Engineering Bacteriophage To Target KPC+ K. pneumoniae

FARE Award Winner

Authors

• J Jensen
• D Court
• A Parks

Abstract

KPC+ Klebsiella pneumoniae are extensively resistant to commonly used antibiotics. We have developed a system for rapidly and efficiently modifying bacteriophages for use as therapeutic agents. The Red-Recombinase system was used to modify phage T7 as a model system for determining the conditions necessary to generally modify lytic phage genomes. These experiments focus on exchanging tail fiber genes, since they are the primary players in bacterial cell recognition. Genomic DNA from bacteriophage T7 was co-eletroporated into cells, along with synthetically produced oligonucleotides that encode a desired genetic change. Cells that expressed Red-Recombinase showed a 1000-fold increase in phage transfection frequency over controls that do not express the recombinase. Phage particles that infect Klebsiella can also be recovered from Red-expressing E. coli cells that have been transfected with Klebsiella specific phage genomic DNA, indicating that E. coli may be used as a general-purpose recombination host. Oligonucleotide recombination frequencies up to ~10% have been achieved to make simple point mutational changes, and deletions of phage genes are made with 0.1-0.5% efficiency. We have also recombined tail fiber genes onto the E. coli chromosome. Fibers made can assemble onto T7 phage defective for their own tail fiber production when expressed from the E. coli chromosome. The series of bacterial and phage strains that we have generated will be useful in generating diverse phages that lyse antibiotic resistant Klebsiella, and the tools developed in this study can be broadly applied to phages of other intractable pathogens.

Scientific Focus Area: Microbiology and Infectious Diseases

This page was last updated on Friday, March 26, 2021