Phenotypic Heterogeneity Shapes Phage Resistance and Cocktail Efficacy in Klebsiella pneumoniae

The emergence of phage-resistant bacteria poses a significant challenge to the success of phage therapy. Although phage cocktails can delay resistance, their efficacy relies on the ability to target the full spectrum of resistant variants, which are often more diverse than that represented by clonal isolates. In this study, we investigated how phenotypic diversity within phage-resistant Klebsiella pneumoniae influences susceptibility to newly isolated phages and the effectiveness of phage cocktails. We isolated phages from three cultures resistant to a capsule-dependent phage: a heterogeneous acapsular population, an acapsular mutant with a stable phenotype, and a capsule-reverted isolate that regained capsule expression upon removal of phage pressure. These phages differed in host tropism and in their capacity to delay resistance emergence when combined with the capsule-dependent phage. Phages targeting acapsular variants, particularly those isolated from the heterogeneous population, were the most effective, exhibiting strong synergy. Single-cell analyses further revealed that sustained selective pressure from the capsule-dependent phage prevents capsule reversion and maintains cocktail efficacy. Overall, our results highlight the importance of accounting for phenotypic heterogeneity when designing phage therapies and support population-level approaches for optimizing phage cocktail composition. ImportancePhage therapy is a promising alternative to antibiotics, but its success is often limited by the rapid emergence of phage-resistant bacteria. These resistant populations can be highly heterogeneous, comprising both stable mutants and variants with reversible, non-genetic resistance. In this study, we explore how this phenotypic diversity influences the effectiveness of phage cocktails. By isolating new phages and testing them in combination, we demonstrate that the selective pressure exerted by specific phages can prevent the reversion in transiently resistant variants, thereby sustaining treatment efficacy. Our findings highlight the need to consider not only the range of bacterial targets but also how phage pressure shapes bacterial population dynamics. This work offers a more refined strategy for designing phage cocktails with improved clinical potential.