Cutibacterium acnes biofilm formation is influenced by bone microenvironment, implant surfaces and bacterial internalization.

BACKGROUND: The bacterial persistence, responsible for therapeutic failures, can arise from the biofilm formation, which possesses a high tolerance to antibiotics. This threat often occurs when a bone and joint infection is diagnosed after a prosthesis implantation. Understanding the biofilm mechanism is pivotal to enhance prosthesis joint infection (PJI) treatment and prevention. However, little is known on the characteristics of Cutibacterium acnes biofilm formation, whereas this species is frequently involved in prosthesis infections. METHODS: In this study, we compared the biofilm formation of C. acnes PJI-related strains and non-PJI-related strains on plastic support and textured titanium alloy by (i) counting adherent and viable bacteria, (ii) confocal scanning electronic microscopy observations after biofilm matrix labeling and (iii) RT-qPCR experiments. RESULTS: We highlighted material- and strain-dependent modifications of C. acnes biofilm. Non-PJI-related strains formed aggregates on both types of support but with different matrix compositions. While the proportion of polysaccharides signal was higher on plastic, the proportions of polysaccharides and proteins signals were more similar on titanium. The changes in biofilm composition for PJI-related strains was less noticeable. For all tested strains, biofilm formation-related genes were more expressed in biofilm formed on plastic that one formed on titanium. Moreover, the impact of C. acnes internalization in osteoblasts prior to biofilm development was also investigated. After internalization, one of the non-PJI-related strains biofilm characteristics were affected: (i) a lower quantity of adhered bacteria (80.3-fold decrease), (ii) an increase of polysaccharides signal in biofilm and (iii) an activation of biofilm gene expressions on textured titanium disk. CONCLUSION: Taken together, these results evidenced the versatility of C. acnes biofilm, depending on the support used, the bone environment and the strain.

Development of an antibody fused with an antimicrobial peptide targeting Pseudomonas aeruginosa: A new approach to prevent and treat bacterial infections.

The increase in emerging drug resistant Gram-negative bacterial infections is a global concern. In addition, there is growing recognition that compromising the microbiota through the use of broad-spectrum antibiotics can impact long term patient outcomes. Therefore, there is the need to develop new bactericidal strategies to combat Gram-negative infections that would address these specific issues. In this study, we report and characterize one such approach, an antibody-drug conjugate (ADC) that combines (i) targeting the surface of a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of an antimicrobial peptide. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance: Pseudomonas aeruginosa. To target this organism, we designed an ADC by fusing an antimicrobial peptide to the C-terminal end of the VH and/or VL-chain of a monoclonal antibody, VSX, that targets the core of P. aeruginosa lipopolysaccharide. This ADC demonstrates appropriately minimal levels of toxicity against mammalian cells, rapidly kills P. aeruginosa strains, and protects mice from P. aeruginosa lung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study might result in a broadly useful strategy for targeting specific pathogenic microorganisms without further augmenting antibiotic resistance.

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.

Staphylococcus aureus Behavior on Artificial Surfaces Mimicking Bone Environment.

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.

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.

Comparison of Two Cutibacterium acnes Biofilm Models.

The 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 (×2.04 ± 0.53) and of dead bacteria proportion (×3.5 ± 1.03) 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.

Staphylococcus aureus Strain-Dependent Biofilm Formation in Bone-Like Environment.

Staphylococcus aureus species is an important threat for hospital healthcare because of frequent colonization of indwelling medical devices such as bone and joint prostheses through biofilm formations, leading to therapeutic failure. Furthermore, bacteria within biofilm are less sensitive to the host immune system responses and to potential antibiotic treatments. We suggested that the periprosthetic bone environment is stressful for bacteria, influencing biofilm development. To provide insights into S. aureus biofilm properties of three strains [including one methicillin-resistant S. aureus (MRSA)] under this specific environment, we assessed several parameters related to bone conditions and expected to affect biofilm characteristics. We reported that the three strains harbored different behaviors in response to the lack of oxygen, casamino acids and glucose starvation, and high concentration of magnesium. Each strain presented different biofilm biomass and live adherent cells proportion, or matrix production and composition. However, the three strains shared common responses in a bone-like environment: a similar production of extracellular DNA and engagement of the SOS response. This study is a step toward a better understanding of periprosthetic joint infections and highlights targets, which could be common among S. aureus strains and for future antibiofilm strategies.

Interaction of implant infection-related commensal bacteria with mesenchymal stem cells: a comparison between Cutibacterium acnes and Staphylococcus aureus.

Staphylococcus aureus and Cutibacterium acnes are involved in several tissue infections and can encounter mesenchymal stem cells (MSCs) during their role in tissue regenerative process. C. acnes and S. aureus internalization by three types of MSCs derived from bone marrow, dental pulp and Wharton's jelly; and bacterial biofilm production were compared. Internalization rates ranged between 1.7-6.3% and 0.8-2.7% for C. acnes and S. aureus, respectively. While C. acnes strains exhibited limited cytotoxic effect on MSCs, S. aureus were more virulent with marked effect starting after only 3 h of interaction. Both bacteria were able to produce biofilms with respectively aggregated and monolayered structures for C. acnes and S. aureus. The increase in C. acnes capacity to develop biofilm following MSCs' internalization was not linked to the significant increase in number of live bacteria, except for bone marrow-MSCs/C. acnes CIP 53.117 with 79% live bacteria compared to the 36% before internalization. On the other hand, internalization of S. aureus had no impact on its ability to form biofilms composed mainly of living bacteria. The present study underlined the complexity of MSCs-bacteria cross-interaction and brought insights into understanding the MSCs behavior in response to bacterial infection in tissue regeneration context.

Cutibacterium acnes Biofilm Study during Bone Cells Interaction.

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.

Biopolymers-calcium phosphate antibacterial coating reduces the pathogenicity of internalized bacteria by mesenchymal stromal cells.

A multifunctional material system that kills bacteria and drives bone healing is urgently sought to improve bone prosthesis. Herein, the osteoinductive coating made of calcium phosphate/chitosan/hyaluronic acid, named Hybrid, was proposed as an antibacterial substrate for stromal cell adhesion. This Hybrid coating possesses a contact-killing effect reducing by 90% the viability of Gram-positive Staphylococcus aureus (S. aureus) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa) strains after 48 h of contact. In addition to the production of immunomodulatory mediators, Wharton's jelly (WJ-SCs), dental pulp (DPSCs) and bone marrow (BM-MSCs) derived stromal cells were able to release antibacterial and antibiofilm agents effective against S. aureus and P. aeruginosa strains, respectively. Studying the effect of the Hybrid coating on the internalization of S. aureus by the stromal cells, in acute-mimicking bone infection, highlighted an increase in the bacteria internalization by DPSCs and BM-MSCs when cultured on the Hybrid coating versus uncoated glass. Despite the internalization, Hybrid coating showed a beneficial effect by reducing the pathogenicity of the internalized bacteria. The formation of biofilm was reduced by at least 50% in comparison to internalized bacteria by stromal cells on uncoated glass. This work opens the route for the development of innovative antibacterial coatings by taking into account the internalization of bacteria by stromal cells.

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.