From phenotype to receptor: validating physiological clustering of

Understanding the relationship between bacteriophage (phage) classification and target receptors is crucial for phage ecology and applied research. In this study, we compared 13 previously isolated IMPORTANCE: Phage therapy is gaining attention as an alternative treatment for antibiotic-resistant bacteria. Developing effective phage cocktails requires combining phages with different target receptors, but traditional methods for identifying target receptors are labor-intensive. This study demonstrates that E. coli phages targeting the TK001 strain with different target receptors can be grouped based on their physiological characteristics, tail fiber sequences, or whole genomes. Our approach was enhanced through systematic bacterial panel selection for host range assessment, optimized distance metrics for physiological characteristics, and objective cluster determination using silhouette coefficient analysis for all three classifications. This insight can be used to more efficiently create diverse phage cocktails. Additionally, we identified phages targeting diverse sites, including different regions of LPS, membrane proteins, and flagella. These findings deepen our understanding of phage host recognition mechanisms, enabling the rapid preparation of effective phage cocktails and contributing to new advancements in bacterial infection treatment.