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 micro 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.

FIG. 1.

z-Axis projections from a CLSM time-lapse series showing oral biofilm 1 after exposure to 0.2% CHX in the presence of BacLight LIVE/DEAD stain. Green represents the viable channel; blue represents the nonviable channel.

FIG. 2.

Sagittal projections through biofilm 1 showing contraction after exposure to 0.2% CHX (viable channel only).

FIG. 3.

Contraction of biofilm 1 as measured by object tracking after exposure to 0.2% CHX. The z-axis positions of three features within the image stack were tracked over time; the diamonds represent the uppermost optical section containing biofilm. The total image depth was 79.35 μm; the rate of contraction was 1.176 μm min⁻¹. Total image fluorescence values were adjusted to compensate for biofilm contraction in a time-lapse series with the equation [d − (Δct)/d] × f, where Δc is the rate of biofilm contraction (micrometers per minute), t is the time point (minutes), d is the total image depth (micrometers), and f is the total image fluorescence (units).

FIG. 4.

Total image stack fluorescence over time for oral biofilm 1 after exposure to 0.2% CHX. (A) Raw data. (B) Total image fluorescence adjusted to compensate for biofilm contraction with the equation given in the legend to Fig. 3. Trend lines are included for reference only.

FIG. 5.

(a) Total image stack fluorescence over time for oral biofilm 3 after exposure to 0.05% CHX. The rate of biofilm contraction was not incorporated into these data since the contraction was slight and nonuniform. (b) Control (no CHX).

FIG. 6.

Viable (a) and nonviable (b) fluorescence profiles through biofilm 2 after exposure to 0.2% CHX. The peak fluorescence value for both channels shifts from an initial depth of 21 μm to 45 μm after 10 min. The fluorescence values for these samples (viable, 0 to 10; nonviable, 0 to 45) cannot be compared because the PMT settings are user definable for each channel. These data must first be normalized (i.e., maximum value, 1 unit) in order to compare spatial trends in image fluorescence.

FIG. 7.

Viable (a) and nonviable (b) fluorescence profiles through biofilm 3 after exposure to 0.05% CHX.

FIG. 8.

Time lapse of viability profiles through oral biofilm 2 after exposure to 0.2% CHX, corresponding to the data shown in Fig. 6.

FIG. 9.

Time lapse of viability profiles through oral biofilm 3 after exposure to 0.05% CHX, corresponding to the data shown in Fig. 7.