Mellado, E. | Search bioRxiv

bioRxiv2025

Establishing thresholds for azole tolerance and persistence in Aspergillus fumigatus to study their impact on voriconazole treatment in vivo

Antimicrobial tolerance and persistence are phenomena that enable pathogenic microbes to survive for extended periods in the presence of high concentrations of cidal drugs. Current evidence suggests that these phenomena may contribute to treatment failure and could even lead to the development of resistance. However, our understanding of antifungal tolerance and persistence in Aspergillus fumigatus, as well as their potential role in therapeutic failure, remains limited. In this study, we present an optimized, easy-to-perform method for detecting A. fumigatus tolerance and persistence to azole antifungals, based on a single colony-forming unit (CFU) measurement. Additionally, we developed a microscopic approach to investigate the dynamics of conidial killing in medium-throughput assays. Using these methods, we established epidemiological threshold values to classify strains as tolerant or persister and applied them to screen and categorize a collection of clinical isolates. Furthermore, we demonstrate that tolerance--but not persistence--negatively impacts the efficacy of voriconazole treatment in a Galleria mellonella infection model. Based on these findings, we propose that tolerance and persistence should be monitored in clinical isolates and potentially considered when determining therapeutic strategies.

microbiology↗

bioRxiv2022

Aspergillus fumigatus can exhibit persistence to the fungicidal drug voriconazole

Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung-diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non- mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A sub-population of the persister isolates can survive for extended periods and even grow at slow rates in the presence of supra-MIC (Minimum Inhibitory Concentration) of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seems to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. IMPORTANCEThe phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge about antifungal tolerance and, particularly, persistence are still very scarce. In this study, we have characterised the response of the prominent fungal pathogen Aspergillus fumigatus to the first line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and identify various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.

microbiology↗