Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn₂-Cys₆ type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B). Better survival of chronically ill patients has produced an increased number of cases involving fungal infectious disease. This is partly due to a larger number of immunocompromised patients. Meanwhile, the catalogue of antifungal drugs remains quite limited and the appearance of resistant isolates is becoming more common. The filamentous fungus Aspergillus fumigatus is the main causative pathogen for deep-seated aspergillosis. For this fungus, resistance to azoles, which are the most commonly utilized drug for treatment of aspergillosis, has exhibited an alarming increase linked with elevated mortality. We know relatively little of the molecular details underpinning azole resistance in A. fumigatus other than alterations in the target enzyme. Here, we identified and characterized a novel transcription factor AtrR that is required for wild-type azole resistance in this fungus as well as other Aspergillus fungi, such as Aspergillus oryzae and Aspergillus nidulans. We discovered that AtrR co-regulates the target and pump, both of which are essential for azole resistance. More importantly, deletion of the atrR gene could sensitize a clinically isolated multi-azole resistant strain and exhibited a virulence defect in a mouse model. This success in overcoming an existing resistance mutation provides a new avenue for the prevention and treatment of aspergillosis.

Aspergillus fumigatus is known to produce various immunosuppressive mycotoxins including gliotoxin. However, none of these mycotoxins has been confirmed as being directly related to the pathogenesis of aspergilli. Recent studies have made substantial progress in the determination of mycotoxins as virulence factors. Gliotoxin was found to be produced much faster than previously believed under certain culture conditions, such as at 37°C and under high oxygen content, which is close to the environment in the host. Gliotoxin was also found to be detectable in the sera of aspergillosis mice and of aspergillosis patients. Based on these findings, it is becoming evident that gliotoxin is produced in the infected organs of patients of aspergillosis at a significant level. In addition to these known mycotoxins, A. fumigatus produces many mycotoxins apparently different from known toxins. From the aspect of gene analysis, the deletion of laeA was found to block the expression of metabolic gene clusters such as sterigmatocystin, and the gene is also expected to be related to the production of gliotoxin. The significance of mycotoxins as virulence factors will hopefully be clarified in the near future.

From 2006 to 2013, an increasing incidence of fusariosis was observed in the hematologic patients of our University Hospital. We suspected of an environmental source, and the indoor hospital air was investigated as a potential source of the fungemia. Air samplings were performed in the hematology and bone marrow transplant (BMT) wards using an air sampler with pre-defined air volumes. To study the molecular relationship among environmental and clinical isolates, 18 Fusarium spp. recovered from blood cultures were included in the study. DNA sequencing of a partial portion of TEF1α gene was performed for molecular identification. Molecular typing was carried out by multi-locus sequence typing (MLST) using a four-gene scheme: TEF1α, rDNA, RPB1 and RPB2. One hundred four isolates were recovered from the air of the hematology (n = 76) and the BMT (n = 28) wards. Fusarium isolates from the air were from five species complexes: Fusarium fujikuroi (FFSC, n = 56), Fusarium incarnatum-equiseti (FIESC, n = 24), Fusarium solani (FSSC, n = 13), Fusarium chlamydosporum (FCSC, n = 10), and Fusarium oxysporum (FOSC, n = 1). Fifteen Fusarium isolates recovered from blood belonged to FSSC, and three to FFSC. MLST identified the same sequence type (ST) in clinical and environmental isolates. ST1 was found in 5 isolates from blood and in 7 from the air, both identified as FSSC (Fusarium petroliphilum). STn1 was found in one isolate from blood and in one from the air, both identified as FFSC (Fusarium napiforme). F. napiforme was isolated from the air of the hospital room of the patient with fungemia due to F. napiforme. These findings suggested a possible clonal origin of the Fusarium spp. recovered from air and bloodcultures. In conclusion, our study found a diversity of Fusarium species in the air of our hospital, and a possible role of the air as source of systemic fusariosis in our immunocompromised patients.

Scedosporium apiospermum is increasingly recognized as a cause of localized and disseminated mycotic infections in near-drowning victims.

This study was designed to analyze the interaction of 21 antifungal combinations consisting of seven major antifungal agents against 11 echinocandin- susceptible and six-resistant C. glabrata isolates. The combinations were divided into five major groups and were evaluated by checkerboard, disc diffusion, and time-killing assays. Synergy based on the fractional inhibitory concentration index of ≤0.50 was observed in 17.65–29.41% of the cases for caspofungin combinations with azoles or amphotericin B. Amphotericin B combination with azoles induced synergistic interaction in a range of 11.76–29.41%. Azole combinations and 5-flucytosine combinations with azoles or amphotericin B did not show synergistic interactions. None of the 21 combinations showed antagonistic interactions. Interestingly, 90% of the detected synergism was among the echinocandin-resistant isolates. Disk diffusion assays showed that the inhibition zones produced by antifungal combinations were equal to or greater than those produced by single drugs. The time-killing assay showed the synergistic action of caspofungin combination with fluconazole, voriconazole, and posaconazole, and the amphotericin B-5-flucytosine combination. Furthermore, for the first time, this assay confirmed the fungicidal activity of caspofungin-voriconazole and amphotericin B-5-flucytosine combinations. The combination interactions ranged from synergism to indifference and, most importantly, no antagonism was reported and most of the synergistic action was among echinocandin-resistant isolates.