Antibacterial effects of nanopillar surfaces are mediated by cell impedance, penetration and induction of oxidative stress

Abstract

Some insects, such as dragonflies, have evolved nanoprotrusions on their wings that rupture bacteria on contact. This has inspired the design of antibacterial implant surfaces with insect-wing mimetic nanopillars made of synthetic materials. Here, we characterise the physiological and morphological effects of mimetic titanium nanopillars on bacteria. The nanopillars induce deformation and penetration of the Gram-positive and Gram-negative bacterial cell envelope, but do not rupture or lyse bacteria. They can also inhibit bacterial cell division, and trigger production of reactive oxygen species and increased abundance of oxidative stress proteins. Our results indicate that nanopillars' antibacterial activities may be mediated by oxidative stress, and do not necessarily require bacterial lysis.

Fig. 1. Characterisation of TiO₂ nanopillars.

Scanning electron micrographs of TiO₂ nanopillar surface NW-850-5 from a top view a and 40° stage tilt b. NW-850-5 was generated at 850 °C for a duration of 5 min. Scale bars: 1 µm. Micrographs are representative of three thermal oxidation batches (n = 3), each batch containing 25 surfaces.

Fig. 2. Determining bacterial morphology on nanopillar surface NW-850-5 using SEM.

Scanning electron micrographs of Gram-positive (S. aureus) or Gram-negative (E. coli and K. pneumoniae) bacteria following 3-h static incubation on flat titanium alloy (control) and TiO₂ nanopillar surface NW-850-5. White arrows highlight regions of nanopillar-induced envelope deformation. Micrographs are representative of three surfaces (n = 3), with each surface visualised in at least three different areas.

Fig. 3. Nanopillar-induced envelope deformation and penetration in Gram-negative and Gram-positive bacteria.

TEM micrographs of S. aureus, E. coli and K. pneumoniae following incubation on control surfaces and NW-850-5 for 3 h. Regions where nanopillars had deformed or penetrated the bacterial envelope have been marked by white arrows. Cross section thickness: 70 nm. Micrographs are representative of three independent surfaces (n = 3).

Fig. 4. Tomogram reconstruction of nanopillar-induced envelope deformation in E. coli.

A tilt series was acquired of E. coli a, where a single nanopillar appeared to be penetrating the envelope (dashed white circle). Analysis of tomographic slices from the start b, middle c and point of nanopillar contact d revealed that the nanopillar had clearly indented the envelope but had not pierced through. The interaction was therefore categorised as nanopillar-induced envelope deformation, localised at the point of nanopillar contact. This interaction is highlighted in the 3D reconstruction shown in e. Electron tomography was performed on three E. coli cells interacting with TiO₂ nanopillars, these were acquired in two tilt series.

Fig. 5. Tomogram reconstruction of nanopillar-induced envelope penetration in K. pneumoniae.

A tilt series was acquired of K. pneumoniae under bright-field TEM a, where multiple nanopillars appeared to be penetrating the envelope (1–6). Analysis of nanopillar tip coordinates in x, y and z revealed that nanopillars 1–4 and 6 had penetrated the envelope, while nanopillar 5 was only deforming the cell. A 3D reconstruction of the tomogram revealed all nanopillars had penetrated the bacterial envelope by at least 100 nm b. The dark bands seen within nanopillars are bend contours; these spatial contrasts arise from local bending or deformation of nanopillars. Electron tomography was performed on five K. pneumoniae cells interacting with TiO₂ nanopillars, these were acquired in four tilt series.

Fig. 6. 3D FIB-SEM reconstruction of E. coli.

Automated FIB-SEM cross sectional analysis was performed on an E. coli cell a that was pinned between two nanopillars b, c. The focused ion beam produced 120 cross sections (30 nm each) that were imaged, and reconstructed in Avizo d, e. Analysis of E. coli cross section #30 showed that nanopillar 1 had forced E. coli into nanopillar 2, resulting in a 189 nm reduction in cell width f. While nanopillar 1 had induced significant envelope deformation, no evidence of envelope penetration was found, and the width of E. coli cell quickly restored to normal either side of the contact point, indicating that the cell had not lost turgor pressure g, h. FIB-SEM analysis was performed on four E. coli cells interacting with TiO₂ nanopillars.

Fig. 7. 3D FIB-SEM reconstruction of S. aureus.

Automated FIB-SEM cross sectional analysis was performed on an S. aureus cell interacting with three nanopillars a, b. The focused ion beam produced 57 cross sections (30 nm each) that were imaged, and reconstructed in Avizo c. Analysis of S. aureus cross section #40 showed the tip of nanopillar 2 located ~50 nm into the cell, providing confirmation of cell wall and inner envelope penetration d, e. FIB-SEM analysis was performed on one S. aureus cell interacting with TiO₂ nanopillars.

Fig. 8. Determining bacterial viability on nanopillar surfaces.

Luminescence signals and CFU of S. aureus a, E. coli b and K. pneumoniae c incubated on control or nanopillar TiO₂ surfaces for up to 10 h, as determined by real-time or endpoint assays, respectively. Values are given as mean ± standard deviation and individual data points for each mean are shown. *** indicates P ≤ 0.001 relative to control, as determined by one-way ANOVA and Tukey HSD post hoc test. BacTiter-Glo experiments n = 3; RealTime-Glo experiments n = 6 (S. aureus), n = 3 (E. coli) and n = 4 (K. pneumoniae). Total surface CFU were determined from disc areas of 0.64 cm². Exact P-values are indicated for each significant time point.

Fig. 9. S. aureus and E. coli proteomic response to nanopillar surface NW-850-5.

The predicated functional partners among S. aureus a and E. coli b DEPs are shown, where individual proteins are represented in circles (nodes), and protein–protein interactions (PPI) are represented by the connecting lines (edges). DEPs have been coloured according to the log2 fold change in protein abundance (pink = decreased abundance and blue = increased abundance), and node colour indicates the confidence of supporting interaction evidence (0.15 = low confidence, 0.40 = medium confidence, 0.70 = high confidence and 0.9 = highest confidence). For S. aureus, 60 DEPs displayed at least one interaction at a confidence level between 0.46 and 1.00, and the PPI enrichment P-value was 1.0e−16. For E. coli, six proteins had at least one interaction at a medium to high confidence and had a PPI enrichment P-value of 9.54e−06. PPI enrichment P-values were determined using a hypergeometric test and corrected for multiple testing using the method of Benjamini and Hochber. Data are representative of one experimental replicate, performed in triplicate.

Fig. 10. Determination of ROS production in response to nanopillar surfaces.

S. aureus or E. coli were incubated for 24 h on control or NW-850-5 surfaces under the same growth conditions used for proteomic analysis. Following 24-h incubation, the levels of H₂O₂ were determined by ROS-Glo a. Values given are mean ± standard deviation and individual data points for each mean are shown. ** indicates P ≤ 0.01 relative to control for S. aureus (P = 0.003) and E. coli (P = 0.01), as determined by a two-sided Student’s t-test; n = 3 performed in duplicate. Representative SEM micrographs of S. aureus b, c and E. coli d, e incubated on control or NW-850-5 are shown. Insets show higher magnification SEM images of each surface.