Optically Trapped Bacteria Pairs Reveal Discrete Motile Response to Control Aggregation upon Cell–Cell Approach

Abstract Aggregation of bacteria plays a key role in the formation of many biofilms. The critical first step is cell– cell approach, and yet the ability of bacteria to control the likelihood of aggregation during this primary phase is unknown. Here, we use optical tweezers to measure the force between isolated Bacillus subtilis cells during approach. As we move the bacteria towards each other, cell motility (bacterial swimming) initiates the generation of repulsive forces at bacterial separations of *3 lm. Moreover, the motile response displays spatial sensitivity with greater cell–cell repulsion evident as inter-bacterial distances decrease. To examine the environmental influ-ence on the inter-bacterial forces, we perform the experi-ment with bacteria suspended in Tryptic Soy Broth, NaCl solution and deionised water. Our experiments demonstrate that repulsive forces are strongest in systems that inhibit biofilm formation (Tryptic Soy Broth), while attractive forces are weak and rare, even in systems where biofilms develop (NaCl solution). These results reveal that bacteria are able to control the likelihood of aggregation during the approach phase through a discretely modulated motile response. Clearly, the force-generating motility we observe during approach promotes biofilm prevention, rather than biofilm formation