Coevolution of a phage cocktail constrains reversible phenotypic resistance in K. pneumoniae

The therapeutic potential of phages is frequently compromised by rapid bacterial adaptation. Strategies such as phage cocktails and phage training are widely used to counteract bacterial resistance. Although phage receptor mutations are highly common, bacteria can also exploit flexible non-genetic strategies to evade phage recognition. Overcoming such phenotypic resistance remains particularly challenging due to its transient and reversible nature. Here, we investigated the coevolutionary dynamics between Klebsiella pneumoniae and a phage cocktail with complementary host tropisms: a capsule-dependent phage and an acapsular-targeting phage, coevolved with bacteria either individually or in combination. The coevolutionary phage training demonstrated that evolution of the acapsular-targeting phage was the main driver of the enhanced resistance delay observed. Notably, while bacteria could evade individual phages through non-mutational mechanisms, the combined selective pressure imposed by the phage cocktail constrained this reversible escape route, forcing bacteria to evolve mutation-based resistance mechanisms. Together, these findings highlight the importance of elucidating phage-bacteria coevolutionary dynamics to optimize phage-based therapeutics design and reshape bacterial evolutionary trajectories toward favourable outcomes. ImportancePhage therapy is re-emerging as a promising alternative to antibiotics, yet its long-term efficacy is often limited by the rapid evolution of bacterial resistance. While phage cocktails and experimental phage training have been widely proposed to mitigate this problem, most studies focus on genetically fixed resistance, overlooking reversible phenotypic resistance mechanisms. Here, we show that phenotypic resistance based on capsule regulation enables bacteria to evade single-phage treatments without acquiring mutations. In contrast, coevolutionary training of a two-phage cocktail targeting complementary bacterial phenotypes constrains this potentially low-cost escape route, forcing bacteria to adopt a mutation-based resistance, typically associated with higher fitness costs. These findings highlight the importance of phage cocktails not only as therapeutic tools, but also as ecological drivers that redirect bacterial evolutionary trajectories.