Dwibedy, S. K. | Search bioRxiv

bioRxiv2026

Insights into the Klebsiella pneumoniae adaptive response mechanisms to colistin exposure using a label-free quantitative proteomics approach

The rise of MDR Klebsiella pneumoniae and its resistance to the last-resort antibiotic colistin poses a significant threat to global healthcare. While genomic studies have identified several resistance mutations, the transient proteomic shifts that occur during the initial exposure of sensitive strains to lethal antibiotic doses remain poorly characterised. In this study, we employed a label-free quantitative proteomics approach to investigate the protein expression profile of K. pneumoniae strain ATCC 13883 treated with colistin at its MIC. Membrane proteins were extracted at critical growth stages, and differentially abundant proteins (DAPs) were analysed using Gene Ontology and KEGG pathway enrichment analysis. Our proteomic analysis identified 718 DAPs (339 upregulated and 379 downregulated). The cellular response was characterised primarily by outer membrane remodelling and a significant upregulation of the capsule-associated kinase Wzc and the ArnBCADTEF operon, which facilitates lipid A modification with L-Ara4N moiety. Paradoxically, while RND-family efflux pumps (AcrAB) were significantly induced, the global activator RamA and major porins (OmpA, OmpX, LamB) were downregulated, possibly to minimise antibiotic entry. KEGG pathway enrichment analysis further revealed a synchronised metabolic shift, characterised by an intensified TCA cycle flux to fuel high-energy resistance processes despite a general slowdown in carbohydrate metabolism. Our findings demonstrate that K. pneumoniae responds to colistin stress through a rapid, multifaceted proteomic reorganisation involving charge neutralisation, structural reinforcement of the cell envelope, and metabolic re-routing. These results provide a molecular blueprint of the early adaptive response, identifying several proteins as potential therapeutic targets.

microbiology↗

bioRxiv2024

Fitness trade-off and the discovery of a novel missense mutation in the PmrB sensor kinase of a colistin-resistant Pseudomonas aeruginosa strain developed by adaptive laboratory evolution

Pseudomonas aeruginosa is a prominent bacterial pathogen that causes several nosocomial infections and is notorious for its environmental resilience and rapid development of resistance to frontline antibiotics. A major cause of mortality and morbidity among cystic fibrosis patients, multidrug-resistant P. aeruginosa is often targeted with the antibiotic colistin as a last option. However, increasing reports of colistin resistance among P. aeruginosa is a matter of significant concern. Though the molecular mechanisms responsible for the development of colistin resistance are well known, the evolutionary trajectory to colistin resistance is an important area of investigation. In this work, using the adaptive laboratory evolution (ALE) approach we have evolved a colistin-sensitive P. aeruginosa ancestral strain to a resistant one. During the process of laboratory evolution in 106 generations, colistin MIC was increased 32-fold. The evolved strain had lower fitness than the ancestral strain as evidenced by a lower growth rate and higher doubling time. Moreover, the evolved strain produced more biofilm and less pyocyanin. Interestingly, the evolved strain showed collateral sensitivity to several antibiotics such as co-trimoxazole, rifampicin, kanamycin, tigecycline, penicillin, ampicillin, and teicoplanin. On analysing various TCS modules involved in the development of colistin resistance a novel missense mutation (V136G) was detected in the PmrB sensor kinase. Bioinformatics prediction indicated that the mutation could be deleterious, though the functionality of the PmrB mutant remains to be validated experimentally.

microbiology↗