Bacterial Adhesion and Biofilm Formation of Enterococcus faecalis on Zwitterionic Methylmethacrylat and Polysulfones

Abstract

Biofilm-associated implant infections represent a major challenge for healthcare systems around the world due to high patient burden and enormous costs incurred. Enterococcus faecalis (E. faecalis) is the most prevalent enterococcal species identified in biofilm-associated infections. The steadily growing areas of application of implants demand a solution for the control of bacterial infections. Therefore, the development of modified anti-microbial implant materials and the testing of the behavior of different relevant bacterial strains towards them display an indispensable task. Recently, we demonstrated an anti-microbial effect of zwitterionic modified silicone rubber (LSR) against Staphylococcus aureus. The aim of this study was to evaluate bacterial colonization and biofilm formation of another clinically relevant strain, E. faecalis, on this material in comparison to two of the most commonly used thermoplastic polyurethanes (TPUs) and other modified LSR surfaces. By generating growth curves, crystal violet, and fluorescence staining, as well as analyzing the expression of biofilm-associated genes, we demonstrated no anti-microbial activity of the investigated materials against E. faecalis. These results point to the fact that anti-microbial effects of novel implant materials do not always apply across the board to all bacterial strains.

Keywords: Enterococcus faecalis; biofilm; methylmethacrylat; polysulfone; zwitterion.

Figure 1

The effects of the investigated surface modifications of the materials on E. faecalis growth. (A) Growth behavior of E. faecalis (ATCC 29212) was assessed at 37°C with shaking at 150 rpm in liquid BHI medium on the different material disks. Growth was monitored hourly by measuring OD₆₀₀. Results are presented as averages + SEM of three independent experiments performed in duplicates. (B) Investigations of inhibition of bacterial growth by substances released by the material were performed using disk diffusion test. 1 x 10⁸ CFU/ml of the bacterial culture were seeded on agar plates. The different material disks were placed in the middle of the plates and incubated at 37°C for 6 h Representative images of the plates with the different materials are shown.

Figure 2

Viability and biofilm development of E. faecalis at two different time points. (A) After 6 and 24 h, bacterial viability was determined using LIVE/DEAD™ BacLight™ Bacterial Viability Kit two-color fluorescence assay. Fluorescence intensity was measured using a microplate reader (FLUOstar Omega, BMG Labtech) to calculate the percentage of living cells using the adjusted dye ratio equation as follows: % living cells = (100 x SYTO^®/PI)/(1+ SYTO^®/PI) in accordance to Ou et al. (2016). Results are presented as averages of three independent experiments with two technical replicates + SEM. (B–H) The fluorescence was visualized using a fluorescence microscope (Axio Scope.A1, Zeiss, Germany) equipped with a camera (AxioCam MRc, Zeiss, Jena, Germany). SYTO^® 9 green-fluorescent nucleic acid (SYTO^®) stain labels all bacteria in a population (live) and the red-fluorescent nucleic acid stain, propidium iodide (PI), penetrates only bacteria with damaged membranes (dead). The scale for all images shown is 50 µm.

Figure 3

The effects of the investigated surface modifications of the materials on E. faecalis biofilm formation. E. faecalis (1 x 10⁸ CFU/ml) was cultured on the different material discs and incubated at 37°C for 6 and 24 h. Biofilm formation was assessed by crystal violet staining. Results are presented as averages of three independent experiments + SEM. (A) 6 and (B) 24 h.

Figure 4

The mRNA expression levels of biofilm-associated genes of E. faecalis. E. faecalis (1 x 10⁸ CFU/ml) was cultured on the different material discs and incubated at 37°C for 6 and 24 h. Relative gene expression of (A) GelE, (B) ebpA, (C) efaA and (D) AS was determined by real-time PCR. Data are represented as mean + SEM of 6 independent experiments. Significant results are indicated as *p < 0.05.