Capsule mutations serve as a key strategy of phage resistance evolution of K54 hypervirulent Klebsiella pneumoniae.

Phage therapy is a promising antibacterial strategy against the antibiotic resistance crisis. The evolved phage resistance could pose a big challenge to clinical phage therapy. Therefore, it is necessary to conduct a comprehensive analysis of phage resistance mechanisms during treatment. Here, we characterize 37 phage-resistant mutants of hypervirulent K. pneumoniae strain SCNJ1 under phage-imposed selection in both in vitro and in vivo experiments. We show that 97.3% (36/37) of phage-resistant clones possessed at least one mutation in genes related to the CPS biosynthesis. Notably, the wcaJ gene emerges as a mutation hotspot, as mutations in this gene are detected at a high frequency under both conditions. In contrast, mutations in wzc exhibit more association with in vivo samples. These CPS-related mutants all exhibit compromised bacterial fitness and attenuated virulence in mice. Strain CM8 is the only non-CPS-related mutant, which has a bglA mutation that confers phage resistance and retains full fitness and virulence. This study highlights that laboratory characterization of phage resistance evolution can give useful insights for clinical phage therapy.