Measuring growth dynamics in three-dimensional bacterial aggregates

Three-dimensional non-surface-attached bacterial aggregates are widespread in nature, occurring in soil, wastewater, marine snow, and chronic infections. However, the vast majority of studies on bacterial growth have focused on 2D colonies or surface-attached biofilms. We have developed an in vitro model system for generating and imaging 3D bacterial aggregates that makes use of a normally zebrafish-gut-native microbe (genus Aeromonas) whose adhesion is triggered by the sugar N-acetylglucosamine (GlcNAc). Following initial seeding in a GlcNAc solution, aggregates are embedded in 2% agarose gel and immersed in M9 minimal media with 0.4% GlcNAc. Using light sheet fluorescence microscopy, we capture 3D time-lapse images of growing aggregates. We find an overall growth rate that decreases with aggregate size, indicating surface-dominated growth. This platform also enables co-culture experiments. For example, co-aggregates formed between this Aeromonas strain and a (normally) non-aggregating species maintain a stable 1:1 ratio of species abundance, even when the non-aggregator is dominant in the initial culture. Structural analysis of these aggregates reveals spatial signatures of interspecies cooperation and competition.