Bactericidal Efficacy of Nanostructured Surfaces Increases under Flow Conditions

Abstract

Bacterial colonization on solid surfaces creates enormous problems across various industries causing billions of dollars' worth of economic damages and costing human lives. Biomimicking nanostructured surfaces have demonstrated a promising future in mitigating bacterial colonization and related issues. The importance of this non-chemical method has been elevated due to bacterial evolvement into antibiotic and antiseptic-resistant strains. However, bacterial attachment and viability on nanostructured surfaces under fluid flow conditions has not been investigated thoroughly. In this study, attachment and viability of Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) on a model nanostructured surface were studied under fluid flow conditions. A wide range of flow rates resulting in a broad spectrum of fluid wall shear stress on a nanostructured surface representing various application conditions were experimentally investigated. The bacterial suspension was pumped through a custom-designed microfluidic device (MFD) that contains a sterile Ti-6Al-4V substrate. The surface of the titanium substrate was modified using a hydrothermal synthesis process to fabricate the nanowire structure on the surface. The results of the current study show that the fluid flow significantly reduces bacterial adhesion onto nanostructured surfaces and significantly reduces the viability of adherent cells. Interestingly, the bactericidal efficacy of the nanostructured surface was increased under the flow by ∼1.5-fold against P. aeruginosa and ∼3-fold against S. aureus under static conditions. The bactericidal efficacy had no dependency on the fluid wall shear stress level. However, trends in the dead-cell count with the fluid wall shear were slightly different between the two species. These findings will be highly useful in developing and optimizing nanostructures in the laboratory as well as translating them into successful industrial applications. These findings may be used to develop antibacterial surfaces on biomedical equipment such as catheters and vascular stents or industrial applications such as ship hulls and pipelines where bacterial colonization is a great challenge.

Figure 1

Simplified fluid domain in the microfluidic device based on the parallel-plate flow cell (PPFC) principle.

Figure 2

(A) 3D model of the assembled microfluidic device (MFD) with the front plate made transparent. (B) Cross-sectional plan view (horizontal plane through mid-height of the MFD). (C) Isolated fluid volume with the two substrates (nanostructured on one side and the flat substrate on the other side). (D) Photograph of the actual MFD with outer dimensions. (E) Schematic illustration of the experimental setup. Components: [1] Nanostructured surface, [2] flat (control) surface, [3] front plate of the MFD, [4] back plate of the MFD, [5] bacterial suspension reservoir, [6] MFD connected using isoprene tubing, [7] peristaltic pump, [8] waste reservoir, and [9] rubber O-rings.

Figure 3

Scanning electron microscope (SEM) images of (A) P. aeruginosa cells on the flat Ti-6Al-4V titanium alloy surface under no-flow conditions, (B) P. aeruginosa cells on the nanostructured Ti-6Al-4 V titanium alloy surface under no-flow conditions, (C) P. aeruginosa cells on the nanostructured titanium surface after flowing the bacterial suspension under 10.00 Pa fluid wall shear stress, (D) S. aureus cells on the smooth titanium surface under no-flow conditions, (E) S. aureus cells on the nanostructured titanium surface under no-flow conditions, and (F) S. aureus cells on the nanostructured titanium surface after flowing the bacterial suspension under 10.00 Pa fluid wall shear stress [scale bar: 1 μm].

Figure 4

Fluorescence images of P. aeruginosa and S. aureus cells on nanostructured and control surfaces by staining with SYTO9 and propidium iodide under different fluid wall shear stress levels. Dead cells are shown in red color and live cells are shown in green color. The fluid wall shear stress is computed for the centroid of substrate [scale bar: 100 μm].

Figure 5

Total cell adhesions of (A) P. aeruginosa on the nanostructured surface, (B) P. aeruginosa on the flat surface, (C) S. aureus on the nanostructured surface, and (D) S. aureus on the flat surface under varying flow rates. Number of live and dead cells on treated and untreated surfaces under different flow conditions were quantified to compute the total cell adhesion on the surface. The cells were stained with a mixture of SYTO9 and PI then imaged using a fluorescence microscope with FITC and CY3 filters. Cells were quantified by counting pixels of each image above the threshold level. Data are the mean of 45 images of three independent experiments ± the standard error of means. * shows statistical significance with Student’s t test. ns: P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, and ****: P ≤ 0.0001.

Figure 6

Dead counts of (A) P. aeruginosa on the nanostructured surface, (B) P. aeruginosa on the flat surface, (C) S. aureus on the nanostructured surface, and (D) S. aureus on the flat surface under varying flow rates. The number of dead cells on treated and untreated surfaces under different flow conditions was quantified. Live counts of (E) P. aeruginosa on the nanostructured surface, (F) P. aeruginosa on the flat surface, (G) S. aureus on the nanostructured surface, and (H) S. aureus on the flat surface under varying flow rates. The cells were stained with a mixture of SYTO9 and PI then imaged using a fluorescence microscope with FITC and CY3 filters. Cells were quantified by counting pixels of each image above the threshold level. Data are means of 45 images of three independent experiments ± standard error of means. * shows statistical significance with Student’s t test. ns: P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, and ****: P ≤ 0.0001.

Figure 7

Bactericidal efficacy of the nanostructured surface against P. aeruginosa and S. aureus species at varying fluid wall shear stress levels. The number of live and dead cells on treated and untreated surfaces under different flow conditions was quantified to compute the bactericidal efficacy of the surface. The cells were stained with a mixture of SYTO9 and PI then imaged using a fluorescence microscope with FITC and CY3 filters. Cells were quantified by counting pixels of each image above the threshold level. Data are means of 45 images of three independent experiments ± standard error of means.