Galleria mellonella larva model in evaluating the effects of biofilm in Candida albicans

Abstract Biofilm-related infections are chronic infections that cause serious increase in morbidity and mortality as well as significant economic loss. Galleria mellonellalarva is shown as a reliable animal model for in vivo toxicology and pathogenicity tests due to its large size, ease of practice, ability to survive at 15-37°C and its similarity to mammals' natural immune system. The aim of this study was to evaluate the effects biofilm activity of Candida albicans in a G.mellonella larva model. Two C.albicans strains isolated as a disease agent were used for the model, where one was positive (BP), and the other one was negative (BN) for biofilm production. Eighty healthy G.mellonella larvae, all in the last larval stage and 2-2.5 cm long, were divided into 4 groups of equal size. Group 1 was set as the control group. Group 2 was injected with sterile phosphate buffer (PBS) group. Group 3 was injected with BP C.albicans strain and group 4 with BN C.albicans strain. A 5 μL volume of C.albicans prepared at 5 × 10(5) cfu/ml concentration with PBS was injected into the last left rear-legs of the larvae. The larvae were kept in sterile petri dishesat 37°C. They were observed for a total of 96 hours, for 4 hours in the first 24 hours, then in 12 hours intervals. Melanization, survival, total hemocytecount and fungal burden were evaluated as infection indicators. Melanizationand death were not observed throughout the study period in group 1. One larva died in group 2. Small melanizationspots (dark spots) and subsequent progressive melanizationwere observed from 3rd hour in the larvae infected with C.albicans. When compared with the BN C.albicans infected group, survival rate was 20% for BP C.albicans infected larvae at the end of 24 hours. Total hemocytecount was very low in the infected groups compared to groups 1 and 2, also significantly lower in group 3 than in group 4. In quantitative cultures, growth of C.albicans was detected in groups 3 and 4 while not in groups 1 and 2. Fungal load was significantly higher in BP C.albicans infected group than BN C.albicans infected group. In this study, G.mellonella larvae were used as live hosts to demonstrate the effects of biofilm activity of C.albicans. Our results suggest that larval models can be used to investigate the effects of fungal infections and biofilm like virulence factors on host cells, and invertebrate animal models can be widely used and can bridge between in vitro studies and mammalian models.