1. Introduction

Chlorhexidine (CHX) is a bisbiguanide with bacteriostatic and bactericidal effects ^[1]. It is the most studied and most effective anti-plaque and anti-gingivitis agent and is considered the “gold standard” anti-plaque agent ^[2]. CHX is a broad-spectrum antiseptic agent effective against gram-positive and gram-negative bacteria, yeasts, and viruses ^[3]. It is a cationic molecule and binds non-specifically to negatively charged membrane phospholipids of bacteria ^[4]. The mechanism of action of CHX is dose-dependent. It is bacteriostatic at very low concentrations (0.02–0.06%) and bactericidal at higher concentrations (0.12–0.20%) ^[5]. In addition to its immediate bactericidal effect, CHX also binds to the oral mucosa resulting in a slow and prolonged antibacterial effect ^[6][7].

CHX is widely used in dentistry. It is available as oral rinses (0.02–0.3%), gels (0.12–1%), sprays (0.12–0.2%), and dental varnishes (1%, 10%, 40%). It is also found in toothpastes and mouthwashes ^[2][8]. CHX is used most widely as a gluconate compound in disinfectant formulations ^[9].

The long-term use of CHX is associated with local adverse effects of temporary alteration of taste (dysgeusia) and tooth pigmentation. Unaesthetic brownish pigments accumulate on teeth, tongue, as well as on prosthetic crowns which affects patient compliance ^[10][11]. It is also shown to have cytotoxic activity against human cells in vitro which can cause apoptosis and necrotic cell death ^[12].

2. Recent Findings

A comparative analysis of three mouthwashes containing CHX showed that a 0.2% CHX mouthwash resulted in a significantly better prevention of supragingival plaque and lower plaque scores than 0.12% and 0.06% CHX mouthwashes after 21 days of use. No significant difference was observed in the plaque inhibitory effects between 0.12% and 0.06% CHX mouthwashes. Furthermore, no differences were seen in gingivitis or the gingival index between the three mouthwashes after three weeks of rinsing ^[13]. Additionally, the 0.2% CHX mouthwash was observed to significantly reduce plaque, gingival inflammation, and gingival scores when used both with and without alcohol. Rinsing with 10 mL of either solution one time a day for a period of six weeks showed to be more effective in controlling both plaque and gingivitis compared to brushing alone. After six weeks of use, the CHX levels in saliva were higher in both groups using CHX with or without alcohol, with a similar amount of CHX retained in the oral cavity for both groups ^[14].

The number of studies on CHX has increased over the years and confirms the significant effect of CHX on plaque and dental biofilm. All systematic reviews on CHX confirm it to provide statistically significant improvements of plaque and gingival scores. It has a better anti-dental biofilm and anti-gingivitis properties than the Listerine mouthwash containing essential oils (EO). The relative differences in dental biofilm control were 31.6% and 36% for CHX and 24% and 35% for EO at three months and six months, respectively ^[15]. Another study showed that a 0.2% CHX mouthwash is more effective than Listerine against the aerobic and facultative bacteria in the supragingival plaque samples from gingivitis patients. CHX produced a zone of inhibition (ZOI) with the average diameter of 18.38 mm after 24 h while Listerine showed no inhibition after 24 h. Furthermore, the mean bacterial count was reduced by 23.13 CFU after using CHX for two weeks while Listerine produced a mean reduction of 19.75 CFU. The antimicrobial effect of CHX persists longer than that of Listerine ^[16].

Recent studies on CHX have also demonstrated its significant antibacterial effect on periodontal pathogens associated with peri-implantitis. A 0.2% CHX mouth rinse resulted in large zones of growth inhibition on Aggregatibacter actinomycetemcomitans species isolated from subgingival plaque samples from peri-implantitis lesions ^[17]. Another study showed the use of a 0.2% CHX gel during different stages of implant placement to significantly reduce the Porphyromonas gingivalis load on the healing abutment. CHX also controls the inflammatory infiltrate in the peri-implant soft tissue which is linked to the bacterial load. Thus, the use of CHX improves clinical outcomes of implant-supported restoration by reducing the presence of P. gingivalis and peri-implant inflammation ^[18]. The use of CHX inside a dental implant abutment connection has also shown to reduce peri-implant marginal bone loss which is caused by bacteria present in the implant connection ^[19].

A preliminary study of a new oral gel formulation, ADC, named after its active ingredients—Ag+ ions (silver-2-mercaptobenzoate), CHX digluconate, and didecyldimethylammonium chloride, showed its good clinical efficacy when used in daily oral hygiene. The results show a statistically significant reduction in the total bacterial load without any noticeable side effects ^[20]. The topical application of both the gingiva-adhering and the soluble form of a CHX gel has also shown to improve the clinical parameters after scaling and root planing when compared to no application ^[21]. Another study showed that when used as an adjunct to non-surgical periodontal treatment in patients with chronic periodontitis, a xanthan-based CHX gel containing 1.5% CHX significantly reduced periodontal pocket depths at selected sites (Mesiodistal: 0.15 mm). The use of CHX alone was not effective due to the high clearance of CHX within the pockets ^[22].

A recent study assessing the efficacy of a mouthwash containing CHX with fluoride showed that the CHX + Fl combination has a better anti-plaque efficacy than CHX alone. The combination has a better impact on accumulation of plaque while resulting in a drop in plaque pH equal to CHX. These results combined with the previous evidence that a CHX–Fl mouthwash resulted in a better reduction of caries with no reported side effects ^[23] suggest it to be a promising alternative to CHX mouth rinses ^[24]. The prominent side effect of teeth staining caused by CHX is also significantly reduced by the addition of an anti-discoloration system (ADS) without affecting the antiplaque activity of CHX ^[25]. ADS provided significant reduction of stain scores while no differences were seen in the plaque, gingivitis, and bleeding scores ^[26].

Results of a recent study suggest that CHX in combination with an anti-biofilm peptide 1018 can be used for effective control of oral biofilm growth. The combined use of CHX and a broad-spectrum peptide showed a strong additive effect in killing bacterial cells. Though no difference in residual biofilm volume was seen, the combination resulted in a higher percentage of dead cells compared to the treatment with either CHX or peptide 1018 alone. The proportion of dead cells increased significantly with increasing time of exposure to treatment ^[27]. According to another recent study, topical CHX, when combined with systemic amoxicillin (AMX) and metronidazole (MET), shows promising results as an alternative approach to intensive mechanical therapy in patients with a severe form of periodontitis. An enhanced non-surgical mechanical therapy combining extensive use of topical CHX with the systemic antibiotics is shown to be effective in the treatment of severe generalized aggressive periodontitis (GAP). The combined therapeutic approaches showed improved clinical parameters and reduced periodontal pathogens. A transitory increase in the minimum inhibitory concentration (MIC) of the subgingival biofilm to CHX and AMX was also seen ^[28].

In recent years, the use of newer approaches and technology to improve the efficacy of CHX have been studied. Low-intensity direct current (DC) has shown to promote CHX antimicrobial efficacy against P. gingivalis within a biofilm. A significant increase in the 0.2% CHX efficacy against P. gingivalis was seen when applying 10 mA current. This effect is called a bioelectric phenomenon. No effect of electric current was seen with 1.5 mA ^[29]. Nanotechnology is also being used to enhance the anti-biofilm efficacy of CHX. A study assessing two forms (spherical and wire) of CHX-encapsulated mesoporous silica nanoparticles (MSNs) show the spherical nanoparticle encapsulated CHX to have a greater antibiofilm capacity than the wire form or the CHX-free form. This is attributed to the effective releasing mode and the close interactions of the spherical form with the microbes ^[30]. The use of magnetic nanoparticles (MNPs) as a carrier of CHX also shows a great potential in the development of antiseptic nanosystems. CHX attached to MNPs shows an increased ability to inhibit the growth of multispecies biofilms compared to free CHX. The CHX-functionalized nanoparticles did not affect the host cell proliferation or the release of the proinflammatory cytokine, IL8. Findings from the study suggest that MNPs with their unique properties (size, magnetic moment) may be used as a new approach in the treatment of infections caused by drug-resistant pathogenic bacteria ^[31].

Many recent studies assess the effect of CHX use on the oral microbiome. The use of a CHX mouthwash for seven days exhibited a major shift in salivary microbiome with a significant increase in the abundance of Firmicutes and Proteobacteria species and a reduced amount of Bacteriodetes, phyla SR1 and TM7, and Fusobacteria. This shift was associated with a significant decrease in saliva pH and buffering capacity resulting in more acidic oral conditions favorable for increased dental caries. In addition, the use of CHX reduced the amount of oral nitrate-reducing bacteria which contribute to cardiovascular health. These findings suggest a more careful consideration of the applications of the CHX mouthwash. ^[32]. In another study, the impact of short-term exposure of CHX on two types of oral biofilms, a human tongue microbiota and a 14-species community, was explored. Both biofilms treated with CHX showed a pattern of inactivation (>3 log units) and rapid regrowth to the initial bacterial concentrations. Profound shifts in microbiota composition and metabolic activity were also seen. The study suggests the need for alternative treatments that selectively target the disease-associated bacteria in the biofilm without affecting the commensal bacteria ^[33]. A comparative study on the recovery of multispecies oral biofilms following treatment with chlorhexidine gluconate (CHX) and CHX with surface modifiers (CHX-Plus) showed CHX-Plus to be more effective in killing bacteria in biofilms than the regular 2% CHX. Though the cells continued to be killed for up to a week after treatment with both CHX solutions, the biofilms returned fully to the pre-treatment levels after eight weeks. The results indicate the presence of persister cells as the main reason for relapse. These persister cells remain in the dormant state and promote tolerance to high concentrations of CHX. Thus, the study highlights the need to identify compounds that synergize with CHX to prevent regrowth of bacteria while limiting its negative side effects ^[34]. According to a recent review on evidence for resistance in oral bacteria to CHX, though it is not fully clear if there is a reason for concern regarding enhanced tolerance or resistance in oral bacteria towards CHX, the dental community must be aware about the potential risk of CHX resistance ^[35].