Mechanical Engineering Department, Embry-Riddle Aeronautical University, Daytona Beach, FL, USA.
Virginia Tech Hampton Roads Agriculture Research and Extension Center, Virginia Tech, Blacksburg, VA, USA.
Life Sci Space Res (Amst). 2022 Aug;34:1-8. doi: 10.1016/j.lssr.2022.04.003. Epub 2022 Apr 18.
Understanding the impacts of microgravity on bacteria is vital for successful long duration space missions. In this environment, bacteria have been shown to become more virulent, more resistant to antibiotics and to regulate biofilm formation. Since the study of these phenomena under true microgravity is cost- and time challenging, the use of ground-based analogs might allow researchers to test hypotheses before planning and executing experiments in the space environment. We designed and developed a 2D clinostat with capabilities robust enough for bacterial studies to allow for multiple simultaneous replicates of treatment and control conditions, thus permitting the generation of growth curves, in a single run. We used computational fluid dynamics (CFD), biofilm growth measurement and differential gene expression analysis on Escherichia coli cultures grown to late exponential phase (24 h) to validate the system's ability to simulate microgravity conditions. The CFD model with a rotational speed of 8 rpm projected cells growing homogeneously distributed along the tube, while the static condition showed the accumulation of the cells at the bottom of the container. These results were empirically validated with cultures on nutrient broth. Additionally, crystal violet assays showed that higher biofilm biomass grew on the internal walls of the gravity control tubes, compared to the simulated microgravity treatment. In contrast, when cells from both treatments were grown under standard conditions, those exposed to simulated microgravity formed significantly more biofilms than their gravity counterparts. Consistent with this result, transcriptome analysis showed the upregulation of several gene families related to biofilm formation and development such as cells adhesion, aggregation and regulation of cell motility, which provides a potential transcriptional explanation for the differential phenotype observed. Our results show that when operated under parameters for simulated microgravity, our 2D clinostat creates conditions that maintain a proportion of the cells in a constant free-falling state, consistent with the effect of microgravity. Also, the high-throughput nature of our instrument facilitates, significantly, bacterial experiments that require multiple sampling timepoints and small working volumes, making this new instrument extremely efficient.
了解微重力对细菌的影响对于成功进行长期太空任务至关重要。在这种环境下，细菌已经表现出更强的毒性、对抗生素的更强抗性以及生物膜形成的调节能力。由于在真正的微重力下研究这些现象具有成本和时间上的挑战性，因此使用地面模拟设备可能使研究人员能够在计划和执行太空环境中的实验之前，测试假设。我们设计和开发了一种 2D 回转仪，其功能强大到足以进行细菌研究，可以同时进行多个处理和对照条件的重复实验，从而可以在单次运行中生成生长曲线。我们使用计算流体动力学（CFD）、生物膜生长测量和大肠杆菌培养物的差异基因表达分析，这些培养物在晚期指数期（24 小时）生长，以验证该系统模拟微重力条件的能力。以 8 rpm 的转速旋转的 CFD 模型预测细胞均匀分布在管中生长，而静态条件则显示细胞在容器底部积累。这些结果通过在营养肉汤中培养的细菌得到了经验验证。此外，结晶紫测定表明，与模拟微重力处理相比，重力控制管的内壁上生长了更多的生物膜生物量。相比之下，当两种处理的细胞在标准条件下生长时，暴露于模拟微重力下的细胞比其重力对照物形成的生物膜多得多。与该结果一致，转录组分析显示，与生物膜形成和发育相关的几个基因家族的上调，例如细胞粘附、聚集和细胞运动性调节，这为观察到的差异表型提供了潜在的转录解释。我们的结果表明，当在模拟微重力的参数下运行时，我们的 2D 回转仪创造了一种条件，使一部分细胞保持在恒定的自由落体状态，这与微重力的影响一致。此外，我们仪器的高通量特性极大地促进了需要多个采样时间点和小工作体积的细菌实验，使这种新仪器非常高效。
