Characterization of a Klebsiella pneumoniae mutant strain wGF 1–2 with attenuated virulence, altered morphology, and reduced biofilm formation

Introduction The global rise of antimicrobial resistance has positioned multidrug-resistant Klebsiella pneumoniae as a critical health threat, necessitating alternative therapeutic strategies such as phage therapy. However, the long-term evolutionary consequences of phage-bacteria interactions remain poorly understood. This study characterizes a unique attenuated mutant, wGF 1-2, derived from a hypervirulent K. pneumoniae strain (GF) during phage isolation efforts. Methods The wGF 1-2 mutant was serendipitously isolated during attempts to obtain lytic phages against the parental GF strain. We performed an integrated multi-omics and phenotypic characterization, including genomic sequencing, proteomic profiling, and transcriptomic analysis. Host-pathogen interactions were assessed using a murine infection model (evaluating survival and tissue colonization), and the impact on the gut microbiota was analyzed via metagenomics. Results Compared to the parental strain, wGF 1-2 exhibited a significant reduction in biofilm formation and distinct morphological alterations. In a murine model, the mutant was avirulent, resulting in 100% survival even at a high challenge dose (10⁶ CFU), with minimal tissue colonization. Multi-omics analysis revealed extensive genomic structural variations (81 insertions and 64 deletions). Proteomic shifts included the downregulation of proteins involved in metal ion binding and metabolic pathways. Furthermore, infection with wGF 1-2 led to host inflammatory suppression and a restructuring of the gut microbiota characterized by an increase in beneficial Bacteroidota . Discussion This study provides a comprehensive characterization of an attenuated K. pneumoniae mutant, wGF 1-2. The extensive genomic and phenotypic alterations observed highlight the significant evolutionary potential of bacterial pathogens during phage interactions. These findings underscore the necessity of thorough safety assessments, including evolutionary risk evaluations, for the future development of phage-based therapies.