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

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

Influence of the periprosthetic environment on Staphylococcus aureus biofilm formation

Les infections ostéo-articulaires liées à l’implantation d’une prothèse orthopédique sont des complications post-opératoires difficiles à traiter. Staphylococcus aureus (S. aureus) est la bactérie majoritairement impliquée dans ces infections et plus généralement dans les infections de l’os. Dans l’environnement périprothétique, différents réservoirs bactériens ont été mis en évidence, notamment le développement de biofilm qui sont des communautés bactériennes complexes et entourées d’une matrice sécrétée par les bactéries. Malgré différentes stratégies mêlant antibiothérapies et traitements chirurgicaux, le taux d’échec thérapeutique est élevé quand Staphylococcus aureus est impliqué. L’objectif de ce travail est d’identifier les paramètres de l’environnement périprothétique influençant le développement des biofilms de S. aureus et de caractériser le phénotype des biofilms formés selon ces conditions. Cela est primordiale pour développer de nouvelles stratégies thérapeutiques efficaces et ciblées. Ainsi, le manque de nutriments et d’oxygène, une forte concentration en magnésium, le sécrétome des ostéoblastes ainsi que la présence de surfaces de nature diverses (titane recouvert de fibronectine, phosphate de calcium, explants osseux ou os minéral de banque) ont démontré une influence sur le phénotype des biofilms formés par trois souches de S. aureus (deux souches sensibles à la méthicilline : CIP 53.154 et SH1000, et une souche résistante à la méthicilline : USA300). Ces résultats démontrent la forte influence de l’environnement infectieux sur le comportement des agents pathogènes, ainsi que l’hétérogénéité de ce comportement selon les souches étudiées. L’ensemble de cette étude a permis la conception d’un modèle d’IOAP in vitro pour mieux cribler de nouvelles stratégies thérapeutiques.Prosthetic joint infections are postoperative complications and difficult-to-treat infections. Staphylococcus aureus (S. aureus) is the bacterium mainly involved and more globally for bone infections. In the periprosthetic environment, different distinct reservoirs of bacteria were described, including biofilms, which are complex bacterial communities surrounded by a matrix. Despite antibiotic therapies and surgical management, the reinfection rate remains high, especially if S. aureus is involved. The aim of this thesis is to identify the conditions of the periprosthetic environment influencing the development of S. aureus biofilms and to analyse the phenotype of the biofilms in these conditions (biofilm biomass, number of biofilm-embedded bacteria, biofilm matrix, gene regulation). It is of utmost importance to develop more efficient strategies to combat biofilm-related infections. Thus, the lack of nutrients (amino acids and glucose) and the lack of oxygen, a high concentration of magnesium, the osteoblast secretome (from cell line Saos-2 and from primary osteoblast cultures) as well as the presence of different surfaces (titanium, fibronectin-coated titanium, calcium phosphate coating, and bone samples or mineral bone from bank) were found to influence the biofilm phenotype of three S. aureus strains (including two methicillin-susceptible strains: CIP 53.154, SH1000; and one methicillin-resistant strain: USA300). These results highlight the strong influence of the infectious environment over the pathogen, as well as the strain-dependent response. Together, these results allowed to conceive an in vitro periprosthetic infection model to better evaluate novel therapeutic strategies

Antibiotic Tolerance of Staphylococcus aureus Biofilm in Periprosthetic Joint Infections and Antibiofilm Strategies

International audienceThe need for bone and joint prostheses is currently growing due to population aging, leading to an increase in prosthetic joint infection cases. Biofilms represent an adaptive and quite common bacterial response to several stress factors which confer an important protection to bacteria. Biofilm formation starts with bacterial adhesion on a surface, such as an orthopedic prosthesis, further reinforced by matrix synthesis. The biofilm formation and structure depend on the immediate environment of the bacteria. In the case of infection, the periprosthetic joint environment represents a particular interface between bacteria, host cells, and the implant, favoring biofilm initiation and maturation. Treating such an infection represents a huge challenge because of the biofilm-specific high tolerance to antibiotics and its ability to evade the immune system. It is crucial to understand these mechanisms in order to find new and adapted strategies to prevent and eradicate implant-associated infections. Therefore, adapted models mimicking the infectious site are of utmost importance to recreate a relevant environment in order to test potential antibiofilm molecules. In periprosthetic joint infections, Staphylococcus aureus is mainly involved because of its high adaptation to the human physiology. The current review deals with the mechanisms involved in the antibiotic resistance and tolerance of Staphylococcus aureus in the particular periprosthetic joint infection context, and exposes different strategies to manage these infections

Comparison of Two Cutibacterium acnes Biofilm Models

International audienceThe study of biofilms in vitro is complex and often limited by technical problems due to simplified models. Here, we compared C. acnes biofilm formation, from species involved in bone and prosthesis infection, in a static model with a dynamic model. Using similar parameters, the percentage of live bacteria within the biofilm was higher in dynamic than in static approach. In both models, bacterial internalization in osteoblast-like cells, playing the role of stress factor, affected this proportion but in opposite ways: increase of live bacteria proportion in the static model and of dead bacteria proportion in the dynamic model. This work highlights the huge importance in the selection of a relevant biofilm model in accordance with the environmental or clinical context to effectively improve the understanding of biofilms and the development of better antibiofilm strategies

Approaching prosthesis infection environment: Development of an innovative in vitro Staphylococcus aureus biofilm model

The major role and implication of bacterial biofilms in the case of bone and prosthesis infections have been highlighted and often linked to implant colonization. Management strategies of these difficult-to-treat infections consist in surgeries and antibiotic treatment, but the rate of relapse remains high, especially if Staphylococcus aureus, a high-virulent pathogen, is involved. Therapeutic approaches are not adapted to the specific features of biofilm in bone context whereas infectious environment is known to importantly influence biofilm structure. In the present study, we aim to characterize S. aureus SH1000 (methicillin-sensitive strain, MSSA) and USA300 (methicillin-resistant strain, MRSA) biofilm on different surfaces mimicking the periprosthetic environment. As expected, protein adsorption on titanium enhanced the number of adherent bacteria for both strains. On bone explant, USA300 adhered more than SH1000. The simultaneous presence of two different surfaces was also found to change the bacterial behaviour. Thus, proteins adsorption on titanium and bone samples (from bank or directly recovered after an arthroplasty) were found to be key parameters that influence S. aureus biofilm formation: adhesion, matrix production and biofilm-related gene regulation. These results highlighted the need for new biofilm models, more relevant with the infectious environment by using adapted culture medium and presence of surfaces that are representative of in situ conditions to better evaluate therapeutic strategies against biofilm

Human Osteoblast-Conditioned Media Can Influence Staphylococcus aureus Biofilm Formation

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

Cutibacterium acnes Biofilm Study during Bone Cells Interaction

International audienceCutibacterium 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

Pyridazinone Derivatives Limit Osteosarcoma-Cells Growth In Vitro and In Vivo

International audienceOsteosarcoma is a rare primary bone cancer that mostly affects children and young adults. Current therapeutic approaches consist of combining surgery and chemotherapy but remain unfortunately insufficient to avoid relapse and metastases. Progress in terms of patient survival has remained the same for 30 years. In this study, novel pyridazinone derivatives have been evaluated as potential anti-osteosarcoma therapeutics because of their anti-type 4 phosphodiesterase activity, which modulates the survival of several other cancer cells. By using five—four human and one murine osteosarcoma—cell lines, we demonstrated differential cytotoxic effects of four pyridazinone scaffold-based compounds (mitochondrial activity and DNA quantification). Proapoptotic (annexin V positive cells and caspase-3 activity), anti-proliferative (EdU integration) and anti-migratory effects (scratch test assay) were also observed. Owing to their cytotoxic activity in in vitro conditions and their ability to limit tumor growth in a murine orthotopic osteosarcoma model, our data suggest that these pyridazinone derivatives might be hit-candidates to develop new therapeutic strategies against osteosarcoma