Flagellum-driven motility enhances Pseudomonas aeruginosa biofilm formation by altering cell orientation

Bacterial motility plays a crucial role in biofilm development, yet the underlying mechanism remains not fully understood. Here, we demonstrate that the flagellum-driven motility of Pseudomonas aeruginosa enhances biofilm formation by altering the orientation of bacterial cells, an effect controlled by shear stress rather than shear rate. By tracking wild-type P. aeruginosa and its non-motile mutants in a microfluidic channel, we demonstrate that while non-motile cells align with the flow, many motile cells can orient toward the channel sidewalls, enhancing cell surface attachment and increasing biofilm cell density by up to 10-fold. Experiments with varying fluid viscosities further demonstrate that bacterial swimming speed decreases with increasing fluid viscosity, and the cell orientation scales with the shear stress rather than shear rate. Our results provide a quantitative framework to predict the role of motility in the orientation and biofilm development under different flow conditions and viscosities.IMPORTANCEBiofilms are ubiquitous in rivers, water pipes, and medical devices, impacting the environment and human health. While bacterial motility plays a crucial role in biofilm development, a mechanistic understanding remains limited, hindering our ability to predict and control biofilms. Here, we reveal how the motility of Pseudomonas aeruginosa, a common pathogen, influences biofilm formation through systematically controlled microfluidic experiments with confocal and high-speed microscopy. We demonstrate that the orientation of bacterial cells is controlled by shear stress. While non-motile cells primarily align with the flow, many motile cells overcome the fluid shear forces and reorient toward the channel sidewalls, increasing biofilm cell density by up to 10-fold. Our findings provide insights into how bacterial transition from free-swimming to surface-attached states under varying flow conditions, emphasizing the role of cell orientation in biofilm establishment. These results enhance our understanding of bacterial behavior in flow environments, informing strategies for biofilm management and control.