Single-cell segmentation in bacterial biofilms with an optimized deep learning method enables tracking of cell lineages and measurements of growth rates.

Bacteria often grow into matrix-encased three-dimensional (3D) biofilm communities, which can be imaged at cellular resolution using confocal microscopy. From these 3D images, measurements of single-cell properties with high spatiotemporal resolution are required to investigate cellular heterogeneity and dynamical processes inside biofilms. However, the required measurements rely on the automated segmentation of bacterial cells in 3D images, which is a technical challenge. To improve the accuracy of single-cell segmentation in 3D biofilms, we first evaluated recent classical and deep learning segmentation algorithms. We then extended StarDist, a state-of-the-art deep learning algorithm, by optimizing the post-processing for bacteria, which resulted in the most accurate segmentation results for biofilms among all investigated algorithms. To generate the large 3D training dataset required for deep learning, we developed an iterative process of automated segmentation followed by semi-manual correction, resulting in >18,000 annotated Vibrio cholerae cells in 3D images. We demonstrate that this large training dataset and the neural network with optimized post-processing yield accurate segmentation results for biofilms of different species and on biofilm images from different microscopes. Finally, we used the accurate single-cell segmentation results to track cell lineages in biofilms and to perform spatiotemporal measurements of single-cell growth rates during biofilm development.

Shared biophysical mechanisms determine early biofilm architecture development across different bacterial species.

Bacterial biofilms are among the most abundant multicellular structures on Earth and play essential roles in a wide range of ecological, medical, and industrial processes. However, general principles that govern the emergence of biofilm architecture across different species remain unknown. Here, we combine experiments, simulations, and statistical analysis to identify shared biophysical mechanisms that determine early biofilm architecture development at the single-cell level, for the species Vibrio cholerae, Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa grown as microcolonies in flow chambers. Our data-driven analysis reveals that despite the many molecular differences between these species, the biofilm architecture differences can be described by only 2 control parameters: cellular aspect ratio and cell density. Further experiments using single-species mutants for which the cell aspect ratio and the cell density are systematically varied, and mechanistic simulations show that tuning these 2 control parameters reproduces biofilm architectures of different species. Altogether, our results show that biofilm microcolony architecture is determined by mechanical cell-cell interactions, which are conserved across different species.

VxrB Influences Antagonism within Biofilms by Controlling Competition through Extracellular Matrix Production and Type 6 Secretion.

The human pathogen Vibrio cholerae grows as biofilms, communities of cells encased in an extracellular matrix. When growing in biofilms, cells compete for resources and space. One common competitive mechanism among Gram-negative bacteria is the type six secretion system (T6SS), which can deliver toxic effector proteins into a diverse group of target cells, including other bacteria, phagocytic amoebas, and human macrophages. The response regulator VxrB positively regulates both biofilm matrix and T6SS gene expression. Here, we directly observe T6SS activity within biofilms, which results in improved competition with strains lacking the T6SS. VxrB significantly contributes to both attack and defense via T6SS, while also influencing competition via regulation of biofilm matrix production. We further determined that both Vibrio polysaccharide (VPS) and the biofilm matrix protein RbmA can protect cells from T6SS attack within mature biofilms. By varying the spatial mixing of predator and prey cells in biofilms, we show that a high degree of mixing favors T6SS predator strains and that the presence of extracellular DNA in V. cholerae biofilms is a signature of T6SS killing. VxrB therefore regulates both T6SS attack and matrix-based T6SS defense, to control antagonistic interactions and competition outcomes during mixed-strain biofilm formation. IMPORTANCE This work demonstrates that the Vibrio cholerae type six secretion system (T6SS) can actively kill prey strains within the interior of biofilm populations with substantial impact on population dynamics. We additionally show that the response regulator VxrB contributes to both T6SS killing and protection from T6SS killing within biofilms. Components of the biofilm matrix and the degree of spatial mixing among strains also strongly influence T6SS competition dynamics. T6SS killing within biofilms results in increased localized release of extracellular DNA, which serves as an additional matrix component. These findings collectively demonstrate that T6SS killing can contribute to competition within biofilms and that this competition depends on key regulators, matrix components, and the extent of spatial population mixture during biofilm growth.