WO2020130652A9 - Novel bacteriophage for lysing bacteria - Google Patents

Abstract

The present invention relates to a bacteriophage exhibiting bactericidal activity specifically against Acinetobacter sp. or Klebsiella sp. bacteria.

Description

New bacteriophage that lyses bacteria

The present invention relates to a novel bacteriophage that lyses bacteria, particularly bacteria that are resistant to antibiotics.

Bacterial infection is one of the most common and fatal causes of human disease. Since penicillin, numerous types of antibiotics have been developed and have been used to combat external invasion bacteria. However, in recent years, strains resistant to these antibiotics have appeared and are considered a big problem. Bacterial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa , which can be life-threatening, are resistant to all known antibiotics to date. Has been raised (Stuart B. Levy, Scientific American, (1988): 46-53).

Tolerance to antibiotics is a phenomenon that is distinct from resistance to antibiotics, and was first discovered in Pneumococcus sp. in the 1970s and provided important clues about the mechanism of action of penicillin (Tomasz). et al., Nature, 227, (1970): 138-140). Conventional chemical antibiotics such as penicillin and cephalosporin exhibit an antibiotic action by inhibiting the synthesis of a cell wall or protein of a microorganism. However, resistant species stop growing in the presence of conventional concentrations of antibiotics, but do not die as a result. Resistance occurs because when antibiotics inhibit cell wall synthase, autolytic enzymes such as autolysin do not activate. This is because penicillin activates the endogenous hydrolytic enzyme. By killing the bacteria, the bacteria also inhibit their activity, resulting in survival even when treated with antibiotics. Therefore, the development of antibiotics having a new mechanism of action capable of combating these resistant strains is urgent, and antibiotic peptides exhibiting different antibiotic mechanisms from conventional chemical antibiotics are attracting attention as a new concept of next-generation antibiotics (Zasloff M.Curr). Opin Immunol 4 (1992): 3-7; Boman, HG, Cell, 65.205 (1991); Boman, HG J Intern Med. 254.3 (2003): 197-215; Hancock, RE, & Scott, MG, Proc. Natl. Acad. Sci. USA 97 (2000): 8856-8861, Zasloff, M., Nature 415 (2002): 389-395).

On the other hand, Acinetobacter baumannii is a Gram-negative aerobic bacterium and is an important cause of nosocomial infections in many hospitals. In particular, recently, aminoglycosides, sepharoseporin, fluoroquinolone, beta lactamase Infections caused by beta-lactamase inhibitors (beta-lactamase inhibitors) and multidrug-resistant Acinetobacter Baumani (MRAB), which are resistant to carbapenem, are increasing.

In 2010, 46 people were infected with the Acinetobacter bacteria infection at the University of Tokyo Hospital, and 10 of them died, arousing awareness about MRAB, which is highly resistant to antibiotics, which is rapidly increasing worldwide in the last 10 years, and spurred the development of antibiotics. have. Acinetobacter bacteria themselves are also common in water, soil or human skin, and in healthy people, infection does not occur. However, if people with weakened immunity are infected, they can die of pneumonia or sepsis, and from the 1990s, they began to increase in the United States and Europe, and from 2000, almost no antibiotics were used.

In addition, the bacteria of the genus Klebsiella is a genus name of the intestinal bacteria family, and is a gram-negative bacillus. There is no flagella and is characterized by producing mucus while passing through the capsular membrane. It obtains a carbon source from citrate and produces acids and gases other than hydrogen sulfide from various carbohydrates. It is widely present in nature, is detected in human respiratory, intestinal, and urinary tract, and is known as the causative agent of acute pneumonia. Recently, various bacteria of the genus Klebsiella have been diagnosed and detected, including Klebsiella pneumoniae, Klebsiella ozaenae, and Klebsiella rhinoscleromatis.

Klebsiella pneumoniae, which is known to account for most of the bacteria of the genus Klebsiella in clinical terms, is a Gram-negative bacillus, anaerobe, or aerobic bacteria that resides in the human intestine, skin, oral cavity, and respiratory tract. It is said that 10-20% of pneumonia is caused by this fungus. In addition, it is sometimes isolated from urinary tract infections, and is reported as the main causative agent of sepsis and intraperitoneal infection, and it is the causative agent of bacteremia that occurs in intensive care unit patients, showing a high infection rate.

Meanwhile, various antimicrobial agents that can effectively kill microorganisms are currently used, and beta-lactam-based antibiotics account for about 50% or more of currently used antimicrobial agents. However, it is reported that many Gram-negative bacteria exhibit resistance to these antibiotics by producing beta-lactam-degrading enzymes against these beta-lactam-based antibiotics. Among the currently discovered bacteria of the genus Klebsiella, pneumococcal, in particular, K. pneumoniae carbaphenemase (KPC)-producing K. pneumoniae (KPC-Kp), which is an antibiotic-resistant bacteria, spreads worldwide and increases the mortality rate of infected patients. Raising it. Accordingly, in Korea, a new strategy is needed to inhibit the spread and increase of K. pneumoniae carbaphenemase (KPC)-producing K. pneumoniae (KPC-Kp).

Bacteriophage refers to a bacteria-specific virus that inhibits and inhibits the growth of infected bacteria by infecting specific bacteria. Bacteriophages have the ability to kill bacteria by proliferating inside bacterial cells after infection with bacteria, and destroying the cell walls of host bacteria when progeny bacteriophages come out of the bacteria after proliferation. The bacterial infection method of bacteriophage is very specific, so the types of bacteriophage that can infect specific bacteria are limited to some. That is, a specific bacteriophage can infect only a specific category of bacteria, and because of this, a specific bacteriophage kills only specific bacteria and does not affect other bacteria. Therefore, the use of bacteriophage as a countermeasure for recent bacterial diseases is receiving great attention. In particular, after 2000, the limitations of existing antibiotics appeared due to the increase in antibiotic-resistant bacteria, and the possibility of development as a substitute for the existing antibiotics emerged.

Until now, studies on novel bacteriophages for preventing or treating infections caused by bacteria of the genus Acinetobacter and genus Klebsiella are still insufficient. Therefore, it is necessary to develop a technology for bacteriophage and its application, which has a specific lytic activity for bacteria of the genus Klebsiella.

It is an object of the present invention to provide a novel bacteriophage having specific infection and killing ability against bacteria, particularly Acinetobacter genus and Klebsiella genus bacteria having resistance to antibiotics.

Another object of the present invention is a composition for the prevention and treatment of infectious diseases caused by novel bacteriophages having specific infection and killing ability against bacteria of the genus Acinetobacter, in particular, bacteria of the genus Acinetobacter having resistance to antibiotics It is to provide a food composition for improving diseases and diseases.

According to an embodiment of the present invention, a bacteriophage having a specific killing ability against bacteria of the genus Acinetobacter or Klebsiella is provided.

In the present invention, the term "bacteriophage" is a bacterial-specific virus that inhibits and inhibits the growth of the bacteria by infecting a specific bacterium, and refers to a virus containing a single or double-stranded DNA or RNA as a genetic material. .

In the Acinetobacter the invention bacteria are Acinetobacter baumannii (Acinetobacter baumannii), Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus), Acinetobacter H. Mori T kusu (Acinetobacter haemolyticus), Acinetobacter gave (Acinetobacter junii ), Acinetobacter johnsonii , Acinetobacter lwoffii , Acinetobacter radioresistens , Acinetobacter ursingii , Acinetobacter ursingii , Acinetobacter lwoffii Acinetobacter schindleri), Acinetobacter Parque booth (Acinetobacter parvus), Acinetobacter Bay Li (Acinetobacter baylyi), Acinetobacter Bow Betty (Acinetobacter bouvetii), Acinetobacter tow Nourishing (Acinetobacter towneri), Acinetobacter tandoyi (Acinetobacter tandoii ), Acinetobacter grimontii , Acinetobacter tjernbergiae , and Acinetobacter gerneri.It may be any one or more selected from the group, but is not limited thereto.

In the present invention, the bacteriophage has a specific killing ability against bacteria of the genus Acinetobacter, but also has specific killing ability against bacteria of the genus Acinetobacter having antibiotic resistance among the bacteria of the genus Acinetobacter.

In the present invention, the "antibiotic resistance" means that the drug does not work because of resistance to a specific antibiotic, and for the purposes of the present invention, the antibiotics are colistin, erythromycin, ampicillin, and MP Cylin-Sulbectam (Ampicillin-s µlbactam), Vancomycin, Linezolid, Methicillin, Oxacillin, Ceotaxime, Rifampicin, Amikacin (Amikacin), Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Doripenem, Doripenem, Imipenem Imipenem), Imipenem/Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Aztreonam (Aztreonam), Piperacillin, Polymyxin B, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Gatifloxacin, Piperacillin, Piperacillin piperacillin-tazobactam), minocycline, Tigecycline, Cotrimoxa, combinations thereof, and derivatives thereof, but may be any one or more selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, the bacteriophage is a bacteriophage separated by collecting a sample from a sewage treatment plant in a hospital, named bacteriophage YMC14/01/P262_ABA_BP, and on November 15, 2018, the deposit number KFCC11798P in the Korea Microbiology Conservation Center. It may have been deposited.

It was confirmed that the bacteriophage YMC14/01/P262_ABA_BP of the present invention belongs to the family Myoviridae, which has a long tail with a hexagonal head, and as a result of total nucleotide sequence analysis, it has a size of 44,597 bp and the total number of ORFs is 79. Confirmed.

In addition, the bacteriophage YMC14/01/P262_ABA_BP in the present invention may include the nucleotide sequence represented by SEQ ID NO: 1 as all or part of the entire gene.

In addition, the bacteriophage YMC14/01/P262_ABA_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 1, and a functional equivalent of the nucleotide sequence. The functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 1 as a result of modification or substitution of the base sequence. Having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 6.

In addition, the bacteriophage YMC14/01/P262_ABA_BP provided by the present invention may include any one of SEQ ID NOs: 2 to 4. In the present invention, each of SEQ ID NOs: 2 to 4 may be an ORF (Open reading frame) of the bacteriophage, and the protein represented by SEQ ID NO: 2 may be an amino acid sequence of a protein presumed to be endolysin, and the SEQ ID NO: The protein represented by 3 may be an amino acid sequence of a lysozyme-like domain, and the protein represented by SEQ ID NO: 4 may be an amino acid sequence of a putative tail-fiber/lysosomal protein. In more detail, SEQ ID NO: 2 may be the amino acid sequence of ORF38, SEQ ID NO: 3 is the amino acid sequence of ORF50, and SEQ ID NO: 4 may be the amino acid sequence of ORF51.

In addition, the bacteriophage YMC14/01/P262_ABA_BP provided by the present invention may include a genome represented by any one of SEQ ID NOs: 5 to 7. Here, SEQ ID NO: 5 is the nucleotide sequence of the genome encoding ORF38, SEQ ID NO: 6 is the nucleotide sequence of the genome encoding ORF50, and SEQ ID NO: 7 may be the nucleotide sequence of the genome encoding ORF51.

In another embodiment of the present invention, the bacteriophage is a bacteriophage separated by collecting a sample from a sewage treatment plant in a hospital, named as bacteriophage YMC15/02/T28_ABA_BP, and as deposit number KFCC11799P at the Korea Microbiology Conservation Center on November 15, 2018. It may have been deposited.

It was confirmed that the bacteriophage YMC15/02/T28_ABA_BP of the present invention belongs to the family Myobiri family, which has a hexagonal head and a long tail, and as a result of total nucleotide sequence analysis, it has a size of 44,580 bp and the total number of ORFs is 77.

In addition, in the present invention, the bacteriophage YMC15/02/T28_ABA_BP may include the nucleotide sequence represented by SEQ ID NO: 8 as all or part of the entire gene.

In addition, the bacteriophage YMC15/02/T28_ABA_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 8, and a functional equivalent of the nucleotide sequence. The functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 8 as a result of modification or substitution of the base sequence. Having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 8.

In addition, the bacteriophage YMC15/02/T28_ABA_BP provided by the present invention may include any one of SEQ ID NOs: 9 to 11. In the present invention, each of SEQ ID NOs: 9 to 11 is an ORF (open reading frame) of the bacteriophage, and the protein represented by SEQ ID NO: 9 may be an amino acid sequence of a lysozyme-like domain, and the SEQ ID NO: The protein represented by 10 may be an amino acid sequence of a putative tail-fiber/lysosomal protein, and the protein represented by SEQ ID NO: 11 may be an amino acid sequence of an endolysin putative protein. In more detail, SEQ ID NO: 9 may be the amino acid sequence of ORF7, SEQ ID NO: 10 may be the amino acid sequence of ORF8, and SEQ ID NO: 11 may be the amino acid sequence of ORF73.

In addition, the bacteriophage YMC15/02/T28_ABA_BP provided by the present invention may include a genome represented by any one of SEQ ID NOs: 12 to 14. Here, SEQ ID NO: 12 is the nucleotide sequence of the genome encoding ORF7, SEQ ID NO: 13 is the nucleotide sequence of the genome encoding ORF8, and SEQ ID NO: 14 may be the nucleotide sequence of the genome encoding ORF73.

In another embodiment of the present invention, the bacteriophage is a bacteriophage separated by collecting a sample from a sewage treatment plant in a hospital, named as bacteriophage YMC15/09/R1869_ABA_BP, and deposited number KFCC11802P to the Korea Microbiology Conservation Center on November 15, 2018. It may have been deposited as.

It was confirmed that the bacteriophage YMC15/09/R1869_ABA_BP of the present invention belongs to the family Myobiri family, which has a hexagonal head and a long tail, and as a result of total nucleotide sequence analysis, it has a size of 42,555 bp and the total number of ORFs is 77.

In addition, the bacteriophage YMC15/09/R1869_ABA_BP in the present invention may include the nucleotide sequence represented by SEQ ID NO: 15 as all or part of the entire gene.

In addition, the bacteriophage YMC15/09/R1869_ABA_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 15, and a functional equivalent of the nucleotide sequence. The functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 15 as a result of modification or substitution of the base sequence. Having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 15.

In addition, the bacteriophage YMC15/09/R1869_ABA_BP provided in the present invention may include any one of SEQ ID NOs: 16 and 17. In the present invention, each of SEQ ID NOs: 16 and 17 is an ORF (Open reading frame) of the bacteriophage, and SEQ ID NO: 16 may be an amino acid sequence of a lysozyme-like domain, and SEQ ID NO: 17 is a lysozyme family protein It may be an amino acid sequence of a protein estimated to be. In more detail, each of SEQ ID NOs: 16 and 17 may be an amino acid sequence of a lysozyme-like domain as ORF7 and ORF73.

In addition, the bacteriophage YMC15/09/R1869_ABA_BP provided by the present invention may include a genome represented by any one of SEQ ID NOs: 18 and 19. Here, SEQ ID NO: 18 may be a nucleotide sequence of a genome encoding ORF7, and SEQ ID NO: 19 may be a nucleotide sequence of a genome encoding ORF73.

In the present invention, the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/09/R1869_ABA_BP has excellent stability against heat and pH.

The bacteriophage YMC14/01/P262_ABA_BP of the present invention; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/09/R1869_ABA_BP maintains lytic activity within the range of 4 to 60° C., but is not limited thereto.

In addition, the bacteriophage YMC14/01/P262_ABA_BP of the present invention; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/09/R1869_ABA_BP maintains lytic activity in the range of pH 3.0 to pH 11.0, preferably pH 5.0 to pH 10.0, but is not limited thereto.

Acinetobacter genus bacteria-specific lytic activity, acid resistance and basic resistance as described above, the bacteriophage YMC14/01/P262_ABA_BP of the present invention; Bacteriophage YMC15/02/T28_ABA_BP; And bacteriophage YMC15/09/R1869_ABA_BP composition for the prevention and treatment of infectious diseases caused by bacteria of the genus Acinetobacter, and the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And in applying to a variety of products containing the bacteriophage YMC15/09/R1869_ABA_BP as an active ingredient, it enables the application of a variety of pH ranges.

In another embodiment of the present invention, the bacteria of the genus Klebsiella are Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella planticola (Klebsiella planticola), and Klebsiella terrigena (Klebsiella terrigena) can be any one or more selected from the group consisting of, and from a clinical point of view as pneumococcal Klebsiella pneumoniae, which accounts for most of the bacteria in the genus Klebsiella, is known as a bacterium representing the bacteria in the genus Klebsiella.

In the present invention, the bacteriophage has a specific killing ability against bacteria of the genus Klebsiella, but also has a specific killing ability against bacteria of the genus Klebsiella having antibiotic resistance among bacteria of the genus Klebsiella.

In the present invention, the "antibiotic resistance" means that the drug does not work because of resistance to a specific antibiotic, and for the purposes of the present invention, the antibiotic may be an antibiotic having a structure of carbapenem. Specifically, Amikacin, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Ceftazidime, Cefazoline, Imipenem ), Ertapenem, Cefepime, Cefoxitin, Cefotaxime, Gentamicine, Levoflocacin, Meropenem, Piperacillin / Tazobactam (Piperacillin/Tazobactam), Cortrimoxa, and Tigecyline may be one or more selected from the group consisting of, but is not limited thereto. For the purposes of the present invention, the Klebsiella pneumoniae may have antibiotic resistance, and the antibiotic resistance may be generated by producing a carbapenemase enzyme that degrades the carbapenem and inhibits the exertion of the effect. .

In the present invention, the bacteriophage YMC17/01/P6_KPN_BP may include the nucleotide sequence represented by SEQ ID NO: 20 as all or part of the entire gene. In addition, the bacteriophage YMC17/01/P6_KPN_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 20, and a functional equivalent of the nucleotide sequence. The functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 20 as a result of modification or substitution of the base sequence. By having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 20.

In addition, the bacteriophage provided by the present invention may be one containing any one of SEQ ID NOs: 21 and 22 protein. In the present invention, each of SEQ ID NOs: 21 and 22 is an ORF (Open reading frame) of the bacteriophage, and among proteins that perform adsorption and lysis functions in the genus Klebsiella, in particular, holin or anti-holin ) May be the amino acid sequence of the protein, and more specifically, SEQ ID NOs: 21 and 22 may be the amino acid sequences of ORF57 and ORF59, respectively.

In addition, the bacteriophage provided by the present invention may include a genome represented by any one of SEQ ID NOs: 23 and 24. Here, SEQ ID NO: 23 is the nucleotide sequence of the genome encoding ORF57, and SEQ ID NO: 24 is the nucleotide sequence of the genome encoding ORF59.

The bacteriophage YMC17/01/P6_KPN_BP of the present invention has excellent lytic activity against bacteria of the genus Klebsiella, particularly carbapenem-based antibiotic-resistant pneumoniae. It was confirmed that the bacteriophage YMC17/01/P6_KPN_BP belongs to the family Siphoviridae, which is a form having an angled head and tail, and as a result of total nucleotide sequence analysis, it has a size of 54,880 bp and the total number of ORFs is 87. Confirmed.

The bacteriophage YMC17/01/P6_KPN_BP of the present invention is a bacteriophage separated by collecting samples from a sewage treatment plant in a hospital, named bacteriophage YMC17/01/P6_KPN_BP, and deposited with the Korea Microbiology Conservation Center as the deposit number KFCC11804P on November 15, 2018. I did.

The bacteriophage YMC17/01/P6_KPN_BP of the present invention maintains lytic activity in the range of 4° C. to 60° C., but is not limited thereto.

The bacteriophage YMC17/01/P6_KPN_BP of the present invention maintains lytic activity in the range of pH 3.0 to pH 11.0, preferably pH 5.0 to pH 10.0, but is not limited thereto.

In the present invention, the bacteriophage YMC17/01/P6_KPN_BP of the genus Klebsiella has specific lytic activity, acid resistance and basic resistance, and the bacteriophage of the present invention is a composition for preventing and treating infectious diseases caused by bacteria of the genus Klebsiella. , In applying to a variety of products containing the bacteriophage as an active ingredient, it enables the application of a variety of pH ranges.

According to another embodiment of the present invention, bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/09/R1869_ABA_BP; Or it provides a composition for preventing, improving or treating diseases caused by Acinetobacter genus bacteria or Klebsiella bacteria comprising bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

In the composition of the present invention, the bacteriophage and the Acinetobacter genus bacterium or the Klebsiella bacterium are duplicated as described in the bacteriophage, and detailed descriptions thereof will be omitted below.

In the present invention, the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/09/R1869_ABA_BP specifically kills bacteria of the genus Acinetobacter, particularly antibiotic-resistant bacteria of the genus Acinetobacter, and thus shows an effect in the treatment of various diseases caused by the bacteria of the genus Acinetobacter.

In the present invention, the infectious diseases caused by the bacteria of the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondylom, vancomycin-resistant yellow staphylococcus aureus infection, and vancomycin endocytic fungal infection. , Methicillin-resistant yellow aeruginosa infection, multi-drug-resistant Pseudomonas aeruginosa infection, multi-drug-resistant asinetobacter baumani infection, carbapenem intragrowth mycobacterial infection, intestinal infection, acute respiratory tract infection, and enterovirus infection However, it is not limited thereto.

The composition of the present invention may include 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL of bacteriophage, and preferably 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL of bacteriophage. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the bacteriophage to form plaque.

In the present invention, the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumococcal bacteria having resistance to carbapenem antibiotics, and thus has an effect on the treatment of various diseases caused by the bacteria of the genus Klebsiella. Show.

In the present invention, the infectious diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media. , Bacteremia, endocarditis, cholecystitis or parotitis, which is particularly a disease caused by pneumococcal, but is not limited thereto.

The composition of the present invention may include 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL of bacteriophage, and preferably 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL of bacteriophage. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the bacteriophage to form plaque.

According to another embodiment of the present invention, bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/09/R1869_ABA_BP; Or it provides a composition for preventing, improving or treating diseases caused by Acinetobacter genus bacteria or Klebsiella bacteria comprising bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

In the composition of the present invention, the bacteriophage and the Acinetobacter genus bacterium or the Klebsiella bacterium are duplicated as described in the bacteriophage, and detailed descriptions thereof will be omitted below.

In the present invention, the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/09/R1869_ABA_BP specifically kills bacteria of the genus Acinetobacter, particularly antibiotic-resistant bacteria of the genus Acinetobacter, and thus shows an effect in the treatment of various diseases caused by the bacteria of the genus Acinetobacter.

In the present invention, the infectious diseases caused by the bacteria of the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondylom, vancomycin-resistant yellow staphylococcus aureus infection, and vancomycin endocytic fungal infection. , Methicillin-resistant yellow aeruginosa infection, multi-drug-resistant Pseudomonas aeruginosa infection, multi-drug-resistant asinetobacter baumani infection, carbapenem intragrowth mycobacterial infection, intestinal infection, acute respiratory tract infection, and enterovirus infection However, it is not limited thereto.

The composition of the present invention may include 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL of bacteriophage, and preferably 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL of bacteriophage. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the bacteriophage to form plaque.

In the present invention, the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumococcal bacteria having resistance to carbapenem antibiotics, and thus has an effect on the treatment of various diseases caused by the bacteria of the genus Klebsiella. Show.

In the present invention, the infectious diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media. , Bacteremia, endocarditis, cholecystitis or parotitis, which is particularly a disease caused by pneumococcal, but is not limited thereto.

The composition of the present invention may include 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL of bacteriophage, and preferably 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL of bacteriophage. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the bacteriophage to form plaque.

According to another embodiment of the present invention, bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/09/R1869_ABA_BP; Or it provides a composition for preventing, improving or treating diseases caused by Acinetobacter genus bacteria or Klebsiella bacteria comprising bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

In the composition of the present invention, the bacteriophage and the Acinetobacter genus bacterium or the Klebsiella bacterium are duplicated as described in the bacteriophage, and detailed descriptions thereof will be omitted below.

In the present invention, the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/09/R1869_ABA_BP specifically kills bacteria of the genus Acinetobacter, particularly antibiotic-resistant bacteria of the genus Acinetobacter, and thus shows an effect in the treatment of various diseases caused by the bacteria of the genus Acinetobacter.

In the present invention, the infectious diseases caused by the bacteria of the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondylom, vancomycin-resistant yellow staphylococcus aureus infection, and vancomycin endocytic fungal infection. , Methicillin-resistant yellow aeruginosa infection, multi-drug-resistant Pseudomonas aeruginosa infection, multi-drug-resistant asinetobacter baumani infection, carbapenem intragrowth mycobacterial infection, intestinal infection, acute respiratory tract infection, and enterovirus infection However, it is not limited thereto.

The composition of the present invention may include 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL of bacteriophage, and preferably 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL of bacteriophage. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the bacteriophage to form plaque.

In the present invention, the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumococcal bacteria having resistance to carbapenem antibiotics, and thus has an effect on the treatment of various diseases caused by the bacteria of the genus Klebsiella. Show.

In the present invention, the infectious diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media. , Bacteremia, endocarditis, cholecystitis or parotitis, which is particularly a disease caused by pneumococcal, but is not limited thereto.

The composition of the present invention may include 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL of bacteriophage, and preferably 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL of bacteriophage. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the bacteriophage to form plaque.

According to another embodiment of the present invention, the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/09/R1869_ABA_BP; Or it provides a cleaning agent, including bacteriophage YMC17/01/P6_KPN_BP.

The bacteriophage YMC14/01/P262_ABA_BP of the present invention; Bacteriophage YMC15/02/T28_ABA_BP; Alternatively, the bacteriophage YMC15/09/R1869_ABA_BP has a specific killing ability for bacteria of the genus Acinetobacter or Klebsiella, so the skin surface or body of an individual exposed to or likely to be exposed to the genus Acinetobacter or Klebsiella It can also be used for washing each area.

In addition, the present invention is a bacteriophage YMC14/01/P262_ABA_BP for use for the manufacture of a cleaning agent; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/09/R1869_ABA_BP; Or it may be to use bacteriophage YMC17/01/P6_KPN_BP.

According to another embodiment of the present invention, the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/09/R1869_ABA_BP; Or it provides a pharmaceutical composition for the prevention or treatment of diseases caused by bacteria of the genus Acinetobacter or Klebsiella genus, comprising the bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

In the present invention, diseases caused by bacteria of the genus Acinetobacter or genus Klebsiella are duplicated as described above, and thus detailed descriptions thereof will be omitted.

According to another embodiment of the present invention, Acinetobacter genus comprising administering any one of the bacteriophages selected from claims 1 to 25 as an active ingredient to a subject in need of prevention or treatment Alternatively, it may be a method of preventing or treating diseases caused by bacteria of the genus Klebsiella.

In the present invention, the pharmaceutical composition may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that for humans.

The pharmaceutical composition of the present invention is not limited thereto, but is formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injectable solutions according to conventional methods. I can. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections. Agents, solubilizers, isotonic agents, stabilizers, and the like can be mixed and used, and in the case of topical administration, a base agent, excipient, lubricant, preservative, etc. can be used. The formulation of the pharmaceutical composition of the present invention may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc.In the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.

On the other hand, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like may additionally be included.

The route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local , Sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention depends on a number of factors including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day. It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention. The pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

In the present invention, the cosmetic composition is a lotion, nutritional lotion, nutritional essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, Suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory {cosmetic}, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, Bath soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap (non-medical), cream soap, facial wash, body cleaner, scalp cleaner, hair rinse, makeup soap, tooth whitening gel, toothpaste, etc. It can be manufactured in a form. To this end, the composition of the present invention may further include a solvent or a suitable carrier, excipient, or diluent that is commonly used in the manufacture of cosmetic compositions.

The type of solvent that can be further added to the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used. As a carrier, excipient or diluent, purified water, oil, wax , Fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like, but are not limited thereto. In addition, whitening agents, moisturizing agents, vitamins, sunscreen agents, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.

Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm seed oil, jojoba oil, and avocado oil may be used as the oil. Can be used.

Stearic acid, linoleic acid, linolenic acid, and oleic acid may be used as the fatty acid, and cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol may be used as fatty acid alcohols. And as the fatty acid ester, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As the surfactant, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as far as possible.

In addition, hygroscopic agents, thickeners, antioxidants, and the like, which are widely known in the cosmetic field, may be included, and the types and amounts thereof are as known in the art.

The food composition of the present invention can be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, and bread. Since the food composition of the present invention is composed of plant extracts with little toxicity and side effects, it can be safely used even when taken for a long time for prophylactic purposes.

When the bacteriophage of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 50% of the total weight.

Here, when the food composition is prepared in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and as an additional component, various flavoring agents or natural carbohydrates, etc. may be included as in a conventional beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are included. can do. Examples of the flavoring agent include natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

In addition, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.

These components may be used independently or in combination. The proportion of these additives is not so important, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

The novel bacteriophage provided by the present invention has a specific killing ability for Acinetobacter genus or Klebsiella bacteria, and Acinetobacter genus or Klebsiella bacteria resistant to antibiotics compared to chemical substances such as conventional antibiotics. Have.

In addition, since the bacteriophage of the present invention does not infect other hosts other than bacteria such as humans, animals, plants, etc., it has the advantage of solving the problems of antibiotic-resistant bacteria due to abuse of antibiotics, the problem of residual antibiotics in food, and problems of a wide host range have.

Therefore, the bacteriophage of the present invention can be used in various fields in the field of prevention or treatment of infectious diseases caused by Acinetobacter genus or Klebsiella bacteria, antibiotic compositions, feed addition compositions, feed, disinfectants, or cleaning agents.

1 shows an electron microscope photograph of a bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention.

Figure 2 is a graph showing the adsorption capacity of the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention against bacteria in Acinetobacter genus having antibiotic resistance.

Figure 3 shows a one-stage proliferation curve of the lytic bacteriophage YMC14/01/P262_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

Figure 4 is a graph showing the lytic ability of the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention against bacteria in Acinetobacter genus having antibiotic resistance in vitro.

Figure 5 is a graph showing the change in survival rate of the larva after treating the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention to the larvae of the bees infected with bacteria of the genus Acinetobacter having antibiotic resistance. .

6 is a graph showing the pH stability of the lytic bacteriophage YMC14/01/P262_ABA_BP against bacteria in the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

7 is a graph showing the temperature stability of the lytic bacteriophage YMC14/01/P262_ABA_BP against bacteria in the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

8 shows the result of analyzing the entire genome sequence of the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention.

9 shows an electron microscope photograph of the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention.

10 is a graph showing the adsorption capacity of the bacteriophage YMC15/02/T28_ABA_BP against bacteria in Acinetobacter genus having antibiotic resistance according to an embodiment of the present invention.

FIG. 11 shows a one-stage proliferation curve of the lytic bacteriophage YMC15/02/T28_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

12 is a graph showing the lytic ability of the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention against bacteria in Acinetobacter genus having antibiotic resistance in vitro.

13 is a graph showing the change in the survival rate of the larva after treatment with the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention to the larvae of the honey bee infected with bacteria of the genus Acinetobacter having antibiotic resistance. .

14 is a graph showing the pH stability of the lytic bacteriophage YMC15/02/T28_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

15 is a graph showing the temperature stability of the lytic bacteriophage YMC15/02/T28_ABA_BP against bacteria in the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

Figure 16 shows the result of analyzing the entire genome sequence of the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention.

Figure 17 shows an electron microscope photograph of the bacteriophage YMC15/09/R1869_ABA_BP according to an embodiment of the present invention.

18 is a graph showing the adsorption capacity of the bacteriophage YMC15/09/R1869_ABA_BP against bacteria in Acinetobacter genus having antibiotic resistance according to an embodiment of the present invention.

FIG. 19 shows a one-stage proliferation curve of the lytic bacteriophage YMC15/09/R1869_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

FIG. 20 is a graph showing the change in survival rate of the larva after treatment with the bacteriophage YMC15/09/R1869_ABA_BP according to an embodiment of the present invention to bee larvae infected with Acinetobacter Baumani having antibiotic resistance. .

Figure 21 shows the number of bacteria of the Acinetobacter Baumani in the lungs of the mouse after treatment with the bacteriophage YMC15/09/R1869_ABA_BP according to an embodiment of the present invention to a mouse infected with an antibiotic-resistant Acinetobacter Baumani It shows the change in a graph.

22 is a graph showing the pH stability of the lytic bacteriophage YMC15/09/R1869_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

23 is a graph showing the temperature stability of the lytic bacteriophage YMC15/09/R1869_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.

FIG. 24 shows the results of analyzing the entire genome sequence of the bacteriophage YMC15/09/R1869_ABA_BP according to an embodiment of the present invention.

25 shows an electron microscope photograph of a bacteriophage according to an embodiment of the present invention.

26 is a graph showing the adsorption capacity of a bacteriophage according to an embodiment of the present invention to bacteria of the genus Klebsiella having antibiotic resistance.

FIG. 27 shows a one-stage proliferation curve of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.

FIG. 28 is a graph showing the lytic ability of bacteriophage against bacteria of genus Klebsiella having antibiotic resistance in vitro according to an embodiment of the present invention.

29 is a graph showing the pH stability of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.

30 is a graph showing the temperature stability of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.

FIG. 31 shows the result of analyzing the entire genome sequence of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.

The present invention relates to a bacteriophage having a specific killing ability for bacteria of the genus Acinetobacter or Klebsiella.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

[Example 1] Bacteriophage YMC14/01/P262_ABA_BP

1. Isolation of clinical specimens and selection of antibiotic resistant strains

As shown in Table 1 below, Acinetobacter baumannii ( Acinetobacter baumannii ) bacteria were isolated and cultured from ICU (intensive care unit) blood and clinical samples of university hospitals. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Thereafter, the antibiotic susceptibility test was performed using a CLSI disc diffusion test method in which Mueller-Hinton agar was used for overnight culture at 37° C. outside air, and the test antibiotics were amicacin, ampicillin-sulfur. Bectam (ampicillin-sulbactam), ceftazidime, ciprofloxacin, colistin, cefepime, cefotaxime, gentamicine, imipenem, levofloxacin levofloxacin), meropenem, minocycline, piperacillin, piperacillin-tazobactam, cotrimoxa and tigecycline were used. The sensitivity results were read based on the Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profiles of the collected Acinetobacter baumannii strains are shown in Tables 2 to 4 below. However, in Tables 2 to 4 below, S, I, and R are the results of evaluating the sensitivity to antimicrobial agents, where'S' is sensitive (Susceptible),'I' is intermediate (Intermediate), and'R' is resistant (Resistant). Means.

Host strain Sample origin Host strain Sample origin YMC15/01/P186 Aspirate Head YMC15/04/R1148 Tracheal tube tips YMC15/01/R2319 Phlegm (pneumonia) YMC15/04/R68 Phlegm (pneumonia) YMC15/01/P760 Swap or drain tube YMC15/04/P369 YMC15/01/R3872 Tracheal inhalation (pneumonia) YMC15/04/R663 Phlegm (pneumonia) YMC15/02/R923 Phlegm (pneumonia) YMC15/04/T132 YMC15/02/R830 Phlegm (pneumonia) YMC15/04/R1427 Phlegm (pneumonia) YMC15/02/R1418 Phlegm (pneumonia) YMC15/04/U2285 Random Urine YMC15/02/R2403 Phlegm (pneumonia) YMC15/04/R2498 Phlegm (pneumonia) YMC15/02/R3331 Phlegm (pneumonia) YMC15/05/R1603 Phlegm (pneumonia) YMC15/02/P701 Swap or drainage bridge YMC15/05/R1818 Phlegm (pneumonia) YMC15/03/R2284 Phlegm (pneumonia) YMC15/05/R2554 Tracheal inhalation (pneumonia) YMC15/03/R2817 Phlegm (pneumonia) YMC15/05/R3556 Phlegm (pneumonia) YMC15/03/P828 Swab or drainage tube pelvis YMC15/06/R675 Phlegm (pneumonia) YMC15/03/B10119 Blood YMC15/08/R1402 Tracheal inhalation (pneumonia) YMC15/03/R3835 Phlegm (pneumonia) YMC15/08/R1398 Phlegm (pneumonia)) YMC15/03/R4022 Phlegm (pneumonia) YMC15/08/R1719 Tracheal tube tips

As shown in Tables 2 to 4, the collected Acinetobacter baumannii 32 strains were found to be multi-resistance strains having resistance to various antibiotics.

2. Collection of bacteriophage samples

2-1. Collecting samples to build a phage bank

Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours. The filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.

2-2. Selection of lytic phage and measurement of lysis titer

Separation and purification of lytic phage was performed by a spot test method (Mazzocco A et al. In Bacteriophages, Clokie and Kropinski AM, eds. Humana Press. 2009). The obtained strain was inoculated on McConkie agar medium and then incubated overnight at 35° C. outside air. After cultivation, strains susceptible to phage were selected to see the formation of transparent plaques. The susceptible strain was inoculated on Macconky agar medium and cultured at 35° C. for 12 hours. A suspension of each strain was prepared with McFarland 0.5 turbidity in a 1 ml saline tube, and H top agar (3 ml), 100 µl of sensitive bacteria, and phage solution (1 µl, 10 µl and 50 µl, respectively) were mixed and applied to LB agar, Incubated for 12 hours at 35 ℃. After observing the plaque, the plaque was collected with a Pasteur pipette, diluted in SM buffer solution, and purified again three times using a susceptible strain suspension. The pure bacteriophage thus obtained YMC14/01/P262_ABA_BP was diluted in SM buffer solution and purified three times again using a susceptible strain suspension. The pure bacteriophage thus obtained YMC14/01/P262_ABA_BP was diluted and stored in SM buffer solution.

Antibiotic-resistant Acinetobacter baumannii ( Acinetobacter baumannii ) 32 strains identified in 1. above were inoculated and cultured on Macconki agar medium, and then the bacteriophage YMC14/01/P262_ABA_BP purified by the above process was smeared with each resistance. Plaque formation was confirmed by inoculating the strain with 5 µl, and the titer range was checked, and lytic properties are shown in Table 5 below. However, in Table 5 below, + and-are the evaluation of plaque activity for the collected strains,'+' means clear plaque, and'-' means that lysis has not occurred.

Host strain Lysis Host strain Lysis YMC15/01/P186 ++ YMC15/04/R1148 ++ YMC15/01/R2319 ++ YMC15/04/R68 ++ YMC15/01/P760 ++ YMC15/04/P369 ++ YMC15/01/R3872 ++ YMC15/04/R663 ++ YMC15/02/R923 ++ YMC15/04/T132 ++ YMC15/02/R830 ++ YMC15/04/R1427 ++ YMC15/02/R1418 ++ YMC15/04/U2285 ++ YMC15/02/R2403 ++ YMC15/04/R2498 - YMC15/02/R3331 ++ YMC15/05/R1603 ++ YMC15/02/P701 ++ YMC15/05/R1818 ++ YMC15/03/R2284 - YMC15/05/R2554 ++ YMC15/03/R2817 ++ YMC15/05/R3556 ++ YMC15/03/P828 ++ YMC15/06/R675 - YMC15/03/B10119 ++ YMC15/08/R1402 ++ YMC15/03/R3835 ++ YMC15/08/R1398 ++ YMC15/03/R4022 ++ YMC15/08/R1719 ++

As shown in Table 5, it was confirmed that the bacteriophage YMC14/01/P262_ABA_BP according to the present invention lyses a significant number of antibiotic-resistant Acinetobacter Baumani strains.

3. Electron microscopic analysis of lytic bacteriophage against antibiotic-resistant Acinetobacter Baumani strains

Bacteriophage YMC14/01/P262_ABA_BP purified by the method of 2 above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added to the supernatant. After addition in an amount of 10% (w/v), it was stored refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage YMC14/01/P262_ABA_BP was analyzed using an energy-filtering transmission electron microscope, and the results are shown in FIG. Done.

As shown in Figure 1, when viewed as a standard for classifying the YMC14/01/P262_ABA_BP bacteriophage according to the present invention by shape, it was classified as belonging to the family Myoviridae, which has a hexagonal head and a long tail.

4. Analysis of adsorption capacity and one-step growth curve of bakperiophage

After incubating the antibiotic-resistant Acinetobacter Baumani strain to have an OD value of 0.5, add the bacteriophage YMC14/01/P262_ABA_BP purified in 2. to the Acinetobacter Baumani strain at an MOI of 0.001 and incubate at room temperature. , 100 µl sample was collected at 1 ml each in 1, 2, 3, 4, and 5 minutes, diluted in LB medium, and then evaluated for adsorption capacity of the bacteriophage through plaque analysis, and the results are shown in FIG. 2.

In addition, the antibiotic-resistant Acinetobacter Baumani strain was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium, The bacteriophage YMC14/01/P262_ABA_BP purified in 2. above was added to an MOI of 0.001 (titer 10 ⁸ pfu/cell) and incubated at 37° C. for 5 minutes. A pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 3.

As shown in Figure 2, within 5 minutes after inoculation of the bacteriophage YMC14/01/P262_ABA_BP, about 99% of the bacteriophage was adsorbed to the Acinetobacter Baumani strain (4 minutes: 6.9%, 10 minutes: 1.3%, 15 minutes : 0%).

In addition, as shown in FIG. 3, the result of the single-stage proliferation curve showed a high burst size of approximately 79 PFU/infected cells (0 min: 8 PFU/ml, 100 min: 636 PFU/ml).

Through the above results, the bacteriophage YMC14/01/P262_ABA_BP according to the present invention can be adsorbed in a relatively short time to the Acinetobacter Baumani strain having antibiotic resistance, and exhibits a high burst size of 79 PFU/infected cells. It can be seen that the dissolution effect of

5. Verification of the lytic ability of bacteriophage against in vitro antibiotic-resistant Acinetobacter bacteria

Antibiotic-resistant Acinetobacter Baumani strain 1 X 10 ⁹ CFU / ml prepared bacteriophage YMC14/01/P262_ABA_BP 1 X 10 ⁸ CFU / ml (MOI: 0.1), 1 X 10 ⁹ PFU / ml (MOI: 1), Each treatment was performed in an amount of 1 X 10 ¹⁰ PFU/ml (MOI: 10), and the OD value (wavelength 600 nm) was measured for each time. However, as a negative control, PBS + SM buffer was treated, and the values are shown in FIG. 4.

As shown in FIG. 4, when the bacteriophage YMC14/01/P262_ABA_BP was treated for the Acinetobacter Baumani strain, the OD value decreased, and the OD value decreased further as the MOI value increased. The ability was high.

Through the above results, it can be seen that the bacteriophage YMC14/01/P262_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain.

6. Verification of the lytic ability of bacteriophage against bacteria in antibiotic-resistant Acinetobacter genus in vivo

After preparing 200 larvae of Galleria mellonella larvae aged 3 to 4 years old, 10 animals were classified for each group. After injecting the Acinetobacter Baumani strain, which is resistant to colistin to each larva, through the proleg of the larva at a minimum lethal concentration (MLD), colistin and the bacteriophage purified in 2 above YMC14/01/P262_ABA_BP After the mixed inoculation with MOI 10 or MOI 100, the survival rate of the larva was checked every 12 or 24 hours until 72 hours, and the results are shown in FIG. 5.

As shown in FIG. 5, when the bacteriophage YMC14/01/P262_ABA_BP according to the present invention was treated to the larvae injected with the Acinetobacter Baumani strain having resistance to colistin, the survival rate of the larva increased, and as the MOI value increased It was confirmed that the survival rate of larvae was further increased. In addition, even when only the bacteriophage YMC14/01/P262_ABA_BP was injected without injecting the Acinetobacter Baumani strain having resistance to colistin, it was confirmed that there was no toxicity when comparing the survival rate with the healthy control group.

Through the above results, the bacteriophage YMC14/01/P262_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.

7. Stability evaluation of bacteriophage against antibiotic-resistant Acinetobacter Baumani strain

Bacteriophage according to the present invention It was confirmed that the bacteriophage YMC14/01/P262_ABA_BP maintains stability without being destroyed in temperature and alkali.

1 µl of the bacteriophage YMC14/01/P262_ABA_BP purified by the method of 2 above was added to 40 µl of SM buffer adjusted to a pH of 4, 5, 6, 7, 8, 9 and 10, and then incubated at 37°C for 1 hour After that, plaque analysis was performed with the antibiotic-resistant Klebsiella pneumoniae by the method of 4 above, and the results are shown in FIG. 6.

In addition, the bacteriophage YMC14/01/P262_ABA_BP solution was cultured at 4° C., 37° C., 50° C., 60° C., and 70° C. for 1 hour, and each sample was sampled with the Acinetobacter Baumani strain 4 Plaque analysis was performed by the method of. And the results are shown in FIG. 7.

As shown in Figure 6, the bacteriophage YMC14/01/P262_ABA_BP according to the present invention exhibited high stability in both acidic, neutral and alkaline, and the bacteriophage YMC14/01/P262_ABA_BP was particularly neutral (pH 7-8) for 30 days. ) Showed relatively stability.

In addition, as shown in Figure 7, the bacteriophage YMC14/01/P262_ABA_BP showed very high stability up to a high temperature of 70 ℃.

8. Whole genome sequence analysis of bacteriophage against antibiotic-resistant Klebsiella sp.

In order to identify the characteristics of the bacteriophage YMC14/01/P262_ABA_BP according to the present invention, the entire gene sequence analysis was performed through the Illumina sequencer (Roche) based on the method of analyzing the entire genome sequence, which is obvious to the skilled person, and the results are shown. 8 and Tables 6 to 11.

8 and Tables 6 to 11, the bacteriophage YMC14/01/P262_ABA_BP contains a linear dsDNA (linear dsDNA) and was composed of 79 ORFs.

As a result of comparing the sequence of the bacteriophage YMC14/01/P262_ABA_BP according to the present invention with the sequence of the existing bacteriophage, the bacteriophage having similarity to the bacteriophage according to the present invention was not detected. Through the above results, it can be seen that the bacteriophage YMC14/01/P262_ABA_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.

[Example 2] Bacteriophage YMC15/02/T28_ABA_BP

1. Isolation of clinical specimens and selection of antibiotic resistant strains

As shown in Table 12 below, Acinetobacter baumannii bacteria were isolated and cultured from ICU (intensive care unit) blood and clinical specimens of university hospitals. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Subsequently, the antibiotic susceptibility test was performed using a CLSI disc diffusion test method in which Mueller-Hinton agar was used for overnight culture at 37° C., and the test antibiotics were imipenem, piperacillin-tazobactam. (piperacillin-tazobactam), ampicillin-s ulbactam, aztreonam, ceftazidime, cefepime, cefotaxime, gentamicine , Amicacin, ciprofloxacin, levofloxacin, tigecyline and colistin were used. The sensitivity results were read based on the Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profiles of the collected Acinetobacter baumannii strains are shown in Tables 13 to 15 below. However, in Tables 13 to 15 below, S, I, and R are the results of evaluating the sensitivity to antimicrobial agents, where'S' is sensitive,'I' is intermediate, and'R' is resistant. Means.

Host strain Sample origin Host strain Sample origin YMC15/01/P186 Other Catheter Tip YMC15/04/R1148 Phlegm (pneumonia) YMC15/01/R2319 Swap or drain pipe YMC15/04/R68 Phlegm (pneumonia) YMC15/01/P760 Phlegm (pneumonia) YMC15/04/P369 Phlegm (pneumonia) YMC15/01/R3872 Gluteal pressure sores YMC15/04/R663 Phlegm (pneumonia) YMC15/02/R923 blood YMC15/04/T132 Tracheal tube tips YMC15/02/R830 Phlegm (pneumonia) YMC15/04/R1427 Tracheal inhalation (pneumonia) YMC15/02/R1418 Gluteal pressure sores YMC15/04/U2285 Tracheal inhalation (pneumonia) YMC15/02/R2403 Intravenous catheter tips YMC15/04/R2498 Swap or drain pipe, etc. YMC15/02/R3331 Phlegm (pneumonia) YMC15/05/R1603 bedsore YMC15/02/P701 Intravenous catheter tips YMC15/05/R1818 Bile, PTBD

As shown in Tables 13 to 15, the collected 20 strains of Acinetobacter baumannii were found to be multi-resistance strains having resistance to various antibiotics.

2. Collection of bacteriophage samples

2-1. Collecting samples to build a phage bank

Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours. The filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.

2-2. Selection of lytic phage and measurement of lysis titer

Separation and purification of lytic phage was performed by a spot test method (Mazzocco A et al. In Bacteriophages, Clokie and Kropinski AM, eds. Humana Press. 2009). The obtained strain was inoculated on Macconky agar medium and then incubated overnight at 35° C. outside air. After cultivation, strains susceptible to phage were selected to see the formation of transparent plaques. The susceptible strain was inoculated on Macconki agar medium and cultured at 35° C. for 12 hours. A suspension of each strain was prepared in a 1 ml saline tube with a turbidity of 0.5 McFarland, H top agar (3 ml), 100 μl of sensitive bacteria and a phage solution (1 μl, 10 μl and 50 μl, respectively) were mixed and applied to LB agar, Incubated for 12 hours at 35 ℃. After observing the plaques, the plaques were collected with a Pasteur pipette, diluted in SM buffer solution, and purified again three times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC15/02/T28_ABA_BP was diluted in SM buffer solution and purified again three times using a susceptible strain suspension. The pure bacteriophage thus obtained YMC15/02/T28_ABA_BP was diluted and stored in SM buffer solution.

After inoculating and culturing the antibiotic-resistant Acinetobacter baumannii strain identified in 1. above on McConkie agar medium, the bacteriophage YMC15/02/T28_ABA_BP purified by the above process was applied to each of the stained resistant strains 5 Plaque formation was confirmed by inoculation with µl, and the titer range was confirmed, and lysis was shown in Table 16 below.

However, in Table 16 below, + and-refer to the evaluation of plaque activity for the collected strains, and'+' means clear plaque, and'-' means that lysis has not occurred.

Host strain Lysis Host strain Lysis YMC15/02/T28 + YMC13/05/R2199 - YMC13/02/R669 + YMC13/05/R3526 + YMC13/02/R1380 + YMC13/06/R42 - YMC13/02/P386 + YMC13/06/R633 + YMC13/05/T180 + YMC13/12/P154 +

As shown in Table 16, it was confirmed that the bacteriophage YMC15/02/T28_ABA_BP according to the present invention lyses a significant number of antibiotic-resistant Acinetobacter Baumani strains.

3. Electron microscopic analysis of lytic bacteriophage against antibiotic-resistant Acinetobacter Baumani strains

The bacteriophage YMC15/02/T28_ABA_BP purified by the method of 2 above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added to the supernatant. After addition in an amount of 10% (w/v), it was stored in the refrigerator overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage YMC15/02/T28_ABA_BP was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. Done.

As shown in Figure 9, when viewed as a criterion for classifying the YMC15/02/T28_ABA_BP bacteriophage according to the present invention by shape, it was classified as belonging to the family Myobiridae having a long tail with a hexagonal head.

4. Analysis of adsorption capacity and one-step growth curve of bakperiophage

After incubating the antibiotic-resistant Acinetobacter Baumani strain to an OD value of 0.5, add the bacteriophage YMC15/02/T28_ABA_BP purified in 2. above to the Acinetobacter Baumani strain at an MOI of 0.001 and incubate at room temperature. , 100 µl sample was collected at 1 ml each in 1, 2, 3, 4, and 5 minutes, diluted in LB medium, and then the adsorption capacity of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 10.

In addition, the antibiotic-resistant Acinetobacter Baumani strain was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium, The bacteriophage YMC15/02/T28_ABA_BP purified in 2. above was added to an MOI of 0.001 (titer 10 ⁸ pfu/cells) and incubated at 37° C. for 5 minutes. A pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 11.

As shown in Figure 10, within 5 minutes after inoculation of the bacteriophage YMC15/02/T28_ABA_BP, about 99% of the bacteriophage was adsorbed to the Acinetobacter Baumani strain (10 minutes: 0.24%).

In addition, as shown in FIG. 11, the result of the single-stage proliferation curve showed a high burst size of 424 PFU/infected cells (10 min: 0.24 PFU/ml).

Through the above results, the bacteriophage YMC15/02/T28_ABA_BP according to the present invention can be adsorbed in a relatively fast time to the Acinetobacter Baumani strain having antibiotic resistance, and exhibits a high burst size of 424 PFU/infected cells. It can be seen that the dissolution effect of

5. Verification of the lytic ability of bacteriophage against in vitro antibiotic-resistant Acinetobacter bacteria

Bacteriophage YMC15/02/T28_ABA_BP prepared in antibiotic-resistant Acinetobacter Baumani strain 1 X 10 ⁹ CFU/ml 1 X 10 ⁸ CFU/ml (MOI: 0.1), 1 X 10 ⁹ PFU/ml (MOI: 1), Each treatment was performed in an amount of 1 X 10 ¹⁰ PFU/ml (MOI: 10), and the OD value (wavelength 600 nm) was measured for each time. However, PBS + SM buffer was treated as a negative control, and the values are shown in FIG. 12.

As shown in FIG. 12, when the bacteriophage YMC15/02/T28_ABA_BP was treated for the Acinetobacter Baumani strain, the OD value decreased, and the OD value further decreased as the MOI value increased. The ability was high.

Through the above results, it can be seen that the bacteriophage YMC15/02/T28_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain.

6. Verification of the lytic ability of bacteriophage against bacteria in antibiotic-resistant Acinetobacter genus in vivo

After preparing 200 larvae (Galleria mellonella larvae) aged 3 to 4 years old, 10 animals were classified for each group. After injecting the Acinetobacter Baumani strain, which is resistant to colistin to each larva, through the reprint of the larva at a minimum lethal concentration (MLD), the bacteriophage YMC15/02/T28_ABA_BP purified in 2. Alternatively, after mixed inoculation with MOI 100, the survival rate of the larva was checked every 12 or 24 hours until 72 hours, and the results are shown in FIG. 13.

As shown in Figure 13, when the bacteriophage YMC15/02/T28_ABA_BP according to the present invention was treated to the larvae injected with the Acinetobacter Baumani strain having resistance to colistin, the survival rate of the larva increased, and the MOI value increased It was confirmed that the survival rate of larvae was further increased. In addition, even when only the bacteriophage YMC15/02/T28_ABA_BP was injected without injecting the Acinetobacter Baumani strain resistant to colistin, it was confirmed that there was no toxicity when comparing the survival rate with a healthy control group.

Through the above results, the bacteriophage YMC15/02/T28_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.

7. Stability evaluation of bacteriophage against antibiotic-resistant Acinetobacter Baumani strain

It was confirmed that the bacteriophage YMC15/02/T28_ABA_BP according to the present invention was not destroyed at temperature and alkali and maintained stability.

1 µl of the bacteriophage YMC15/02/T28_ABA_BP purified by the method of 2 above was added to 40 µl of SM buffer adjusted to a pH of 4, 5, 6, 7, 8, 9 and 10, and then incubated for 1 hour at 37°C. After that, plaque analysis was performed with the antibiotic-resistant Klebsiella pneumoniae by the method of 4 above, and the results are shown in FIG. 14.

In addition, the bacteriophage YMC15/02/T28_ABA_BP solution was cultured at 4° C., 37° C., 50° C., 60° C., and 70° C. for 1 hour, and each sample was sampled with the Acinetobacter Bowmani strain 4 Plaque analysis was performed by the method of.

As shown in FIG. 14, the bacteriophage YMC15/02/T28_ABA_BP according to the present invention exhibited the most stability at neutrality corresponding to pH 7.5, and the bacteriophage YMC15/02/T28_ABA_BP was relatively stable in neutral/alkaline for 30 days. Indicated.

In addition, as shown in Fig. 15, the bacteriophage YMC15/02/T28_ABA_BP showed very high stability up to a high temperature of 50°C.

8. Whole genome sequence analysis of bacteriophage against antibiotic-resistant Klebsiella sp.

In order to identify the characteristics of the bacteriophage YMC15/02/T28_ABA_BP according to the present invention, the entire gene sequence analysis was performed through the Illumina sequencer (Roche), based on the method of analyzing the entire genome sequence, which is obvious to the skilled person, and the results are shown. 16 and Tables 17 to 22.

As shown in Fig. 16 and Tables 17 to 22, the bacteriophage YMC15/02/T28_ABA_BP contains a linear dsDNA (linear dsDNA) and was composed of 77 ORFs.

As a result of comparing the sequence of the bacteriophage YMC15/02/T28_ABA_BP according to the present invention with the sequence of the existing bacteriophage, the bacteriophage having similarity to the bacteriophage according to the present invention was not detected. Through the above results, it can be seen that the bacteriophage YMC15/02/T28_ABA_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.

[Example 3] Bacteriophage YMC15/09/R1869_ABA_BP

1. Isolation of clinical specimens and selection of antibiotic resistant strains

As shown in Table 23 below, Acinetobacter baumannii strain was isolated and cultured from ICU (intensive care unit) blood and clinical specimens of university hospitals. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Thereafter, the antibiotic susceptibility test was performed using a CLSI disc diffusion test method in which Mueller-Hinton agar was used for overnight culture at 37° C. outside air, and the test antibiotics were amicacin, ampicillin-sulfur. Bectam (ampicillin-s μlbactam), ceftazidime, ciprofloxacin, colistin, cefepime, cefotaxime, gentamicine, imipenem, Levofloxacin, meropenem, minocycline, piperacillin, piperacillin-tazobactam, cotrimoxa and tigecycline were used. . The sensitivity results were read based on the Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profiles of the bacteria of the genus Acinetobacter are shown in Tables 24 to 28 below. However, in Tables 24 to 28 below, S, I, and R are the results of evaluating the sensitivity to antibacterial agents, where'S' is sensitive,'I' is intermediate, and'R' is resistant. Means.

Host strain origin Host strain Lysis YMC16/12/R12914 Phlegm (pneumonia) YMC16/01/R198 Tracheal inhalation (pneumonia YMC16/12/B11422 Catheter blood YMC16/01/R353 Phlegm (pneumonia) YMC16/12/B11449 blood YMC16/01/R405 Phlegm (pneumonia) YMC16/12/B10832 blood YMC16/01/R397 Phlegm (pneumonia) YMC16/12/B13325 Catheter blood YMC16/01/R380 Phlegm (pneumonia) YMC17/01/P518 Swab or drainage tube YMC16/12/R4637 Phlegm (pneumonia) YMC17/01/B8053 Catheter blood YMC17/01/R2812 Phlegm (pneumonia) YMC17/01/B10087 Catheter blood YMC17/02/R541 Phlegm (pneumonia) YMC17/01/B12075 Catheter blood YMC17/02/R2392 Phlegm (pneumonia) YMC17/02/B14 blood YMC17/03/R348 Phlegm (pneumonia) YMC17/01/B13454 blood YMC17/03/R5305 YMC17/02/B87 blood YMC17/03/R3095 YMC17/02/B721 blood YMC17/03/R3428 YMC17/02/B4520 Catheter blood YMC17/03/R4607 Phlegm (pneumonia) YMC17/02/B4039 blood YMC17/03/P971 Swab or drainage tube YMC17/02/B4864 blood YMC16/03/R4461 Tracheal inhalation (pneumonia YMC17/02/P523 Gluteal pressure sores YMC16/05/R2210 Phlegm (pneumonia) YMC17/02/B8414 Peritoneal blood disease YMC16/07/R2512 Bronchial lavage YMC17/03/R585 Phlegm (pneumonia) YMC16/09/R2471 Tracheal inhalation (pneumonia YMC17/03/B4730 Catheter blood YMC16/10/R2537 Phlegm (pneumonia) YMC17/03/B5000 Catheter blood YMC16/12/P503 Swab or drain tube YMC17/03/R1888 Phlegm (pneumonia) YMC15/02/T28 Other Catheter Tip YMC17/03/R3279 Phlegm (pneumonia) YMC15/02/R436 Tracheal inhalation (pneumonia YMC17/03/R4077 Tracheal inhalation (pneumonia YMC15/03/R1604 Tracheal inhalation (pneumonia YMC17/04/R488 Phlegm (pneumonia) YMC15/09/R1869 Phlegm (pneumonia) YMC17/04/R640 Phlegm (pneumonia) YMC14/06/R2359 Phlegm (pneumonia) YMC/17/05/R1095 Tracheal inhalation (pneumonia YMC14/08/T90 Catheter tip YMC16/01/P11 Swab or drain tube YMC14/08/R1169 Phlegm (pneumonia) YMC16/01/R123 Tracheal tube tips

As shown in Tables 24 to 28, the collected Acinetobacter baumannii 57 strains were found to be multi-resistance strains having resistance to various antibiotics.

2. Collection of bacteriophage samples

2-1. Collecting samples to build a phage bank

Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours. The filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.

2-2. Selection of lytic phage and measurement of lysis titer

Separation and purification of lytic phage was performed by a spot test method (Mazzocco A et al. In Bacteriophages, Clokie and Kropinski AM, eds. Humana Press. 2009). The obtained strain was inoculated on Macconky agar medium and then incubated overnight at 35° C. outside air. After cultivation, strains susceptible to phage were selected to see the formation of transparent plaques. The susceptible strain was inoculated on Macconky agar medium and cultured at 35° C. for 12 hours. A suspension of each strain was prepared in a 1 ml saline tube with a turbidity of 0.5 McFarland, H top agar (3 ml), 100 μl of sensitive bacteria and a phage solution (1 μl, 10 μl and 50 μl, respectively) were mixed and applied to LB agar, Incubated for 12 hours at 35 ℃. After observing the plaques, the plaques were collected with a Pasteur pipette, diluted in SM buffer solution, and purified again three times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC15/09/R1869_ABA_BP was diluted in SM buffer solution and purified again 3 times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC15/09/R1869_ABA_BP was diluted and stored in SM buffer solution.

After inoculating and culturing each of 57 strains of antibiotic-resistant Acinetobacter baumannii identified in 1. above on McConkie agar medium, each resistance plated with bacteriophage YMC15/09/R1869_ABA_BP purified by the above process Plaque formation was confirmed by inoculating the strain with 5 µl, and the titer range was checked, and lytic properties are shown in Table 29 below. However, in Table 29 below, + and-are evaluated for plaque activity against the collected strains,'+' means a clear plaque, and'-' means that no lysis has occurred.

Host strain Lysis Host strain Lysis YMC17/01/B10087 + YMC17/03/R348 + YMC17/02/B4520 + YMC17/03/R3095 + YMC17/03/R585 + YMC17/03/R3428 ++ YMC17/03/R3279 + YMC17/03/P971 + YMC/17/05/R1095 + YMC16/03/R4461 ++ YMC16/01/P11 + YMC16/05/R2210 ++ YMC16/01/R198 + YMC16/07/R2512 + YMC16/01/R353 + YMC16/09/R2471 ++ YMC16/01/R405 + YMC15/03/R1604 ++ YMC16/01/R397 + YMC15/09/R1869 + YMC16/12/P503 + YMC14/06/R2359 + YMC15/02/T28 + YMC14/08/T90 + YMC14/08/R1169 +

As shown in Table 29, it was confirmed that the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention lyses a significant number of antibiotic-resistant Acinetobacter Baumani strains.

3. Electron microscopic analysis of lytic bacteriophage against antibiotic-resistant Acinetobacter Baumani strains

The bacteriophage YMC15/09/R1869_ABA_BP purified by the method of 2 above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added to the supernatant. After addition in an amount of 10% (w/v), it was stored refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage YMC15/09/R1869_ABA_BP was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. Done.

As shown in FIG. 17, when viewed as a criterion for classifying the YMC15/09/R1869_ABA_BP bacteriophage according to the present invention by shape, it was classified as belonging to the family Myobiridae having a long tail with a hexagonal head.

4. Analysis of adsorption capacity and one-step growth curve of bakperiophage

After incubating the antibiotic-resistant Acinetobacter Baumani strain to have an OD value of 0.5, add the bacteriophage YMC15/09/R1869_ABA_BP purified in 2. above to the Acinetobacter Baumani strain at an MOI of 0.001 and incubate at room temperature. , 100 µl sample was collected at 1 ml each in 1, 2, 3, 4, 5 minutes, diluted in LB medium, and then the adsorption capacity of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 18.

In addition, the antibiotic-resistant Acinetobacter Baumani strain was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium, The bacteriophage YMC15/09/R1869_ABA_BP purified in 2. above was added to an MOI of 0.001 (titer 10 ⁸ pfu/cells) and incubated at 37° C. for 5 minutes. A pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 19.

As shown in Figure 18, within 5 minutes after inoculation of the bacteriophage YMC15/09/R1869_ABA_BP, about 99% of the bacteriophage was adsorbed to the Acinetobacter Baumani strain (5 minutes: 2.9%).

In addition, as shown in FIG. 19, a single-stage proliferation curve resulted in a high burst size of 78 PFU/infected cells (0 min: 14 PFU/ml, 50 min: 1096 PFU/ml).

Through the above results, the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention can be adsorbed in a relatively short time to the Acinetobacter Baumani strain having antibiotic resistance, and exhibited a high burst size of 78 PFU/infected cells, resulting in an antibiotic resistant strain. It can be seen that the dissolution effect of

5. Verification of the lytic ability of bacteriophage against bacteria in antibiotic-resistant Acinetobacter genus in vivo

After preparing 200 larvae of Galleria mellonella larvae aged 3 to 4 years old, 10 animals were classified for each group. After injecting the Acinetobacter Baumani strain, which is resistant to colistin to each larva, through the reprint of the larva at a minimum lethal concentration (MLD), the bacteriophage YMC15/09/R1869_ABA_BP purified in 2. Alternatively, after mixed inoculation with MOI 100, the survival rate of larvae was checked every 12 or 24 hours until 72 hours, and the results are shown in FIG. 20.

As shown in FIG. 20, when the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention was treated to the larva injected with the Acinetobacter Baumani strain having resistance to colistin, the survival rate of the larva increased, and the MOI value increased. It was confirmed that the survival rate of larvae was further increased. In addition, even when only the bacteriophage YMC15/09/R1869_ABA_BP was injected without injecting the Acinetobacter Baumani strain having resistance to colistin, it was confirmed that there was no toxicity when comparing the survival rate with a healthy control group.

Through the above results, the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.

6. Verification of the lytic ability of bacteriophage against bacteria in antibiotic-resistant Acinetobacter genus in vivo

After intranasal administration of the Acinetobacter Baumani strain (30 μL) resistant to colistin to mice for 2 hours, the bacteriophage YMC15/09/R1869_ABA_BP (30 μL) or colistin antibiotic (30 μL) purified in 2. ) Was administered intranasally in the same way. After the 1st and 5th days, 4 animals were autopsied for each experimental group, and lung tissue was removed. By culturing the bacteria with a sample obtained by grinding a part of the mouse lung, the change in the actual number of bacteria was measured to evaluate the synergistic effect with bacteriophage or antibiotics.

As shown in FIG. 21, when the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention is administered to a mouse injected with a colistin-resistant Acinetobacter Baumani strain, the Acinetobacter Baumani in the lungs of the mouse It could be seen that the number of bacteria was significantly decreased, and as the MOI value increased, the number of bacteria of the Acinetobacter Baumani was further decreased. In addition, when the bacteriophage YMC15/09/R1869_ABA_BP and colistin were administered together, it was confirmed that the number of bacteria in the lungs of the mouse Acinetobacter Baumani was further reduced compared to the case where only the bacteriophage YMC15/09/R1869_ABA_BP was administered alone.

Through the above results, the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.

7. Stability evaluation of bacteriophage against antibiotic-resistant Acinetobacter Baumani strain

Bacteriophage according to the present invention It was confirmed that the bacteriophage YMC15/09/R1869_ABA_BP maintains stability without being destroyed in temperature and alkali.

1 µl of the bacteriophage YMC15/02/T28_ABA_BP purified by the method of 2 above was added to 40 µl of SM buffer adjusted to a pH of 4, 5, 6, 7, 8, 9 and 10, and then incubated for 1 hour at 37°C. After that, plaque analysis was performed with the antibiotic-resistant Klebsiella pneumoniae by the method of 4 above, and the results are shown in FIG. 22.

In addition, the bacteriophage YMC15/09/R1869_ABA_BP solution was cultured at 4° C., 37° C., 50° C., 60° C., and 70° C. for 1 hour, and each sample was sampled with the Acinetobacter Bowmani strain 4 Plaque analysis was performed by the method of. And the results are shown in FIG. 23.

As shown in Fig. 22, the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention exhibited stability in both acidic, neutral and alkaline, but for 30 days, the bacteriophage YMC15/09/R1869_ABA_BP showed relatively stability in neutral/alkaline properties. Done.

In addition, as shown in Figure 23, the bacteriophage YMC15/09/R1869_ABA_BP showed very high stability up to a high temperature of 70°C.

8. Whole genome sequence analysis of bacteriophage against antibiotic-resistant Klebsiella sp.

In order to identify the characteristics of the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention, the entire gene sequence analysis was performed through the Illumina sequencer (Roche) based on the method of analyzing the whole genome sequence, which is obvious to the skilled person, and the results are shown. 24 and Tables 30 to 35.

As shown in Fig. 24 and Tables 30 to 35, the bacteriophage YMC15/09/R1869_ABA_BP contains a linear dsDNA (linear dsDNA) and was composed of 77 ORFs.

As a result of comparing the sequence of the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention with the sequence of the existing bacteriophage, the bacteriophage having similarity to the bacteriophage according to the present invention was not detected. Through the above results, it can be seen that the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.

[Example 4] Bacteriophage YMC17/01/P6_KPN_BP

1. Isolation of clinical specimens and selection of antibiotic resistant strains

As shown in Table 36 below, Klebsiella pneumoniae bacteria were cultured and isolated from Severance Hospital patients or their feces. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Then, for the antibiotic susceptibility test, a CLSI disk diffusion test method was used in which Mueller-Hinton agar was used and cultured overnight at 37° C. outside air. Test antibiotics for Klebsiella pneumoniae bacteria are Amikacin, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Ceftazidime, Cefazoline, Imipenem, Ertapenem, Cefepime, Cefoxitin, Cefotaxime, Gentamicine, Levofloxacin, Meropenem (Meropenem), piperacillin/tazobactam, Cortrimoxa, and Tigecyline were used. The sensitivity results were read based on the Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profiles of the collected Klebsiella pneumoniae 47 strains are shown in Tables 37 to 40 below. However, in Tables 37 to 40 below, S, I and R are the results of evaluating the sensitivity to antimicrobial agents, where'S' is sensitive (Susceptible),'I' is intermediate (Intermediate), and'R' is resistant (Resistant). Means.

Host strain Sample origin Host strain Sample origin YMC16/12/N708 Feces YMC17/05/N331 Feces YMC16/12/N681 Feces YMC17/05/N355 Feces YMC17/01/N115 Feces YMC17/05/R3201 Phlegm (pneumonia) YMC17/01/P6 Swab or drainage tube pelvis YMC17/05/N405 Feces YMC17/01/N167 Feces YMC17/05/N500 Feces YMC17/01/N132 Feces YMC17/05/N424 Feces YMC17/01/N270 Feces YMC17/05/N421 Feces YMC17/01/N189 Feces YMC17/06/U687 Random urine YMC17/02/N103 Feces YMC17/06/N182 Feces YMC17/02/N97 Feces YMC17/06/N196 Feces YMC17/02/N84 Feces YMC17/06/N263 Feces YMC17/02/N151 Feces YMC17/06/N297 Feces YMC17/02/N183 Feces YMC17/06/R4267 Phlegm (pneumonia) YMC17/02/N180 Feces YMC17/06/N445 Feces YMC17/02/N189 Feces YMC17/07/N293 Feces YMC17/02/N232 Feces YMC17/07/N393 Feces YMC17/02/N227 Feces YMC17/07/R3882 Phlegm (pneumonia) YMC17/02/N245 Feces YMC17/08/N34 Feces YMC17/02/N254 Feces YMC17/07/U6299 Random urine YMC17/02/R2881 Tracheal inhalation (pneumonia) YMC17/08/N153 Feces YMC17/02/N312 Feces YMC17/08/N243 Feces YMC17/05/N213 Feces YMC17/08/N456 Feces YMC17/05/R1069 Phlegm (pneumonia) YMC17/10/N291 Feces YMC17/05/N300 Feces

As shown in Tables 37 to 40, the collected Klebsiella pneumoniae 47 bacteria were found to be multi-resistance strains resistant to various carbapenem antibiotics.

2. Collection of bacteriophage samples

2-1. Collecting samples to build a phage bank

Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours. The filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.

2-2 Selecting lytic phage and measuring lysis titer

Separation and purification of lytic phage was performed by a spot test method (Mazzocco A et al. In Bacteriophages, Clokie and Kropinski AM, eds. Humana Press. 2009). The obtained strain was inoculated on Macconky agar medium and then incubated overnight at 35° C. outside air. After cultivation, strains susceptible to phage were selected to see the formation of transparent plaques. The susceptible strain was inoculated on Macconky agar medium and cultured at 35° C. for 12 hours. Prepare a suspension of each strain in a 1 ml saline tube with a turbidity of 0.5 McFarland, H top agar (3 ml), 100 µl of sensitive bacteria, and a phage solution (1 µl, 10 µl, and 50 µl, respectively) were mixed and applied to LB agar. Incubated at °C for 12 hours. After observing the plaque, the plaque was collected with a Pasteur pipette, diluted in SM buffer solution, and purified again three times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC17/01/P6_KPN_BP was diluted in SM buffer solution and purified again three times using a susceptible strain suspension. The pure bacteriophage thus obtained YMC17/01/P6_KPN_BP was diluted and stored in SM buffer solution.

Each of the 47 antibiotic-resistant Klebsiella pneumoniae strains identified in the above 1. was inoculated and cultured on McConkie agar medium, and then the bacteriophage YMC17/01/P6_KPN_BP purified by the above process was smeared. Plaque formation was confirmed by inoculating the resistant strain with 5 μl, and the titer range was confirmed, and the lytic properties are shown in Table 41 below. However, in Table 41 below, + and-are the evaluation of plaque activity for the collected strains,'+' means clear plaque, and'-' means that lysis has not occurred.

Host strain Lysis Host strain Lysis YMC16/12/N708 ++ YMC17/05/N331 + YMC16/12/N681 ++ YMC17/05/N355 + YMC17/01/N115 + YMC17/05/R3201 + YMC17/01/P6 + YMC17/05/N405 + YMC17/01/N167 + YMC17/05/N500 + YMC17/01/N132 + YMC17/05/N424 - YMC17/01/N270 + YMC17/05/N421 - YMC17/01/N189 + YMC17/06/U687 - YMC17/02/N103 ++ YMC17/06/N182 - YMC17/02/N97 ++ YMC17/06/N196 - YMC17/02/N84 ++ YMC17/06/N263 - YMC17/02/N151 ++ YMC17/06/N297 - YMC17/02/N183 ++ YMC17/06/R4267 - YMC17/02/N180 ++ YMC17/06/N445 - YMC17/02/N189 ++ YMC17/07/N293 + YMC17/02/N232 ++ YMC17/07/N393 + YMC17/02/N227 ++ YMC17/07/R3882 + YMC17/02/N245 ++ YMC17/08/N34 - YMC17/02/N254 ++ YMC17/07/U6299 + YMC17/02/R2881 ++ YMC17/08/N153 - YMC17/02/N312 ++ YMC17/08/N243 - YMC17/05/N213 + YMC17/08/N456 + YMC17/05/R1069 + YMC17/10/N291 + YMC17/05/N300 +

As shown in Table 41, it was confirmed that the bacteriophage YMC17/01/P6_KPN_BP according to the present invention lyses 35 strains (74%) of 47 strains of antibiotic-resistant Klebsiella pneumoniae.

3. Electron microscopic analysis of lytic bacteriophage against antibiotic-resistant Klebsiella pneumoniae

The bacteriophage purified by the method of 2. above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added 10% (w/) to the supernatant. After addition in the amount of v), it was kept refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage shape was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. 25.

As shown in Figure 25, when viewed as a criterion for classifying the YMC17/01/P6_KPN_BP bacteriophage according to the present invention by shape, it was classified as belonging to the family Siphoviridae having an angled head and tail.

4. Analysis of adsorption capacity and one-step growth curve of bakperiophage

After incubating the antibiotic-resistant Klebsiella pneumoniae to an OD value of 0.5, add the bacteriophage YMC17/01/P6_KPN_BP purified in 2. above to Klebsiella pneumoniae at an MOI of 0.001 and incubate at room temperature. , 100 µl sample was collected at 1 ml each in 1, 2, 3, 4, 5 minutes, diluted in LB medium, and then the adsorption capacity of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 26.

In addition, antibiotic-resistant Klebsiella pneumoniae was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium, The bacteriophage purified in Example 2 was added with an MOI of 0.001 (titer 10 ⁸ pfu/cells) and incubated at 37° C. for 5 minutes. A pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 27.

As shown in FIG. 26, about 99% of the bacteriophage was adsorbed to Klebsiella pneumoniae within 5 minutes after inoculation of the bacteriophage YMC17/01/P6_KPN_BP (10 minutes: 1.05%).

In addition, as shown in FIG. 27, a single-stage proliferation curve resulted in a high burst size of 43 PFU/infected cells (0 min: 20 PFU/ml, 95 min: 872 PFU/ml).

Through the above results, the bacteriophage YMC17/01/P6_KPN_BP according to the present invention can be adsorbed to Klebsiella pneumoniae having antibiotic resistance within a relatively short time, and exhibited a high burst size of 43 PFU/infected cells, resulting in antibiotic resistance Kleb It can be seen that it exerts a lytic effect on Siella pneumoniae.

5. Verification of the lytic ability of bacteriophage against in vitro antibiotic-resistant Klebsiella genus bacteria

Bacteriophage YMC17/01/P6_KPN_BP prepared in antibiotic-resistant Klebsiella pneumoniae 1 X 10 ⁹ CFU/ml 1 X 10 ⁸ CFU/ml (MOI: 0.1), 1 X 10 ⁹ PFU/ml (MOI: 1), Each treatment was performed in an amount of 1 X 10 ¹⁰ PFU/ml (MOI: 10), and the OD value (wavelength 600 nm) was measured for each time. However, PBS + SM buffer was treated as a negative control, and the values are shown in FIG. 28.

As shown in FIG. 28, when compared with the negative control group, when the bacteriophage was treated with Klebsiella pneumoniae, the OD value decreased, and the OD value further decreased as the MOI value increased. The dissolution ability was high.

Through the above results, it can be seen that the bacteriophage according to the present invention has lytic properties against antibiotic-resistant Klebsiella pneumoniae.

6. Stability evaluation of bacteriophage against antibiotic-resistant Klebsiella sp.

It was confirmed that the bacteriophage YMC17/01/P6_KPN_BP according to the present invention maintains stability without being destroyed in temperature and alkali.

1 µl of the bacteriophage purified by the method of 2 above was added to 40 µl of SM buffer adjusted to a pH of 4, 5, 6, 7, 8, 9 and 10, and then incubated at 37°C for 1 hour, and then antibiotic resistance Plaque analysis was performed by the method of 4. above with Klebsiella pneumoniae, and the results are shown in FIG. 29.

In addition, the bacteriophage solution was cultured at 4° C., 37° C., 60° C., and 70° C. for 1 hour, and each sample was analyzed with Klebsiella pneumoniae by the method of 4. The results are shown in FIG. 30.

As shown in FIG. 29, the bacteriophage YMC17/01/P6_KPN_BP according to the present invention exhibited stability in both acidic, neutral and alkaline corresponding to pH 7, and for 30 days, the bacteriophage was particularly stable in neutral/alkaline. Indicated.

In addition, as shown in Figure 30, the bacteriophage YMC17/01/P6_KPN_BP showed very high stability up to a high temperature of 60 ℃.

7. Whole genome sequence analysis of bacteriophage against antibiotic-resistant Klebsiella sp.

In order to identify the characteristics of the bacteriophage YMC17/01/P6_KPN_BP according to the present invention, the entire gene sequence analysis was performed through the Illumina sequencer (Roche) based on the method of analyzing the entire genome sequence, which is obvious to the skilled person, and the results are shown. 31 and Tables 42 to 46.

31 and Tables 42 to 46, the bacteriophage YMC17/01/P6_KPN_BP contains a linear dsDNA (linear dsDNA) and was composed of 87 ORFs.

As a result of comparing the sequence of the bacteriophage YMC17/01/P6_KPN_BP according to the present invention with the sequence of the existing bacteriophage, the bacteriophage having similarity to the bacteriophage according to the present invention was not detected. Through the above results, it can be seen that the bacteriophage YMC17/01/P6_KPN_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention described in the claims. It will be self-evident to those who have the knowledge of.

[Accession number (1)]

Bacteriophage YMC14/01/P262_ABA_BP

Depositary institution name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11798P

Consignment Date: 20181115

[Accession number (2)]

Bacteriophage YMC15/02/T28_ABA_BP

Depositary institution name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11799P

Consignment Date: 20181115

[Accession number (3)]

Bacteriophage YMC15/09/R1869_ABA_BP

Depositary institution name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11802P

Consignment Date: 20181115

[Accession number (4)]

Bacteriophage YMC17/01/P6_KPN_BP

Depositary institution name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11804P

Consignment Date: 20181115

The present invention relates to a novel bacteriophage that lyses bacteria, particularly bacteria that are resistant to antibiotics.

Claims (41)

Acinetobacter (Acinetobacter) or Klebsiella (Klebsiella) genus bacteriophage having a specific killing ability of bacteria. The method of claim 1, The bacteria of the genus Acinetobacter are Acinetobacter baumannii , Acinetobacter calcoaceticus , Acinetobacter haemolyticus , Acinetobacter junii , Acinetobacter johnsonii , Acinetobacter lwoffii , Acinetobacter radioresistens , Acinetobacter ursingii , Acinetobacter lwoffii , Acinetobacter schindleri , Acinetobacter parvus , Acinetobacter baylyi , Acinetobacter bouvetii , Acinetobacter towneri , Acinetobacter tandoii , Acinetobacter tandoii , Acinetobacter so Monty (Acinetobacter grimontii), Acinetobacter shale reunbeo Jia (Acinetobacter tjernbergiae) and Acinetobacter germanium Nourishing (Acinetobacter gerneri) 1 jong or more, a bacteriophage selected from the group consisting of. The method of claim 1, The bacteria of the genus Acinetobacter are Acinetobacter baumannii, a bacteriophage. The method of claim 1, The bacteria of the Acinetobacter genus are antibiotic-resistant bacteria, bacteriophage. The method of claim 4, The antibiotics include Colistin, Erythromycin, Ampicillin, Vancomycin, Linezolid, Methicillin, Oxacillin, and Cefotaxime, Rifampicin, Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Aztreonam, Piperacillin, Polymyxin B, Ciprofloxacin, Levofloxacin, Moxifloxacin, Moxifloxacin, Gatifloxacin, Ticyclxacin, Tigecyclacin ), any one or more selected from the group consisting of combinations thereof and derivatives thereof, bacteriophage. The method of claim 1, The bacteria of the genus Klebsiella are Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca. , Klebsiella planticola (Klebsiella planticola) and Klebsiella terrigena (Klebsiella terrigena) that is one or more selected from the group consisting of, bacteriophage. The method of claim 1, The bacteria of the genus Klebsiella are Klebsiella pneumoniae, a bacteriophage. The method of claim 1, The bacteria of the genus Klebsiella are antibiotic-resistant bacteria, bacteriophage. The method of claim 8, The antibiotic is carbapenem (Carbapenem) antibiotic, bacteriophage. The method of claim 1, The name of the bacteriophage is YMC14/01/P262_ABA_BP, and the deposit number is KFCC11798P, bacteriophage. The method of claim 1, The bacteriophage consists of a nucleotide sequence represented by SEQ ID NO: 1, bacteriophage. The method of claim 1, The bacteriophage comprises any one of the protein of SEQ ID NO: 2 to 4, bacteriophage. The method of claim 1, The bacteriophage will comprise a genome represented by any one of SEQ ID NOs: 5 to 7, bacteriophage. The method of claim 1, The name of the bacteriophage is YMC15/02/T28_ABA_BP, and the deposit number is KFCC11799P, bacteriophage. The method of claim 1, The bacteriophage consists of a nucleotide sequence represented by SEQ ID NO: 8, bacteriophage. The method of claim 1, The bacteriophage comprises any one protein of SEQ ID NO: 9 to 11, bacteriophage. The method of claim 1, The bacteriophage will contain a genome represented by any one of SEQ ID NOs: 12 to 14, bacteriophage. The method of claim 1, The name of the bacteriophage is YMC15/09/R1869_ABA_BP, and the accession number is KFCC11802P, bacteriophage. The method of claim 1, The bacteriophage consists of a nucleotide sequence represented by SEQ ID NO: 15, bacteriophage. The method of claim 1, The bacteriophage is a bacteriophage comprising any one of the protein of SEQ ID NO: 16 and 17. The method of claim 1, The bacteriophage will contain the genome represented by any one of SEQ ID NO: 18 and 19, bacteriophage. The method of claim 1, The name of the bacteriophage is YMC17/01/P6_KPN_BP, and the deposit number is KFCC11804P bacteriophage. The method of claim 1, The bacteriophage will consist of a nucleotide sequence represented by SEQ ID NO: 20, bacteriophage. The method of claim 1, The bacteriophage is a bacteriophage comprising any one of the protein of SEQ ID NO: 21 and 22. The method of claim 1, The bacteriophage will comprise the genome represented by any one of SEQ ID NO: 23 and 24, bacteriophage. A composition for antibiotics comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient. A composition for feed addition comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient. A drinking water additive comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient. A disinfectant comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient. A cleaning agent comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient. A pharmaceutical composition for the prevention or treatment of diseases caused by bacteria of the genus Acinetobacter or Klebsiella, comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient. The method of claim 31, Diseases caused by the bacteria of the genus Acinetobacter include hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondilom, vancomycin-resistant yellow staphylococcus aureus infection, vancomycin-resistant yellow staphylococcus aureus infection, methicillin resistance. Staphylococcus aureus infection, multi-drug-resistant Pseudomonas aeruginosa infection, multi-drug-resistant Acinetobacter baumani infection, carbapenem intragrowth mycobacterium infection, intestinal infection, acute respiratory tract infection, and enterovirus infection. Composition. The method of claim 31, Diseases caused by the Klebsiella genus include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media, bacteremia, endocarditis , A disease selected from the group consisting of cholecystitis and mumps, a pharmaceutical composition. The use of any one of the bacteriophages selected from claims 1 to 25 for administering an antibiotic composition to a subject in need of treatment or prevention. The use of any one of the bacteriophage selected from claim 1 to claim 25 for use in the preparation of a composition for feed addition. Use of any one of the bacteriophages selected from claims 1 to 25 for use in the preparation of drinking water additives. Use of any one of the bacteriophages selected from claims 1 to 25 for use in the manufacture of disinfectants. The use of any one of the bacteriophages selected from claims 1 to 25 for use in the manufacture of cleaning agents. Acinetobacter genus or Klebsiella genus bacteria comprising administering any one of the bacteriophages selected from claims 1 to 25 as an active ingredient to a subject in need of prophylaxis or treatment Prevention or treatment of diseases caused by. The method of claim 39, Diseases caused by the bacteria of the genus Acinetobacter include hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondilom, vancomycin-resistant yellow staphylococcus aureus infection, vancomycin-resistant yellow staphylococcus aureus infection, methicillin resistance. Staphylococcus aureus infection, multi-drug-resistant Pseudomonas aeruginosa infection, multi-drug-resistant Acinetobacter Baumani infection, carbapenem intragrowth mycobacterium infection, intestinal infection, acute respiratory tract infection, and enterovirus infection, which is a disease selected from the group consisting of, prevention or Method of treatment. The method of claim 39, Diseases caused by the Klebsiella genus include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media, bacteremia, endocarditis , Cholecystitis and mumps, which is a disease selected from the group consisting of, prevention or treatment method.

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