Evaluation of in vitro antifungal activity of osthole against Talaromyces marneffei in yeast phase

Abstract

Objective To evaluate the in vitro antifungal activity of osthole against Talaromyces marneffei (TM) in yeast phase, in order to provide an experimental reference for the clinical treatment of TM infection with Chinese medicine. Methods There were 20 TM strains, including 1 standard strain, 2 fluconazole-spontaneously resistant strains, 11 clinical isolates, and 6 isolates from wild bamboo rats. A microdilution method was used to prepare 96-well antifungal sensitivity test plates containing osthole, fluconazole, amphotericin B, itraconazole and voriconazole at different concentrations, which were incubated with (1-5) × 103 CFU/ml of tested TM strain suspensions at 37 ℃ for 48 hours. Meanwhile, TM strains cultured in the media without antifungal drugs served as positive (growth) control group, and culture media served as negative group. The minimum inhibitory concentrations (MICs) of antifungal drugs against yeasts were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M27-A2 Document) . Fluconazole MIC was defined as the lowest drug concentration that resulted in ≥ 80% growth inhibition, and MICs of other antifungal drugs were the lowest drug concentrations that resulted in 100% growth inhibition, compared with growth control wells. Results The MICs among the quality control strains were all within the reference range, and TM grew well in the positive control wells. The MIC ranges of fluconazole, amphotericin B, itraconazole and voriconazole against TM strains were 2.0-8.0 mg/L, 1.0-4.0 mg/L, 0.03-0.25 mg/L and 0.06-0.25 mg/L respectively, and the MIC of fluconazole against fluconazole-spontaneously resistant strains was 128 mg/L. The MICs of osthole against the TM standard strain (FRR2161) , fluconazole-spontaneously resistant strains and 1 isolate from wild bamboo rats were 16, 32 and 128 mg/L respectively, and the MIC range of osthole against other 16 TM strains was 16-64 mg/L. The MICs of osthole at which 90% and 50% of the TM strains were inhibited were 64 and 32 mg/L respectively. Conclusion Osthole exhibits the antifungal activity against the yeast form of TM. Key words: Talaromyces; Osthole; Microbial sensitivity tests; Talaromyces marneffei