Sequential Use of Two Capsule-Targeting Klebsiella Phages Reveals Order-Dependent Efficacy and Distinct Resistance Pathways in vitro and in vivo

Bacteriophage cocktails are widely assumed to improve treatment of multidrug-resistant Klebsiella pneumoniae, yet many therapeutic phages target capsular polysaccharides (CPS), potentially promoting antagonism and shared resistance. Here, we investigated how receptor usage, resistance evolution, and dosing order influence the activity of two K1-specific phages, Loop and Spear, against a hypervirulent ST23 strain. Using in vitro killing assays and Bliss analysis, we show that a 1:1 Loop and Spear cocktail did not improve bacterial suppression compared to Loop alone and instead exhibits multiplicity-of-infection-dependent antagonism, consistent with competition for a shared CPS receptor. Sequential dosing revealed strong order dependence: treatment with Spear followed by Loop qualitatively altered growth trajectories in a subset of cultures, whereas the reverse order or repeated dosing, provided little additional benefit. Resistance profiling showed that mutants resistant to both phages predominantly carried mutations in capsule synthesis or export genes, whereas Spear specific resistance was associated with mutations in fkpA, encoding a periplasmic chaperone for outer membrane protein biogenesis. Adsorption assays confirmed that capsule associated mutations abolished Loop attachment, while {Delta}fkpA mutants retained Loop binding, supporting CPS to be a primary receptor with Spear additionally requiring an FkpA-dependent secondary receptor. In a Galleria mellonella infection model, a capsule-mutant resistant isolate showed reduced virulence, and only sequential therapy with Spear followed by Loop improved survival beyond monotherapy. These findings show that receptor sharing can render phage cocktails antagonistic and highlight sequential, order-aware regimens as a strategy to exploit resistance-virulence trade-offs while limiting the emergence of double-resistant mutants.