WO2009087356A1 - Phage de changement de gamme d'hôtes - Google Patents

Phage de changement de gamme d'hôtes Download PDF

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Publication number
WO2009087356A1
WO2009087356A1 PCT/GB2008/004302 GB2008004302W WO2009087356A1 WO 2009087356 A1 WO2009087356 A1 WO 2009087356A1 GB 2008004302 W GB2008004302 W GB 2008004302W WO 2009087356 A1 WO2009087356 A1 WO 2009087356A1
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WO
WIPO (PCT)
Prior art keywords
phage
sai
staphylococcus aureus
host range
host
Prior art date
Application number
PCT/GB2008/004302
Other languages
English (en)
Inventor
Nicholas Mann
Original Assignee
Novolytics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novolytics Limited filed Critical Novolytics Limited
Publication of WO2009087356A1 publication Critical patent/WO2009087356A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/445—Staphylococcus aureus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to methods of producing compositions utilising host range change phage, for use to treat bacterial infection. Strains of bacteria used in the methods are also provided.
  • Antibacterial agents in the form of chemically-based antibiotics (i.e. non-viral agents), such as penicillin or tetracycline, are well known.
  • the problem with such antibiotics is that resistance to them is becoming increasingly common. Mutations conferring antibiotic resistance, or genes encoding antibiotic resistance enzymes, such as penicillinases, are becoming increasingly common in pathogenic bacteria world-wide.
  • Methicillin-resistant Staphylococcus aureus (MRSA) bacteria for example, are an increasingly common form of infection, often acquired during surgery for other causes at hospitals. MRSA infections are extremely difficult to treat using conventional antibiotics.
  • bacteriophage therapy was first developed early in the twentieth century, but has been little used in the West since the advent of antibiotics in the 1940s. More extensive work has been carried out in Eastern Europe.
  • Bacteriophages are specific to specific kinds of bacterial cells. They cannot infect the cells of more complex organisms because of major differences in key intracellular machinery, as well as in cell-surface components. Most bacteriophages have structures, such as tail fibres, which enable the bacteriophages to bind to specific molecules on the surface of their target bacteria. Viral DNA within the bacteriophages, or RNA in some bacteriophages, is then injected, usually through the tail, into the host cell, which then directs the production of progeny bacteriophage.
  • bacteriophages which infect different bacteria. Conventionally, they can be isolated from the environment in which the particular bacterium grows, for example from sewage or faeces.
  • the presence of a bacteriophage in a sample may be determined by passing the sample through a filter with pores small enough to prevent the bacteria getting through the filter.
  • the filtered extract is usually mixed with growth medium, suitable host bacteria added, and then spread on, for example, an agar plate.
  • the presence of clear spots, called plaques, on the resulting lawn of bacteria indicates the presence of one or more bacteriophages, which cause the bacteria to lyse.
  • Lytic bacteriophages Bacteriophages which can only kill bacteria are known as "lytic" bacteriophages. Lytic bacteriophages exist outside the bacterial cell in the form of nucleic acid material, usually DNA, surrounded by a protein coat. The protein coat usually has one or more molecules attached to it which allow the bacteriophages to attach to specific molecules on the surface of the bacteria. Upon binding to the bacteria the DNA gains entry into the bacterial host where it is transcribed and translated into various proteins necessary for replication and assembly of new bacteriophage. The DNA is also replicated and is packaged into new bacteriophage which are released upon lysis of the bacterial cell.
  • temperate or lysogenic bacteriophages.
  • These temperate bacteriophages have two life cycles, one in which they lyse the infected cell, and the other in which they enter the prophage state. Lytic bacteriophages always have to infect from outside, reprogram the host cell and release a burst of bacteriophage through breaking open or lysing the infected cell.
  • "Lysogenic" bacteriophages may integrate their DNA into the host bacterial DNA leading to a virtually permanent association as a prophage within a specific bacterium and its progeny. Some prophages do not physically integrate into the chromosome, but exist as an autonomous replicon.
  • the prophage directs the synthesis of a repressor which blocks the expression of its own genes and also those of any closely-related lysogenic bacteriophages. Occasionally, the prophage may escape regulation by its repressor.
  • the prophage DNA may then be cut out of the genome by site-specific recombination, replicated, and the progeny released from the host cell, in most cases by lysis.
  • Lytic bacteriophage have been used to treat bacterial infections. Isolated lytic bacteriophages have been applied to wounds or injected intravenously where they kill bacteria. The advantage of bacteriophages is that they are self-replicating, with as few as one hundred or so bacteriophages being able to kill as many as one hundred million bacteria. The bacteriophages simply replicate themselves by killing bacteria until they have eliminated them from the individual or the environment.
  • WO 01/51066 discloses a method of treating a patient with one or more lytic bacteriophages.
  • US 4,957,686 discloses a method of treating dental caries with bacteriophage.
  • US 5,660,812, US 5,688,501 and US 5,766,892 all show methods of selecting bacteriophages to improve the bacteriophage half-life within the blood of a patient to be treated.
  • US 5,81 1,093 discusses selecting a modified gene encoding one of the capsid (coating) proteins (capsid E) so that the bacteriophages survive in an animal's circulatory system for longer. In the case of the latter patent, the modification was identified as a point mutation within a gene.
  • phages are often bacterial strain-specific.
  • the presence of, for example, a prophage within a bacterium may block the expression of genes from an infectious phage, thus preventing replication of the infectious phage and preventing lysis and killing of the bacterium.
  • a prophage may also cause the destruction of incoming phage DNA.
  • MRSA Methicillin-resistant Staplylococcus aureus
  • Staplylococcus aureus can cause systemic infections or abscesses and ulcers, especially in sick, elderly or immune-compromised patients. It is increasingly a major cause of, or contribution to, death in hospitals. MRSA may reside in the nasal cavity of doctors or visitors without any apparent disease symptoms. However, the bacteria may be spread from person to person, including to patients. Accordingly, killing the bacteria assists in the prevention of the disease.
  • the inventor has realised that being able to change the range of bacteria that a phage can infect so that it can infect an increased number of strains (or hosts) of bacteria would improve the ability to treat or prevent bacterial infections. It is generally known in the art that growing, or culturing, a phage in different strains of bacteria can alter the properties of the phage. This alteration may be epigenetic. That is it is not due to changes in the nucleic acid sequence encoded by the genome of the phage, but instead appears to be due to chemical changes modifying the bases in the DNA. Changes include, for example, changes in the methylation of the nucleic acid in the phage due to DNA methylases.
  • the inventor has found that culturing phage in different strains of bacteria can be used to increase the host range of bacterial strains that can be infected by a phage. This reduces the number of different phage that need to be included in a panel to ensure a broad coverage of different bacterial strains.
  • the invention provides a method producing an antibacterial composition comprising culturing a first form of phage in a first bacterial host strain to produce multiplied phage having a first host range, culturing the multiplied phage in a second bacterial host strain to produce an altered host range phage having an altered host range and mixing the altered host range phage with a first form of phage, to produce a panel of phages.
  • the first form of phage is preferably a phage which is capable of infecting the same species of bacterium as the second form of phage, but which is capable of infecting one or more different strains of the same species of bacterium compared with the second form of phage.
  • the second form of phage may be capable of infecting a different bacterial species.
  • the phage are preferably lytic phage for at least one strain of bacterium.
  • temperate phage or vir mutants of temperate phage may also be used. Vir mutants are described in WO03/080823.
  • the first form of phage are cultured, that is multiplied, using two different strains of bacteria to extend the number of bacterial strains that the phage is capable of infecting and therefore lysing and killing.
  • the second form of phage may also be cultured on two different strains of bacteria to extend its host range.
  • the two forms of phage may be selected so that the host ranges complement each other to improve the overall number of bacterial strains that the antibacterial composition is capable of infecting and killing. There may be some overlap in the host range of the two forms of phage.
  • More than two forms of phage such as three, four, five or more forms of phage may be used in the composition.
  • the phage may be cultured on still further bacterial host strains to further alter the host range.
  • the host range change caused by culturing in the different bacterial host strains is usually epigenetic. Hence, at least some of the altered host range is often lost once the phage has infected, multiplied, lysed and been released from a different bacterium.
  • the method is particularly useful for the preparation of compositions where the composition is put in contact with bacteria, such as on a surface such as mammalian (especially human) skin. In such a situation the initial concentration of the phage is enough to disinfect the lyse bacteria present on the surface.
  • the surface may, for example, be medical equipment, bedding, furniture, walls or floors in a hospital.
  • the surface to be treated is the external skin of a mammal, for example the nasal cavity or the surface of a wound or cut in the surface of a mammal.
  • the mammal is a human.
  • the disinfectant composition may be made in the form of a spray or liquid wash for the surface.
  • the composition may be a hand wash.
  • the composition may take the form of a lotion, cream, ointment, paste, gel, foam, or any other physical form known for topical administration.
  • Such thickened topical formulations are particularly advantageous because the formulations adhere to the area of the skin on which the material is placed, thus allowing a localised high concentration of phage to be introduced to the particular area to be disinfected.
  • the method may comprise adding a paraffin or lanolin-based cream, which are particularly useful for the application of product to the nasal cavity, are generally known in the art.
  • a paraffin or lanolin-based cream which are particularly useful for the application of product to the nasal cavity, are generally known in the art.
  • other thickeners such as polymer thickeners, may be added.
  • the method may also comprise the addition of one or more of the following: water, preservatives, active surfactants, emulsif ⁇ ers, anti-oxidants, or solvents.
  • the phage is a pathogen of a bacterium selected from Staphylococcus, Helicobacter, Klebsiella, Listeria, Mycobacterium, Escherichia, Meningococcus, Campylobacter, Streptococcus, Enterococcus, Shigella, Pseudomonas, Burkholderia, Clostridium, Legionella, Acetinobacter, or Salmonella.
  • a bacterium selected from Staphylococcus, Helicobacter, Klebsiella, Listeria, Mycobacterium, Escherichia, Meningococcus, Campylobacter, Streptococcus, Enterococcus, Shigella, Pseudomonas, Burkholderia, Clostridium, Legionella, Acetinobacter, or Salmonella.
  • the phage is a pathogen of Staphylococcus aureus, especially an MRSA, C. difficile or Pseudomonas aeruginosa.
  • composition may comprise one or more generally known phages which are capable of infecting MRSA such phage P68.
  • the first or second bacterial host strain is preferably selected from Staphylococcus aureus, strains SAI 656 or SAI 661. These have been deposited under The Budapest Treaty at the NCTC (National Collection of Type Cultures), London, United Kingdom as accession numbers NCTC 13426 and NCTC 13425 respectively. They were deposited on 9 November 2007. Such strains were selected because they were observed Io extend the host ranges of phages cultured on them. They are both EMRSA type 15 strains. They were also selected because they were not observed to liberate detectable temperate phage. This later property is an especially preferred property of the phage used in the methods of the invention.
  • One or both of the bacterial host strains on which the first form of phage is cultured may be SAI 656 and/or SAI 661.
  • the second bacterial host strain is SAI 656 or SAI 661.
  • the other bacterial strain used may be SAI 653 (ATCC 19685)
  • Staphylococcus aureus strains SAI 656 (NCTC 13426) and SAI 661 (NCTC 13425) are also included in the scope of the invention.
  • Isolated phage obtainable by culturing a phage on one or more of the strains are also provided.
  • Antibacterial compositions comprising the isolated phage are also provided by the invention.
  • compositions are preferably as described above for any aspect of the invention.
  • the invention also provides methods of killing bacteria on a surface comprising applying a disinfectant composition made by a method of the invention or according to any aspect of the invention, to the surface.
  • a disinfectant composition made by a method of the invention or according to any aspect of the invention, to the surface.
  • This may be used as a disinfectant, for example to prevent the spread of a particular bacterium. It also may be used as a way of inhibiting a bacterial infection on the surface of, for example, skin.
  • the surface is the skin of a mammal, such as a human.
  • the surface may be the nasal cavity of a mammal, or skin on the hands of a human.
  • compositions according to the invention for use to treat a bacterial infection.
  • the disinfectant composition may be applied to a bandage or wound dressing.
  • Methods of making wound dressings or bandages comprising applying a composition made by or according to any aspect of the invention are also provided.
  • the wound dressing may be a pad or sticking plaster-type dressing.
  • the phage and/or concentrations used are preferably as defined above for the previous aspects of the invention.
  • the phage may be applied to the wound dressing or bandage as a disinfectant formulation or topical cream, prior to applying to the wound dressing or bandage.
  • the wound dressing or bandage may be soaked in a carrier containing the phage and dried to leave the phage impregnated within the dressing or bandage.
  • Phage may also be adsorbed onto the surface of the bandage or wound dressing using techniques generally known in the art.
  • Methods of inhibiting or treating bacteria by applying a bandage or wound dressing to a patient are also provided.
  • Bacteriophage P68 and phages derived from it is preferably used in the compositions of the invention. These induce Iysis-from-within in a wide range of MRSA strains.
  • P68 may be obtained from the Felix d'Herelle Reference Center for Bacterial Viruses from the Universite Laval (HER49). Other phage may also be used.
  • a stock preparation of phage P68 was serially 10- fold diluted to 10 ⁇ 8 with phosphate- buffered saline containing 10 mJVI MgCl 2 (PBS-Mg buffer) (prepared from NaCl 8 g, KCl 2 g, Na 2 HPO 4 .12H 2 O 3.58 g, KH 2 PO 4 0.27 g and 10 ml 1 M MgSO 4 .7H 2 O, all dissolved in 1 1 deionized water). Aliquots (lOO ⁇ l) of each dilution were incubated with 100 ⁇ l of SAI (Staphylococcus aureus isolate) 653 (S.
  • SAI Staphylococcus aureus isolate
  • APS-Mg-Ca top agar for phage plaque titration was prepared by the addition of 0.7% (w/v) agar and Ca(NO 3 ) 2 and MgSO 4 .7H 2 O to concentrations of 10 mM.
  • the resulting titre of P68 was approximately 1 x 10 n PFU (plaque-forming units) ml "1 .
  • the host range of P68 which had been propagated on SAI653 (10 3 PFU) was then assessed by measuring its plaque forming activity on a range of EMRSA 13, 15 and 16 strains as described above.
  • SAI661 like SAI656, doesn't release spontaneously induced prophages during culture and therefore would be a preferred host over SAI654.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
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  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • Animal Behavior & Ethology (AREA)
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Abstract

L'invention porte sur un procédé de production d'une composition antibactérienne consistant à : (i) cultiver une première forme de phage dans une première souche hôte bactérienne pour produire un phage multiplié ayant une première gamme d'hôtes ; (ii) cultiver le phage multiplié dans une seconde souche hôte bactérienne pour produire un phage amélioré de gamme d'hôtes ayant une gamme d'hôtes étendue ; et (iii) mélanger le phage amélioré de gamme d'hôtes avec une seconde forme de phage pour produire un panel de phages. De préférence, les souches de Staphylococcus aureus SAI 656 et SAI 661 sont utilisées (NCTC 13426 et NCTC 134250).
PCT/GB2008/004302 2008-01-04 2008-12-29 Phage de changement de gamme d'hôtes WO2009087356A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0800149.7 2008-01-04
GBGB0800149.7A GB0800149D0 (en) 2008-01-04 2008-01-04 Improved host range phage

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WO2009087356A1 true WO2009087356A1 (fr) 2009-07-16

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3372085A1 (fr) * 2017-03-08 2018-09-12 Pherecydes Pharma Thérapie phagique
CN109234240A (zh) * 2018-08-22 2019-01-18 中国科学院南京土壤研究所 一种噬菌体组合物及其在灭活抗生素抗性致病细菌中的应用
CN109988753A (zh) * 2019-04-04 2019-07-09 中国科学院南京土壤研究所 肺炎克雷伯氏菌噬菌体的冻干保护剂及其制备方法和应用
US10806769B2 (en) 2016-03-31 2020-10-20 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
US10874700B2 (en) 2016-03-31 2020-12-29 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
US11564879B2 (en) 2016-11-23 2023-01-31 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
EP4144839A1 (fr) 2021-09-03 2023-03-08 Universitat Pompeu Fabra Procédé de criblage de modifications dans la plage d'infectivité de bactériophages en raison de l'empreinte épigénétique
US11998575B2 (en) 2020-11-20 2024-06-04 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient

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CA2304123A1 (fr) * 2000-04-19 2001-10-19 Mount Sinai Hospital Traitement contre les bacteries resistantes aux antibiotiques
WO2003080823A2 (fr) * 2002-03-25 2003-10-02 University Of Warwick Agents anti-bacteriens

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11633451B2 (en) 2016-03-31 2023-04-25 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
US10806769B2 (en) 2016-03-31 2020-10-20 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
US10874700B2 (en) 2016-03-31 2020-12-29 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
US11564879B2 (en) 2016-11-23 2023-01-31 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
WO2018162566A1 (fr) * 2017-03-08 2018-09-13 Pherecydes Pharma Phagothérapie
US11690885B2 (en) 2017-03-08 2023-07-04 Pherecydes Pharma Anti-bacterial compositions and uses thereof
IL268905A (en) * 2017-03-08 2019-10-31 Pherecydes Pharma Faji treatment
CN110545670A (zh) * 2017-03-08 2019-12-06 飞瑞赛德斯制药公司 噬菌体疗法
EP3372085A1 (fr) * 2017-03-08 2018-09-12 Pherecydes Pharma Thérapie phagique
CN110545670B (zh) * 2017-03-08 2021-11-23 飞瑞赛德斯制药公司 噬菌体疗法
CN109234240A (zh) * 2018-08-22 2019-01-18 中国科学院南京土壤研究所 一种噬菌体组合物及其在灭活抗生素抗性致病细菌中的应用
CN109988753B (zh) * 2019-04-04 2021-03-02 中国科学院南京土壤研究所 肺炎克雷伯氏菌噬菌体的冻干保护剂及其制备方法和应用
CN109988753A (zh) * 2019-04-04 2019-07-09 中国科学院南京土壤研究所 肺炎克雷伯氏菌噬菌体的冻干保护剂及其制备方法和应用
US11998575B2 (en) 2020-11-20 2024-06-04 Gojo Industries, Inc. Sanitizer composition with probiotic/prebiotic active ingredient
WO2023031342A1 (fr) 2021-09-03 2023-03-09 Universitat Pompeu Fabra Méthode de criblage de modifications du taux d'infectivité de bactériophages dues à l'empreinte épigénétique
EP4144839A1 (fr) 2021-09-03 2023-03-08 Universitat Pompeu Fabra Procédé de criblage de modifications dans la plage d'infectivité de bactériophages en raison de l'empreinte épigénétique

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