Monitoring Bioluminescent Pseudomonas aeruginosa on Mechano-Bactericidal Zinc Oxide Nanopillars: Implications for Self-Cleaning Antibacterial Coatings

Mechano-bactericidal nanopillars represent a class of antibacterial surfaces that rely on nanoscale topographical features to inflict damage on attached bacteria. A potential application for mechano-bactericidal nanopillars is their use as self-cleaning antibacterial coatings for surfaces that are exposed to inadvertent splashes or cough droplets containing infectious bacteria. In this work, we prepared two types of zinc oxide nanopillars: nanopillars with an average width of 194 nm and an average spacing of 163 nm (referred to as low-surface-density nanopillars) and nanopillars with an average width of 88 nm and an average spacing of 91 nm (referred to as high-surface-density nanopillars). To assess their antibacterial capabilities, we deposited small aqueous droplets containing bioluminescent Pseudomonas aeruginosa onto the nanopillars and monitored the loss of the bioluminescence signal to quantify acute bacterial inactivation in real time. To investigate possible recovery of bacterial viability after acute inactivation, the nanopillars were submerged in nutrient-rich media and incubated for another 24 h, while the bioluminescence signal was monitored. Combining the results from bioluminescence monitoring with scanning electron microscopy, optical density measurements, and plate counting of colony-forming units after the recovery period, we found that nanopillars with an average width of 194 nm and an average spacing of 163 nm were indeed mechano-bactericidal and efficacy was dependent on the bacterial concentration within the droplets. Nanopillars with an average width of 88 nm and an average spacing of 91 nm were ineffective as bactericidal surfaces regardless of the bacterial concentration. This work further supports the use-case of mechano-bactericidal nanopillars as antibacterial coatings for dry surfaces and demonstrates bioluminescence monitoring as a promising tool to complement existing assessment techniques for mechano-bactericidal nanopillars as well as other antibacterial coatings.