Department of Biosystems Science and Engineering, ETH Zurich, CH-4056 Basel, Switzerland.
The University of Tokyo, Department of Physics, Tokyo 113-0033, Japan.
Lab Chip. 2024 Mar 26;24(7):2049-2057. doi: 10.1039/d3lc00707c.
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.
细菌分泌细胞外囊泡（EVs），也称为细菌膜囊泡，其中携带脂质、核酸和毒力因子等化合物。最近的研究强调了 EVs 在抗生素耐药性出现中的作用，作为药物的载体和吸收颗粒，以保护细胞，或作为传播耐药元件的途径。在这项研究中，我们有兴趣在单个细菌水平上描述 EVs 的分泌，以最终了解细胞如何对抗生素治疗做出反应。我们引入了一种微流控装置，该装置能够在单个细菌细胞的水平上培养和捕获从这些个体分泌的 EVs。该装置包含平行的狭窄缠绕通道，可在入口处捕获单个杆状细胞。通过在陷阱的开口侧连续流动，立即将子细胞除去，因此陷阱中始终只包含单个细胞。细胞在这些陷阱中生长 24 小时，倍增时间为 25 分钟。在抗生素处理下，倍增时间没有改变，但我们观察到被困细胞的细胞长度发生了微小变化（在 0 和 250ng/mL 多粘菌素 B 下分别从 4.0μm 减小到 3.6μm），并且细胞在数小时内停止生长，这取决于药物浓度。与批量培养相比，结果表明在芯片上培养的细胞对药物的敏感性更高（在芯片上培养的细胞 250ng/mL >500ng/mL），这可能是由于细胞陷阱中的空间限制和剪切力等原因造成的。在培养过程中，被困细胞分泌的 EVs 进入缠绕通道。我们开发了一种选择性地用聚-L-赖氨酸涂覆这些通道的程序，导致表面带正电荷，从而能够静电捕获带负电荷的 EVs。随后，用亲脂性染料固定化 EVs 并用荧光显微镜检测。我们的研究结果证实了单个细菌之间 EV 分泌的巨大差异，并表明在抗生素处理下 EV 分泌的相对较高速率。所提出的方法可以扩展到检测其他感兴趣的分泌物质，并可能有助于阐明细菌中未知的异质性。
