Abstract
OBJECTIVES: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major clinical and public health threat due to its high mortality in sepsis and the complexity of its resistance mechanisms, which greatly limit therapeutic effectiveness. Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria harbor various virulence factors and resistance determinants, and may facilitate long-distance pathogenic communication. However, the OMV protein composition of CRKP isolated from sepsis patients remains poorly understood. This study aimed to characterize the protein cargo of OMVs derived from clinical CRKP isolates and to identify differential proteins and pathways associated with bacterial pathogenicity and carbapenem resistance. METHODS: Three CRKP and three carbapenem-sensitive K. pneumoniae (CSKP) were isolated from blood cultures of patients with sepsis, and then their OMVs were extracted. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and SDS-PAGE were used for characterization. Quantitative proteomic profiling was performed using LC-MS/MS, followed by differential expression analysis, Gene Ontology (GO), KEGG pathway enrichment, and protein-protein interaction (PPI) network analysis. RESULTS: A total of 1,193 OMV proteins were identified, with CRKP-OMVs containing substantially more unique proteins (140 vs. 5 in CSKP-OMVs) and significantly increased overall protein abundance. Among the 199 differential proteins, 180 were upregulated in CRKP-OMVs. Most differential proteins were localized to the membrane or cytoplasm, and were enriched in enzymatic functions and pathways including β-alanine metabolism, O-antigen nucleotide sugar biosynthesis, Lipopolysaccharide biosynthesis, and β-lactam resistance. Key proteins such as adhE, NDK, treA, and ackA were markedly elevated, while galE and Ter family proteins were uniquely present in CRKP-OMVs, indicating potential roles in resistance and pathogenicity. CONCLUSIONS: CRKP-derived OMVs from sepsis patients exhibit distinct protein enrichment patterns associated with membrane functions, metabolic remodeling, and antibiotic resistance. These findings provide insights into CRKP pathogenesis and highlight candidate OMV-associated proteins that may serve as targets for antimicrobial strategies or vaccine development.