Figure 1.
Comparison of the virulence of C. krusei and C. albicans in G. mellonella.
(A) Survival curve of G. mellonella infected with different inocula of C. krusei ATCC 6258 • PBS; ▪ 107 cells/larva; ▴ 5×106 cells/larva;▾ 2.5×106 cells/larva incubated at 37°C (B). Survival curve of G. mellonella infected with different inocula of C. albicans SC5314 • PBS; ▪ 106 cells/larva; ▴ 5×105 cells/larva; ▾105 cells/larvae (C) Survival of G. mellonella infected with inactivated yeast. Control dead cells • PBS; ▪ C. krusei ATCC 6258 5×106 cells/larva; ▴ C. krusei ATCC 6258 5×106 cells/larva (dead); ▾ C. albicans SC5314 106 cells/larva; ♦ C. albicans SC5314 106 cells/larva (dead) (D); Effect of the incubation temperature on the virulence of C. albicans and C. krusei. • PBS; ▴ C. krusei ATCC 6258 (37°C); ▾C. krusei ATCC 6258 (30°C); ♦ C. albicans SC5314 (37°C); ▪C. albicans SC5314 (30°C); Growth curves of C. albicans (E) and C. krusei (F) at different temperatures. ○ 37°C; ▴ 30°C.
Figure 2.
Melanization of G. mellonella infected with C. krusei.
(A) Visual appearance of G. mellonella larvae infected with different C. krusei doses. (B, C and D) Optical Density (OD) of the haemolymph of G. mellonella infected with C. krusei ATCC 6258 (B), clinical isolate CL8053 (C) and CL80317 (D) with 5×105, 106, 5×106 cells/larva. The different size inoculum reveals dose-response melanization (* p<0.05). All the experiments in this figure were performed at 37°C.
Figure 3.
Interaction between C. krusei and haemocytes.
(A) Changes in haemocyte density during C. krusei infection. The haemolymph of infected larvae with C. neoformans, C. albicans SC5314, C. krusei ATCC 6258, CL8053 and CL80317 clinical isolates and PBS was collected and the concentration of haemocytes was estimated using a haemocytometer (B). Phagocytosis percentage of C. neoformans, C. albicans SC5314, C. krusei ATCC 6258, CL8053 and CL80317 clinical isolates. Asterisks denote differences statistically significant (p<0.05).
Figure 4.
Antifungal susceptibility profile of C. krusei and C. albicans.
A) Distribution of MIC values (n = 10) to amphotericin B, caspofungin, fluconazole and voriconazole of C. albicans (white bars) and C. krusei (black bars). B) Description of antifungal susceptibility of C. albicans and C. krusei to different antifungals. N = 10. The geometric mean (GM), mode, minimum (Min) and maximum (Max) are shown.
Figure 5.
Efficacy of fluconazole during G. mellonella infection with C. krusei or C. albicans.
Effect of fluconazole during infection of larvae with 5×106 cells of C. krusei (ATCC 6258) per larvae (A and B) and 5×105 cells of C. albicans cells (SC5314) per larva (C and D) in G. mellonella. Fluconazole treatment with 4 or 12 mg/kg (A and C); 32, 64 or 128 mg/kg (B and D).
Figure 6.
Efficacy of voriconazole, amphotericin B or caspofungin during C. krusei and C. albicans infection in G. mellonella.
A and B) Voriconazole treatment efficacy (7 and 10 mg/kg) in G. mellonella infected with C. albicans SC5314 (A) or C. krusei ATCC 6258 (B). C and D) Amphotericin B treatment efficacy (1, 2, 4 mg/kg) in G. mellonella infected with C. albicans SC5314 (C) or C. krusei ATCC 6258 (D). E and F) Caspofungin treatment efficacy (1, 2, 4 mg/kg) in G. mellonella infected with C. albicans SC5314 (E) or Candida krusei ATCC 6258 (F). In all the cases, the larvae were infected with 5×105 C. albicans cells/larva and 5×106 C. krusei cells/larva.
Figure 7.
Effect of antifungal treatment on fungal burden in G. mellonella infected with C. albicans or C. krusei.
Galleria mellonella larvae were infected with C. krusei ATCC 6258 (A, 5×106 cells/larva) or C. albicans SC5314 (B, 5×105 cells/larva) and CFUs recovered from G. mellonella. Black bars, no treatment, white bars, fluconazole (12 mg/kg), grey bars, amphotericin B (4 mg/kg).
Figure 8.
Histopathology of G. mellonella infected with C. krusei and C. albicans and treated with different antifungals.
Galleria mellonella was infected with 5×105 cells/larva of C. albicans SC5314 (C–H), or with 5×106 cells/larva of C. krusei ATCC 6258 (K–P). After 96 hours of infection, larvae were processed for histopathology as described in Material and Methods. (A, B, I, J), uninfected controls; (C, D, K and L), untreated controls; (E, F, M and N), larvae treated with fluconazole (12 mg/kg); (G, H, O and P), larvae treated with amphotericin B (4 mg/kg). (A, C, E, G, I, K, M, O), low magnification; (B, D, F, H, J, L, N and P), high magnification.
Figure 9.
Effect of antifungal treatment of haemocyte density and melanization of G. mellonella infected with C. krusei or C. albicans.
(A) Hemocytic density of G. mellonella infected with C. albicans SC5314 or C. krusei ATCC 6258 treated with amphotericin B (4 mg/kg) or fluconazole (64 mg/kg). (B) Optical Density (OD) of the haemolymph of G. mellonella infected with C. albicans or C. krusei treated with amphotericin B (4 mg/kg) or with fluconazole (64 mg/kg). Black bars, no treatment; grey bars, fluconazole; white bars, amphotericin B. * p<0.05.
Figure 10.
Virulence of C. krusei and C. albicans in C. elegans and antifungal efficacy.
Caenorhabditis elegans was infected as described in material and methods with C. krusei (ATCC 6258), C. albicans (SC5314) and E. coli OP50. (A) Visual appearance of infected worms (50× magnification). (B) Antifungal efficacy in C. elegans infected with C. albicans. ♦ OP50, • C. albicans, ▪ C. albicans and treated with 2 µg/mL amphotericin B (p<0.0001), ▴ Fluconazole 12 µg/mL (p<0.0001); ▾Caspofungin 2 µg/mL (p<0.0001). (C) Antifungal efficacy during C. krusei infection ♦ OP50; • C. krusei; ▪ C. krusei treated with amphotericin B 2 µg/mL; (p<0.0001); ▴Fluconazole 12 µg/mL (p = 0.1207); ▾Caspofungin 2 µg/mL (p<0.0001). (D) Effect of voriconazole on survival of C. elegans worms infected with C. albicans (•, C. albicans, ▴, voriconazole 0.25 mg/L (p<0.0001); ▪, voriconazole, 7.5 mg/L (p<0.0001); ▾ voriconazole 10 mg/L (p<0.0001)). (E), Efficacy of voriconazole during infection of C. elegans by C. krusei (•C. krusei; ▴voriconazole 0.25 mg/L (p = 0.1217); ▾ voriconazole 7.5 mg/L (9<0.0001); ▪ voriconazole 10 mg/L (p<0.0001)).