DeFrates, K. G. | Search bioRxiv

bioRxiv2026

Mechanofiltration Enables High-Throughput Measurements of Bacterial Cell Mechanics

Bacteria experience diverse mechanical forces throughout their natural environments, yet quantitative measurements of bacterial biomechanics remain challenging because most existing techniques require specialized instrumentation, such as atomic force microscopy (AFM) or microfluidic devices. Here, we introduce mechanofiltration, a simple, high-throughput assay that estimates whole-cell mechanics using standard laboratory equipment. In mechanofiltration, bacterial suspensions are centrifuged through porous membranes in multiwell plates. Pressure generated during centrifugation drives cells toward pores that are smaller than cell width, requiring cells to deform to transit through the membrane. By combining recovery on the opposite side of the filter with measurements of cell size, the assay estimates maximum cell deformation, a size-corrected proxy for whole-cell stiffness. We show that mechanofiltration detects established reductions in Escherichia coli cell stiffness caused by genetic disruption of load-bearing cell envelope components, treatment with outer membrane-destabilizing agents, and sublethal exposure to antibiotics. Fold changes in maximum deformation closely agree with published measurements obtained using AFM, cell-bending assays, and osmotic shock experiments. Mechanofiltration also reproduces previously reported mechanical differences among bacterial species with distinct morphologies and envelope architectures. Together, these results establish mechanofiltration as an accessible, inexpensive, and scalable approach for identifying genetic and environmental determinants of bacterial mechanics and for high-throughput screening of bacterial biomechanical phenotypes.

microbiology↗

bioRxiv2025

Physical mechanisms that enable bacteria to traverse channels half their width

Bacteria colonize surfaces in the environment and can also penetrate tissues and materials by entering micro- and nanoscale cracks and pores. Staphylococcus aureus has been observed within nanoscale channels in bone that are 2-3 times smaller than cell diameter. Once inside bone, bacteria are protected from host immunity and systemic antibiotics, potentially contributing to chronic and recurrent infections. The physical mechanisms that enable bacteria to enter channels smaller than cell width are unclear. It has been proposed that bacteria traverse narrow passages through division - such that daughter cells form within small channels and eventually create a chain of cells extending down the channel length. Here we use microfluidics to test the idea that S. aureus can traverse bone-like nanochannels through growth. We examined 322 individual cells trapped within tapered nanochannels ([~]1.5 to 0.4 mm in width). When cells were deformed below 606 nm (65% original width), growth and division were slowed or completely inhibited. When cell division did occur in nanochannels, daughter cells were more likely to travel towards the wider side of the channel. Hence, it is unlikely that cell division would preferentially enable transit of S. aureus into nanoscale channels. However, the magnitudes of fluid pressure needed to deform S. aureus to widths similar to that seen in bone (1 to 6.5 kPa), were small relative to fluid pressure in bone generated by physical forces in vivo (8-20 kPa). Thus, our findings suggest that colonization of nanoscale channels in bone or other tissues is more likely due to moderate fluid pressure rather than growth. ImportanceBacteria that colonize materials and tissues within the body can be difficult to remove, even with thorough cleaning and application of antibiotics. Recent studies show that bacteria not only colonize the surfaces of tissues in the body but can also squeeze into naturally occurring pores and channels and thereby gain protection from immune cells and antibiotics. Here we ask how physical forces and cell growth might enable bacteria to enter small pores within materials. We use microfluidic devices to study the growth and migration of the human pathogenic bacteria, S. aureus, which is the leading cause of chronic bone infections.

microbiology↗