• bla SHV-12 amplification confers ceftazidime/avibactam resistance in CRKP. • tet (A) amplification and mutation mediate tigecycline resistance under pressure. • phoPQ, pmrAB , and mgrB mutations drive stable colistin resistance evolution. • Antibiotic pressure leads to capsule loss and attenuated virulence in CRKP. • Transcriptomics reveals global gene reprogramming during resistance evolution. Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly the ST11-KL64 clone carrying bla KPC-2 , is a critical global health threat. This study investigates the adaptive evolution of CRKP under last-line antibiotic pressure, focusing on resistance mechanisms, genetic alterations, and associated phenotypic changes. A serial passage laboratory evolution model was established using a triple-antibiotic combination—ceftazidime/avibactam (CZA), tigecycline (TGC), and colistin (COL) in a 4:2:1 ratio—against the ST11-KL64 CRKP strain. Six independent evolutionary lineages were generated and analyzed for resistance phenotypes, genomic mutations, and changes in fitness and virulence. Antimicrobial susceptibility testing, whole-genome sequencing, and transcriptomic analysis were employed to characterize resistance mechanisms and physiological adaptations. Evolved populations exhibited markedly increased resistance to CZA, TGC, and COL, reaching maximal antibiotic concentrations within 19-45 days. Fixed mutations were identified in the pmrAB and phoPQ two-component systems and the mgrB regulator. Plasmid-borne gene amplifications of bla SHV-12 and tet (A) were observed. Transcriptomic profiling revealed widespread gene expression reprogramming associated with resistance. Notably, evolved strains showed increased fitness costs, altered colony morphology, reduced capsule production, and attenuated virulence. This study demonstrates the complex adaptive mechanisms employed by CRKP to enhance multidrug resistance. Mutations, gene amplifications, and altered gene expression underpin resistance to key antibiotics, while virulence factors are modified, likely due to fitness costs. These findings highlight the need for more effective strategies to combat the evolving threat of CRKP in clinical settings.
Ding et al. (2026) studied this question.