High-throughput 3D tracking reveals the importance of near wall swimming and initial attachment behaviors of P. aeruginosa for biofilm formation on a vertical wall

Studying the swimming behaviour of bacteria in 3 dimensions (3D) allows us to understand critical biological processes, such as biofilm formation. It is still unclear how near wall swimming behaviour may regulate the initial attachment and biofilm formation. It is challenging to address this as visualizing the movement of bacteria with reasonable spatial and temporal resolution in a high-throughput manner is technically difficult. Here, we compared the near wall (vertical) swimming behaviour of P. aeruginosa (PAO1) and its mutants {Delta}dipA (reduced in swarming motility and increased in biofilm formation) and {Delta}fimX (deficient in twitching motility and reduced in biofilm formation) using our new imaging technique based on light sheet microscopy. We found that P. aeruginosa (PAO1) increases its speed and changes its swimming angle drastically when it gets closer to a wall. In contrast, {Delta}dipA mutant moves toward the wall with steady speed without changing of swimming angle. The near wall behavior of {Delta}dipA allows it to be more effective to interact with the wall or wall-attached cells, thus leading to more capture events and a larger biofilm volume during initial attachment when compared with PAO1. Furthermore, we found that {Delta}fimX has a similar near wall swimming behavior as PAO1, however, it has a higher dispersal frequency and smaller biofilm formation when compared with PAO1 which can be explained by its poor twitching motility. Together, we propose that near wall swimming behavior of P. aeruginosa plays an important role in the regulation of initial attachment and biofilm formation. ImportanceBacterial biofilm is a community of bacteria on surfaces which leads to serious problems in medical devices, food industry, and aquaculture. The initial attachment and subsequent microcolony formation play critical roles in bacterial biofilm formation. However, it is unclear how the initial attachment is regulated, in particular, on a vertical surface. To study this, we have developed a novel imaging technique based on light sheet microscopy, which overcame the limitations of other imaging techniques, to understand how 3D bacterial motility near a wall may regulate initial attachment during biofilm formation. Using our technique, we discovered that near wall swimming behavior of the bacteria, P. aeruginosa, plays an important role in the regulation of biofilm formation during initial attachment.