Resolving physical interactions between bacteria and nanotopographies with focused ion beam scanning electron microscopy

To robustly assess the antibacterial mechanisms of nanotopographies, it is critical to analyze the bacteria-nanotopography adhesion interface. Here, we utilize focused ion beam milling combined with scanning electron microscopy to generate three-dimensional reconstructions of Staphylococcus aureus or Escherichia coli interacting with nanotopographies. For the first time, 3D morphometric analysis has been exploited to quantify the intrinsic contact area between each nanostructure and the bacterial envelope, providing an objective framework from which to derive the possible antibacterial mechanisms of synthetic nanotopographies. Surfaces with nanostructure densities between 36 and 58 per μm2 and tip diameters between 27 and 50 nm mediated envelope deformation and penetration, while surfaces with higher nanostructure densities (137 per μm2) induced envelope penetration and mechanical rupture, leading to marked reductions in cell volume due to cytosolic leakage. On nanotopographies with densities of 8 per μm2 and tip diameters greater than 100 nm, bacteria predominantly adhered between nanostructures, resulting in cell impedance.