398 Phage Wars: Uncovering the resistance strategies of <i>Escherichia coli</i> O157:H7

Objectives/Goals: The goal of this work is to understand the physiological profile of phage susceptibility and identify candidate phage defense mechanisms. Additionally, it aims to determine the host receptors targeted by bacteriophages to infect E. coli O157:H7 through random bar code transposon-site sequencing (RB-TnSeq). Methods/Study Population: A collection of 109 E. coli O157:H7 strains from environmental, food, and animal sources were analyzed, representing phylogenetic lineages corresponding to clades 2, 3, 5, 6, 7, and 8. Phage susceptibility profiles were determined using 23 bacteriophages, assessing plaque morphology. Using the O157:H7 genomes, a genomic analysis was conducted with the Prokaryotic Antiviral Defense Locator (PADLOC), which identified putative phage defense systems through sequence homology. Additionally, 5 RB-TnSeq libraries were generated in representative strains to study loss-of-function mutations. These libraries will be screened against a subset of diverse phage to identify the receptors involved in phage adsorption. Results/Anticipated Results: The phage resistance patterns showed susceptibility varied across clades, suggesting distinct mechanisms. Several defense systems were identified using PADLOC, including restriction-modification, Cas, Lamassu, and Druantia. Phage defense candidate (PDC) systems were identified, showing homology to known systems, though their specific function remains unknown. Clade 7.2 exhibited higher phage resistance and a greater presence of PDCs compared to the other clades. Five saturated RB-TnSeq libraries were constructed in O157:H7, achieving 84.5–89% gene coverage. These libraries will facilitate the identification of receptors involved in phage adsorption and resistance. Discussion/Significance of Impact: This study deepens our understanding of phage resistance in E. coli O157:H7 by identifying key defense systems and receptors. The discovery of novel antiviral mechanisms offers promising targets for phage-based interventions, potentially enhancing strategies for controlling this dangerous pathogen.