Oral Microbiol Immunol 1991: 6: 284-287
Mutans streptococci in saiiva and interciental spaces after topical applications of an antibacteriai varnish in schoolchiidren
Lars G. Petersson\ Yoshinobu Maki'^ Svante Twetman^", Stig Edwardsson^ 'Department of Preventive Dentistry, Medical and Dental Health Center, Lanssjukhuset, Halmstad, ^Department of Oral Microbiology, School of Dentistry, Lund University, Malmo, Sweden, 'Department of Preventive and Community Dentistry, Tokyo Dental College, Chiba, Japan, ^Department of Pedodontics, Karolinska Institute, Huddinge, Sweden
Petersson LG, Maki Y, Twetman S, Edwardsson S. Mutans streptococci in saliva and interdental spaces after topical applications of an antibacterial varnish in schoolchildren. Oral Microbiol Immunol 1991: 6: 284-287. The effect of a chlorhexidine-containing varnish on the levels of mutans streptococci in saliva and in interdental spaces was investigated in 33 15-year-old schoolchildren. Each child was treated with an active (1% chlorhexidine; 1% thytnol) and a placebo varnish preparation following the split-tnouth technique. The varnishes were applied twice in small amounts into upper interdental areas with a 2-d interval, Mutans streptococci in saliva and interdental plaque was sampled and enutnerated during 3 months. The results showed an immediate reduction of the number of interdental mutans streptococci on both test and placebo side after the varnish applications. The levels, however, were significantly lower in the test quadrants compared with the placebo-treated sides after 8, 30 and 90 d. Thus, the findings indicate a slower recolotiization in interdental spaces treated with the aetive preparation. The levels of mutans streptococci in saliva were signifieantly reduced 1 and 3 months after varnish treattnent, suggesting a longterm effect of the antibacterial varnish.
Several studies have emphasized the specific role of mutans streptococci in the initiation, progression and prevalence of dental caries (5, 14). It is also well established that tnutans streptococci atid other cariogenic tnicroorganisms are highly sensitive to chlorhexidine (2, 3), Suppression of these microorganistns in the oral cavity results iti caries reduction, detnonstrated both in children (21) and in adults (16), The effect of topical treattnents with chlorhexidine rinses and gels, however, seetns to be temporary, and recolonization occurs within a few weeks (6), To achieve prolonged antibacterial action, varnishes for sustained release of chlorhexidine have been developed (1), An effective long-term elitnination of mutans streptococci after repeated treatments with a 20% chlorhexidine varnish has been reported (17), Still higher eoncentrations of chlorhexidine (40-50%) in varnish preparations have been used in attetnpts to suppress or eliminate tnutans streptococci after single applications (18, 19), In the human permanent dentition.
high levels of mutans streptococci are most often found in interdental spaces and particularly between posterior teeth (6, 10, 12), Therefore, efforts to eliminate or suppress bacteria in those areas are of importance. Recently, an antimicrobial vartiish contaitiitig a low concentration of chlorhexidine (1%) was described by Huizinga et al, (9), A recent study has suggested that this preparation has an antimicrobial effect against caries- and periodontitis-associated bacteria (Petersson & Edwardsson, submitted), and an experimental study has indicated that it may prevent root caries in vivo (9), The aim of this study was to evaluate the effect of this varnish on the nutnber of tnutans streptococci present in ititerdental spaces and saliva from schoolchildren during a 3-tnonth period. Material and methods Subjects
To select a suitable study group, 140 15year-old schoolchildren were screened
Key words: antimicrobial varnish; dental plaque: mutans streptococci; saliva; schoolchildren Dr. Lars G. Petersson, Department of Preventive Dentistry, Lanssjukhuset, S-301 85 Halmstad, Sweden Accepted for publication December 4, 1990
for their levels of mutans streptococci in saliva. After infonned consent, 40 children with high levels of tnutans streptococci (> 100 colony-fortning units (CFU), corresponding to > 10^ per tnl saliva (13)) in the saliva were invited to participate in the study. Of these children, 33 complied for the whole experimental period. The reasons for dropout were vacation and illness. Data on the caries experience (DFS) on proximal surfaces were collected frotn bite-wing radiographs not older than 6 tnonths. Initial lesions were defined as radiolucent areas within the enamel that could not be related to nortnal anatotny or hypoplasia. Treatment procedures
Each subject was treated with 2 varnishes, one containing 1 % chlorhexidine and 1% thymol (9) and a placebo varnish without the 2 active ingredients. The test varnish was randotnly applied in the right upper side of the tnouth in half of the children and on the left upper
Antibacterial varnish and streptococci Table L Caries experience (mean atid standard deviation) of proximal surface of test and Placebo
Test mean
Index
O.I 09
DFS Initial*
SD 0.3 1.0
range 0-1 0^
mean
SD
range
0,2 0.9
0.5 0.8
0-2 0-2
*See Material and tnethods for definition.
side in the others. The placebo varnish was applied on the opposite side following the split-tnouth technique. The varnishes were applied in small amounts with brushes from both the buccal and lingual side of each interdental space between the upper tnolars and premolars. Prior to application the interdental areas were fiossed and carefully dried with air. The varnish was allowed to set for 10-15 s with help of a gentle air fiow. The treattnent was repeated in the same manner after 2 d though without previous fiossing. The children were instructed to refrain frotn oral hygiene procedures for 12 h after the varnish applications. Bacterial sampling and cuitivation
For the quantitative determination of mutans streptococci in saliva, the spatula method described by Kohler & Bratthall (13) was used. A wooden spatula was rotated on the tongue after paraffin chewing for 1 tnin. The spatula was subsequently pressed on a selective MSB-agar (8), The plates were incubated anaerobically in Gas-pak (BBL Microbiology Systetns, Cockeysville,
MD) jars at 37°C for 48 h. The number of CFU resembling mutans streptococci were counted using a stereomicroscope (x 12) and grouped into the following scores: 0 = 0-10 CFU; 1 = 11-99 CFU; 2= > 100 CFU, Salivary samplitig was perfortned at baseline and after 1 and 3 months respectively. Interdental enumeration of mutans streptococci was performed at 5 differetit occasions; at baselitie, after 2 d (before second varnish application), and subsequently 8, 30 and 90 d after the varnish treatments using the method described by Kristoffersson & Bratthall (11), The sampling was performed using soft wooden triangular toothpicks (TePe, Ekelund & Petersson, Malmo, Sweden), which were pressed from the lingual side into each interdental area until firtn contact was obtained with both teeth. Both sides of the toothpick were thereafter itnmediately pressed against the selective agar plates. The number of mutans streptococci was calculated frotn each interdental space after 48 h of tnicroaerophilic incubation in nitrogen and 5% CO, at 37°C, The plates were analysed as described above for the saliva samples. The following
[ 10 CFU ^ 1 - 2 0 CFU 21-100 CFU 100 CFU
BL
DAYS
DAYS
DAY30
DAY90
Fig I Quantitative distribution of mutans streptococci scores in interproximal spaces of test (T, « = 99) and placebo quadrants (P, « = 99) before and after varnish treatments. Differences between the quadrants were obtained at day 8 (P 100 CFU) at the baseline exatninThe results disclosed a decrease in that the varnish per se resembles and ation. Seventeen satnples (53%) of the number of mutans streptococci in inter- aets as a "shedding surface" with subareas on test sides had lower levels than dental spaces as well as in saliva. Our sequetit elitnination of tnicroorbaseline. The corresponding tiutnber for findings are thus in agreement with pre- gansistns. Nevertheless, a significant difthe placebo-treated interproximal vious reports on reduction and recolon- ference in favour of the test side was spaces was 11 satnples (32%), ization of mutans streptococci in inter- recorded at days 8, 30 and 90, indicating The number of proximal surfaces per dental areas after topical application a slower recolonization in sites where individual without presence of tnutans with chlorhexidine varnish (17, 19), No the test varnish was applied. The sucstreptococci at the start and during the side effects were noticed atnong the par- cessive increase on both sides is presutn3-month experitnental period is pre- ticipants. The taste was well tolerated ably caused by regrowth frotn retention sented in Table 3, Ati iticrease by all participants and none objected sites (6), Moreover, it must be con(P
Mutans streptococci in saiiva and interciental spaces after topical applications of an antibacteriai varnish in schoolchiidren
Lars G. Petersson\ Yoshinobu Maki'^ Svante Twetman^", Stig Edwardsson^ 'Department of Preventive Dentistry, Medical and Dental Health Center, Lanssjukhuset, Halmstad, ^Department of Oral Microbiology, School of Dentistry, Lund University, Malmo, Sweden, 'Department of Preventive and Community Dentistry, Tokyo Dental College, Chiba, Japan, ^Department of Pedodontics, Karolinska Institute, Huddinge, Sweden
Petersson LG, Maki Y, Twetman S, Edwardsson S. Mutans streptococci in saliva and interdental spaces after topical applications of an antibacterial varnish in schoolchildren. Oral Microbiol Immunol 1991: 6: 284-287. The effect of a chlorhexidine-containing varnish on the levels of mutans streptococci in saliva and in interdental spaces was investigated in 33 15-year-old schoolchildren. Each child was treated with an active (1% chlorhexidine; 1% thytnol) and a placebo varnish preparation following the split-tnouth technique. The varnishes were applied twice in small amounts into upper interdental areas with a 2-d interval, Mutans streptococci in saliva and interdental plaque was sampled and enutnerated during 3 months. The results showed an immediate reduction of the number of interdental mutans streptococci on both test and placebo side after the varnish applications. The levels, however, were significantly lower in the test quadrants compared with the placebo-treated sides after 8, 30 and 90 d. Thus, the findings indicate a slower recolotiization in interdental spaces treated with the aetive preparation. The levels of mutans streptococci in saliva were signifieantly reduced 1 and 3 months after varnish treattnent, suggesting a longterm effect of the antibacterial varnish.
Several studies have emphasized the specific role of mutans streptococci in the initiation, progression and prevalence of dental caries (5, 14). It is also well established that tnutans streptococci atid other cariogenic tnicroorganisms are highly sensitive to chlorhexidine (2, 3), Suppression of these microorganistns in the oral cavity results iti caries reduction, detnonstrated both in children (21) and in adults (16), The effect of topical treattnents with chlorhexidine rinses and gels, however, seetns to be temporary, and recolonization occurs within a few weeks (6), To achieve prolonged antibacterial action, varnishes for sustained release of chlorhexidine have been developed (1), An effective long-term elitnination of mutans streptococci after repeated treatments with a 20% chlorhexidine varnish has been reported (17), Still higher eoncentrations of chlorhexidine (40-50%) in varnish preparations have been used in attetnpts to suppress or eliminate tnutans streptococci after single applications (18, 19), In the human permanent dentition.
high levels of mutans streptococci are most often found in interdental spaces and particularly between posterior teeth (6, 10, 12), Therefore, efforts to eliminate or suppress bacteria in those areas are of importance. Recently, an antimicrobial vartiish contaitiitig a low concentration of chlorhexidine (1%) was described by Huizinga et al, (9), A recent study has suggested that this preparation has an antimicrobial effect against caries- and periodontitis-associated bacteria (Petersson & Edwardsson, submitted), and an experimental study has indicated that it may prevent root caries in vivo (9), The aim of this study was to evaluate the effect of this varnish on the nutnber of tnutans streptococci present in ititerdental spaces and saliva from schoolchildren during a 3-tnonth period. Material and methods Subjects
To select a suitable study group, 140 15year-old schoolchildren were screened
Key words: antimicrobial varnish; dental plaque: mutans streptococci; saliva; schoolchildren Dr. Lars G. Petersson, Department of Preventive Dentistry, Lanssjukhuset, S-301 85 Halmstad, Sweden Accepted for publication December 4, 1990
for their levels of mutans streptococci in saliva. After infonned consent, 40 children with high levels of tnutans streptococci (> 100 colony-fortning units (CFU), corresponding to > 10^ per tnl saliva (13)) in the saliva were invited to participate in the study. Of these children, 33 complied for the whole experimental period. The reasons for dropout were vacation and illness. Data on the caries experience (DFS) on proximal surfaces were collected frotn bite-wing radiographs not older than 6 tnonths. Initial lesions were defined as radiolucent areas within the enamel that could not be related to nortnal anatotny or hypoplasia. Treatment procedures
Each subject was treated with 2 varnishes, one containing 1 % chlorhexidine and 1% thymol (9) and a placebo varnish without the 2 active ingredients. The test varnish was randotnly applied in the right upper side of the tnouth in half of the children and on the left upper
Antibacterial varnish and streptococci Table L Caries experience (mean atid standard deviation) of proximal surface of test and Placebo
Test mean
Index
O.I 09
DFS Initial*
SD 0.3 1.0
range 0-1 0^
mean
SD
range
0,2 0.9
0.5 0.8
0-2 0-2
*See Material and tnethods for definition.
side in the others. The placebo varnish was applied on the opposite side following the split-tnouth technique. The varnishes were applied in small amounts with brushes from both the buccal and lingual side of each interdental space between the upper tnolars and premolars. Prior to application the interdental areas were fiossed and carefully dried with air. The varnish was allowed to set for 10-15 s with help of a gentle air fiow. The treattnent was repeated in the same manner after 2 d though without previous fiossing. The children were instructed to refrain frotn oral hygiene procedures for 12 h after the varnish applications. Bacterial sampling and cuitivation
For the quantitative determination of mutans streptococci in saliva, the spatula method described by Kohler & Bratthall (13) was used. A wooden spatula was rotated on the tongue after paraffin chewing for 1 tnin. The spatula was subsequently pressed on a selective MSB-agar (8), The plates were incubated anaerobically in Gas-pak (BBL Microbiology Systetns, Cockeysville,
MD) jars at 37°C for 48 h. The number of CFU resembling mutans streptococci were counted using a stereomicroscope (x 12) and grouped into the following scores: 0 = 0-10 CFU; 1 = 11-99 CFU; 2= > 100 CFU, Salivary samplitig was perfortned at baseline and after 1 and 3 months respectively. Interdental enumeration of mutans streptococci was performed at 5 differetit occasions; at baselitie, after 2 d (before second varnish application), and subsequently 8, 30 and 90 d after the varnish treatments using the method described by Kristoffersson & Bratthall (11), The sampling was performed using soft wooden triangular toothpicks (TePe, Ekelund & Petersson, Malmo, Sweden), which were pressed from the lingual side into each interdental area until firtn contact was obtained with both teeth. Both sides of the toothpick were thereafter itnmediately pressed against the selective agar plates. The number of mutans streptococci was calculated frotn each interdental space after 48 h of tnicroaerophilic incubation in nitrogen and 5% CO, at 37°C, The plates were analysed as described above for the saliva samples. The following
[ 10 CFU ^ 1 - 2 0 CFU 21-100 CFU 100 CFU
BL
DAYS
DAYS
DAY30
DAY90
Fig I Quantitative distribution of mutans streptococci scores in interproximal spaces of test (T, « = 99) and placebo quadrants (P, « = 99) before and after varnish treatments. Differences between the quadrants were obtained at day 8 (P 100 CFU) at the baseline exatninThe results disclosed a decrease in that the varnish per se resembles and ation. Seventeen satnples (53%) of the number of mutans streptococci in inter- aets as a "shedding surface" with subareas on test sides had lower levels than dental spaces as well as in saliva. Our sequetit elitnination of tnicroorbaseline. The corresponding tiutnber for findings are thus in agreement with pre- gansistns. Nevertheless, a significant difthe placebo-treated interproximal vious reports on reduction and recolon- ference in favour of the test side was spaces was 11 satnples (32%), ization of mutans streptococci in inter- recorded at days 8, 30 and 90, indicating The number of proximal surfaces per dental areas after topical application a slower recolonization in sites where individual without presence of tnutans with chlorhexidine varnish (17, 19), No the test varnish was applied. The sucstreptococci at the start and during the side effects were noticed atnong the par- cessive increase on both sides is presutn3-month experitnental period is pre- ticipants. The taste was well tolerated ably caused by regrowth frotn retention sented in Table 3, Ati iticrease by all participants and none objected sites (6), Moreover, it must be con(P
