Biofilm Formation of Candida Albicans On Nanofiber Textured Surfaces

Introduction: Medical implants are susceptible to infection caused by microorganisms that adhere to the surface and form biofilms. Biofilms consist of extracellular polymeric substance which protects the embedded microbial cells from the antimicrobials. Due to the high cost of surgical replacements and ineffectiveness of long-term antibiotic as treatments, a new method is needed. The new research direction for anti-biofilm surfaces have been prompted by the anti-fouling topographies of marine organisms. Our research involves interaction between microorganism and bio-inspired submicron topographic features on surface. In the work presented here, we investigate the effect of surface topography feature size and shear stress on biofilm formation and differentiation of Candida albicans, a model organism for fungal infection.

Materials and Methods:

Sample Fabrication- Nanofiber-textured model surfaces were fabricated by depositing a single layer of highly aligned and isodiametric polystyrene nanofibers on 45 mm² flat polystyrene substrates. The Spinneret based Tunable Engineered Parameters (STEP) nanofiber manufacturing platform developed by Nain et al. was utilized to make the polystyrene nanofibers (diameter = 750 nm; separation distance = 2000 nm – 3000 nm). Three different types of surfaces were manufactured for the experiment; smooth (control), single layer, and crisscross double layer. The scanning electron microscopy (SEM) images of single and double layer fiber surfaces are shown in Figure 1.

Biofilm Growth Assay- All samples were soaked in fetal bovine serum (FBS) overnight prior to the biofilm growth assay in order to condition the surface to mimic the natural environment of the microorganism. Candida albicans (C. albicans) SC5314, a model human fungal pathogen, was used in this study. Dynamic retention assay was conducted by placing the smooth (control) and the nanofiber-textured surfaces on the sample holder bars of a center for disease control (CDC) biofilm reactor and exposing them to a suspension of 1•10⁵ CFU/ml of C. albicans in yeast nitrogen base (YNB) media with 50 mM dextrose. After 24 hours of growth at 37°Æ C and 80 RPM (1.6 dyne/cm²), the cells are detached from the surfaces and counted using serial dilution and standard plate count method.

Results and Discussion: Our preliminary experimental results shown in Figure 2 suggest that the minimum experimental adhesion density occurs for samples with single layer nanofiber texture. When compared to the smooth surface, the adhesion density for these samples is reduced by approximately 24%.  Unlike the single layer surface, the double layer nanofiber surface increased the cell adhesion by 170% when compared to the smooth surface.

 Conclusions: Our results indicate that a highly aligned single layer of nanofiber texture significantly delays biofilm formation of C. albicans, and is therefore a potentially effective way to minimize the pathogenic effect of this organism. The shear stress of common implant devices that are affected by C. albicans are shown in Table 1. Our current and future experiments are focused on conducting pathogenicity assays and optimizing the nanofiber texture parameters for maximum delay in biofilm formation under different physiologically relevant shear stresses.

 

Figure 1. Scanning electron microscopy images of A- single layer fibrous surface and B- double layer fibrous surface is shown. The double layer surface is manufactured by repeating the STEP method on a single layer fibrous surface in a perpendicular direction. The scale bar represents A- 10mm, B- 50mm.

 

Figure 2. The average cell density (cells per squared mm) is shown for each type of surface. The single layer fiber coated surface shows approximately 24% reduction in cell density when compared to the bare (control) surface. The crisscross double layer fiber coated surface, unlike single layer, showed increase in adhesion by approximately 170%.

 

Table 1. Implant devices affected by yeast infections

Device Physiological shear stress (dyne/cm2) Corresponding rotational velocity (RPM) Oral environment 0.6 8 Pacemaker 25 322 Catheter 200 2580 Artificial heart valve 1000 12900