Engineered enzymatically active bacteriophage and methods for dispersing biofilms

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.

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 , wherein the strong promoter is T7φ10. 
     
     
         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 , wherein the strong promoter is T7 φ10. 
     
     
         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.