A Modular Workflow for Quantitative, Structural and Functional Analysis of <em>Leptospira</em> Biofilms

Here, we present an integrated suite of protocols for the cultivation, quantitative monitoring, structural and functional characterization of Leptospira spp. biofilms over time. This workflow combines crystal violet microtiter assays to measure biofilm biomass at multiple time points, with a time-resolved fractionation approach that distinguishes attached (biofilm) and non-attached (liquid-phase) bacterial populations, time-lapse phase-contrast imaging for non-destructive kinetic observation, confocal laser-scanning microscopy to generate full 3D reconstructions with matrix-probe readouts, and membrane-supported scanning electron microscopy for ultrastructural analysis. In parallel, we detail a standardized procedure for harvesting intact biofilm aggregates and preparing them for intraperitoneal injection into the susceptible golden Syrian hamster model, enabling direct assessment of biofilm-associated virulence in vivo alongside matched planktonic controls. Optimized for the pathogenic strain Leptospira interrogans Manilae L495, each module is readily transferable to other Leptospira species and mutant libraries to compare biofilm-forming capacity. Together, these coordinated modules provide a robust foundation for screening anti-biofilm strategies, probing genetic determinants, and clarifying the contribution of biofilms to Leptospira persistence and pathogenesis.