Proteome-wide remodelling in Klebsiella pneumoniae during step-wise adaptive evolution against colistin

Short-duration exposure (SDE) of antibiotics is among the major drivers of the emergence of antimicrobial resistance (AMR) among bacterial pathogens. Klebsiella pneumoniae is a critical priority bacterial pathogen and rapidly acquiring MDR/XDR traits even against major antibacterial classes of antibiotics. Since colistin is a last-resort antibiotic for treating drug-resistant K. pneumoniae infections and the development of new antibiotics is resource-intensive, understanding how antibiotic-resistance emerges following SDE is crucial for devising strategies to preserve antibiotics for future use. With this aim, we developed colistin (1x, 2x, and 4x MICs)-resistant K. pneumoniae using SDE of colistin and protein profiled to understand the emergence of SDE-induced colistin resistance. Interestingly, 1xMIC-adapted cells showed an ~ 8-fold increase in MIC for colistin, with no further rise at 2x or 4x MIC-adapted cells, indicating that the emergence of AMR can be a non-linear adaptive response and SDE of even 1x MIC-dose alone can trigger rapid physiological remodelling and target modification. Proteomic profiling identified 1379 proteins, including 245, 383, and 175 differentially expressed proteins (DEPs) in 1x, 2x, and 4x MIC-adapted cells, respectively. Among these, 75 DEPs were common across all conditions, representing a core proteomic signature consistently altered upon exposure and represented valS, arnA, arnB, pheT, and thrS the top upregulated proteins, and gjJ18, kphS, B5L96, potD, and phoU the top downregulated proteins. Functional enrichment highlighted catalytic, antioxidant, and binding activities, whereas protein-protein interaction (PPI) analysis revealed metabolic reprogramming, involving central carbon/amino acid metabolism and transcription/translation machinery associated with reduced antibiotic sensitivity. Lipopolysaccharide (LPS) modification (arnA, arnB, and phoU) and biomolecular synthesis (valS, pheT, and thrS) were the key functions altered during colistin adaptation. Since elevated arnA, arnB and phoU are known to reduce colistin binding to the bacterial surface, they may be facilitating the emergence of antibiotic resistance. Taken together, this research provides insight into SDE-induced colistin resistance in K. pneumoniae and may help to identify potential therapeutic and diagnostic interventions.