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

Abstract

Chlorhexidine (Cx) augmented with beta-cyclodextrin (β-cd) inclusion compounds, termed Cx:β-cd complexes, have been developed for use as antiseptic agents. The aim of this study was to examine the interactions of Cx:β-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 Cx:β-cd complexes have with the yeast membrane and assessment of membrane morphology following exposure to Cx:β-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 1:1, 1:2, 1:3, and 1:4, 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 Cx:β-cd complexes. Cell disorganization increased significantly as the proportion of β-cyclodextrin present in the complex increased. Morphology of cells exposed to complexes with 1:3 and 1:4 molar ratios of Cx:β-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 Cx:β-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.

Cyclodextrin modulates the cytotoxic effects of chlorhexidine on microrganisms and cellsin vitro

“…In this study, the effects of a variety of Cx-cyclodextrin inclusion complexes on cell viability were tested and compared in order to develop new potential therapies that may minimize these problems. The effectiveness of these complexes was verified, as previously reported (Teixeira et al, 2012(Teixeira et al, , 2013. In all groups, 1:3 and 1:4 Cx:Cd molar ratios improved the antimicrobial activity of the inclusion complexes, irrespective of the identity of the Cd.…”

“…Based on this concept, we hypothesized that the activity of a drug/Cd complex can be tailored by varying the identity and concentration of the Cd. This study corroborated data from a preliminary study of Cx-b-Cd inclusion complexes (Teixeira et al, 2012) and compared the effectiveness of these complexes to that of a-Cd-and Hp-b-Cd-containing complexes. Hp-b-Cd and Cx-b-Cd complexes exhibited a lower ability to extract ergosterol when compared with a-Cd complexes.…”

“…Sterols can be removed from cells by extraction, forming small unilamellar vesicles. Similarly, chlorhexidine induces changes in the fluidity of the yeast membrane (Teixeira et al, 2012). Antibiotic molecules must compete with phospholipids in order to interact with cholesterol and form pores (Pankov et al, 2006;Shaw et al, 2008).…”

“…It is a well-known inactivating agent of many non-sporulated bacteria, and exhibits some sporostatic and mycobacteriostatic action. It is also effective against yeasts and protozoa, with a minimum inhibitory concentration comparable to most antiseptics (Adriaanse et al, 1996;Best et al, 2007;Teixeira et al, 2012;Salim et al, 2013). The mechanism of action of Cx has been well described, and its main target has been identified as the bacterial membrane (Gianelli et al, 2008;Faria et al, 2009).…”

“…The enhanced antimicrobial activity of Cx was more evident at 1:3 and 1:4 molar ratios of drug-cyclodextrin complexes. In addition, sterol solubilization was also evident when 1:3 and 1:4 Cx:b-Cd complexes were analyzed using Candida albicans (C.a) as an experimental model (Teixeira et al, 2012).…”