## 1 Introduction  
Dental plaque is a complex multispecies biofilm growing on the tooth surface, in which bacteria are embedded and proliferate, protected from environmental stresses and host defense [1, 2]. Even though several microorganisms contribute to dental plaque development and establishment, _Streptococcus mutans_ (_S. mutans_) is considered to play a pivotal role in creating an optimal microenvironment in terms of pH and extracellular polysaccharide matrix, thus promoting the growth of other cariogenic bacteria. _S. mutans_ is able to use a large variety of carbohydrates as an energy source, and glycolysis is its exclusive route for energy production [1]. As a facultative anaerobe, _S. mutans_ is specialized in converting carbohydrates into organic acids through anaerobic fermentation and glycolysis. This feature, defined as acidogenicity, leads to a pH reduction below 4.0 in the microenvironment, ultimately causing tooth demineralization [1]. _S. mutans_ is able to use aerobic respiration and bears detoxification mechanisms of reactive oxygen species generated during oxygen exposure [3]. Genome sequencing and annotation of different _S. mutans_ strains highlighted the presence of two main ABC-type transporters for the uptake of multiple sugars and around 280 genes associated with sugar phosphotransferase systems [4; 5]. Sucrose (\(\beta\)-1,2-linked disaccharide of glucose and fructose) is one of the main sugars used as an energy source in human nutrition, and it has been recognized that several _S. mutans_ extracellular glycosyl transferase uses this molecule to synthesize extracellular polymers rich in dextran, \(\alpha\)-1,3 and \(\alpha\)-1,6 glucans [6]. These polysaccharides share a central role in mediating the tooth adhesion and virulence of the bacterium, which renders _S. mutans_ one of the principal actors in virulent plaque formation and one of the model organisms to investigate for the study of dental plaque development. Daily use of mouthwash helps control dental plaque, mainly by limiting further adhesion of oral bacteria and plaque's uncontrolled development [7]. Chlorhexiidine (CHG), a bis-biguanide compound including two chloroguanide chains carrying positive charges at physiological pH, is currently the most used antiseptic agent in mouthwashes [8; 9]. Its mechanism of action relies on its charged moieties, which interact with negatively charged membrane phospholipids in bacteria, ultimately leading to membrane damage and cytoplasmic leakage [10]. Generally, Gram-positive bacteria such as _S. mutans_ show high sensitivity to CHG, while in Gram-negative bacteria, the outer membrane layer acts as a protection, binding chlorhexiidine functional groups and shielding the plasma inner membrane from chlorhexiidine action. A Chlorhexiidine digluconate concentration of around 0.2% is currently used in mouthwashes. This concentration was proven to exert an effective bactericidal action against most microorganisms, and CHG has been considered for a long time by dental clinicians as the gold-standard antiseptic for dental plaque and gingivitis control [11]. However, little is still known about the effect of CHG at sub-inhibitory concentrations against complex oral biofilms. In this case, in fact, a plethora of events may occur, including, among others, osmoregulation impairment and alteration in transport and respiratory activity [12]. All these aspects are worth further consideration since during treatments in the oral cavity with CHG-containing products, drug concentration may easily fall in subinhibitory concentration, thus resulting in the onset and dissemination of resistant strains [12]. Acquired resistance mechanisms toward CHG often involve an increase in multidrug efflux pump activity and cell membrane changes [13]. Inside oral biofilms, the situation is even more favorable for the emergence of resistant strains since lowered CHG concentration in deeper biofilm layers creates subinhibitory microenvironments with a strong selective pressure favoring CHG-resistant strains [14].