Surface-associated bacterial communities, known as biofilms, are responsible for a broad spectrum of infections in humans. Recent studies have indicated that surfaces containing nanoscale protrusions, like those in dragonfly wings, create a hostile niche for bacterial colonization and biofilm growth. This functionality has been mimicked on metals and semiconductors by creating nanopillars and other high aspect ratio nanostructures at the interface of these materials. However, bactericidal topographies have not been reported on clinically relevant hydrogels and highly compliant polymers, mostly because of the complexity of fabricating nanopatterns in hydrogels with precise control of the size that can also resist aqueous immersion. Here, we report the fabrication of bioinspired bactericidal nanostructures in bacterial cellulose (BC) hydrogels using low-energy ion beam irradiation. By challenging the currently accepted view, we show that the nanostructures grown in BC affect preferentially stiff membranes like those of the Gram-positive bacteria Bacillus subtilis in a time-dependent manner and, to a lesser extent, the more deformable and softer membrane of Escherichia coli. Moreover, the nanostructures in BC did not affect the viability of murine preosteoblasts. Using single-cell analysis, we demonstrate that indeed B. subtilis requires less force than E. coli to be penetrated by nanoprobes with dimensions comparable to those of the nanostructured BC, providing the first direct experimental evidence validating a mechanical model of membrane rupture via a tension-induced mechanism within the activation energy theory. Our findings bridge the gap between mechano-bactericidal surfaces and low-dimensional materials, including single-walled carbon nanotubes and graphene nanosheets, in which a higher bactericidal activity toward Gram-positive bacteria has been extensively reported. Our results also demonstrate the ability to confer bactericidal properties to a hydrogel by only altering its topography at the nanoscale and contribute to a better understanding of the bacterial mechanobiology, which is fundamental for the rational design bactericidal topographies.
Keywords: AFM; bacterial mechanics; bactericidal nanostructures; cell wall; cellulose.