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WO2024007055A1 - Procédé de production de phage modifié et procédé de traitement l'utilisant - Google Patents

Procédé de production de phage modifié et procédé de traitement l'utilisant Download PDF

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WO2024007055A1
WO2024007055A1 PCT/AU2023/050622 AU2023050622W WO2024007055A1 WO 2024007055 A1 WO2024007055 A1 WO 2024007055A1 AU 2023050622 W AU2023050622 W AU 2023050622W WO 2024007055 A1 WO2024007055 A1 WO 2024007055A1
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Prior art keywords
phage
bacteria
species
infection
modified
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PCT/AU2023/050622
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English (en)
Inventor
Peter-John Wormald
Sarah Sylvia Joseph VREUGDE
Sha Liu
Alkiviadis James PSALTIS
George Spyro BOURAS
Keith Edward SHEARWIN
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The University Of Adelaide
Aushealth Corporate Pty Ltd
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Priority claimed from AU2022901885A external-priority patent/AU2022901885A0/en
Application filed by The University Of Adelaide, Aushealth Corporate Pty Ltd filed Critical The University Of Adelaide
Priority to AU2023304968A priority Critical patent/AU2023304968A1/en
Publication of WO2024007055A1 publication Critical patent/WO2024007055A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • 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
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    • 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
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • 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
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2795/00032—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00051—Methods of production or purification of viral material
    • 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
    • C12N2795/00071—Demonstrated in vivo effect
    • 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
    • C12N2795/10121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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

Definitions

  • the present disclosure relates to the field of bacteriophage therapy, particularly in the circumstance of the treatment of a drug-resistant bacteria which is also bacteriophage non-sensitive (insensitive). Among other things, the present disclosure relates to a method of producing modified bacteriophage that are capable of infecting and causing lysis of a parent host bacteria that is insensitive to the unmodified bacteriophage.
  • PRIORITY DOCUMENT [0002] The present application claims priority from Australian Provisional Patent Application No.
  • bacteria antibiotic resistance include either a reduction in the affinity of the antibiotic agent to bind to its bacterial target protein or a reduction in the bacterial intracellular drug concentration.
  • the former is primarily caused by the emergence of mutation(s) in the genes encoding the target protein of the antibiotic, while the latter is mainly achieved through alterations in the plasma membrane permeability, expression of degradation/modification enzymes, or overexpression of efflux pumps.
  • bacteria can increase their tolerance and resistance to antibiotics through the formation of a biofilm, which then acts as a natural barrier to prevent the absorption of antibacterial drugs such as antibiotics.
  • the biofilm drug-resistance mechanism is different from that of individual bacteria and, compared with planktonic bacteria, this drug-resistance mechanism can increase the bacterial antibiotic tolerance 10- to 1000-fold, and is one of the main causes of current bacterial drug resistance.
  • the extracellular polymeric substance (EPS) secreted by bacteria one of the main components of a biofilm, forms a dense and negatively charged natural barrier, which prevents the penetration of antibiotics and other antibacterial drugs into the membrane, thus reducing the concentration of drugs inside the membrane. Additionally, the lack of enough nutrients inside of the biofilm will inhibit the bacterial metabolic activity, and subsequently promote bacteria to enter a dormant state, making them more resistant to the drug during the reproduction period as many antibiotics rely on proliferation of the bacteria for effectiveness.
  • Phage were first applied to anti-bacterial infection treatments in clinics over 100 years ago, and they were utilised specifically to kill and target bacterial hosts causing infection. Due to the discovery and widespread use of antibiotic agents, the use of phage has diminished. However, due to the ongoing problem of drug-resistant bacteria (and especially, the multidrug-resistant (MDR) bacteria), phage therapy has now returned to the spotlight. Further, it is now more fully appreciated that phage possess unique advantages that may mean that phage targeting specific pathogenic bacteria can be isolated to meet clinical needs more quickly than the development of novel antibacterial drugs. Phage are also very abundant in nature, lack cross-resistance with antibiotics, and are highly specific (compared to antibiotics).
  • phage can also be effective in disrupting bacterial biofilms, and offer enormous potential to serve as the basis of therapies providing an effective and sustainable solution for the treatment of bacteria, including treatment for superbugs and refractory MDR bacterial infections.
  • the utility of phage in the clinic has been limited. Part of the reason for the lack of adoption of phage therapy is the narrow antibacterial spectrum of many phage. Further, many clinical isolates are resistant to known phage. In such conditions, a patient may have a considerable waiting period until a suitable phage is available for treating their specific bacterial infection and/or there are no phage suitable to infect a particular resistant bacterial infection.
  • the present disclosure provides a method of producing a modified phage, said method comprising the steps of: (i) treating a host bacteria insensitive to a selected phage strain (ie a phage non-sensitive, or phage insensitive, or phage resistant bacteria) with a suitable amount of the said phage strain and at least one antibacterial agent comprising protein synthesis inhibitor activity, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage; and (ii) recovering and expanding progeny phage which are phage with a modified capability to infect and cause lysis of said host bacteria.
  • a host bacteria insensitive to a selected phage strain ie a phage non-sensitive, or phage insensitive, or phage resistant bacteria
  • a suitable amount of the said phage strain and at least one antibacterial agent comprising protein synthesis inhibitor activity culturing the bacteria for a period and under conditions suitable for the phage
  • the host bacteria is a sample of a bacteria causative of an infection being suffered by a subject
  • the phage is a phage selected from a "phage bank" and is one that is normally regarded as infectious for the host bacteria if not for the circumstance that the bacteria has become insensitive to the phage (ie is phage non-sensitive).
  • the progeny phage have a modified capability to infect and cause lysis of said host bacteria, and may therefore be useful in treating the infection in the subject.
  • the present disclosure provides a method of treating a bacterial infection in a subject, said method comprising the steps of: 1. obtaining a sample of a bacteria causative of an infection in said subject, 2.
  • the bacteria with a suitable amount of a selected phage strain to which the bacteria is insensitive (ie the bacteria is phage non-sensitive) and at least one antibacterial agent comprising protein synthesis inhibitory activity, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage; 3. recovering and expanding progeny phage which are phage with a modified capability to infect and cause lysis of said host bacteria; and 4. administering to the subject an effective amount of the progeny phage, or a further propagated form thereof, to treat the infection.
  • a suitable amount of a selected phage strain to which the bacteria is insensitive ie the bacteria is phage non-sensitive
  • at least one antibacterial agent comprising protein synthesis inhibitory activity
  • the present disclosure provides a modified phage produced in accordance with the method of the first aspect, or a further propagated form thereof.
  • the present disclosure provides a pharmaceutical composition comprising a modified phage produced in accordance with the method of the first aspect, or a further propagated from thereof, optionally in combination with at least one pharmaceutically acceptable additive, carrier, diluent and/or excipient.
  • the present disclosure provides the use of a modified phage produced in accordance with the method of the first aspect, or a further propagated form thereof, in the manufacture of a medicament for treating an infection in a subject.
  • the present disclosure provides a modified phage, produced in accordance with the method of the first aspect, or a further propagated form thereof, for treating an infection in a subject.
  • the present disclosure provides a method of killing bacteria, said method comprising contacting the bacteria with a modified phage produced in accordance with the method of the first aspect or a further propagated form thereof.
  • the present disclosure provides an isolated phage lysin protein (an endolysin) derived from a modified phage produced in accordance with the method of the first aspect.
  • Figure 1 provides the results of experimentation to determine any synergy between phage and various antibiotic agents on bacterial viability.
  • Protein synthesis inhibitors PSI
  • E erythromycin
  • C clindamycin
  • Az azithromycin
  • V cell wall actives amoxicillin
  • CI3 vancomycin
  • Significant reductions were observed, compared to treatment with phage or antibiotic alone, for all combinations of phage and PSIs.
  • Figure 2 provides the results of experiments to assess whether enhanced bacterial killing by the combination of clindamycin and phage was dose dependent.
  • Black bars represent S. aureus ATCC51650 and CI11 treated with medium (TSB), J- Sa36 (Sa36) or clindamycin (1 ⁇ 2 MIC or 0.1 ⁇ g/ml) as control.
  • Grey bars represent S. aureus ATCC51650 and CI11 treated with J-Sa36 combined with different concentrations of clindamycin;
  • Figure 3 provides results showing that the phage J-Sa36, Sa83 or Sa87 combined with clindamycin reduces the viability of ATCC51650 and CI11 biofilms. In particular, the reduction of biofilm viability of S.
  • aureus ATCC51650 or CI11 were grown in the absence (control) or presence of J- Sa36 (Sa36; MOI 0.2) or 1 ⁇ 2 MIC clindamycin for 3.5 hours, followed by the addition of 1 ⁇ 2 MIC clindamycin (Sa36+ 1 ⁇ 2 MIC clindamycin added at 3.5 hrs) or phage J-Sa36 (1 ⁇ 2 MIC clindamycin + J- Sa36 added at 3.5 hrs) with OD600 recorded at 3.5 (black bars) and 24 hours (grey bars); (C, D) ATCC51650 and CI11 respectively were pre-treated for different times with either 1 ⁇ 2 MIC clindamycin (black bar) or TSB only (grey) before J-Sa36 (MOI 0.2) was added at 1, 2, 3, 3.5, 4, 6 and 24 hrs.
  • Colony Forming Unit (CFU) counts CFU/ml (A) and biofilm biomass ( ⁇ m 3 / ⁇ m 2 ) (B) of a fresh piece of rat sinonasal mucosa from rats treated with 0.5 x 10 10 PFU/ml Sa87 in combination with 1 ⁇ 2 MIC clindamycin [Sa87 + C], 0.5 x 10 10 PFU/ml Sa87 in combination with 1 ⁇ 2 MIC azithromycin [Sa87 + Az], 0.5 x 10 10 PFU/ml Sa87 [Sa87], 1 ⁇ 2 MIC clindamycin [C], 1 ⁇ 2 MIC azithromycin [Az], saline [Saline].
  • CFU Colony Forming Unit
  • FIG. 6 provides graphical results showing that the combination of S. aureus phage and 1 ⁇ 2 MIC clindamycin kills non-sensitive S. aureus strains: (A) S. aureus C43 treated with APTC-SA-2 and 1 ⁇ 2 MIC clindamycin; (B) S.
  • aureus C330 treated with APTC-SA-2 and 1 ⁇ 2 MIC clindamycin
  • C S. aureus ATCC25923 treated with APTC-SA-2 and 1 ⁇ 2 MIC clindamycin
  • D S. aureus C285 treated with APTC-SA-12 and 1 ⁇ 2 MIC clindamycin
  • E S. aureus C319 treated with APTC-SA-12 and 1 ⁇ 2 MIC clindamycin
  • F S. aureus ATCC25923 treated with APTC-SA-12 and 1 ⁇ 2 MIC clindamycin. Significance was determined compared to untreated control. Data expressed as mean ⁇ SEM for three independent experiments.
  • Figure 7 provides the results of bactericidal activity tests performed on exit phage. The exit phage's bactericidal activity was tested in its own host, S.
  • Figure 8 shows graphical results of tests for the exit phage specificity and lytic activity.
  • FIG. 9 provides characterising (stability and growth) results for entry phage and exit phage:
  • FIG. 10 shows the results of a representative PHEARLESS Assay to determine the lytic activity of exit phage produced in accordance with the method of the disclosure.
  • aureus with lytic phage in combination with a sub-inhibitory concentration (MIC) of erythromycin, clindamycin and azithromycin can significantly reduce the growth of the bacteria.
  • Characterisation of the phage isolated after the treatment (“exit phage") then, surprisingly, revealed that the phage were able to re-infect their "parent” bacterial host without the need (or “help") of the antibiotic, and moreover, with up to a 1000-fold higher level of lytic activity as compared to that of the "entry phage" (ie the initial phage used).
  • the dosage of exit phage to have significant antibacterial effects against the parent bacterial host was less than 1000 th the dosage of the entry phage and the entry phage could only infect the parent bacteria in the presence of protein synthesis inhibitor (PSI) antibiotics whilst the exit phage could infect the parent bacterial strains also in the absence of PSI antibiotics.
  • PSI protein synthesis inhibitor
  • the stability (ie temperature and pH stability) of the exit phage and their growth curve were, however, substantially unchanged and, in particular, showed no significant difference when compared with the corresponding entry phage.
  • the burst size of the exit phage was higher than the entry phage (ie the burst size (calculated at 30min) for entry phage APTC- SA-2 was 30 PFUs/infected cell; while for exit phage APTC-SA-2-43, the burst size was 40 PFUs/infected cell, and for exit phage APTC-SA-2-330, 42 PFUs/infected cell.
  • the burst size was 14 PFUs/infected cell, in comparison to burst sizes of 33 and 38 PFUs/infected cell, respectively, for the exit phage APTC-SA-12-C285 and exit phage APTC-SA- 12-319).
  • exit phage showed a significantly lower BIM frequency compared to entry phage.
  • the Applicants have identified a novel method for the production of a modified phage, derived from a phage for which a host bacterial strain is or has become insensitive (ie the bacteria is phage non- sensitive), which is capable of infecting the host bacteria strain and, preferably, also shows an enhanced level of lytic activity with a reduction in dosage required to achieve bacterial eradication, an increase in the burst size of the exit phage, and/or achieves a reduction in the frequency of emergence of bacteriophage insensitive mutants (BIM) when bacteria are treated with the exit phage compared to when they are treated with the parent entry phage (in the presence of sub-inhibitory PSI antibiotics).
  • BIM bacteriophage insensitive mutants
  • the present disclosure provides a method of producing a modified phage, said method comprising the steps of: (i) treating a host bacteria insensitive to a selected phage strain (ie a phage non-sensitive, or phage insensitive, or phage resistant bacteria) with a suitable amount of the said phage strain and at least one antibacterial agent comprising protein synthesis inhibitor activity, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage; and (ii) recovering and expanding progeny phage which are phage with a modified capability to infect and cause lysis of said host bacteria.
  • a host bacteria insensitive to a selected phage strain ie a phage non-sensitive, or phage insensitive, or phage resistant bacteria
  • a suitable amount of the said phage strain and at least one antibacterial agent comprising protein synthesis inhibitor activity culturing the bacteria for a period and under conditions suitable for the phage
  • the present disclosure provides a method of producing phage with improved lytic activity against a phage insensitive bacteria, by exposing the insensitive host bacterium to a selected infecting phage and an agent having protein synthesis inhibitory activity, and producing a phage with improved lytic activity against the insensitive bacteria.
  • the host bacteria used in step (i) may be any phage insensitive bacteria strain, but preferably, will be a phage insensitive bacteria strain of medical and/or veterinary significance.
  • the host bacteria is selected from the group consisting of Staphylococcus species, Streptococcus species, Enterococcus species, Corynebacterium species, Bacteroides species, Bacillus species, Yersinia species, Legionella species, Francisella species, Salmonella species, Gordonia species, Mycobacterium species, Acinetobacter species, Clostridium species, Pseudomonas species, and Enterobacteriaceae species. [0032] In some embodiments, the host bacteria is selected from the group consisting of S. aureus, S. epidermidis, S.
  • the host bacteria is a drug-resistant bacteria, such as an antibiotic- resistant bacteria, and especially a multidrug-resistant (MDR) bacteria or superbug, such as methicillin-resistant Staphylococcus aureus (MRSA).
  • MDR multidrug-resistant
  • the host bacteria is a bacteria having a characteristic of the ability to form a biofilm.
  • the host bacteria is an S.
  • aureus bacteria may otherwise be of a species such as Acinetobacter baumannii (which can cause life-threatening infections in the blood, lungs, urinary tract and in wounds), Clostridium difficile (which can cause life-threatening infections and damage to the intestinal mucosa), Pseudomonas aeruginosa (which is a common cause of chronic airway infections in cystic fibrosis patients, and which can cause serious infections in the ear, skin, blood and parts of the body following surgery), and Enterobacteriaceae species (such as E.coli, E. faecium such as vancomycin-resistant E. faecium, and Klebsiella spp. such as K. pneumoniae).
  • Acinetobacter baumannii which can cause life-threatening infections in the blood, lungs, urinary tract and in wounds
  • Clostridium difficile which can cause life-threatening infections and damage to the intestinal mucosa
  • Pseudomonas aeruginosa which is
  • the host bacteria is selected from the group consisting of multidrug resistant (MDR) strains of bacteria.
  • MDR multidrug resistant
  • examples of MDR strains of A. baumannii, E. coli, Klebsiella spp. and P. aeruginosa are also particularly well known and widespread.
  • the host bacteria used in step (i) may, preferably, be a clinical isolate and, in particular, a clinical isolate from a subject requiring treatment for a bacterial infection; that is, the host bacteria is a sample of the bacteria causative of an infection being suffered by a subject.
  • the host bacteria is a clinical isolate from a bacterial infection characterised by the presence of a bacterial biofilm.
  • the bacteria are gram-negative bacteria, while in some other embodiments, the bacteria are gram-positive bacteria.
  • suitable host bacteria include bacteria from the following species: Bacillus (eg Bacillus anthracis), Brochothrix, Corynebacterium, Enterococcus, Fingoldia (eg Fingoldia magna), Francisella (eg Francisella tularensis) Geobacillus, Gordonia, Lactobacillus, Leuconostoc, Lactococcus, Legionella, Listeria, Microcystis, Mycobacterium (eg Mycobacterium abscessus, Mycobacterium tuberculosis), Nocardia, Pediococcus, Propionibacterium, Rhodococcus, Staphylococcus (eg Staphylococcus aureus; Staphylococcus epidermidis, Staphylococcus lugdunensis) Streptococcus, Aeromonas, Azospirillum, Aggregatibacter, Bacteroides,
  • the bacteria are selected from bacteria of the following genus: Citrobacter Cronobacter
  • the host bacteria is treated in step (i) with a suitable amount of a phage that would normally be regarded as infectious for the bacteria if not for the circumstance that the particular bacterial strain concerned has become insensitive to the phage.
  • the phage may be a phage that is normally infectious for S. aureus.
  • Staphylococcus phage examples include phage from the Myoviridae, Siphoviridae, Podoviridae, Picovirinae, Spounavirinae, and Twortlikevirus families.
  • Staphylococcus phage examples include Staphylococcus phage SA1, Staphylococcus phage phi13, Staphylococcus phage phi2958PVL, Staphylococcus phage phiETA2, Staphylococcus phage phiETA3, Staphylococcus phage phiMR11, Staphylococcus phage phiMR25, Staphylococcus phage phiNM, Staphylococcus phage phiNM3, Staphylococcus phage phiPVL108, Staphylococcus phage phiPVL-CN125, Staphylococcus phage phiSauS-IPLA35, Sta
  • the phage used in step (i) may be selected from examples of Enterococcus phage such as those from the Siphoviridae and Myoviridae families.
  • Enterococcus phage include Enterococcus phage EFAP-1, Enterococcus phage phiEf11, Enterococcus phage phiEF24C, Enterococcus phage phiFL1A, Enterococcus phage phiFL2A, Enterococcus phage phiFL3A, Enterococcus phage phiFL4A, and Enterococcus phage SAP6.
  • the phage used in step (i) may be selected from examples of Corynebacterium phage such as those of the Siphoviridae family.
  • Particular examples of Corynebacterium phage include Corynebacterium phage BFK20 and Corynebacterium phage P1201.
  • the phage used in step (i) may be selected from examples of Pseudomonas phage including phage of the Podoviridae, Autographivirinae, Siphoviridae, Podoviridae, Myoviridae and Cystoviridae families.
  • Pseudomonas phage include Pseudomonas phage H105/1, Pseudomonas phage LKA1, Pseudomonas phage B3, Pseudomonas phage D3, Pseudomonas phage F10, Pseudomonas phage F116, Pseudomonas phage F8, Pseudomonas phage gh-1, Pseudomonas phage LBL3, Pseudomonas phage LKD16, Pseudomonas phage LMA2, Pseudomonas phage LUZ19, Pseudomonas phage PA11, Pseudomonas phage PAJU2, Pseudomonas phage PaP3, Pseudomonas phage PB1, Pseudomona
  • the phage used in step (i) may be selected from examples of Bacteroides phage include phage of the Siphoviridae family such as Bacteroides phage B40-8 and Bacteroides phage B124-14.
  • the phage used in step (i) may be selected from examples of Propionibacterium phage include phage of the Siphoviridae family such as Propionibacterium phage PA6, Propionibacterium phage PAD20, and Propionibacterium phage PAS50.
  • the phage used in step (i) may be selected from examples of Mycobacterium include phage of the Siphoviridae and Myoviridae families.
  • Mycobacterium phage include Mycobacterium phage Muddy, Mycobacterium phage Baee, Mycobacterium phage Girr, Mycobacterium phage Hamulus, Mycobacterium phage Mozy, Mycobacterium phage Omega, Mycobacterium phage Ramsey, Mycobacterium phage Soul22, and Mycobacterium phage St Annes.
  • the phage used in step (i) may be selected from examples of Yersinia such as those of the Myoviridae family. Particular examples of Yersinia phage include Yersinia phage phiR1-37.
  • the phage used in step (i) may be selected from examples of Gordonia such as those of the Siphoviridae family. Particular examples of Gordonia phage include Gordonia phage Smoothie.
  • Further examples of suitable specific phage for use in step (i), and their respective bacterial "pair" (ie host bacteria) are shown in Table 1 below. [0056] Table 1 Bacteriophage Host genus Enterococcus phage vB_EfaP_Ef6.3 Enterococcus
  • the phage may be selected from available stocks of phage such as may be found in a "phage bank", or identified by screening natural sources of phage.
  • the phage used to treat the host bacteria are referred to as the "entry phage”.
  • the phage will be provided in an amount corresponding to a multiplicity of infection (MOI) value in the range of 0.05 to 2.0, more preferably about 0.5 to 1.2, and most preferably, of about 1.0.
  • MOI multiplicity of infection
  • the MOI may be 1.0 or more, 0.5 or more, 0.1 or more, or 0.001 or more, or less than 1.0, less than 0.1, or less than 0.01.
  • the antibacterial agent used to treat the host bacteria in step (i) comprises protein synthesis inhibitor activity.
  • Protein synthesis inhibitors may act via a mechanism of an interruption of peptide chain elongation, the blocking of the decoding site (A site) of ribosomes, the misreading of the genetic code or the prevention of the attachment of oligosaccharide side chains to glycoproteins.
  • Antibacterial agents such as PSI antibiotics are well known to those skilled in the art.
  • the antibacterial agent comprising protein synthesis inhibitory activity comprises an agent which binds to the 50S ribosomal subunit and inhibits protein synthesis by preventing peptide chain elongation. Examples include macrolides such as clindamycin, erythromycin, clarithromycin and azithromycin.
  • the antibacterial agent comprising protein synthesis inhibitory activity comprises an agent that can inhibit protein synthesis by blocking the A site of the 30S ribosomal subunit.
  • examples include kanamycin and streptomycin.
  • protein synthesis inhibitors suitable for use in step (i) include spectinomycin, tetracycline, clindamycin, azithromycin, kanamycin, gentamicin, netilmicin, linezolid, chloramphenicol, capreomycin, mupirocin, dactinomycin, telithromycin, neomycin, lincomycin, fusidic acid, cethromycin, viomycin, sisomycin, puromycin and streptomycin.
  • the host bacteria is treated in step (i) with an amount of the antibacterial agent that is equal to or greater than the MIC, for example 2 x MIC, or 1 x MIC. [0063] In some embodiments, the host bacteria is treated in step (i) with a sub-inhibitory amount of at least one antibacterial agent.
  • the sub-inhibitory amount will be in the range of about 0.125 to 0.95 x MIC (minimum inhibitory concentration), more preferably about 0.125 to 0.65 x MIC and most preferably, about 0.5 x MIC ("1 ⁇ 2 MIC"). Other concentrations are, however, also contemplated.
  • the present disclosure provides a method of producing a modified phage, said method comprising the steps of: (i) treating a host bacteria insensitive to a selected phage strain (ie a phage non-sensitive, or phage insensitive, or phage resistant bacteria) with a suitable amount of the said phage strain and a sub- inhibitory amount of at least one antibacterial agent comprising protein synthesis inhibitor activity, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage; and (ii) recovering and expanding progeny phage which are phage with a modified capability to infect and cause lysis of said host bacteria.
  • a host bacteria insensitive to a selected phage strain ie a phage non-sensitive, or phage insensitive, or phage resistant bacteria
  • a suitable amount of the said phage strain and a sub- inhibitory amount of at least one antibacterial agent comprising protein synthesis inhibitor activity and cul
  • the treatment with the antibacterial agent may be performed concurrently with the phage treatment, before treatment with the phage, and/or after treatment with the phage.
  • the treatment with the antibacterial agent is performed before the treatment with the phage (ie before the phage is added) in what is effectively a pre-treatment with the antibacterial agent.
  • the pre-treatment with the antibacterial agent may be commenced up to 10 hours, more preferably up to 6 hours, more preferably up to 4 hours, more preferably up to 3 hours, prior to contacting the host bacteria with the entry phage.
  • the phage may be added first, then followed by the addition of the antibacterial agent.
  • the host bacteria treated in accordance with step (i) are cultured for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage.
  • the culture will be performed on standard growth media for 24 hours at 37 o C.
  • the progeny phage are also referred to as the "exit phage”.
  • Exit phage may be readily purified from the culture of step (i), if desired, by standard methodologies known to those skilled in the art including, for example, a purification process comprising centrifugation of the culture (to remove residual bacteria and bacterial cell walls etc.
  • exit phage progeny phage
  • exit phage that have a modified capability to infect and cause lysis and killing of the host bacteria
  • exit phage which show a modified, enhanced, level of bactericidal activity against the host bacteria or "parent" used in step (i) relative to the entry phage
  • the number of phage ie the number of phage particles is increased
  • a method of treating a subject requiring treatment for a bacterial infection or for storage in, for example, a phage bank are recovered and propagated to expand the number of phage (ie the number of phage particles is increased) for subsequent use in, for example, a method of treating a subject requiring treatment for a bacterial infection or for storage in, for example, a phage bank.
  • This may involve one or more steps (preferably, two or more steps) of culturing the host bacteria in the presence of the exit phage, or in other words, one or more steps of passaging the exit phage through the host bacteria.
  • These culturing/passaging steps preferably do not include any treatment with the antibacterial agent as used in step (i) of the method, but may do so. Otherwise, the exit phage may be propagated by culturing/passaging with other sensitive bacteria to expand the number of phage. Phage particles that are present after these culturing/passaging steps represent modified exit phage that are capable of infecting and causing lysis of the host bacteria.
  • these exit phage may undergo one or more selection or analysis steps including, but not limited to: identifying exit phage with markedly enhanced lytic activity (eg to identify modified phage capable of infecting the parent bacteria only, but with at least a 500-fold or, more preferably, 1000-fold higher level of lytic activity; identifying exit phage with stability and/or growth (replication) characteristics that are substantially unchanged from that of the entry phage, except for a higher burst size; and/or identifying exit phage which encode and produce a lysin protein with markedly enhanced lytic activity which may involve, for example, assessing the relative lytic activity of the lysin protein produced by the modified phage (eg by expressing an isolated nucleic acid molecule encoding the lysin protein in a host bacteria: see for example the PHEARLESS assay described in the examples hereinafter) or through the sequencing of at least a lysin-encoding nucleotide sequence within the exit phage genome.
  • the method of the first aspect may be performed in a multiple (parallel) format where different entry phage are separately used to treat different samples of host bacteria to generate different progeny exit phage; and the use of the optional one or more selection or analysis steps may then be used to identify a preferred exit phage from amongst the separately generated exit phage candidates.
  • the exit phage may be further propagated using the host bacteria or other sensitive phage to further expand the number of phage, such as for the purpose of storage in a phage bank.
  • the method of the first aspect may be advantageous inasmuch as the use of repeated steps for the generation of modified phage may be avoided.
  • step (i) preferably consists of no more than three steps, or no more than two steps, and more preferably only a single step of treating the insensitive host bacteria with a suitable amount of the selected phage strain and at least one antibacterial agent, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage.
  • the steps of culturing/passaging are preferably not conducted using the host bacteria in combination with at least the antibacterial agent that was used in step (i) or, more preferably, any other antibacterial agent comprising protein synthesis inhibitor activity.
  • the method of the first aspect may produce a modified exit phage to which the host bacteria has been re-sensitised, and which may be of use in (for example) treating a subject requiring treatment for a bacterial infection (ie in a phage therapy).
  • the method of the first aspect may be used to produce a modified exit phage which, unlike the selected entry phage, is capable of infecting and causing lysis of a host bacteria (such as a clinical isolate) causative of an infection being suffered by a subject.
  • the method of the first aspect is for producing a modified phage for use in a phage therapy of a subject's infection, and comprises the steps of: (i) treating a host bacteria that has been isolated from the said subject and which is insensitive to a selected phage strain (ie a phage non-sensitive bacteria) with a suitable amount of the said phage strain and at least one antibacterial agent comprising protein synthesis inhibitory activity, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage; (ii) recovering and expanding progeny phage which are phage with a modified capability to infect and cause lysis of said host bacteria; and (iii) providing the modified phage in a pharmaceutical composition for administration to the said subject.
  • a selected phage strain ie a phage non-sensitive bacteria
  • the amount of the antibacterial agent comprising protein synthesis inhibitory is activity is a sub-inhibitory amount.
  • Such embodiments enable a "personalised" approach to phage therapy which may be employed, for example, where a phage with an effective level of bactericidal activity against a bacteria causative of an infection in a subject, has either not been identified or is not readily available.
  • the method may enable the selection of an available phage that would normally be regarded as infectious for the bacteria if not for the circumstance that the particular bacterial strain concerned has become insensitive to the phage, and to use that phage as an entry phage from which to produce a modified exit phage that may then enable an effective phage therapy of the infection in the subject.
  • the present disclosure provides a method of treating a bacterial infection in a subject, said method comprising the steps of: 1. obtaining a sample of a bacteria causative of an infection in said subject, 2.
  • the bacteria with a suitable amount of a selected phage strain to which the bacteria is insensitive (ie the bacteria is phage non-sensitive) and at least one antibacterial agent comprising protein-synthesis inhibitory activity, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage; 3. recovering and expanding progeny phage which are phage with a modified capability to infect and cause lysis of said host bacteria; and 4. administering to the subject an effective amount of the progeny phage, or a further propagated form thereof, to treat the infection.
  • a suitable amount of a selected phage strain to which the bacteria is insensitive ie the bacteria is phage non-sensitive
  • at least one antibacterial agent comprising protein-synthesis inhibitory activity
  • the present disclosure provides a method of treating a bacterial infection in a subject, the method comprising producing phage with improved lytic activity against a phage insensitive bacteria by exposing the insensitive host bacterium to a selected infecting phage and an agent having protein synthesis inhibitory activity and treating the infection with an effective amount of the phage with improved lytic activity.
  • the present disclosure provides a method of treating a bacterial infection in a subject, said method comprising the steps of: 1. obtaining a sample of a bacteria causative of an infection in said subject, 2.
  • the bacteria with a suitable amount of a selected phage strain to which the bacteria is insensitive (ie the bacteria is phage non-sensitive) and a sub-inhibitory amount of at least one antibacterial agent comprising protein-synthesis inhibitory activity, and culturing the bacteria for a period and under conditions suitable for the phage to infect the bacteria and replicate to generate progeny phage; 3. recovering and expanding progeny phage which are phage with a modified capability to infect and cause lysis of said host bacteria; and 4. administering to the subject an effective amount of the progeny phage, or a further propagated form thereof, to treat the infection.
  • a suitable amount of a selected phage strain to which the bacteria is insensitive ie the bacteria is phage non-sensitive
  • a sub-inhibitory amount of at least one antibacterial agent comprising protein-synthesis inhibitory activity comprising protein-synthesis inhibitory activity
  • the method of the second aspect may, particularly, be used to treat an infection in the subject which is not responding to antibiotic-based therapy (thereby indicating that the infectious bacteria is antibiotic drug-resistant) and/or treatment with phage (thereby indicating that the infectious bacteria is phage resistant or phage insensitive).
  • the bacterial sample may be obtained from (eg isolated from), for example, a suitable body sample from the subject such as blood, urine, faecal sample, swab, or a tissue biopsy (eg a biopsy from a tissue at the site of infection). Bacteria present in the body sample may be isolated and expanded according to any of the standard methodologies known to those skilled in the art.
  • the bacteria may optionally be identified by using standard methodologies including microscopy techniques, tests such as latex agglutination tests (see, for example, Essers L., J Clin Microbiol 12(5):641-643, 1980), MALDI and/or 16 S rRNA gene sequencing such as is well known to those skilled in the art.
  • a phage that would normally be regarded as infectious for the bacteria may be selected from, for example, a phage bank for use as the "entry phage", however more typically, the selected phage will be a phage previously employed in a phage therapy on the subject (which was unsuccessful) or to which the causative bacteria has been found to be insensitive in, for example, a standard in vitro phage sensitivity test.
  • the bacteria may be treated with the phage and antibacterial agent as described above in relation to the method of the first aspect. The treatment of the bacteria with the antibacterial agent is typically performed before the phage treatment.
  • the step of recovering and expanding the progeny "exit phage" may be carried out as described above in relation to the method of the first aspect.
  • the modified exit phage may also be further propagated using, for example, a sensitive bacterial strain, as required (eg for storage in a phage bank).
  • the modified exit phage (or a further propagated form thereof) will typically be provided as a pharmaceutical composition comprising the phage in combination with at least one pharmaceutically acceptable carrier, diluent and/or excipient.
  • the modified exit phage may be administered to the subject by any suitable route such as topical administration (eg for treatment of a surface wound), intramuscular administration (im) (eg by injection), intravenous administration (iv) (especially systemic infusion), oral administration or airway administration (eg by rinsing, nebulisation, spraying into the nose and paranasal sinuses and into the lungs in pulmonary administration).
  • topical administration eg for treatment of a surface wound
  • intramuscular administration eg by injection
  • intravenous administration iv
  • oral administration eg by rinsing, nebulisation, spraying into the nose and paranasal sinuses and into the lungs in pulmonary administration.
  • the modified phage (or a further propagated form thereof) may be administered with one or more additional phage strain(s) (ie in a "phage cocktail") which may contribute to the effectiveness of the phage therapy in treating the infection, particularly in the case of a polymicrobial infection (ie where more than one bacteria strain is present in the infection).
  • the modified phage (or a further propagated form thereof) may be used in a combination therapy with a suitable antibacterial agent.
  • the method of the second aspect may be used to treat a bacterial infection in a subject, and especially a bacterial infection caused by bacteria showing drug-resistance and/or which is phage insensitive.
  • the method of the second aspect may be used to treat an infection in a subject caused by methicillin-resistant Staphylococcus aureus (MRSA) (eg a skin or lung infection, diabetic foot ulcer, sinus infection or sepsis), A. baumannii (eg an infection of the blood (bacteremia), lungs or urinary tract, or a wound infection by A. baumannii), C. difficile (eg a colon infection), P. aeruginosa (eg a lung infection, bacteremia, bone and joint infections, post-surgery infection, diabetic foot ulcers, sinus infection, ear infection), MDR strains of E.coli (eg an urinary tract infection (UTI)), an E.
  • MRSA methicillin-resistant Staphylococcus aureus
  • the method of the second aspect may be used to treat sinusitis or rhinosinusitis (acute rhinosinusitis and chronic rhinosinusitis) which is commonly caused by S. aureus and/or P. aeruginosa.
  • sub-inhibitory amount refers to an amount (concentration) of an antibacterial agent which is below that required to detectably inhibit the growth and replication of a particular bacteria.
  • the sub-inhibitory amount is a non-lethal amount for the particular bacteria.
  • minimum inhibitory concentration is to be understood as it is to those skilled in the art. Therefore, as used herein, the term refers to the lowest amount (concentration) of an antibacterial agent that detectably inhibits the growth of a particular bacteria.
  • phage non-sensitive phage insensitive
  • phage resistant are used interchangeably to describe a characteristic of a particular bacteria to be insensitive to a certain phage or phages, such that phage(s) when contacted with the bacteria is unable to substantially infect and/or cause lysis of the bacteria (eg as may be detected by standard in vitro phage sensitivity test where lysis zones are assessed after spotting of phage onto a bacterial lawn on a standard petri dish or plate, and overnight culture).
  • Such bacteria may be partially or fully resistant to the particular phage or phages.
  • phage contacted with "sensitive" bacteria will infect and/or cause lysis of the bacteria.
  • lysis refers to a process whereby, for example, a phage or chemical agent causes the breaking down of the wall or membrane of a cell such as a bacterial cell leading to cell death.
  • the modified exit phage may show enhanced “lytic activity", meaning that the modified phage has an enhanced (increased) capability to cause lysis and/or killing of the host bacteria as compared to the entry phage.
  • This lytic activity may be achieved, wholly or in part, by phage proteins known as "endolysins" (lysins) which are peptidoglycan-degrading or endopeptidase enzymes which can breakdown bacterial cell walls.
  • the term “treating” includes prophylaxis as well as the alleviation of established symptoms of an infectious disease or condition.
  • the act of "treating" an infection in a subject therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the infectious disease or condition developing in a subject afflicted with the disease or condition; (2) inhibiting the infectious disease or condition (ie arresting, reducing or delaying the development of a bacterial infection or a relapse thereof (in case of a maintenance treatment) or at least one clinical or subclinical symptom thereof); and (3) relieving or attenuating the infectious disease or condition (ie causing regression of the bacterial infection or at least one of its clinical or subclinical symptoms).
  • Treatment encompasses single or multiple treatments.
  • the phrase "manufacture of a medicament” includes the use of the modified phage directly as a medicament, in combination with other additives, or in any stage of the manufacture of a medicament.
  • the term "effective amount” refers to an amount sufficient to effect beneficial or desired clinical results (ie it is a “therapeutically effective amount”).
  • a therapeutically effective amount can be administered in one or more administrations.
  • a therapeutically effective amount is sufficient for treating an infectious disease or condition, or otherwise to palliate, ameliorate, stabilise, reverse, slow or delay the progression of such a disease or condition.
  • a therapeutically effective amount of a modified phage produced in accordance with the method of the first aspect may comprise 10 4 to 10 12 PFU, and more preferably 10 8 to 10 10 PFU.
  • the therapeutically effective amount may vary and depend upon a variety of factors including the activity of particular phage(s), the age, body weight, sex and health of the subject, the route and time of administration of the modified phage, and the severity of, for example, the infectious disease or condition, and the type or types of bacteria causative of the infection.
  • the modified phage may be used in a combination therapy with a suitable antibacterial agent, in which case, the phage and antibacterial agent (eg an antibiotic) may be administered to the subject consecutively, together or in either order.
  • a suitable antibacterial agent eg an antibiotic
  • the phage and antibacterial agent may be administered one after another with practically no time interval (ie one is administered effectively immediately after the other) or, otherwise, after an interval of 1 to 5 minutes or more (eg 10 minutes, 30 minutes, 60 minutes, 4 hours or 6 hours).
  • the phage and the antibacterial agent are provided in combination for administration in a single pharmaceutical composition.
  • Such a pharmaceutical composition may further comprise, for example, a pharmaceutically acceptable carrier, diluent and/or excipient, and may be suitable for, for example, topical administration, intramuscular administration (im), oral administration or intravenous administration (iv).
  • a pharmaceutically acceptable carrier diluent and/or excipient
  • the phage and the antibacterial agent may each be formulated for administration in a pharmaceutical composition form including a pharmaceutically acceptable carrier, diluent and/or excipient, which may be the same or different.
  • the pharmaceutical compositions may be suitable for, for example, topical administration, intramuscular administration (im), oral administration or intravenous administration (iv).
  • the present disclosure provides a modified phage produced in accordance with the method of the first aspect.
  • the phage may be further propagated using, for example, a sensitive bacterial strain, as required.
  • the present disclosure provides a pharmaceutical composition comprising a modified phage produced in accordance with the method of the first aspect, or a further propagated from thereof, optionally in combination with at least one pharmaceutically acceptable additive, carrier, diluent and/or excipient.
  • Such a pharmaceutical composition may also comprise one or more additional phage strain(s) (ie in a "phage cocktail") and/or a suitable antibacterial agent (ie for a combination therapy).
  • the pharmaceutical composition may optionally comprise other substances such as absorption enhancers including surfactants (eg sodium lauryl sulphate, laureth-9, sodium dodecylsulphate, sodium taurodihydrofusidate and poly oxyethylene ethers), chelating agents (eg EDTA, citric acid and salicylates), and preservatives (eg sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid) and/or suitable binders, lubricants, suspending agents, coating agents and solubilising agents.
  • absorption enhancers including surfactants (eg sodium lauryl sulphate, laureth-9, sodium dodecylsulphate, sodium taurodihydrofusidate and poly oxyethylene ethers), chelating agents (eg EDTA, citric acid and salicylates), and preservatives (eg sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid) and/or suitable binders
  • binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • the at least one pharmaceutically acceptable additive, carrier, diluent and/or excipient may be selected from any of the suitable additives, carriers, diluents and excipients that are well known to those skilled in the art, and which are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1995.
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • suitable excipients may be found in the Handbook of Pharmaceutical Excipients, 2 nd Edition, (1994), Edited by A Wade and PJ Weller.
  • compositions may be intended for single daily administration, multiple daily administration, or controlled or sustained release, as needed to achieve the most effective results.
  • Examples of pharmaceutical compositions include compositions suitable for delivery by injection, compositions suitable for intravenous administration, compositions suitable for delivery by topical administration, compositions suitable for delivery via a gel, and compositions suitable for pulmonary delivery.
  • a suitable composition for topical delivery may comprise a requisite concentration of phage in PBS, in water or in SM buffer (To prepare: NaCl, 5.8 g, 100 mM ; MgSO4•7H2O 2 g, 8 mM ; Tris-Cl (1 M, pH 7.5), 50 ml, 50 mM ; H 2 O to 1 liter).
  • the present disclosure provides the use of a modified phage produced in accordance with the method of the first aspect, or a further propagated form thereof, in the manufacture of a medicament for treating an infection in a subject. Methods for manufacturing medicaments are known in the art.
  • the present disclosure provides a modified phage, produced in accordance with the method of the first aspect, or a further propagated form thereof, for treating an infection in a subject.
  • diseases or conditions suitable for treatment with a modified phage include sinus conditions (eg acute rhinosinusitis and chronic rhinosinusitis), respiratory conditions (eg lung infections, cystic fibrosis), ear conditions (infections of the external ear canal or of the middle ear), diabetic ulcers (eg diabetic foot ulcers), sepsis or a skin infection (eg Staphylococcus skin infections).
  • sinus conditions eg acute rhinosinusitis and chronic rhinosinusitis
  • respiratory conditions eg lung infections, cystic fibrosis
  • ear conditions infections of the external ear canal or of the middle ear
  • diabetic ulcers eg diabetic foot ulcers
  • sepsis or a skin infection
  • Other diseases or conditions are contemplated.
  • the modified phage of the present disclosure is typically applied to the treatment of an infectious disease or condition in a human subject.
  • the subject may also be selected from, for example, livestock animals (eg cows, horses, pigs, sheep and goats), companion animals (eg dogs and cats) and exotic animals (eg non-human primates, tigers, elephants etc).
  • livestock animals eg cows, horses, pigs, sheep and goats
  • companion animals eg dogs and cats
  • exotic animals eg non-human primates, tigers, elephants etc.
  • the method of the seventh aspect may be conducted in, for example, a laboratory setting where, for example, the method may be used to prevent bacterial contamination, and other situations where bacterial infections or contaminations can occur (eg industrial and environmental situations).
  • the method of the seventh aspect may be used for decontaminating surfaces and equipment such as food preparation surfaces and equipment, and medical and surgical surfaces and/or equipment etc.
  • the bacteria for killing may be present in a planktonic or biofilm form.
  • the method of the seventh aspect may also be used for the treatment of a subject.
  • the modified phage produced in accordance with the method of the first aspect are capable of infecting and causing lysis of the parent host bacteria.
  • the modified phage may show markedly enhanced lytic activity of a level which is at least 1000-fold higher than that of the entry phage.
  • Such modified phage may express a novel lysin protein with markedly enhanced lytic activity.
  • the present disclosure provides an isolated phage lysin protein (an endolysin) derived from a modified phage produced in accordance with the method of the first aspect.
  • the lysin protein may also be formulated into a composition, such as a pharmaceutical composition for use in treating a bacterial infection in a subject (such as those described above), or for killing bacteria in other situations such as environmental contaminations, or for decontaminating surfaces and equipment such as some of those mentioned above including food preparation surfaces and equipment, surgical equipment and air conditioning systems etc.
  • Standard techniques and equipment may be used for recombinant DNA technology, DNA sequencing, oligonucleotide synthesis, molecular biology, cell biology and enzymatic reactions, which may be generally performed according to methods known in the art and/or as commercially available, and are as described for example in Sambrook et al.
  • aureus ATCC51650 and ATCC25923 were obtained from the American Type Culture Collection (Manassas, VA, United States of America) and used as reference strains. In addition, a total of 15 S. aureus clinical isolates (CIs) were isolated from the sinonasal cavities of chronic sinusitis (CRS) patients. In each case, S. aureus was cultured by an independent diagnostic laboratory and the antibiotic susceptibility determined by disc diffusion on Mueller-Hinton agar per Clinical and Laboratory Standards Institute (CLSI) recommendations (Adelaide Pathology Partners, Mile End, SA, Australia).
  • CCSI Clinical and Laboratory Standards Institute
  • aureus strains was assessed using the broth microdilution technique as previously described (Wiegand I et al., Nature protocols 3(2):163-175, 2008) and breakpoints defined according to CSLI guidelines. Clinical strains resistant to at least one antibiotic in three or more antibiotic categories were defined as multidrug resistant (MDR).
  • MDR multidrug resistant
  • Phage sensitivity test The sensitivity of S. aureus isolates (15 clinical isolates and 2 ATCC strains) to phages J-Sa36, Sa83 and Sa87 was determined as previously described (Drilling A et al., Am J Rhinol Allergy 28(1):3-11, 2014). Briefly, 0.5 McFarland Units of S.
  • aureus CIs diluted 1:100 in 10 ml Tryptic soy broth (TSB) were grown overnight at 37°C with shaking.200 ⁇ L of overnight broth cultures were then spread onto Columbian blood agar plates evenly (Oxoid). Plates were incubated at 37°C for 1 hour and 5 ⁇ L [10 6 plaque forming units (PFU)] of 3 phages were spotted in triplicates onto the plates and incubated at 37°C overnight.
  • Phage buffer solution (SM buffer) alone was spotted in the centre of the plates as negative control. Phage sensitivity was determined according to the protocol described in Zhang G et al., editors.
  • Single colonies were transferred to Tryptone Soya Broth (TSB) to generate a culture with a starting concentration of 5 x 10 5 CFU/ml.
  • Phage J-Sa36, Sa83 and Sa87 were added to the cultures at multiplicity of infection (MOI) of 0.2 according to predetermined phage titers (described below).
  • Antibiotics were added to the cultures at 1 ⁇ 2 MIC (according to the pretest MIC of antibiotics) if required.
  • Optical density (OD) readings at 600 nm were recorded at 24 hours to assess the growth of bacteria co-inoculated with phage unless otherwise specified. Uninoculated, fresh media was included as a sterility control.
  • Phage titers were determined against S. aureus RN4220 (ATCC) using the double layer spot assay technique. Briefly, 100 ⁇ l of a S. aureus overnight culture was mixed with 4 mL of 0.4% TSA and poured onto a 1.5 % TSA plate. After 20 minutes, serially diluted samples of phage J-Sa36, Sa83 or Sa87 were spotted onto the double layer agar plates in 5 ⁇ l spot volumes in triplicates. After the plates were incubated in a humidified incubator at 37°C overnight, plaques were counted, counts were averaged, and titers of the filtrate were calculated based on the dilution.
  • Biofilms were then treated with 8 x10 6 plaque forming units (PFU) of phage J-Sa36, Sa83 or Sa87 and clindamycin added with a final concentration of 1 ⁇ g/ml, 0.5 ⁇ g/ml, 0.2 ⁇ g/ml, 0.1 ⁇ g/ml, or 0.05 ⁇ g/ml, in TSB. Wells were treated for 24 hours at 37°C with gentle shaking.
  • PFU plaque forming units
  • the AlamarBlue viability assay (Thermo Fisher Scientific, Waltham, MA, United States of America) was used as previously described (Richter K et al., editors. Mind “De GaPP”: in vitro efficacy of deferiprone and gallium ⁇ protoporphyrin against Staphylococcus aureus biofilms. International Forum of Allergy & Rhinology; 2016: Wiley Online Library to determine the anti-biofilm activity of the phage. Fluorescence was measured in a microplate reader (FLUOstar OPTIMA, BMG LABTECH, Offenburg, Germany) and the percent reduction of biofilm after treatment determined relative to untreated controls.
  • S. aureus clinical isolate was selected for in vivo experiments that was sensitive to both antibiotics to be used, insensitive to Sa87 phage on planktonic inhibition assays and could be sensitised to phage in the presence of both antibiotics in vitro.
  • a fresh culture of CI3 on 1.5% TSA plates was prepared two days before inoculation.
  • a single colony was resuspended in 0.9 % saline (ca. 2ml) to turbidity reading 0.5 McFarland Units and then a 1 in 100 dilution in TSB was cultured overnight one day before the in vivo experiment.
  • Treatments were administered intranasally twice a day for 21 days, the general wellbeing of the rat (including its appetite, behaviour, and any weight change) being closely monitored.
  • the rats were then sacrificed and the sinus mucosal tissue, lungs, heart, liver, kidneys, spleen and brain harvested. Tissue was divided and kept fresh or 10% formalin-fixed, the latter then processed to paraffin wax, and 6 ⁇ m sections cut and stained with haematoxylin and eosin (H&E) for histopathological examination. Formalin fixed tissue was embedded in paraffin and processed to undergo H&E staining and histopathological analysis.
  • H&E haematoxylin and eosin
  • the fresh sinus mucosa, lungs, heart, liver, kidneys, spleen, and brain were all analysed for phage titers. Faecal samples were collected from all the rats prior to the administration of the first treatment, as well as every day thereafter for phage titers.500 ⁇ l blood samples were collected from the rat tail vein prior to the first treatment and again at 1, 2, 4 and 6 hours following the morning treatment on day 7 and 14 of treatment. This was repeated at 1, 2, 4, 6, 12, 24 and 48 hours following the last treatments on Day 21.
  • Phage titers in faeces and organ tissues 1 g of faeces was suspended in 10 mL of SM buffer, centrifuged at 3214 ⁇ g for 10 minutes (Eppendorf 5810 centrifuge, North Ryde, NSW, Australia),filtered (0.22 ⁇ m), and titrated for phage concentration. 1g of rat faeces was collected from every rat at day 0 and daily after the first treatment, and directly placed into 5 mL SM buffer.
  • the rat frontal sinus mucosa was dissected and placed directly into 5 mL SM buffer.1cm x 1cm pieces of organ were collected from the brain, lungs, heart, liver, kidney and spleen and were also placed into 5 mL SM buffer. Phage titers and enrichment were performed as previously described (Drilling et al., 2014 supra). Briefly, organ and sinus tissues were ground in 5mL SM buffer using a Qiagen TissueRuptor (Qiagen, Hilden, Germany).
  • tissue samples were then centrifuged at 3000 g for 10 minutes, filtered through a 0.22 ⁇ m of syringe filter (Pall Corporation, San Diego, CA, United States of America), and titrated for phage.1g of faeces was suspended in 5 mL of SM buffer, centrifuged at 3214 ⁇ g for 10 minutes (Eppendorf 5810 centrifuge),filtered using a 0.22 ⁇ m filter, and titrated for phage concentration. For phage enrichment, 100 ⁇ L of 0.5 McFarland Units of S. aureus RN4220 diluted 1:100 with 10 ml TSB was grown overnight at 37 ⁇ C with shaking.
  • Phage titer in the blood sample 500 ⁇ l of blood was collected from the rat tail vein. Blood tubes were centrifuged at 1000 rpm for 20 minutes at 4°C. The plasma was then harvested and filtered through a 0.22 ⁇ m syringe filter. Phage enrichment was then performed, as above.
  • a two-day recovery was given to the rat before treatments begun.
  • Treatments were administered intranasally twice a day for 14 days.
  • Rats were monitored for weight and respiratory alterations (respiration rate; nasal discharge, hyperaemia or pruritus; and sneezing) during treatment, and, 14 days post-treatment initiation, the rats were humanely sacrificed. Their sinus mucosa was harvested for CFU counting, biofilm quantification, histopathology, and phage enumeration. [00122] Colony-forming unit (CFU) counting of sinus mucosa A fresh (0.2 x 0.2cm) piece of sinus mucosa was harvested and directly placed in 250 ⁇ l 0.9% saline. The mucosa was then homogenised on the highest setting for 30 seconds, or underwent vortex mixing on the highest setting for two minutes.
  • CFU Colony-forming unit
  • Biofilm biomass quantification on sinus mucosa The quantification of biofilm biomass on sinus mucosa was determined using an established protocol (Singhal D et al., editors. Quantitative analysis of in vivo mucosal bacterial biofilms. International Forum of Allergy & Rhinology; 2012: Wiley Online Library (27).
  • the frontal sinus mucosa was harvested and placed into Dulbecco’s Modified Eagle’s Medium (Life Technologies, Carlsbad, CA, United States of America) on ice.
  • Dulbecco’s Modified Eagle’s Medium (Life Technologies, Carlsbad, CA, United States of America) on ice.
  • One piece of mucosa from each frontal sinus was stained with a LIVE/DEAD® BacLight Bacterial Viability Kit (Life Technologies Australia). The stained mucosa was then examined at 20X magnification using a confocal laser scanning microscope (Zeiss LSM700, Carl Zeiss AG, Oberkochen, Germany).
  • the z-stack images (21 slices, interval 1 ⁇ m) were then taken from each sample and, COMSTAT Version 2.1 software was used to measure the biomass of biofilms, the threshold setting being applied so as to minimise background staining (Heydorn A et al., Microbiology 146(10):2395-2407, 2000).
  • Post-treatment sinus microbiology analysis A fresh (0.2 x 0.2 cm) piece of mucosa from each rat sinus was harvested and homogenised in 0.9% saline. The supernatant was then harvested and transferred into a new tube without centrifugation. The collected supernatant was serially diluted and plated on blood agar plates in triplicates.
  • a further three isolates exhibited resistance to only erythromycin, two isolates exhibited resistance to azithromycin and two exhibited multidrug resistance.
  • the isolates were also tested for susceptibility to the three phages, J-Sa36, Sa83 and Sa87.
  • J-Sa36 was active in vitro against only 2/17 strains tested (sensitive strains); 2/17 strains were semi-sensitive and 13/17 strains were insensitive (resistant).
  • the in vitro activity observed for Sa83 and Sa87 was considerably higher; for Sa83, 11/17 isolates were sensitive or semi-sensitive and 6/17 insensitive and for Sa87, 12/17 isolates were sensitive or semi-sensitive and 5/17 insensitive.
  • a synergistic effect was defined as a statistically significant decrease in optical density (OD) that was larger than the combined decrease in OD of antibiotic and phage only controls. None of the combinations of phage with either of the cell wall actives (amoxicillin and vancomycin) showed any significant additive or synergistic effects. However, in contrast, all nine isolates showed synergism with the combination of a PSI antibiotic (erythromycin, clindamycin and azithromycin) and at least one phage. The results for one representative isolate, namely S. aureus CI3, are shown in Figure 1.
  • aureus Effects were characterised as resistant (-) if no significant difference in OD was observed compared to untreated control, semi-sensitive ( ⁇ ) if a significant difference was observed but OD remained ⁇ 10% of untreated control, sensitive (+) if growth remained ⁇ 10% of untreated control and synergistic (+) with shading if both sensitive and significantly reduced compared to antibiotic and phage only controls. Dark grey represents synergistic effects against phage resistant strains; light grey represents synergistic effects against semi-sensitive strains. [00131] Dose-dependence of clindamycin in combination with phage J-Sa36 to kill S.
  • Incubation with 0.025 ⁇ g/ml (1/8 MIC), 0.05 ⁇ g/ml (1/4 MIC) or 0.1 ⁇ g/ml (1 ⁇ 2 MIC) in combination with J-Sa36 equally reduced OD values compared to positive controls after 24 hours incubation (p ⁇ 0.01).
  • aureus biofilms with and without clindamycin Clindamycin alone significantly reduced the viability of preformed ATCC51650 and CI11 biofilms at a concentration of 0.5 ⁇ g/ml (MIC 0.2 ⁇ g/ml) and above, with lower concentrations having no significant effect on biofilm viability.
  • Treatment with phages J-Sa36, Sa83 or Sa87 (PFU 8 x 10 6 /ml) alone did not affect CI11 biofilm viability, and J-Sa36 did not affect ATCC51650 biofilm viability.
  • aureus phage insensitive and semi-sensitive strains both ATCC51650 and CI11 in liquid culture were incubated with either 1 ⁇ 2 MIC clindamycin or phage J- Sa36 (MOI 0.2) for 3.5 hours. Following this, the alternate treatment was added before continuous incubation with 1 ⁇ 2 MIC clindamycin and phage J-Sa36 for 24 hours. As shown in Figure 4 (A-B), incubation with 1 ⁇ 2 MIC clindamycin or J-Sa36 only did not significantly affect bacterial viability at 24 hours.
  • significantly reduced OD600 values were observed for cultures where J-Sa36 was added up to 4 hours post-clindamycin challenge but not when J-Sa36 was added 6 and 24 hours after challenge (see Figure 4 (C-D)).
  • Sub-inhibitory (below MIC concentrations) clindamycin enhances phage activity To determine whether clindamycin enhanced phage activity or induced an endogenous prophage, phage titers were determined from cultures of ATCC51650, CI11 and CI8 after incubation for 48 hours with 1 ⁇ 2 MIC clindamycin, phage or both.100 ⁇ l of the resulting culture was then chloroform treated to release phage and the supernatant used to determine phage titers using ATCC51650, CI11 and CI8 as host.
  • the initial phage inoculum of 10 4 PFU/ml at 0 hours generated plaques only against ATCC51650 in the presence of clindamycin in the agar overlay, but no plaques were observed for CI11 or CI8. This indicates that a high MOI might be required in addition to clindamycin to facilitate phage lysis in these strains.
  • the supernatant of the isolates exposed to 1 ⁇ 2 MIC clindamycin only for ATCC51650, CI11 and CI8 produced no plaques in any of the strains after 48 hours incubation, indicating that no intact prophages that could lyse those isolates were induced by clindamycin treatment.
  • J-Sa36 added to ATCC51650 produced no significant increase in phage titer and CI11 produced no plaques, indicating that J-Sa36 cannot infect and replicate in these isolates in the absence of clindamycin. Similar results were observed for phage Sa83; with phage only treatment of CI11 for 48 hours not producing any plaques. In contrast, co-treatment of 1 ⁇ 2 MIC clindamycin and phage led to increased phage titers in all cases.
  • ATCC51650 treated with J-Sa36 and 1 ⁇ 2 MIC clindamycin showed a concentration increase from ⁇ 10 4 PFU/ml to 7.73 ⁇ 2.14 ⁇ 10 6 PFU/ml.
  • This result indicates that a clindamycin-mediated phage infection is capable of producing new phage particles, not simply leading to host cell death.
  • the same result was observed for CI11 treated with J-Sa36 and 1 ⁇ 2 MIC clindamycin (1.9 ⁇ 1.3 ⁇ 10 9 PFU/ml), and Sa83 and 1 ⁇ 2 MIC clindamycin (6.4 ⁇ 2.6 ⁇ 10 7 PFU/ml).
  • aureus CFU counts/ml compared to rats treated with 0.5 x 10 10 PFU/ml Sa87, 1 ⁇ 2 MIC clindamycin or 1 ⁇ 2 MIC azithromycin (p > 0.05). Sensitivity testing indicated similar sensitivity to the antibiotics and insensitivity to Sa87 for the post- and pre-treatment S. aureus isolates. In contrast, no S. aureus were cultured in rats treated with 0.5 x 10 10 PFU/ml Sa87 in combination with 1 ⁇ 2 MIC clindamycin or azithromycin and were significantly lower than control saline-treated rats or rats treated with Sa87 or 1 ⁇ 2 MIC clindamycin or azithromycin (p 0.0086) (see Figure 5A). However, bacterial colonies that were not S.
  • aureus were grown from the nasal mucosa of those rats (CFU counts of ⁇ 500 CFU/mL).
  • MALDI-TOF analysis identified those bacteria to be E. coli and E. faecalis.
  • Biofilm biomass quantitation showed similar biomass quantities in controls, Sa87 and 1 ⁇ 2 MIC clindamycin treated rats and a significant increase in biomass in 1 ⁇ 2 MIC azithromycin treated rats as compared to control.
  • histopathology of the maxilloturbinates by a pathologist showed normal ciliated pseudostratified respiratory epithelium with no evidence of inflammation or glandular hyperplasia in rats treated with Sa87 in combination with 1 ⁇ 2 MIC clindamycin or azithromycin.
  • rats treated with Sa87, 1 ⁇ 2 MIC clindamycin, 1 ⁇ 2 MIC azithromycin or saline only there was evidence of cilial denudation and surface epithelial erosion, with desquamated necrotic luminal cellular debris admixed with neutrophils.
  • Example 2 [00137] The study described in this example aimed to provide further evidence that sub-inhibitory concentrations of PSI antibiotics can sensitise phage non-sensitive S. aureus to phage infection, and additionally, provide some characterisation of the progeny phage (exit phage).
  • Materials and Methods [00138] Bacterial strains and growth conditions S. aureus strains frozen stocks were thawed and streaked on 1.5% trypticase soy agar (TSA) (Oxoid) plates at 37°C overnight.
  • TSA trypticase soy agar
  • E4643 E. coli expression strain was an E. coli made in Shearwin Laboratory from E4640+ (Shearwin K., School of Biological Sciences, University of Sydney, Sydney, SA, Australia).
  • Phages and phage titer determination Two strictly lytic S. aureus phages (APTC-SA-2 and APTC-SA-12; Sydney Phage Therapy Centre, Sydney, SA, Australia) were used for all experiments. The phages were identified as being of the genus Kayvirus, subfamily Twortvirinae, family Herelleviridae. Phage titers were determined against S. aureus RN4220 (ATCC) using the double layer spot assay (DLSA).
  • Phage sensitivity test Sensitivity tests of S. aureus clinical isolates was performed as described previously (Drilling et al., 2014 supra). Briefly, 200 ⁇ l of an overnight S.
  • aureus culture was spread onto Columbian blood agar plates (Oxoid) and then the plates were incubated at 37°C for 1 hour.5 ⁇ l diluted phage (10 6 PFU/ml) were spotted onto the plates in triplicates. Phage buffer solution (SM buffer and TSB) was spotted in the centre of the plates as a negative control. Phage sensitivity was determined as previously described after overnight incubation at 37°C (see Zhang G et al., 2018 supra).
  • Results were interpreted as follows: Sensitive: ⁇ 0.2 ⁇ g/mL; Intermediate: 0.4 ⁇ g/ml; Resistant: ⁇ 0.6 ⁇ g/mL.
  • the S. aureus strains sensitive to clindamycin were selected for further tests. The MIC was performed three times.
  • Phage and clindamycin combination treatment A phage and clindamycin synergy assay was performed using 96 well plates. In brief, selected S. aureus strains (phage non-sensitive) were grown on 1.5 % TSA at 37°C overnight. A single colony was transferred to TSB to generate a culture with a starting concentration of 5 x 10 5 CFU/ml.
  • One-step growth curve was performed as detailed previously (Chang Y et al., Viruses 7(10):5225- 5242, 2015). Briefly, 100 ⁇ L phages (1 ⁇ 10 8 PFU/mL) were mixed with 1 mL S. aureus (1.5 x 10 8 CFU/mL) culture at a MOI of 0.1 in 8.9 mL TSB and incubated at 37 °C for 5 min. The mixture was then centrifuged, and the pellets were resuspended in 9.9 mL fresh TSB. The resuspended pellets were incubated at 37 °C with shaking at 180 rpm.100 ⁇ L mixture was taken every 30 min.
  • BIMs bacteriophage-insensitive mutants
  • E. coli expressing phage proteins and PHEARLESS assay A system, termed PHEARLESS (phage-based expression, amplification, and release of lytic enzyme species), was used to express phage proteins of interest in E. coli comprising three components engineered into the E. coli expression strain (E4643), namely (1) the integrated cumate inducible 186tum induction module, (2) the engineered 186 ⁇ tum prophage genome and (3) the protein expression plasmid.
  • the E.coli containing the target protein expression plasmids were streaked out on LB agar plates with spectinomycin (50 ⁇ g/mL; Sigma Aldrich, Bayswater, VIC, Australia) and the S. aureus strains C319, C244, C330, C43, C259 and ATCC29923, were streaked out on LB agar plates. The plates were incubated at 37 °C degrees overnight. A single colony of the E. coli expression strain was picked to grow in LB broth with spectinomycin (50 ⁇ g/mL) overnight to generate a fresh bacterial culture. Similarly, a single colony of each of the S. aureus strains was picked to grow in LB broth overnight.
  • Phage sensitivity test A total of 60 S. aureus clinical isolates were tested for their phage sensitivity in order to select those that were insensitive to both lytic APTC-SA-2 and APTC-SA-12 S. aureus phage.
  • aureus strains (C43, C330, C285 and C319) were selected to be treated with the APTC-SA-2 and APTC-SA-12 phage combined with 1 ⁇ 2 MIC clindamycin.
  • the combination treatment could kill the bacteria after 24 hours and was significantly more effective than control (ie bacteria with TSB), or treatment with 1 ⁇ 2 MIC clindamycin and phage only.
  • the growth of S. aureus bacteria was similar among control, 1 ⁇ 2 MIC clindamycin treated, and phage only treated groups (see Figure 6A-B, D-E).
  • ATCC 25923 was sensitive to both phage types, and phage in combination with 1 ⁇ 2 MIC clindamycin were equally effective at killing this strain compared to no-treatment control and to 1 ⁇ 2 MIC clindamycin ( Figure 6C and F).
  • Exit phage bactericidal activity To evaluate whether the progeny phage particles (exit phage), produced after sensitisation of non- sensitive S.
  • aureus strains with 1 ⁇ 2 MIC clindamycin and adding the entry phage were effective at killing their own parent strain, exit phage particles were purified for both APTC-SA-2 or APTC-SA-12 entry phage after they went through an infection cycle of two representative non-sensitive S. aureus strains.
  • TSB positive control
  • entry phage treatment APTC-SA-2 or APTC-SA-12
  • PHEARLESS assay The PHEARLESS assay described above was conducted to investigate the lytic activity of the exit phage. In particular, the assay was conducted to investigate and compare the lytic activity of the lysin protein of the entry phage (LysK) with that of the exit phage (denoted mLysK). Briefly, exit phage mLysK expression constructs were prepared from APTC-SA-2-C330 and from APTC-SA-2-C319 and an entry phage LysK expression construct was prepared from APTC-SA-2. All expression constructs were transformed into the PHEARLESS system and tested against the S.
  • aureus phage non-sensitive strains C319, C285, C330 and C43 and also against the phage sensitive ATCC25923 to evaluate their ability to facilitate bacterial lysis.
  • the entry phage LysK only showed weak lytic activity against ATCC25923 as a zone of clearing was observed at the highest dosage only, whilst all other strains remained unaffected by the entry phage LysK.
  • the expressed exit phage mLysK from APTC-SA-2-C330 (298) and from APTC-SA-12-C319 (294) showed lytic activity against all of the tested strains in a dose-dependent way (zone of clearing appearance clear to blurred from highest dosage to lowest dosage).

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Abstract

L'invention concerne un procédé de production d'un bactériophage modifié ("phage") capable d'infecter et de provoquer la lyse d'une bactérie hôte parente qui est insensible au phage non modifié. Le procédé comprend les étapes suivantes : traitement d'une bactérie hôte insensible à une souche phagique sélectionnée avec une quantité appropriée de ladite souche phagique et au moins un agent antibactérien comprenant une activité d'inhibition de la synthèse protéique, et culture de la bactérie pendant une période et dans des conditions permettant au phage d'infecter la bactérie et de se répliquer pour générer une descendance phagique ; et récupération et multiplication de la descendance phagique constituée de phages présentant une capacité modifiée pour infecter et provoquer la lyse de ladite bactérie hôte. L'invention concerne également des méthodes de traitement impliquant l'utilisation du phage modifié, ainsi que des compositions pharmaceutiques le comprenant.
PCT/AU2023/050622 2022-07-05 2023-07-05 Procédé de production de phage modifié et procédé de traitement l'utilisant WO2024007055A1 (fr)

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CN118792265A (zh) * 2024-09-14 2024-10-18 临沂大学 一种噬菌体a149及其在拮抗耐万古霉素粪肠球菌中的应用

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