Journal: iScience

Article Title: Identification of proteotoxic and proteoprotective bacteria that non-specifically affect proteins associated with neurodegenerative diseases

doi: 10.1016/j.isci.2024.110828

Figure Lengend Snippet:

Article Snippet: Enterococcus faecium , BEI Resources , HM-968.

Techniques: Virus, Recombinant, Membrane, Western Blot, Software

Journal: Journal of medicinal chemistry

Article Title: Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity In Vitro and In Vivo against Vancomycin-resistant Enterococci

doi: 10.1021/acs.jmedchem.6b01780

Figure Lengend Snippet: Minimum inhibitory concentration (MIC 50 ) (in μg/mL) and minimum bactericidal concentration (MBC 50 ) of phenylthiazole compounds 1–3 , vancomycin, and linezolid against 50% of vancomycin-sensitive (VSE) and vancomycin-resistant Enterococcus faecalis or E. faecium (VRE) strains.

Article Snippet: Clinical isolates of E. faecalis and E. faecium were obtained through BEI Resources ( Table S1 ).

Techniques: Concentration Assay

Journal: Journal of medicinal chemistry

Article Title: Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity In Vitro and In Vivo against Vancomycin-resistant Enterococci

doi: 10.1021/acs.jmedchem.6b01780

Figure Lengend Snippet: Time-kill analysis of phenylthiazole compounds 1, 2, 3, and linezolid (all tested at 4 × MIC) over a 24 hour incubation period at 37 °C against A) vancomycin-resistant Enterococcus faecium ATCC 700221 and B) vancomycin-resistant Enterococcus faecalis HM-201. DMSO served as a negative control. The error bars represent standard deviation values obtained from triplicate samples used for each compound/antibiotic studied.

Article Snippet: Clinical isolates of E. faecalis and E. faecium were obtained through BEI Resources ( Table S1 ).

Techniques: Incubation, Negative Control, Standard Deviation

Journal: Journal of medicinal chemistry

Article Title: Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity In Vitro and In Vivo against Vancomycin-resistant Enterococci

doi: 10.1021/acs.jmedchem.6b01780

Figure Lengend Snippet: Multi-step resistance selection of compounds 1, 2, 3, and linezolid against A) vancomycin-resistant Enterococcus faecium HM-968 and B) vancomycin-resistant Enterococcus faecalis ATCC 51299. Bacteria were serially passaged over a fourteen-day period and the broth microdilution assay was used to determine the minimum inhibitory concentration of each compound against VRE after each successive passage. A four-fold shift in the MIC is defined as resistance to the test agent.

Article Snippet: Clinical isolates of E. faecalis and E. faecium were obtained through BEI Resources ( Table S1 ).

Techniques: Selection, Bacteria, Microdilution Assay, Concentration Assay

Journal: International Journal of Environmental Research and Public Health

Article Title: Interference in Pheromone-Responsive Conjugation of a High-Level Bacitracin Resistant Enterococcus faecalis Plasmid of Poultry Origin

doi: 10.3390/ijerph10094245

Figure Lengend Snippet: Interference in the transfer frequencies of plasmid encoding high-level bacitracin resistance of the donor E. faecalis strain 543 to the recipient strain E. faecalis JH2-2 tested by pheromone-responsive mating experiments with or without addition of antibodies.

Article Snippet: Membranes were washed and incubated with a goat anti-rabbit horseradish peroxidase conjugate (Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA) for 1 h. Proteins extract from E. faecalis JH2-2 and pre-immune sera (1:1,000) (GenScript Corporation) were used as negative controls.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Extracellular vesicles and their RNA cargo facilitate bidirectional cross-kingdom communication between human and bacterial cells

doi: 10.1101/2025.07.10.664124

Figure Lengend Snippet: ( A-E ) Characterization of BEVs. Typical size distribution of purified BEVs of L. casei MVs ( A ), E. faecalis MVs ( B ), and P. mirabilis OMVs ( C ) measured by nanoparticle tracking analysis. Representative cryo-TEM images of L. casei MVs ( D ), E. faecalis MVs ( E ), and P. mirabilis OMVs ( F ) showed round shaped particles. ( G-L ) Flow cytometry analysis of the uptake of fluorescence-labeled BEVs in Caco-2 cells. Percentage of PE (Phycoerythrin)-positive cells and representative histograms compared to the control (PBS) after 4 h, 12 h, 24 h, or 48 h incubation with the DiI-labeled BEVs of L. casei ( G and H ), E. faecalis ( I and J ), or P. mirabilis ( K and L ). Results are presented as mean ± standard deviation (SD) from 3-4 independent experiments. ( M-O ) Visualization of the internalization of BEVs into Caco-2 cells using CLSM. Caco-2 cells were incubated for 12 h, 24 h, or 48 h with DiI-labeled L. casei MVs ( M ), E. faecalis MVs ( N ), or P. mirabilis OMVs ( O ). After the incubation, the cells were washed, fixed, and permeabilized. F-Actin was stained with phalloidin, and the cell nuclei were stained with DAPI. DAPI was visualized using a diode laser at 405 nm (blue), phalloidin with an argon laser at 488 nm (green), and DiI with a DPSS laser at 561 nm (red). The white arrow indicates an area where weak signals for DiI-labeled L. casei MVs were detected. Scale bar: 100 µm.

Article Snippet: L. casei (DSM 20011) was grown in deMan, Rogosa and Sharpe (MRS) broth (Carl Roth), E. faecalis (DSM 20478) and P. mirabilis (DSM 4479) were grown in brain heart infusion (BHI) broth (Merck Millipore).

Techniques: Purification, Flow Cytometry, Fluorescence, Labeling, Control, Incubation, Standard Deviation, Staining

Journal: bioRxiv

Article Title: Extracellular vesicles and their RNA cargo facilitate bidirectional cross-kingdom communication between human and bacterial cells

doi: 10.1101/2025.07.10.664124

Figure Lengend Snippet: ( A-F ) Measurement of cell viability and cytotoxicity after incubation of Caco-2 cells with different concentrations of L. casei MVs ( A and D ), E. faecalis MVs ( B and E ), or P. mirabilis OMVs ( C and F ) for 24 h ( E. faecalis MVs or P. mirabilis OMVs) or 48 h ( L. casei MVs). 1 % Triton X-100 was used as dead control, whereas PBS was used as live control. Results are presented as mean ± SD from 3-4 independent experiments. ( G and H ) Analysis of BEV-RNA. ( G ) Displayed is the amount of RNA present in 5x10 10 vesicles in box-whisker plots. Results shown are from 6-7 independent experiments. ( H ) Representative electropherograms show size distribution of L. casei MV-RNA, E. faecalis MV-RNA, and P. mirabilis OMV-RNA analyzed using Agilent RNA 6000 Pico Chip. ( I-N ) Measurement of cell viability and cytotoxicity after transfection of Caco-2 cells with different amounts of L. casei MV-RNA ( I and L ), E. faecalis MV-RNA ( J and M ), or P. mirabilis OMV-RNA ( K and N ) for 24 h ( E. faecalis MV-RNA or P. mirabilis OMV-RNA) or 48 h ( L. casei MV-RNA). 1 % Triton X-100 was used as dead control, whereas Lipofectamine™ 3000 transfection reagent mixed with nuclease-free water (vehicle) was used as live control. Results are presented as mean ± SD from 3-4 independent experiments. For all experiments, a two-tailed unpaired t-test was conducted to determine statistical significance (p < 0.01 **, p < 0.001 ***).

Article Snippet: L. casei (DSM 20011) was grown in deMan, Rogosa and Sharpe (MRS) broth (Carl Roth), E. faecalis (DSM 20478) and P. mirabilis (DSM 4479) were grown in brain heart infusion (BHI) broth (Merck Millipore).

Techniques: Incubation, Control, Whisker Assay, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Extracellular vesicles and their RNA cargo facilitate bidirectional cross-kingdom communication between human and bacterial cells

doi: 10.1101/2025.07.10.664124

Figure Lengend Snippet: (A) Illustration of experimental workflow during the analysis of the transcriptome (Created with BioRender). Caco-2 cells have been incubated with BEVs ( L. casei MVs: 1.5x10 11 particle, E. faecalis MVs: 7.5x10 9 particle, P. mirabilis OMVs: 6.0x10 9 particle) or transfected with BEV-RNA ( L. casei MV-RNA: 100 ng, E. faecalis MV-RNA: 5 ng, P. mirabilis OMV-RNA: 2 ng) for 10 h, 24 h, or 48 h. ( B-D ) Upset plots highlight the number of differentially expressed genes between the different time points and treatments. ( E-J ) Differentially expressed genes were used to perform an over-representation analysis (ORA) using GeneTrail 3.2. Shown are the top 20 Gene Ontology (GO) - biological process in which the differentially expressed genes were enriched in. The dashed line marks the significance level of p = 0.05. Biological processes that showed an enrichment after incubation with BEVs and transfection of BEV-RNA derived from the same bacteria at the same timepoint are highlighted in orange.

Article Snippet: L. casei (DSM 20011) was grown in deMan, Rogosa and Sharpe (MRS) broth (Carl Roth), E. faecalis (DSM 20478) and P. mirabilis (DSM 4479) were grown in brain heart infusion (BHI) broth (Merck Millipore).

Techniques: Incubation, Transfection, Derivative Assay, Bacteria

Journal: bioRxiv

Article Title: Extracellular vesicles and their RNA cargo facilitate bidirectional cross-kingdom communication between human and bacterial cells

doi: 10.1101/2025.07.10.664124

Figure Lengend Snippet: ( A and B ) Characterization of Caco-2 EVs. ( A ) Typical size distribution of purified Caco-2 (-FCS) EVs measured by nanoparticle tracking analysis. ( B ) Representative cryo-TEM images of Caco-2 (-FCS) EVs showed round shaped particles. ( C-E ) Growth of L. casei ( C ), E. faecalis ( D ), and P. mirabilis ( E ) in the presence of different amounts of Caco-2 (-FCS) EVs or PBS as a control over a period of 48 h or 36 h at 37 °C. Results are presented as mean ± SD from 4 independent experiments. ( F-H ) Visualization of the interaction of L. casei ( F ), E. faecalis ( G ) or P. mirabilis ( H ) with Caco-2 (-FCS) EVs. Bacteria were incubated for 4 h, 8 h or 24 h with PKH26-labeled Caco-2 (-FCS) EVs. After the incubation, bacteria were stained with SYTO™ 9 and fixed. SYTO™ 9 was visualized using an argon laser at 488 nm, and PKH26 with a DPSS laser at 561 nm. For better illustration, the bacteria are shown in blue, and EVs are displayed in yellow. The white arrows indicate areas where PKH26-labeled Caco-2 (-FCS) EVs were detected. Scale bar: 10 µm. For all experiments, a two-tailed unpaired t-test was conducted to determine statistical significance (p < 0.05 *, p < 0.001 ***).

Article Snippet: L. casei (DSM 20011) was grown in deMan, Rogosa and Sharpe (MRS) broth (Carl Roth), E. faecalis (DSM 20478) and P. mirabilis (DSM 4479) were grown in brain heart infusion (BHI) broth (Merck Millipore).

Techniques: Purification, Control, Bacteria, Incubation, Labeling, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Extracellular vesicles and their RNA cargo facilitate bidirectional cross-kingdom communication between human and bacterial cells

doi: 10.1101/2025.07.10.664124

Figure Lengend Snippet: ( A-C ) Growth of L. casei ( A ), E. faecalis ( B ), and P. mirabilis ( C ) in the presence different concentrations of synthetic miR-192-5p or PBS as a control over a period of 48 h or 36 h at 37 °C. Results are presented as mean ± SD from 3-4 independent experiments. ( D-F ) Flow cytometry analysis of the uptake of fluorescence-labeled synthetic miRNA by various bacteria. All bacteria were cultivated in the presence of 4 μM free Cy3-labeled synthetic miR-192-5p in PBS or DMEM, or 4 μM liposome-packaged Cy3-labeled synthetic miR-192-5p for 24 h ( E. faecalis or P. mirabilis ) or 48 h ( L. casei ) at 37 °C. After the incubation, bacteria were stained with SYTO™ 9 and fixed. The percentage of SYTO™ 9/PE (Phycoerythrin)-positive bacteria compared to the respective control (PBS, DMEM or liposomes). Results are presented as mean ± SD from 4 independent experiments. ( G-I ) Visualization of the interaction of L. casei ( G ), E. faecalis ( H ) or P. mirabilis ( I ) with free or liposome-packaged fluorescently labeled synthetic miRNA. Bacteria were cultivated in the presence of 4 μM free Cy3-labeled synthetic miR-192-5p in PBS or DMEM, or 4 μM liposome-packaged Cy3-labeled synthetic miR-192-5p for 24 h ( E. faecalis or P. mirabilis ) or 48 h ( L. casei ) at 37 °C. After the incubation, bacteria were stained with SYTO™ 9 and fixed. SYTO™ 9 was visualized using an argon laser at 488 nm, and Cy3 with a DPSS laser at 561 nm. For better illustration, the bacteria are shown in blue, and synthetic miRNA is displayed in yellow. The white arrows indicate areas where weak signals for Cy3-labeled synthetic miR-192-5p were detected. Scale bar: 10 µm. For all experiments, a two-tailed unpaired t-test was conducted to determine statistical significance (p < 0.05 *, p < 0.01 **, p < 0.001 ***).

Article Snippet: L. casei (DSM 20011) was grown in deMan, Rogosa and Sharpe (MRS) broth (Carl Roth), E. faecalis (DSM 20478) and P. mirabilis (DSM 4479) were grown in brain heart infusion (BHI) broth (Merck Millipore).

Techniques: Control, Flow Cytometry, Fluorescence, Labeling, Bacteria, Incubation, Staining, Liposomes, Two Tailed Test