Staphylococcus aureus Behavior on Artificial Surfaces Mimicking Bone Environment

Abstract

Infections, which interfere with bone regeneration, may be a critical issue to consider during the development of biomimetic material. Calcium phosphate (CaP) and type I collagen substrates, both suitable for bone-regeneration dedicated scaffolds, may favor bacterial adhesion. Staphylococcus aureus possesses adhesins that allow binding to CaP or collagen. After their adhesion, bacteria may develop structures highly tolerant to immune system attacks or antibiotic treatments: the biofilms. Thus, the choice of material used for scaffolds intended for bone sites is essential to provide devices with the ability to prevent bone and joint infections by limiting bacterial adhesion. In this study, we compared the adhesion of three different S. aureus strains (CIP 53.154, SH1000, and USA300) on collagen- and CaP-coating. Our objective was to evaluate the capacity of bacteria to adhere to these different bone-mimicking coated supports to better control the risk of infection. The three strains were able to adhere to CaP and collagen. The visible matrix components were more important on CaP- than on collagen-coating. However, this difference was not reflected in biofilm gene expression for which no change was observed between the two tested surfaces. Another objective was to evaluate these bone-mimicking coatings for the development of an in vitro model. Thus, CaP, collagen-coatings, and the titanium-mimicking prosthesis were simultaneously tested in the same bacterial culture. No significant differences were found compared to adhesion on surfaces independently tested. In conclusion, these coatings used as bone substitutes can easily be colonized by bacteria, especially CaP-coating, and must be used with an addition of antimicrobial molecules or strategies to avoid bacterial biofilm development.

Keywords: Staphylococcus aureus; adhesion; biofilm; bone substitutes.

Figure 1

S. aureus adhesion on glass coated with collagen or calcium phosphate. (A) Results represent the number of viable adherent bacteria per cm² for CIP 53.154, SH1000, and USA300 on different supports, n = 4 to 8 (green dots represent independent biological replicates). Wilcoxon–Mann–Whitney test **, p < 0.01. (B) Representative images of scanning electron microscopy acquisitions (magnification ×1500 and ×15,000). Red arrows show the fibrous matrix; blue arrows show pili-like structures. The scale bars indicate 1 µm, n = 2.

Figure 2

Characterization of S. aureus biofilms on different supports. (A) Volumes of fluorescent staining with repartition of SYTO^TM 9 and PI staining. The graph shows the fluorescence volumes overlapping on top of each other. (B) Percentage of SYPRO^® Ruby (purple, protein), WGA (blue, PIA), and TOTO^TM-3 (orange, extracellular DNA) acquired by confocal microscopy on glass coated in calcium phosphate. (C) Representative 3D views of matrix biofilm composition on glass coated in calcium phosphate. Scale bar indicates 50 µm, n = 2.

Figure 3

S. aureus gene relative expression on different surfaces. Results represent relative mRNA expressions of bacteria within biofilm vs. planktonic bacteria. Expression of stress-related genes for CIP 53.154 (A), SH1000 (B), and USA300 (C). Expression of genes related to biofilm formation for CIP 53.154 (D), SH1000 (E), and USA300 (F). Baseline = 1; n = 2 to 3 (green dots represent independent biological replicates).

Figure 4

S. aureus adhesion in the simultaneous presence of different coatings. Results represent the number of viable adherent bacteria per cm² in presence of both supports: titanium coated with fibronectin, glass coated with collagen, and glass coated with calcium phosphate. (A) CIP 53.154, (B) SH1000, (C) USA300, n = 2 to 5 (green dots represent independent biological replicates). Wilcoxon–Mann–Whitney test * p < 0.05, ** p < 0.01.