Capturing of extracellular vesicles derived from single cells of .

Bacteria secrete extracellular vesicles (EVs), also referred to as bacterial membrane vesicles, which carry, among other compounds, lipids, nucleic acids and virulence factors. Recent studies highlight the role of EVs in the emergence of antibiotic resistance, as carrier and absorbent particles of the drug to protect the cells, or as a pathway to disseminate resistance elements. In this study, we are interested in characterizing the secretion of EVs at the single bacterial level to ultimately understand how cells respond to antibiotic treatment. We introduce a microfluidic device that enables culture of single bacterial cells and capture of EVs secreted from these individuals. The device incorporates parallel, narrow winding channels to trap single rod-shaped cells at their entrances. The daughter cells are immediately removed by continuous flow on the open side of the trap, so that the trap contains always only a single cell. Cells grew in these traps over 24 h with a doubling time of 25 minutes. Under antibiotic treatment, the doubling time did not change, but we observed small changes in the cell length of the trapped cells (decrease from 4.0 μm to 3.6 μm for 0 and 250 ng mL polymyxin B, respectively), and cells stopped growing within hours, depending on the drug concentration. Compared to bulk culture, the results indicate a higher susceptibility of on-chip-cultured cells (250 ng mL >500 ng mL in bulk), which may be caused, among other reasons, by the space limitation in the cell trap and shear forces. During the culture, EVs secreted by the trapped cells entered the winding channel. We developed a procedure to selectively coat these channels with poly-L-lysine resulting in a positively charged surface, which enabled electrostatic capture of negatively charged EVs. Subsequently, the immobilized EVs were stained with a lipophilic dye and detected by fluorescence microscopy. Our findings confirm large variations of EV secretion among individual bacteria and indicate a relative high rate of EV secretion under antibiotic treatment. The proposed method can be extended to the detection of other secreted substances of interest and may facilitate the elucidation of unknown heterogeneities in bacteria.