In the fungal pathogen Aspergillus fumigatus, resistance to azole antifungals is often linked to mutations in CYP51A, a gene that encodes the azole antifungal drug target lanosterol 14α-demethylase. The aim of this study was to investigate whether similar changes could be associated with azole resistance in a Malaysian Fusarium solani species complex (FSSC) isolate collection. Most (11 of 15) clinical FSSC isolates were Neocosmospora keratoplastica and the majority (6 of 10) of environmental isolates were Neocosmospora suttoniana strains. All 25 FSSC isolates had high minimum inhibitory concentrations (MICs) for itraconazole and posaconazole, low MICs for amphotericin B, and various (1 to >32 mg/l) voriconazole susceptibilities. There was a tight association between a 23 bp CYP51A promoter deletion and high (>32 mg/l) voriconazole MICs; of 19 FSSC strains sequenced, nine isolates had voriconazole MICs > 32 mg/l, and they all contained the 23 bp CYP51A promoter deletion, although it was absent in the ten remaining isolates with low (≤12 mg/l) voriconazole MICs. Surprisingly, this association between voriconazole resistance and the 23 bp CYP51A promoter deletion held true across species boundaries. It was randomly distributed within and across species boundaries and both types of FSSC isolates were found among environmental and clinical isolates. Three randomly selected N. keratoplastica isolates with low (≤8 mg/l) voriconazole MICs had significantly lower (1.3-7.5 times) CYP51A mRNA expression levels than three randomly selected N. keratoplastica isolates with high (>32 mg/l) voriconazole MICs. CYP51A expression levels, however, were equally strongly induced (∼6,500-fold) by voriconazole in two representative strains reaching levels, after 80 min of induction, that were comparable to those of CYP51B. Our results suggest that FSSC isolates with high voriconazole MICs have a 23 bp CYP51A promoter deletion that provides a potentially useful marker for voriconazole resistance in FSSC isolates. Early detection of possible voriconazole resistance is critical for choosing the correct treatment option for patients with invasive fusariosis.

Fusarium keratoplasticum is arguably the most common Fusarium solani species complex (FSSC) species associated with human infections. Invasive fusariosis is a life-threatening fungal infection that is difficult to treat with conventional azole antifungals. Azole drug resistance is often caused by the increased expression of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG sub-family. Most investigations of Fusarium ABC transporters associated with azole antifungal drug resistance are limited to plant pathogens. Through the manual curation of the entire ABCG protein family of four FSSC species including the fully annotated genome of the plant pathogen Nectria haematococca we identified PDR transporters ABC1 and ABC2 as the efflux pump candidates most likely to be associated with the innate azole resistance phenotype of Fusarium keratoplasticum. An initial investigation of the transcriptional response of logarithmic phase F. keratoplasticum cells to 16 mg/L voriconazole confirmed strong upregulation (372-fold) of ABC1 while ABC2 mRNA levels were unaffected by voriconazole exposure over a 4 h time-period. Overexpression of F. keratoplasticum ABC1 and ABC2 in the genetically modified Saccharomyces cerevisiae host ADΔΔ caused up to ∼1,024-fold increased resistance to a number of xenobiotics, including azole antifungals. Although ABC1 and ABC2 were only moderately (20% and 10%, respectively) expressed compared to the Candida albicans multidrug efflux pump CDR1, overexpression of F. keratoplasticum ABC1 caused even higher resistance levels to certain xenobiotics (e.g., rhodamine 6G and nigericin) than CDR1. Our investigations suggest an important role for ABC1 orthologues in the innate azole resistance phenotype of FSSC species.

Neoscytalidium dimidiatum is an opportunistic fungus causing cutaneous infections mostly, which are difficult to treat due to antifungal resistance. In Malaysia, N. dimidiatum is associated with skin and nail infections, especially in the elderly. These infections may be mistaken for dermatophyte infections due to similar clinical appearance. In this study, Neoscytalidium isolates from cutaneous specimens, identified using morphological and molecular methods (28 Neoscytalidium dimidiatum and 1 Neoscytalidium sp.), were evaluated for susceptibility towards antifungal agents using the CLSI broth microdilution (M38-A2) and Etest methods. Amphotericin B, voriconazole, miconazole and clotrimazole showed high in vitro activity against all isolates with MIC ranging from 0.0313 to 1 µg/mL. Susceptibility towards fluconazole and itraconazole was noted in up to 10% of isolates, while ketoconazole was inactive against all isolates. Clinical breakpoints for antifungal drugs are not yet available for most filamentous fungi, including Neoscytalidium species. However, the results indicate that clinical isolates of N. dimidiatum in Malaysia were sensitive towards miconazole, clotrimazole, voriconazole and amphotericin B, in vitro.

Fusarium infections in humans (fusariosis) and in economically important plants involve species of several Fusarium species complexes. Species of the Fusarium solani species complex (FSSC) are the most frequent cause of human fusariosis. The FSSC comprises more than 60 closely related species that can be separated into three major clades by multi-locus sequence typing (MLST) using translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II (RPB2) DNA sequences. The MLST nomenclature for clade 3 of the FSSC assigns numbers to species types (e.g., FSSC 2) and lowercase letters to identify unique haplotypes. The aim of this study was to analyse the genotypic and phenotypic characteristics of 15 environmental and 15 clinical FSSC isolates from Malaysia. MLST was used for the genotypic characterisation of FSSC isolates from various locations within Malaysia, which was complemented by their morphological characterisation on potato dextrose and carnation leaf agar. MLST identified eight different FSSC species: thirteen Fusarium keratoplasticum (i.e., FSSC 2), six Fusarium suttonianum (FSSC 20), five Fusarium falciforme (FSSC 3+4), two Fusarium cyanescens (FSSC 27), and one each of Fusarium petroliphilum (FSSC 1), Fusarium waltergamsii (FSSC 7), Fusarium sp. (FSSC 12), and Fusarium striatum (FSSC 21). Consistent with previous reports from Malaysia, most (11 of 15) clinical FSSC isolates were F. keratoplasticum and the majority (9 of 15) of environmental isolates were F. suttonianum (5) or F. falciforme (4) strains. The taxonomic relationships of the isolates were resolved phylogenetically. The eight Fusarium species also showed distinct morphological characteristics, but these were less clearly defined and reached across species boundaries. Although TEF1-α and RPB2 sequences were sufficient for the species identification of most FSSC isolates, a more precise MLST scheme needs to be established to reliably assign individual isolates of the species-rich FSSC to their geographically-, epidemiologically-, and host-associated sub-lineages.