Abstract
Enterococcus faecium is a clinically important pathogen associated with opportunistic infection and multi-drug resistance. E. faecium has been shown to produce membrane vesicles (MVs), but MV production by E. faecium under antibiotic stress conditions and the pathogenic traits thereof have yet to be determined. This study investigated the production of MVs in E. faecium ATCC 700221 cultured with sub-minimum inhibitory concentrations (MICs) of vancomycin or linezolid and determined their pathologic effects on colon epithelial Caco-2 cells. E. faecium ATCC 700221 cultured with 1/2 MIC of vancomycin or linezolid produced 3.0 and 1.5 times more MV proteins than bacteria cultured without antibiotics, respectively. Totals of 438, 461, and 513 proteins were identified in MVs from E. faecium cultured in brain heart infusion broth (MVs/BHI), BHI broth with 1/2 MIC of vancomycin (MVs/VAN), or BHI broth with 1/2 MIC of linezolid (MVs/LIN), respectively. Intact MVs/BHI induced cytotoxicity and the expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells in a dose-dependent manner, but proteinase K-treated MVs significantly suppressed these pro-inflammatory responses. MVs/LIN were more cytotoxic toward Caco-2 cells than MVs/BHI and MVs/VAN, whereas MVs/VAN stimulated more pro-inflammatory cytokine gene expression in Caco-2 cells than MVs/BHI and MVs/LIN. Overall results indicated that antibiotics modulate the biogenesis and proteomes of MVs in E. faecium at subinhibitory concentrations. MVs produced by E. faecium cultured under antibiotic stress conditions induce strong host cell responses that may contribute to the pathogenesis E. faecium.
Highlights
Enterococci, gram-positive, facultative anaerobic bacteria, are among the most abundant commensal flora of the human and animal gut microbiomes (Shepard and Gilmore, 2002; Heidari et al, 2016)
To determine whether E. faecium ATCC 700221 produced membrane vesicles (MVs), bacteria were cultured in brain heart infusion (BHI) broth to late exponential phase (Figure S1) and MVs were isolated from the culture supernatant
E. faecium were cultured in BHI broth with 1/2 minimum inhibitory concentrations (MICs) of vancomycin (256 μg/ml) or linezolid (1 μg/ml), and MVs were isolated from the culture supernatants to determine the effect of subinhibitory concentrations of antibiotics on MV production and their protein profiles
Summary
Enterococci, gram-positive, facultative anaerobic bacteria, are among the most abundant commensal flora of the human and animal gut microbiomes (Shepard and Gilmore, 2002; Heidari et al, 2016). These microorganisms are of medical importance as opportunistic pathogens, especially in severely ill or immunocompromised hosts (Carmeli et al, 2002; da Silva et al, 2014). In addition to antimicrobial resistance, enterococci harbor several virulence factors, including major autolysin (AtlA), enterococcal leucine-rich repeat-containing protein (ElrA), enterococcal surface protein (Esp), cytolysin (CylA), and collagen-binding protein (Acm), which lead to them being opportunistic pathogens (Shankar et al, 2002; Eckert et al, 2006; Emirian et al, 2009; Arias and Murray, 2012; Dumoulin et al, 2013; Hendrickx et al, 2013; Heidari et al, 2016, 2017)
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