Australian Dental Journal, October, 1976
383
Volume 21,
No. 5
A clinical study of the effect of a cetylpyridinium chloride-based mouth wash on the concentration of Streptococcus mutans in dental plaque" J. D. Holbeche** M.
K. Ruljancich AND
P. C. Reade
ABSTRACT-A commercially available mouthwash containing 0.05 per cent weight/volume cetylpyridinium chloride when used regularly in a clinical trial had no effect on the concentration of Streptococcus mutans in plaque. (Received for publication September, 1975)
Introduction It is generally accepted that the initiation of both dental caries and periodontal disease is directly associated with the accumulation on teeth of bacterial plaque'. In the search for effective methods of controlling dental plaque emphasis has focused on the use of chemical agents, especially those substances with antibacterial activity. Various antibacterial compounds, including antibiotics and antiseptics, have been tested clinically for their ability t o inhibit plaque accumulation. Several compounds such as the antibiotic, vancomycin, and the antiseptic, chlorhexidine, have been shown to significantly inhibit plaque formationz. 3.
Most studies on chemical plaque control agents have been of a purely clinical nature and in only a few cases has the effect of the antibacterial compound on the oral flora been assessed. The reductions in plaque accumulation produced by chlorhexidine and vancomycin have been reported to be accompanied by significant changes in the plaque flora including specific inhibition of Streptococcus mutans39 I,an organism thought to play an important role in the initiation of smooth surface caries5. It was recently reported that regular rinsing after meals with the commercially available mouthwash, Cepacol, containing 0.05 per cent w/v of the quaternary ammonium compound, cetylpyridinium chloride (CPC), significantly reduced the thickness and extent of accumulated plaque6. As part of
* %ppor.tcd
by a grant from Richardson-Merrell Pty. Ltd. b a scholarship provided by RichardsonMerrell Pty. Ltd.
** Supported
Oibbons, R. J., and van Houte, J . 4 n the formation of dental plaques. J. Periodontol., 44:6, 347-360 (June) 1973. 2 Loe, H.-Does chlorhexidine have a place in the prophylaxis of dental diseases? J. Periodontol. Res., 8: Suppl. 12, 93-99. 1973. 3 Jordan, H. V.-A systematic approach to antibiotic control of dental caries. J. Canad. D. A.. 39:10, 703-708 (Oct.) 1973. 1
B., Mikkelsen, L., Loe H. and Rindom Schiott. C.-Influence of chlorhexidine 'on 'Streptococcus mutans !%-_human plaque. J. D. Res., 51:s. 1309 (Sept.-Oct.)
4 Jensen, 9.
A clinical trie! of the efficacy nf n cetvlnvridiniiirn chloride-hased mouthwnsh I Fffe
384
Australian Dental Journal, October, 1976
this clinical trial a study was undertaken to assess the effect of rinsing with this mouthwash on the concentration of S. mutans in plaque. The results of this study are presented here. Materials end methods Details of the clinical trial and the procedures used have been described previously6. Briefly the trial was a three-week double-blind crossover study in which each of 100 subjects rinsed regularly with the proprietary mouthwash* (containing 0.05 per cent w/v CPC as the active ingredient), and a placebo mouthwash for a period of one week. The initial week of the trial was a control period in which no mouthwash was used. The sequence of use of the mouthwashes and their mode of use (before or after meals) was randomly distributed throughout the subjects; thus in effect there were four separate groups of subjects: (a) those rinsing before meals and using the placebo mouthwash during the first test period, (b) those rinsing before meals and using the CPC-containing mouthwash during the first test period, (c) those rinsing after meals and using the placebo mouthwash during the first test period, and (d) those rinsing after meals and using the CPCcontaining mouthwash during the first test period. Each subject was examined four times: at the commencement of the trial, and at the end of each of the three subsequent weeks. At each examination plaque was collected and cultured for S . mutans.
Collection of plaque Plaque was taken from each subject using a sterile, preweighed flat plastic instrument. The wet weight of plaque obtained was determined by a second weighing which was carried out immediately after collection of the plaque to avoid drying. Before collecting the plaque the surface of the tooth was carefully mopped free of excess saliva with sterile gauze. The plaque obtained was a composite sample taken from the lingual surface of both the lower right second standing molar and the lower left second standing molar. Culturing plaque to enumerate S. mucane After weighing, the plaque was immediately transferred to a small sterile tissue grinder and thoroughly homogenized in peptone water containing 1 per cent w/v Tween 80. The volume
* Cepmol.
Richardson-Merrell Pty. Ltd., Sydney, Australia.
of this neutralizing diluent was calculated so that the final homogenized suspension contained 1 mg of plaque/ml. After allowing to stand for 30 minutes to ensure that any residual CPC had been completely inactivated, the plaque suspension was thoroughly mixed and ten-fold serial dilutions were prepared in peptone water. Using glass spreaders, duplicate 0.1 ml aliquots of the dilu10-3 and 10-4 mg plaque/ml tions containing were spread onto the surface of Mitis Salivarius agar (B.B.L.) plates containing 0.1 per cent w /v sulphadimidine. The plates were incubated for 48 hours at 37°C in an atmosphere of hydrogen 4-5 per cent carbon dioxide. The plaque samples from 25 randomly selected subjects were also cultured on plain Mitis Salivarius agar plates and incubated in the same atmosphere. Identification of S. mutans Using a stereoscopic microscope the plates were examined by reflected light for colonies morphologically resembling S. mutans (the typical colonial morphology of Mitis Salivarius agar plates incubated anaerobically has been described in detail by Krasse7). The number of such colonies on appropriate duplicate plates was counted and the mean obtained. From this figure the concentration of S. mutans in the original plaque sample was calculated. To confirm the identity of colonies distinguished as S. mutans, and to test suspect colonies with atypical morphology, representative colony types from each sample were tested for their ability to ferment mannitol and sorbitol. Fermentation of these carbohydrates provided good presumptive evidence that the organism was S. mutans8. The original count was amended if this was indicated by the results of the fermentation reactions. Results The concentration of S. mutans in each of the four plaque samples was determined for 96 of the 100 subjects. Complete results for the remaining four subjects were not obtained due to technical errors. Normal plaque The plaque samples obtained at examinations 1 and 2 (i.e. before the use of any mouthwash) were controls and thus 192 examinations of normal plaque were performed. From these examinations some information was obtained about the
7 Krasse, B.-Human 8
streptococci and experimental caries in hamsters. Arch. Oral Biol., 11:4, 429436 (April) 1966. Colman, G., and Williams, R . E. 0.-Taxonomy of some human viridans streptococci. In. Streptococci and strep tococcal diseases. Recognition, understanding and management. Edits. Wannamaker, L. W., and Matsen, J. M. London, Academic Press, 1972 (pp. 281-299).
Australian Dental Journal, October, 1976
385
TABLE 1
Concentration of Streptococcus mutans in the plaque samples obtained at the control examinations (based on 192 samples from 96 subjects) ~~
Concentration of S. mutans.
Number of samples in range
Frequency (Per cent)
383
Volume 21,
No. 5
A clinical study of the effect of a cetylpyridinium chloride-based mouth wash on the concentration of Streptococcus mutans in dental plaque" J. D. Holbeche** M.
K. Ruljancich AND
P. C. Reade
ABSTRACT-A commercially available mouthwash containing 0.05 per cent weight/volume cetylpyridinium chloride when used regularly in a clinical trial had no effect on the concentration of Streptococcus mutans in plaque. (Received for publication September, 1975)
Introduction It is generally accepted that the initiation of both dental caries and periodontal disease is directly associated with the accumulation on teeth of bacterial plaque'. In the search for effective methods of controlling dental plaque emphasis has focused on the use of chemical agents, especially those substances with antibacterial activity. Various antibacterial compounds, including antibiotics and antiseptics, have been tested clinically for their ability t o inhibit plaque accumulation. Several compounds such as the antibiotic, vancomycin, and the antiseptic, chlorhexidine, have been shown to significantly inhibit plaque formationz. 3.
Most studies on chemical plaque control agents have been of a purely clinical nature and in only a few cases has the effect of the antibacterial compound on the oral flora been assessed. The reductions in plaque accumulation produced by chlorhexidine and vancomycin have been reported to be accompanied by significant changes in the plaque flora including specific inhibition of Streptococcus mutans39 I,an organism thought to play an important role in the initiation of smooth surface caries5. It was recently reported that regular rinsing after meals with the commercially available mouthwash, Cepacol, containing 0.05 per cent w/v of the quaternary ammonium compound, cetylpyridinium chloride (CPC), significantly reduced the thickness and extent of accumulated plaque6. As part of
* %ppor.tcd
by a grant from Richardson-Merrell Pty. Ltd. b a scholarship provided by RichardsonMerrell Pty. Ltd.
** Supported
Oibbons, R. J., and van Houte, J . 4 n the formation of dental plaques. J. Periodontol., 44:6, 347-360 (June) 1973. 2 Loe, H.-Does chlorhexidine have a place in the prophylaxis of dental diseases? J. Periodontol. Res., 8: Suppl. 12, 93-99. 1973. 3 Jordan, H. V.-A systematic approach to antibiotic control of dental caries. J. Canad. D. A.. 39:10, 703-708 (Oct.) 1973. 1
B., Mikkelsen, L., Loe H. and Rindom Schiott. C.-Influence of chlorhexidine 'on 'Streptococcus mutans !%-_human plaque. J. D. Res., 51:s. 1309 (Sept.-Oct.)
4 Jensen, 9.
A clinical trie! of the efficacy nf n cetvlnvridiniiirn chloride-hased mouthwnsh I Fffe
384
Australian Dental Journal, October, 1976
this clinical trial a study was undertaken to assess the effect of rinsing with this mouthwash on the concentration of S. mutans in plaque. The results of this study are presented here. Materials end methods Details of the clinical trial and the procedures used have been described previously6. Briefly the trial was a three-week double-blind crossover study in which each of 100 subjects rinsed regularly with the proprietary mouthwash* (containing 0.05 per cent w/v CPC as the active ingredient), and a placebo mouthwash for a period of one week. The initial week of the trial was a control period in which no mouthwash was used. The sequence of use of the mouthwashes and their mode of use (before or after meals) was randomly distributed throughout the subjects; thus in effect there were four separate groups of subjects: (a) those rinsing before meals and using the placebo mouthwash during the first test period, (b) those rinsing before meals and using the CPC-containing mouthwash during the first test period, (c) those rinsing after meals and using the placebo mouthwash during the first test period, and (d) those rinsing after meals and using the CPCcontaining mouthwash during the first test period. Each subject was examined four times: at the commencement of the trial, and at the end of each of the three subsequent weeks. At each examination plaque was collected and cultured for S . mutans.
Collection of plaque Plaque was taken from each subject using a sterile, preweighed flat plastic instrument. The wet weight of plaque obtained was determined by a second weighing which was carried out immediately after collection of the plaque to avoid drying. Before collecting the plaque the surface of the tooth was carefully mopped free of excess saliva with sterile gauze. The plaque obtained was a composite sample taken from the lingual surface of both the lower right second standing molar and the lower left second standing molar. Culturing plaque to enumerate S. mucane After weighing, the plaque was immediately transferred to a small sterile tissue grinder and thoroughly homogenized in peptone water containing 1 per cent w/v Tween 80. The volume
* Cepmol.
Richardson-Merrell Pty. Ltd., Sydney, Australia.
of this neutralizing diluent was calculated so that the final homogenized suspension contained 1 mg of plaque/ml. After allowing to stand for 30 minutes to ensure that any residual CPC had been completely inactivated, the plaque suspension was thoroughly mixed and ten-fold serial dilutions were prepared in peptone water. Using glass spreaders, duplicate 0.1 ml aliquots of the dilu10-3 and 10-4 mg plaque/ml tions containing were spread onto the surface of Mitis Salivarius agar (B.B.L.) plates containing 0.1 per cent w /v sulphadimidine. The plates were incubated for 48 hours at 37°C in an atmosphere of hydrogen 4-5 per cent carbon dioxide. The plaque samples from 25 randomly selected subjects were also cultured on plain Mitis Salivarius agar plates and incubated in the same atmosphere. Identification of S. mutans Using a stereoscopic microscope the plates were examined by reflected light for colonies morphologically resembling S. mutans (the typical colonial morphology of Mitis Salivarius agar plates incubated anaerobically has been described in detail by Krasse7). The number of such colonies on appropriate duplicate plates was counted and the mean obtained. From this figure the concentration of S. mutans in the original plaque sample was calculated. To confirm the identity of colonies distinguished as S. mutans, and to test suspect colonies with atypical morphology, representative colony types from each sample were tested for their ability to ferment mannitol and sorbitol. Fermentation of these carbohydrates provided good presumptive evidence that the organism was S. mutans8. The original count was amended if this was indicated by the results of the fermentation reactions. Results The concentration of S. mutans in each of the four plaque samples was determined for 96 of the 100 subjects. Complete results for the remaining four subjects were not obtained due to technical errors. Normal plaque The plaque samples obtained at examinations 1 and 2 (i.e. before the use of any mouthwash) were controls and thus 192 examinations of normal plaque were performed. From these examinations some information was obtained about the
7 Krasse, B.-Human 8
streptococci and experimental caries in hamsters. Arch. Oral Biol., 11:4, 429436 (April) 1966. Colman, G., and Williams, R . E. 0.-Taxonomy of some human viridans streptococci. In. Streptococci and strep tococcal diseases. Recognition, understanding and management. Edits. Wannamaker, L. W., and Matsen, J. M. London, Academic Press, 1972 (pp. 281-299).
Australian Dental Journal, October, 1976
385
TABLE 1
Concentration of Streptococcus mutans in the plaque samples obtained at the control examinations (based on 192 samples from 96 subjects) ~~
Concentration of S. mutans.
Number of samples in range
Frequency (Per cent)
