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 (H(2)O(2)) to the human pathogen Pseudomonas aeruginosa. By treating cells with levels of flow and H(2)O(2) that commonly co-occur in human host tissues,(13)(,)(14)(,)(15)(,)(16)(,)(17)(,)(18) we discover that previous reports significantly overestimate the H(2)O(2) levels required to block bacterial growth. Specifically, we establish that flow increases H(2)O(2) effectiveness 50-fold, explaining why previous studies lacking flow required much higher concentrations. Using natural H(2)O(2) levels, we identify the core H(2)O(2) regulon, characterize OxyR-mediated dynamic regulation, and demonstrate that multiple H(2)O(2) scavenging systems have redundant roles. By examining single-cell behavior, we serendipitously discover that the combined effects of H(2)O(2) and flow block pilus-driven surface migration. Thus, our results counter previous studies and reveal that natural levels of H(2)O(2) 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.