Hyphae of Rhizopus arrhizus and Lichtheimia corymbifera Are More Virulent and Resistant to Antifungal Agents Than Sporangiospores In Vitro and in Galleria mellonella

Abstract

Mucorales 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, Galleria mellonella larvae were infected with either sporangiospores or hyphae of Rhizopus arrhizus and Lichtheimia corymbifera. Hyphal infections resulted in greater and more rapid larval lethality than sporangiospores, with an approximate 10–16-fold decrease in LD50 of hyphae for R. arrhizus (p = 0.03) and L. corymbifera (p = 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 Mucorales species for virulence testing and antifungal drug screening in vitro and in G. mellonella for studies of wound mucormycosis.

Development of a Combat-Relevant Murine Model of Wound Mucormycosis: A Platform for the Pre-Clinical Investigation of Novel Therapeutics for Wound-Invasive Fungal Diseases

“…It is unlikely that such a heavy conidial contamination of wounds of human patients could come from airborne sporangiospores. Lastly, we have recently shown that hyphae of R. arrhizus and L. corymbifera are 10 and 16 times more lethal in a G. mellonella infection model compared to sporangiospores [33]. Thus, due to a strong hypothesis of wound infection development with hyphae, these fungal elements were used in our study.…”

“…Glycerol stocks of the strains were initiated for sporangiospore production on synthetic modified mucor agar (SMMA) or potato dextrose agar (PDA) plates and incubated at 37 • C for 7 days. Sporangiospores were isolated using a previously described method [33].…”

“…Hyphae are a common element of fungal mycelial colonies inhabiting the soil and have been proposed to be the cause of WIFDs [15]. We recently demonstrated that hyphae have a 10-to 16-fold increase in virulence compared to sporangiospores in a Galleria mellonella model of traumatic mucormycosis [33]. Furthermore, we sought to validate our animal model and evaluate the efficacy of L-AmB against R. arrhizus, as it is typically utilized as an antifungal agent to mitigate the immediate threats posed by WIFDs.…”

Alternative in-vivo models of mucormycosis

“…One of the limitations of in vitro antifungal susceptibility testing is, that in standardized protocols, drug efficacy is tested against spores, a morphology rarely found on the site of infection. Lately, a single dose of amphotericin B (10 mg/kg) against spores or germlings of R. arrhizus and L. corymbifera was shown to be efficient only against infections with spores but not germlings, where all larvae died 24 h after infection, independently of receiving amphotericin B or not ( Samdavid Thanapaul et al., 2023 ).…”