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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230873	3/8	Enterococcus faecium	E155	(d)(U)C DG Filtration UF	Wagner T	2018	57%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 57% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Exponential growth in BHI Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells E155 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per MV/CFU Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 37 EM EM-type Atomic force microscopy Image type Close-up, Wide-field

	EV230873	1/8	Enterococcus faecium	DO	(d)(U)C DG Filtration UF	Wagner T	2018	43%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Exponential growth in BHI Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells DO EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per MV/CFU Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 42

	EV230873	2/8	Enterococcus faecium	DO	(d)(U)C DG Filtration UF	Wagner T	2018	43%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Stationary growth in LB Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells DO EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230873	4/8	Enterococcus faecium	E155	(d)(U)C DG Filtration UF	Wagner T	2018	43%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Stationary growth in LB Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells E155 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230873	5/8	Enterococcus faecium	K59-68	(d)(U)C DG Filtration UF	Wagner T	2018	43%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Exponential growth in BHI Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells K59-68 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per MV/CFU Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 83

	EV230873	6/8	Enterococcus faecium	K59-68	(d)(U)C DG Filtration UF	Wagner T	2018	43%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Stationary growth in LB Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells K59-68 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230873	7/8	Enterococcus faecium	K60-39	(d)(U)C DG Filtration UF	Wagner T	2018	43%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Exponential growth in BHI Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells K60-39 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Protein Yield (µg) per MV/CFU Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 51

	EV230873	8/8	Enterococcus faecium	K60-39	(d)(U)C DG Filtration UF	Wagner T	2018	43%
Study summary Full title Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. All authors Wagner T, Joshi B, Janice J, Askarian F, Škalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M Journal J Proteomics Abstract Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resi (show more...)Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Stationary growth in LB Focus vesicles Membrane vesicles (MVs) Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Ultrafiltration Adj. k-factor 17410 (washing) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Enterococcus faecium Sample Type Cell culture supernatant EV-producing cells K60-39 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 20000 Wash: time (min) 180 Wash: Rotor Type SW 40 Ti Wash: speed (g) 20000 Wash: adjusted k-factor 17410 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 30% Orientation Bottom-up Speed (g) 100000 Duration (min) 180 Fraction volume (mL) 0.2 Fraction processing Ultrafiltration Pelleting: adjusted k-factor TDB Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV230873
species	Enterococcus faecium
sample type	Cell culture
cell type	E155	DO	DO	E155	K59-68	K59-68	K60-39	K60-39
condition	Exponential growth in BHI	Exponential growth in BHI	Stationary growth in LB	Stationary growth in LB	Exponential growth in BHI	Stationary growth in LB	Exponential growth in BHI	Stationary growth in LB
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration
Exp. nr.	3	1	2	4	5	6	7	8
EV-METRIC %	57	43	43	43	43	43	43	43

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