Objectives: Recent increases in triazole resistance in Aspergillus fumigatus have been attributed primarily to target site (cyp51A) mutations. A recent survey of resistant isolates in Manchester showed that .50% of resistant isolates had no mutation in cyp51A or its promoter. We investigated the mechanisms of resistance in clinical azole-resistant isolates without cyp51A mutations.Methods: Twelve azole-resistant isolates, 10 of which were itraconazole resistant, were studied. Bioinformatic comparisons between Candida albicans efflux genes and A. fumigatus genome data identified 20 putative azole transporter genes. Basal and azole-induced expression of these genes and cyp51A was quantified using RT-PCR with comparison with clinical azole-susceptible isolates. Function of high basal or itraconazole-induced expression transporters was tested by gene knockout in azole-susceptible and azole-resistant isolates.Results: All susceptible strains showed minimal basal expression of cdr1B compared with 8 of 10 azole-resistant strains with high basal expression of this gene (.5-fold), 3 of which showed .30-fold increased expression. Knockout of this gene resulted in a 4-fold reduction in itraconazole, posaconazole and voriconazole MICs for a susceptible clinical isolate and a 4-fold reduction in itraconazole susceptibility in a clinical resistant isolate. One strain showed a .500-fold induction of cyp51A. No increase in basal expression or expression after induction was seen for the 18 remaining putative transporters. Conclusions:The reasons behind the shift away from target site mutation in azole-resistant isolates from Manchester are unknown. The modest change in expression of cdr1B in azole-susceptible strains implies that only study of resistant isolates will lead to further understanding of resistance mechanisms in A. fumigatus.

The very low organism burdens of fungi causing infection have previously prevented direct culture and detection of antifungal resistance in clinical samples. These findings have major implications for the sustainability of triazoles for human antifungal therapy.

The frequency of antifungal resistance, particularly to the azole class of ergosterol biosynthetic inhibitors, is a growing global health problem. Survival rates for those infected with resistant isolates are exceptionally low. Beyond modification of the drug target, our understanding of the molecular basis of azole resistance in the fungal pathogen Aspergillus fumigatus is limited. We reasoned that clinically relevant antifungal resistance could derive from transcriptional rewiring, promoting drug resistance without concomitant reductions in pathogenicity. Here we report a genome-wide annotation of transcriptional regulators in A. fumigatus and construction of a library of 484 transcription factor null mutants. We identify 12 regulators that have a demonstrable role in itraconazole susceptibility and show that loss of the negative cofactor 2 complex leads to resistance, not only to the azoles but also the salvage therapeutics amphotericin B and terbinafine without significantly affecting pathogenicity.