Mechanoadaptation of bacterial physiology and antibiotic tolerance in confined environments

Bacteria encounter mechanical forces in nearly every environment they inhabit, and these forces can regulate their physiology and behavior. In many settings, they grow in dense, surface-associated communities where close packing and limited space create physical constraints. These conditions protect them from external stresses, but within infections they can turn bacteria into far more resilient and persistent pathogens. This is a serious concern in clinical and industrial contexts. It is well known that resident cells rely on biochemical signals, but the role of mechanical forces within biofilm-like populations remains poorly understood. Emerging evidence suggests that bacteria can sense and respond to mechanical cues, much like higher organisms. However, the lack of precise tools to probe and manipulate these forces has slowed progress, especially in infection biology. Uncovering this mechanobiology layer could reshape how we control bacterial physiology, opening new strategies to combat persistent infections with more effective antimicrobial therapies. In Chapter 2, I demonstrated that the constrained growth of P. aeruginosa deforms surrounding synthetic hydrogels. This growth-induced pressure regulates population size and alters the physiology of individual cells within them. Under these conditions, encapsulation confers high resilience to bacteria, even to the last-resort antibiotic colistin. The stiffness of the surrounding material modulates this protection. Finally, we identified genomic signatures that drive bacterial adaptation to confinement, leading to increased antibiotic tolerance. In Chapter 3, I investigated the transcriptional adaptation of bacteria to mechanical confinement. To do this, I developed a new method for extracting bacterial transcripts while preserving RNA integrity. RNA-seq analysis of embedded cells showed broad gene expression changes. Mechanical confinement influenced factors that regulate twitching motility, biofilm regulation, and quorum sensing. In the end, we used fluorescent transcriptional reporters to confirm that cells actively respond to physical confinement, and that substrate stiffness defines their gene expression patterns. Overall, our results demonstrate that spatial confinement shapes bacterial physiology by controlling proliferation and cell morphology within confined populations. This mechanically induced phenotype influences infection outcomes by modulating antibiotic susceptibility, either through adaptation or increased virulence. Thus, mechanical forces drive a feedback loop within biofilm-resident cells, ultimately shaping cellular behavior and maintaining biofilm homeostasis.