Eumycetoma is a chronic granulomatous subcutaneous infectious disease, endemic in tropical and subtropical regions and most commonly caused by the fungus Madurella mycetomatis. Interestingly, although grain formation is key in mycetoma, its formation process and its susceptibility towards antifungal agents are not well understood. This is because grain formation cannot be induced in vitro; a mammalian host is necessary to induce its formation. Until now, invertebrate hosts were never used to study grain formation in M. mycetomatis. In this study we determined if larvae of the greater wax moth Galleria mellonella could be used to induce grain formation when infected with M. mycetomatis. Three different M. mycetomatis strains were selected and three different inocula for each strain were used to infect G. mellonella larvae, ranging from 0.04 mg/larvae to 4 mg/larvae. Larvae were monitored for 10 days. It appeared that most larvae survived the lowest inoculum, but at the highest inoculum all larvae died within the 10 day observation period. At all inocula tested, grains were formed within 4 hours after infection. The grains produced in the larvae resembled those formed in human and in mammalian hosts. In conclusion, the M. mycetomatis grain model in G. mellonella larvae described here could serve as a useful model to study the grain formation and therapeutic responses towards antifungal agents in the future.

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter ؊1 of malate at a yield of 0.42 mol (mol glucose) ؊1 in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO 3 , was required for efficient C 4 dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO 3 dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose) ؊1, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO 2 , calcium, and O 2 ). Under optimized conditions, a malate yield of 0.48 ؎ 0.01 mol (mol glucose) ؊1 was obtained in bioreactors, a 19% increase over yields in shake flask experiments.

Mycetoma is a tropical neglected disease characterized by large subcutaneous lesions in which the causative organisms reside in the form of grains. The most common causative agent is Madurella mycetomatis. Antifungal therapy often fails due to these grains, but to identify novel treatment options has been difficult since grains do not form in vitro. We recently used Galleria mellonella larvae to develop an in vivo grain model. In the current study, we set out to determine the therapeutic efficacy of commonly used antifungal agents in this larval model. Pharmacokinetics of ketoconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and terbinafine were determined in the hemolymph of G. mellonella larvae. Antifungal therapy was given either therapeutically or prophylactic on three consecutive days in therapeutically equivalent dosages. Survival was monitored for 10 days and colony-forming units (cfu) and melanization were determined on day 3. Measurable concentrations of antifungal agents were found in the hemolymph of the larvae. None of the azole antifungal agents prolonged survival when given therapeutically or prophylactically. Amphotericin B and terbinafine did prolong survival, even at concentrations below the minimal inhibitory concentration of M. mycetomatis. The cfu and melanization did not differ between any of the treated groups and phosphate-buffered saline (PBS) treated groups. Grains were still present in surviving larvae but appeared to be encapsulated. This study demonstrated for the first time a comparison between the efficacy of different antifungal agents toward grains of M. mycetomatis. It appeared that amphotericin B and terbinafine were able to prolong larval survival.

The current treatment of eumycetoma utilizing ketoconazole is unsatisfactory because of high recurrence rates, which often leads to complications and unnecessary amputations, and its comparatively high cost in endemic areas. Hence, an effective and affordable drug is required to improve therapeutic outcome. E1224 is a potent orally available, broad-spectrum triazole currently being developed for the treatment of Chagas disease. E1224 is a prodrug that is rapidly converted to ravuconazole. Plasma levels of E1224 are low and transient, and its therapeutically active moiety, ravuconazole is therapeutically active. In the present study, the in vitro activity of ravuconazole against Madurella mycetomatis, the most common etiologic agent of eumycetoma, was evaluated and compared to that of ketoconazole and itraconazole. Ravuconazole showed excellent activity with MICs ranging between ≤0.002 and 0.031 µg/ml, which were significantly lower than the MICs reported for ketoconazole and itraconazole. On the basis of our findings, E1224 with its resultant active moiety, ravuconazole, could be an effective and affordable therapeutic option for the treatment of eumycetoma.

Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.