J. Periodontal Res. 12: 323-330, 1977
Some effects of chiorhexidine acetate on the adherence of oral bacteria to dental enamel m vitro D. G. TUSTIAN AND R. P. ElXEN
Departments of Periodontoiogy and Clinical Sciences, University of Toronto, Facalty of Dentistry, 124 Edward Street, Toronto, Ontario, Canada The effects of chlorhexidine acetate on the ability of some oral Streptococcus and Actinomyces strains to attach to bovine enamel were evaluated in an in vitro system. The numbers of bacteria adhering per unit area to enamel blocks immersed in bacterial suspensions were enomerated using an incident light microscope. Pre-incubation of the enamel in whole saliva to form an in vitro pellicle enhanced tlie adsorption of some strains and reduced the adsorption of others. Pretreatment of uncoated enamel with mouthwash concentrations of chiorhexidine significantly impaired the adsorption of two of the fonr test strains and enhanced the adsorption of one of the strains. Chiorhexidine pretreatment of saliva-coated enamel enhanced the adherence of all bacterial strains tested. These changes in bacterial adsorption may reflect chlorhexidine-mediated alterations in the properties of peUiclecovered enamel. Pretreatmeut of the bacterial celis with subinhibitory doses of chiorhexidine acetate or sodium acetate did not significantly reduce the ability of the bacteria to attach to the saliva-coated enamel surface. Together, the data suggest that the reduction in piaque accumulation associated with chiorhexidine mouthrinsing may be independent of a direct inhibition of initial bacterial adsorption to pellicle. (Accepted for publication December 3, 1976)
Introduction Bacterial adsorption to salivary proteins coating enamel surfaces is considered to be the initial stage of dental plaque formation (Gibbons & van Houte 1973, 1975). The relative abilities of specific salivary bacteria to colottize clean tooth surfaces are influenced by both their natural affinities for salivary pellicle proteins and by their numbers available in saliva (van Hoate, Gibbons & Banghart 1970, van Houte, Gibbons & Pulkkinen 1971, van Houte & Green 1974). Chlorhaxidine, a potent antibacterial agent (Davies et al. 1954, Hennessey 1973) which has been used successfully in moiithwashes to prevent
dental plaque accumulation (Davies et al. ^^JQ^ j^g^ ^ Schiott 1970), may affect both of these factors. The use of chiorhexidine moBthriases resuits in markedly reduced numbers of salivary bacteria (Schiott et al. 1970). This effeet has been related to the abUity of chlorhexidine to be retained in the mouth through reversible binding to mucous membranes (Rolla, Loe & Schiott 1970, 1971, Bonesvoi et al. 1974, Gjermo, BoaesvoU & Rolla 1974). Subsequent gradual desorption of chiorhexidine molecules over a period of hours results in a prolonged antibacterial effect (Davies 1973, Gjermo et al. 1975, RoUa & Kaae 1975). However, the plaqtie-
324
TUSTIAN
preventing effects of cUorhexidine seem to be greater than can be explained solely by the observed decrease of bacterial populations in saliva. It has been suggested that chlorhexidine exerts specific antiplaque effects at the tooth surface (Davies et al. 1970, Davies 1974). Chlorhexidine may be able to alter the bacterial cell surface and/or the exposed tooth surface properties thereby influencing the adsorption of bacteria to dental surfaces (Rolla & Melsen 1975b, Quintana, Fisher & Lasslo 1972). It has also been suggested that the chlorhexidine molecule may exert a "specific" surfactant activity at hydroxyapatite-water interfaces (Heard & Ashworth 1968) which would impair bacterial adherence in general (Fisher, Quintana & Bouleware 1975), The present study was designed to investigate the possibility that chlorhexidine, apart from its antibacterial properties, may alter the ability of oral bacteria to adsorb to dental surfaces. An attempt was made to investigate separately the effects of chlorhexidine at the bacterial surface and its effects at the tooth surface.
AMD
ELLEN
soy agar {Difco) slants at 4° C and in fltiid thioglycollate medium (Difco). The thioglycollate cultures were transferred fortnightly.
Assay for Bacterial Adherence to Enamel The naoibers of bacteria adhering to enamel surfaces were evaluated by incident iight microscopy (0rstavik, Kraus & Henshaw 1974). Bacteria were harvested by centrifugatton from 20-22 hour aerobic coltares in tr3/pticase salts broth (Gibbons & Fitzgerald 1969) and were washed twice in 0.01 M phosphate buffered saline (PBS) at pH 7.2. Bacterial suspension containing 1.5—3.0 X 10* organisms per mi were prepared in the same buffer. One or two clean, polished enamel slabs were incobated in the bacterial suspensions for one hour at 37° C in a shaking water bath. The enamel siabs were washed free of unattached bacteria in PBS for one minute and allowed to dry. The adsorbed bacteria were fixed in ethanol and stained using a modified Gram strain (0rstavils et al. 1974). For these experiments, bovine teeth were sectioned into 1 cm square blocks approximately 2-3 mm thick. The labial surfaces were poiished flat and smooth with a series Material and Metliod of abrasive papers. Before each experiment, the enamel was pumiced thoroughly, washBacterial Strains and Cultural Conditions The foilowing oral bacterial strains were ed, and stored in sterile distilled water. tested: Streptococcus saUvarius strain SALA microscope equipped with an incident 1, Streptococcus sanguis strains JAL-8 and iight vertical illuminator, a 60-power oil obFl, Actinomyces naeslundii strains Ig and jective lens (Emst Leitz, Ortholux), and a CI, and Actinomyces viscosus strains C12 reticule grid in the ocular lens was employed and T14. A!l strains were isolated from la- to quantify the bacteria adsorbed to the boratory personnel at the Uoiversity of To- enamel surfaces (0rstavik et al. 1974). Nuronto, Faculty of Dentistry except A. visco- merical data for each experimental treatsus strain T14, which was obtained from the ment were obtained by counting the bacteria culture collection of Forsyth Dental Center, in a set grid pattern of 40-160 randomly seand A. naeslundii strain Ig, which was iso- lected fields. The values obtained represent lated from the feces of germfree rats mono- the number of visible bacteria adhering to infected with A. naeslundii strain I (S. S. So- 4.9 X 10~^ mm^ of enamel. Large enamel cransky, personal communication). AU defects or cracks were avoided in the selecstrains were maintained as stock cultures on tion of fields for the counting procedure.
CHLORHEXIDINE AND BACTERIAL
The examiner was unaware of the identity of the sample under observation. Bacterial Adherence to Saliva-Coated Enamel The effect of saliva-coating the enamel surface on bacterial adherence was evaluated. A two-hour in vitro pellicle (0rstavik et al. 1974) was formed by immersing clean enamel slabs in 2.0 ml samples of stimulated, whole, cleared saliva which was pooled from the same two individuals for each experiment. The slabs were washed in distilled water prior to incubation with bacteria. Effect of Chlorhexidine Treatment of Enamel Surfaces on Bacterial A dherence The most recently isolated strains of the four species which had been tested as described above were chosen to study the effects of chlorhexidine on adherence. Clean enamel slabs and slabs with a salivary pellicle were exposed to typical mouthwash concentrations (0.2 %, 3.2 X 10^= M) of chlorhexidine acetate (Gjermo et al. 1970) at pH 7.0 for either 5 or 10 minutes. The enamel was washed for 5 minutes in large volumes of distilled water before incubation with bacterial suspensions. Effect of Chlorhexidine Treatment of Bacteria on their Adherence to Enamel Minimum inhibitory concentrations of chlorhexidine acetate were determined for the bacterial strains studied. Duplicate tubes of 5.0 ml glucose-supplemented (1.0 %, w/v) ttyptic soy broth (Difco) containing various concentrations of cMorhexidine acetate or sodium acetate were inoculated with approximately 5.0 X 10'' washed bacteria. Bacterial growth was estimated by measuring the optical density (550 nm) of the cultures at intervals for up to 48 hours. A chlorhexidine concentration of 1.0 ug/ml was selected as the treatment dose for bacteria in the adherence studies because it was found to be
ADHERENCE
325
below the minimum inhibitory concentrations for the test strains, and because it was within the range of salivary concentrations reported in clinical trials of chlorhexidine mouth rinses (Jensen & Christensen 1971). The effect of pretreating the bacteria with chlorhexidine on their abilities to adhere to enamel was evaluated by exposing washed cells to pH 7.0 PBS solutions containing 1.0 ug chlorhexidine acetate per ml (1.6 X 10"^ M) for 5 minutes. Control bacteria were treated similarly vrith the same molar concentration of sodium acetate. The bacteria were washed twice in PBS at pH 7.2 before Incubation with enamel slabs. Statistical Analysis The numerical data representing counts of bacteria per unit area of identical treatments of individual enamel blocks from different experiments were grouped together. The skewness of the data (from histogram distributions) precluded quantitative comparisons of the different experimental treatments. Therefore, qualitative methods for determining significance were employed. The data for different treatments of the bacteria or enamel were divided into groups of high and low values using the mean of all data for a particular strain as the dividing criterion. Comparisons of different treatments were carried out from 2 X 2 association tables using the chi-square test with one degree of freedom. Yates' correction for continuity was applied (Maxwell 1961). Probabilities were ascertained from chi-square tables; P values greater than 0.05 were assumed to be not significant. Results
Microscope observation of polished enamel slabs revealed smooth sturfaces with occasional cracks. Interprismatic material was lightly stained. Exposing the enamel to suspensions of bacteria resulted in the adher-
326
TUSTIAN
AiND
ELLEN
Table 1
Table 3
Comparison of bacterial adherence to salivacoated and uncoated (control) enamel
Comparison of bacterial adherence to Chiorhexidine-treated and untreated (control)
Bacterial Spscies snd strEtn
Mean number of P adherent bacteria" (range ot values} cbj-square uncoated sa!iva-coat. test enamel enamet
S. salivarius SAL-1 S. sanguis JAL-8 S. sanguis
2B0 (43-1055) 180 (2-55D) 23
95 (0^00) 61 (0-245) 82
Ft A. naeslndii
(0-70) 234
(0-575) 95
(8-665) 196 (25-760) 96 (10-225)
(0-280) 98 (0-405) 135 (0-635)
lii
A. viscosus T14 A. viscosus C12
< 0.05 < 0.05 < 0.001
Some effects of chiorhexidine acetate on the adherence of oral bacteria to dental enamel m vitro D. G. TUSTIAN AND R. P. ElXEN
Departments of Periodontoiogy and Clinical Sciences, University of Toronto, Facalty of Dentistry, 124 Edward Street, Toronto, Ontario, Canada The effects of chlorhexidine acetate on the ability of some oral Streptococcus and Actinomyces strains to attach to bovine enamel were evaluated in an in vitro system. The numbers of bacteria adhering per unit area to enamel blocks immersed in bacterial suspensions were enomerated using an incident light microscope. Pre-incubation of the enamel in whole saliva to form an in vitro pellicle enhanced tlie adsorption of some strains and reduced the adsorption of others. Pretreatment of uncoated enamel with mouthwash concentrations of chiorhexidine significantly impaired the adsorption of two of the fonr test strains and enhanced the adsorption of one of the strains. Chiorhexidine pretreatment of saliva-coated enamel enhanced the adherence of all bacterial strains tested. These changes in bacterial adsorption may reflect chlorhexidine-mediated alterations in the properties of peUiclecovered enamel. Pretreatmeut of the bacterial celis with subinhibitory doses of chiorhexidine acetate or sodium acetate did not significantly reduce the ability of the bacteria to attach to the saliva-coated enamel surface. Together, the data suggest that the reduction in piaque accumulation associated with chiorhexidine mouthrinsing may be independent of a direct inhibition of initial bacterial adsorption to pellicle. (Accepted for publication December 3, 1976)
Introduction Bacterial adsorption to salivary proteins coating enamel surfaces is considered to be the initial stage of dental plaque formation (Gibbons & van Houte 1973, 1975). The relative abilities of specific salivary bacteria to colottize clean tooth surfaces are influenced by both their natural affinities for salivary pellicle proteins and by their numbers available in saliva (van Hoate, Gibbons & Banghart 1970, van Houte, Gibbons & Pulkkinen 1971, van Houte & Green 1974). Chlorhaxidine, a potent antibacterial agent (Davies et al. 1954, Hennessey 1973) which has been used successfully in moiithwashes to prevent
dental plaque accumulation (Davies et al. ^^JQ^ j^g^ ^ Schiott 1970), may affect both of these factors. The use of chiorhexidine moBthriases resuits in markedly reduced numbers of salivary bacteria (Schiott et al. 1970). This effeet has been related to the abUity of chlorhexidine to be retained in the mouth through reversible binding to mucous membranes (Rolla, Loe & Schiott 1970, 1971, Bonesvoi et al. 1974, Gjermo, BoaesvoU & Rolla 1974). Subsequent gradual desorption of chiorhexidine molecules over a period of hours results in a prolonged antibacterial effect (Davies 1973, Gjermo et al. 1975, RoUa & Kaae 1975). However, the plaqtie-
324
TUSTIAN
preventing effects of cUorhexidine seem to be greater than can be explained solely by the observed decrease of bacterial populations in saliva. It has been suggested that chlorhexidine exerts specific antiplaque effects at the tooth surface (Davies et al. 1970, Davies 1974). Chlorhexidine may be able to alter the bacterial cell surface and/or the exposed tooth surface properties thereby influencing the adsorption of bacteria to dental surfaces (Rolla & Melsen 1975b, Quintana, Fisher & Lasslo 1972). It has also been suggested that the chlorhexidine molecule may exert a "specific" surfactant activity at hydroxyapatite-water interfaces (Heard & Ashworth 1968) which would impair bacterial adherence in general (Fisher, Quintana & Bouleware 1975), The present study was designed to investigate the possibility that chlorhexidine, apart from its antibacterial properties, may alter the ability of oral bacteria to adsorb to dental surfaces. An attempt was made to investigate separately the effects of chlorhexidine at the bacterial surface and its effects at the tooth surface.
AMD
ELLEN
soy agar {Difco) slants at 4° C and in fltiid thioglycollate medium (Difco). The thioglycollate cultures were transferred fortnightly.
Assay for Bacterial Adherence to Enamel The naoibers of bacteria adhering to enamel surfaces were evaluated by incident iight microscopy (0rstavik, Kraus & Henshaw 1974). Bacteria were harvested by centrifugatton from 20-22 hour aerobic coltares in tr3/pticase salts broth (Gibbons & Fitzgerald 1969) and were washed twice in 0.01 M phosphate buffered saline (PBS) at pH 7.2. Bacterial suspension containing 1.5—3.0 X 10* organisms per mi were prepared in the same buffer. One or two clean, polished enamel slabs were incobated in the bacterial suspensions for one hour at 37° C in a shaking water bath. The enamel siabs were washed free of unattached bacteria in PBS for one minute and allowed to dry. The adsorbed bacteria were fixed in ethanol and stained using a modified Gram strain (0rstavils et al. 1974). For these experiments, bovine teeth were sectioned into 1 cm square blocks approximately 2-3 mm thick. The labial surfaces were poiished flat and smooth with a series Material and Metliod of abrasive papers. Before each experiment, the enamel was pumiced thoroughly, washBacterial Strains and Cultural Conditions The foilowing oral bacterial strains were ed, and stored in sterile distilled water. tested: Streptococcus saUvarius strain SALA microscope equipped with an incident 1, Streptococcus sanguis strains JAL-8 and iight vertical illuminator, a 60-power oil obFl, Actinomyces naeslundii strains Ig and jective lens (Emst Leitz, Ortholux), and a CI, and Actinomyces viscosus strains C12 reticule grid in the ocular lens was employed and T14. A!l strains were isolated from la- to quantify the bacteria adsorbed to the boratory personnel at the Uoiversity of To- enamel surfaces (0rstavik et al. 1974). Nuronto, Faculty of Dentistry except A. visco- merical data for each experimental treatsus strain T14, which was obtained from the ment were obtained by counting the bacteria culture collection of Forsyth Dental Center, in a set grid pattern of 40-160 randomly seand A. naeslundii strain Ig, which was iso- lected fields. The values obtained represent lated from the feces of germfree rats mono- the number of visible bacteria adhering to infected with A. naeslundii strain I (S. S. So- 4.9 X 10~^ mm^ of enamel. Large enamel cransky, personal communication). AU defects or cracks were avoided in the selecstrains were maintained as stock cultures on tion of fields for the counting procedure.
CHLORHEXIDINE AND BACTERIAL
The examiner was unaware of the identity of the sample under observation. Bacterial Adherence to Saliva-Coated Enamel The effect of saliva-coating the enamel surface on bacterial adherence was evaluated. A two-hour in vitro pellicle (0rstavik et al. 1974) was formed by immersing clean enamel slabs in 2.0 ml samples of stimulated, whole, cleared saliva which was pooled from the same two individuals for each experiment. The slabs were washed in distilled water prior to incubation with bacteria. Effect of Chlorhexidine Treatment of Enamel Surfaces on Bacterial A dherence The most recently isolated strains of the four species which had been tested as described above were chosen to study the effects of chlorhexidine on adherence. Clean enamel slabs and slabs with a salivary pellicle were exposed to typical mouthwash concentrations (0.2 %, 3.2 X 10^= M) of chlorhexidine acetate (Gjermo et al. 1970) at pH 7.0 for either 5 or 10 minutes. The enamel was washed for 5 minutes in large volumes of distilled water before incubation with bacterial suspensions. Effect of Chlorhexidine Treatment of Bacteria on their Adherence to Enamel Minimum inhibitory concentrations of chlorhexidine acetate were determined for the bacterial strains studied. Duplicate tubes of 5.0 ml glucose-supplemented (1.0 %, w/v) ttyptic soy broth (Difco) containing various concentrations of cMorhexidine acetate or sodium acetate were inoculated with approximately 5.0 X 10'' washed bacteria. Bacterial growth was estimated by measuring the optical density (550 nm) of the cultures at intervals for up to 48 hours. A chlorhexidine concentration of 1.0 ug/ml was selected as the treatment dose for bacteria in the adherence studies because it was found to be
ADHERENCE
325
below the minimum inhibitory concentrations for the test strains, and because it was within the range of salivary concentrations reported in clinical trials of chlorhexidine mouth rinses (Jensen & Christensen 1971). The effect of pretreating the bacteria with chlorhexidine on their abilities to adhere to enamel was evaluated by exposing washed cells to pH 7.0 PBS solutions containing 1.0 ug chlorhexidine acetate per ml (1.6 X 10"^ M) for 5 minutes. Control bacteria were treated similarly vrith the same molar concentration of sodium acetate. The bacteria were washed twice in PBS at pH 7.2 before Incubation with enamel slabs. Statistical Analysis The numerical data representing counts of bacteria per unit area of identical treatments of individual enamel blocks from different experiments were grouped together. The skewness of the data (from histogram distributions) precluded quantitative comparisons of the different experimental treatments. Therefore, qualitative methods for determining significance were employed. The data for different treatments of the bacteria or enamel were divided into groups of high and low values using the mean of all data for a particular strain as the dividing criterion. Comparisons of different treatments were carried out from 2 X 2 association tables using the chi-square test with one degree of freedom. Yates' correction for continuity was applied (Maxwell 1961). Probabilities were ascertained from chi-square tables; P values greater than 0.05 were assumed to be not significant. Results
Microscope observation of polished enamel slabs revealed smooth sturfaces with occasional cracks. Interprismatic material was lightly stained. Exposing the enamel to suspensions of bacteria resulted in the adher-
326
TUSTIAN
AiND
ELLEN
Table 1
Table 3
Comparison of bacterial adherence to salivacoated and uncoated (control) enamel
Comparison of bacterial adherence to Chiorhexidine-treated and untreated (control)
Bacterial Spscies snd strEtn
Mean number of P adherent bacteria" (range ot values} cbj-square uncoated sa!iva-coat. test enamel enamet
S. salivarius SAL-1 S. sanguis JAL-8 S. sanguis
2B0 (43-1055) 180 (2-55D) 23
95 (0^00) 61 (0-245) 82
Ft A. naeslndii
(0-70) 234
(0-575) 95
(8-665) 196 (25-760) 96 (10-225)
(0-280) 98 (0-405) 135 (0-635)
lii
A. viscosus T14 A. viscosus C12
< 0.05 < 0.05 < 0.001
