Galleria mellonella as an alternative in vivo model to study implant-associated fungal infections.

Fungal implant-associated bone infections are rare but difficult to treat and often associated with a poor outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play a major role to study biofilm development and potential new treatment options, however, there are only a very few in vivo models to study fungi-associated biofilms. Furthermore, mammalian infection models are replaced more and more due to ethical restrictions with other alternative models in basic research. Recently, we developed an insect infection model with Galleria mellonella larvae to study biofilm-associated infections with bacteria. Here, we further expanded the G. mellonella model to study in vivo fungal infections using Candida albicans and Candida krusei. We established a planktonic and biofilm-implant model to test different anti-fungal medication with amphotericin B, fluconazole and voriconazole against the two species and assessed the fungal biofilm-load on the implant surface. Planktonic infection with C. albicans and C. krusei showed the killing of the G. mellonella larvae at 5x10 5 CFU. Treatment of larvae with anti-fungal compounds with amphotericin B and fluconazole showed significant survival improvement against planktonic C. albicans infection, but voriconazole had no effect. Titanium and stainless steel K-wires were pre-incubated with C. albicans and implanted inside the larvae to induce the biofilm infection on the implant surface. The survival analysis revealed significantly reduced survival of the larvae with Candida spp. infection compared to non-infected implants. The treatment with antifungal amphotericin B and fluconazole resulted in a slight and non-significant improvement survival of the larvae. The treatment with the anti-fungal compounds in the biofilm-infection model was not as effective as in the planktonic infection model, which highlights the resistance of fungal biofilms to anti-fungal compounds like in bacterial biofilms. Scanning electron microscopy (SEM) analysis revealed the formation of a fungal biofilm with hyphae and spores associated with larvae tissue on the implant surface. Thus, our study highlights the use of G. mellonella larvae as alternative in vivo model to study biofilm-associated implant fungal infections and that fungal biofilms exhibit high resistance profiles comparable to bacterial biofilms. The model can be used in the future to test anti-fungal treatment options for fungal biofilm infections. This article is protected by copyright. All rights reserved.