Elizabethkingia anophelis, a nonfermenting Gram-negative bacterium, causes life-threatening health care-associated infections. E. anophelis harbors multidrug resistance (MDR) genes and is intrinsically resistant to various classes of antibiotics. Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and contain materials involved in bacterial survival and pathogenesis. OMVs specialize and tailor their functions by carrying different components to challenging environments and allowing communication with other microorganisms or hosts. In this study, we sought to understand the characteristics of E. anophelis OMVs under different antibiotic stress conditions. An extensively drug-resistant clinical isolate, E. anophelis C08, was exposed to multiple antibiotics in vitro, and its OMVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and proteomic analysis. Protein functionality analysis showed that the OMVs were predominantly involved in metabolism, survival, defense, and antibiotic resistance processes, such as the Rag/Sus family, the chaperonin GroEL, prenyltransferase, and an HmuY family protein. Additionally, a protein-protein interaction network demonstrated that OMVs from imipenem-treated E. anophelis showed significant enrichments in the outer membrane, adenyl nucleotide binding, serine-type peptidase activity, the glycosyl compound metabolic process, and cation binding proteins. Collectively, the OMV proteome expression profile indicates that the role of OMVs is immunologically relevant and related to bacterial survival in antibiotic stress environments rather than representing a resistance point. IMPORTANCE Elizabethkingia anophelis is a bacterium often associated with nosocomial infection. This study demonstrated that imipenem-induced E. anophelis outer membrane vesicles (OMVs) are immunologically relevant and crucial for bacterial survival under antibiotic stress conditions rather than being a source of antibiotic resistance. Furthermore, this is the first study to discuss the protein-protein interaction network of the OMVs released by E. anophelis, especially under antibiotic stress. Our findings provide important insights into clinical antibiotic stewardship.

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