Pioneering preclinical models to study mixed triazole-susceptible and triazole-resistant Aspergillus fumigatus infections

Author:

Agustin Resendiz Sharpe (BE)

Abstract:

Background: Invasive aspergillosis from Aspergillus fumigatus is a significant clinical challenge, with mortality rates of 20-35%, exacerbated by rising triazole resistance leading to more treatment failures. The coexistence of triazole-susceptible (WT) and triazole-resistant (TR) A. fumigatus strains in the environment, driven by triazole fungicide use, raises concerns about mixed infections resulting from inhalation of both strain types. Despite clinical reports, research on mixed A. fumigatus infections is limited and our understanding on the dynamic interplay between triazole-susceptible and resistant strains and their roles in disease development and treatment responses remains largely unknown. Therefore, to address these research needs, we introduce an innovative preclinical framework to investigate mixed A. fumigatus infections using an in vitro and an in vivo model, namely Galleria mellonella.
Methods: The developed in vitro and Galleria mellonella models for studying mixed A. fumigatus infections incorporated various quantitative methodologies for monitoring the collective and individual burdens of WT and TR A. fumigatus while tracking disease progression. To achieve this, we modified conventional single-time assessment methods, including colony-forming unit (CFU) counts (utilizing agar plates with or without triazole antifungals to isolate resistant fungi only) and qPCR (focused on the quantification of either all A. fumigatus or exclusively TR A. fumigatus, correlated to a standard curve). Additionally, we introduced a novel noninvasive bioluminescence imaging (BLI) approach to monitor mixed fungal burdens over time. Experimental conditions included single or mixed scenarios involving WT and TR strains: WT or TR only, or a combination of WT with either 0.5 log10 (33%), 1 log10 (10%) or 2 log10 (1%) of TR strain within the total inoculum using bioluminescent (BL) and no-BL strains as depicted in Figure 1. The in vitro assay followed the EUCAST broth microdilution methodology (medium and inoculum specifications).
Results: BLI proved highly effective in vitro and in vivo, offering unique insights into fungal burden kinetics and TR A. fumigatus dynamics. It excelled in distinguishing TR from WT strains with a wide dynamic detection range (Figures 2A-B, 3A-B). Our novel qPCR method also successfully discriminated TR A. fumigatus, especially with pronounced log-differences in conidia equivalents (CE) concentration between WT and TR strains (Figure 2C and 3C). In contrast, in vitro CFU counts were unreliable in mixed samples, with high variability (Figure 2D). In Galleria mellonella, CFU counts showed reduced variability (Figure 3D), but log-differences between WT and TR strains were again less robust as in the other methodologies were log-differences could be easily observed. Notably, WT A. fumigatus exhibited growth on itraconazole-containing plates, suggesting limited interaction between larval samples and the antifungal, allowing both WT and TR conidia proliferation. Thus, CFU counts were unsuitable for distinguishing strains in this model.
Conclusions: Our study presents innovative in vitro and in vivo mixed models of A. fumigatus infection featuring BLI and qPCR, which successfully discriminate between wild-type and triazole-resistant strains. Our models provide a unique platform to gain in-depth insights into the interactions between triazole-susceptible and resistant A. fumigatus, disease development, and antifungal treatment responses offering valuable methodologies readily translated to mouse models.

Abstract Number: 6

Conference Year: 2024

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