Heterogeneous multicopy of bla_CTX-M variants on the same plasmid enhances evolutionary adaptability in clinical...

Pathogenic bacteria continually evolve under antimicrobial pressure through acquired resistance genes, making it crucial to understand their evolutionary strategies. We identify a clinical Klebsiella pneumoniae isolate resistant to ceftazidime/avibactam (CZA), harboring heterogeneous multicopy bla_CTX-M, among which a bla_CTX-M-249 variant mediates CZA resistance. Both bla_CTX-M-249 and its closely related allele bla_CTX-M-65 are dominant within the clonal population and are located at two loci on the same plasmid, with their proportions shifting under antibiotic pressure. Using experimental and mathematical models, we demonstrate that the heterogeneous arrangement of bla_CTX-M variants on the same plasmid confers greater stability and competitive advantage than that across separate plasmids, particularly during drug switching. Re-analysis of large genomic datasets supports the universality of this phenomenon. Our findings reveal an evolutionary strategy in which β-lactamase genes, through multicopy heterogeneity on a single plasmid, ensure stable inheritance of resistance and enhance bacterial adaptability under fluctuating clinical antibiotic pressures.