Synergistic Effect of Pseudolaric Acid B with Fluconazole Against Resistant Isolates and Biofilm of Candida tropicalis

PURPOSE: Candida tropicalis (C. tropicalis) has emerged as an important fungal pathogen due to its increasing resistance to conventional antifungal agents, especially fluconazole (FLC). Pseudolaric acid B (PAB), a herbal-originated diterpene acid from Pseudolarix kaempferi Gordon, has been reported to possess inhibitory activity against fungus. The present study aims to investigate the antifungal effect of PAB alone and in combination with FLC on planktonic and biofilm cells of C. tropicalis. METHODS: The antifungal activity of PAB against planktonic isolates was evaluated alone and in combination with FLC using the chequerboard microdilution method and growth curve assay. The anti-biofilm effects were quantified by tetrazolium (XTT) reduction assay, which were further confirmed by scanning electron microscopy (SEM) and fluorescent microscope to observe morphological changes of biofilm treated with PAB and FLC. RESULTS: It was revealed that PAB alone exhibited similar inhibitory activity against FLC-resistant and FLC-susceptible strains with median MIC ranging from 8 to 16 µg/mL. When administered in combination, synergism was observed in all (13/13) FLC-resistant and (2/9) FLC-susceptible strains with FICI ranging from 0.070 to 0.375. Moreover, the concomitant use of PAB and FLC exhibited a strong dose-dependent synergistic inhibitory effect on the early and mature biofilm, eliminating more than 80% biofilm formation. SEM found that PAB, different from azoles, could significantly inhibit spore germination and destroy the cell integrity causing cell deformation, swelling, collapse and outer membrane perforation. CONCLUSION: PAB was highly active against FLC-resistant isolates and biofilm of C. tropicalis, particularly when combined with FLC. These findings suggest that PAB may have potential as a novel antifungal agent with different targets from azole drugs.