Systematic Discovery of Salmonella Phage-Host Interactions via High-Throughput Genome-Wide Screens

species comprise a chronic threat to human health, with over 1.35 million infections and 420 deaths occurring in the US per year due to non-typhoidal, foodborne infection. With the rise of antimicrobial resistant (AMR) infections, it is imperative to develop alternative prevention and treatment strategies, such as utilization of lytic bacteriophage as pathogen countermeasures. However, phage-host interactions remain poorly understood, impeding widespread practice. Here, we employed high-throughput, functional analyses to discover the genetic determinants of phage-host interactions between a model enteric species, Typhimurium () and its phages. To rapidly assess genetic contributions to phage sensitivity, we created a 66,996 member randomly barcoded transposon (RB-TnSeq) library in MS1868. Using this library, we compared fitness across eleven diverse lytic phages. Consistent with other loss of function studies against bacteriophage predation, this approach efficiently identified receptors essential to adsorption as well as their regulation. While many of the tested phages bound directly to the lipopolysaccharide (LPS) layer, we report a highly resolved view of differential structural LPS layer requirements for diverse phages, including novel adsorption strategies. We also uncover unique routes to phage resistance, including phage-specific metabolic requirements, ion flow, and global transcription factor interplay, difficult to find through traditional approaches. constitutes a major challenge and opportunity. We highlight several examples of how the scale of barcoded screens allowed systems-level hypotheses to be efficiently formulated. These include discovery of a number of cases of cross-resistance and collateral sensitivity among the diverse phage tested. We anticipate that the phage-resistance landscape presented here will faciliate better design of phage-based biocontrol treatments and phage-antibiotic combination therapies.