In vitro activity of caspofungin combined with sulfamethoxazole against clinical isolates of Aspergillus spp

Abstract

Caspofungin (CAS) inhibits fungal cell wall synthesis. Sulfamethoxazole (SMX) inhibits folate biosynthesis and is active in vitro against Aspergillus spp. We studied the activities of the combination of CAS and SMX against 31 Aspergillus isolates and compared them with that of SMX combined with amphotericin B (AMB) or itraconazole (ITC). MICs and minimal effective concentrations (MECs) were determined by the NCCLS broth microdilution method. With MIC endpoints, the combination of SMX and CAS showed synergy or synergy to additivity against 29 of 31 isolates. With MEC endpoints, synergy to additivity was found against 12 of 31 isolates and indifference was displayed against the rest of them. SMX in combination with AMB or ITC was not truly synergistic, while synergy to additivity was found for SMX-AMB and SMX-ITC against 17 of 31 and 3 of 12 isolates, respectively. No antagonism was found with any of the drug combinations. Further analysis of the synergy of CAS and SMX was performed by detailed measurement of hyphal length by microscopy and time-dependent 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based hyphal damage experiments. With MEC endpoints, the combination of CAS and SMX was characterized by a greater than 50% decrease in hyphal length compared to the hyphal lengths achieved with double the concentration of each drug alone. The XTT-based hyphal damage studies showed a statistically significant (P < 0.05) reduction in viability with CAS and SMX in combination compared to the viabilities achieved with double the concentration of each drug alone. These findings support the synergy results found by using MIC endpoints and suggest that visual MEC measurements may not be sufficient to identify the synergistic interactions seen by more sensitive, quantitative methods. Animal models are required to validate the significance of the synergy of CAS and SMX against Aspergillus spp. observed in vitro.

FIG. 1.

(A) Microscopic analysis of A. fumigatus 7, A. niger 6, A. terreus 5, and A. flavus 1 in the presence of the MECs of CAS (CASx1; A. terreus 5 = 1 μg/ml, A. fumigatus 7, A. niger 6 = 0.25 μg/ml, A. flavus 1 = 0.06 μg/ml), the MECs of SMX (SMXx1; A. niger 6 = 62 μg/ml, A. terreus 5, A. flavus 1 = 62 μg/ml, A. fumigatus 7 = 31 μg/ml); and SMX and CAS together at the concentrations indicated above and at double the concentrations indicated above (CAS x 2 and SMX x 2, respectively). The experiments were repeated three times, with similar results each time. (B) Mean hyphal length measurements (in micrometers) taken from the experiment described for panel A. Each column represents average measurements for approximately 50 randomly selected germlings. Error bars denote standard deviations. P values calculated by comparison between Aspergillus strains treated with both drugs combined at the MEC and the same strains receiving each drug alone at two times the MEC were all statistically significant (P < 0.01). The bars represent A. niger 6, A. terreus 5, A. fumigatus 7, and A. flavus 1 from left to right, respectively.

FIG. 1.

(A) Microscopic analysis of A. fumigatus 7, A. niger 6, A. terreus 5, and A. flavus 1 in the presence of the MECs of CAS (CASx1; A. terreus 5 = 1 μg/ml, A. fumigatus 7, A. niger 6 = 0.25 μg/ml, A. flavus 1 = 0.06 μg/ml), the MECs of SMX (SMXx1; A. niger 6 = 62 μg/ml, A. terreus 5, A. flavus 1 = 62 μg/ml, A. fumigatus 7 = 31 μg/ml); and SMX and CAS together at the concentrations indicated above and at double the concentrations indicated above (CAS x 2 and SMX x 2, respectively). The experiments were repeated three times, with similar results each time. (B) Mean hyphal length measurements (in micrometers) taken from the experiment described for panel A. Each column represents average measurements for approximately 50 randomly selected germlings. Error bars denote standard deviations. P values calculated by comparison between Aspergillus strains treated with both drugs combined at the MEC and the same strains receiving each drug alone at two times the MEC were all statistically significant (P < 0.01). The bars represent A. niger 6, A. terreus 5, A. fumigatus 7, and A. flavus 1 from left to right, respectively.