| Summary: | <i>Mucorales</i> species cause debilitating, life-threatening sinopulmonary diseases in immunocompromised patients and penetrating wounds in trauma victims. Common antifungal agents against mucormycosis have significant toxicity and are often ineffective. To evaluate treatments against mucormycosis, sporangiospores are typically used for in vitro assays and in pre-clinical animal models of pulmonary infections. However, in clinical cases of wound mucormycosis caused by traumatic inoculation, hyphal elements found in soil are likely the form of the inoculated organism. In this study, <i>Galleria mellonella</i> larvae were infected with either sporangiospores or hyphae of <i>Rhizopus arrhizus</i> and <i>Lichtheimia corymbifera</i>. Hyphal infections resulted in greater and more rapid larval lethality than sporangiospores, with an approximate 10–16-fold decrease in LD<sub>50</sub> of hyphae for <i>R. arrhizus</i> (<i>p</i> = 0.03) and <i>L. corymbifera</i> (<i>p</i> = 0.001). Liposomal amphotericin B, 10 mg/kg, was ineffective against hyphal infection, while the same dosage was effective against infections produced by sporangiospores. Furthermore, in vitro, antifungal susceptibility studies show that minimum inhibitory concentrations of several antifungal agents against hyphae were higher when compared to those of sporangiospores. These findings support using hyphal elements of <i>Mucorales</i> species for virulence testing and antifungal drug screening in vitro and in <i>G. mellonella</i> for studies of wound mucormycosis.
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