Synergistic Effect of Berberine Hydrochloride and Fluconazole Against Candida albicans Resistant Isolates

Abstract

The emergence of resistant <i>Candida albicans</i> has made clinical fluconazole (FLC) treatment difficult. Improving sensitivity to FLC is an effective way to treat resistant isolates. Berberine hydrochloride (BBH) is a commonly used traditional Chinese medicine with antimicrobial effects, especially in resistant isolates. We investigated the molecular mechanisms underlying BBH and FLC synergism on biofilm-positive FLC-resistant <i>C. albicans</i> inhibition. Checkerboard microdilution assays and time-kill assays showed a strong synergistic effect between BBH and FLC in resistant <i>C. albicans</i> isolates, causing a significant 32-512-fold reduction in minimum inhibitory concentrations. BBH combined with FLC inhibited intracellular FLC efflux due to key efflux pump gene <i>CDR1</i> downregulation, whereas FLC alone induced high <i>CDR1</i> transcription in resistant strains. Further, BBH + FLC inhibited yeast adhesion, morphological hyphae transformation, and biofilm formation by downregulating the hyphal-specific genes <i>ALS3</i>, <i>HWP1</i>, and <i>ECE1</i>. BBH caused cytoplasmic Ca²⁺ influx, while FLC alone did not induce high intracellular Ca²⁺ levels. The vacuolar calcium channel gene <i>YVC1</i> was upregulated, while the vacuolar calcium pump gene <i>PMC1</i> was downregulated in the BBH + FLC and BBH alone groups. However, vacuolar calcium gene expression after FLC treatment was opposite in biofilm-positive FLC-resistant <i>C. albicans</i>, which might explain why BBH induces Ca²⁺ influx. These results demonstrate that BBH + FLC exerts synergistic effects to increase FLC sensitivity by regulating multiple targets in FLC-resistant <i>C. albicans</i>. These findings further show that traditional Chinese medicines have multi-target antimicrobial effects that may inhibit drug-resistant strains. This study also found that the vacuolar calcium regulation genes <i>YVC1</i> and <i>PMC1</i> are key BBH + FLC targets which increase cytoplasmic Ca²⁺ in resistant isolates, which might be critical for reversing biofilm-positive FLC-resistant <i>C. albicans</i>.