Differences in chlorhexidine mouthrinses formulations influence the quantitative and qualitative changes in in-vitro...

Chlorhexidine mouthrinses are marketed in different formulations. This study aimed at investigating qualitative and quantitative changes in in-vitro multispecies oral biofilms, induced by different chlorhexidine-containing mouthrinses.

Earlier studies comparing chlorhexidine mouthrinses are either clinical studies or in-vitro studies assessing the antimicrobial efficacy of the mouthrinses. However, no clear investigations are available regarding ecological impact of different chlorhexidine formulations on in-vitro multispecies oral biofilms after rinsing with different chlorhexidine formulations.

Nine commercially available chlorhexidine mouthrinses were selected. Multispecies oral communities (14 species) were grown for 48 h in a Biostat-B Twin bioreactor. After that, they were used to develop biofilms on the surface of hydroxyapatite disks in 24-well pates for 48 h. Biofilms were then rinsed once or multiple times with the corresponding mouthrinse. Biofilms were collected before starting the rinsing experiment and every 24 h for 3 days and vitality quantitative PCR was performed. The experiment was repeated 3 independent times on 3 different days and the results were analyzed using a linear mixed model.

The mouthrinses provoked different effects in terms of change in total viable bacterial load (VBL), ecology, and community structure of the multispecies biofilms. There was no relation between chlorhexidine concentrations, presence, or absence of cetylpyridinium chloride and/or alcohol, and the observed effects. Some tested chlorhexidine mouthrinses (MC, HG, HH, and HI) strongly lowered the total VBL (≈10⁰⁷ Geq/ml), but disrupted biofilm symbiosis (≥40% of the biofilms communities are pathobionts). On the other hand, other tested chlorhexidine mouthrinses (MD, ME, and HF) had limited impact on total VBL (≥10¹⁰ Geq/ml), but improved the biofilm ecology and community structure (≤10% of the biofilms communities are pathobionts).

Not all chlorhexidine mouthrinses have the same effect on oral biofilms. Their effect seems to be strongly product dependent and vary according to their compositions and formulations.