Antifungal susceptibility tests
We assessed the activity of cabanillasin against various fungal pathogens of
humans: ascomycetes yeasts (Candida albicans ATCC 90029, Candida glabrata
ATCC 90030, Candida krusei ATCC 6258 and Candida lusitaniae CBS 6936),
basidiomycete yeasts (Cryptococcus neoformans, clinical strain), ascomycete
filamentous fungi (Aspergilus fumigatus, clinical strain; Fusarium oxysporum,
clinical strain) and zygomycete filamentous fungi (Rhizopus oryzae, environmental
strain). The CLSI M27-A2 broth microdilution method and the CLSI
M38-A microdilution method with RPMI 1640 medium (recommended in the
Clinical and Laboratory Standards Institute M23-A document)22 were used for
the testing of yeasts and filamentous fungi, respectively.23,24
Inocula of yeasts and filamentous fungi were prepared on YPD medium
(10 g yeast extract, 10 g peptone and 20 g glucose in 1.0 l tap water) and malt
medium (20 g malt extract in 1.0 l tap water), respectively. The density of each
inoculum was adjusted by dilution in RPMI 1640 broth to between 5102
and 5103 cfuml1 for yeast isolates and 5103 and 5104 cfuml1 for
filamentous fungi. We added 10 ml of cabanillasin solution to 190 ml of fungal
inoculum in microdilution trays (96 U-bottomed plates; final concentration of
50–0.098 mgml1 for all fungi). The inoculated microdilution trays were
incubated at 35 1C in ambient air. The absorbance of the culture was measured
at 450 nm, after 24 and 48 h of incubation for the yeasts and after 48 and 72 h
of incubation for filamentous fungi and Cryptococcus neoformans. Activity was
assessed by determining the percentage growth inhibition ((1A450 nm culture
with cabanillasin)/A450 nm culture without cabanillasin)100.