Orthogonal microfluidic approaches reveal force-enhanced migration of bacterial populations on surfaces.

Host-generated flow is expected to oppose bacterial migration by sweeping cells downstream, yet some bacteria can migrate upstream against the direction of flow. However, it is unclear how the magnitude of shear force influences upstream bacterial migration. Here, we use microfluidics to examine upstream migration of the human pathogen Pseudomonas aeruginosa under host-relevant shear forces (0.1-10 pN). By independently varying flow rate and solution viscosity, we discover that increasing shear force counterintuitively enhances population-level upstream migration. Single-cell tracking reveals that force-enhanced migration is driven by an increase in twitching motility speed. Using a microfluidic-based trigonometry approach, we link the increase in speed to the cell-surface angle. At low shear forces, type IV pilus retraction generates a torque that tilts cells toward a vertical orientation, geometrically constraining forward movement and decreasing twitching speed. In contrast, higher shear forces push cells toward a horizontal orientation, increasing twitching speed. Collectively, our results reveal how shear force can enhance bacterial motility and upstream migration, providing a framework to understand how host shear forces may promote the spread of bacterial infections.