Yee, W.-X.; Banta, A. B.; Ward, R. D.; Musunuri, S.; Liu, M.; Huiting, E.; Gordeeva, J.; Letham, S. C.; Bharat, T.; Peters, J. M.; Bondy-Denomy, J.

Bacteriophage therapy is needed to treat antibiotic resistant infections; however, when a clinical isolate resists a given phage, it is often unclear why. It is therefore currently unknown how to rationally fortify phage therapies to circumvent a priori resistance. Using a family of broad host range therapeutic Pseudomonas aeruginosa phages (Pbunaviruses), we show that cell surface receptor masking and intracellular defenses are both common barriers in distinct clinical isolates. In some cases these barriers can be bypassed by intrafamily phage engineering. Using unbiased genome-wide CRISPRi screens, we reveal that the broadly conserved L-Rhamnose in the core polysaccharide is the receptor for Pbunavirus family. This molecule is often masked by diverse O-antigen structures. In other isolates with the L-Rha receptor accessible, internal defense mechanisms commonly prevent Pbunavirus DNA replication. A single anti-defense locus often encoding 8-11 different genes within the Pbunavirus family is required for optimal host range, providing anti-defense genes that enable replication of both Pbunavirus phages and phages of other families. Our work demonstrates the importance of both internal and surface defense mechanisms in clinical isolates causally antagonizing a commonly used phage therapeutic and presents phage engineering strategies to circumvent a priori resistance.