Cutibacterium acnes Biofilm Study during Bone Cells Interaction

Abstract

Cutibacterium acnes is an opportunistic pathogen involved in Bone and Prosthesis Infections (BPIs). In this study, we observed the behavior of commensal and BPI C. acnes strains in the bone environment through bacterial internalization by osteoblast-like cells and biofilm formation. For the commensal strains, less than 1% of the bacteria were internalized; among them, about 32.7 ± 3.9% persisted intracellularly for up to 48 h. C. acnes infection seems to have no cytotoxic effect on bone cells as detected by LDH assay. Interestingly, commensal C. acnes showed a significant increase in biofilm formation after osteoblast-like internalization for 50% of the strains (2.8-fold increase). This phenomenon is exacerbated on a titanium support, a material used for medical devices. For the BPI clinical strains, we did not notice any increase in biofilm formation after internalization despite a similar internalization rate by the osteoblast-like cells. Furthermore, fluorescent staining revealed more live bacteria within the biofilm after osteoblast-like cell interaction, for all strains (BPIs and commensal). The genomic study did not reveal any link between their clinical origin and phylotype. In conclusion, we have shown for the first time the possible influence of internalization by osteoblast-like cells on commensal C. acnes.

Keywords: Cutibacterium acnes; biofilms; host–pathogen interactions; internalization; joint infections.

Figure 1

Behaviors of the commensal C. acnes strains. (a) Biofilm forming capacity by crystal violet quantification before (full bars) and after internalization (hatched bars) by osteoblast-like cells ($; p < 0.05 compared to initial biofilm quantification); (b) internalization rate (%) by osteoblast-like cells (#; p < 0.05; different to all other strains).

Figure 2

The living bacterial proportion in the commensal C. acnes strain biofilms. Repartition of Syto9 (green bars) and PI (red bars) staining within biofilms formed by the commensal C. acnes strains before (a) and after internalization (b) by osteoblast-like cells. Acquisition of images by fluorescent microscopy and calculation by Image J software.

Figure 3

Fate of the internalized commensal C. acnes. (a) Stability of biofilm increase for intracellular C. acnes strains. Each time, red histograms represent the quantification of the initial biofilm formed before internalization. Other histograms have a number corresponding to the number of cultures after internalization ($, p < 0.05); (b) percentage of viable intracellular and extracellular bacteria following 48 h post-infection; 100% corresponding to intracellular C. acnes strains at 3 h of interaction (red dot line); (c) LDH release measurement normalized on cells without bacteria, in supernatant after 48 h of C. acnes/osteoblast-like cells interaction; 100% corresponding to the measurement of cells without bacteria.

Figure 4

Behaviors of the C. acnes BPI strains and comparison with the commensal strains. (a) Biofilm forming capacity by crystal violet quantification without interaction (full bars) and after internalization (hatched bars) by osteoblast-like cells by crystal violet quantification $; p < 0.05 compared to initial biofilm quantification; (b) internalization rate (%) by osteoblast-like cells; (c) comparison commensal and BPI strains compiled results: behaviors of C. acnes commensal “C” strains and strains isolated from BPIs: biofilm quantification by crystal violet assay (left graph); internalization rate (%) by osteoblast-like cells (middle graph); and biofilm quantification after internalization (right graph).

Figure 5

The living bacterial proportion in the BPI C. acnes strain biofilms and comparison with the commensal strain biofilms. Repartition of Syto9 (green bars) and PI (red bars) staining within biofilm formed by the C. acnes strains isolated from BPIs before (a) and after internalization (b) by osteoblast-like cells; (c) comparison of commensal and BPI strains: live dead proportion quantification within biofilm of C. acnes commensal “C” strains and isolated from BPIs. Images acquisition by fluorescent microscopy and calculation by Image J software.

Figure 6

Comparison of the commensal strains’ adhesion (compiled results of the four strains) between plastic and titanium surfaces before and after internalization by osteoblast-like cells. Biofilm-forming capacity by crystal violet quantification ($, significant difference with formed biofilm before internalization, p < 0.01).