Species-specific mechanical coupling dictates biofilm persistence in E. coli, S. aureus, and P. aeruginosa

Bacterial biofilms pose a formidable challenge in clinical medicine, accounting for over 80% of chronic infections and exhibiting profound resistance to both antibiotics and host immune clearance. While extensive research has focused on the biochemical and genetic determinants of biofilm formation, the physical principles governing species-specific persistence remain poorly understood. Here we employed single-cell atomic force microscopy force spectroscopy and optical tweezers to systematically investigate the nanomechanical properties-adhesion force, cell wall stiffness, and work of detachment-across planktonic, biofilm, and small colony variant (SCV) phenotypes of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa recovered from pus samples of chronic wound infections. Our findings reveal three distinct mechanical strategies that correlate with clinical persistence. E. coli exhibits weak mechanical coupling (μ = 4.8), prioritizing rapid growth and dispersal over permanent surface commitment. S. aureus demonstrates moderate coupling (μ = 6.9), balancing high initial adhesion with reduced detachment work in its SCV phenotype. In striking contrast, P. aeruginosa displays exceptionally tight mechanical coupling (μ = 8.9); its SCVs achieve the highest mean adhesion force (478 ± 45 pN) and work of detachment (258 ± 28 aJ) while minimizing metabolic energy dissipation to an astonishingly low 0.018 fW/cell. Thermodynamic analysis reveals that surface locking in P. aeruginosa SCVs is strongly enthalpy-driven (ΔG° = -41.2 kJ/mol), with a substantial entropic penalty (-219 J/mol·K) overcome by favorable enthalpy changes. The mechanical coupling coefficient introduced herein provides a novel biophysical framework for predicting biofilm recalcitrance, demonstrating that pathogens actively remodel their surface architectures to manipulate envelope rigidity and viscoelasticity for niche-specific survival. These findings reframe biofilm persistence as a mechanobiological property amenable to targeted physical disruption, opening avenues for therapeutic strategies such as cell-wall-softening agents designed to weaken and eradicate chronic bacterial infections.