Investigation of the therapeutic efficacy and resistance mechanisms of lytic phages targeting ST218 KL57 CR-hvKP

Carbapenem-resistant hypervirulent <i>Klebsiella pneumoniae</i> (CR-hvKP) infection is gradually increasing globally. Phage therapy is a viable application as an alternative to antibiotics. However, clinical application of phage therapy is restricted by phage resistance. To further explore the mechanism underlying phage resistance, particularly the difference observed between <i>in vivo</i> and <i>in vitro</i>, we employed a mouse intra-abdominal infection model to assess the antibacterial properties of two lytic phages and further isolate and characterize phage-resistant mutants. We identified that the majority of the mutation sites in the phage-resistant <i>K. pneumoniae</i> mutants were located in the capsular polysaccharide (CPS) gene cluster, as determined through genomic and transcriptomic analysis. However, some <i>K. pneumoniae</i> phage-resistant mutants, including RM01, RM02, and RM12, developed phage resistance by downregulating CPS and the respective transcriptional regulators without any mutations in the CPS gene. In summary, these findings provide further evidence supporting phage therapy, particularly addressing the issue of CR-hvKP infections.IMPORTANCEThe global rise in antibiotic resistance has rekindled interest in utilizing bacteriophage therapy as a potential solution. In this study, we explored the therapeutic potential of two novel bacteriophages, with a focus on their <i>in vivo</i> efficacy using mouse models, and analyzed the probable mechanisms of phage resistance in bacteria. Our results indicated that in a murine infection model, phages JLBP1001 and JLBP1002 for <i>Klebsiella pneumoniae</i> were highly effective, significantly improving mouse survival. We further characterized and analyzed phage-resistant <i>K. pneumoniae</i> isolated from the mice and found that the resistance mechanisms in an <i>in vivo</i> environment are primarily concentrated in the capsular polysaccharide gene cluster. In RM01, RM02, and RM12, <i>putA</i> contributes to phage resistance through point mutations. These insights are important for optimizing phage-based therapies, particularly in the context of multidrug-resistant bacterial infections.