Journal: Frontiers in cellular and infection microbiology

Article Title: Production of Membrane Vesicles by Enterococcus faecium Cultured With or Without Subinhibitory Concentrations of Antibiotics and Their Pathological Effects on Epithelial Cells.

doi: 10.3389/fcimb.2019.00295

Figure Lengend Snippet: FIGURE 2 | Proteomic analysis of MVs produced by E. faecium ATCC 700221. MVs were isolated from E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 µg/ml vancomycin (MVs/VAN), or BHI broth with 1 µg/ml linezolid (MVs/LIN). (A,B) A total of 438 proteins in the MVs/BHI were analyzed based on cellular localization (A) and Gene Ontology (B). (C) A Venn diagram of proteins identified in the MVs/BHI, MVs/VAN, and MVs/LIN.

Article Snippet: MVs produced by E. faecium ATCC 700221 were isolated from bacterial culture supernatants as previously described (Gurung et al., 2011; Kim et al., 2016; Yun et al., 2018).

Techniques: Produced, Isolation, Cell Culture

Journal: Frontiers in cellular and infection microbiology

Article Title: Production of Membrane Vesicles by Enterococcus faecium Cultured With or Without Subinhibitory Concentrations of Antibiotics and Their Pathological Effects on Epithelial Cells.

doi: 10.3389/fcimb.2019.00295

Figure Lengend Snippet: FIGURE 1 | Membrane vesicles (MVs) produced by E. faecium ATCC 700221 cultured with or without antibiotics. (A) Transmission electron micrographs of MVs from E. faecium cultured in BHI broth. (B) SDS-PAGE analysis of proteins obtained from bacterial lysates (lane 1), culture supernatant (lane 2), and MVs (lane 3). Bacteria were cultured in BHI broth to late exponential phase. Lane M, molecular weight marker. (C) Production of MVs from E. faecium cultured with or without antibiotics. MVs were isolated from E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 µg/ml vancomycin (MVs/VAN), or BHI broth with 1 µg/ml linezolid (MVs/LIN). The protein concentration of MVs isolated from 1 L of bacterial culture was measured using a modified BCA assay. Data are presented as the mean ± SD of three independent experiments. **P < 0.01 compared to MVs/BHI. (D) SDS-PAGE analysis of MV proteins. Lane M, molecular weight marker; 1, MVs/BHI; 2, MVs/VAN; 3, MVs/LIN. (A,B,D) represent one of three independent experiments.

Article Snippet: MVs produced by E. faecium ATCC 700221 were isolated from bacterial culture supernatants as previously described (Gurung et al., 2011; Kim et al., 2016; Yun et al., 2018).

Techniques: Membrane, Produced, Cell Culture, Transmission Assay, SDS Page, Bacteria, Molecular Weight, Marker, Isolation, Protein Concentration, BIA-KA

Journal: Frontiers in cellular and infection microbiology

Article Title: Production of Membrane Vesicles by Enterococcus faecium Cultured With or Without Subinhibitory Concentrations of Antibiotics and Their Pathological Effects on Epithelial Cells.

doi: 10.3389/fcimb.2019.00295

Figure Lengend Snippet: FIGURE 3 | Cytotoxicity of Caco-2 cells treated with MVs from E. faecium ATCC 700221. MVs were isolated from culture supernatants of E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 µg/ml vancomycin (MVs/VAN), or BHI broth with 1 µg/ml linezolid (MVs/LIN). Cells were treated with various concentrations of E. faecium MVs for 24 h. (A) Cell viability was determined using an MTT assay. Data are presented as the mean ± SD of three independent experiments. +P < 0.05, ++P < 0.01 compared to untreated control cells. *P < 0.05, **P < 0.01 among the same concentrations of MVs/BHI, MVs/VAN, or MVs/LIN. (B) Flow cytometric analysis of Caco-2 cell death induced by E. faecium MVs. Cells were treated with 20 µg/ml of E. faecium MVs for 24 h. Cells were stained with FITC-Annexin V and propidium iodide, and 104 cells were counted. The figure represents one of three independent experiments that yielded similar results.

Article Snippet: MVs produced by E. faecium ATCC 700221 were isolated from bacterial culture supernatants as previously described (Gurung et al., 2011; Kim et al., 2016; Yun et al., 2018).

Techniques: Isolation, Cell Culture, MTT Assay, Control, Staining

Journal: Frontiers in cellular and infection microbiology

Article Title: Production of Membrane Vesicles by Enterococcus faecium Cultured With or Without Subinhibitory Concentrations of Antibiotics and Their Pathological Effects on Epithelial Cells.

doi: 10.3389/fcimb.2019.00295

Figure Lengend Snippet: FIGURE 5 | Host cell responses to proteinase K (PK)-treated E. faecium MVs. (A) Caco-2 cells were incubated with intact or PK-treated MVs/BHI for 24 h and cell viability was determined using an MTT assay. Data are presented as the mean ± SD of three independent experiments. (B) Cells were incubated with intact or PK-treated MVs/BHI for 3 h and gene expression was assessed via qPCR. Data are presented as the mean ± SD of three independent experiments. **P < 0.01 comparing intact and PK-treated MVs.

Article Snippet: MVs produced by E. faecium ATCC 700221 were isolated from bacterial culture supernatants as previously described (Gurung et al., 2011; Kim et al., 2016; Yun et al., 2018).

Techniques: Incubation, MTT Assay, Gene Expression

Journal: Frontiers in cellular and infection microbiology

Article Title: Production of Membrane Vesicles by Enterococcus faecium Cultured With or Without Subinhibitory Concentrations of Antibiotics and Their Pathological Effects on Epithelial Cells.

doi: 10.3389/fcimb.2019.00295

Figure Lengend Snippet: FIGURE 4 | Expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells treated with MVs from E. faecium ATCC 700221. MVs were isolated from culture supernatants of E. faecium cultured in BHI broth (MVs/BHI), BHI broth with 256 µg/ml vancomycin (MVs/VAN), or BHI broth with 1 µg/ml linezolid (MVs/LIN). Cells were treated with various concentrations of E. faecium MVs for 3 h and gene expression was assessed via qPCR. Data are presented as the mean ± SD of three independent experiments. +P < 0.05, ++P < 0.01 compared to untreated control cells. *P < 0.05, **P< 0.01 among the same concentrations of MVs/BHI, MVs/VAN, or MVs/LIN.

Article Snippet: MVs produced by E. faecium ATCC 700221 were isolated from bacterial culture supernatants as previously described (Gurung et al., 2011; Kim et al., 2016; Yun et al., 2018).

Techniques: Expressing, Isolation, Cell Culture, Gene Expression, Control