This study showed that CHX mouthwash significantly changed the oral microbiome towards greater abundance of Firmicutes and Proteobacteria species, with lower abundance of Bacteroidetes, TM7, SR1 and Fusobacteria. These changes were associated with an increase in oral acidic conditions, represented by lower salivary pH. Saliva lactate and glucose concentrations were also elevated after using CHX. Additionally, CHX disrupted the ability of oral bacteria to reduce nitrate into nitrite, which may support our finding of lower circulatory nitrite bioavailability.

To the best of our knowledge, this is the first study showing the impact of 7-day use of CHX on the oral microbiome. A large body of literature suggests that mouthwashes with CHX are effective in reducing dental plaque accumulation, gingival inflammation and bleeding2,3,4. However, the view about oral bacteria and oral health has changed substantially over the last few years12. Current approaches, using genome sequencing to identify and quantify the microorganisms in dental biofilms, have revealed a much more complex ecosystem than previously appreciated13. Results from this study showed that CHX led to an increase in the abundance of some genera such as Neisseria, Streptococcus and Granulicatella, and lowered the abundance of Actinomyces, but did not affect the abundance of Veillonella. However, it remains difficult to determine whether these microbial changes suggest a shift towards a healthy oral environment, or whether they may increase the risk of oral disease, as both increases and decreases in the bacteria associated with caries and periodontal disease have been reported14. Consequently, additional studies are required to investigate bacterial communities during different disease states, with and without CHX. Nevertheless, in the current study, we were able to associate genome sequencing measurements with other general markers of oral health, which allowed us to analyse more in detail the impact of mouthwash containing CHX on oral and systemic health.

Lower microbial diversity as represented by the Shannon’s index was found after using CHX. This result is in agreement with another recent study showing lower diversity of bacteria colonizing the tongue7. These findings are relevant from a dental viewpoint since lower diversity of oral bacteria has been related to greater risk of oral diseases13. This may reflect the ecological pressure of lowered environmental pH. Healthy biofilms are associated with an active balance between slow rates of acid production and compensatory alkali generation, resulting in an environment with a broadly neutral pH12. Surprisingly, the effect of CHX in salivary pH has only been investigated acutely in both in vivo15 and in vitro conditions16, but, no previous study analysed the impact of this antibacterial compound over a period of days in healthy individuals. Our results showing lower saliva pH after using CHX are relevant, since decreased pH in saliva is associated with demineralization of tooth enamel and risk of caries, tooth loss and other dental problems17. Oral pH may differ between different oral conditions: whilst saliva pH is more alkaline in chronic gingivitis, it tends to be more acidic in chronic periodontitis18. Thus, in terms of salivary pH, CHX could therefore be more useful for managing gingivitis than periodontitis.

Saliva’s composition is another factor to pay attention when analysing the antimicrobial effectiveness of CHX. Several in vitro studies have indicated that saliva has a neutralizing effect on CHX19,20,21. Since CHX is a strongly cationic molecule it can react with anionic chemicals, resulting in inactivation of antimicrobial activity. We did not analyse the antimicrobial interaction between saliva and CHX in this study, but, we investigated the effect of CHX in several saliva markers. We found that CHX increased saliva lactate concentration and reduced its buffering capacity. These changes are commonly associated with greater risk of oral disease22. Regarding bacteria, we found a negative correlation between the phylum Actinobacteria and saliva lactate concentration. This phylum comprises a large variety of Gram-positive bacteria and is known for its high production of bioactive compounds, including those with antimicrobial activity such as lantibiotics23. For instance, Nisin is one of the best known antibiotics for its highly effective bactericidal activity against most lactic acid bacteria24. Another important bacterial change associated with CHX administration was an increase of the major phyla Firmicutes, mainly comprised of an increase of the genus Streptococcus. This genus contains several families of lactic acid bacteria that are able to produce large quantities of this compound in the mouth25. On the other hand, we also found a significant decrease in the abundance of Bacteroidetes after using CHX. This was the second most abundant phyla in the oral cavity and some genera from this phyla such as Veillonella has been shown to be important in maintaining the acid/base conditions in the mouth26. Overall, these findings indicate that CHX promotes acidification of saliva by changing the ratio abundance of different families of bacteria that are essential to maintain the acid/base conditions in the mouth of healthy people.

Oral nitrite synthesis is another factor to take into account, with regards to the acid/base conditions of the oral cavity27. Nitrite is a nitrogen compound that forms naturally in the mouth by the action of oral bacteria that can use exogenous (diet) or endogenous (nitric oxide synthesis) nitrate sources28. Species within the genus Veillonella and Actinomyces have been suggested to lead this reaction in the oral cavity29. Importantly, CHX had a detrimental effect lowering the abundance of bacteria from these groups and reducing nitrite availability. Thus, the detrimental effect of CHX on oral nitrite synthesis is another key point requiring further attention by dental professionals, since nitrite has been shown to have an inhibitory effect in the growth of periodontal bacteria which can also help to reduce the acid production from these strains27,30.

On the other hand, nitrite synthesis in the mouth has been shown to play a key role in cardiovascular control by enhancing circulatory nitrite availability. The vasodilatory effects of nitrite are well described by previous studies using intra-arterial infusions or dietary supplements with this anion31,32. Some recent studies, but not all9,33, have also found that the use of CHX mouthwash from 3 to 7 days led to higher blood pressure in healthy8 and hypertensive individuals34. Participants from these studies had higher values of blood pressure compared to participants in our current study. In agreement with our results, Sundqvist et al.33 did not show a raise in blood pressure in a young and healthy group of females after using CHX for three days. Additional studies are required to improve our understanding about the hypertensive effect of CHX in males and females with different resting blood pressure levels and physiological status, especially, after new evidence has shown that CHX raised the mortality rate in hospitalized patients35. Overall, current studies seem to indicate that the use of CHX mouthwash leads to an increase of blood pressure, and this may be more accentuated in people with high blood pressure levels8,9,11,33.

This study has some limitations. For instance, it would be interesting to analyse the effect of CHX in patients with different oral health conditions such as gingivitis or periodontitis. We assessed the oral health status of participants using a medical questionnaire, but it would be useful to undertake a full oral and dental examination, to analyse more in detail the concurrent effect of CHX on markers of periodontal health. Treatments were not randomized in this study due to the lack of available data indicating the time needed for the full recovery of the oral microbiome after one-week use of CHX. Consequently, there was not a wash out period between treatments. Furthermore, we analysed the microbiome in saliva as it provides an average of the oral microbiome, but bacterial communities can significantly differ among sites in distinct microbial niches in the oral cavity, therefore where the effects of CHX may also differ.

In conclusion this study indicates that a 7-day use of CHX mouthwash has a significant impact on the oral microbiome, as well as shift to an acidic environment, favourable for increased dental caries, and a reduction of the amount of oral nitrate-reducing bacteria, which contribute to cardiovascular health. Thus, these findings add to the growing body of evidence that the applications of CHX mouthwash should be more carefully considered, and that CHX could have detrimental effects on the healthy microbiome, and in turn cardiovascular health, requiring further investigation.