Effects of chlorhexidine on proteolytic and glycosidic enzyme activities of dental plaque bacteria

Chlorhexidine was tested for its ability to inhibit a wide range of glycosidic and proteolytic enzyme activities produced by Treponema denticola, Porphyromonas gingivalis, Bacteroides intermedius, Actinobacillus actinomycemcomitans, Capnocytophaga sputigena, Capnocytophaga gingivalis, Capnocytophaga orchracea, Capnocytophaga sp., Actinomyces viscosus, Streptococcus mitior, Streptococcus mutans, Streptococcus sobrinus, Streptococcus mitis, Streptococcus anginosus, Streptococcus oralis and Streptococcus sanguis. The enzymes produced by Capnocytophaga spp. were the most resistant to inhibition by chlorhexidine while the hydrolysis of proteolytic substrates by all the other species was markedly susceptible to inhibition with less than 0.125 mM chlorhexidine inhibiting enzyme activities by greater than or equal to 50%. Glycosidase activities, of all species, were generally more resistant to inhibition, especially neuraminidase activity. Chlorhexidine at less than 0.032 mM inhibited the degradation of bovine serum albumin by suspensions of dental plaque bacteria. These observations support an hypothesis that chlorhexidine exerts a bacteristatic effect in vivo, in part, by reducing the ability of dental plaque bacteria to degrade host-derived proteins and glycoproteins which normally provide essential nutrients for growth.     

The effect of some cationic antiseptics on the acidogenicity of dental plaque in vivo

Two series of experiments were performed in order to compare the ability of different cationic antiseptics to inhibit the acid production in plaque. In addition an attempt was made to evaluate the influence of oral retention on the acid-inhibiting properties of these agents. In one series of experiments acid production, following sucrose applications on plaque, was measured in situ prior to and at given time intervals after rinsing with the individual agents. In a second series the effect of eluting the antiseptics retained in the oral cavity by means of 5 consecutive acetic acid (6 mM) rinses was evaluated. The results showed that chlorhexidine (0.5 mM) was more effective than benzalkonium chloride (1 mM) and piperazine (1 mM). Cetylpyridinium chloride (1 mM) was the least effective. Acidic elution markedly reduced the inhibitory effect of single rinses of chlorhexidine (0.5 mM), benzalkonium chloride (1 mM) and the cetylpyridinium chloride (1 mM). This effect was less pronounced with a higher concentration (2.2 mM) of chlorhexidine. The results gave support to the view that retention of an agent in the mouth and in plaque is of significance for its ability to inhibit acid production of dental plaque.     

The effect of oxidising mouthrinses compared with chlorhexidine on salivary bacterial counts and plaque regrowth

Abstract For various clinical indications, oxidising agents have been used in dentistry for many years. Little is known, however, of their antibacterial activity and their ability to inhibit plaque formation. In this study, 2 mouthrinses containing peroxyborate (Bocasan) and peroxycarbonate (Kavosan) were compared alongside a negative control saline rinse and a positive control chlorhexidine rinse (Corsodyl) for their ability to inhibit plaque reformation. Employing a randomised four replicate 4x4 latin square cross over design and, whilst omitting all other oral hygiene, plaque was measured by area and index after rinsing for 4 days. In a second study, in vivo antibacterial effects of the rinses were assessed by measuring salivary bacterial counts following single rinses with the preparations at various time intervals over 7 h, Plaque inhibition by chlorhexidine was significantly greater than the other rinses. All rinses were significantly better than the saline rinse at inhibiting plaque. For plaque area, the peroxycarbonate rinse was significantly better than the peroxyborate rinse at inhibiting plaque. Salivary bacterial count reductions were significantly greater compared to saline with chlorhexidine at all time intervals up to 7 h. Whilst both peroxyborate and peroxycarbonate rinses produced greater reductions in bacterial counts than saline up to 3 h, at no time interval were the differences significant. The findings of these studies would suggest oxidising mouthrinses may inhibit plaque formation not by a direct antibacterial effect, but by some other mechanism. The magnitude of plaque reductions obtained with the peroxyborate and more so peroxycarbonate rinses would suggst a need for further study of these preparations when used as adjuncts to normal toothbrushing.     

Inhibition of intergeneric coaggregation among oral bacteria by cetylpyridinium chloride, chlorhexidine digluconate and octenidine dihydrochloride

The potential inhibitory effect of chlorhexidine digluconate on the intergeneric coaggregation of 11 pairs of Gram-positive organisms was compared to its ability to inhibit coaggregations of 14 pairs comprised of both a Gram-positive and a Gram-negative cell type. Dramatic differences in the inhibitory effectiveness of the antimicrobial compound on the two kinds of coaggregation pairs were found. Gram-positive pairs were not inhibited at a concentration of 0.25%, whereas the coaggregations involving a Gram-negative partner were usually completely blocked at concentrations as low as 0.01%. Similar effects to chlorhexidine digluconate were found with octenidine dihydrochloride and cetylpyridinium chloride, while sodium dodecylsulfate was inhibitory only at 10- to 50-fold higher concentrations. These results suggest that chlorhexidine digluconate, octenidine dihydrochloride, and cetylpyridinium chloride may be effective inhibitors of later microbial colonizers of dental plaque but may not disturb a normal healthy indigenous flora.     

The effect of chlorhexidine and some other detergents on the activity of dextransucrase from Streptococcus mutans

The inhibitory effect of chlorhexidine and other bis-biguanides on the formation of dental plaque is not fully understood. The present paper describes the effect of chlorhexidine and some selected detergents on the activity of dextransucrase (EC 2.4.1.5.), an enzyme involved in the formation of important components of dental plaque. All detergents examined exerted an inhibitory effec⁺ on dextransucrase activity, to some degree dependent on the presence of charged groups and their characters. The high concentrations of chlorhexidine necessary to inhibit dextransucrase activity seem to exclude the possibility that chlorhexidine exerts its plaque inhibiting effect by means of an effect on dextransucrase.     

The effect of chlorhexidine and some other detergents on the activity of dextransucrase from Streptococcus mutans.

The inhibitory effect of chlorhexidine and other bis-biguanides on the formation of dental plaque is not fully understood. The present paper describes the effect of chlorhexidine and some selected detergents on the activity of dextransucrase (EC 2.4.1.5.), an enzyme involved in the formation of important components of dental plaque. All detergents examined exerted an inhibitory effect on dextransucrase activity, to some degree dependent on the presence of charged groups and their characters. The high concentrations of chlorhexidine necessary to inhibit dextransucrase activity seem to exclude the possibility that chlorhexidine exerts its plaque inhibiting effect by means of an effect on dextransucrase.     

Inhibition of superoxide generation by human polymorphonuclear leukocytes with chlorhexidine. Its possible relation to periodontal disease

During the acute phase of periodontal disease, as many as 60% of the cells of the junctional epithelium may be accounted for by polymorphonuclear cells (PMNs). It is generally accepted that these cells play a dominant role in the destruction of connective tissue by virtue of the proteinases they release and the free oxygen radicals they generate. Modulation of the proteolytic activity and free radical production seems to be essential for the inhibition of tissue destruction. We have therefore studied the effect of chlorhexidine on the generation of free oxygen radicals and luminol-dependent chemiluminescence (LDCL) by stimulated human PMNs. Nontoxic chlorhexidine concentrations (0.1-1 microgram/ml) were found to inhibit superoxide production but did not affect LDCL. We therefore suggest that in addition to its antiseptic effect, chlorhexidine may also modulate the generation of free radicals by activated PMN cells in the inflamed gingiva.     

Dose response of chlorhexidine against plaque and comparison with triclosan

Abstract The optimum dose of chlorhexidine delivered by mouthrinse, which balances efficacy against local side-effects, is generally considered to be in the region of 20 mg 2 × daily. Unfortunately, there have been few dose-response studies for chlorhexidine mouthrinses and for these, only limited details are published. The aims of this study were to determine the dose response of chlorhexidine to plaque inhibition and position a 0.1% triclosan rinse within this model. 28 subjects took part in this 7-treatment, double-blind, randomised cross-over 4-day plaque regrowth study. The rinses were 0.01%, 0.05%, 0.1% and 0.2% chlorhexidine, 0.1% triclosan and minus active controls for chlorhexidine and triclosan. On day 1 from a zero plaque baseline, volunteers suspended tooth-cleaning and commenced supervised 2 × daily rinsing with 10 ml volumes of the allocated rinses. On Day 5, plaque was scored by index and area. Treatment differences between the 7 rinses were highly significant. A clear dose-response pattern was seen for chlorhexidine with mean plaque scores decreasing with increasing dose. Even at 0.01%. chlorhexidine showed considerable and significant plaque inhibition compared to control. Triclosan at 0.1% showed limited plaque inhibition and less than 0.01% chlorhexidine. The findings of this study suggest that consideration could be given to low concentration chlorhexidine rinses as adjuncts to oral hygiene.     

Effect of chlorhexidine on animal cells in vitro

abstract – When cultures of human epithelial cells were treated for 5 min at 37°C with chlorhexidine in Eagle's medium without serum added, concentrations from 0.05 mM were found to be toxic as measured by growth inhibition and differential staining. About 20 times higher concentrations were needed to obtain a toxic effect, however, when the cells were treated with chlorhexidine dissolved in calf serum. Human whole saliva collected from a single subject had no such protective effect. The intracellular activities of 5'-nucleotidase, alkaline phosphatase, and NADPH₂+ NADH₂-diaphorases decreased upon treatment of the cells with concentrations of chlorhexidine at 0.2 and 2 mM, whereas 0.02 mM had no measurable effect on these enzymes. Treatment with chlorhexidine at 10⁻⁴mM had no effect on the hypotonic hemolysis of human erythrocytes, 0.001–0.1 mM stabilized the cells, but increasing the concentration to 1 mM gave 100% hemolysis. A concentration-dependent inhibition of the Na⁺–K⁺–ATPase activity was found when erythrocyte membranes were incubated with chlorhexidine in the range of 0.002–0.2 mM.     

A clinical trial to assess the efficacy of sanguinarine-zinc mouthrinse (Veadent) compared with chlorhexidine mouthrinse (Corsodyl)

Abstract. A single-blind crossover study was used to compare the ability of a 0.2% chlorhexidine mouthrinse (Corsodyl) with a sanguinarine-zinc mouthrinse (Veadent) to inhibit plaque and gingivitis. 14 volunteers starting with plaque-free mouths and optimal gingival health, rinsed with the preparations over two 19-day periods whilst refraining from all other oral hygiene procedures. At days 8, 15 and 19 of the trial, Corsodyl was significantly more effective at inhibiting both plaque and gingivitis. The Findings of this study would suggest that the Veadent mouthrinse would at most only have a limited rôle as an inhibitor of plaque and gingivitis.     

Comparison of the effect of chlorhexidine and CuSO4 on plaque formation and development of gingivitis

Recent studies have shown that several metal ions inhibit plaque formation and reduce the acidogenicity of dental plaque. The aim of the present study was to compare the effect on plaque and gingivitis of 2.2 mM CuSO4 with an equimolar solution of chlorhexidine in a modified experimental gingivitis model. The study was performed according to a double-blind cross-over design. Before each experimental period the GI was recorded and the participants' teeth were scaled and polished to remove all supragingival deposits. All mechanical oral hygiene was suspended during the experimental periods. The mean PII after rinsing with chlorhexidine, CuSO4 and water was 0.29, 0.79 and 1.25, respectively. The corresponding GI values were 0.57, 0.83 and 1.02. All differences were statistically significant. The results showed that CuSO4 inhibits plaque formation and development of gingivitis, but not to the same degree as chlorhexidine.     

A comparision between chlorhexidine and some quaternary ammonium compounds with regard to retention, salivary concentration and plaque-inhibiting effect in the human mouth after mouth rinses.

The oral retention of chlorhexidine, cetylpyridinium chloride and hexadecyltrimethylammonium bromide (a component of cetrimide) was measured by means of ¹⁴C-labelled compounds in 7 subjects after 10-ml 2.2 mM mouth rinses for 1 min. The oral retention of chlorhexidine was 32 ± 6 per cent, of cetylpyridinium chloride 65 ± 5 per cent and of hexadecyltrimethylammonium bromide 70 ± 7 per cent of the administered dose. The salivary concentration was measured after similar mouth rinses in 3 subjects and calculated according to the ¹⁴C-activity of saliva samples from 0.5 to 24 h after the rinsing. Although the concentrations of the quaternary ammonium compounds were usually higher than those of chlorhexidine shortly after rinsing, their concentrations were significantly lower (p < 0.001) than those of chlorhexidine from 4 h and onwards. The plaque-inhibiting effect was assessed in subjects who rinsed with 2.2 mM test solutions twice daily for 3 days, using sucrose rinses to provoke plaque formation. The plaque-inhibiting effect of the quaternary ammonium compounds was also tested when used in a mouth rinse four times daily. A moderate degree of plaque inhibition was obtained when the quaternary ammonium compounds were used twice daily. When the frequency was increased to four times daily, the plaque-inhibiting effect of the quaternary ammonium compounds seemed to approach that of chlorhexidine.     

The in vitro inhibition of microbial growth and plaque formation by surfactant drugs

Several cationic, mixed and amphoteric surfactants were tested for their antimicrobial activity and ability to inhibit the formation of in vitro plaque by oral microorganisms. All had antimicrobial activity against Actinomyces viscosus. Actinomyces naeslundii and Streptococcus mutans. Cationic surfactants were comparable to chlorhexidine in antimicrobial activity but were less effective in inhibiting plaque formation. Amphoteric surfactants were less effective than other detergents in antibacterial activity and had very limited capacity for the inhibition of plaque formation. Comparison of drug structure provides evidence that surfactant substantivity to saliva-coated enamel is a cation active process. Saliva was found to have an antagonistic effect on the activity of cetylpyridinium chloride but not on Triburon.     

Effect of saccharin on antibacterial activity of chlorhexidine gel

Although chlorhexidine is the most effective agent against dental plaque it is extremely bitter. To prepare formulations, it is necessary to use flavoring and sweetening, which can inhibit the antibacterial effect of chlorhexidine. Saccharin has been considered a compatible substance to use in chlorhexidine rinse or gel preparations; however, the effect of a range of concentrations has not been studied. To evaluate the effect of different concentrations of saccharin on the antibacterial activity of chlorhexidine gel, hydroxy-ethyl-cellulose gels containing 1.0% chlorhexidine digluconate and 0.0 to 1.0% sodium saccharin were prepared. Activity against Streptococcus mutans was evaluated using the agar diffusion method and determination of MIC values. The inhibitory zones of growth were 7.83 +/- 0.54 mm when no saccharin was added to the chlorhexidine gel and 7.75 +/- 0.50, 7.63 +/- 0.48, 6.21 +/- 0.40, 4.13 +/- 0.38, when the concentrations of saccharin in the gels were 0.02, 0.10, 0.5, and 1.0%, respectively. The range of MIC values was 1-2 micrograms/ml, with saccharin concentrations of 0%, 0.02, and 0.1%. In contrast, the MIC values were 4-8 and 8-16 micrograms/ml with saccharin concentrations of 0.5% and 1.0%, respectively. The paired "t" test showed that 0.5 and 1.0% sodium saccharin inhibit the antibacterial activity of 1% digluconate chlorhexidine gel. These in vitro results suggest that saccharin may inhibit the efficacy of chlorhexidine against mutans streptococci, depending on the concentration.     

Antimicrobial properties of 2 aliphatic amines and chlorhexidine in vitro and in saliva

The surfactants tetradecylamine, hexadecylamine and chlorhexidine have been compared with regard to their ability to inhibit microbial growth. Antibacterial activity was assessed by tube dilution methods. Tetradecylamine and chlorhexidine were similar in antibacterial activity, being effective at low concentrations against most organisms tested. Hexadecylamine also inhibited growth but at higher concentrations. Viable counts of salivary organisms were monitored in volunteers over 48 h after one rinse with the agents. The initial reduction in numbers of total viable salivary bacteria and streptococci by tetradecylamine and of streptococci by hexadecylamine had disappeared 3 h after a single mouth rinse, but the reduction in numbers of all salivary bacteria by chlorhexidine was more prolonged.     

Studies on the effect of toothpaste rinses on plaque regrowth

A number of compounds have been added to toothpastes to inhibit plaque regrowth. The inclusion of cationic antiseptics, such as chlorhexidine, poses formulation difficulties because of interactions with other ingredients particularly anionic detergents. More recently, Triclosan/zinc citrate formulations have been shown effective plaque inhibitors. The aim of this study was to compare a commercially available 0.2% Triclosan/0.5% zinc citrate toothpaste with a number of experimental 0.5% chlorhexidine/detergent toothpastes for effects on plaque regrowth over 4 days. Subjects rendered plaque free at each baseline rinsed twice a day with toothpaste slurries and disclosed plaque was scored at the end of each period. All toothpastes significantly reduced plaque by comparison with a control toothpaste, but were significantly less effective than a 0.2% chlorhexidine rinse. Some significant differences in favour of one chlorhexidine toothpaste were noted but these were small in magnitude. Whether the plaque inhibition obtained with Triclosan/zinc citrate toothpaste was greater than would be expected from other commercially available preparations cannot be determined from this study and is the subject of a further investigation.     

Inhibition of Porphyromonas gingivalis proteinases (gingipains) by chlorhexidine: synergistic effect of Zn(II)

BACKGROUND/AIMS: Gingipains, proteolytic enzymes produced by the periodontal pathogen Porphyromonas gingivalis, are regarded as virulence factors in the pathogenesis of periodontitis. Inhibition of gingipain activity therefore may have therapeutic potential, and it has been suggested that chlorhexidine may inhibit the activities of these enzymes. The purposes of the present study were to examine systematically the inhibitory effects of chlorhexidine on three purified gingipains and to determine the effect of Zn(II) on chlorhexidine inhibition. METHODS: The activities of lys-gingipain (Kgp) and two forms of arg-gingipain (RgpB and HRgpA) were measured in the presence of varying concentrations of chlorhexidine and with chlorhexidine supplemented with Zn(II). Inhibition constants (K(i)'s) were determined for chlorhexidine alone and in the presence of Zn(II). Fractional inhibitory constant indices were calculated to assess the synergy of the chlorhexidine-Zn(II) inhibition. RESULTS: RgpB, HRgpA, and Kgp were all inhibited by chlorhexidine with K(i)'s in the micromolar range. For RgpB and HRgpA, the inhibitory effects of chlorhexidine were enhanced 3-30-fold by Zn(II). The chlorhexidine-Zn(II) interaction was synergistic for inhibition of HRgpA and RgpB. For Kgp, the effect of Zn(II) on chlorhexidine inhibition was antagonistic. CONCLUSIONS: Chlorhexidine is an effective inhibitor of gingipains, and the inhibition of R-gingipains is enhanced by Zn(II). A mixture of chlorhexidine and Zn(II) may be useful as an adjunct in the treatment of periodontitis and in the post-treatment maintenance of periodontitis patients.     

Microbiological aspects of an in situ model to study effects of antimicrobial agents on dental plaque ecology

This study validates an in situ model for ecological studies of dental plaque exposed to various antimicrobial agents with different modes of action on plaque bacteria. Eleven subjects wore two acrylic appliances, each containing two bovine enamel discs, during two 1-wk test periods. Using a split-mouth crossover design, the appliances were dipped twice daily for 1 min into water (control; treatment A), fluoride (26.3 mM NaF; B), zinc acetate (20.0 mM; C), or fluoride plus zinc acetate (D). Four of the subjects used also chlorhexidine diacetate (2.2 mM; E) and chlorhexidine plus fluoride (F). At the end of each period, plaque was collected from the discs, after which the microbiota were analyzed by culture, automated quantitative immunofluorescence, and a viability fluorescence stain. As compared to control, treatments B, C, and D resulted in a significant reduction of individual taxa as detected by immunofluorescence, whereas similar bacterial viability and total bacterial numbers were observed. In contrast, chlorhexidine significantly reduced bacterial viability, total cell numbers, and the abundance of most of the enumerated taxa. We conclude that this in situ model is well suited to study effects of antimicrobial agents on dental plaque ecology. Combined with viability testing, immunofluorescence is obviously superior to culture in detecting taxa-specific shifts caused by antimicrobial agents.     

The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man

Twenty-four male dental students with healthy gingivae and clean teeth ceased all oral hygiene procedures. (A) Four subjects rinsed, twice daily, with a 0.2 per cent solution of chlorhexidine gluconate; (B) eight students rinsed, once daily, with the same solution; (C) six students did not rinse and formed the control group and (D) six students received one daily application of a 2 per cent solution of chlorhexidine gluconate. The study confirmed previous observations (Löe and Rindom Schiött 1969, 1970) that two daily mouth rinses with a 0.2 per cent solution of chlorhexidine effectively prevent plaque formation. One daily rinse did not inhibit plaque formation in all areas of the dentition. One daily topical application of a 2 per cent solution of chlorhexidine gluconate prevented plaque formation completely. Upon discontinuation of the chlorhexidine treatment plaque formed at normal rates, suggesting that there is no appreciable effect beyond a 24 hour period. It is concluded that complete inhibition of plaque and prevention of gingivitis may be achieved by daily application of chlorhexidine, provided the agent is administered in such a way that it reaches all tooth surfaces.     

Studies on the effect of polyvinyl pyrrolidone on the activity of chlorhexidine mouthrinses: plaque and stain

BACKGROUND, AIMS: Polyvinyl pyrrolidone (PVP) was shown in vitro to reduce chlorhexidine induced, dietary staining without affecting the uptake of the antiseptic to the test substrate. The aim of these studies in vivo was to determine whether PVP affected plaque and dietary staining by a low concentration chlorhexidine rinse. METHODS: The plaque and stain studies used a double blind, randomised 6, treatment crossover design involving healthy subjects with a high standard of oral hygiene and gingival health. The rinse formulations under test were: (A) aqueous alcohol (placebo control), (B) 0.03% chlorhexidine, (C) 0.06% chlorhexidine, (D) 0.06% chlorhexidine+1.2% PVP, (E) 0.06% chlorhexidine+5% PVP, (F) 0.06% chlorhexidine+10% PVP. In the plaque study, on day 1 of each period, subjects were rendered plaque free and then rinsed with 15 ml of the test rinse for 60 s. No further tooth cleaning was performed and subjects returned 24 h later for plaque scoring by area. In the stain study, on day 1 of each period, the tongue and teeth of each subject were rendered stain free. Subjects then rinsed under supervision for 60 s with 15 ml of the allocated rinse 8 x a day between 09:00 h and 17:00 h for 3 days. Immediately after each rinse with the test formulation, subjects rinsed for 120 s with 15 ml of warm black tea. Subjects were requested to also drink at least 5 cups of tea or coffee per day. On day 4, stain was scored by area and intensity from designated teeth and dorsum of the tongue. Washout periods were at least 7 days in both studies. RESULTS: Plaque areas were greatest with placebo and least with 0.06% chlorhexidine. Plaque scores increased with increasing concentrations of PVP in the 0.06% chlorhexidine rinse and were significantly higher than 0.06% chlorhexidine without PVP rinse. Tooth stain areas were comparable for placebo, 0.03% and 0.06% chlorhexidine rinses, but significantly reduced with the PVP/chlorhexidine rinses compared to the 0.06% chlorhexidine rinse. Tooth stain intensity was significantly increased with 0.06% chlorhexidine rinses compared to placebo and chlorhexidine/PVP rinses. Tongue stain area and intensity were significantly reduced with 5% and 10% PVP/chlorhexidine rinses compared to 0.06% chlorhexidine rinse. CONCLUSION: PVP, at the concentrations tested, reduced the stain propensity of a 0.06% chlorhexidine rinse but at the expense of some loss of plaque inhibition.