Opposite evolution of pathogenicity driven by in vivo wzc and wcaJ mutations in ST11-KL64 carbapenem-resistant Klebsiella pneumoniae

Aims: To investigate the in vivo evolution of the mucoid-phenotype of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from the same patients and gain insights into diverse evolution and biology of these strains.Methods: Whole genome sequencing and bioinformatic analysis were used to determine the mutation involved in the mucoid phenotype of ST11-KL64 CRKP. Gene knockout, bacterial morphology and capsular polysaccharides (CPS) extraction were used to verify the role of wzc and wcaJ in the mucoid phenotypes. Antimicrobial sus-ceptibility, growth assay, biofilm formation, host cell adhesion and virulence assay were used to investigate the pleiotropic role of CPS changes in ST11-KL64 CRKP strains.Results: Mutation of wzc S682N led to hypermucoid phenotype, which had negative impact on bacterial fitness and resulted in reduced biofilm formation and epithelial cell adhesion; while enhanced resistance to macrophage phagocytosis and virulence. Mutations of wcaJ gene led to non-mucoid phenotype with increased biofilm for-mation and epithelial cell adhesion, but reduced resistance of macrophage phagocytosis and virulence. Using virulence gene knockout, we demonstrated that CPS, rather than the pLVPK-like virulence plasmid, has a greater effect on mucoid phenotypic changes. CPS could be used as a surrogate marker of virulence in ST11-KL64 CRKP strains.Conclusions: ST11-KL64 CRKP strains sacrifice certain advantages to develop pathogenicity by changing CPS with two opposite in vivo evolution strategies.