Three-Dimensional Bacterial Motions near a Surface Investigated by Digital Holographic Microscopy: Effect of Surface Stiffness

Abstract

Surface stiffness plays a critical role in bacterial adhesion, but the mechanism is unclear since the bacterial motion before adhesion is overlooked. Herein, the three-dimensional (3D) motions of <i>Escherichia coli</i> and <i>Pseudonomas</i> sp. nov 776 onto poly(dimethylsiloxane) (PDMS) surfaces with varying stiffness before adhering were monitored by digital holographic microscopy (DHM). As Young's modulus (<i>E</i>) of the PDMS surface decreases from 278.1 to 3.4 MPa, the adhered <i>E. coli</i> and <i>Pseudonomas</i> sp. decrease in number by 40.4 and 34.9%, respectively. Atomic force microscopy (AFM) measurements show that the adhesion force of bacteria to the surface declines with the decreased surface stiffness. In contrast, a nontumbling mutant of adhered <i>E. coli</i> (HCB1414 with the adaptive function being partially deficient) decreases much less (by 18.4%). On the other hand, the tumble frequency (<i>F</i>_t) of <i>E. coli</i> HCB1 and flick frequency (<i>F</i>_f) of <i>Pseudomonas</i> sp. increase as the surface stiffness decreases, and the motion bias (<i>B</i>_θ) of <i>Pseudomonas</i> sp. also increases. These facts clearly indicate that the bacteria have adapted responses to the surface stiffness. RNA sequencing (RNA-seq) reveals that the downregulated Cph2 and CsrA as well as the upregulated GcvA of swimming <i>E. coli</i> HCB1 in bulk near the softer surface promote the bacterial motility.