Antimicrobial Mechanisms and Preparation of Antibiotic-impregnated Cement-coated Locking Plates in the Treatment of Infected Non-unions.

Background: Antibiotic-impregnated cement-coated plates (ACPs) have been used successfully for temporary internal fixation between stages in the two-stage treatment of infected non-unions. We describe our approach of using an ACP in the staged treatment of a methicillin-resistant Staphylococcus aureus (MRSA)-infected distal femoral non-union below a total hip prosthesis. In addition, we present the results of an in vitro experiment to provide an in-depth insight into the capacity of ACPs in (i) treating residual biofilm and (ii) preventing bacterial recolonisation. Materials and methods: In the first stage, we used a titanium LISS plate coated with hand-mixed PALACOS with vancomycin (PAL-V) for temporary internal fixation combined with commercially prepared COPAL with gentamicin and vancomycin (COP-GV) to fill the segmental defect. In the second stage, the non-union was treated with double-plate fixation and bone grafting.A Kirby-Bauer agar disc diffusion assay was performed to determine the antimicrobial activity of both ACPs and a drug-release assay to measure antibiotic release over time. A biofilm killing assay was also carried out to determine if the antibiotic released was able to reduce or eradicate biofilm of the patient's MRSA strain. Results: At one-year follow-up, there was complete bone-bridging across the previous non-union. The patient was pain-free and ambulatory without need for further surgery. Both ACPs with COP-GV and PAL-V exerted an antimicrobial effect against the MRSA strain with peak concentrations of antibiotic released within the first 24 hours. Concentrations released from COP-GV in the first 24 hours in vitro caused a 7.7-fold log reduction of colony-forming units (CFU) in the biofilm. At day 50, both COP-GV and PAL-V still released concentrations of antibiotic above the respective minimal inhibitory concentrations (MIC), likely contributing to the positive clinical outcome. Conclusion: The use of an ACP provides stability and infection control in the clinical scenario of an infected non-union. This is confirmed in vitro where the release of antibiotics from ACPs is characterised by an early burst followed by a prolonged sustained release above the MIC until 50 days. The burst release from COP-GV reduces CFU in the biofilm and prevents early recolonisation through synergistic activity of the released vancomycin and gentamicin. Clinical significance: An antibiotic-impregnated cement-coated plate is a useful addition to the surgeon's armamentarium to provide temporary internal fixation without the disadvantages of external fixation and contribute to infection control in an infected non-union. How to cite this article: Wagner RK, Guarch-Pérez C, van Dam AP, et al. Antimicrobial Mechanisms and Preparation of Antibiotic-impregnated Cement-coated Locking Plates in the Treatment of Infected Non-unions. Strategies Trauma Limb Reconstr 2023;18(2):73-81.

Monitoring local delivery of vancomycin from gelatin nanospheres in zebrafish larvae.

BACKGROUND: Infections such as biomaterial-associated infection and osteomyelitis are often associated with intracellular survival of bacteria (eg, Staphylococcus aureus). Treatment of these infections remains a major challenge due to the low intracellular efficacy of many antibiotics. Therefore, local delivery systems are urgently required to improve the therapeutic efficacy of antibiotics by enabling their intracellular delivery. PURPOSE: To assess the potential of gelatin nanospheres as carriers for local delivery of vancomycin into macrophages of zebrafish larvae in vivo and into THP-1-derived macrophages in vitro using fluorescence microscopy. MATERIALS AND METHODS: Fluorescently labeled gelatin nanospheres were prepared and injected into transgenic zebrafish larvae with fluorescent macrophages. Both the biodistribution of gelatin nanospheres in zebrafish larvae and the co-localization of vancomycin-loaded gelatin nanospheres with zebrafish macrophages in vivo and uptake by THP-1-derived macrophages in vitro were studied. In addition, the effect of treatment with vancomycin-loaded gelatin nanospheres on survival of S. aureus-infected zebrafish larvae was investigated. RESULTS: Internalization of vancomycin-loaded gelatin nanospheres by macrophages was observed qualitatively both in vivo and in vitro. Systemically delivered vancomycin, on the other hand, was hardly internalized by macrophages without the use of gelatin nanospheres. Treatment with a single dose of vancomycin-loaded gelatin nanospheres delayed the mortality of S. aureus-infected zebrafish larvae, indicating the improved therapeutic efficacy of vancomycin against (intracellular) S. aureus infection in vivo. CONCLUSION: The present study demonstrates that gelatin nanospheres can be used to facilitate local and intracellular delivery of vancomycin.

Nebulized antithrombin limits bacterial outgrowth and lung injury in Streptococcus pneumoniae pneumonia in rats.

INTRODUCTION: Disturbed alveolar fibrin turnover is a cardinal feature of severe pneumonia. Clinical studies suggest that natural inhibitors of coagulation exert lung-protective effects via anticoagulant and possibly also anti-inflammatory pathways. Intravenous infusion of the natural anticoagulants increases the risk of bleeding. Local administration may allow for higher treatment dosages and increased local efficacy while at the same time reducing the risk of bleeding. We evaluated the effect of nebulized anticoagulants on pulmonary coagulopathy and inflammation in a rat model of Streptococcus pneumoniae pneumonia. METHODS: In this randomized controlled in vivo laboratory study rats were challenged intratracheally with S. pneumoniae, inducing pneumonia, and randomized to treatment with normal saline (placebo), recombinant human activated protein C (rh-APC), plasma-derived antithrombin (AT), heparin or danaparoid, by means of nebulization. RESULTS: S. pneumoniae infection increased pulmonary levels of thrombin-antithrombin complexes and fibrin degradation products. All nebulized anticoagulants significantly limited pulmonary coagulopathy. None of the agents except danaparoid resulted in changes in systemic coagulopathy. Treatment with plasma-derived AT reduced outgrowth of S. pneumoniae and histopathologic damage in lungs. In vitro experiments confirmed outgrowth was reduced in bronchoalveolar lavage fluid (BALF) from rats treated with plasma-derived AT compared with placebo. Neutralizing of cationic components in BALF diminished the inhibitory effects on bacterial outgrowth of BALF, suggesting a role for cationic antimicrobial proteins. CONCLUSIONS: Nebulization of anticoagulants attenuates pulmonary coagulopathy during S. pneumoniae pneumonia in rats while only danaparoid affects systemic coagulation. Nebulized plasma-derived AT reduces bacterial outgrowth and exerts significant lung-protective effects.