In situ neutralisation of the antibacterial effect of 0.2% Chlorhexidine on salivary microbiota: Quantification of substantivity

•The immediate antibacterial effect of a CHX mouthwash is very powerful.•The bacterial population needs more than 3 h to return to baseline vitality.•The CHX substantivity is a property that increases its antibacterial activity.•The CHX substantivity prolongs its antibacterial effect by at least double.

ObjectiveTo quantify the substantivity of a single 0.2% Chlorhexidine mouthwash in saliva after its neutralisation with tooth-brushing and 1% acetic acid, in order to identify the effect of Chlorhexidine substantivity in regard to the re-growing period of the salivary bacteria.MethodsUnstimulated saliva samples were collected from a group of 15 healthy individuals at baseline (BS), and then 30 s and 1, 3, 5 and 7 h after the following protocols were performed: a single sterile water mouthwash (M-WATER) (negative control), a single 0.2% Chlorhexidine mouthwash (M-0.2% CHX) (positive control) and a single 0.2% Chlorhexidine mouthwash followed by a complete and detailed tooth-brushing, and a single 1% acetic acid mouthwash (M-0.2% CHX + NP). The samples were analysed using an epifluorescence microscope in combination with LIVE/DEAD® BacLight™ fluorescence solution.ResultsAfter the M-0.2% CHX treatment, the bacterial vitality was significantly lower than BS until 7 h (87.6 ± 6.5% vs. 73.6 ± 8.8%; p < 0.001). However, after M-0.2% CHX + NP, the bacterial vitality remained significantly lower until 3 h with regard to BS (81.4 ± 3.8% vs. 68.1 ± 10.6%; p = 0.001), increasing at 5 and 7 h (no differences from BS).ConclusionThe immediate antibacterial effect of a single 0.2% Chlorhexidine mouthwash is so potent that the bacterial population needs more than 3 h to return to baseline bacterial vitality levels. The substantivity of a 0.2% Chlorhexidine mouthwash is a property that significantly increases its antibacterial activity from the first hour and contributes to extend the duration of its effect by at least double.