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Objectives and questions: Periprosthetic infections caused by vancomycin-resistant pathogens, such as vancomycin-resistant Enterococcus faecalis (VRE), pose a significant clinical challenge. This study aimed to utilize the Galleria mellonella larvae implant infection model to determine the optimal daptomycin dosage for incorporation into different PMMA cements and evaluate its efficacy.

Material and methods: Daptomycin (1 g or 2 g) was added to different commercially available PMMA cements, including Simplex T and PALACOS R+G. Mechanical stability was assessed by four-point bending strength, modulus, and compressive strength according to ISO 5833, along with bending and impact strength per DIN 53435. Additionally, in vitro assays, including bacterial proliferation and inhibition zone tests, were conducted.

For in vivo analysis, rod-shaped bone cement implants were produced using a Teflon mould. In a preventive approach, implants were inserted into G. mellonella larvae, followed by VRE infection one hour later. Antimicrobial efficacy was also evaluated in a treatment model, where larvae were first injected with VRE, followed by implant insertion 3 h post infection. Larval survival was monitored over time, and bacterial loads in larval tissue and on the implant surface were quantified after 24 h.

Results: Adding more than 1.5 g of daptomycin to Simplex T reduced its mechanical strength below the ISO standard of 50 MPa for four-point bending strength. In vitro, both 1 g and 2 g of daptomycin effectively inhibited VRE growth on day 1, with the largest inhibition zone observed for PALACOS R+G with 2 g of daptomycin. By day 42, significant bacterial growth inhibition was seen only with 2 g of daptomycin. Inhibition zone test results correlated with bacterial proliferation assay findings.

Survival and bacterial burden analyses in both models demonstrated that adding 1 g or 2 g of daptomycin effectively prevented VRE infections, significantly improving larval survival and reducing bacterial numbers in larval tissue and on the implant surface, regardless of PMMA bone cement type.

Discussion and conclusions: Daptomycin incorporation into ALBC successfully prevented VRE infections in an <em>in vivo</em> model. These findings highlight the G. mellonella implant infection model as a valuable tool for evaluating ALBC efficacy against multidrug-resistant bacteria, thereby accelerating preclinical research and advancing strategies to combat periprosthetic infections.