Combining multiple stressors blocks bacterial migration and growth

In nature, organisms experience combinations of stressors. However, laboratory studies use batch cultures, which simplify reality and focus on population-level responses to individual stressors.^1,2,3,4,5 In recent years, bacterial stress responses have been examined with single-cell resolution using microfluidics.^{6,7,8,9,10,11,12} Here, we use a microfluidic approach to simultaneously provide a physical stressor (shear flow) and a chemical stressor (H₂O₂) to the human pathogen Pseudomonas aeruginosa. By treating cells with levels of flow and H₂O₂ that commonly co-occur in human host tissues,^{13,14,15,16,17,18} we discover that previous reports significantly overestimate the H₂O₂ levels required to block bacterial growth. Specifically, we establish that flow increases H₂O₂ effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H₂O₂ levels, we identify the core H₂O₂ regulon, characterize OxyR-mediated dynamic regulation, and demonstrate that multiple H₂O₂ scavenging systems have redundant roles. By examining single-cell behavior, we serendipitously discover that the combined effects of H₂O₂ and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H₂O₂ and flow synergize to restrict bacterial motility and survival. By studying two stressors at once, our research highlights the limitations of oversimplifying nature and demonstrates that physical and chemical stress can combine to yield unpredictable effects.