Enhanced clearance of phage-resistant Klebsiella pneumoniae strains by the innate immune system

Phage therapy has emerged as a promising method to target antibiotic-resistant bacterial strains. Despite its potential, phage therapy has many challenges, including the development of phage resistance whose role in the context of pathogen virulence needs to be fully elucidated. In this study, the interactions between phage-resistant Klebsiella pneumoniae strains and the innate immune system were evaluated. We generated a first-generation phage-resistant mutant, BO-FR-1, using the parental KKBO-1 clinical isolate (Sequence Type 258). Further, starting from BO-FR-1 strain, we isolated three second-generation phage-resistant mutants by using a different phage: FR-GP7-4, FR-GP7-7, and FR-GP7-10. Parental as well as mutant bacterial strains were sequenced and subjected to bioinformatic analysis. Results show the presence of mutations in phage resistant mutants leading to capsule depletion and lipopolysaccharide alteration. Subsequently, human macrophages were infected with these five Klebsiella pneumoniae strains and differences between the parental strain and first- and second-generation phage-resistant mutants were analysed in terms of internalization index and intracellular bacterial killing capability by CFU assay. Additionally, we assessed each strain’s susceptibility to the complement system by serum bactericidal assay. Results indicate that BO-FR-1 strain is more susceptible to phagocytosis, intracellular killing and complement system compared to KKBO-1. Conversely, FR-GP7-4, FRGP7- 7, and FR-GP7-10, although seem to better evade phagocytosis by macrophages compared to BO-FR-1 strain, are more effectively killed by the complement system. Altogether, these results support the hypothesis that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host.