Mapping bacterial extracellular vesicle research: insights, best practices and knowledge gaps

Abstract

Bacterial extracellular vesicles (BEVs) enable communication between bacteria and their natural habitats, including multicellular organisms such as humans. Consequently, the study of BEVs has rapidly gained attention with recent research raising the prospect of developing BEVs as biomarkers and treatments to manage (mal)functioning of natural habitats. Although diverse technologies are available, the composition of their source, their heterogeneity in biophysical and biochemical features, and their multifaceted cargo composition challenges the analysis of BEVs. To map current practices in BEV research, we analyzed 845 publications released in 2015-2021, reporting 3338 BEV-related experiments. The extracted data are accessible via the publicly available EV-TRACK knowledgebase ( https://evtrack.org/ ). We identify the need for transparent reporting, delineate knowledge gaps, outline available best practices and define areas in need of guidance to ensure advances in BEV research and accelerate BEV applications.

Fig. 1. Search reveals a heterogeneous reporting landscape in BEV research.

Binary heatmap showing the reported experimental parameters (rows of the heatmap, selection of 102 out of 233 parameters for binarity and relevance to experimental results) for each experiment (columns of the heatmap, total n = 3338). The heatmap is divided horizontally into three sections of parameters (preparation, biophysical characterization, and biochemical characterization; indicated in purple, green, and blue and including 57, 32, and 13 parameters, respectively). It is divided vertically in eight blocks according to source (cell culture supernatant (n = 3123, dark green), blood (n = 58, red), feces (n = 53, dark brown), intestinal tissue (n = 19, light brown), milk (n = 3, light blue), saliva (n = 13, dark blue), urine (n = 26, dark yellow) and other (n = 43, dark pink) and in seven blocks according to publication year: 2015 (n = 80, light red), 2016 (n = 73, orange), 2017 (n = 93, light yellow), 2018 (n = 110, light green), 2019 (n = 123, cyan), 2020 (n = 184, purple), and 2021 (n = 182, light pink). For each section, columns (experiments) are sorted according to descending total number of reported experimental parameters. Parameters that were not reported in an experiment appear as a white space in its corresponding column. Ab antibody, BEV bacterial extracellular vesicle, DC density cushion, DG density gradient, DLS dynamic light scattering, DUC differential (ultra-)centrifugation, ELISA enzyme linked immune sorbent assay, FC flow cytometry, NTA nanoparticle tracking analysis, RNA ribonucleic acid, SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis, SEC size exclusion chromatography, TRPS tunable resistive pulse sensing, UF ultrafiltration.

Fig. 2. BEV practices: source, preparation, and characterization.

a Source: BEVs are prepared from bacterial cell culture (93.6%; colors correspond to phyla in cladogram) or other sources (6.4%; gray/white; blood, feces, intestinal tissue, milk, saliva, urine and other) (right). Circular cladogram indicating the taxonomy of studied bacterial species. The outer circle shows studied phyla, with each color representing one phylum. Number of experiments per phylum is represented by the color of the cladogram species level (gray vs. blue for most abundant phylum Pseudomonadota). Number of experiments per species is indicated by the size of the circles on the outer layer of the species level. The three most abundant species are indicated in purple (and with arrow): E. coli, S. aureus, and P. aeruginosa (left). b Preparation: Bar plot indicating number of implemented methods: one (6%), two (48%), or more than two (46%). Chord diagram shows combination of reported preparation methods, with DUC (green) and filtration (blue) as most implemented methods. Other method includes commercial method, tangential flow filtration (TFF), density cushion and precipitation. c Characterization: Bar plot indicating number of implemented methods combined: zero (28.6%), one (38.4%) two (20%), or three or more (13%). Chord diagram shows combination of reported characterization methods, with most studies performing no biochemical (orange) or biophysical characterization (pink). Other method includes PAMP reporter assays, multi-angle light scattering (MALS), confocal microscopy, spectrophotometry. BEV bacterial extracellular vesicle, DLS dynamic light scattering, DUC differential (ultra-)filtration, E. coli Escherichia coli, ELISA enzyme linked immune sorbent assay, NTA nanoparticle tracking analysis, P. aeruginosa Pseudomonas aeruginosa, S. aureus Staphylococcus aureus, SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis, SEC size exclusion chromatography, TRPS tunable resistive pulse sensing.

Fig. 3. Biophysical and biochemical BEV characteristics: size, concentration, density, and molecules.

a Size: Average particle size (nm) per experiment for most abundant phylum Pseudomonadota (left) and top three studied species E. coli, S. aureus, and P. aeruginosa (right). Colors indicate method implemented for size measurement. For statistical analysis, a two-sided Mann‐Whitney U test was used. P values smaller than 0.05 were considered statistically significant (**p < 0.01, ****p < 0.0001). Exact p values are provided in the Source data file. b Concentration: Average particle concentration (particles/mL of starting sample) per experiment, plotted on a Log10 scale, for most abundant phyla Pseudomonadota and Bacillota (left) and top two studied species: E. coli and S. aureus (right). Particle concentration is not plotted for Bacteroidota or P. aeruginosa as concentration is only reported for 8 and 3 experiments, respectively. Colors indicate method implemented for concentration measurement. For statistical analysis, a two-sided Mann‐Whitney U test was used. P values smaller than 0.05 were considered statistically significant (**p = 0.0013). c Density: Average particle density (g/mL) per source type: bacterial cell culture (colors indicate the studied phylum; n = 7) or other sources (gray; n = 6). d Molecules: Treemaps indicating the most studied BEV-enriched (left; purple) and BEV-depleted (right; blue) proteins and other molecules for all species studied to recover BEVs from bacterial cells under laboratory conditions, and for the top three studied species: E. coli, S. aureus, and P. aeruginosa. BEV-depleted proteins are not plotted for S. aureus and P. aeruginosa as zero and two proteins are reported, respectively. GroEL (red) and flagellin (orange) are implemented as both BEV-enriched and BEV-depleted. BEV bacterial extracellular vesicle, Crp Cytoplasmic cAMP receptor protein, DLS dynamic light scattering, DnaK chaperone protein, E. coli Escherichia coli, EM electron microscopy, ExoA/U exotoxin A/U, FimA Type-1 fimbrial protein, A, FlaB Flagella filament protein, Gp100 glycoprotein 100, IgIC pathogenicity island protein, Lpp outer membrane prolipoprotein lpp, LUKF-PV F component of Panton-Valentine leucocidin, LPS lipopolysaccharide, LTA lipoteichoic acid, NTA nanoparticle tracking analysis, OmpA/C/F/T outer membrane protein A/C/F/T, P. aeruginosa Pseudomonas aeruginosa, Pba peptidoglycan-binding anchor, Pic protein involved in intestinal colonization, RNAP RNA polymerase, RpoA DNA-directed RNA polymerase subunit alpha, S. aureus Staphylococcus aureus, SepA Shigella extracellular protein A, SigA Shigella IgA protease-like homologue, Stx2a Shiga toxin 2 subunit A, TRP2 tyrosinase-related protein 2, TseF hypothetical protein.

Fig. 4. Using the EV-METRIC to evaluate reporting transparency.

a Evolution of average EV-METRIC (%) for EEV (cyan) and BEV (purple) studies from 2015 to 2021. Violin and dot plot show EV-METRIC for each included BEV study; number of studies per year are indicated on the x-axis. The highest EV-METRIC is 87%; 36% achieve an EV-METRIC of 0%. b–e Radar charts representing the percentage of experiments that adhere to each of the respective EV-METRIC components according to b source type, c gram staining of studied bacteria, d species type (top three studied species E. coli S. aureus, and P. aeruginosa), and e domain (included experiments for BEVs compared to EEVs). BEV bacterial extracellular vesicle, EEV eukaryotic extracellular vesicle, E. coli Escherichia coli, EM electron microscopy, Pseudomonas aeruginosa, S. aureus Staphylococcus aureus.