Development of molecular approaches to enhance bacteriophage studies in Escherichia coli

Abstract

Urinary tract infections (UTIs) are among the most common bacterial infections globally, affecting millions each year. Uropathogenic Escherichia coli (UPEC) is the leading cause of UTIs and includes strains increasingly resistant to antibiotics. As multidrug-resistant UPEC continues to emerge, there is a growing need for alternative treatment strategies such as phage therapy. This thesis focused on developing molecular tools for the genetic engineering of bacteriophages that target UPEC. Central to this work was the establishment of protocols to introduce modified phage genomes into bacterial hosts through electroporation. Several approaches were tested to improve electroporation efficiency, including refining genome purification methods, adjusting host growth conditions, and reducing DNA degradation by removing one of the restriction-modification systems in the clinical UPEC strain CFT073 via mutagenesis. To enable real-time tracking of phage activity, fluorescent E. coli strains were constructed, attempting to monitor bacterial lysis via fluorescence. In parallel, Tn5-based transposition techniques were developed to facilitate insertional mutagenesis of phage genomes, both in vitro and in vivo. Collectively, the methods and tools developed in this study offer a foundation for more effective phage genome manipulation and lay the groundwork for advancing bacteriophage-based therapies against UPEC infections.