Capturing of extracellular vesicles derived from single cells of Escherichia coli

Bacteria are not just solitary entities but are capable of sophisticated communication and interaction through the secretion of extracellular vesicles (EVs). These tiny vesicles, also known as bacterial membrane vesicles, carry various compounds, including lipids, nucleic acids, and virulence factors. Recent research has highlighted the critical role of EVs in the development of antibiotic resistance, posing a significant challenge in combatting bacterial infections. In this study, scientists from ETH Zurich delve into the intricacies of EV secretion at the individual bacterial cell level to gain deeper insights into bacterial responses to antibiotic treatment.

Microfluidic Device Design

To investigate EV secretion at the single bacterial cell level, the researchers designed a specialized microfluidic device. This innovative device allows for the cultivation of individual bacterial cells and captures the EVs secreted by these cells. The device features parallel, narrow winding channels designed to trap single rod-shaped Escherichia coli (E. coli) cells at their entrances. Importantly, the continuous flow within the device swiftly removes daughter cells, ensuring that each trap contains only a single bacterial cell.

Design of the isolated mother machine (iMM) device for capturing
bacterial cells and collecting EVs from individual cells

(A) The microfluidic device enables the isolation of individual bacteria and continuous removal of their daughter cells. The process of EV collection using the iMM device is as follows: step 1. Bacterial cells are loaded into the device through a wide trench by medium flow; step 2. A single bacterial cell is captured by a cell trap at the entrance of a narrow channel; step 3. During culture, daughter cells from the isolated cell are quickly removed from the cell trap by flow, so that the isolated single cell remains singular; step 4. EVs secreted from the single cell are carried towards the end of a narrow channel by flow; step 5. Staining of the EVs for detection. (B) Enlarged view of the narrow channels illustrating the cell trap and the winding structure for EV capturing. (C) Schematic side view of the narrow channel surface to immobilize EVs by electrostatic forces. The lipid-staining dye (FM4-64) integrates into the EV membrane and thereby increases the emission of fluorescence. All the sketches are not to scale.

Experimental Findings

Over a 24-hour period, the trapped bacterial cells exhibited rapid growth, with a doubling time of approximately 25 minutes. Interestingly, under antibiotic treatment, although the doubling time remained unchanged, there were observable changes in the length of the trapped cells. Additionally, bacterial growth ceased within hours of antibiotic exposure, depending on the drug concentration. Notably, the on-chip-cultured cells displayed higher susceptibility to antibiotics compared to bulk culture conditions, possibly due to space limitation within the cell trap and shear forces.

Detection of EV Secretion

Throughout the bacterial culture period, EVs secreted by the trapped cells entered the winding channels of the microfluidic device. To selectively capture these EVs, the researchers developed a novel procedure involving the coating of channels with poly-L-lysine, creating a positively charged surface. This modification facilitated the electrostatic capture of negatively charged EVs, which were subsequently stained with a lipophilic dye and visualized using fluorescence microscopy.

The study’s findings highlight significant variations in EV secretion among individual bacteria, with a relatively high rate observed under antibiotic treatment. The developed microfluidic device and detection method offer a powerful tool for exploring bacterial heterogeneities and understanding the dynamics of EV secretion. Moreover, these insights could pave the way for novel strategies to combat antibiotic resistance and enhance our understanding of bacterial communication and adaptation mechanisms.

Yokoyama F, Kling A, Dittrich PS. (2024) Capturing of extracellular vesicles derived from single cells of Escherichia coli. Lab Chip [Epub ahead of print]. [article]

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