Emergence of Polymyxin Resistance Driven by a PhoQ Mutation in KPC-2-Producing Klebsiella pneumoniae

Background: The emergence of polymyxin-resistant, carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a critical challenge to clinical management. This study aimed to delineate the molecular mechanisms driving the acquisition of polymyxin resistance in CRKP. Methods: We analyzed polymyxin-susceptible and polymyxin-resistant CRKP isolates obtained from a single patient. Antimicrobial susceptibility testing was performed to determine the minimum inhibitory concentrations. Whole genome sequencing was employed to identify variations in two-component systems and to screen for mcr genes, which were involved in polymyxin resistance. Differential gene expression was assessed using RNA sequencing and validated by quantitative real-time PCR. Furthermore, site-directed mutagenesis was utilized to confirm the causal role of specific mutations in conferring the resistant phenotype. Results: An L96P mutation in the PhoQ protein was found in the polymyxin-resistant CRKP isolate. Compared with the PhoQ wild-type, this mutation significantly upregulated expression of phoP/Q, pmrD, and arnBCADTEF operon-related genes. A corresponding L96P mutant was subsequently constructed in the polymyxin-susceptible ATCC 13883 strain via site-directed mutagenesis. Antimicrobial susceptibility testing confirmed that the PhoQ L96P mutation elevates the minimal inhibitory concentrations of colistin and polymyxin B to 64 mg/L and >32 mg/L, respectively, from a baseline of 0.5 mg/L. Conclusions: The PhoQ L96P mutation is a pivotal driver of polymyxin resistance in CRKP, primarily mediated through the upregulation of the arnBCADTEF operon.