Substrate stiffness modulates bacterial adhesion and diversity of adherent phenotypes across growth stages

TL;DR

This study develops a novel AFM-based method to quantify how substrate stiffness influences bacterial adhesion, revealing species-specific and growth stage-dependent variations that affect biofilm formation.

Contribution

It introduces a cell-level Force Distance Spectroscopy technique to measure bacterial adhesion relative to substrate stiffness, highlighting the role of mechanical cues in bacterial colonization.

Findings

Adhesion force increases with substrate stiffness in Chromatium okenii.

E. coli shows weak dependence of adhesion on substrate stiffness.

Bacterial populations diversify into weak and strong adherent phenotypes over growth stages.

Abstract

Surface-adhesion and stiffness of underlying substrates mediate geometry, mechanics and self-organization of bacterial colonies. Recent studies have qualitatively indicted that stiffness may impact bacterial attachment, yet the variation of cell-to-surface adhesion with substrate stiffness remains to be quantified. Here, by developing a cell-level Force Distance Spectroscopy (FDS) technique based on Atomic Force Microscopy (AFM), we simultaneously quantify the cell-surface adhesion alongside stiffness of the underlying substrates to reveal stiffness-dependent adhesion in phototrophic bacterium Chromatium okenii. As stiffness of the soft substrate, modelled via low-melting-point (LMP) agarose pad, was varied between 20 kPa and 120 kPa by changing agarose concentrations, we observe a progressive increase of the mean adhesion force by over an order of magnitude, from 0.21 (+/-0.10) nN to 2.42 (+/-1.16) nN. In contrast, passive polystyrene (PS) microparticles of comparable dimensions showed no perceptible change in their surface adhesion. Furthermore, for Escherichia coli, the cell-surface adhesion varied between 0.29 (+/-0.17) nN to 0.39 (+/-0.20) nN, showing a weak dependence on the substrate stiffness, thus suggesting that the stiffness-modulated adhesion is a species-specific trait. Finally, by quantifying the adhesion of C. okenii populations across growth stages, we report an emergent co-existence of weak and strongly adherent sub-populations, demonstrating a diversification of adherent phenotypes over time. Taken together, these findings suggest that bacteria, depending on the species and their physiological stage, actively modulate cell-to-surface adhesion in response to substrate stiffness, and leverage it as a functional trait to modulate initial attachment and colonization on soft substrates during early stages of biofilm development.