Biofilm disruption by an air bubble reveals heterogeneous age-dependent detachment patterns dictated by initial extracellular matrix distribution.

Bacteria often adhere to surfaces, where they form communities known as biofilms. Recently, it has been shown that biofilm formation initiates with the microscopically heterogeneous deposition of a skeleton of extracellular polymeric substances (EPS) by individual cells crawling on the surface, followed by growth of the biofilm into a surface-covering continuum. Here we report microfluidic experiments with Pseudomonas aeruginosa biofilms showing that their "hidden" heterogeneity can affect the later dynamics of their disruption. Using controlled air bubbles as a model for mechanical insult, we demonstrate that biofilm disruption is strongly dependent on biofilm age, and that disruption to early-stage biofilms can take the shape of a semi-regular pattern of ~15 µm diameter holes from which bacteria have been removed. We explain hole formation in terms of the rupture and retreat of the thin liquid layer created by the long bubble, which scrapes bacteria off the surface and rearranges their distribution. We find that the resulting pattern correlates with the spatial distribution of EPS: holes form where there is less EPS, whereas regions with more EPS act as strongholds against the scraping liquid front. These results show that heterogeneity in the microscale EPS skeleton of biofilms has profound consequences for later dynamics, including disruption. Because few attached cells suffice to regrow a biofilm, these results point to the importance of considering microscale heterogeneity when designing and assessing the effectiveness of biofilm removal strategies by mechanical forces.