suspension, Staphylococcus aureus was not grown at all (10 minutes later: OD600=0.1, 60 minutes later: OD600=0.05). It was confirmed from the above results that the bacteriophage SAP-2 isolated in Example 1 was very effective in the prevention of the infection of Staphylococcus aureus.
Staphylococcus aureus killing activity (OD600)
10 dairy cows infected with mastitis caused by Staphylococcus aureus were selected to investigate the effect of bacteriophage SAP-2 isolated in Example 1 on treatment of mastitis. The cows were divided into two groups (5 cows per group). 10 ml of bacteriophage SAP-2 suspension (108 pfu/ml) was sprayed on the lesion of dairy cows in one group every day and 10 ml of PBS without the bacteriophage was sprayed on the other group every day with same manner, particularly on the infected regions. The spray was continued for 10 days. As a result, significant treatment effect was observed in the group sprayed with the bacteriophage suspension. From the result, it was confirmed that bacteriophage SAP-2 isolated in Example 1 was very effective in the treatment of the infectious disease caused by Staphylococcus aureus.
The present inventors compared bacteriolytic activities between the two Staphylococcus aureus specific bacteriophages (KACC 97001P and KCTC 11154BP). The target Staphylococcus aureus strains were provided from Culture Collection of Antimicrobial Resistant Microbes, Seoul Women's University, Seoul 139-774, Korea. And the results are as shown below.
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
In the Table, CCARM No. indicates reference number used at Culture Collection of Antimicrobial Resistant Microbes, “□” indicates bacteriolytic activity confirmed and “x” indicates no bacteriolytic activity. “A” illustrates the result of the bacteriophage deposited under accession number of KACC 97001P and “B” illustrates the result of the bacteriophage deposited under the accession number of KCTC 11154BP.
As shown in the above results, two different bacteriophages exhibited bacteriolytic activity against different kinds of Staphylococcus aureus. In the case when they showed equal bacteriolytic activity, the activity level was quite different. So, two different bacteriophages above were complementary each other and thus the cocktail of the two was believed to increase antimicrobial effect. In most cases, bacteriophages had different bacteriolytic mechanisms.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
