Phage heteroresistance in clinical Klebsiella pneumoniae isolates: impact of differentiated capsule expression on phage efficacy

Abstract Heteroresistance, commonly described in the context of antibiotics, refers to the presence of resistant subpopulations within a bacterial strain, and is recognized as a likely contributor to treatment failure. Whether population heterogeneity affects phage activity remains poorly understood. We investigated whether heterogeneity in clinical, mostly multidrug-resistant Klebsiella pneumoniae (Kpn) isolates contributes to phage heteroresistance, focusing on the role of capsule-deficient subpopulations. We isolated and characterized 23 strictly lytic phages against diverse clinical Kpn strains. Host range revealed phages with varying spectra, one able to infect up to four different sequence types and two capsule loci. Most phages surprisingly led to longer bacterial growth inhibition at lower multiplicity of infection (MOI). Half of the Kpn samples harboured capsule-deficient variants. Sequencing revealed the presence of insertion sequences in capsule biosynthesis or regulation genes suggesting impaired capsule production. Spot-test assays showed complete loss or reduced activity on these variants. Interestingly, reduced activity by spot-test translated into a delayed onset of infection in kinetic assays, suggesting only partial capsule-dependence for successful infection. Our findings show that lower MOI led to higher virulence in most phages, that capsule-deficient Kpn subpopulations were common, and that capsule loss significantly altered phage activity. Although the clinical implications of phage heteroresistance remain unclear, this phenomenon should be considered when designing personalized phage therapies, as it may explain treatment failure or predict the early emergence of phage-resistant mutants.