Abstract
Polymyxin B (PB) is introduced into the clinic as the last-line therapy against carbapenem-resistant Klebsiella pneumoniae (CRKP). Unfortunately, increased resistance to PB in Klebsiella pneumoniae (K. pneumoniae) has threatened global health. Resistance of K. pneumoniae to PB was induced by passaging in serial concentrations of PB and determined by microbroth dilution method. Growth characteristics of induced strains including growth curve, reversibility of resistance, and biofilm formation (crystal violet staining method) were measured. This study employed TMT-labeled quantitative proteomics and LC-MS/MS metabolomics analysis to investigate the key biological processes associated with PB resistance in K. pneumoniae. A total of 315 differentially expressed proteins (DEPs) were identified, of which 133 were upregulated and 182 were downregulated in the PB-resistant K. pneumoniae. KEGG enrichment analysis revealed that the DEPs were mainly involved in ATP-binding cassette (ABC) transporters and cationic antimicrobial peptide (CAMP) resistance. Proteins related to central carbon metabolism were inhibited in the PB-resistant K. pneumoniae, but proteins mediating LPS modification were activated. Transcriptional levels of CAMP resistance-related proteins were significantly different between PB-susceptible and -resistant K. pneumoniae. PB treatment led to an increase in reactive oxygen species (ROS) levels of K. pneumoniae. Metabolomics data demonstrated that 23 metabolites were significantly upregulated in PB-resistant K. pneumoniae and 5 were downregulated. The differential metabolites were mainly lipids, including glycerophospholipids, sphingolipids, and fatty acids. Exposure to PB resulted in increased level of phospholipid transport gene mlaF in K. pneumoniae. Our study suggested that membrane remodeling and inhibited central carbon metabolism are conducive to the development of PB resistance in K. pneumoniae.
Highlights
Klebsiella. pneumoniae is the main cause of hospital-acquired infection, including urinary tract infections, pneumonia, and bacteremia (Kontopidou et al, 2014; Balkhair et al, 2019; Marques et al, 2019)
Among these ten carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates, strain KPWT showed the strongest Polymyxin B (PB) resistance after induction, so it was selected for subsequent studies
The KPWT clinical isolate used in this study rapidly developed strong and stable resistance under the concentrations of PB ranging from 0.5 × MIC to 4 × MIC
Summary
Klebsiella. pneumoniae is the main cause of hospital-acquired infection, including urinary tract infections, pneumonia, and bacteremia (Kontopidou et al, 2014; Balkhair et al, 2019; Marques et al, 2019). The bactericidal activity of polymyxin is mainly achieved by electrostatic interaction between the cationic L-α, γ- diaminobutyric acid (Dab) residues of the polymyxin molecule and anionic phosphate groups on the lipid A section of lipopolysaccharide (LPS) in the outer membrane (OM) (Velkov et al, 2010). This interaction will cause insertion of PB molecule into the fatty acyl layer of the OM and damage of LPS (Velkov et al, 2010; Poirel et al, 2017). Resistance to polymyxin in CRE has been increasingly observed due to the growing use of polymyxin, with polymyxin-resistant CRKP accounting for the majority (Macesic et al, 2020)
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