Nanopillared surfaces have emerged as a promising strategy to combat bacterial infections on medical devices. However, the mechanisms that underpin nanopillar-induced rupture of the bacterial cell membrane remain speculative. In this study, we have tested three medically relevant poly(ethylene terephthalate) (PET) nanopillared-surfaces with well-defined nanotopographies against both Gram-negative and Gram-positive bacteria. Focused ion beam scanning electron microscopy (FIB-SEM) and contact mechanics analysis were utilised to understand the nanobiophysical response of the bacterial cell envelope to a single nanopillar. Given their importance to bacterial adhesion, the contribution of bacterial surface proteins to nanotopography-mediated cell envelope damage was also investigated. We found that, whilst cell envelope deformation was affected by the nanopillar tip diameter, the nanopillar density affected bacterial metabolic activities. Moreover, three different types of bacterial cell envelope deformation were observed upon contact of bacteria with the nanopillared surfaces. These were attributed to bacterial responses to cell wall stresses resulting from the high intrinsic pressure caused by the engagement of nanopillars by bacterial surface proteins. Such influences of bacterial surface proteins on the antibacterial action of nanopillars have not been previously reported. Our findings will be valuable to the improved design and fabrication of effective antibacterial surfaces.
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.