Acoustic signatures uncover multistage mechanical maturation of bacterial biofilms

Bacterial biofilm formation is commonly viewed as a gradual increase in surface-bound biomass; however, the mechanical transitions that govern maturation remain insufficiently characterized. Here, we integrate quartz crystal microbalance with dissipation monitoring (QCM-D), field-emission scanning electron microscopy (FESEM), and bulk biofilm assays to investigate the multistage mechanical development of bacterial biofilms using a controlled heterologous expression platform for the Pseudomonas aeruginosa adhesin CdrA. This protein enhances biomass accumulation while accelerating structural and mechanical biofilm maturation. QCM-D measurements reveal discrete mechanical regimes, culminating in acoustic harmonic inversion associated with the emergence of a mechanically cohesive viscoelastic film. This inversion is observed only upon CdrA induction. FESEM imaging demonstrates that mechanical consolidation coincides with reduced spatial heterogeneity and increased structural connectivity. These findings support acoustic harmonic inversion as a candidate acoustic marker of late-stage mechanical consolidation and highlight combined acoustic and structural imaging as a powerful experimental approach for studying biofilm integrity and mechanically relevant transitions during biofilm development.