Location of chlorhexidine in DMPC model membranes: a neutron diffraction study

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.

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“…Additionally, the cells appeared to lose their characteristic shape and to allow leakage of intracellular substances (Fig. 3 C-F) (11, 20, 38). …”

Section: Resultsmentioning

confidence: 99%

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

Teixeira¹,

Araújo²,

Sinisterra³

et al. 2012

Braz. J. Microbiol.

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“…Because it has fully saturated acyl chains, DMPC bilayers are robust and do not require special preparatory environments. Over the years, DMPC bilayers have been well characterized using a variety of physical techniques (Ku9 cerka et al, 2011;Jämbeck and Lyubartsev, 2012;Aussenac et al, 2003), and as a result, they have been used to study problems ranging from drug-membrane interactions (Komljenovic et al, 2010), to the enhancement of cholesterol flipflop (Ku9 cerka et al, 2010), to the difference between bulk water and interfacial polarity and pH (Voinov et al, 2009), to examining the location, behavior and antioxidant properties of a-tocopherol in biomimetic membranes (Takahashi et al, 1989;Serbinova et al, 1991;Wassall et al, 1991;Afri et al, 2004a;Fukuzawa, 2008). Despite its broad use, there have been some authors who have questioned DMPC's biological relevance due to its oxidative stability (Marquardt et al, 2014) and the low level of 14:0 fatty acid-containing phospholipids in biological systems (e.g., <1% in lipoproteins) (Ruíz-Gutiérrez et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer

Afri

Alexenberg

Aped

et al. 2014

Chemistry and Physics of Lipids

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“…Chlorhexidine is amphipathic, and it likely interacts with both phospholipids and proteins on the bacterial cell surface (15,16). Its interaction with the membrane is reported to be similar to that of antimicrobial peptides (15). These interactions disrupt membrane integrity and potential, leading to leakage of cytoplasmic constituents; at high chlorhexidine concentrations, cytoplasm congealing and complete breakdown of the cell membrane occur, conferring a bactericidal effect (17)(18)(19).…”

mentioning

confidence: 99%

“…The chlorhexidine remains on the skin, providing an antimicrobial coating that is replenished with each bathing. Chlorhexidine is amphipathic, and it likely interacts with both phospholipids and proteins on the bacterial cell surface (15,16). Its interaction with the membrane is reported to be similar to that of antimicrobial peptides (15).…”

mentioning

confidence: 99%

Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci

Bhardwaj

Ziegler

Palmer

2016

Antimicrob Agents Chemother

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Chlorhexidine is a bisbiguanide antiseptic used for infection control. Vancomycin-resistant E. faecium (VREfm) is among the leading causes of hospital-acquired infections. VREfm may be exposed to chlorhexidine at supra-and subinhibitory concentrations as a result of chlorhexidine bathing and chlorhexidine-impregnated central venous catheter use. We used RNA sequencing to investigate how VREfm responds to chlorhexidine gluconate exposure. Among the 35 genes upregulated >10-fold after 15 min of exposure to the MIC of chlorhexidine gluconate were those encoding VanA-type vancomycin resistance (vanHAX) and those associated with reduced daptomycin susceptibility (liaXYZ). We confirmed that vanA upregulation was not strain or species specific by querying other VanA-type VRE. VanB-type genes were not induced. The vanH promoter was found to be responsive to subinhibitory chlorhexidine gluconate in VREfm, as was production of the VanX protein. Using vanH reporter experiments with Bacillus subtilis and deletion analysis in VREfm, we found that this phenomenon is VanR dependent. Deletion of vanR did not result in increased chlorhexidine susceptibility, demonstrating that vanHAX induction is not protective against chlorhexidine. As expected, VanA-type VRE is more susceptible to ceftriaxone in the presence of sub-MIC chlorhexidine. Unexpectedly, VREfm is also more susceptible to vancomycin in the presence of subinhibitory chlorhexidine, suggesting that chlorhexidine-induced gene expression changes lead to additional alterations in cell wall synthesis. We conclude that chlorhexidine induces expression of VanA-type vancomycin resistance genes and genes associated with daptomycin nonsusceptibility. Overall, our results indicate that the impacts of subinhibitory chlorhexidine exposure on hospital-associated pathogens should be further investigated in laboratory studies.

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Location of chlorhexidine in DMPC model membranes: a neutron diffraction study

Cited by 40 publications

References 43 publications

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

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

NMR-based molecular ruler for determining the depth of intercalants within the lipid bilayer

Chlorhexidine Induces VanA-Type Vancomycin Resistance Genes in Enterococci

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