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

Abstract

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 antifungal 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 5 × 105 colony forming units (CFU). Treatment of larvae with antifungal 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 preincubated with C. albicans and implanted inside the larvae to induce biofilm infection on the implant surface. The survival analysis revealed significantly reduced survival of the larvae with Candida spp. infection compared to noninfected implants. The treatment with antifungal amphotericin B and fluconazole resulted in a slight and nonsignificant improvement survival of the larvae. The treatment with the antifungal compounds in the biofilm‐infection model was not as effective as in the planktonic infection model, which highlights the resistance of fungal biofilms to antifungal 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 antifungal treatment options for fungal biofilm infections.

Galleria mellonella as a Model for the Study of Fungal Pathogens: Advantages and Disadvantages

“…G. mellonella has been used to study many fungi, including Aspergillus spp., Candida spp., Cryptococcus spp., Conidiobolus coronatus , Histoplasma capsulatum , Madurella mycetomatis , Malassezia spp. and Paraccocidioides brasiliensis [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 ].…”

“…Categorising studies using G. mellonella larvae to assess virulence or drug efficacy is a challenging task. It is more appropriate to evaluate the work based on different fungal genera since some species have undergone extensive research in recent years [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 ].…”

“…The research team utilised a planktonic and biofilm-implant model to test various antifungal drugs, namely amphotericin B, fluconazole and voriconazole, against the two species and assessed the fungal biofilm load on the implant surface. This investigation aimed to evaluate the efficacy of antifungal drugs in treating fungal infections associated with implants [ 72 ].…”

A promising antibiotic candidate, brevinin-1 analogue 5R, against drug-resistant bacteria, with insights into its membrane-targeting mechanism

“…Effects of the peptide on the in vivo biofilm-associated infection model were evaluated according to the method in Gopala et al's work [33,34]. Titanium K-wires with a diameter of 0.8 mm (Sigma, UK) were cut and sharpened to a length of 4-5 mm as the implant.…”

The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae

“…G. mellonella serves as a model host for human pathogens such as Bacillus cereus ( 5 ) , Candida albicans ( 6 , 7 ), Listeria monocytogenes ( 8 ), and Staphylococcus aureus ( 9 ). Additionally, this insect is a popular model in research on entomopathogenic fungi activity ( 10 ) like Conidiobolus coronatus (Entomophthorales), a soil fungus pathogenic to insects and occasionally humans.…”