The Effect of Different Concentrations of Chlorhexidine Mouthwash on Oral Bacteria, Vascular and Metabolic Functions in Healthy Individuals in a Placebo Controlled Randomised Crossover Study Design

The oral nitrate-nitrite-nitric oxide pathway is a fundamental mechanism in vascular regulation and metabolic health (Chai et al., 2024). This pathway involves the conversion of dietary and endogenous nitrate into nitrite by oral bacteria, which is then further reduced to nitric oxide (NO) in the body (Fukuto et al., 2000). NO plays a critical role in maintaining vascular tone (Modin et al., 2001), regulating blood pressure (Lundberg et al., 2004; Kapil et al., 2013), and supporting metabolic functions such as glucose uptake (Higaki et al., 2001; Wang et al., 2013). The oral microbiome is essential for nitrate reduction, making it a key player in this pathway (Bermúdez et al., 2022). However, this microbiome is susceptible to disruption by external factors, including the use of antiseptic mouthwashes (Brookes et al., 2023). Chlorhexidine (CHX) mouthwash is a widely used antiseptic in dental care due to its ability to reduce bacterial load and control oral infections (Moore et al., 2005). However, emerging evidence suggests that CHX may have unintended effects on the oral microbiome’s nitrate-reducing capacity (Bescos et al., 2020). Several studies have shown that the use of antibacterial mouthwash containing chlorhexidine inhibits the nitrate-reducing activity of oral bacteria leading to lower nitrite availability in saliva and plasma. This has also been related to an increase in systolic blood pressure in healthy and hypertensive individuals. We have also found that chlorhexidine mouthwash caused changes such as lower salivary pH and increased salivary lactate and glucose that led to lower diversity and activity of the oral microbiome. (observations made in an earlier study and will be published). Overall, this new evidence is not supporting the use of chlorhexidine to promote oral health. In addition to its effects on vascular and oral health, CHX may also influence metabolic markers such as post-prandial glucose levels and salivary α-amylase activity (Kazemi et al., 2019). Salivary α-amylase is a critical enzyme in carbohydrate digestion, and alteration in its activity can serve as a marker for oral and systemic metabolic changes induced by CHX such as diabetes and stress-related disorders. (Gachons et al., 2019). Chlorhexidine (CHX) is typically used in various concentrations in clinical and personal oral hygiene settings, with the most common being 0.12% and 0.2% (Deus et al., 2022). While higher concentrations are associated with increased antimicrobial efficacy (Solderer et al., 2019), they may also lead to greater disruption of the oral microbiome (Kelhoffer et al., 2022), including nitrate-reducing bacteria, and potentially exacerbate adverse systemic effects (Richards et al., 2015; Pignatelli et al., 2020). Conversely, lower concentrations may reduce these disruptions but could compromise antimicrobial effectiveness (Chye et al., 2019). Exploring the effects of different CHX concentrations allows for an understanding of the balance between its antimicrobial benefits and its impact on oral and systemic health (Alrashdan et al., 2023). This is particularly important for optimising CHX use to maintain oral health while minimising potential metabolic and vascular side effects (Tribble et al., 2023; Alrashdan et al., 2023). The use of varying concentrations in this study will provide insights into dose-dependent effects on the oral microbiome's nitrate-reducing capacity and salivary α-amylase activity. Understanding these effects across concentrations can help identify the most effective and least disruptive CHX concentration for maintaining both oral and systemic health. This evidence will contribute to informed recommendations for CHX use in clinical and personal care settings. The primary objective of this study is to assess the acute effects of different concentrations of CHX mouthwash on the oral microbiome's nitrate-reducing activity, and salivary α-amylase activity. This will be examined in a healthy population to evaluate the potential impact of CHX mouthwash on oral health and metabolic responses. By investigating these interactions, this study will provide valuable insights into the broader implications of CHX mouthwash on systemic and oral health, informing evidence-based recommendations for its use.