ENGINEERED ENZYMATICALLY ACTIVE BACTERIOPHAGE AND METHODS FOR DISPERSING BIOFILMS

• US 20090155215A1
• Filed: 12/18/2008
• Published: 06/18/2009
• Est. Priority Date: 12/18/2007
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology, Trustees of Boston University
• Original Assignee: Massachusetts Institute of Technology, Trustees of Boston University
• Inventors: Collins, James J., Lu, Timothy Kuan-Ta
• Granted Patent: US 8,153,119 B2
• Time in Patent Office: Days
• US Class Current: 424/93.200
• CPC Class Codes: A01N 63/40 Viruses, e.g. bacteriophages
• CPC Class Codes: A01N 63/50 Isolated enzymes; Isolated proteins peptides A01N37/46
• CPC Class Codes: C12N 2795/10211 Podoviridae
• CPC Class Codes: C12N 2795/10221 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2795/10232 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12Y 207/07006 DNA-directed RNA polymerase (2.7.7.6)
• CPC Class Codes: C12Y 302/01052 Beta-N-acetylhexosaminidase (3.2.1.52)

Abstract

The present invention is directed to engineered enzymatically active bacteriophages that are both capable of killing the bacteria by lysis and dispersing the bacterial biofilm because they have been also engineered to express biofilm-degrading enzymes, particularly dispersin B (DspB), an enzyme that hydrolyzes β-1,6-N-acetyl-D-glucosamine, a crucial adhesion molecule needed for biofilm formation and integrity in Staphylococcus and E. coli, including E. coli K-12, as well as clinical isolates.

Claims

1. An engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B enzyme or an β

-1,6-N-acetyl-D-glucosamine degrading enzymatically active fragment thereof operably linked to a strong promoter and further comprising a nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage.

2. The engineered lytic T7 bacteriophage of claim 1, wherein the a nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage encodes T3 1.2 gene.

3. The engineered lytic T7 bacteriophage of claim 1 or 2, wherein the strong promoter is T7 φ

10.

4. A method of dispersing bacterial biofilm comprising administering to a surface infected with biofilm the method comprising administering to the surface an engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B enzyme or an β

-1,6-N-acetyl-D-glucosamine degrading enzymatically active fragment thereof operably linked to a strong promoter and further comprising a nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage.

5. The method of claim 4, wherein the a nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage encodes T3 1.2 gene.

6. The method of claim 4 or 5, wherein the strong promoter is T7 φ

10.

7. The method of claim 4, wherein the biofilm is a mature biofilm.

8. The method of claim 4, wherein the biofilm comprises β

-1,6-N-acetyl-D-glucosamine.

9. The method of claim 8 further comprising a step of prior to administering the bacteriophage, determining if the biofilm comprises β

-1,6-N-acetyl-D-glucosamine, and if it does, then administering the engineered lytic bacteriophage.

10. The method of claim 4, wherein the biofilm is formed by bacteria selected from the group consisting of Staphylococcus and E. coli, including E. coli K-12 strain, and clinical isolates of E. coli.

11. The method of claim 4, wherein the administering is performed once.

12. The method of claim 4, wherein the administering is performed before, after or concurrently with an antibiotic or antimicrobial agent.

13. The method of claim 4, wherein the administering is performed before, after or concurrently with a biofilm degrading chemical.