Chlorhexidine: beta-cyclodextrin inhibits yeast growth by extraction of ergosterol

ABSTRACT

Chlorhexidine (Cx) augmented with beta-cyclodextrin (β-cd) inclusion compounds, termed Cxβ-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cxβ-cd complexes, prepared at different molecular ratios, with sterol and yeast membranes. The Minimal Inhibitory Concentration (MIC) against the yeast Candida albicans (C.a.) was determined for each complex; the MICs were found to range from 0.5 to 2 µg/mL. To confirm the MIC data, quantitative analysis of viable cells was performed using trypan blue staining. Mechanistic characterization of the interactions that the Cxβ-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cxβ-cd complexes were performed using Sterol Quantification Method analysis (SQM) and scanning electron microscopy (SEM). SQM revealed that sterol extraction increased with increasing β-cd concentrations (1.71 × 10³; 1.4 × 10³; 3.45 × 10³, and 3.74 × 10³ CFU for 11, 12, 13, and 14, respectively), likely as a consequence of membrane ergosterol solubilization. SEM images demonstrated that cell membrane damage is a visible and significant mechanism that contributes to the antimicrobial effects of Cxβ-cd complexes. Cell disorganization increased significantly as the proportion of β-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 13 and 14 molar ratios of Cxβ-cd were observed to have large aggregates mixed with yeast remains, representing more membrane disruption than that observed in cells treated with Cx alone. In conclusion, nanoaggregates of Cxβ-cd complexes block yeast growth via ergosterol extraction, permeabilizing the membrane by creating cluster-like structures within the cell membrane, possibly due to high amounts of hydrogen bonding.