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Role of sucrose-dependent exopolysaccharides in the biofilm development of Streptococcus mutans revealed at the microscale level | Applied and Environmental Microbiology Streptococcus mutans is a major pathogen in caries development due to its ability to rapidly metabolize sucrose into EPS. EPS serves as a major component of the S. mutans biofilm matrix, and previous studies mostly explored the effects of EPS on the macroscale. However, how EPS shapes S. mutans biofilm formation at the microscale is not well understood. By combining single-cell tracking with fluorescence staining techniques, we demonstrate that sucrose-dependent EPS governs the transition from 2D growth to 3D biofilm architecture and facilitates the formation of a liquid region at the bottom of the biofilm. These findings bridge a fundamental knowledge gap between the microscale organization and macroscale attributes of biofilms, offering novel perspectives for developing targeted anti-caries strategies. https://journals.asm.org/doi/abs/10.1128/aem.00009-26