The Effects of Subcellular Nanograting Geometry on the Formation and Growth of Bacterial Biofilms

Biofilm formation by bacteria protects them against environmental stresses such as desiccation, shear forces and antimicrobial agents, making them much harder to remove and increasing their virulence and persistence in industrial water systems and biomedical equipment. One promising method of disrupting biofilm formation and growth is to employ passive surface structures to inhibit bacterial adhesion and aggregation. However, most studies thus far have mainly focused on the early stages of biofilm formation and it is unclear if the influence of surface topography in the early phase will propagate to later stages. Here, we attempt to address this with an investigation into the biofilm formation of Pseudomonas aeruginosa on 25 different nanograting geometries, with dimensions that were systematically varied from subcellular to cellular sizes. The biofilms were characterized from the exponential growth phase to the decline phase, in intervals of 24 H over 4 days, using confocal scanning laser microscopy. Comparing the maximum volume of biofilm formed on each surface over 96 H, it was found that approximately 1/3 of the nanograting geometries exhibited 72 ± 16 % lower biovolume density than a flat surface. Bacteria on these nanogratings were also observed to form 40 ± 11 % smaller microcolonies that were 17 ± 6 % less compact than that found on the control surface. The majority of these nanogratings had deep trenches (i.e. depth ≥ 70% of the cell diameter). Furthermore, P. aeruginosa cells were observed to multiply at approximately twice the rate on almost all the nanogratings compared to flat surfaces, but these cell populations also began to decline 24 H earlier than those on a flat surface. Using available literature on P. aeruginosa, a qualitative model was put forth, attributing the results to increased cell motility, decreased exopolysaccharide formation and disrupted psl adhesin/signal trails on nanogratings. These factors, together, led to the net effects of reduced attachment, increased scattering of cells and rapid decline of the biofilms on nanogratings. The insights derived from this study suggest that passive surface geometries can be designed and optimized to successfully control/inhibit biofilm formation and growth.