WO2020130652A2 - 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

A new bacteriophage that dissolves bacteria

The present invention relates to a novel bacteriophage that lyses bacteria, especially 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 used to combat bacteria from external invasion into the living body. However, recently, strains resistant to these antibiotics have appeared, which is 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. (Stuart B. Levy, ScientificAmerican, (1988): 46-53).

Tolerance of antibiotics is a phenomenon distinct from resistance to antibiotics. It was first discovered in Pneumococcus sp. in the 1970s and provided important clues to the mechanism of action of penicillin (Tomasz et al., Nature, 227, (1970): 138-140). Conventional chemical antibiotics, such as penicillin and cephalosporin, exhibit antibiotic activity by inhibiting the cell wall of microorganisms or the synthesis of proteins. However, resistant species stop growing in the presence of antibiotics at normal concentrations, but do not die as a result. Resistance occurs because the activity of bacterial autolytic enzymes, such as autolysin, does not occur when antibiotics inhibit cell wall synthase, and this is because penicillin activates endogenous hydrolytic enzymes. By killing 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 that exhibit different antibiotic mechanisms than 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).

Meanwhile, Acinetobacter baumannii is a Gram-negative aerobic bacillus, which is an important cause of infection in hospitals in many hospitals, especially aminoglycosides, cephalosporins, fluoroquinolones, and beta lactamases. Infection with beta-lactamase inhibitors and multidrug-resistant acinetobacter baumani (MRAB), which is resistant to carbapenem, is increasing.

In 2010, at the University of Tokyo Hospital, 46 people were infected and 10 of them died from the infection of Acinetobacter bacteria, which has sparked awareness of MRAB, an antibiotic-resistant drug that has rapidly increased worldwide in the last decade, and accelerated the development of antibiotics. have. The Acinetobacter bacteria themselves are common in water, soil, or human skin, and in healthy people, they do not develop when infected. However, people with reduced immunity may die from pneumonia or sepsis when infected, and began to increase in the United States and Europe since the 1990s, and from 2000 to almost no antibiotics.

In addition, the bacteria of the genus Klebsiella is a Gram-negative bacilli as a genus of intestinal bacteria. It has no flagella and is characterized by producing mucus as it passes through the stenosis. Carbon sources are obtained from citrate, and various carbohydrates generate acids and gases excluding hydrogen sulfide. It is widely present in nature and is detected in human respiratory, intestinal, and urinary tract and is known as a causative agent of acute pneumonia. Recently, various bacteria of the genus Klebsiella have been diagnosed and detected, such as Klebsiella pneumoniae, Klebsiella ozaenae, and Klebsiella rhinoscleromatis.

Klebsiella pneumoniae, which is known to occupy most of the bacteria of the genus Klebsiella in clinical terms, is a Gram negative bacillus, aerobic or aerobic bacteria, and is present in the human intestinal tract, skin, oral cavity, respiratory system, etc. It is said that 10-20% of pneumonia is caused by the bacteria. In addition, it may be isolated from urinary tract infection, and is reported as the main causative agent such as sepsis and intraperitoneal infection, and has a high infection rate as the causative agent of bacteremia occurring in patients in the intensive care unit.

On the other hand, various antibacterial agents that can effectively kill microorganisms are currently used. Betalactam-based antibiotics account for about 50% or more of currently used antibacterial agents. However, many Gram-negative bacteria have been reported to exhibit resistance to the antibiotics by generating beta-lactamase against these beta-lactam-based antibiotics. Among the currently found bacteria of the genus Klebsiella pneumonia, K. pneumoniae carbapene maize (KPC)-producing K. pneumoniae (KPC-Kp), which is an antibiotic-resistant bacterium, increases the mortality rate of infected patients with global spread. Raising. Therefore, in Korea, a new strategy is needed to suppress the spread and increase of K. pneumoniae carbapene maize (KPC)-producing K. pneumoniae (KPC-Kp).

Bacteriophage refers to a bacterial specific virus that inhibits and inhibits the growth of infected bacteria by infecting certain bacteria. The bacteriophage has the ability to kill bacteria by destroying the cell wall of the host bacteria when the progeny bacteriophages come out of the bacteria after proliferation inside the bacterial cells after infection. The method of bacterial infection of bacteriophage is very specific, so the type of bacteriophage capable of infecting certain bacteria is limited to a part. That is, a specific bacteriophage can infect only a specific category of bacteria, thereby killing a specific bacteriophage and not affecting other bacteria. Therefore, the recent use of bacteriophage (bacteriophage) as a countermeasure against bacterial diseases has attracted much attention. In particular, after 2000, due to the increase in antibiotic-resistant bacteria, the limitations of existing antibiotics appeared, and the possibility of development of alternatives to existing antibiotics emerged, and bacteriophage has attracted attention as an anti-bacterial agent.

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

One object of the present invention is to provide a novel bacteriophage having specific infection and killing ability against bacteria of the genus Acinetobacter and Klebsiella that are resistant to antibiotics, especially antibiotics.

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

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

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

In the present invention, the bacteria of the genus Acinetobacter are Acinetobacter baumannii , Acinetobacter calcoaceticus , Acinetobacter haemolyticus , Acinetobacter haemolyticus , Acinetobacter junii ), Acinetobacter johnsonii , Acinetobacter lwoffii , Acinetobacter radioresistens , Acinetobacter urresingens , Acinetobacter ursingii , Acinetobacter ursingii 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 may be any one or more selected from the group, but are not limited thereto.

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

In the present invention, the "antibiotic resistance" means resistance to a specific antibiotic and thus does not hear the effect, and for the purpose of the present invention, the antibiotic is colistin, erythromycin, ampicillin, ampicillin Ampicillin-s μlbactam, Vancomycin, Linezolid, Methicillin, Oxacillin, Cefataxime, Rifampicin, Amikacin (Amikacin), Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Dorippenem, Iripenem Imipenem, Imipenem/Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Aztreonam (Aztreonam), Piperacillin, Polymyxin B, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Piperactillin It may be any one or more selected from the group consisting of piperacillin-tazobactam, minocycline, Tigecycline, Cotrimoxa, combinations thereof and derivatives thereof, but is not limited thereto.

In one embodiment of the present invention, the bacteriophage is a bacteriophage separated by taking a sample from a sewage treatment plant in a hospital, named bacteriophage YMC14/01/P262_ABA_BP, and deposited with KFCC11798P as deposit number KFCC11798P on November 15, 2018. It may be deposited.

It was confirmed that the bacteriophage YMC14/01/P262_ABA_BP of the present invention belongs to the Myoviridae family having a long tail on the hexagonal head, and the total sequencing analysis showed a size of 44,597 bp and the total number of ORFs was 79. Confirmed.

In addition, in the present invention, the bacteriophage YMC14/01/P262_ABA_BP 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 the base sequence represented by SEQ ID NO: 1, and a functional equivalent of the base sequence. As a result of modification and substitution of the base sequence, the functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% with the base sequence represented by SEQ ID NO:1. As having the above sequence homology, it means a sequence exhibiting substantially the same physiological activity as the base 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: 2-4. In the present invention, each of SEQ ID NOs: 2 to 4 is 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. More specifically, SEQ ID NO: 2 is 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 base sequence of the genome encoding ORF38, SEQ ID NO: 6 is the base sequence of the genome encoding ORF50, and SEQ ID NO: 7 may be the base sequence of the genome encoding ORF51.

In another embodiment of the present invention, the bacteriophage is a bacteriophage separated by taking a sample from a sewage treatment plant in a hospital, named bacteriophage YMC15/02/T28_ABA_BP, and deposited with KFCC11799P as deposit number KFCC11799P on November 15, 2018. It may be deposited.

It was confirmed that the bacteriophage YMC15/02/T28_ABA_BP of the present invention belongs to the family Mioviridae having a long tail on the hexagonal head, and the total sequencing analysis confirmed that it had a size of 44,580 bp and the total number of ORFs was 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 the base sequence represented by SEQ ID NO: 8, and a functional equivalent of the base 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. As having the above sequence homology, it means a sequence exhibiting substantially the same physiological activity as the base sequence represented by SEQ ID NO: 8.

In addition, the bacteriophage YMC15/02/T28_ABA_BP provided in 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 the amino acid sequence of the putative tail-fiber/lysosomal protein, and the protein represented by SEQ ID NO: 11 may be the amino acid sequence of the endorisin putative protein. More specifically, 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 base sequence of the genome encoding ORF7, the SEQ ID NO: 13 is the base sequence of the genome encoding ORF8, and SEQ ID NO: 14 may be the base sequence of the genome encoding ORF73.

In another embodiment of the present invention, the bacteriophage is a bacteriophage separated by taking a sample from a sewage treatment plant in a hospital, named bacteriophage YMC15/09/R1869_ABA_BP, and deposited with the Korea Microbial Conservation Center on November 15, 2018, KFCC11802P It may be deposited with.

It was confirmed that the bacteriophage YMC15/09/R1869_ABA_BP of the present invention belongs to the family Mioviridae having a long tail on the hexagonal head, and the total sequencing analysis confirmed that the size of the total ORF was 42,555 bp and the total number of ORFs was 77.

In addition, in the present invention, the bacteriophage YMC15/09/R1869_ABA_BP 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 be composed 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. As having the above sequence homology, it means a sequence exhibiting substantially the same physiological activity as the base sequence represented by SEQ ID NO: 15.

In addition, the bacteriophage YMC15/09/R1869_ABA_BP provided by 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 the amino acid sequence of the protein estimated to be. More specifically, 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 base sequence of a genome encoding ORF7, and SEQ ID NO: 19 may be a base sequence of a genome encoding ORF73.

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

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

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

Bacterial specific lytic activity, acid resistance and basic resistance in the genus Acinetobacter as described above, the bacteriophage of the present invention YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And a bacteriophage YMC15/09/R1869_ABA_BP composition for preventing and treating infectious diseases caused by bacteria in the genus Acinetobacter, and the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And bacteriophage YMC15/09/R1869_ABA_BP in various products, including as an active ingredient, to enable application of various pH ranges.

In another embodiment of the present invention, the bacteria of the genus Klebsiella are Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, It may be any one or more selected from the group consisting of Klebsiella oxytoca, Klebsiella planticola and Klebsiella terrigena, and may be used as a pneumonia bacterium in clinical terms. Klebsiella pneumoniae, which accounts for most of the bacteria of the genus Klebsiella, is known as a representative bacteria of the genus Klebsiella.

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

In the present invention, the “antibiotic resistance” means resistance to a specific antibiotic and thus does not listen to the medicinal effect. For the purpose of the present invention, the antibiotic may be an antibiotic having a structure of carbapenem. Specifically, Amikacin, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Ceftazidime, Cefazolin, Cefazolin, Imipenem ), Ertapenem, Cefepime, Cefoxitin, Cefotaxime, Gentamicine, Levoflocacin, Meropenem, Piperacillin / Tazobactam (Piperacillin/Tazobactam), may be one or more selected from the group consisting of Cotrimoxa (Cortrimoxa) and tigecycline (Tigecyline), 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 decomposing the carbapenem and producing a carbapenemase that 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 the base sequence represented by SEQ ID NO: 20, and a functional equivalent of the base 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. As having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the base sequence represented by SEQ ID NO: 20.

In addition, the bacteriophage provided by the present invention may include any one of SEQ ID NOs: 21 and 22. In the present invention, each of SEQ ID NOs: 21 and 22 is an open reading frame (ORF) of the bacteriophage, among proteins that adsorb and bind to the genus Klebsiella, particularly 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.

Further, 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 base sequence of the genome encoding ORF57, and SEQ ID NO: 24 is the base 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 pneumococcus. It was confirmed that the bacteriophage YMC17/01/P6_KPN_BP belongs to the Siphoviridae family, which has an angled head and tail, and the total sequencing analysis has a size of 54,880 bp and the total number of ORFs is 87. Confirmed.

The bacteriophage of the present invention YMC17/01/P6_KPN_BP is a bacteriophage obtained by collecting and separating samples from a sewage treatment plant in a hospital. Did.

The bacteriophage YMC17/01/P6_KPN_BP of the present invention maintains lytic activity within a 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 within a 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, bacteriophage specific lytic activity, acid resistance and basicity of bacteriophage, is a composition for the prevention and treatment of infectious diseases caused by bacteria of the genus Klebsiella of the present invention, or , In applying to the various products containing the bacteriophage as an active ingredient, it is possible to apply a variety of pH range.

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 provides a composition for the prevention, improvement or treatment of diseases caused by bacteria in the genus Acinetobacter or Klebsiella containing bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

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

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

In the present invention, the infectious disease caused by the bacteria in the genus Acinetobacter is hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, soft sensation, genital herpes simplex, sphincter condilom, vancomycin resistant yellow staphylococcal infection, vancomycin intragrowth infection , A disease selected from the group consisting of methicillin-resistant Staphylococcus aureus infection, multidrug-resistant Pseudomonas aeruginosa infection, multidrug-resistant acinetobacter baumanii infection, growth invasion within carbapenem, intestinal infection, acute aerobic infection and enterovirus infection However, it is not limited thereto.

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

In the present invention, since the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumonia bacterium having carbapenem antibiotic resistance, it is effective in treating various diseases caused by the bacteria of the genus Klebsiella. Shows.

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

The composition of the present invention may include a bacteriophage of 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL, preferably a bacteriophage of 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the formation of a bacteriophage 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 provides a composition for the prevention, improvement or treatment of diseases caused by bacteria in the genus Acinetobacter or Klebsiella containing bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

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

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

In the present invention, the infectious disease caused by the bacteria in the genus Acinetobacter is hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, soft sensation, genital herpes simplex, sphincter condilom, vancomycin resistant yellow staphylococcal infection, vancomycin intragrowth infection , A disease selected from the group consisting of methicillin-resistant Staphylococcus aureus infection, multidrug-resistant Pseudomonas aeruginosa infection, multidrug-resistant acinetobacter baumanii infection, growth invasion within carbapenem, intestinal infection, acute aerobic infection and enterovirus infection However, it is not limited thereto.

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

In the present invention, since the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumonia bacterium having carbapenem antibiotic resistance, it is effective in treating various diseases caused by the bacteria of the genus Klebsiella. Shows.

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

The composition of the present invention may include a bacteriophage of 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL, preferably a bacteriophage of 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the formation of a bacteriophage 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 provides a composition for the prevention, improvement or treatment of diseases caused by bacteria in the genus Acinetobacter or Klebsiella containing bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

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

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

In the present invention, the infectious disease caused by the bacteria in the genus Acinetobacter is hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, soft sensation, genital herpes simplex, sphincter condilom, vancomycin resistant yellow staphylococcal infection, vancomycin intragrowth infection , A disease selected from the group consisting of methicillin-resistant Staphylococcus aureus infection, multidrug-resistant Pseudomonas aeruginosa infection, multidrug-resistant acinetobacter baumanii infection, growth invasion within carbapenem, intestinal infection, acute aerobic infection and enterovirus infection However, it is not limited thereto.

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

In the present invention, since the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumonia bacterium having carbapenem antibiotic resistance, it is effective in treating various diseases caused by the bacteria of the genus Klebsiella. Shows.

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

The composition of the present invention may include a bacteriophage of 1 X 10 ³ to 1 X 10 ¹⁰ PFU/mL, preferably a bacteriophage of 1 X 10 ⁶ to 1 X 10 ⁹ PFU/mL. The term used in the present invention, PFU (plaque forming unit) is a unit that quantifies the formation of a bacteriophage 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 bacteriophage YMC17/01/P6_KPN_BP.

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

In addition, the present invention is a bacteriophage YMC14/01/P262_ABA_BP for use as a cleaning agent; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/09/R1869_ABA_BP; Or it may be to use the 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 (Acinetobacter) or Klebsiella (Bacteria) containing the bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.

In the present invention, the diseases caused by bacteria of the genus Acinetobacter or Klebsiella are overlapped with those described above, and detailed description thereof will be omitted below.

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

The pharmaceutical composition of the present invention is not limited to these, but may be used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. when administered orally, and buffers, preservatives, and analgesics for injections. Agents, solubilizers, isotonic agents, stabilizers, etc. can be used in combination. For topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. may be prepared, and in the case of injections, unit dosage ampoules or multiple doses may be prepared. have. Others can be formulated as solutions, suspensions, tablets, capsules, and sustained release preparations.

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

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

In the present invention, “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, epidural, 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, age, weight, general health, sex, formulation, administration time, route of administration, rate of discharge, drug combination and severity of the particular disease to be prevented or treated, of the specific compound used. It may vary, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, disease severity, dosage form, administration route and duration, but can 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 into several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions.

The cosmetic composition in the present invention includes lotion, nutrient lotion, nutrient essence, massage cream, beauty bath water 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, hair loss agent {for cosmetics}, 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 cleaner (hand cleaner), medicinal soap (for non-medical), cream soap, facial wash, whole body cleaner, scalp cleaner, hair rinse, cosmetic soap, tooth whitening gel, toothpaste, etc. It can be produced in the form. To this end, the composition of the present invention may further include a solvent or a suitable carrier, excipient or diluent, which is commonly used in the preparation of cosmetic compositions.

The type of the solvent that can be further added to the cosmetic composition of the present invention is not particularly limited, for example, water, saline, DMSO, or a combination thereof can be used, and as 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. In addition, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as necessary.

Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil may be used as the oil, and waxes include beeswax, mellow, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin. Can be used.

As the fatty acid, stearic acid, linoleic acid, linolenic acid, and oleic acid may be used, and cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol may be used as the fatty acid alcohol. 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, if possible.

In addition, it may include a hygroscopic agent, a thickener, an antioxidant, etc., which are widely known in the cosmetic field, and their types and amounts are as known in the art.

The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, cookies, rice cakes, breads, and the like. Since the food composition of the present invention is composed of plant extracts having little toxicity and side effects, it can be safely used even for a long period of time for prevention purposes.

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

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

Other food compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, and protective colloidal thickeners , pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like.

These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally selected from 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 against the genus of Acinetobacter or Klebsiella, and the Acinetobacter genus or Klebsiella bacteria resistant to antibiotics, compared to chemicals 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 problems of antibiotic-resistant bacteria due to misuse of antibiotics, residual problems of antibiotics in food, and problems of a wide range of hosts. have.

Accordingly, the bacteriophage of the present invention can be used in various fields in the field of Acinetobacter or prevention or treatment of infectious diseases caused by Klebsiella bacteria, composition for antibiotics, composition for adding feed, feed, disinfectant, or detergent.

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

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

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

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

5 is a graph showing the change in the survival rate of the larvae after treatment with the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention to a honeybee beetle moth caterpillar infected with bacteria of the genus Acinetobacter having antibiotic resistance. .

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

Figure 8 shows the overall genome sequence analysis results of the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention.

9 is an electron micrograph of a bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention.

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

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

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

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 a honeybee beetle moth caterpillar infected with bacteria of the genus Acinetobacter having antibiotic resistance. .

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

15 is a graph showing the temperature stability of 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 overall genome sequence analysis results of bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention.

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

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

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

20 is a graph showing the change in the survival rate of the larva after treatment with the bacteriophage YMC15/09/R1869_ABA_BP according to an embodiment of the present invention to a honeybee beetle moth caterpillar infected with antibiotic-resistant Acinetobacter baumani .

Figure 21 is treated with bacteriophage YMC15/09/R1869_ABA_BP according to an embodiment of the present invention to mice infected with antibiotic-resistant acinetobacter baumani, the number of bacteria in the lungs of the mouse. It is a graphical representation of the change.

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

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

Figure 24 shows the overall genome sequence analysis 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 bacteria of the genus Klebsiella having antibiotic resistance of bacteriophage according to an embodiment of the present invention.

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

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

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

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

Figure 31 shows the overall genome sequence analysis results of the lytic bacteriophage against Klebsiella genus bacteria having antibiotic resistance according to an embodiment of the present invention.

The present invention relates to a bacteriophage having a specific killing ability to 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 merely 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. Separation of clinical samples and selection of antibiotic-resistant strains

As shown in Table 1 below, Acinetobacter baumannii bacteria were isolated and cultured from ICU (intensive care unit) blood and clinical specimens of a university hospital. 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 disk diffusion test method incubated overnight at 37° C. using a Mueller-Hinton agar, and the test antibiotics were amicacin, ampicillin-sulfur. Ampicillin-sulbactam, ceftazidime, ciprofloxacin, colistin, cefepime, cefotaxime, gentamicine, imipenem, levofloxacin Levofloxacin, meropenem, minocycline, piperacillin, piperacillin-tazobactam, cotrimoxa and tigecycline were used. Susceptibility results were read based on Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profile of the collected Acinetobacter baumannii strains is shown in Tables 2 to 4 below. However, in Tables 2 to 4, S, I and R are the results of evaluating susceptibility to antibacterial agents,'S' is susceptible,'I' is intermediate, and'R' is resistance. 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 pipe	YMC15/04/P369
YMC15/01/R3872	Trachea 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	Trachea inhalation (pneumonia)
YMC15/03/R2817	Phlegm (pneumonia)	YMC15/05/R3556	Phlegm (pneumonia)
YMC15/03/P828	Swap or drain pelvis	YMC15/06/R675	Phlegm (pneumonia)
YMC15/03/B10119	Blood	YMC15/08/R1402	Trachea 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, it was found that the collected 32 Acinetobacter baumannii strains are multi-resistant strains resistant to various antibiotics.

2. Collecting bacteriophage samples

2-1. Sample collection for phage banking

Raw water from which suspended solids and sediments were removed was secured after the first sedimentation at a sewage treatment facility at Severance Hospital. This was limited to sewage at all stages of the chemical treatment facility. 58 g of sodium chloride per 1 L was added to the collected sample, and then centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm millipore filter. To the obtained filtrate, polyethylene glycol (PEG, molecular weight 8000) was added at 10% W/V and stored refrigerated at 4° C. for 12 hours. The filtrate stored for 12 hours was centrifuged at 12,000 g for 20 minutes to resuspend the precipitate in phage dilution buffer (SM buffer), and then stored frozen by adding the same amount of chloroform. This was repeated 3 times to collect 300 mL of bacteriophage suspension.

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

Separation and purification of lytic phage was performed by the Spot Test method (Mazzocco A et al. In Bacteriophages, Clokie and Kropinski AM, eds. Humana Press. 2009). The inoculated strain was inoculated on a McConkie agar medium and cultured overnight at 35°C outside. After cultivation, strains sensitive to phage were selected by seeing the formation of transparent plaques. The susceptible strains were inoculated in McConkie agar medium and cultured at 35°C for 12 hours. After preparing a suspension of each strain with McFarland 0.5 turbidity in a 1 ml tube, 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 at 35° C. for 12 hours. After observing the plaque, the plaque was collected with a Pasteur pipette, diluted in SM buffer solution, and purified again 3 times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC14/01/P262_ABA_BP was diluted in SM buffer solution and purified again 3 times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC14/01/P262_ABA_BP was stored diluted in SM buffer solution.

After inoculating and incubating each of the 32 strains of antibiotic-resistant Acinetobacter baumannii identified in 1. above in McConkie agar, each resistant plated bacteriophage YMC14/01/P262_ABA_BP purified by the above process was smeared. The strain was inoculated with 5 μl to confirm plaque formation, and the titer range was confirmed, and solubility was shown in Table 5 below. However, in Table 5 below, + and-are evaluation of plaque activity for the collected strains,'+' means clear plaque, and'-' means that no lysis 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 above, it was confirmed that the bacteriophage YMC14/01/P262_ABA_BP according to the present invention lysed a significant number of antibiotic-resistant Acinetobacter Baumani strains.

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

After inoculating and culturing the bacteriophage YMC14/01/P262_ABA_BP purified by the method of 2. above in a sensitive strain culture medium (20 ml LB medium), filtered with a 220 nm millipore filter, and polyethylene glycol (MW 8,000) in the supernatant. After adding in an amount of 10% (w/v), it was refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the shape of the bacteriophage YMC14/01/P262_ABA_BP was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. 1. Did.

As shown in FIG. 1, when viewed as a criterion for classifying the YMC14/01/P262_ABA_BP bacteriophage according to the present invention, it was classified as belonging to the Myoviridae family having a long tail on a hexagonal head.

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

After incubating the antibiotic-resistant Acinetobacter baumani strain with an OD value of 0.5, the bacteriophage YMC14/01/P262_ABA_BP purified in 2. above was added to the Acinetobacter baumani strain as MOI 0.001 and cultured at room temperature. , 100 μl samples were collected at 1, 2, 3, 4, and 5 minutes for 1 ml, diluted in LB medium, and then the plaque analysis was conducted to evaluate the adsorption capacity of the bacteriophage, and the results are shown in FIG. 2.

In addition, after incubating the antibiotic resistant Acinetobacter baumani strain with an OD value of 0.3, the cells were precipitated by centrifugation at 7,000 g for 5 minutes at 4° C., and then diluted in 0.5 ml of LB medium, The bacteriophage YMC14/01/P262_ABA_BP purified in 2. was put into MOI 0.001 (titer 10 ⁸ pfu/cell) and cultured at 37°C for 5 minutes. The pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and incubated at 37°C. During the culture, samples were taken every 10 minutes to evaluate the 1-stage proliferation curve of the bacteriophage through plaque analysis, and the results are shown in FIG. 3.

As shown in Figure 2, within about 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 min: 6.9%, 10 min: 1.3%, 15 min). : 0%).

In addition, as shown in Figure 3, the results 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 to the Acinetobacter baumani strain having antibiotic resistance within a relatively fast time, and exhibits a high burst size of 79 PFU/infected cells. It can be seen that exhibits the lytic effect of.

5. In vitro antibiotic resistance bacteriophages for bacteriostasis validation in bacteria of Acinetobacter

Bacteriophage YMC14/01/P262_ABA_BP prepared in antibiotic resistant Acinetobacter baumani strain 1 X 10 ⁹ CFU/ml is 1 X 10 ⁸ CFU/ml (MOI: 0.1), 1 X 10 ⁹ PFU/ml (MOI: 1), Each was treated with an amount of 1 X 10 ¹⁰ PFU/ml (MOI: 10) and OD values (wavelength 600 nm) were measured by time. However, PBS+SM buffer was treated as a negative control, and the values are shown in FIG. 4.

As shown in FIG. 4, when the bacteriophage YMC14/01/P262_ABA_BP was treated with respect to the Acinetobacter baumani strain, the OD value was decreased, and as the MOI value was increased, the OD value was further decreased, especially when it was MOI 10. 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 solubility against the antibiotic resistant Acinetobacter baumani strain.

6. Verification of bacteriophage lytic capacity against bacteria in antibiotic-resistant Acinetobacter in vivo

After preparing 3 to 4-year-old honeybee beetle moth caterpillars (Galleria mellonella larvae), they were classified into 10 animals for each group. After injecting costine-resistant Acinetobacter baumani strain to each larva through the larval proleg at the minimum lethal concentration (MLD), colistin and bacteriophage purified in 2. above YMC14/01/P262_ABA_BP After inoculation with MOI 10 or MOI 100, the survival rate of larvae 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 larvae injected with acinetobacter baumani strain resistant to colistin, the survival rate of the larva increased and the MOI value increased. It was confirmed that the survival rate of the larva increased further. In addition, it was confirmed that when the bacteriophage YMC14/01/P262_ABA_BP was injected without injecting the Acinetobacter Baumani strain resistant to colistin, 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 solubility against antibiotic resistant Acinetobacter Baumani strain in vivo, effectively preventing infectious diseases caused by the Acinetobacter Baumani strain , It can be seen that can be improved or treated.

7. Evaluation of stability of bacteriophage against antibiotic-resistant Acinetobacter baumani strain

It was confirmed that the bacteriophage bacteriophage YMC14/01/P262_ABA_BP according to the present invention does not break at temperature and alkali and maintains stability.

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

In addition, each sample was incubated at 4° C., 37° C., 50° C., 60° C., and 70° C. for each of the bacteriophage YMC14/01/P262_ABA_BP solutions at 10° C. for 10 minutes, and each sample was added together with the Acinetobacter Baumani strain. 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 showed high stability in both acidic, neutral and alkaline, and for 30 days the bacteriophage YMC14/01/P262_ABA_BP was particularly neutral (pH 7-8) ).

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

8. Analysis of the entire genome sequence of bacteriophage against antibiotic-resistant Klebsiella genus

In order to characterize the bacteriophage YMC14/01/P262_ABA_BP according to the present invention, the entire gene sequence analysis is analyzed based on the whole genome sequencing method obvious to a person skilled in the art through an Illumina sequencer (Roche), and the results are shown. 8 and Tables 6 to 11.

As shown in FIG. 8 and Tables 6 to 11, the bacteriophage YMC14/01/P262_ABA_BP includes linear dsDNA (dsDNA) and consists 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, a 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 new bacteriophage not previously found.

[Example 2] Bacteriophage YMC15/02/T28_ABA_BP

1. Separation of clinical samples 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 a university hospital. 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 incubated overnight at 37°C using a Mueller-Hinton agar, and the test antibiotics were imipenem, piperacillin-tazobactam (piperacillin-tazobactam), ampicillin-s μlbactam, aztreonam, ceftazidime, cefepime, cefotaxime, gentamicin , Amicacin, ciprofloxacin, levofloxacin, tigecyline and colistin were used. Susceptibility results were read based on Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profile of the collected Acinetobacter baumannii strains is shown in Tables 13 to 15 below. However, in Tables 13 to 15, S, I and R are the results of evaluating susceptibility to antibacterial agents,'S' is susceptible,'I' is intermediate, and'R' is resistance. 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 drainage	YMC15/04/R68	Phlegm (pneumonia)
YMC15/01/P760	Phlegm (pneumonia)	YMC15/04/P369	Phlegm (pneumonia)
YMC15/01/R3872	Buttocks bedsores	YMC15/04/R663	Phlegm (pneumonia)
YMC15/02/R923	blood	YMC15/04/T132	Tracheal tube tips
YMC15/02/R830	Phlegm (pneumonia)	YMC15/04/R1427	Trachea inhalation (pneumonia)
YMC15/02/R1418	Buttocks bedsores	YMC15/04/U2285	Trachea inhalation (pneumonia)
YMC15/02/R2403	Intravenous catheter tips	YMC15/04/R2498	Swap or drain pipe
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, it was found that the collected 20 strains of Acinetobacter baumannii were multi-resistant strains resistant to various antibiotics.

2. Collecting bacteriophage samples

2-1. Sample collection for phage banking

Raw water from which suspended solids and sediments were removed was secured after the first sedimentation at a sewage treatment facility at Severance Hospital. This was limited to sewage at all stages of the chemical treatment facility. 58 g of sodium chloride per 1 L was added to the collected sample, and then 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 for 12 hours was centrifuged at 12,000 g for 20 minutes to resuspend the precipitate in phage dilution buffer (SM buffer), and then stored frozen by adding the same amount of chloroform. This was repeated 3 times to collect 300 mL of bacteriophage suspension.

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

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

After inoculating and incubating the antibiotic-resistant Acinetobacter baumannii strain identified in 1. above in the McConkie agar medium, the bacteriophage purified by the above process YMC15/02/T28_ABA_BP was stained with each resistant strain 5 Plasma formation was confirmed by inoculation with µl, and the titer range was confirmed, and solubility was shown in Table 16 below.

However, in Table 16 below, + and-are evaluation of plaque activity for the collected strains,'+' means transparent plaque, and'-' means that no lysis 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 lysed a significant number of antibiotic-resistant Acinetobacter Baumani strains.

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

After inoculating and culturing the bacteriophage YMC15/02/T28_ABA_BP purified by the method of the above in a sensitive strain culture medium (20 ml LB medium), filtered with a 220 nm millipore filter, and polyethylene glycol (MW 8,000) in the supernatant. After adding in an amount of 10% (w/v), it was refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the shape of the bacteriophage YMC15/02/T28_ABA_BP was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. 9. Did.

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

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

After incubating the antibiotic-resistant Acinetobacter baumani strain with an OD value of 0.5, after adding the bacteriophage YMC15/02/T28_ABA_BP purified in 2. to the acinetobacter baumani strain as MOI 0.001, and then incubating at room temperature , 100 µl samples were collected at 1, 2, 3, 4, and 5 minutes for 1 ml, 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, after incubating the antibiotic resistant Acinetobacter baumani strain with an OD value of 0.3, the cells were precipitated by centrifugation at 7,000 g for 5 minutes at 4° C., and then diluted in 0.5 ml of LB medium, Bacteria phage YMC15/02/T28_ABA_BP purified in 2. was put into MOI 0.001 (titer 10 ⁸ pfu/cells) and incubated at 37°C for 5 minutes. The pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and incubated at 37°C. During the culture, samples were taken every 10 minutes to evaluate the 1-stage proliferation curve of the bacteriophage through plaque analysis, and the results are shown in FIG. 11.

As shown in Figure 10, within about 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 min: 0.24%).

In addition, as shown in Figure 11, the results 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 to the Acinetobacter baumani strain having antibiotic resistance within a relatively fast time, and exhibits a high burst size of 424 PFU/infected cells. It can be seen that exhibits the lytic effect of.

5. In vitro antibiotic resistance bacteriophages for bacteriostasis validation in bacteria of Acinetobacter

Bacteriophage YMC15/02/T28_ABA_BP prepared in antibiotic resistant Acinetobacter baumani strain 1 X 10 ⁹ CFU/ml is 1 X 10 ⁸ CFU/ml (MOI: 0.1), 1 X 10 ⁹ PFU/ml (MOI: 1), Each was treated with an amount of 1 X 10 ¹⁰ PFU/ml (MOI: 10) and OD values (wavelength 600 nm) were measured by 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 with respect to the Acinetobacter baumani strain, the OD value was decreased, and as the MOI value was increased, the OD value was further decreased, especially when it was MOI 10. 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 solubility against the antibiotic-resistant Acinetobacter baumani strain.

6. Verification of bacteriophage lytic capacity against bacteria in antibiotic-resistant Acinetobacter in vivo

After preparing 3 to 4-year-old honeybee beetle moth caterpillars (Galleria mellonella larvae), 10 animals were classified for each group. After inoculating each larva with a coccistin-resistant acinetobacter baumani strain through dip larval reproduction at a minimum lethal concentration (MLD), colistin and the bacteriophage purified in step 2. YMC15/02/T28_ABA_BP are MOI 10 Alternatively, after 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. 13.

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

Through the above results, the bacteriophage YMC15/02/T28_ABA_BP according to the present invention has solubility against antibiotic resistant Acinetobacter Baumani strain in vivo, effectively preventing infectious diseases caused by the Acinetobacter Baumani strain , It can be seen that can be improved or treated.

7. Evaluation of stability of bacteriophage against antibiotic-resistant Acinetobacter baumani strain

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

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

In addition, each sample was cultured at 4° C., 37° C., 50° C., 60° C., and 70° C. for each of the bacteriophage YMC15/02/T28_ABA_BP solutions at 10° C. for 10 minutes, and each sample was added together with the Acinetobacter Baumani strain. Plaque analysis was performed by the method of. And the results are shown in FIG. 15.

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

In addition, as shown in Figure 15, the bacteriophage YMC15/02/T28_ABA_BP showed very high stability even at a high temperature of 50 ℃.

8. Analysis of the entire genome sequence of bacteriophage against antibiotic-resistant Klebsiella genus

In order to characterize the bacteriophage YMC15/02/T28_ABA_BP according to the present invention, the whole gene sequence analysis is performed based on the whole genome sequencing method obvious to the skilled person through the Illumina sequencer (Roche), 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 includes linear dsDNA (dsDNA), and consists 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, no bacteriophage having similarity to the bacteriophage according to the present invention was 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 new bacteriophage not previously found.

[Example 3] Bacteriophage YMC15/09/R1869_ABA_BP

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

As shown in Table 23 below, Acinetobacter baumannii strains were isolated and cultured from ICU (intensive care unit) blood and clinical specimens of a university hospital. 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 disk diffusion test method incubated overnight at 37° C. using a Mueller-Hinton agar, and the test antibiotics were amicacin, ampicillin-sulfur. Ampicillin-s μlbactam, ceftazidime, ciprofloxacin, colistin, cefepime, cefotaxime, gentamicine, imipenem, Levofloxacin, meropenem, minocycline, piperacillin, piperacillin-tazobactam, cotrimoxa and tigecycline were used. . Susceptibility results were read based on Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profile of the collected bacteria in Acinetobacter is shown in Tables 24 to 28 below. However, in Tables 24 to 28, S, I and R are the results of evaluating susceptibility to antibacterial agents,'S' is susceptible,'I' is intermediate, and'R' is resistance. Means

Host strain	origin	Host strain	Lysis
YMC16/12/R12914	Phlegm (pneumonia)	YMC16/01/R198	Trachea 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	Swap or drainage	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	Swap or drainage
YMC17/02/B4864	blood	YMC16/03/R4461	Trachea inhalation (pneumonia
YMC17/02/P523	Buttocks bedsores	YMC16/05/R2210	Phlegm (pneumonia)
YMC17/02/B8414	Peritoneal blood disease	YMC16/07/R2512	Bronchial wash
YMC17/03/R585	Phlegm (pneumonia)	YMC16/09/R2471	Trachea inhalation (pneumonia
YMC17/03/B4730	Catheter blood	YMC16/10/R2537	Phlegm (pneumonia)
YMC17/03/B5000	Catheter blood	YMC16/12/P503	Swap or drainage chest
YMC17/03/R1888	Phlegm (pneumonia)	YMC15/02/T28	Other Catheter Tip
YMC17/03/R3279	Phlegm (pneumonia)	YMC15/02/R436	Trachea inhalation (pneumonia
YMC17/03/R4077	Trachea inhalation (pneumonia	YMC15/03/R1604	Trachea 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	Trachea inhalation (pneumonia	YMC14/08/T90	Catheter tips
YMC16/01/P11	Swap or drainage abdomen	YMC14/08/R1169	Phlegm (pneumonia)
YMC16/01/R123	Tracheal tube tips

As shown in Tables 24 to 28, it was found that the collected 57 strains of Acinetobacter baumannii were multi-resistant strains resistant to various antibiotics.

2. Collecting bacteriophage samples

2-1. Sample collection for phage banking

Raw water from which suspended solids and sediments were removed was secured after the first sedimentation at a sewage treatment facility at Severance Hospital. This was limited to sewage at all stages of the chemical treatment facility. 58 g of sodium chloride per 1 L was added to the collected sample, and then 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 for 12 hours was centrifuged at 12,000 g for 20 minutes to resuspend the precipitate in phage dilution buffer (SM buffer), and then stored frozen by adding the same amount of chloroform. This was repeated 3 times to collect 300 mL of bacteriophage suspension.

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

Separation and purification of lytic phage was performed by the Spot Test method (Mazzocco A et al. In Bacteriophages, Clokie and Kropinski AM, eds. Humana Press. 2009). The inoculated strains were inoculated on a McConkie agar medium and incubated overnight at 35°C. After cultivation, strains sensitive to phage were selected by seeing the formation of transparent plaques. The susceptible strains were inoculated in McConkie agar medium and cultured at 35°C for 12 hours. Prepare a suspension of each strain with McFarland 0.5 turbidity in a 1 ml tube, and mix with H top agar (3 ml), 100 µl of susceptible bacteria and phage solution (1 µl, 10 µl and 50 µl, respectively) and apply to the LB agar. Incubated at 35° C. for 12 hours. After observing the plaque, the plaque was collected with a Pasteur pipette, 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 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 stored diluted in SM buffer solution.

After inoculating each of the 57 strains of the antibiotic-resistant Acinetobacter baumannii identified in 1. above in a McConkie agar medium and incubating them, each of the resistant strains of the bacteriophage YMC15/09/R1869_ABA_BP purified by the above procedure was smeared. The strain was inoculated with 5 μl to confirm plaque formation, and the titer range was confirmed, and solubility was shown in Table 29 below. However, in Table 29 below, + and-are evaluation of plaque activity for the collected strains,'+' means clear plaque, and'-' means that no lysis 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 lysed a significant number of antibiotic-resistant Acinetobacter Baumani strains.

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

After inoculating and incubating the bacteriophage YMC15/09/R1869_ABA_BP purified by the method of 2 above in a sensitive strain culture medium (20 ml LB medium), filtered with a 220 nm millipore filter, and polyethylene glycol (MW 8,000) in the supernatant. After adding in an amount of 10% (w/v), it was refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the shape of the bacteriophage YMC15/09/R1869_ABA_BP was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. 17. Did.

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

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

After incubating the antibiotic-resistant Acinetobacter baumani strain with an OD value of 0.5, after adding the bacteriophage YMC15/09/R1869_ABA_BP purified in 2. above to the acinetobacter baumani strain as MOI 0.001, and then incubating at room temperature , 100 μl samples were collected at 1, 2, 3, 4, and 5 minutes for 1 ml, diluted in LB medium, and then the plaque analysis was used to evaluate the adsorption capacity of the bacteriophage, and the results are shown in FIG. 18.

In addition, after incubating the antibiotic resistant Acinetobacter baumani strain with an OD value of 0.3, the cells were precipitated by centrifugation at 7,000 g for 5 minutes at 4° C., and then diluted in 0.5 ml of LB medium, The bacteriophage purified in step 2. YMC15/09/R1869_ABA_BP was added with MOI 0.001 (titer 10 ⁸ pfu/cells) and incubated at 37° C. for 5 minutes. The pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and incubated at 37°C. During the culture, samples were taken every 10 minutes to evaluate the 1-stage proliferation curve of the bacteriophage through plaque analysis, and the results are shown in FIG. 19.

As shown in Fig. 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 min: 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 to the Acinetobacter baumani strain having antibiotic resistance within a relatively fast time, and exhibits a high burst size of 78 PFU/infected cells, thus showing antibiotic resistance strain It can be seen that exhibits the lytic effect of.

5. Verification of bacteriophage lytic capacity against bacteria in antibiotic-resistant Acinetobacter in vivo

After preparing 3 to 4-year-old honeybee beetle moth caterpillars (Galleria mellonella larvae), they were classified into 10 animals for each group. After inoculating each larva with an acinetobacter Baumani strain resistant to colistin through a larval dip at a minimum lethal concentration (MLD), colistin and the bacteriophage purified in step 2. YMC15/09/R1869_ABA_BP are MOI 10 Alternatively, after 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 larvae injected with acinetobacter baumani strain resistant to colistin, the survival rate of the larva increased and the MOI value increased. It was confirmed that the survival rate of the larva increased further. In addition, it was confirmed that when the bacteriophage YMC15/09/R1869_ABA_BP was injected without injecting the Acinetobacter Baumani strain resistant to colistin, there was no toxicity when comparing the survival rate with the healthy control group.

Through the above results, the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention has solubility against antibiotic resistant Acinetobacter Baumani strain in vivo, effectively preventing infectious diseases caused by the Acinetobacter Baumani strain , It can be seen that can be improved or treated.

6. Verification of bacteriophage lytic capacity against bacteria in antibiotic-resistant Acinetobacter in vivo

After intranasally administering to the mouse an acinetobacter Baumani strain (30 μl) resistant to colistin for 2 hours, the bacteriophage YMC15/09/R1869_ABA_BP (30 μl) purified in 2. above or colistin antibiotic (30 μl) ) Was administered in the same way. After 1 and 5 days, lung tissue was removed by autopsy of 4 animals per each experimental group. By culturing the bacteria with a sample obtained by grinding a part of the mouse lung, the change in 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 was administered to mice injected with acinetobacter baumani strain resistant to colistin, the acinetobacter baumani of the mouse was in the lung. It could be seen that the number of bacteria was significantly reduced, and it was confirmed that the number of bacteria of the Acinetobacter baumani decreased further as the MOI value increased. In addition, when the bacteriophage YMC15/09/R1869_ABA_BP and colistin were administered together, it was confirmed that the bacteriophage YMC15/09/R1869_ABA_BP alone showed a further decrease in the number of acinetobacter baumani in the lungs of mice.

Through the above results, the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention has solubility against antibiotic resistant Acinetobacter Baumani strain in vivo, effectively preventing infectious diseases caused by the Acinetobacter Baumani strain , It can be seen that can be improved or treated.

7. Evaluation of stability of bacteriophage against antibiotic-resistant Acinetobacter baumani strain

It was confirmed that the bacteriophage bacteriophage YMC15/09/R1869_ABA_BP according to the present invention does not break at temperature and alkali and maintains stability.

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

In addition, each sample was incubated at 4° C., 37° C., 50° C., 60° C., and 70° C. for the bacteriophage YMC15/09/R1869_ABA_BP solution at 4° C., 37° C., and 70° C., respectively. Plaque analysis was performed by the method of. And the results are shown in FIG. 23.

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

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 ℃.

8. Analysis of the entire genome sequence of bacteriophage against antibiotic-resistant Klebsiella genus

In order to characterize the bacteriophage YMC15/09/R1869_ABA_BP according to the present invention, the whole gene sequence analysis is performed based on the whole genome sequencing method obvious to the skilled person through the Illumina sequencer (Roche), 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 includes linear dsDNA (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, a 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 new bacteriophage not previously found.

[Example 4] Bacteriophage YMC17/01/P6_KPN_BP

1. Separation of clinical samples and selection of antibiotic-resistant strains

As shown in Table 36 below, Klebsiella pneumoniae bacteria were cultured and separated from patients at Severance Hospital or their feces. 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 incubated overnight at 37° C. using a Mueller-Hinton agar. Test antibiotics for Klebsiella pneumoniae bacteria include Amicacin, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Cestazidime, Cefazolin, Imipenem, Ertapenem, Cefepime, Cefoxitin, Cefotaxime, Gentamicine, Levofloxacin, Meropenem (Meropenem), Piperacillin/Tazobactam, Cortrimoxa, and Tigecyline. Susceptibility results were read based on Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profiles of 47 strains of Klebsiella pneumoniae collected are shown in Tables 37 to 40 below. However, in Tables 37 to 40, S, I, and R are the results of evaluating susceptibility to antibacterial agents,'S' is susceptible,'I' is intermediate, and'R' is resistance. 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	Swap or drain 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	Trachea 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-resistant strains resistant to various carbapenem-based antibiotics.

2. Collecting bacteriophage samples

2-1. Sample collection for phage banking

Raw water from which suspended solids and sediments were removed was secured after the first sedimentation at a sewage treatment facility at Severance Hospital. This was limited to sewage at all stages of the chemical treatment facility. 58 g of sodium chloride per 1 L was added to the collected sample, and then 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 for 12 hours was centrifuged at 12,000 g for 20 minutes to resuspend the precipitate in phage dilution buffer (SM buffer), and then stored frozen by adding the same amount of chloroform. This was repeated 3 times to collect 300 mL of bacteriophage suspension.

2-2 Screening of lytic phage and measurement of lytic titer

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

After inoculating and incubating each of the 47 strains of antibiotic-resistant Klebsiella pneumoniae identified in 1. in McConkie agar medium, each of the bacteriophage purified by the above process was smeared YMC17/01/P6_KPN_BP Tolerant strains were inoculated with 5 μl to confirm plaque formation, and to confirm the titer range, solubility was shown in Table 41 below. However, in Table 41 below, + and-are evaluations of plaque activity for the collected strains,'+' means clear plaque, and'-' means that no lysis 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 lysed 35 strains (74%) of 47 strains of antibiotic resistant Klebsiella pneumoniae.

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

After inoculating and incubating the bacteriophage purified by the method of 2. above in a sensitive strain culture medium (20 ml LB medium), filtering with a 220 nm millipore filter, and 10% (w/w) of polyethylene glycol (MW 8,000) in the supernatant. v) and then kept refrigerated overnight. After centrifugation for 20 minutes under the conditions of 12,000 g, the shape of the bacteriophage was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. 25.

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

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

After culturing the antibiotic-resistant Klebsiella pneumoniae to have an OD value of 0.5, the Klebsiella pneumoniae bacteriophage YMC17/01/P6_KPN_BP purified in 2. above was put into MOI 0.001 and cultured at room temperature. , 100 µl samples were collected at 1, 2, 3, 4, and 5 minutes for 1 ml, diluted in LB medium, and then the plaque analysis was performed to evaluate the adsorption capacity of the bacteriophage, and the results are shown in FIG. 26.

In addition, after culturing the antibiotic-resistant Klebsiella pneumoniae to have an OD value of 0.3, the cells were precipitated by centrifugation at 7,000 g for 5 minutes at 4°C, and then diluted in 0.5 ml of LB medium, The bacteriophage purified in Example 2 was added with MOI 0.001 (titer 10 ⁸ pfu/cells) and incubated at 37°C for 5 minutes. The pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and incubated at 37°C. During the culture, samples were taken every 10 minutes to evaluate the 1-stage proliferation curve of the bacteriophage 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 min: 1.05%).

In addition, as shown in Figure 27, the results of the single-stage proliferation curve showed 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 adsorb to antibiotic-resistant Klebsiella pneumoniae within a relatively short time, and exhibits a high burst size of 43 PFU/infected cells. It can be seen that it exerts a lytic effect on the Ciella pneumoniae.

5. In vitro antibiotic-resistant Klebsiella genus bacteriophage validation

Bacteriophage YMC17/01/P6_KPN_BP prepared in antibiotic resistant Klebsiella pneumoniae 1 X 10 ⁹ CFU/ml is 1 X 10 ⁸ CFU/ml (MOI: 0.1), 1 X 10 ⁹ PFU/ml (MOI: 1), Each was treated with an amount of 1 X 10 ¹⁰ PFU/ml (MOI: 10) and OD values (wavelength 600 nm) were measured by 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 to the negative control group, when the bacteriophage was treated with Klebsiella pneumoniae, the OD value decreased, and as the MOI value increased, the OD value further decreased, especially when it was MOI 10 The solubility was high.

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

6. Evaluation of stability of bacteriophage against antibiotic-resistant Klebsiella genus

It was confirmed that the bacteriophage YMC17/01/P6_KPN_BP according to the present invention does not break at temperature and alkali and maintains stability.

After adding 1 µl of the bacteriophage purified by the method of 2. to 40 µl of SM buffer adjusted to pH of 4, 5, 6, 7, 8, 9 and 10, and then incubating at 37° C. for 1 hour, 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, each sample was incubated at 4° C., 37° C., 60° C., and 70° C. for 1 hour in 10-minute increments, and each sample was subjected to plaque analysis by the method of 4. above with Klebsiella pneumoniae Fig. 30 shows the result.

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

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. Total genome sequence analysis of bacteriophage against antibiotic-resistant Klebsiella genus

In order to characterize the bacteriophage YMC17/01/P6_KPN_BP according to the present invention, the entire gene sequence analysis is analyzed based on the whole genome sequencing method apparent to a person skilled in the art through an Illumina sequencer (Roche), and the results are shown. 31 and Tables 42 to 46.

As shown in FIG. 31 and Tables 42 to 46, the bacteriophage YMC17/01/P6_KPN_BP includes linear dsDNA (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, no bacteriophage having similarity to the bacteriophage according to the present invention was 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 new bacteriophage not previously found.

The present invention has been described in detail above, but the scope of the present invention is not limited thereto, and it is common in the art that various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. It will be obvious to those who have the knowledge of

[Accession number (1)]

Bacteriophage YMC14/01/P262_ABA_BP

Depository name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11798P

Date of accession: 20181115

[Accession number (2)]

Bacteriophage YMC15/02/T28_ABA_BP

Depository name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11799P

Date of accession: 20181115

[Accession number (3)]

Bacteriophage YMC15/09/R1869_ABA_BP

Depository name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11802P

Date of accession: 20181115

[Accession number (4)]

Bacteriophage YMC17/01/P6_KPN_BP

Depository name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11804P

Date of accession: 20181115

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

Claims (41)

1. A bacteriophage that has specific killing ability against bacteria of the genus Acinetobacter or Klebsiella.
2. According to claim 1,

   The bacteria of the genus Acinetobacter are Acinetobacter baumannii , Acinetobacter calcoaceticus , Acinetobacter haemolyticus , Acinetobacter junii , Acinetobacter junii Acinetobacter johnsonii , Acinetobacter lwoffii , Acinetobacter radioresistens , Acinetobacter ursingii , Acinetobacter ursingii , Acinetobacter ursingii , Acinetobacter ursingii , Acinetobacter parvus , Acinetobacter baylyi , Acinetobacter bouvetii , Acinetobacter towneri , Acinetobacter towneri , Acinetobacter towneri , Acinetobacter tando 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.
3. According to claim 1,

   The bacteria in the genus Acinetobacter is Acinetobacter baumannii, bacteriophage.
4. According to claim 1,

   The bacteria in the Acinetobacter are antibiotic-resistant bacteria, bacteriophage.
5. According to claim 4,

   The antibiotics include colistin, erythromycin, ampicillin, vancomycin, linezolid, methicillin, oxacillin, cetaxoxime, Rifampicin, Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Doripenem, Imipenem/Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Aztreonam, Piperacillin, Polymyxin B, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Tigecycline Cycline ), a combination thereof and derivatives thereof, any one or more selected from the group consisting of, bacteriophage.
6. According to claim 1,

   The Klebsiella bacteria are Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca , Klebsiella planticola (Klebsiella planticola) and Klebsiella terrigena (Klebsiella terrigena) one or more selected from the group consisting of, bacteriophage.
7. According to claim 1,

   The Klebsiella genus bacteria are Klebsiella pneumoniae, bacteriophage.
8. According to claim 1,

   The Klebsiella bacteria are antibiotic-resistant bacteria, bacteriophage.
9. The method of claim 8,

   The antibiotic is bacteriophage, a carbapenem antibiotic.
10. According to claim 1,

    The name of the bacteriophage is YMC14/01/P262_ABA_BP, the accession number is KFCC11798P, bacteriophage.
11. According to claim 1,

    The bacteriophage is composed of a nucleotide sequence represented by SEQ ID NO: 1, bacteriophage.
12. According to claim 1,

    The bacteriophage comprises any one of SEQ ID NO: 2 to 4, the bacteriophage.
13. According to claim 1,

    The bacteriophage is to include a genome represented by any one of SEQ ID NO: 5 to 7, bacteriophage.
14. According to claim 1,

    The name of the bacteriophage is YMC15/02/T28_ABA_BP, the accession number is KFCC11799P, bacteriophage.
15. According to claim 1,

    The bacteriophage is made of a nucleotide sequence represented by SEQ ID NO: 8, bacteriophage.
16. According to claim 1,

    The bacteriophage comprises a protein of any one of SEQ ID NO: 9 to 11, bacteriophage.
17. According to claim 1,

    The bacteriophage is to include a genome represented by any one of SEQ ID NO: 12 to 14, bacteriophage.
18. According to claim 1,

    The name of the bacteriophage is YMC15/09/R1869_ABA_BP, the accession number is KFCC11802P, bacteriophage.
19. According to claim 1,

    The bacteriophage consists of a nucleotide sequence represented by SEQ ID NO: 15, bacteriophage.
20. According to claim 1,

    The bacteriophage comprises a protein of any one of SEQ ID NO: 16 and 17, bacteriophage.
21. According to claim 1,

    The bacteriophage is to contain a genome represented by any one of SEQ ID NO: 18 and 19, bacteriophage.
22. According to claim 1,

    The name of the bacteriophage is YMC17/01/P6_KPN_BP, and the accession number is KFCC11804P.
23. According to claim 1,

    The bacteriophage is composed of a nucleotide sequence represented by SEQ ID NO: 20, bacteriophage.
24. According to claim 1,

    The bacteriophage comprises a protein of any one of SEQ ID NO: 21 and 22, bacteriophage.
25. According to claim 1,

    The bacteriophage is to include a genome represented by any one of SEQ ID NO: 23 and 24, bacteriophage.
26. Claim 1 to 25, wherein any one of the selected bacteriophage as an active ingredient, antibiotic composition.
27. Claim 1 to claim 25, wherein any one of the bacteriophage selected from the active ingredient, feed composition.
28. Claim 1 to 25, wherein any one of the bacteriophage selected from the active ingredient, drinking water additive.
29. A disinfectant comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient.
30. A cleaning agent comprising the bacteriophage of any one of claims 1 to 25 as an active ingredient.
31. 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.
32. The method of claim 31,

    The diseases caused by the bacteria in the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, soft sensation, genital herpes simplex, sphincter condylom, vancomycin resistant yellow grape bacterial infection, vancomycin intragrowth infection, methicillin resistance Staphylococcus aureus infection, multidrug-resistant Pseudomonas aeruginosa infection, multidrug-resistant acinetobacter baumannii infection, carbapenem growth endocytoma infection, intestinal infection, acute respiratory infection and enterovirus infection, a disease selected from the group, pharmaceutical Composition.
33. The method of claim 31,

    Diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, intraocular inflammation, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media, bacteremia, endocarditis , Cholecystitis and parotitis is a disease selected from the group consisting of, pharmaceutical composition.
34. Use of the bacteriophage of any one of claims 1 to 25 for administering an antibiotic composition to a subject in need of treatment or prevention.
35. Use of the bacteriophage of any one of claims 1 to 25 for use in the preparation of a composition for feed addition.
36. Use of the bacteriophage of any one of claims 1 to 25 for use in the manufacture of drinking water additives.
37. Use of the bacteriophage of any one of claims 1 to 25 for use in the manufacture of disinfectants.
38. Use of the bacteriophage of any one of claims 1 to 25 for use in the manufacture of cleaning agents.
39. A bacteriophage of any one of claims 1 to 25 to a subject in need of prevention or treatment, comprising the step of administering as an active ingredient, Acinetobacter genus or Klebsiella genus bacteria Prevention or treatment of diseases caused by.
40. The method of claim 39,

    The diseases caused by the bacteria in the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, soft sensation, genital herpes simplex, sphincter condylom, vancomycin resistant yellow grape bacterial infection, vancomycin intragrowth infection, methicillin resistance Staphylococcus aureus infections, multidrug-resistant Pseudomonas aeruginosa infections, multidrug-resistant acinetobacter baumanni infections, carbapenem growth-ingrowing bacteriogenic infections, intestinal infections, acute respiratory infections and enterovirus infections Methods of treatment.
41. The method of claim 39,

    Diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, intraocular inflammation, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media, bacteremia, endocarditis , Cholecystitis and parotitis is a disease selected from the group consisting of, prevention or treatment methods.

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