Bioactive glass (BAG) S53P4 is a clinically approved bone substitute with antibacterial, osteoconductive and osteostimulatory properties. Its antibacterial effect is associated with ion release, local pH elevation and osmolality, but the precise biochemical and biophysical mode-of-action is unclear. This study investigates the antibacterial mechanism of BAG S53P4 eluates. BAG eluates, collected at 2, 4, 8, and 24 h, eradicated Staphylococcus aureus. Elemental analysis revealed an early increase in concentrations of Si and Na, a later rise in Ca, depletion of P over time and rapid loss of Mg. Membrane disturbances occurred within 5 min, evident by permeability for SYTOX, aligning with time-kill kinetics for S. aureus and Bacillus subtilis. In B. subtilis, 2h-BAG-eluate induced rapid delocalization of marker proteins for cell division and DNA repair, signaling membrane potential collapse and nucleoid condensation. Transcriptomics revealed early transcription remodeling reflecting ionic and energetic imbalance, including disruption of central metabolism, redox homeostasis, and translational stability. Scanning electron microscopy revealed severe cell surface damage and particulate deposits on S. aureus. Transmission electron microscopy showed cell envelop disruptions and cytoplasmic leakage. Energy dispersive X-ray analysis identified Si on bacterial cell surface at 4 h and intracellular accumulation in punctured, empty cells at 24 h. Overall, BAG ionic dissolution products kill bacteria through a stepwise mechanism involving membrane damage, protein delocalization and metabolic impairment, accompanied by Si deposition on bacterial surfaces and loss of Mg. This finally leads to cell wall degradation, cytoplasmic content leakage and further Si deposition on the cells and inside cell ghosts.

Introduction: Orthopedic implant-associated infections, predominantly caused by S. aureus, pose significant challenges due to biofilm formation and antibiotic resistance. Bioactive Glass (BAG) S53P4 is a unique material with antimicrobial and bone regenerative properties. We aimed to characterize a novel BAG S53P4 cream, consisting of BAG powder and a binder, for its capacity to kill Staphylococcus aureus in suspension and biofilms in the absence or presence of titanium implant material. Since the BAG antimicrobial activity depends on ions eluted, we also analyzed the eluates of the cream and of powder and binder. Methods: BAG cream, BAG powder, and binder were evaluated for antimicrobial activity against planktonic S. aureus in the presence or absence of titanium implant material, and against S. aureus biofilms. Eluates collected at different time points were tested against a panel of bacterial and fungal pathogens. Elemental ion release and pH changes were measured over time. Time-kill and biofilm assays were performed. Additionally, the applicability and antimicrobial efficacy of BAG cream were evaluated in a cadaver mouse bone defect model. Results: The BAG cream and BAG powder applied to titanium implant material, as well as their respective eluates eradicated planktonic S. aureus. Elemental release from BAG cream and powder showed time-dependent shifts in levels of silicon, sodium, calcium and phosphorous together with stable alkaline pH levels, reflecting continuous ion release from the glass network and concurrent precipitation of calcium phosphate and silica phases. BAG cream and powder eluates collected as early as at 2 h were highly effective against S. aureus, the ESKAPE panel of multidrug resistant pathogens, colistin-resistant Escherichia coli and Cutibacterium acnes, and against the fungi Candidozyma auris and Candida albicans. The eluates displayed time-dependent bactericidal activity with significant bacterial killing starting already at 30 min and increasing with longer exposure times. Moreover, significant reduction in S. aureus biofilm was observed with the cream and powder eluates. BAG cream was easy to apply to the bone defect of a cadaver mouse using a syringe and it effectively prevented S. aureus growth. Conclusion: These findings show the potential of BAG cream as an innovative application form of BAG S53P4 offering a promising approach against orthopedic implant-associated infections.

Orthopedic implant-associated infections (OIAIs), predominantly caused by Staphylococcus aureus , pose significant challenges due to biofilm formation and antibiotic resistance, leading to persistent and recurrent infections. Bioactive Glass (BAG) S53P4 is a unique material with antimicrobial and bone regenerative properties. A novel BAG cream formulation can be used as an advanced coating solution to prevent or treat OIAIs. Aim: In this study we aimed to characterize a novel BAG S53P4 cream, consisting of antimicrobial BAG powder and binder, for its bactericidal and biofilm killing activity in vitro and efficacy in a cadaver mouse bone infection model. Since the bactericidal effects is dependent on eluted compounds, we analysed the bactericidal activity of BAG cream, BAG powder, binder and their eluates against S. aureus. Method: To study the influence of the elution time, S. aureus was exposed to eluates collected at 2, 4, 8, and/or 24 hours. The bactericidal and biofilm-killing activity were quantitatively analysed. A time kill assay was performed to assess the speed of killing of S. aureus . The pH was measured to study its relevance for the bactericidal action of BAG. In a cadaver mouse bone infection model, BAG cream was applied to an S. aureus infected femoral bone defect. Results: The BAG cream, BAG powder and their eluates eradicated planktonic S. aureus and significant killing of S. aureus biofilms was observed with the cream and powder eluates. All eluates, including the eluate collected at the earliest elution time of 2 hours, were highly effective against S. aureus , indicating rapid release of bactericidal activity from BAG cream and powder. Time-kill experiments revealed rapid bactericidal activity of BAG cream and powder eluates, with killing starting as early as 30 minutes and increasing with longer exposure times. BAG cream applied to an S. aureus cadaver mouse bone infection model inhibited bacterial growth effectively. Conclusion: These findings show the potential of BAG cream as an innovative solution for managing OIAIs. As a next step we intend to investigate the in vivo antibacterial efficacy and safety of BAG cream applied in a mouse bone fixation plate infection model.

Introduction: Orthopedic implant-associated infections are difficult to treat due to bacterial persistence in biofilms on implant surfaces and in surrounding tissues. Bioactive glass S53P4 (BAG) is a clinically used bone graft substitute with antibacterial properties resulting from surface reactivity and dissolution-mediated changes in the local microenvironment; however, its antibacterial efficacy against established infections with a high bacterial burden is limited. In this study we therefore developed BAG putty and cream formulations loaded with synthetic antimicrobial and antibiofilm peptide SAAP-148 to enhance local antibacterial activity. Methods: Eluates from SAAP-148-loaded and unloaded BAG putty and cream formulations were tested in vitro against planktonic bacteria and biofilms of Staphylococcus aureus and Acinetobacter baumannii . Antibacterial efficacy was further assessed in cadaver mouse subcutaneous implant infection model as an intermediate validation step before evaluation in an in vivo mouse subcutaneous implant infection model using S. aureus . Results: In vitro , SAAP-148-loaded BAG putty and BAG cream eluates eradicated planktonic bacteria as well as young and mature biofilms of S . aureus and A . baumannii . Eluates of BAG formulations without SAAP-148 had limited bactericidal activity, with BAG cream eluates slightly more bactericidal than BAG putty eluates. The enhanced antibacterial effects of SAAP-148-loaded BAG formulations were confirmed in a cadaver mouse subcutaneous implant infection model, where S. aureus was eradicated both on the implant surface and in the surrounding tissue, whereas BAG-only formulations showed limited antibacterial effect. In a subsequent in vivo mouse subcutaneous implant infection model, SAAP-148-loaded BAG putty eradicated S. aureus on the implant surface and markedly reduced bacterial burden in tissue, whereas BAG putty-only failed to reduce bacterial numbers. Conclusion: Loading SAAP-148 into BAG putty and cream enhanced BAG antibacterial and antibiofilm activity, supporting its potential as a clinically relevant adjunct to the BAG’s antimicrobial activity for treating implant-associated infections.