Identification of the Genetic Basis of Phage Resistance in Sequentially Generated Phage-Resistant Klebsiella pneumoniae Using an Established Phage Library

<b><i>Objectives:</i></b> To explore the genetic basis of phage resistance in sequentially generated capsular mutants of phage-resistant <i>Klebsiella pneumoniae</i> using an established phage library. <b><i>Methods:</i></b> Sequential induction strategies were employed to obtain phage-resistant <i>K. pneumoniae</i> capsular mutants by exposing ST11-K64 <i>K. pneumoniae</i> Kp2325 to different single phages. Whole genome sequencing and bioinformatic analysis were used to elucidate the capsular-related genetic changes in phage-resistant mutants. Phenotypic changes were assessed through gene complementation, growth assays, phage cleavage spectrum analysis, TEM for phage morphology, CPS analysis, biofilm formation, and virulence assays. <b><i>Results:</i></b> Three sequentially generated phage-resistant <i>K. pneumoniae</i> capsular mutants were obtained, designated R1, R2 and R3. The narrowing of the phage cleavage spectrum and the evolutionary trade-offs of biological phenotypes were observed. Key genetic changes included: (1) IS<i>Kpn26</i> insertion disrupting <i>wcaJ</i> in R1; (2) combined <i>wcaJ</i> insertion and 9-bp deletion in <i>waaH</i> in R2; and (3) CPS gene cluster deletion in R3 were identified as key mechanisms of phage resistance in <i>K. pneumoniae</i> mutants R1, R2 and R3, respectively. <b><i>Conclusions:</i></b> Sequential exposure to different single phages led to rapid evolution of phage resistance in <i>K. pneumoniae</i> via genetic mutations that disrupt capsular synthesis. These findings highlight the critical role of bacterial capsule in phage-host interactions and emphasize the need to use phage cocktails targeting different types of receptors to counteract the evolution of bacterial defense mechanisms in phage therapy.