Mutations in EnvZ, KPC, and OmpK36 contribute to the evolution from ceftazidime-avibactam heteroresistance to stable resistance in carbapenem-resistant Klebsiella pneumoniae

Antibiotic heteroresistance, an unstable phenotype wherein a minority-resistant subpopulation coexists with a majority-susceptible population, can evolve to stable resistance following in vivo antibiotic exposure, thereby substantially increasing the risk of treatment failure. However, the properties of this evolutionary process remain poorly understood. The present study aimed to investigate the characteristics and mechanisms underlying the evolution from ceftazidime-avibactam (CZA) heteroresistance to stable resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP). Twelve resistant subpopulations isolated from CZA heteroresistant parental strains by population analysis profiling were subjected to in vitro selection experiment to obtain stable CZA-resistant strains. All resistant subpopulations rapidly evolved to stable CZA-resistant strains (MIC ≥ 256/4 mg/L) under CZA pressure. Resistant subpopulations were unstable under antibiotic-free conditions and reverted to CZA-susceptible strains, while stable CZA-resistant strains maintained stable resistance in the absence of antibiotics. Resistant subpopulations exhibited significantly lower growth capacity than their CZA heteroresistant parental strains, but their growth capacity was restored upon evolution to stable CZA-resistant strains. Elevated avibactam concentrations restored CZA susceptibility in resistant subpopulations but had no effect on stable CZA-resistant strains. Tigecycline and polymyxin B retained excellent antimicrobial activity against both resistant subpopulations and stable CZA-resistant strains. Additionally, KPC expression and copy number were upregulated in resistant subpopulations, while a combination of mutations in KPC, OmpK36, EnvZ, PBP2, and LamB was detected and KPC expression was restored in stable CZA-resistant strains. Complementation with the wildtype ompK 36 or envZ in stable CZA-resistant strains significantly reduced the CZA MIC and expressing mutant bla KPC in CZA heteroresistant parental strains substantially increased the CZA MIC. Fitness cost analysis revealed that resistant subpopulations exhibit high fitness costs. Whether resistant subpopulations evolved into stable CZA-resistant strains or susceptibility-reverted strains, the process was accompanied by decreased fitness costs, downregulation of KPC expression, and restored growth capacity. Our study illustrates that CZA heteroresistance easily evolves into stable resistance under antibiotic pressure, accompanied by enhanced resistance stability and growth capacity. The evolution mechanism may involve various combinations of mutations in EnvZ, KPC, and OmpK36.