Programmable 3D Microfluidics Reveals Motility-Driven Antibiotic Resistance Evolution in Gradients.

Antibiotic resistance has become a serious threat to global health. It frequently emerges in real-world environments such as biofilms, where antibiotic gradients impose selective pressures that drive bacterial adaptation. However, the mechanisms underlying resistance development remain poorly understood, largely due to challenges in experimentally controlling confounding parameters. Here, we report a 3D microfluidic platform that enables real-time monitoring of bacterial dynamics under well-defined antibiotic gradients, along with programmable cell retrieval to identify key factors in resistance development. We show that motile Escherichia coli migrated randomly within the gradients regardless of antibiotic class (i.e., gentamicin, ciprofloxacin, and ampicillin). Resistance emerged only when a critical mass of bacteria sustained migration through the gradient for a sufficiently duration, revealing a previously unrecognized migration-duration threshold for adaptive evolution. Whole-genome resequencing revealed gene mutations in the resistant cells, including mutations in ATP synthase-related genes (i.e., atpG, atpD, and atpF). A trade-off between resistance and cell growth was also observed. Similar phenomena were observed in other motile bacteria species, including Serratia marcescens. Overall, we demonstrate that cell motility is a critical determinant of resistance development in heterogeneous antibiotic landscapes. Targeting cell motility and growth could offer effective strategies to combat antibiotic resistance in motile bacteria.