Aspergillus fumigatus Can Display 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 subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed 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. IMPORTANCE The 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 of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized 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 propose 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.

Horizontal Gene Transfer of Triazole Resistance in Aspergillus fumigatus.

Aspergillus fumigatus is the primary mold pathogen in humans. It can cause a wide range of diseases in humans, with high mortality rates in immunocompromised patients. The first-line treatments for invasive A. fumigatus infections are the triazole antifungals that inhibit Cyp51 lanosterol demethylase activity, blocking ergosterol biosynthesis. However, triazole-resistant strains of A. fumigatus are increasingly encountered, leading to increased mortality. The most common triazole resistance mechanisms in A. fumigatus are alterations in the cyp51A gene or promoter. We tested the hypothesis that A. fumigatus can acquire triazole resistance by horizontal gene transfer (HGT) of resistance-conferring gene cyp51A. HGT has not been experimentally analyzed in filamentous fungi. Therefore, we developed an HGT assay containing donor A. fumigatus strains carrying resistance-conferring mutated cyp51A, either in its chromosomal locus or in a self-replicating plasmid, and recipient strains that were hygromycin resistant and triazole sensitive. Donor and recipient A. fumigatus strains were cocultured and transferred to selective conditions, and the recipient strain tested for transferred triazole resistance. We found that chromosomal transfer of triazole resistance required selection under both voriconazole and hygromycin, resulting in diploid formation. Notably, plasmid-mediated transfer was also activated by voriconazole or hypoxic stress alone, suggesting a possible route to HGT of antifungal resistance in A. fumigatus, both in the environment and during host infection. This study provides, for the first time, preliminary experimental evidence for HGT mediating antifungal resistance in a pathogenic fungus. IMPORTANCE It is well known that bacteria can transfer antibiotic resistance from one strain to another by horizontal gene transfer (HGT), leading to the current worldwide crisis of rapidly emerging antibiotic-resistant bacteria. However, in fungi, HGT events have only been indirectly documented by whole-genome sequencing. This study directly examined fungal HGT of antibiotic resistance in a laboratory setting. We show that HGT of antifungal triazole resistance occurs in the important human fungal pathogen Aspergillus fumigatus. Importantly, we show a plasmid-mediated transfer of triazole resistance occurs under conditions likely to prevail in the environment and in infected patients. This study provides an experimental foundation for future work identifying the drivers and mechanistic underpinnings of HGT in fungi.

Triazole-Resistant Aspergillus fumigatus in an Israeli Patient with Chronic Cavitary Pulmonary Aspergillosis Due to a Novel E306K Substitution in Hmg1.

Triazole resistance in the pathogenic mold Aspergillus fumigatus has increased worldwide, posing a growing therapeutic challenge. Recently, mutations in the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase gene (hmg1) have been associated with triazole resistance. Here, we describe a novel E306K triazole resistance-conferring mutation in the HMG-CoA reductase gene from an Israeli patient with chronic cavitary pulmonary aspergillosis (CCPA).