WO2020130652A9 - Nouveau bactériophage pour la lyse de bactéries - Google Patents

Nouveau bactériophage pour la lyse de bactéries Download PDF

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WO2020130652A9
WO2020130652A9 PCT/KR2019/018053 KR2019018053W WO2020130652A9 WO 2020130652 A9 WO2020130652 A9 WO 2020130652A9 KR 2019018053 W KR2019018053 W KR 2019018053W WO 2020130652 A9 WO2020130652 A9 WO 2020130652A9
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bacteriophage
acinetobacter
bacteria
ymc15
klebsiella
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PCT/KR2019/018053
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Korean (ko)
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WO2020130652A2 (fr
WO2020130652A3 (fr
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용동은
전종수
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연세대학교 산학협력단
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Priority claimed from KR1020180164212A external-priority patent/KR102142018B1/ko
Priority claimed from KR1020180164181A external-priority patent/KR102189126B1/ko
Application filed by 연세대학교 산학협력단 filed Critical 연세대학교 산학협력단
Publication of WO2020130652A2 publication Critical patent/WO2020130652A2/fr
Publication of WO2020130652A3 publication Critical patent/WO2020130652A3/fr
Publication of WO2020130652A9 publication Critical patent/WO2020130652A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to a novel bacteriophage that lyses bacteria, particularly bacteria that are resistant to antibiotics.
  • Bacterial infection is one of the most common and fatal causes of human disease. Since penicillin, numerous types of antibiotics have been developed and have been used to combat external invasion bacteria. However, in recent years, strains resistant to these antibiotics have appeared and are considered a big problem. Bacterial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa , which can be life-threatening, are resistant to all known antibiotics to date. Has been raised (Stuart B. Levy, Scientific American, (1988): 46-53).
  • Tolerance to antibiotics is a phenomenon that is distinct from resistance to antibiotics, and was first discovered in Pneumococcus sp. in the 1970s and provided important clues about the mechanism of action of penicillin (Tomasz). et al., Nature, 227, (1970): 138-140).
  • Conventional chemical antibiotics such as penicillin and cephalosporin exhibit an antibiotic action by inhibiting the synthesis of a cell wall or protein of a microorganism.
  • resistant species stop growing in the presence of conventional concentrations of antibiotics, but do not die as a result. Resistance occurs because when antibiotics inhibit cell wall synthase, autolytic enzymes such as autolysin do not activate. This is because penicillin activates the endogenous hydrolytic enzyme.
  • Acinetobacter baumannii is a Gram-negative aerobic bacterium and is an important cause of nosocomial infections in many hospitals.
  • aminoglycosides, sepharoseporin, fluoroquinolone, beta lactamase Infections caused by beta-lactamase inhibitors (beta-lactamase inhibitors) and multidrug-resistant Acinetobacter Baumani (MRAB), which are resistant to carbapenem are increasing.
  • the bacteria of the genus Klebsiella is a genus name of the intestinal bacteria family, and is a gram-negative bacillus. There is no flagella and is characterized by producing mucus while passing through the capsular membrane. It obtains a carbon source from citrate and produces acids and gases other than hydrogen sulfide from various carbohydrates. It is widely present in nature, is detected in human respiratory, intestinal, and urinary tract, and is known as the causative agent of acute pneumonia. Recently, various bacteria of the genus Klebsiella have been diagnosed and detected, including Klebsiella pneumoniae, Klebsiella ozaenae, and Klebsiella rhinoscleromatis.
  • Klebsiella pneumoniae which is known to account for most of the bacteria of the genus Klebsiella in clinical terms, is a Gram-negative bacillus, anaerobe, or aerobic bacteria that resides in the human intestine, skin, oral cavity, and respiratory tract. It is said that 10-20% of pneumonia is caused by this fungus. In addition, it is sometimes isolated from urinary tract infections, and is reported as the main causative agent of sepsis and intraperitoneal infection, and it is the causative agent of bacteremia that occurs in intensive care unit patients, showing a high infection rate.
  • beta-lactam-based antibiotics account for about 50% or more of currently used antimicrobial agents.
  • many Gram-negative bacteria exhibit resistance to these antibiotics by producing beta-lactam-degrading enzymes against these beta-lactam-based antibiotics.
  • pneumococcal in particular, K. pneumoniae carbaphenemase (KPC)-producing K. pneumoniae (KPC-Kp), which is an antibiotic-resistant bacteria, spreads worldwide and increases the mortality rate of infected patients. Raising it. Accordingly, in Korea, a new strategy is needed to inhibit the spread and increase of K. pneumoniae carbaphenemase (KPC)-producing K. pneumoniae (KPC-Kp).
  • Bacteriophage refers to a bacteria-specific virus that inhibits and inhibits the growth of infected bacteria by infecting specific bacteria. Bacteriophages have the ability to kill bacteria by proliferating inside bacterial cells after infection with bacteria, and destroying the cell walls of host bacteria when progeny bacteriophages come out of the bacteria after proliferation.
  • the bacterial infection method of bacteriophage is very specific, so the types of bacteriophage that can infect specific bacteria are limited to some. That is, a specific bacteriophage can infect only a specific category of bacteria, and because of this, a specific bacteriophage kills only specific bacteria and does not affect other bacteria. Therefore, the use of bacteriophage as a countermeasure for recent bacterial diseases is receiving great attention. In particular, after 2000, the limitations of existing antibiotics appeared due to the increase in antibiotic-resistant bacteria, and the possibility of development as a substitute for the existing antibiotics emerged.
  • Another object of the present invention is a composition for the prevention and treatment of infectious diseases caused by novel bacteriophages having specific infection and killing ability against bacteria of the genus Acinetobacter, in particular, bacteria of the genus Acinetobacter having resistance to antibiotics It is to provide a food composition for improving diseases and diseases.
  • a bacteriophage having a specific killing ability against bacteria of the genus Acinetobacter or Klebsiella is provided.
  • bacteriophage is a bacterial-specific virus that inhibits and inhibits the growth of the bacteria by infecting a specific bacterium, and refers to a virus containing a single or double-stranded DNA or RNA as a genetic material. .
  • bacteria are Acinetobacter baumannii (Acinetobacter baumannii), Acinetobacter knife core Shetty kusu (Acinetobacter calcoaceticus), Acinetobacter H. Mori T kusu (Acinetobacter haemolyticus), Acinetobacter gave (Acinetobacter junii ), Acinetobacter johnsonii , Acinetobacter lwoffii , Acinetobacter radioresistens , Acinetobacter ursingii , Acinetobacter ursingii , Acinetobacter lwoffii Acinetobacter schindleri), Acinetobacter Parque booth (Acinetobacter parvus), Acinetobacter Bay Li (Acinetobacter baylyi), Acinetobacter Bow Betty (Acinetobacter bouvetii), Acinetobacter tow Nourishing (Acinetobacter towneri), Acinetobacter tandoyi (Acinetobacter tandoi (Acinetobacter t
  • the bacteriophage has a specific killing ability against bacteria of the genus Acinetobacter, but also has specific killing ability against bacteria of the genus Acinetobacter having antibiotic resistance among the bacteria of the genus Acinetobacter.
  • the "antibiotic resistance” means that the drug does not work because of resistance to a specific antibiotic, and for the purposes of the present invention, the antibiotics are colistin, erythromycin, ampicillin, and MP Cylin-Sulbectam (Ampicillin-s ⁇ lbactam), Vancomycin, Linezolid, Methicillin, Oxacillin, Ceotaxime, Rifampicin, Amikacin (Amikacin), Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Doripenem, Doripenem, Imipenem Imipenem), Imipenem/Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Aztreonam (Aztreonam), Piperacillin, Polymyxin B, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Ga
  • the bacteriophage is a bacteriophage separated by collecting a sample from a sewage treatment plant in a hospital, named bacteriophage YMC14/01/P262_ABA_BP, and on November 15, 2018, the deposit number KFCC11798P in the Korea Microbiology Conservation Center. It may have been deposited.
  • the bacteriophage YMC14/01/P262_ABA_BP of the present invention belongs to the family Myoviridae, which has a long tail with a hexagonal head, and as a result of total nucleotide sequence analysis, it has a size of 44,597 bp and the total number of ORFs is 79. Confirmed.
  • the bacteriophage YMC14/01/P262_ABA_BP in the present invention may include the nucleotide sequence represented by SEQ ID NO: 1 as all or part of the entire gene.
  • the bacteriophage YMC14/01/P262_ABA_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 1, and a functional equivalent of the nucleotide sequence.
  • the functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 1 as a result of modification or substitution of the base sequence. Having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 6.
  • the bacteriophage YMC14/01/P262_ABA_BP provided by the present invention may include any one of SEQ ID NOs: 2 to 4.
  • each of SEQ ID NOs: 2 to 4 may be an ORF (Open reading frame) of the bacteriophage
  • the protein represented by SEQ ID NO: 2 may be an amino acid sequence of a protein presumed to be endolysin
  • the SEQ ID NO: The protein represented by 3 may be an amino acid sequence of a lysozyme-like domain
  • the protein represented by SEQ ID NO: 4 may be an amino acid sequence of a putative tail-fiber/lysosomal protein.
  • SEQ ID NO: 2 may be the amino acid sequence of ORF38
  • SEQ ID NO: 3 is the amino acid sequence of ORF50
  • SEQ ID NO: 4 may be the amino acid sequence of ORF51.
  • 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.
  • SEQ ID NO: 5 is the nucleotide sequence of the genome encoding ORF38
  • SEQ ID NO: 6 is the nucleotide sequence of the genome encoding ORF50
  • SEQ ID NO: 7 may be the nucleotide sequence of the genome encoding ORF51.
  • the bacteriophage is a bacteriophage separated by collecting a sample from a sewage treatment plant in a hospital, named as bacteriophage YMC15/02/T28_ABA_BP, and as deposit number KFCC11799P at the Korea Microbiology Conservation Center on November 15, 2018. It may have been deposited.
  • the bacteriophage YMC15/02/T28_ABA_BP of the present invention belongs to the family Myobiri family, which has a hexagonal head and a long tail, and as a result of total nucleotide sequence analysis, it has a size of 44,580 bp and the total number of ORFs is 77.
  • 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.
  • the bacteriophage YMC15/02/T28_ABA_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 8, and a functional equivalent of the nucleotide sequence.
  • the functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 8 as a result of modification or substitution of the base sequence. Having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 8.
  • the bacteriophage YMC15/02/T28_ABA_BP provided by the present invention may include any one of SEQ ID NOs: 9 to 11.
  • each of SEQ ID NOs: 9 to 11 is an ORF (open reading frame) of the bacteriophage
  • the protein represented by SEQ ID NO: 9 may be an amino acid sequence of a lysozyme-like domain
  • the protein represented by 10 may be an amino acid sequence of a putative tail-fiber/lysosomal protein
  • the protein represented by SEQ ID NO: 11 may be an amino acid sequence of an endolysin putative protein.
  • SEQ ID NO: 9 may be the amino acid sequence of ORF7
  • SEQ ID NO: 10 may be the amino acid sequence of ORF8
  • SEQ ID NO: 11 may be the amino acid sequence of ORF73.
  • 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.
  • SEQ ID NO: 12 is the nucleotide sequence of the genome encoding ORF7
  • SEQ ID NO: 13 is the nucleotide sequence of the genome encoding ORF8
  • SEQ ID NO: 14 may be the nucleotide sequence of the genome encoding ORF73.
  • the bacteriophage is a bacteriophage separated by collecting a sample from a sewage treatment plant in a hospital, named as bacteriophage YMC15/15R1869_ABA_BP, and deposited number KFCC11802P to the Korea Microbiology Conservation Center on November 15, 2018. It may have been deposited as.
  • the bacteriophage YMC15/19R1869_ABA_BP of the present invention belongs to the family Myobiri family, which has a hexagonal head and a long tail, and as a result of total nucleotide sequence analysis, it has a size of 42,555 bp and the total number of ORFs is 77.
  • the bacteriophage YMC15/15R1869_ABA_BP in the present invention may include the nucleotide sequence represented by SEQ ID NO: 15 as all or part of the entire gene.
  • the bacteriophage YMC15/15/15R1869_ABA_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 15, and a functional equivalent of the nucleotide sequence.
  • the functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 15 as a result of modification or substitution of the base sequence. Having the above sequence homology, it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 15.
  • the bacteriophage YMC15/11R1869_ABA_BP provided in the present invention may include any one of SEQ ID NOs: 16 and 17.
  • each of SEQ ID NOs: 16 and 17 is an ORF (Open reading frame) of the bacteriophage
  • SEQ ID NO: 16 may be an amino acid sequence of a lysozyme-like domain
  • SEQ ID NO: 17 is a lysozyme family protein It may be an amino acid sequence of a protein estimated to be.
  • each of SEQ ID NOs: 16 and 17 may be an amino acid sequence of a lysozyme-like domain as ORF7 and ORF73.
  • the bacteriophage YMC15/15R1869_ABA_BP provided by the present invention may include a genome represented by any one of SEQ ID NOs: 18 and 19.
  • SEQ ID NO: 18 may be a nucleotide sequence of a genome encoding ORF7
  • SEQ ID NO: 19 may be a nucleotide sequence of a genome encoding ORF73.
  • the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/19R1869_ABA_BP has excellent stability against heat and pH.
  • the bacteriophage YMC14/01/P262_ABA_BP of the present invention Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/19R1869_ABA_BP maintains lytic activity within the range of 4 to 60° C., but is not limited thereto.
  • the bacteriophage YMC14/01/P262_ABA_BP of the present invention Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/19R1869_ABA_BP maintains lytic activity in the range of pH 3.0 to pH 11.0, preferably pH 5.0 to pH 10.0, but is not limited thereto.
  • the bacteriophage YMC14/01/P262_ABA_BP of the present invention Bacteriophage YMC15/02/T28_ABA_BP; And bacteriophage YMC15/19R1869_ABA_BP composition for the prevention and treatment of infectious diseases caused by bacteria of the genus Acinetobacter, and the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And in applying to a variety of products containing the bacteriophage YMC15/19R1869_ABA_BP as an active ingredient, it enables the application of a variety of pH ranges.
  • the bacteria of the genus Klebsiella are Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella oxytoca (Klebsiella oxytoca), Klebsiella planticola (Klebsiella planticola), and Klebsiella terrigena (Klebsiella terrigena) can be any one or more selected from the group consisting of, and from a clinical point of view as pneumococcal Klebsiella pneumoniae, which accounts for most of the bacteria in the genus Klebsiella, is known as a bacterium representing the bacteria in the genus Klebsiella.
  • the bacteriophage has a specific killing ability against bacteria of the genus Klebsiella, but also has a specific killing ability against bacteria of the genus Klebsiella having antibiotic resistance among bacteria of the genus Klebsiella.
  • the "antibiotic resistance" means that the drug does not work because of resistance to a specific antibiotic
  • the antibiotic may be an antibiotic having a structure of carbapenem.
  • Amikacin, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Ceftazidime, Cefazoline, Imipenem ), Ertapenem, Cefepime, Cefoxitin, Cefotaxime, Gentamicine, Levoflocacin, Meropenem, Piperacillin / Tazobactam (Piperacillin/Tazobactam), Cortrimoxa, and Tigecyline may be one or more selected from the group consisting of, but is not limited thereto.
  • the Klebsiella pneumoniae may have antibiotic resistance, and the antibiotic resistance may be generated by producing a carbapenemase enzyme that degrades the carbapenem and inhibits the exertion of the effect. .
  • 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.
  • the bacteriophage YMC17/01/P6_KPN_BP of the present invention may consist of a nucleotide sequence represented by SEQ ID NO: 20, and a functional equivalent of the nucleotide sequence.
  • the functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the base sequence represented by SEQ ID NO: 20 as a result of modification or substitution of the base sequence.
  • sequence homology it means a sequence that exhibits substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO: 20.
  • the bacteriophage provided by the present invention may be one containing any one of SEQ ID NOs: 21 and 22 protein.
  • each of SEQ ID NOs: 21 and 22 is an ORF (Open reading frame) of the bacteriophage, and among proteins that perform adsorption and lysis functions in the genus Klebsiella, in particular, holin or anti-holin ) May be the amino acid sequence of the protein, and more specifically, SEQ ID NOs: 21 and 22 may be the amino acid sequences of ORF57 and ORF59, respectively.
  • ORF Open reading frame
  • the bacteriophage provided by the present invention may include a genome represented by any one of SEQ ID NOs: 23 and 24.
  • SEQ ID NO: 23 is the nucleotide sequence of the genome encoding ORF57
  • SEQ ID NO: 24 is the nucleotide sequence of the genome encoding ORF59.
  • the bacteriophage YMC17/01/P6_KPN_BP of the present invention has excellent lytic activity against bacteria of the genus Klebsiella, particularly carbapenem-based antibiotic-resistant pneumoniae. It was confirmed that the bacteriophage YMC17/01/P6_KPN_BP belongs to the family Siphoviridae, which is a form having an angled head and tail, and as a result of total nucleotide sequence analysis, it has a size of 54,880 bp and the total number of ORFs is 87. Confirmed.
  • the bacteriophage YMC17/01/P6_KPN_BP of the present invention is a bacteriophage separated by collecting samples from a sewage treatment plant in a hospital, named bacteriophage YMC17/01/P6_KPN_BP, and deposited with the Korea Microbiology Conservation Center as the deposit number KFCC11804P on November 15, 2018. I did.
  • the bacteriophage YMC17/01/P6_KPN_BP of the present invention maintains lytic activity in the range of 4° C. to 60° C., but is not limited thereto.
  • the bacteriophage YMC17/01/P6_KPN_BP of the present invention maintains lytic activity in the range of pH 3.0 to pH 11.0, preferably pH 5.0 to pH 10.0, but is not limited thereto.
  • the bacteriophage YMC17/01/P6_KPN_BP of the genus Klebsiella has specific lytic activity, acid resistance and basic resistance
  • the bacteriophage of the present invention is a composition for preventing and treating infectious diseases caused by bacteria of the genus Klebsiella.
  • bacteriophage YMC14/01/P262_ABA_BP Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/19R1869_ABA_BP; Or it provides a composition for preventing, improving or treating diseases caused by Acinetobacter genus bacteria or Klebsiella bacteria comprising bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.
  • the bacteriophage and the Acinetobacter genus bacterium or the Klebsiella bacterium are duplicated as described in the bacteriophage, and detailed descriptions thereof will be omitted below.
  • the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/19R1869_ABA_BP specifically kills bacteria of the genus Acinetobacter, particularly antibiotic-resistant bacteria of the genus Acinetobacter, and thus shows an effect in the treatment of various diseases caused by the bacteria of the genus Acinetobacter.
  • the infectious diseases caused by the bacteria of the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondylom, vancomycin-resistant yellow staphylococcus aureus infection, and vancomycin endocytic fungal infection.
  • carbapenem intragrowth mycobacterial infection intestinal infection, acute respiratory tract infection, and enterovirus infection
  • enterovirus infection it is not limited thereto.
  • composition of the present invention may include 1 X 10 3 to 1 X 10 10 PFU/mL of bacteriophage, and preferably 1 X 10 6 to 1 X 10 9 PFU/mL of bacteriophage.
  • PFU plaque forming unit
  • PFU plaque forming unit
  • the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumococcal bacteria having resistance to carbapenem antibiotics, and thus has an effect on the treatment of various diseases caused by the bacteria of the genus Klebsiella. Show.
  • the infectious diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media.
  • Bacteremia, endocarditis, cholecystitis or parotitis which is particularly a disease caused by pneumococcal, but is not limited thereto.
  • composition of the present invention may include 1 X 10 3 to 1 X 10 10 PFU/mL of bacteriophage, and preferably 1 X 10 6 to 1 X 10 9 PFU/mL of bacteriophage.
  • PFU plaque forming unit
  • PFU plaque forming unit
  • bacteriophage YMC14/01/P262_ABA_BP Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/19R1869_ABA_BP; Or it provides a composition for preventing, improving or treating diseases caused by Acinetobacter genus bacteria or Klebsiella bacteria comprising bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.
  • the bacteriophage and the Acinetobacter genus bacterium or the Klebsiella bacterium are duplicated as described in the bacteriophage, and detailed descriptions thereof will be omitted below.
  • the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/19R1869_ABA_BP specifically kills bacteria of the genus Acinetobacter, particularly antibiotic-resistant bacteria of the genus Acinetobacter, and thus shows an effect in the treatment of various diseases caused by the bacteria of the genus Acinetobacter.
  • the infectious diseases caused by the bacteria of the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondylom, vancomycin-resistant yellow staphylococcus aureus infection, and vancomycin endocytic fungal infection.
  • carbapenem intragrowth mycobacterial infection intestinal infection, acute respiratory tract infection, and enterovirus infection
  • enterovirus infection it is not limited thereto.
  • composition of the present invention may include 1 X 10 3 to 1 X 10 10 PFU/mL of bacteriophage, and preferably 1 X 10 6 to 1 X 10 9 PFU/mL of bacteriophage.
  • PFU plaque forming unit
  • PFU plaque forming unit
  • the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumococcal bacteria having resistance to carbapenem antibiotics, and thus has an effect on the treatment of various diseases caused by the bacteria of the genus Klebsiella. Show.
  • the infectious diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media.
  • Bacteremia, endocarditis, cholecystitis or parotitis which is particularly a disease caused by pneumococcal, but is not limited thereto.
  • composition of the present invention may include 1 X 10 3 to 1 X 10 10 PFU/mL of bacteriophage, and preferably 1 X 10 6 to 1 X 10 9 PFU/mL of bacteriophage.
  • PFU plaque forming unit
  • PFU plaque forming unit
  • bacteriophage YMC14/01/P262_ABA_BP Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/19R1869_ABA_BP; Or it provides a composition for preventing, improving or treating diseases caused by Acinetobacter genus bacteria or Klebsiella bacteria comprising bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.
  • the bacteriophage and the Acinetobacter genus bacterium or the Klebsiella bacterium are duplicated as described in the bacteriophage, and detailed descriptions thereof will be omitted below.
  • the bacteriophage YMC14/01/P262_ABA_BP; Bacteriophage YMC15/02/T28_ABA_BP; And the bacteriophage YMC15/19R1869_ABA_BP specifically kills bacteria of the genus Acinetobacter, particularly antibiotic-resistant bacteria of the genus Acinetobacter, and thus shows an effect in the treatment of various diseases caused by the bacteria of the genus Acinetobacter.
  • the infectious diseases caused by the bacteria of the genus Acinetobacter are hepatitis C, hand, foot and mouth disease, gonorrhea, chlamydia, dyslexia, genital herpes, cheumgyucondylom, vancomycin-resistant yellow staphylococcus aureus infection, and vancomycin endocytic fungal infection.
  • carbapenem intragrowth mycobacterial infection intestinal infection, acute respiratory tract infection, and enterovirus infection
  • enterovirus infection it is not limited thereto.
  • composition of the present invention may include 1 X 10 3 to 1 X 10 10 PFU/mL of bacteriophage, and preferably 1 X 10 6 to 1 X 10 9 PFU/mL of bacteriophage.
  • PFU plaque forming unit
  • PFU plaque forming unit
  • the bacteriophage YMC17/01/P6_KPN_BP specifically kills bacteria of the genus Klebsiella, particularly pneumococcal bacteria having resistance to carbapenem antibiotics, and thus has an effect on the treatment of various diseases caused by the bacteria of the genus Klebsiella. Show.
  • the infectious diseases caused by bacteria of the genus Klebsiella include pneumonia, urinary tract infection, wound infection, meningitis, osteomyelitis, wound infection, endophthalmitis, endophthalmitis, liver abscess, sore throat, diarrhea, sepsis, sinusitis, rhinitis, otitis media.
  • Bacteremia, endocarditis, cholecystitis or parotitis which is particularly a disease caused by pneumococcal, but is not limited thereto.
  • composition of the present invention may include 1 X 10 3 to 1 X 10 10 PFU/mL of bacteriophage, and preferably 1 X 10 6 to 1 X 10 9 PFU/mL of bacteriophage.
  • PFU plaque forming unit
  • PFU plaque forming unit
  • the bacteriophage YMC14/01/P262_ABA_BP Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/19R1869_ABA_BP; Or it provides a cleaning agent, including bacteriophage YMC17/01/P6_KPN_BP.
  • the bacteriophage YMC14/01/P262_ABA_BP of the present invention Bacteriophage YMC15/02/T28_ABA_BP; Alternatively, the bacteriophage YMC15/19R1869_ABA_BP has a specific killing ability for bacteria of the genus Acinetobacter or Klebsiella, so the skin surface or body of an individual exposed to or likely to be exposed to the genus Acinetobacter or Klebsiella It can also be used for washing each area.
  • the present invention is a bacteriophage YMC14/01/P262_ABA_BP for use for the manufacture of a cleaning agent; Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/19R1869_ABA_BP; Or it may be to use bacteriophage YMC17/01/P6_KPN_BP.
  • the bacteriophage YMC14/01/P262_ABA_BP Bacteriophage YMC15/02/T28_ABA_BP; Bacteriophage YMC15/19R1869_ABA_BP; Or it provides a pharmaceutical composition for the prevention or treatment of diseases caused by bacteria of the genus Acinetobacter or Klebsiella genus, comprising the bacteriophage YMC17/01/P6_KPN_BP as an active ingredient.
  • Acinetobacter genus comprising administering any one of the bacteriophages selected from claims 1 to 25 as an active ingredient to a subject in need of prevention or treatment
  • it may be a method of preventing or treating diseases caused by bacteria of the genus Klebsiella.
  • the pharmaceutical composition may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that for humans.
  • the pharmaceutical composition of the present invention is not limited thereto, but is formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injectable solutions according to conventional methods.
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections.
  • Agents, solubilizers, isotonic agents, stabilizers, and the like can be mixed and used, and in the case of topical administration, a base agent, excipient, lubricant, preservative, etc. can be used.
  • the formulation of the pharmaceutical composition of the present invention may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier as described above.
  • it in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc.In the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like may additionally be included.
  • the route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local , Sublingual or rectal. Oral or parenteral administration is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration.
  • the pharmaceutical composition of the present invention depends on a number of factors including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day. It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.
  • the pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
  • the cosmetic composition is a lotion, nutritional lotion, nutritional essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, Suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory ⁇ cosmetic ⁇ , face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, Bath soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap (non-medical), cream soap, facial wash, body cleaner, scalp cleaner, hair rinse, makeup soap, tooth whitening gel, toothpaste, etc. It can be manufactured in a form.
  • the composition of the present invention may further include a solvent or a suitable carrier, excipient, or diluent that is commonly used in the manufacture of cosmetic compositions.
  • the type of solvent that can be further added to the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used.
  • a carrier excipient or diluent, purified water, oil, wax , Fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like, but are not limited thereto.
  • whitening agents, moisturizing agents, vitamins, sunscreen agents, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.
  • Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm seed oil, jojoba oil, and avocado oil may be used as the oil. Can be used.
  • Stearic acid, linoleic acid, linolenic acid, and oleic acid may be used as the fatty acid
  • cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol may be used as fatty acid alcohols.
  • fatty acid ester isopropyl myristate, isopropyl palmitate, and butyl stearate may be used.
  • surfactant cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as far as possible.
  • hygroscopic agents thickeners, antioxidants, and the like, which are widely known in the cosmetic field, may be included, and the types and amounts thereof are as known in the art.
  • the food composition of the present invention can be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, and bread. Since the food composition of the present invention is composed of plant extracts with little toxicity and side effects, it can be safely used even when taken for a long time for prophylactic purposes.
  • the amount may be added in a proportion of 0.1 to 50% of the total weight.
  • the food composition is prepared in the form of a beverage
  • various flavoring agents or natural carbohydrates, etc. may be included as in a conventional beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are included. can do.
  • flavoring agent examples include natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like.
  • These components may be used independently or in combination.
  • the proportion of these additives is not so important, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.
  • the novel bacteriophage provided by the present invention has a specific killing ability for Acinetobacter genus or Klebsiella bacteria, and Acinetobacter genus or Klebsiella bacteria resistant to antibiotics compared to chemical substances such as conventional antibiotics. Have.
  • the bacteriophage of the present invention does not infect other hosts other than bacteria such as humans, animals, plants, etc., it has the advantage of solving the problems of antibiotic-resistant bacteria due to abuse of antibiotics, the problem of residual antibiotics in food, and problems of a wide host range have.
  • the bacteriophage of the present invention can be used in various fields in the field of prevention or treatment of infectious diseases caused by Acinetobacter genus or Klebsiella bacteria, antibiotic compositions, feed addition compositions, feed, disinfectants, or cleaning agents.
  • FIG. 1 shows an electron microscope photograph of a bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention.
  • Figure 2 is a graph showing the adsorption capacity of the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention against bacteria in Acinetobacter genus having antibiotic resistance.
  • Figure 3 shows a one-stage proliferation curve of the lytic bacteriophage YMC14/01/P262_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • Figure 4 is a graph showing the lytic ability of the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention against bacteria in Acinetobacter genus having antibiotic resistance in vitro.
  • Figure 5 is a graph showing the change in survival rate of the larva after treating the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention to the larvae of the bees infected with bacteria of the genus Acinetobacter having antibiotic resistance. .
  • FIG. 6 is a graph showing the pH stability of the lytic bacteriophage YMC14/01/P262_ABA_BP against bacteria in the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 7 is a graph showing the temperature stability of the lytic bacteriophage YMC14/01/P262_ABA_BP against bacteria in the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 8 shows the result of analyzing the entire genome sequence of the bacteriophage YMC14/01/P262_ABA_BP according to an embodiment of the present invention.
  • FIG. 9 shows an electron microscope photograph of the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention.
  • FIG. 10 is a graph showing the adsorption capacity of the bacteriophage YMC15/02/T28_ABA_BP against bacteria in Acinetobacter genus having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 11 shows a one-stage proliferation curve of the lytic bacteriophage YMC15/02/T28_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 12 is a graph showing the lytic ability of the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention against bacteria in Acinetobacter genus having antibiotic resistance in vitro.
  • FIG. 13 is a graph showing the change in the survival rate of the larva after treatment with the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention to the larvae of the honey bee infected with bacteria of the genus Acinetobacter having antibiotic resistance. .
  • FIG. 14 is a graph showing the pH stability of the lytic bacteriophage YMC15/02/T28_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 15 is a graph showing the temperature stability of the lytic bacteriophage YMC15/02/T28_ABA_BP against bacteria in the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • Figure 16 shows the result of analyzing the entire genome sequence of the bacteriophage YMC15/02/T28_ABA_BP according to an embodiment of the present invention.
  • Figure 17 shows an electron microscope photograph of the bacteriophage YMC15/19R1869_ABA_BP according to an embodiment of the present invention.
  • FIG. 18 is a graph showing the adsorption capacity of the bacteriophage YMC15/19R1869_ABA_BP against bacteria in Acinetobacter genus having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 19 shows a one-stage proliferation curve of the lytic bacteriophage YMC15/19R1869_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 20 is a graph showing the change in survival rate of the larva after treatment with the bacteriophage YMC15/19R1869_ABA_BP according to an embodiment of the present invention to bee larvae infected with Acinetobacter Baumani having antibiotic resistance. .
  • Figure 21 shows the number of bacteria of the Acinetobacter Baumani in the lungs of the mouse after treatment with the bacteriophage YMC15/19R1869_ABA_BP according to an embodiment of the present invention to a mouse infected with an antibiotic-resistant Acinetobacter Baumani It shows the change in a graph.
  • FIG. 22 is a graph showing the pH stability of the lytic bacteriophage YMC15/19R1869_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 23 is a graph showing the temperature stability of the lytic bacteriophage YMC15/19R1869_ABA_BP against bacteria of the genus Acinetobacter having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 24 shows the results of analyzing the entire genome sequence of the bacteriophage YMC15/19R1869_ABA_BP according to an embodiment of the present invention.
  • 25 shows an electron microscope photograph of a bacteriophage according to an embodiment of the present invention.
  • 26 is a graph showing the adsorption capacity of a bacteriophage according to an embodiment of the present invention to bacteria of the genus Klebsiella having antibiotic resistance.
  • FIG. 27 shows a one-stage proliferation curve of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 28 is a graph showing the lytic ability of bacteriophage against bacteria of genus Klebsiella having antibiotic resistance in vitro according to an embodiment of the present invention.
  • 29 is a graph showing the pH stability of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 30 is a graph showing the temperature stability of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.
  • FIG. 31 shows the result of analyzing the entire genome sequence of lytic bacteriophage against bacteria of the genus Klebsiella having antibiotic resistance according to an embodiment of the present invention.
  • the present invention relates to a bacteriophage having a specific killing ability for bacteria of the genus Acinetobacter or Klebsiella.
  • Acinetobacter baumannii Acinetobacter baumannii bacteria were isolated and cultured from ICU (intensive care unit) blood and clinical samples of university hospitals. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Thereafter, the antibiotic susceptibility test was performed using a CLSI disc diffusion test method in which Mueller-Hinton agar was used for overnight culture at 37° C. outside air, and the test antibiotics were amicacin, ampicillin-sulfur.
  • Bectam (ampicillin-sulbactam), ceftazidime, ciprofloxacin, colistin, cefepime, cefotaxime, gentamicine, imipenem, levofloxacin levofloxacin), meropenem, minocycline, piperacillin, piperacillin-tazobactam, cotrimoxa and tigecycline were used.
  • the sensitivity results were read based on the Clinical and Laboratory Standards Institute (CLSI, 2016).
  • the antibiotic resistance profiles of the collected Acinetobacter baumannii strains are shown in Tables 2 to 4 below.
  • S, I, and R are the results of evaluating the sensitivity to antimicrobial agents, where'S' is sensitive (Susceptible),'I' is intermediate (Intermediate), and'R' is resistant (Resistant). Means.
  • the collected Acinetobacter baumannii 32 strains were found to be multi-resistance strains having resistance to various antibiotics.
  • Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours.
  • PEG polyethylene glycol
  • the filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.
  • SM buffer phage dilution buffer
  • a suspension of each strain was prepared with McFarland 0.5 turbidity in a 1 ml saline tube, and H top agar (3 ml), 100 ⁇ l of sensitive bacteria, and phage solution (1 ⁇ l, 10 ⁇ l and 50 ⁇ l, respectively) were mixed and applied to LB agar, Incubated for 12 hours at 35 °C. After observing the plaque, the plaque was collected with a Pasteur pipette, diluted in SM buffer solution, and purified again three times using a susceptible strain suspension. The pure bacteriophage thus obtained YMC14/01/P262_ABA_BP was diluted in SM buffer solution and purified three times again using a susceptible strain suspension. The pure bacteriophage thus obtained YMC14/01/P262_ABA_BP was diluted and stored in SM buffer solution.
  • Antibiotic-resistant Acinetobacter baumannii ( Acinetobacter baumannii ) 32 strains identified in 1. above were inoculated and cultured on Macconki agar medium, and then the bacteriophage YMC14/01/P262_ABA_BP purified by the above process was smeared with each resistance. Plaque formation was confirmed by inoculating the strain with 5 ⁇ l, and the titer range was checked, and lytic properties are shown in Table 5 below. However, in Table 5 below, + and-are the evaluation of plaque activity for the collected strains,'+' means clear plaque, and'-' means that lysis has not occurred.
  • Bacteriophage YMC14/01/P262_ABA_BP purified by the method of 2 above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added to the supernatant. After addition in an amount of 10% (w/v), it was stored refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage YMC14/01/P262_ABA_BP was analyzed using an energy-filtering transmission electron microscope, and the results are shown in FIG. Done.
  • the antibiotic-resistant Acinetobacter Baumani strain was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium,
  • the bacteriophage YMC14/01/P262_ABA_BP purified in 2. above was added to an MOI of 0.001 (titer 10 8 pfu/cell) and incubated at 37° C. for 5 minutes.
  • a pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 3.
  • the result of the single-stage proliferation curve showed a high burst size of approximately 79 PFU/infected cells (0 min: 8 PFU/ml, 100 min: 636 PFU/ml).
  • the bacteriophage YMC14/01/P262_ABA_BP according to the present invention can be adsorbed in a relatively short time to the Acinetobacter Baumani strain having antibiotic resistance, and exhibits a high burst size of 79 PFU/infected cells. It can be seen that the dissolution effect of
  • Antibiotic-resistant Acinetobacter Baumani strain 1 X 10 9 CFU / ml prepared bacteriophage YMC14/01/P262_ABA_BP 1 X 10 8 CFU / ml (MOI: 0.1), 1 X 10 9 PFU / ml (MOI: 1), Each treatment was performed in an amount of 1 X 10 10 PFU/ml (MOI: 10), and the OD value (wavelength 600 nm) was measured for each time. However, as a negative control, PBS + SM buffer was treated, and the values are shown in FIG. 4.
  • the bacteriophage YMC14/01/P262_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain.
  • the bacteriophage YMC14/01/P262_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.
  • Bacteriophage according to the present invention It was confirmed that the bacteriophage YMC14/01/P262_ABA_BP maintains stability without being destroyed in temperature and alkali.
  • the bacteriophage YMC14/01/P262_ABA_BP solution was cultured at 4° C., 37° C., 50° C., 60° C., and 70° C. for 1 hour, and each sample was sampled with the Acinetobacter Baumani strain 4 Plaque analysis was performed by the method of. And the results are shown in FIG. 7.
  • the bacteriophage YMC14/01/P262_ABA_BP according to the present invention exhibited high stability in both acidic, neutral and alkaline, and the bacteriophage YMC14/01/P262_ABA_BP was particularly neutral (pH 7-8) for 30 days. ) Showed relatively stability.
  • the bacteriophage YMC14/01/P262_ABA_BP showed very high stability up to a high temperature of 70 °C.
  • the bacteriophage YMC14/01/P262_ABA_BP contains a linear dsDNA (linear dsDNA) and was composed of 79 ORFs.
  • the bacteriophage YMC14/01/P262_ABA_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.
  • Acinetobacter baumannii bacteria were isolated and cultured from ICU (intensive care unit) blood and clinical specimens of university hospitals. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Subsequently, the antibiotic susceptibility test was performed using a CLSI disc diffusion test method in which Mueller-Hinton agar was used for overnight culture at 37° C., and the test antibiotics were imipenem, piperacillin-tazobactam.
  • the collected 20 strains of Acinetobacter baumannii were found to be multi-resistance strains having resistance to various antibiotics.
  • Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours.
  • PEG polyethylene glycol
  • the filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.
  • SM buffer phage dilution buffer
  • a suspension of each strain was prepared in a 1 ml saline tube with a turbidity of 0.5 McFarland, H top agar (3 ml), 100 ⁇ l of sensitive bacteria and a phage solution (1 ⁇ l, 10 ⁇ l and 50 ⁇ l, respectively) were mixed and applied to LB agar, Incubated for 12 hours at 35 °C. After observing the plaques, the plaques were collected with a Pasteur pipette, diluted in SM buffer solution, and purified again three times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC15/02/T28_ABA_BP was diluted in SM buffer solution and purified again three times using a susceptible strain suspension. The pure bacteriophage thus obtained YMC15/02/T28_ABA_BP was diluted and stored in SM buffer solution.
  • Host strain Lysis Host strain Lysis Host strain Lysis YMC15/02/T28 + YMC13/2017R2199 - YMC13/02/R669 + YMC13/2017R3526 + YMC13/02/R1380 + YMC13/06/R42 - YMC13/02/P386 + YMC13/06/R633 + YMC13/2017T180 + YMC13/12/P154 +
  • the bacteriophage YMC15/02/T28_ABA_BP purified by the method of 2 above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added to the supernatant. After addition in an amount of 10% (w/v), it was stored in the refrigerator overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage YMC15/02/T28_ABA_BP was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. Done.
  • the antibiotic-resistant Acinetobacter Baumani strain was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium,
  • the bacteriophage YMC15/02/T28_ABA_BP purified in 2. above was added to an MOI of 0.001 (titer 10 8 pfu/cells) and incubated at 37° C. for 5 minutes.
  • a pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 11.
  • the result of the single-stage proliferation curve showed a high burst size of 424 PFU/infected cells (10 min: 0.24 PFU/ml).
  • the bacteriophage YMC15/02/T28_ABA_BP according to the present invention can be adsorbed in a relatively fast time to the Acinetobacter Baumani strain having antibiotic resistance, and exhibits a high burst size of 424 PFU/infected cells. It can be seen that the dissolution effect of
  • Bacteriophage YMC15/02/T28_ABA_BP prepared in antibiotic-resistant Acinetobacter Baumani strain 1 X 10 9 CFU/ml 1 X 10 8 CFU/ml (MOI: 0.1), 1 X 10 9 PFU/ml (MOI: 1), Each treatment was performed in an amount of 1 X 10 10 PFU/ml (MOI: 10), and the OD value (wavelength 600 nm) was measured for each time. However, PBS + SM buffer was treated as a negative control, and the values are shown in FIG. 12.
  • the bacteriophage YMC15/02/T28_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain.
  • the bacteriophage YMC15/02/T28_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.
  • the bacteriophage YMC15/02/T28_ABA_BP solution was cultured at 4° C., 37° C., 50° C., 60° C., and 70° C. for 1 hour, and each sample was sampled with the Acinetobacter Bowmani strain 4 Plaque analysis was performed by the method of.
  • the bacteriophage YMC15/02/T28_ABA_BP according to the present invention exhibited the most stability at neutrality corresponding to pH 7.5, and the bacteriophage YMC15/02/T28_ABA_BP was relatively stable in neutral/alkaline for 30 days. Indicated.
  • the bacteriophage YMC15/02/T28_ABA_BP showed very high stability up to a high temperature of 50°C.
  • the entire gene sequence analysis was performed through the Illumina sequencer (Roche), based on the method of analyzing the entire genome sequence, which is obvious to the skilled person, and the results are shown. 16 and Tables 17 to 22.
  • the bacteriophage YMC15/02/T28_ABA_BP contains a linear dsDNA (linear dsDNA) and was composed of 77 ORFs.
  • the bacteriophage YMC15/02/T28_ABA_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.
  • Acinetobacter baumannii strain was isolated and cultured from ICU (intensive care unit) blood and clinical specimens of university hospitals. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Thereafter, the antibiotic susceptibility test was performed using a CLSI disc diffusion test method in which Mueller-Hinton agar was used for overnight culture at 37° C. outside air, and the test antibiotics were amicacin, ampicillin-sulfur.
  • Host strain origin Host strain Lysis YMC16/12/R12914 Phlegm (pneumonia) YMC16/01/R198 Tracheal inhalation (pneumonia YMC16/12/B11422 Catheter blood YMC16/01/R353 Phlegm (pneumonia) YMC16/12/B11449 blood YMC16/01/R405 Phlegm (pneumonia) YMC16/12/B10832 blood YMC16/01/R397 Phlegm (pneumonia) YMC16/12/B13325 Catheter blood YMC16/01/R380 Phlegm (pneumonia) YMC17/01/P518 Swab or drainage tube YMC16/12/R4637 Phlegm (pneumonia) YMC17/01/B8053 Catheter blood YMC17/01/R2812 Phlegm (pneumonia) YMC17/01/B10087 Catheter blood YMC17/02/R541 Phlegm (pneumonia) Y
  • the collected Acinetobacter baumannii 57 strains were found to be multi-resistance strains having resistance to various antibiotics.
  • Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours.
  • PEG polyethylene glycol
  • the filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.
  • SM buffer phage dilution buffer
  • a suspension of each strain was prepared in a 1 ml saline tube with a turbidity of 0.5 McFarland, H top agar (3 ml), 100 ⁇ l of sensitive bacteria and a phage solution (1 ⁇ l, 10 ⁇ l and 50 ⁇ l, respectively) were mixed and applied to LB agar, Incubated for 12 hours at 35 °C. After observing the plaques, the plaques were collected with a Pasteur pipette, diluted in SM buffer solution, and purified again three times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC15/15R1869_ABA_BP was diluted in SM buffer solution and purified again 3 times using a susceptible strain suspension. The thus obtained pure bacteriophage YMC15/19R1869_ABA_BP was diluted and stored in SM buffer solution.
  • the bacteriophage YMC15/19R1869_ABA_BP purified by the method of 2 above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added to the supernatant. After addition in an amount of 10% (w/v), it was stored refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage YMC15/19R1869_ABA_BP was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. Done.
  • the antibiotic-resistant Acinetobacter Baumani strain was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium,
  • the bacteriophage YMC15/19R1869_ABA_BP purified in 2. above was added to an MOI of 0.001 (titer 10 8 pfu/cells) and incubated at 37° C. for 5 minutes.
  • a pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 19.
  • the bacteriophage YMC15/19R1869_ABA_BP according to the present invention can be adsorbed in a relatively short time to the Acinetobacter Baumani strain having antibiotic resistance, and exhibited a high burst size of 78 PFU/infected cells, resulting in an antibiotic resistant strain. It can be seen that the dissolution effect of
  • the bacteriophage YMC15/19R1869_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.
  • the bacteriophage YMC15/19R1869_ABA_BP according to the present invention has lytic properties against the antibiotic-resistant Acinetobacter Baumani strain even in vivo, and thus effectively prevents infectious diseases caused by the Acinetobacter Baumani strain. , It can be seen that it can be improved or cured.
  • Bacteriophage according to the present invention It was confirmed that the bacteriophage YMC15/19R1869_ABA_BP maintains stability without being destroyed in temperature and alkali.
  • the bacteriophage YMC15/19R1869_ABA_BP solution was cultured at 4° C., 37° C., 50° C., 60° C., and 70° C. for 1 hour, and each sample was sampled with the Acinetobacter Bowmani strain 4 Plaque analysis was performed by the method of. And the results are shown in FIG. 23.
  • the bacteriophage YMC15/19R1869_ABA_BP according to the present invention exhibited stability in both acidic, neutral and alkaline, but for 30 days, the bacteriophage YMC15/19R1869_ABA_BP showed relatively stability in neutral/alkaline properties. Done.
  • the bacteriophage YMC15/19R1869_ABA_BP showed very high stability up to a high temperature of 70°C.
  • the bacteriophage YMC15/19R1869_ABA_BP contains a linear dsDNA (linear dsDNA) and was composed of 77 ORFs.
  • the bacteriophage YMC15/19R1869_ABA_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.
  • Klebsiella pneumoniae bacteria were cultured and isolated from Severance Hospital patients or their feces. Strain identification was performed using a kit/ATB 32 GN system (bioMerieux, Marcy l'Etoile, France). Then, for the antibiotic susceptibility test, a CLSI disk diffusion test method was used in which Mueller-Hinton agar was used and cultured overnight at 37° C. outside air.
  • Test antibiotics for Klebsiella pneumoniae bacteria are Amikacin, Ampicillin, Ampicillin/Sulbactam, Aztreonam, Ceftazidime, Cefazoline, Imipenem, Ertapenem, Cefepime, Cefoxitin, Cefotaxime, Gentamicine, Levofloxacin, Meropenem (Meropenem), piperacillin/tazobactam, Cortrimoxa, and Tigecyline were used. The sensitivity results were read based on the Clinical and Laboratory Standards Institute (CLSI, 2016). The antibiotic resistance profiles of the collected Klebsiella pneumoniae 47 strains are shown in Tables 37 to 40 below.
  • S, I and R are the results of evaluating the sensitivity to antimicrobial agents, where'S' is sensitive (Susceptible),'I' is intermediate (Intermediate), and'R' is resistant (Resistant). Means.
  • the collected Klebsiella pneumoniae 47 bacteria were found to be multi-resistance strains resistant to various carbapenem antibiotics.
  • Raw water from which suspended substances and sediments were removed was secured after the first sedimentation at Severance Hospital's sewage treatment facility. This was limited to the sewage of the pre-chemical treatment plant. After adding 58 g of sodium chloride per 1 L to the collected sample, it was centrifuged at 10,000 g for 10 minutes and filtered through a 220 nm Millipore filter. Polyethylene glycol (PEG, molecular weight 8000) was added to the obtained filtrate at 10% W/V and stored refrigerated at 4° C. for 12 hours.
  • PEG polyethylene glycol
  • the filtrate stored refrigerated for 12 hours was centrifuged at 12,000 g for 20 minutes, and the precipitate was resuspended in phage dilution buffer (SM buffer), and then the same amount of chloroform was added and stored frozen. This was repeated three times to collect 300 mL of bacteriophage suspension.
  • SM buffer phage dilution buffer
  • Each of the 47 antibiotic-resistant Klebsiella pneumoniae strains identified in the above 1. was inoculated and cultured on McConkie agar medium, and then the bacteriophage YMC17/01/P6_KPN_BP purified by the above process was smeared. Plaque formation was confirmed by inoculating the resistant strain with 5 ⁇ l, and the titer range was confirmed, and the lytic properties are shown in Table 41 below. However, in Table 41 below, + and-are the evaluation of plaque activity for the collected strains,'+' means clear plaque, and'-' means that lysis has not occurred.
  • Host strain Lysis Host strain Lysis Host strain Lysis YMC16/12/N708 ++ YMC17/2017N331 + YMC16/12/N681 ++ YMC17/2017N355 + YMC17/01/N115 + YMC17/2017R3201 + YMC17/01/P6 + YMC17/2017N405 + YMC17/01/N167 + YMC17/2017N500 + YMC17/01/N132 + YMC17/2017N424 - YMC17/01/N270 + YMC17/2017N421 - 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/N
  • the bacteriophage purified by the method of 2. above was inoculated and cultured in a sensitive strain culture medium (20 ml LB medium), filtered through a 220 nm Millipore filter, and polyethylene glycol (MW 8,000) was added 10% (w/) to the supernatant. After addition in the amount of v), it was kept refrigerated overnight. After centrifugation for 20 minutes under the condition of 12,000 g, the bacteriophage shape was analyzed using an Energy-Filtering Transmission Electron Microscope, and the results are shown in FIG. 25.
  • antibiotic-resistant Klebsiella pneumoniae was cultured to have an OD value of 0.3, and then centrifuged at 7,000 g for 5 minutes at 4° C. to precipitate the cells, and then diluted in 0.5 ml of LB medium,
  • the bacteriophage purified in Example 2 was added with an MOI of 0.001 (titer 10 8 pfu/cells) and incubated at 37° C. for 5 minutes.
  • a pellet obtained by centrifuging the cultured mixed sample at 13,000 g for 1 minute was diluted in 10 ml of LB medium and cultured at 37°C. Samples were collected every 10 minutes during cultivation, and the one-stage proliferation curve of the bacteriophage was evaluated through plaque analysis, and the results are shown in FIG. 27.
  • the bacteriophage YMC17/01/P6_KPN_BP according to the present invention can be adsorbed to Klebsiella pneumoniae having antibiotic resistance within a relatively short time, and exhibited a high burst size of 43 PFU/infected cells, resulting in antibiotic resistance Kleb It can be seen that it exerts a lytic effect on Siella pneumoniae.
  • Bacteriophage YMC17/01/P6_KPN_BP prepared in antibiotic-resistant Klebsiella pneumoniae 1 X 10 9 CFU/ml 1 X 10 8 CFU/ml (MOI: 0.1), 1 X 10 9 PFU/ml (MOI: 1), Each treatment was performed in an amount of 1 X 10 10 PFU/ml (MOI: 10), and the OD value (wavelength 600 nm) was measured for each time. However, PBS + SM buffer was treated as a negative control, and the values are shown in FIG. 28.
  • the bacteriophage according to the present invention has lytic properties against antibiotic-resistant Klebsiella pneumoniae.
  • the bacteriophage YMC17/01/P6_KPN_BP according to the present invention maintains stability without being destroyed in temperature and alkali.
  • the bacteriophage solution was cultured at 4° C., 37° C., 60° C., and 70° C. for 1 hour, and each sample was analyzed with Klebsiella pneumoniae by the method of 4. The results are shown in FIG. 30.
  • the bacteriophage YMC17/01/P6_KPN_BP according to the present invention exhibited stability in both acidic, neutral and alkaline corresponding to pH 7, and for 30 days, the bacteriophage was particularly stable in neutral/alkaline. Indicated.
  • the bacteriophage YMC17/01/P6_KPN_BP showed very high stability up to a high temperature of 60 °C.
  • the entire gene sequence analysis was performed through the Illumina sequencer (Roche) based on the method of analyzing the entire genome sequence, which is obvious to the skilled person, and the results are shown. 31 and Tables 42 to 46.
  • the bacteriophage YMC17/01/P6_KPN_BP contains a linear dsDNA (linear dsDNA) and was composed of 87 ORFs.
  • the bacteriophage YMC17/01/P6_KPN_BP according to the present invention corresponds to a novel bacteriophage that has not been previously discovered.
  • the present invention relates to a novel bacteriophage that lyses bacteria, particularly bacteria that are resistant to antibiotics.

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Abstract

La présente invention concerne un bactériophage présentant une activité bactéricide dirigée en particulier contre les bactéries Acinetobacter sp. ou Klebsiella sp..
PCT/KR2019/018053 2018-12-18 2019-12-18 Nouveau bactériophage pour la lyse de bactéries WO2020130652A2 (fr)

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KR1020180164181A KR102189126B1 (ko) 2018-12-18 2018-12-18 항생제 내성을 갖는 아시네토박터 속의 균을 용균하는 신규한 박테리오파지

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