Phenotypic Heterogeneity and Genetic Mechanisms of Phage Resistance in Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae

Phage resistance poses a major barrier to the clinical application of phage therapy against multidrug-resistant bacterial pathogens. A comprehensive understanding of the phenotypic heterogeneity and underlying molecular mechanisms of phage resistance is essential for deciphering bacterium-phage interactions and optimizing therapeutic strategies. In this study, we identified four distinct resistance phenotypes in K2-serotype hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) under phage Kpph1 pressure. These resistant strains exhibit complete or partial resistance, possess varying degrees of fitness costs and harbor various genetic mutations, suggesting the multifaceted resistance strategies including receptor masking, growth inhibition, and community resistance. Among these, wcaJ mutations was identified and confirmed as the predominant resistance mechanism, mainly resulting from base insertions and integrations of mobile genetic elements, serving as an efficient pre-adaptive strategy for CR-hvKP to evade phage infection. To circumvent the resistance, we isolated a secondary phage, K2V3, which specifically targets resistant strains with wcaJ mutation. The rationally designed phage cocktail of Kpph1 and K2V3 effectively suppressed all resistant variants and restricted the emergence of resistance. These findings deepen the understanding of phage-bacteria interaction dynamics and provide a key theoretical basis for optimizing clinical phage therapy against multidrug-resistant pathogens.