Human Osteoblast-Conditioned Media Can Influence Staphylococcus aureus Biofilm Formation

Abstract

Osteoblasts are bone-forming and highly active cells participating in bone homeostasis. In the case of osteomyelitis and more specifically prosthetic joint infections (PJI) for which Staphylococcus aureus (S. aureus) is mainly involved, the interaction between osteoblasts and S. aureus results in impaired bone homeostasis. If, so far, most of the studies of osteoblasts and S. aureus interactions were focused on osteoblast response following direct interactions with co-culture and/or internalization models, less is known about the effect of osteoblast factors on S. aureus biofilm formation. In the present study, we investigated the effect of human osteoblast culture supernatant on methicillin sensitive S. aureus (MSSA) SH1000 and methicillin resistant S. aureus (MRSA) USA300. Firstly, Saos-2 cell line was incubated with either medium containing TNF-α to mimic the inflammatory periprosthetic environment or with regular medium. Biofilm biomass was slightly increased for both strains in the presence of culture supernatant collected from Saos-2 cells, stimulated or not with TNF-α. In such conditions, SH1000 was able to develop microcolonies, suggesting a rearrangement in biofilm organization. However, the biofilm matrix and regulation of genes dedicated to biofilm formation were not substantially changed. Secondly, culture supernatant obtained from primary osteoblast culture induced varied response from SH1000 strain depending on the different donors tested, whereas USA300 was only slightly affected. This suggested that the sensitivity to bone cell secretions is strain dependent. Our results have shown the impact of osteoblast secretions on bacteria and further identification of involved factors will help to manage PJI.

Keywords: MRSA; MSSA; biofilms; osteoblast secretion; prosthetic joint infection.

Figure 1

Impact of Saos-2 culture supernatant on S. aureus planktonic growth and biofilm biomass. Results represent planktonic growth (A), percentage of biofilm biomass relative to control media (B), and number of adherent bacteria (C) for SH1000 (white histogram) and USA300 (grey histogram). Control media (50% [DMEM + 10% FBS] and 50% MM), SN 50 (50% Saos-2 culture supernatant and 50% MM), Control media + TNF-α (50% [DMEM + 10% FBS + 20 ng/mL TNF-α] and 50% MM), or SN 50 + TNF-α (50% [Saos-2 challenged with TNF-α culture supernatant + 20 ng/mL] and 50% MM). n = 7 to 18. * = p < 0.05.

Figure 2

Impact of Saos-2 culture supernatant on S. aureus biofilm organization. Yellow arrows indicate aggregates. Control media (50% [DMEM + 10% FBS] and 50% MM), SN 50 (50% Saos-2 culture supernatant and 50% MM), Control media + TNF-α (50% [DMEM + 10% FBS + 20 ng/mL TNF-α] and 50% MM), or SN 50 + TNF-α (50% [Saos-2 challenged with TNF-α culture supernatant + 20 ng/mL] and 50% MM). One representative image of each technical duplicate was acquired for each condition of each batch (for one biological replicate). The scale bars indicate 50 µm.

Figure 3

Impact of Saos-2 culture supernatant on S. aureus biofilm shape and matrix. One representative image for two batches is shown. Control media (50% [DMEM + 10% FBS] and 50% MM), SN 50 (50% Saos-2 culture supernatant and 50% MM), Control media + TNF-α (50% [DMEM + 10% FBS + 20 ng/mL TNF-α] and 50% MM), or SN 50 + TNF-α (50% [Saos-2 challenged with TNF-α culture supernatant + 20 ng/mL] and 50% MM). Orange arrows indicate bacterial matrix. Yellow arrows indicate microvesicles-like structure. One coverslip was imaged for two different batches of Saos-2 culture supernatant and the corresponding control media. The scale bars indicate 1 µm.

Figure 4

Impact of Saos-2 culture supernatant on proportion of dead cells and biofilm matrix components of S. aureus. (A) Proportion of dead or damaged bacteria; volume measurement of SYTO^TM 9 and propidium iodide were acquired by CLSM. (B,C) Volume measurement of SYTO^TM 9 (green histograms, live bacteria), SYPRO^® Ruby (purple, protein), WGA (blue, PIA), and TOTO^TM-3 (orange, extracellular DNA) acquired by CLSM for SH1000 and USA300 biofilms, respectively. (D) Representative reconstructions of S. aureus biofilms are shown. Above each column is indicated the labelled component and the corresponding colour. The scale bars indicate 50 µm. Control media (50% [DMEM + 10% FBS] and 50% MM), SN 50 (50% Saos-2 culture supernatant and 50% MM), Control media + TNF-α (50% [DMEM + 10% FBS + 20 ng/mL TNF-α] and 50% MM), or SN 50 + TNF-α (50% [Saos-2 challenged with TNF-α culture supernatant + 20 ng/mL] and 50% MM). n = 2 with three acquisitions per coverslip; dots represent technical replicates for this figure.

Figure 5

Influence of Saos-2 culture supernatant on biofilm-embedded S. aureus gene regulation. Results represent relative mRNA expressions of bacteria within biofilms for SH1000 and USA300. Control media (Yellow histograms = 50% [DMEM + 10% FBS] and 50% MM), SN 50 (Orange histograms = 50% Saos-2 culture supernatant and 50% MM), Control media + TNF-α (Blue histograms = 50% [DMEM + 10% FBS + 20 ng/mL TNF-α] and 50% MM), or SN 50 + TNF-α (Green histograms = 50% [Saos-2 challenged with TNF-α culture supernatant + 20 ng/mL] and 50% MM). Experiments were performed nine (SH1000) and six (USA300) independent times. n = 6 to 9, * p < 0.05.

Figure 6

Impact of primary osteoblast culture supernatant on S. aureus planktonic growth and biofilm biomass. Results represent planktonic growth (A), biofilm biomass (B), and number of live adherent bacteria (C) for SH1000 (white histogram) and USA300 (grey histogram). Control media (50% [DMEM + 10% FBS] and 50% MM), SN 50 (625, 626, 634, and 636) (50% primary human osteoblast culture supernatant and 50% MM), or All SN 50 (addition of results of SN 50 (625, 626, 634, and 636)). n = 2 to 3.

Figure 7

Impact of primary osteoblast culture supernatant on S. aureus biofilm shape and matrix. Control media (50% [DMEM + 10% FBS] and 50% MM), SN 50 (626) (50% primary human osteoblast culture supernatant and 50% MM), or All SN 50 (addition of results of SN 50 (625, 626, 634 and 636)). Red arrow indicates agglomerated dead bacteria. Two coverslips were imaged for each donor of primary osteoblast culture supernatant and the corresponding control media. The scale bars indicate 1 µm.