Conidial germination in Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans: influence of growth conditions and antifungal susceptibility profiles

Abstract

In the present study, we have investigated some growth conditions capable of inducing the conidial germination in Scedosporium apiospermum, S. aurantiacum, S. minutisporum and Lomentospora prolificans. Germination in Sabouraud medium (pH 7.0, 37ºC, 5% CO2) showed to be a typically time-dependent event, reaching ~75% in S. minutisporum and > 90% in S. apiospermum, S. aurantiacum and L. prolificans after 4 h. Similar germination rate was observed when conidia were incubated under different media and pHs. Contrarily, temperature and CO2 tension modulated the germination. The isotropic conidial growth (swelling) and germ tube-like projection were evidenced by microscopy and cytometry. Morphometric parameters augmented in a time-dependent fashion, evidencing changes in size and granularity of fungal cells compared with dormant 0 h conidia. In parallel, a clear increase in the mitochondrial activity was measured during the transformation of conidia-into-germinated conidia. Susceptibility profiles to itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin varied regarding each morphotype and each fungal species. Overall, the minimal inhibitory concentrations for hyphae were higher than conidia and germinated conidia, except for caspofungin. Collectively, our study add new data about the conidia-into-hyphae transformation in Scedosporium and Lomentospora species, which is a relevant biological process of these molds directly connected to their antifungal resistance and pathogenicity mechanisms.

Fig. 1. : representative drawings of conidia and germinated conidia observed in species of Scedosporium and Lomentospora. Two distinct morphological dimensions (length and width) can be evaluated in each conidium, germinated conidium and germ tube-like projection (germination), as shown in the scheme.

Fig. 2. : time-dependence kinetics of conidial germination in Scedosporium apiospermum, S. minutisporum, S. aurantiacum and Lomentospora prolificans. Conidial cells were incubated in Sabouraud medium at 37ºC in an atmosphere of 5% CO2. After 0, 1, 2, 3 and 4 h of incubation, the number of non-germinated and germinated conidial cells (please, see representative images in Fig. 1) were counted by using an inverted microscope. The results are expressed as percentage of germinated conidia in comparison to remaining conidial cells. The results are shown as the mean ± standard deviation of three independent experiments.

Fig. 3. : morphological dimensions evaluated under light microscopy of the pre-germinative stages from conidial cells of Scedosporium apiospermum, S. minutisporum, S. aurantiacum and Lomentospora prolificans. In this set of experiments, conidial cells were incubated in Sabouraud medium at 37ºC in an atmosphere of 5% CO2 up to 2 h. After 0, 1 and 2 h, the length and width of 50 conidia were measured. The results are shown as the mean ± standard deviation of three independent experiments.

Fig. 4. : morphological parameters and metabolic activity evaluated during the transformation from conidia to germinated conidia of Scedosporium apiospermum, S. minutisporum, S. aurantiacum and Lomentospora prolificans. In this set of experiments, conidial cells were incubated in Sabouraud medium at 37ºC in an atmosphere of 5% CO2 up to 4 h. After 0, 1, 2, 3 and 4 h, fungal cells were processed to estimate the size (forward scatter parameter) and granularity (side scatter parameter) by flow cytometry analysis and the results were expressed as fluorescence arbitrary units (FAU). In parallel, the mitochondrial activity was measured by monitoring the metabolic reduction of XTT at 492 nm using a microplate reader. The results are shown as the mean ± standard deviation of three independent experiments. p values were obtained comparing the dormant 0 h conidial cells to the other time-points in which the conidia were collected, and the diamond symbols indicate that p < 0.05 (Student’s t test).

Fig. 5. : light microscopies showing the dormant (0 h-conidia) and germinated conidial cells of Scedosporium apiospermum, S. minutisporum, S. aurantiacum and Lomentospora prolificans after 4 h of incubation in Sabouraud medium at 37ºC in an atmosphere of 5% CO2. The insets highlighted the different morphologies regarding both conidia and germinated conidia observed in each studied fungus. Bars represent 4 μm.

Fig. 6. : evaluation of conidial germination of Scedosporium apiospermum, S. minutisporum, S. aurantiacum and Lomentospora prolificans in different culture media. Conidial cells were incubated for 4 h in Sabouraud, Dulbecco’s modified Eagle’s medium (DMEM) or fetal bovine serum (FBS) at 37ºC with an atmosphere of 5% CO2. After incubation, fungal cells were counted by using an inverted microscope. The results are shown as the mean ± standard deviation of three independent experiments. There was no statistical difference in the germination of each fungal species when cultured under these different culture media.

Fig. 7. : evaluation of conidial germination of Scedosporium apiospermum, S. minutisporum, S. aurantiacum and Lomentospora prolificans in different pH values. Conidial cells were incubated for 4 h at 37ºC with an atmosphere of 5% CO2 in Sabouraud medium in which the pH was adjusted to 5.0, 7.0 and 9.0. After incubation, fungal cells were counted by using an inverted microscope. The results are shown as the mean ± standard deviation of three independent experiments. There was no statistical difference in the germination of each fungal species when cultured in different pH values.

Fig. 8. : evaluation of conidial germination of Scedosporium apiospermum (Sap), S. minutisporum (Smi), S. aurantiacum (Sau) and Lomentospora prolificans (Lpr) in different temperatures and CO2 tensions. Conidial cells were incubated for 4 h on Sabouraud medium at 21ºC and 37ºC in an atmosphere of 5% CO2 or 0.033% CO2. After incubation, fungal cells were counted by using an inverted microscope. The results are shown as the mean ± standard deviation of three independent experiments. Diamonds represent the significant difference (p < 0.05, Student’s t test) when the germination of each fungus was compared under different CO2 tensions (5% or 0.033% CO2), while stars represent the significant difference (p < 0.05, Student’s t test) when the germination of each fungus was compared under different temperatures (21ºC and 37ºC).