Discovery and functional characterisation of novel combination therapies to combat drug-resistant Candida auris and Aspergillus fumigatus infections

Abstract

Antifungal drug resistance in Candida auris and Aspergillus fumigatus presents significant challenges to treatment due to the limited availability of antifungal drugs. This research explores the potential of combination therapies to enhance treatment efficacy against these pathogens and delay resistance development. Using checkerboard assays, the in vitro combinatorial effects of established antifungals – amphotericin B, voriconazole, itraconazole, anidulafungin, 5- flucytosine – and emerging antifungals – manogepix and olorofim – was tested against isolates of both pathogens having varied antifungal susceptibilities. Promising combinations were further assessed for their impact on fungal growth using microfluidics-assisted live-cell imaging. In vivo efficacy was evaluated in a systemic C. auris infection model and a pulmonary aspergillosis model in mice. Additionally, resistance development under short and long-term exposure to these combinations was compared to monotherapy and transcriptomics analysis was conducted to elucidate the mechanistic basis of the observed synergy. For C. auris, combinations involving amphotericin B with manogepix or 5- flucytosine and combinations of manogepix with voriconazole or 5-flucytosine generally exhibited additive or indifferent interactions, whereas the combination of voriconazole with 5-flucytosine displayed mostly indifferent or antagonistic interactions. A. fumigatus responded additively or indifferently to combinations of olorofim with amphotericin B, manogepix, 5-flucytosine or anidulafungin, while combinations with itraconazole or voriconazole were often antagonistic. Notably, the combinations of anidulafungin with manogepix or 5-flucytosine demonstrated synergy against both pathogens, confirmed by significant inhibition of fungal growth and colony size reduction during microfluidics-assisted live-cell imaging. In vivo, the anidulafungin and 5-flucytosine combination significantly decreased fungal burdens in C. auris-infected mice, whereas results in the aspergillosis model showed mixed outcomes. Both anidulafungin combinations showed a protective effect against the development of anidulafungin resistance under short-term exposure, contrasting with increased resistance to 5-flucytosine and manogepix after extended exposure.