The Effect of beta-Lactam Antibiotics on the Evolution of Ceftazidime/Avibactam and Cefiderocol Resistance in KPC-Producing Klebsiella pneumoniae

In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during beta-lactam antibiotic therapy. Five KPC-Kp isolates were collected from a single patient. Whole-genome sequencing and a comparative genomics analysis were performed on the isolates and all bla(KPC-2)-containing plasmids to predict the population evolution process. Growth competition and experimental evolution assays were conducted to reconstruct the evolutionary trajectory of the KPC-Kp population in vitro. Five KPC-Kp isolates (KPJCL-1 to KPJCL-5) were highly homologous, and all harbor an IncFII bla(KPC)-containing plasmid (pJCL-1 to pJCL-5). Although the genetic structures of these plasmids were almost identical, distinct copy numbers of the bla(KPC-2) gene were detected. A single copy of bla(KPC-2) was presented in pJCL-1, pJCL-2, and pJCL-5, two copies of bla(KPC) (bla(KPC-2) and bla(KPC-33)) were presented in pJCL-3, and three copies of bla(KPC-2) were presented in pJCL-4. The bla(KPC-33)-harboring KPJCL-3 isolate presented resistance to ceftazidime-avibactam and cefiderocol. The bla(KPC-2) multicopy strain KPJCL-4 had an elevated ceftazidime-avibactam MIC. The patient had been exposed to ceftazidime, meropenem, and moxalactam, after which KPJCL-3 and KPJCL-4 were isolated, which both showed a significant competitive advantage under antimicrobial pressure in vitro. Experimental evolution assays revealed that bla(KPC-2) multi-copy-containing cells were increased in the original single-copy bla(KPC-2)-harboring KPJCL-2 population under selection with ceftazidime, meropenem, or moxalactam, generating a low-level ceftazidime-avibactam resistance phenotype. Moreover, bla(KPC-2) mutants with a G532T substitution, G820 to C825 duplication, G532A substitution, G721 to G726 deletion, and A802 to C816 duplication increased in the bla(KPC-2) multicopy-containing KPJCL-4 population, generating high-level ceftazidime-avibactam resistance and reduced cefiderocol susceptibility. Ceftazidime-avibactam and cefiderocol resistance can be selected by beta-lactam antibiotics other than ceftazidime-avibactam. Notably, bla(KPC-2) gene amplification and mutation are important in KPC-Kp evolution under antibiotic selection.