Analysis of the Effects of Chlorhexidine on Oral Biofilm Vitality and Structure Based on Viability Profiling and an Indicator of Membrane Integrity

Abstract

Multispecies biofilms modeling interproximal plaque were grown on a hydroxyapatite substratum in a constant-depth film fermentor and then immersed in a viewing solution containing fluorescent indicators of membrane integrity. Confocal laser scanning microscopy (CLSM) revealed the structure and spatial distribution of cell vitality within the biofilms. Chlorhexidine gluconate (CHX) was added to the viewing solution to achieve concentrations of 0.05 and 0.2% (wt/vol) before further CLSM time-lapse series were captured. Image analysis showed that exposure to 0.2% CHX caused the biofilm to contract at a rate of 1.176 m min ؊1 along the z axis and also effected changes in total fluorescence measurements and viability profiles through the biofilms after a delay of 3 to 5 min. At a concentration of 0.05% CHX, total fluorescence measurements for the biofilm exhibited barely detectable changes after 5 min. Fluorescence profiles (fluorescence versus time versus depth), however, clearly showed that a time-dependent effect was present, but the clearest indicator of the effect of dilute CHX over time was viability profiling. These findings suggest the possibility of using fluorescent indicators of membrane integrity in conjunction with viability profiling to evaluate the penetration of the bactericidal effects of membrane-active antimicrobial compounds into biofilm.

Oral Care Interventions and Oropharyngeal Colonization in Children Receiving Mechanical Ventilation

“…In an in vitro model, 35 the effect of chlorhexidine was time dependent, and exposure times of 30 seconds had little effect on the number of viable bacteria recovered from oral biofilms. Even at 0.2%, chlorhexidine was ineffective against dental plaque after 5 minutes of exposure and required 60 minutes to achieve an effective killing.…”

Synthesis, characterization, and antimicrobial properties of novel double layer nanocomposite electrospun fibers for wound dressing applications

“…This interference causes leakage of cytoplasmic chemicals, membrane destruction, and inhibition of enzyme activity inside the microbe. 52 Chlorhexidine loaded on PEO showed antibacterial inhibition against all tested microorganisms. The strongest activity was against S. aureus (21 mm), followed by E. coli, C. albicans (17 mm), and then P. aeruginosa (10 mm).…”

Community-Level Assessment of the Effects of the Broad-Spectrum Antimicrobial Chlorhexidine on the Outcome of River Microbial Biofilm Development

“…Its mode of action occurs via negatively charged groups on the cell surface, causing an irreversible loss of cytoplasmic constituents, as well as membrane damage and enzyme inhibition. At high concentrations (e.g., 0.5 to 1%), chlorhexidine causes extensive cell damage, coagulation of cytoplasmic constituents, and precipitation of proteins and nucleic acids (15). Chlorhexidine's biocidal activity is influenced by environmental factors, including pH, temperature, and the presence of interfering material (42).…”

“…Chlorhexidine is the active ingredient in many commercially available disinfectants, antiseptics, and oral-care products (41) and enters the environment primarily via sewage treatment plant effluents as a consequence of its significant usage in dental (15), medical (20), and veterinary applications (13). Its mode of action occurs via negatively charged groups on the cell surface, causing an irreversible loss of cytoplasmic constituents, as well as membrane damage and enzyme inhibition.…”