The emergence of antibiotic resistance in clinically significant pathogens, coupled with their ability to form persistent biofilms, presents a severe challenge in the management of burns and wound infections. To help mitigate the overuse of antibiotics and reduce the prevalence of antimicrobial-resistant bacteria, we have developed a modular theranostic system by blending two types of biocompatible copolymer building blocks, a chromogenic and an antimicrobial component, respectively. For this purpose, a parent active ester copolymer, poly[(hydroxy ethyl acrylamide)-co-(4-benzophenone acrylamide)-co-(pentafluorophenyl acrylate)-co- (ECOSURF EH-3 acrylate)] was synthesized via free radical polymerization. Two different chromogenic building blocks for bacterial enzyme detection were obtained from the parent polymer by functionalization of the pentafluorophenyl acrylate units with enzyme-labile chromogenic compounds, either 5-bromo-4-chloro-3-indolyl β-d-glucuronide (X-GLUC) or 4-nitrophenyl-β-d-glucuronide (PNPG). The antimicrobial building block for infection prevention and treatment was prepared from the parent active ester polymer by modification with antimicrobial peptide SAAP-148. The chromogenic-antimicrobial polymer blend responded to the bacterial enzyme β-glucuronidase with the release of chromophores that produce a visible colour change, enabling rapid, equipment-free proof-of-concept chromogenic detection of Escherichia coli, a major cause of healthcare-associated infections. This polymer blend also demonstrated potent antimicrobial activity against E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, including multidrug-resistant strains.

The global rise of antibiotic resistance necessitates novel therapeutic strategies for infectious diseases. Inhibition of bacterial efflux pumps, which contribute to multidrug resistance, represents a promising approach to restore or even increase the efficacy of existing antibiotics. Using fluorescence-based ethidium bromide accumulation, broth microdilution, and checkerboard assays, we evaluated 26 endogenous steroidal hormones and neurosteroids, along with 30 synthetic derivatives, for their ability to enhance antibiotic susceptibility in multidrug-resistant Staphylococcus aureus. Structure-activity relationship analysis identified compounds 13 and 16 as lead candidates, exhibiting strong efflux pump inhibition and marked reductions in the minimum inhibitory concentrations of ciprofloxacin and erythromycin. Both compounds showed additive effects in checkerboard assays. Modifications at C-3 (polar substitution) and C-17 (3α,5β-stereochemistry and nonpolar substitution) were essential for potent efflux inhibition and sensitization, although these modifications were not additive when combined. Transcriptome analysis further revealed that compound 13 significantly downregulated S. aureus virulence-associated genes when administered alone or in combination with antibiotics. Cytotoxicity assessment in human peripheral blood mononuclear cells and receptor transactivation assays for estrogen, androgen, and progesterone receptors indicated that the most active derivatives were non-toxic and lacked detectable endocrine activity, suggesting a favorable safety profile. Overall, these findings support the concept that rationally designed andostane-based steroidal scaffolds can function as competitive bacterial efflux pump inhibitors and serve as potential antibiotic adjuvants to mitigate efflux-mediated resistance.

Fracture non-union represents a complex clinical challenge resulting from an incompletely understood interplay between mechanical and biological factors. Infection frequently contributes to non-union but many cases are misdiagnosed due to a lack of classical clinical symptoms. This study characterized peripheral blood mononuclear cells (PBMCs) from aseptic non-union (NU-AS, n = 24) and fracture-related infected non-union (NU-FRI, n = 20), and compare them to healed controls (H, n = 18). High-dimensional mass cytometry (CyTOF) revealed significant elevations of regulatory T cells (Tregs; p = 0.0028) and T helper 1 (Th1) cells (p = 0.0073), and reduced expression of the activation marker CD38 in CD4+ T cells (p = 0.0016) and Tregs (p = 0.0017) in NU compared to H. In a subgroup analysis between NU-AS and NU-FRI, monocyte and CD38+ Treg cell counts provided excellent diagnostic potential, with the combination achieving a sensitivity of 100% and a specificity of 91.7%. These findings highlight an important role of the activation marker CD38 in diagnosing chronic subclinical infection, which promises earlier identification of appropriate management of these patients.

Background: Antibiotic-loaded bone cement (ALBC) is widely used for local antibiotic delivery in joint arthroplasty to prevent and treat prosthetic joint infections (PJIs). In this study, we evaluated the efficacy of cemented Kirschner (K)-wires coated with various ALBC formulations using a Galleria mellonella infection model against multidrug-resistant (MDR) Staphylococcus aureus and Enterococcus faecalis. Methods: We tested commercially available bone cements, including gentamicin-only formulations (PALACOS R+G) and dual-antibiotic formulations, combining gentamicin with either clindamycin (COPAL G+C) or vancomycin (COPAL G+V), alongside an antibiotic-free control (PALACOS R). In vitro assays-including minimum inhibitory/bactericidal concentration (MIC/MBC) determination, antibiotic release kinetics, agar diffusion, and antibiofilm evaluations-demonstrated effective antibiotic release and significant antimicrobial activity against both planktonic and biofilm-associated bacteria. Results: In vivo, ALBC-coated K-wires were well tolerated in G. mellonella and significantly protected the larvae from S. aureus infection compared to controls. Notably, dual-antibiotic formulations provided superior protection, correlating with substantial reductions in bacterial colonisation on implant surfaces and in surrounding tissues. Conclusions: These findings support the utility of the G. mellonella model as a high-throughput, cost-effective platform for the preclinical evaluation of antimicrobial strategies to prevent and treat PJIs and further demonstrate the effectiveness of dual-loaded ALBC against multidrug-resistant bacteria.

In trauma surgery, the development of biomaterial-associated infections (BAI) is one of the most common complications affecting trauma patients, requiring prolonged hospitalization and the intensive use of antibiotics. Following the attachment of bacteria on the surface of the biomaterial, the biofilm-forming bacteria could initiate a chronic implant-related infection. Despite the use of conventional local and systemic antibiotic therapies, persistent biofilms involve various resistance mechanisms that contribute to therapeutic failures. The development of in vivo chronic BAI models to optimize antibiofilm treatments is a major challenge. Indeed, the biofilm pathogenicity and the host response need to be finely regulated, and compatible with the animal lifestyle. Previously, a Galleria mellonella larvae model for the formation of an early-stage biofilm on the surface of a Kirschner (K)-wire was established. In the present study, two models of mature biofilm using clinical Staphylococcus aureus strains were assessed: one related to contaminated K-wires ( in vitro biofilm maturation) and the second to hematogenous infections ( in vivo biofilm maturation). Rifampicin was used as a standard drug for antibiofilm treatment. In the first model, biofilms were formed following an incubation period (up to 7 days) in the CDC Biofilm Reactor (CBR, BioSurface Technologies). Then, after implantation of the pre-incubated K-wire in the larvae, rifampicin (80 mg/kg) was injected and the survival of the larvae was monitored. In the second model, biofilm formation was achieved after an incubation period (up to 7 days) inside the larvae and then, after removing the K-wires from the host, in vitro rifampicin susceptibility assays were performed (according to EUCAST). The first model indicate that in vitro biofilm maturation affects the bacterial pathogenicity in the host, depending on the S. aureus strain used. Furthermore, the more the biofilm is matured, the more the rifampicin treatment efficiency is compromised. The second model shows that, despite the fast in vivo biofilm formation in the host, the number of bacteria, either attached to the surface of the K-wire surface or in surrounding tissue of the larvae, was not increased over time. Altogether, these results allow the establishment of biofilm models using G. mellonella larvae in order to understand the impact of biofilm maturation on both the bacterial pathogenicity and the efficiency of antibiofilm treatments.

Medical devices contribute to the carbon footprint generated by the healthcare sector. The development of implants and biomaterials using recycled waste materials promotes sustainable advances in tissue engineering. Additively manufactured (AM) bone-substituting biomaterials with multifunctional properties, e.g., biodegradability, antibacterial and osteogenic potential, can contribute to sustainable healthcare. Biodegradable biomaterials eliminate secondary surgeries to remove implants, reduce post-surgical complications, and enhance patient recovery, thus decreasing the energy usage and waste associated with medical treatments. Herein, we present porous iron (Fe) scaffolds incorporating 20 vol% waste-derived eggshell particles for bone substitution. The Fe-eggshell scaffolds were fabricated using direct ink writing (DIW) technique and underwent post-AM heat treatment. During sintering, the eggshell's main component - CaCO3, transformed into CaO. Atomic diffusion between α-Fe and CaO phases resulted in the formation of Ca2Fe2O5 phase at the interface. The scaffolds were 70 % porous and displayed a biodegradation rate of 0.11 mm/year. The mechanical properties were comparable to trabecular bone and the scaffolds endured 3 million loading cycles at 0.7σy in r-SBF. The scaffolds showed apatite-forming ability, evidenced by the formation of (carbonaceous) hydroxyapatite, which are conducive to preosteoblast adhesion, proliferation, and differentiation. RT-qPCR analysis confirmed the osteogenic potential of the specimens as evidenced by the upregulated expression of osteopontin and osteocalcin as compared to Ti6Al4V controls. Furthermore, the scaffolds exhibited bactericidal activity (>3.9-log CFU reduction) against methicillin-sensitive and multidrug-resistant strains of Staphylococcus aureus and delayed their biofilm formation. Our research showcases the exceptional multifunctionality of DIW Fe-eggshell composite scaffolds for the sustainable development of orthopedic biomaterials. STATEMENT OF SIGNIFICANCE: We aim to improve the biofunctionalities and sustainability of biodegradable bone substitutes, by developing the extrusion-based 3D printed porous Fe composite scaffolds containing eggshell-derived CaO bioceramics. Our results demonstrated that Fe-eggshell scaffolds exhibited hydroxyapatite-forming ability in simulated body fluid, having mechanical properties in the range of trabecular bone even after 4 weeks biodegradation, supported the proliferation of preosteoblasts and upregulated the expression of osteogenic genes. Moreover, the scaffolds were bactericidal against methicillin-sensitive and multi-drug resistant strains Staphylococcus aureus and delayed their biofilm formation.

Aerosol delivery represents a rapid and non-invasive way to directly reach the lungs while escaping the hepatic first-pass effect. The development of pulmonary drugs for respiratory diseases such as cystic fibrosis, lung infections, pulmonary fibrosis or lung cancer requires an enhanced understanding of the relationships between the natural physiology of the respiratory system and the pathophysiology of these conditions. This knowledge is crucial to better predict and thereby control drug deposition. Moreover, aerosol administration faces several challenges, including the pulmonary tract, immune system, mucociliary clearance, the presence of fluid on the airway surfaces, and, in some cases, bacterial colonisation. Each of them directly influences on the bioavailability of the active molecule. In addition to these challenges, particle size and the device used to administer the treatment are critical factors that can significantly impact the biodistribution of the drugs. Nanoparticles are very promising in the development of new formulations for aerosol drug delivery, as they can be fine-tuned to reach the entire pulmonary tract and overcome the difficulties encountered along the way. However, to properly assess drug delivery, preclinical studies need to be more thorough to efficiently enhance drug delivery.

Background: The escalating global crisis of antibiotic resistance necessitates the discovery of novel antimicrobial agents. Antimicrobial peptides (AMPs) represent a promising alternative to combat multidrug-resistant (MDR) pathogens. Because traditional AMP discovery is labour-intensive and costly, machine learning (ML) is applied to identify AMPs effective against MDR bacteria and skin infections. Methods: The ML-based CalcAMP model predicts the antimicrobial activity of 16,384 unique 14-amino-acid peptide sequences, resulting in a novel Guided Designed Smart antimicrobial Therapeutic (GDST) peptide catalogue. Parent sequences and retro-inverso (RI) variants of two prime GDST peptides undergo extensive testing against MDR bacteria and in skin infection models. Results: GDST-038 and GDST-045, along with their RI variants, show potent antimicrobial activity against Acinetobacter baumannii and Staphylococcus aureus, rapidly depolarizing the cytoplasmic membrane, exhibiting broad-spectrum bactericidal effects against ESKAPE pathogens, and causing minimal haemolysis. RI variants display superior A. baumannii biofilm killing compared to parent sequences, while all GDST peptides achieve >3-log reductions in S. aureus biofilm CFU within 24 h. Potent efficacy is observed in a 3D human skin epidermal infection model, with elimination of S. aureus at ≥15 μM. No resistance develops after 22 passages. Conclusions: ML-driven screening enables rapid identification of two novel candidate AMPs, highlighting the therapeutic potential of GDST peptides for MDR bacterial infections.

In this work, a copolymer of poly[(hydroxy ethyl acrylamide)-co-(4-benzophenone acrylamide)-co-(hexamethylene diamine acrylamide)-co-(ECOSURF EH-3 acrylate)] was synthesized via free radical polymerization, followed by multi-step modification using click chemistry. The copolymer was subsequently functionalized with either 5-bromo-4-chloro-3-indolyl beta-d-glucuronide (X-GLUC) or 4-nitrophenyl-beta-d-glucuronide (PNPG), two enzyme-labile chromogenic substrates used to visually observe bacterial beta-glucuronidase activity. This enzyme is secreted by over 98% of Escherichia coli strains, a common cause of healthcare-associated infections. The use of two substrates demonstrates the system's versatility in detecting beta-glucuronidase activity across different bacterial species, wherein enzymatic cleavage of the dye-sugar bond produces a visible chromogenic signal. Both copolymers were found to be non-cytotoxic to human lung fibroblasts and were independently crosslinked under UV light to form distinct polymer network structures. Upon water swelling, each hydrogel enabled qualitative detection of beta-glucuronidase-producing bacteria through the release of indigo or yellow dyes, corresponding to the chromogenic response of X-GLUC or PNPG, respectively. This colorimetric response was confirmed both visually and spectroscopically, underscoring the potential of these polymers for development into multiplexed enzyme-based bacterial detection platforms. The technology offers promising applications in microbiological diagnostics, particularly in food safety and medical contexts.

IntroductionInfected chronic wounds present a dual therapeutic challenge, requiring both the eradication of pathogens and the restoration of tissue homeostasis. Often the current treatments are ineffective against multidrug-resistant (MDR) pathogens and fail to promote wound healing. Antimicrobial peptides, such as bovine lactoferricin (LfcinB), offer a promising alternative owing to their broad-spectrum activity and immunomodulatory properties. The branched tetrameric LfcinB-derived peptide (LBT; (RRWQWR)4K2Ahx2-C2) is particularly attractive, as its multivalent architecture enhances antimicrobial potency and provides a tunable branching core for structural modifications.MethodsIn this study, three novel tetrameric variants were designed by substituting the L-lysine branching residues in LBT with non-natural lysine derivatives to alter motif orientation and linker flexibility. Among the novel peptides, the diaminopropionic acid (DAP)-modified variant, (LBT-1; (RRWQWR)4DAP2Ahx2-C2) was selected as best-performing candidate based on antimicrobial and hemolytic activity assessment.ResultsCompared to LBT, the novel LBT-1 demonstrated superior activity against methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii, achieving rapid bactericidal action within 5 minutes. LBT-1 also exhibited potent activity across the ESKAPE(E) panel and against the emerging MDR fungal pathogen Candidozyma auris. Beyond direct antimicrobial effects, LBT-1 enhanced macrophage-mediated bacterial clearance, neutralized endotoxins, and accelerated wound closure in vitro. Importantly, LBT-1 showed superior pro-angiogenic activity in vitro and achieved significantly higher bactericidal activity against MRSA in an ex vivo human skin wound infection model. This study identifies LBT-1 as a multifunctional therapeutic that addresses key pathological features of chronic wounds.ConclusionTogether, these findings validate our peptide design strategy, revealing previously unknown characteristics of the LBT peptide and the enhanced multifunctionality achieved with LBT-1, supporting its continued development for chronic wound management.

Background: Bone and joint infections pose significant clinical challenges, often leading to severe complications and substantial healthcare costs. Traditional antibiotic therapies are becoming increasingly ineffective due to rising antibiotic resistance and the biofilm-forming ability of bacteria such as Staphylococcus aureus (S. aureus). Antimicrobial coatings offer a promising approach for the prevention and treatment of implant-associated and bone infections. Following the demonstrated in vitro efficacy of nisin, a naturally occurring antimicrobial peptide, in preventing S. aureus biofilm formation, this study investigates the in vivo potential of a nisin-enriched coating to prevent biofilm-related infections using the Galleria mellonella larva haematogenous implant infection model. Methods: Methicillin-sensitive S. aureus (MSSA) EDCC 5055 was used to infect larvae implanted with nisin-coated titanium Kirschner wires (K-wires). Survival rates and bacterial loads on both the K-wires and in larval tissue were analysed. Biofilm formation on K-wires was further analysed using scanning electron microscopy. Results: The results showed that nisin-coated K-wires significantly improved larval survival and reduced bacterial burden compared to control groups. Scanning electron microscopy confirmed the absence of biofilm formation on nisin-coated K-wires. Conclusions: These findings suggest that nisin-enriched coatings could be a viable strategy for preventing bone and joint infections. Additionally, this study demonstrates the feasibility of testing implant coatings in a cost-effective and ethically sound alternative in vivo model. Further evaluation and testing of the nisin-enhanced coating in vertebrate animal implant infection models is warranted.

Periprosthetic infections caused by vancomycin-resistant pathogens, such as vancomycin-resistant Enterococcus faecalis (VRE), represent a major clinical challenge. Daptomycin can be incorporated into polymethyl methacrylate (PMMA) cement spacers. This study aimed to utilize the in vivo Galleria mellonella larvae implant infection model to determine the optimal dosage of daptomycin in different PMMA cements and evaluate its efficacy. Daptomycin (1 g or 2 g) was added to different PMMA cements (Cement 1 containing 1 g tobramycin; Cement 2 containing 0.5 g gentamicin), to produce bone cement implants. Control groups included non-loaded Cement 1 and Cement 2, as well as Reference 1 and Reference 2, without antibiotics. The test specimens were implanted into larvae of the greater wax moth ( G. mellonella ), followed by infection with 250 CFU of VRE DSM13591 after 1 hour. The survival of the larvae was monitored over time, and bacterial numbers in the larval tissue and on the implant surface were determined after 24h. These experiments were completed with in vitro proliferation assay and inhibition zone testing. Mechanical stability was measured according to ISO 5833 and DIN 53435. Survival and bacterial burden analysis demonstrated that the addition of 1 g or 2 g of daptomycin was effective in preventing VRE infections. There were significantly improved larval survival and reduced bacterial numbers in both the larval tissue and on the implant surface. In contrast, Cement 1 and Cement 2 alone did not enhance larval survival and showed outcomes comparable to the antibiotic-free control groups. The addition of daptomycin was found to be effective in inhibiting bacterial growth when compared to the references, with the largest inhibition observed for Cement 2 (2 g daptomycin). On day 42, sufficient bacterial growth inhibition was observed only with the addition of 2 g daptomycin. These results correlate with those from the proliferation assay. Adding more than 1.5 g daptomycin resulted in a reduction of mechanical strength, with Cement 1 no longer meeting the ISO standard for four-point bending strength. The incorporation of daptomycin into ALBC successfully prevented VRE infections in an in vivo model. These findings emphasize the potential of the G. mellonella implant infection model as a valuable tool for evaluating the efficacy of ALBC against multidrug-resistant bacteria, thereby accelerating pre-clinical research and advancing strategies to combat PJI. We observed differences in mechanical stability and antimicrobial efficacy between daptomycin-loaded Simplex Tobramycin and Palacos R+G with increased stability and bacterial growth reduction for the latter one.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics for treating skin wound infections. Nonetheless, their short half-life in biological environments restricts clinical applicability. Covalent immobilization of AMPs onto suitable substrates offers a comprehensive solution, creating contact-killing surfaces with long-term functionality. Here, a copolymer of poly[(hydroxy ethyl acrylamide)-co-(4-benzophenone acrylamide)-co-(pentafluorophenyl acrylate)-co-(ECOSURF EH-3 acrylate)], in short poly(HEAAm-co-BPAAm-co-PFPA-co-EH3A), is synthesized by free radical polymerization. Subsequent modification of active ester groups with the amine groups of SAAP-148, results in a copolymer, that is non-cytotoxic to human lung fibroblasts. UV photocrosslinking of the benzophenone units yields a polymer network that forms a hydrogel after swelling with aqueous medium. Both the SAAP-148-modified polymer in solution and the photocrosslinked hydrogels show good antimicrobial activity against strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, including multidrug-resistant strains, frequently found in wound infections. The covalent attachment of SAAP-148 prevents leaching, ensuring sustained antimicrobial activity for at least 48 h in diluted human blood plasma and 14 days in PBS. This prolonged retention of antimicrobial activity in human blood plasma significantly enhances its clinical potential. Overall, this study shows the potential of the AMP-functionalized photocrosslinkable polymer as antimicrobial wound dressings, providing an effective alternative to antibiotics.

AimOrthopedic implants play a tremendous role in fixing bone damages due to aging as well as fractures. However, these implants tend to get colonized by bacteria on the surface, leading to infections and subsequently prevention of healing and osteointegration. Recently, Roupie et al. showed that a nisin layer-by-layer based coating applied on biomaterials has both osteogenic and antibacterial properties. The Galleria mellonella larva is a well-known insect infection model that has been used to test the virulence of bacterial and fungal strains as well as for the high throughput screening of antimicrobial compounds against infections. Recently, we have developed an insect infection model with G. mellonella larvae to study implant-associated biofilm infections using Kirschner (K)-wires as implant material. Here, we would like to test the antibacterial capacity of nisin layer-by-layer based coatings on K-wires against Staphylococcus aureus in the G. mellonella larva implant infection model.MethodPrior to the implantation procedure, G. mellonella larvae are maintained at room temperature on wheat germ in an incubator. The larvae received bare titanium K-wires (uncoated), or either control-coated or nisin-coated K-wires. After one hour, the larvae were injected with 5×105S. aureus bacteria per larva (i.e., hematogenous implant infection model). Next, the larvae were incubated at 37oC in an incubator and the survival of the larvae was monitored for five days. Moreover, the number of bacteria on the implant surface and in the surrounding tissue was determined after 24h of incubation. Further, scanning electron microscopy (SEM) analyses were performed to study the effect of nisin on biofilm formation.ResultsThe larvae receiving the nisin-coated K-wires showed significantly higher survival rates compared to uncoated titanium K-wires, although not when compared to control-coated K-wires. A more than 1-log reduction in number of bacteria on the implant surface and in the surrounding tissue was observed in larvae receiving the nisin-coated K-wires, when compared to uncoated titanium K-wires SEM analysis showed reduced colonization of the bacteria nisin-coated K-wires compared to the controlsConclusionsIn conclusion, the antimicrobial nisin layer-by-layer based coating applied on titanium surfaces is able to prevent implant-related S. aureus biofilm infection in G. mellonella and is a promising antimicrobial strategy to prevent implant-related infections.

Prosthetic joint infections (PJIs) can have disastrous consequences for patient health, including removal of the device, and placement of cemented implants is often required during surgery to eradicate PJIs. In translational research, in vivo models are widely used to assess the biocompatibility and antimicrobial efficacy of antimicrobial coatings and compounds. Here, we aim to utilize Galleria mellonella implant infection models to assess the antimicrobial activity of antibiotic-loaded bone cement (ALBC) implants. Therefore, we used commercially available bone cement loaded with either gentamicin alone (PALACOS R+G) or with a combination of gentamicin and vancomycin (COPAL G+V), compared to bone cement without antibiotics (PALACOS R). Firstly, the in vitro antimicrobial activity of ALBC was determined against Staphylococcus aureus. Next, the efficacy of ALBC implants was analyzed in both the G. mellonella hematogenous and early-stage biofilm implant infection model, by monitoring the survival of larvae over time. After 24 h, the number of bacteria on the implant surface and in the tissue was determined. Larvae receiving dual-loaded COPAL G+V implants showed higher survival rates compared to implants loaded with only gentamicin (PALACOS R+G) and the control implants without antibiotics (PALACOS R). In conclusion, G. mellonella larvae infection models with antibiotic-loaded bone cements are an excellent option to study (novel) antimicrobial approaches.

Biomaterials provoke a foreign body response (FBR) which usually results in formation of fibrous tissue, and surface topography properties are known to influence this process. The current paradigm states that materials that induce monocyte binding in vitro will induce more severe encapsulation in vivo. We employ our TopoChip screening platform to define design principles for monocyte binding in vitro, and subsequently investigate the FBR in vivo on clinically applied polycarbonate urethane (PCU) polymers. We first assess monocyte binding in vitro on 2,176 different PCU topographies, and learn that small pillars with sufficient space in between them results in monocyte binding, whereas large pillars and small spacing consistently results in low attachment. Next, we histologically assess the FBR in a murine subcutaneous implant model with 2 low and 3 high monocyte binding surfaces and a non-patterned control surface at 9 and 60 days after implantation. We observe strong differences in the FBR parameters showing that surface topography can be used as a design tool to control the FBR. However, this did not correlate in any way to monocyte binding in vitro. We propose that to establish an in vitro model which is predictive for topography-induced FBR, we first need to establish a mechanistic molecular and cellular model describing the interaction of the key cellular players with topographical cues.

Aims: Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella. Methods: For the haematogenous infection, G. mellonella larvae were implanted with a Kirschner wire (K-wire), infected with S. aureus, and subsequently phages and/or gentamicin were administered. For the early-stage biofilm implant infection, the K-wires were pre-incubated with S. aureus suspension before implantation. After 24 hours, the larvae received phages and/or gentamicin. In both models, the larvae also received daily doses of phages and/or gentamicin for up to five days. The effect was determined by survival analysis for five days and quantitative culture of bacteria after two days of repetitive doses. Results: In the haematogenous infection, a single combined dose of phages and gentamicin, and repetitive injections with gentamicin or in combination with phages, resulted in significantly improved survival rates. In the early-stage biofilm infection, only repetitive combined administration of phages and gentamicin led to a significantly increased survival. Additionally, a significant reduction in number of bacteria was observed in the larvae after receiving repetitive doses of phages and/or gentamicin in both infection models. Conclusion: Based on our results, a single dose of the combination of phages and gentamicin is sufficient to prevent a haematogenous S. aureus implant-related infection, whereas gentamicin needs to be administered daily for the same effect. To treat early-stage S. aureus implant-related infection, repetitive doses of the combination of phages and gentamicin are required. Cite this article: Bone Joint Res 2024;13(8):383–391.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Delta pvl), or complemented mutant (Delta pvl+pvl). Three days post-surgery, Delta pvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Delta pvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Delta pvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

A polymer microarray based on the supramolecular ureido-pyrimidinone (UPy) moiety is fabricated to screen antimicrobial materials for their ability to support cell adhesion. UPy-functionalized additives, either cell-adhesive, antimicrobial or control peptides, are used, and investigated in different combinations at different concentrations, resulting in a library of 194 spots. These are characterized on composition and morphology to evaluate the microarray fabrication. Normal human dermal fibroblasts are cultured on the microarrays and cell adhesion to the spots is systematically analyzed. Results demonstrate enhanced cell adhesion on spots with combinations including the antimicrobial peptides. This study clearly proves the power of the high throughput approach in combination with supramolecular molecules, to screen additive libraries for desired biological response.