A Madurella mycetomatis Grain Model in Galleria mellonella Larvae

Abstract

Eumycetoma is a chronic granulomatous subcutaneous infectious disease, endemic in tropical and subtropical regions and most commonly caused by the fungus Madurella mycetomatis. Interestingly, although grain formation is key in mycetoma, its formation process and its susceptibility towards antifungal agents are not well understood. This is because grain formation cannot be induced in vitro; a mammalian host is necessary to induce its formation. Until now, invertebrate hosts were never used to study grain formation in M. mycetomatis. In this study we determined if larvae of the greater wax moth Galleria mellonella could be used to induce grain formation when infected with M. mycetomatis. Three different M. mycetomatis strains were selected and three different inocula for each strain were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 4 mg/larvae. Larvae were monitored for 10 days. It appeared that most larvae survived the lowest inoculum, but at the highest inoculum all larvae died within the 10 day observation period. At all inocula tested, grains were formed within 4 hours after infection. The grains produced in the larvae resembled those formed in human and in mammalian hosts. In conclusion, the M. mycetomatis grain model in G. mellonella larvae described here could serve as a useful model to study the grain formation and therapeutic responses towards antifungal agents in the future.

Combining two antifungal agents does not enhance survival of Galleria mellonella larvae infected with Madurella mycetomatis

“…Galleria mellonella larvae were infected with M. mycetomatis isolate Mm55 according to the previously published protocol . In short, M. mycetomatis mycelia were cultured for 2 weeks at 37 °C in colourless RPMI 1640 medium supplemented with L‐glutamine (0.3 g/l), 20 m m morpholinepropanesulfonic acid (MOPS) and chloramphenicol (100 mg/l; Oxoid, Basingstoke, UK).…”

“…Five larvae from each group were sacrificed at day 3 post‐infection to determine the fungal burden. Hemolymph was collected and measured at 405 nm to assess melanisation . To assess the number of colony‐forming units (CFU) per larvae, 10 chrome steal metal balls and 1 ml PBS were added.…”

“…To assess the number of colony‐forming units (CFU) per larvae, 10 chrome steal metal balls and 1 ml PBS were added. Larvae were homogenised in a Qiagen TissueLyser for 5 min at 30 Hz . From each homogenised larvae, 250 μl undiluted, 50 μl undiluted and 50 μl 1:10 diluted suspensions were plated out on Sabouraud‐gentamicin agar.…”

Eumicetoma: actualidades y perspectivas

“…Sin embargo, en la actualidad no podemos considerar que los neutrófilos representen la única célula efectora involucrada, dado que la exposición de M. mycetomatis a los neutrófilos no produce la formación del grano en modelos in vitro. Como una alternativa para inducir la formación del grano se utilizó la larva de la polilla de la cera Galleria mellonella; los granos producidos en esta larva semejan los encontrados en humanos y mamíferos, y este modelo de infección podría utilizarse para el estudio de la formación del grano y las respuestas terapéuticas a los agentes antifúngicos en la infección por M. mycetomatis 3 . Por último, otros hongos descritos en eumicetomas, como especies de Aspergillus, Scedosporium y Fusarium, también reconocidos como agentes causantes de infecciones oportunistas, se estudiaron utilizando Drosophila melanogaster como hospedero y se encontró que el tratamiento con voriconazol protege a las moscas de la infección, a excepción de las infectadas por Scedosporium prolificans, que no mostraron mejoría 71,72 .…”

“…Muchos de los pacientes son campesinos u obreros de bajo nivel socioeconómico. Una característica distintiva del micetoma es la formación de los elementos parasitarios llamados «granos», que están constituidos por los microorganismos, probablemente como parte de un mecanismo de defensa contra el sistema inmunitario del hospedero 3 . Dado que una diversidad de microorganismos puede producir la enfermedad, se encuentra una gran variación morfológica en las caracterís-ticas de los granos, que se relacionan con el agente causal.…”