Bacteriophage therapy: a novel method of lytic phage delivery

Abstract

The widespread emergence of muki-antibiotic resistant bacteria has increased the need for alternatives to conventional antibiotic therapy. Accordingly, a significant amount of effort has been made to investigate the potential use of bacteriophages as prophylactic and therapeutic agents for bacterial infections. In this study, molecular biological techniques were applied to construct a iysogen of lytic bacteriophage X in an attempt to combat with multi-antibiotic resistant bacteria by a novel method of lytic phage delivery. To accomplish this goal, two plasmid-based site-specific recombination (SSR) systems for integration and recovery of DNA constructs from Escherichia coli and Salnumella typhimurium chromosomes were developed. The two systems are mediated by SSR machineries of bacteriophages X. of £1 coli and P22 of S. typhimurium. These systems utilize plasmid vectors with conditional replicating origin of replication and provide stable chromosomal integration of genes at specific bacteriophage attachment sites without disruption of any host gene or a need for antibiotic selection. E. coli contains attachment sites for both bacteriophages. When the two systems are applied consecutively, two different genes can be integrated at two specific locations. The integrated plasmids of both systems can also be completely excised and recovered fi'om the host chromosomes to observe any genetic changes, e.g. by DNA sequencing. Both systems are also very applicable in construction of bacterial strains as well as live E. coli and S. typhimurium recombinant vaccines expressing foreign genes of interest. To construct a Iysogen of lytic bacteriophage X, both SSR systems were applied. A lytic mutant {ct) of bacteriophage X was marked with an antibiotic resistant gene cassette to