Effects of Chlorhexidine mouthwash on the oral microbiome

Abstract

Following a single blind, cross-over and non-randomized design we investigated the effect of 7-day use of chlorhexidine (CHX) mouthwash on the salivary microbiome as well as several saliva and plasma biomarkers in 36 healthy individuals. They rinsed their mouth (for 1 min) twice a day for seven days with a placebo mouthwash and then repeated this protocol with CHX mouthwash for a further seven days. Saliva and blood samples were taken at the end of each treatment to analyse the abundance and diversity of oral bacteria, and pH, lactate, glucose, nitrate and nitrite concentrations. CHX significantly increased the abundance of Firmicutes and Proteobacteria, and reduced the content of Bacteroidetes, TM7, SR1 and Fusobacteria. This shift was associated with a significant decrease in saliva pH and buffering capacity, accompanied by an increase in saliva lactate and glucose levels. Lower saliva and plasma nitrite concentrations were found after using CHX, followed by a trend of increased systolic blood pressure. Overall, this study demonstrates that mouthwash containing CHX is associated with a major shift in the salivary microbiome, leading to more acidic conditions and lower nitrite availability in healthy individuals.

Figure 1

Absolute abundance and Linear Discriminant Analysis (LDA) scores in the main bacterial phyla (A,B), genus (D,E) and family (F,G) after a 7-day treatment with placebo and chlorhexidine (CHX). Figure C shows the ratio between the two main phyla (Firmicutes:Bacteroidetes) following each treatment. Operational Taxonomic Units (OTUs) with an asterisk were statistically significant (False Discovery Rate > 0.05).

Figure 2

Shannon’s index representing alpha-diversity (A) and Bray-Curtis index representing beta-diversity (B) (each dot represents an individual sample and ellipsis represents the 95% confidence regions for group) after a 7-day treatment with placebo and chlorhexidine (CHX).

Figure 3

Saliva pH (A), saliva buffering capacity (B) and concentration of salivary lactate (C), glucose (D), nitrite (F), nitrate (G), as well as the oral nitrate-reducing capacity of bacteria (ONRC) (E) and concentration of plasma nitrite (H) and nitrate (I) after a 7-day treatment with placebo and chlorhexidine (CHX).

Figure 4

Moderate degree and significant Pearson correlations (r > 0.40; P < 0.05) found between abundance of oral bacteria (Operational Taxonomic Units [OTUs] %) at phylum level and salivary markers after the placebo and chlorhexidine treatment. In the placebo condition, abundance of Proteobacteria was negatively correlated to the oral nitrate-reducing capacity of bacteria (ONRC) (A), and with lower levels of diastolic blood pressure (D). Abundance of Bacteroidetes was negatively associated with plasma nitrite (B), while abundance of Actinobacteria was positively correlated (E). Abundance of the phylum SR1 was positively associated with greater salivary pH. Following 7-day use of chlorhexidine, the abundance of Fusobacteria was correlated with greater concentration of glucose in saliva (F). Abundance of Actinobacteria was negatively correlated with saliva lactate (G), and abundance of Proteobacteria was also negatively correlated with saliva nitrite concentration (H).

Figure 5

Changes in systolic (SBP) and diastolic (DBP) blood pressure after 7-day use of placebo and chlorhexidine (CHX).