Spatial concentration gradients of antibiotics are prevalent in the natural environment. Yet, the microbial response in these heterogeneous systems remains poorly understood. We used a microfluidic reactor to create an artificial microscopic ecosystem that generates diffusive gradients of solutes across interconnected microenvironments. With this reactor, we showed that chemotaxis toward a soluble electron acceptor (nitrate) allowed Shewanella oneidensis MR-1 to inhabit and sustain metabolic activity in highly toxic regions of the antibiotic ciprofloxacin (>80× minimum inhibitory concentration, MIC). Acquired antibiotic resistance was not observed for cells extracted from the reactor, so we explored the role of transient adaptive resistance by probing multidrug resistance (MDR) efflux pumps, ancient elements that are important for bacterial physiology and virulence. Accordingly, we constructed an efflux pump deficient mutant (∆mexF) and used resistance-nodulation-division (RND) efflux pump inhibitors (EPIs). While batch results showed the importance of RND efflux pumps for microbial survival, microfluidic studies indicated that these pumps were not necessary for survival in antibiotic gradients. Our work contributes to an emerging body of knowledge deciphering the effects of antibiotic spatial heterogeneity on microorganisms and highlights differences of microbial response in these systems versus well-mixed batch conditions.

抗生素的空间浓度梯度在自然环境中普遍存在。然而，人们对这些异质系统中的微生物反应仍然知之甚少。我们使用微流体反应器创建了一个人工微观生态系统，该生态系统在相互关联的微环境中产生溶质扩散梯度。有了这个反应器，我们发现对可溶性电子受体（硝酸盐）的趋化作用允许Shewanella oneidensisMR-1 在抗生素环丙沙星（>80× 最小抑菌浓度，MIC）的高毒性区域中驻留和维持代谢活动。从反应器中提取的细胞未观察到获得性抗生素耐药性，因此我们通过探测多药耐药性 (MDR) 外排泵（对细菌生理学和毒力很重要的古老元素）来探索瞬时适应性耐药性的作用。因此，我们构建了一个外排泵缺陷突变体（ΔmexF) 和使用电阻结节分裂 (RND) 外排泵抑制剂 (EPI)。虽然批量结果表明 RND 外排泵对微生物存活的重要性，但微流体研究表明这些泵对于抗生素梯度中的存活不是必需的。我们的工作有助于一个新兴的知识体系破译抗生素空间异质性对微生物的影响，并突出这些系统中微生物反应与混合良好的批次条件的差异。