An integrated molecular, phenotypic, and adaptive analysis of polymyxin resistance in KPC-producing Klebsiella pneumoniae

Rising resistance to polymyxins poses a critical challenge in the management of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Klebsiella pneumoniae . This study investigated 50 polymyxin-resistant K. pneumoniae isolates recovered from hospitals in Brazil using an integrated approach combining antimicrobial susceptibility testing, targeted molecular analysis, lipid A profiling, and phenotypic assays. Eighteen isolates were classified as XDR and 32 as MDR. All isolates carried bla KPC , 56% co-harbored bla CTX−M−type genes, and 62% were resistant to ceftazidime–avibactam. Targeted sequencing revealed diverse alterations in polymyxin resistance-associated loci, particularly mgrB , pmrAB , phoQ , and crrB , including amino acid substitutions, premature stop codons, insertions, and deletions. Lipid A analysis by MALDI-TOF MS confirmed structural remodeling, such as the incorporation of 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosphoethanolamine, consistent with polymyxin-resistant phenotypes. Virulence assessment using the Galleria mellonella model showed marked heterogeneity among isolates, with no direct association between resistance-associated alterations and virulence phenotypes. Short-term survival following polymyxin B exposure at pH 5.8 and 7.7 differed in only a subset of isolates, indicating a strain-dependent response to environmental conditions. In addition, the laboratory-evolved Escherichia coli D12 mutant exhibited lipid A remodeling and alterations in PmrAB following polymyxin selective pressure. Overall, these findings support the multifactorial nature of polymyxin resistance and highlight the value of integrating molecular, structural, and phenotypic approaches for antimicrobial resistance surveillance.