Bacteriophages for reducing toxicity of bacteria

• US 8,865,158 B2
• Filed: 05/22/2012
• Issued: 10/21/2014
• Est. Priority Date: 05/22/2012
• Status: Active Grant

• Current Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Original Assignee: Ramot at Tel Aviv University Limited (Tel Aviv University)
• Inventors: Edgar, Rotem, Friedman, Nir, Qimron, Ehud
• Primary Examiner(s): HILL, KEVIN KAI
• Application Number:
  US13/477,213
• Publication Number:
  US 20130315869A1
• Time in Patent Office: 882 Days
• Field of Search: 424/93.6, 435/235.1, 435/252.3, 435/264, 435/320.1
• US Class Current: 424/93.6
• CPC Class Codes: A61K 35/76 Viruses; Subviral particles; Bacteriophages
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C12N 2795/10332 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Abstract

A genetically modified bacteriophage is disclosed which comprises:

• • (i) an exogenous polynucleotide which encodes an agent which reduces the toxicity of a bacterium; and
  • (ii) an exogenous polynucleotide which encodes a selectable marker.

Uses thereof and kits comprising same are also disclosed.

Claims

1. A genetically modified bacteriophage comprising:

(i) an exogenous polynucleotide which encodes at least one agent which reduces the toxicity of a bacterium, is non-toxic to the bacterium and does not affect directly the survival of said bacterium, said polynucleotide is a dominant sensitive antibiotic resistant gene that reverses resistance of said bacterium to an antibiotic, wherein said at least one agent is 30S ribosomal subunit protein S12, encoded by the polynucleotide sequence of SEQ ID NO;

24; and

(ii) an exogenous polynucleotide which encodes a selectable marker that renders a bacterium infected by said phage resistant to a toxic compound selected from the group consisting of potassium-tellurite, chlorhexidine salts, diamidines, acridines, arsenite, arsenate and antimonite.

2. The bacteriophage of claim 1, wherein said polynucleotide further comprises at least one dominant sensitive antibiotic resistant gene selected from the rpsl gene encoding the 30S ribosomal subunit protein S12, the gyrA gene encoding the gyrase protein, the rpoB gene that encodes the RNA Polymerase β

Subunit and the thyA gene that encodes thymidylate synthase.

3. The bacteriophage of claim 2, wherein said resistance is due to a mutated polypeptide of said bacterium selected to be 30S ribosomal subunit protein S12.

4. The bacteriophage of claim 2, wherein said resistance is due to a mutated polypeptide of said bacterium selected to be the gyrase protein.

5. The bacteriophage of claim 2, wherein said at least one dominant sensitive antibiotic resistant gene is a gene encoding a polypeptide selected to be 30S ribosomal subunit protein S12 or the gyrase protein.

6. The bacteriophage of claim 2, wherein said at least one dominant sensitive antibiotic resistant gene is a gene encoding a 30S ribosomal subunit protein S12.

7. The bacteriophage of claim 1, wherein said agent is a polynucleotide agent which down-regulates expression of an antibiotic resistance gene in said bacterium.

8. The bacteriophage of claim 1, wherein said selectable marker comprises a resistance marker to tellurite.

9. The bacteriophage of claim 1, being a lambda temperate phage.

10. An anti-bacterial composition, comprising a carrier and as an active ingredient the bacteriophage of claim 1.

11. The antibacterial composition of claim 10, formulated as a spray, a stick, a paint, a gel, a cream, wash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment or a paste.

12. An isolated population of bacterial cells comprising the bacteriophage of claim 1.

13. A kit comprising the phage of claim 1 and a compound which is toxic to bacteria, wherein said toxic compound is selected from the group consisting of potassium-tellurite, chlorhexidine salts, diamidines, acridines, arsenite, arsenate and antimonite.

14. A method of preventing a bacterial infection which is resistant to an antibiotic in a subject, the method comprising contacting a solid surface with an anti-bacterial composition that includes the bacteriophage of claim 1, thereby preventing the bacterial infection.

15. The method of claim 14, further comprising contacting the solid surface with a compound which is toxic to bacteria, said compound is selected from the group consisting of potassium-tellurite, chlorhexidine salts, diamidines, acridines, arsenite, arsenate and antimonite.

16. A genetically modified bacteriophage comprising:

(i) an exogenous polynucleotide which encodes a polypeptide which reverses resistance of a bacterium to an antibiotic, wherein said polypeptide is 30S ribosomal subunit protein S12, encoded by the polynucleotide sequence of SEQ ID NO;

24; and

(ii) an exogenous polynucleotide which encodes a selectable marker that renders a bacterium infected by said phage resistant to a toxic compound selected from the group consisting of potassium-tellurite, chlorhexidine salts, diamidines, acridines, arsenite, arsenate and antimonite.

17. A genetically modified bacteriophage comprising an exogenous polynucleotide which encodes at least one polypeptide which reverses resistance of a bacterium to an antibiotic and is non-toxic to the bacterium, wherein said wherein said at least one polypeptide is 30S ribosomal subunit protein S12, encoded by the polynucleotide sequence of SEQ ID NO:

24.

18. The bacteriophage of claim 17, wherein said polynucleotide further comprises at least one dominant sensitive antibiotic resistant gene selected from the rpsl gene encoding the 30S ribosomal subunit protein S12, the gyrA gene encoding the gyrase protein, the rpoB gene that encodes the RNA Polymerase β

Subunit and the thyA gene that encodes thymidylate synthase.

19. The bacteriophage of claim 17, wherein said polynucleotide further comprises a dominant sensitive antibiotic resistant gene encoding a gyrase protein.

20. The bacteriophage of claim 19, wherein said exogenous polynucleotide further encodes a selectable marker.

21. An anti-bacterial composition, comprising a carrier and as an active ingredient the bacteriophage of claim 19.

22. A method of preventing a bacterial infection which is resistant to an antibiotic in a subject, the method comprising contacting a solid surface with an anti-bacterial composition that includes the bacteriophage of claim 19, thereby preventing the bacterial infection.

23. A genetically modified bacteriophage comprising an exogenous polynucleotide which encodes a polypeptide which reverses resistance of a bacterium to an antibiotic, wherein said polypeptide is the 30S ribosomal subunit protein S12 and is encoded by the polynucleotide sequence of SEQ ID NO:

24.

24. The bacteriophage of claim 23, wherein said exogenous polynucleotide further encodes a selectable marker.

25. The bacteriophage of claim 24, wherein said selectable marker comprises a resistance marker to tellurite.

26. The bacteriophage of claim 23, being a lambda temperate phage.

27. An anti-bacterial composition, comprising a carrier and as an active ingredient the bacteriophage of claim 23.

28. The antibacterial composition of claim 27, formulated as a spray, a stick, a paint, a gel, a cream, wash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment or a paste.

29. A method of preventing a bacterial infection which is resistant to an antibiotic in a subject, the method comprising contacting a solid surface with an anti-bacterial composition that includes the bacteriophage of claim 23, thereby preventing the bacterial infection.

30. The method of claim 29, further comprising contacting the solid surface with a compound which is toxic to bacteria said compound is selected from the group consisting of potassium-tellurite, chlorhexidine salts, diamidines, acridines, arsenite, arsenate and antimonite.

31. The method of claim 30, wherein said exogenous polynucleotide further encodes a selectable marker that renders a bacterium infected by said phage insensitive to said toxic compound.