Bone Environment Influences Irreversible Adhesion of a Methicillin-Susceptible Staphylococcus aureus Strain

Abstract

Prosthesis and joint infections are an important threat in public health, especially due to the development of bacterial biofilms and their high resistance to antimicrobials. Biofilm-associated infections increase mortality and morbidity rates as well as hospitalization costs. Prevention is the best strategy for this serious issue, so there is an urgent need to understand the signals that could induce irreversible bacterial adhesion on a prosthesis. In this context, we investigated the influence of the bone environment on surface adhesion by a methicillin-susceptible Staphylococcus aureus strain. Using static and dynamic biofilm models, we tested various bone environment factors and showed that the presence of Mg²⁺, lack of oxygen, and starvation each increased bacterial adhesion. It was observed that human osteoblast-like cell culture supernatants, which contain secreted components that would be found in the bone environment, increased bacterial adhesion capacity by 2-fold (p = 0.015) compared to the medium control. Moreover, supernatants from osteoblast-like cells stimulated with TNF-α to mimic inflammatory conditions increased bacterial adhesion by almost 5-fold (p = 0.003) without impacting on the overall biomass. Interestingly, the effect of osteoblast-like cell supernatants on bacterial adhesion could be counteracted by the activity of synthetic antibiofilm peptides. Overall, the results of this study demonstrate that factors within the bone environment and products of osteoblast-like cells directly influence S. aureus adhesion and could contribute to biofilm initiation on bone and/or prosthetics implants.

Keywords: antibiofilm peptides; bacterial starvation; biofilm; bone and joint infections; bone environment.

FIGURE 1

Impact of Ca²⁺ on S. aureus biofilm formation. Planktonic growth normalized on control (A) and fold-increase of live adhered cells (B). Ca²⁺ × 1 and ×2 serological concentration (=1.2 mM). (n = 9). ^∗Statistically significantly different from control (p < 0.05). Fluorescence microscopy (C) with live (green color)/dead (red color) staining. Scale bar = 20 μm. Scanning Electronic Microscopy (SEM) (D). Main panels: scale bar = 1 μm and insert panels showing the homogeneity on a wide field: scale bar = 10 μm.

FIGURE 2

Impact of Mg²⁺ on S. aureus biofilm formation. Planktonic growth normalized to control (A); biofilm biomass quantified by crystal violet staining (B) and fold-increase of live adhered cells (C). Mg²⁺ × 2 (control) and ×20 serological concentration (=1 mM). (n = 9). ^∗Statistically significantly different from control (p < 0.05). Fluorescence microscopy (D) with live (green color)/dead (red color) staining. Scale bar = 20 μm. Scanning Electronic Microscopy (SEM) (E). Main panels: scale bar = 1 μm and insert panels showing the homogeneity on a wide field: scale bar = 10 μm.

FIGURE 3

Hypoxia increased S. aureus biomass biofilm. Planktonic growth normalized on control (A) and biofilm biomass quantified by crystal violet staining (B) under aerobic and anaerobic conditions. (n = 9). Scanning Electronic Microscopy (SEM) (C). Main panels: scale bar = 1 μm and insert panels showing the homogeneity on a wide field: scale bar = 10 μm.

FIGURE 4

Glucose and amino acid starvation increased S. aureus biofilm formation when normalized on planktonic growth. Planktonic growth normalized on control (A) fold-increase of biofilm biomass quantified by crystal violet staining (B) and fold-increase of live adhered cells (C), fold-increase of biomass fraction on planktonic growth (D) and fold-increase of adherent cells fraction on planktonic growth (E). Fluorescence microscopy (F) with live (green color)/dead (red color) staining. Scale bar = 40 μm. no iron, without iron; no CAA, without casamino acids; no Glu, without glucose. (n = 9). ^∗Statistically significantly different from control (p < 0.05).

FIGURE 5

Diluted supernatants of osteoblast culture influenced S. aureus biofilm formation. Planktonic growth normalized on control (A) biomass biofilm quantified by crystal violet staining (B) and fold-increase of live adhered cells (C). Control media = 50% DMEM + 10% FCS and 50% of minimal medium; SN 50 = culture with 50% of osteoblast culture supernatants and 50% of minimal media. (n = 9).

FIGURE 6

Diluted supernatants of osteoblast culture stimulated with TNF-α influenced S. aureus biofilm formation. Planktonic growth normalized on control (A) fold-increase of biomass biofilm quantified by crystal violet staining (B) and fold-increase of live adhered cells (C). Control medium + TNF-α = 50% DMEM, 10% FCS, 20 ng/ml TNF-α, 50% minimal medium; SN 50 + TNF-α = culture with 50% of osteoblast culture supernatants exposed to 20 ng/ml of TNF-α and 50% of minimal media. (n = 9).

FIGURE 7

Dynamic model confirmed the impact of osteoblast culture supernatants on S. aureus biofilm formation. Impact of osteoblast culture on biofilm was tested into the flow-through medium of the flow cell apparatus for 24 h. Each panel shows 3D reconstruction by Imaris software after acquisition in confocal microscopy with live (green color)/dead (red color) staining. Bottom panel represents the quantitative data calculated Imaris software based on acquired images. Control media, 50% DMEM + 10% FCS and 50% of minimal media; SN 50, culture with 50% of osteoblast culture supernatants and 50% of minimal media; Control media + TNF-α = 50% DMEM, 10% FCS, 20 ng/ml TNF-α, 50% minimal media; SN 50+TNF-α, culture with 50% of osteoblast culture supernatants exposed to 20 ng/ml of TNF-α and 50% of minimal media.

FIGURE 8

Synthetic peptides (1018, 1002, 3002, and DJK-5, 5 μg/ml) inhibited osteoblast products effect on S. aureus biofilm formation. Fold-increase of biofilm biomass quantified by crystal violet staining (A,C) and fold-increase of live adhered cells (B,D) under normal conditions of osteoblast-like culture (A,B) and TNF-α stimulated osteoblast-like culture (C,D). SN cell., increase of biofilm formation in contact of osteoblast supernatants. SN cell. TNF-α, increase of biofilm formation in contact of osteoblast supernatants culture after TNF-α exposition. (n = 9). ^∗Statistically significantly different from biofilm formed in the presence of SN cell or SN cell. TNF-α (p < 0.05). #Statistically significantly different from basal level of biofilm formation. Dotted line = basal level of biofilm formed in the absence of SN cell or SN cell. TNF-α.