Presence of Streptococcus mutans and lactobacilli in saliva and on enamel, glass ionomer cement, and composite resin surfaces JAN VAN DIJKEN', STEN PERSSON= AND STAFFAN SJOSTROM' ^Department of Cariology, ^Department of Oral Microbiology artd ^Department of Hygienist Education, Faculty of Odontology, University of Umed, Umed, Sweden
Dijken JWV van, Persson S, Sjostrom S: Presence oi Streptococcus mutans and lactobacilli in saliva and on enamel, glass ionomer cement, and composite resin surfaces. Scand J Dent Res 1991; 99: 13-9. Abstract - The quantity of 6". mutans, total streptococci, and lactobacilli on sound enamel surfaces and 1-yr-old glass ionomer cement and composite resin fillings with the cervical margins placed subgingivally was compared intraindividually. The amount of bacteria was compared to their number in saliva. The evaluation was done in a cross sectional study, where the patients continued to use their customary oral hygiene procedures and during a 14-day period of experimental plaque formation. The number of lactobacilli and S. tnutans recovered from the test surfaces indicated that the critical salivary concentrations necessary for the isolation of 5. mutans and lactobacilli from glass ionomer cement and composite resin surfaces are the same as for the enamel surfaces. The fluoride levels in plaque adjacent to gla.ss iononner cement will not become high enough to inhibit the accumulation of the investigated bacteria. Key words: composite resin; glass ionomer cement; lactobacilli; S. mulans. Jan van Dijken, Department ofCariology, Faculty of Odontology, University of Umea, Umea, 90187 Sweden. Accepted for publication 9 June 1990.
The composition of dental plaque can be affected by the chemical properties of different restorative materials. SKJORLAND showed both in vitro and in vivo that composite resins harbor high proportions of S. mutans, but they are scarcely demonstrable on enamel, amalgam and silicate cement (1-3). The caries preventive effects of fluoride have stimulated the inclusion of fluoride m a host of dental materials (4). One expla-
nation of the cariostatic effect of fluoride is the interference of fluoride on the adherence of bacteria to the enamel pellicle or with the subsequent multiplication and aggregation of plaque bacteria (5). However, several studies suggest that the normal resistance of most of the oral organisms is such that their growth would not be inhibited by the levels of fluoride normafly found in the plaque (6, 7). Plaque fluoride levels adjacent to a fluoride
14
VAN DIJKEN ET AL.
releasiag material like glass ionomer cement can be c^ansiderably higher due to the constantly d^isolubilizing surface of the cement. Plaque formation may be hampered because of the fluoride release and absorption to enamel (^8). Earlier studies have shown a relationship between the salivary concentration of ^S". mutans and lactobacilli and their colonisation on enamel surfaces (9, 10). Both bacterial affinity and the number of bacteria in saliva available for attachment explain differences in plaque formation on different surfaces jn the oral cavity. The purpose of this in vivo study was to compare intraindividually the occurrence of 5. mutans and lactobacilli oti glass ionomer cement, composite resin, a n d sound enamel surfaces, and to relate it t o salivary levels of these bacteria. The evaluation was done 1) in patients who continued to use their customary oral hygiene procedures and 2) during a 14-day period o f experimental plaque formation.
surfaces. Samples of stimulated whole saliva was obtained by parafiin wax chewing. One ml was transferred to 4 ml of a buffered salt solution, which contained per liter 4.3 g NaGl, 0.42 g KCl, 1.0 g NajHPO^, 10 g (3-glycerophosphate, 0.24 g GaCl,, and 0.1 g MgGl,-6H,O (11). A bacterial plaque sample was taken from the experimental teeth after isolation with cotton rolls and a suction device. All tooth surfaces were sprayed with water and carefully dried with compressed air. Plaque samples were obtained by pressing a strip of filter paper of standardized area for 30 s against the test surface. The paper strips were then transferred to 4 ml of the buffered salt solution. Saliva and plaque samples were brought to the laboratory and cultured within 3 h. Experimental plaque formation study - All subjects
were started on an oral hygiene regime in order . to eliminate all marginal inflammation in the experimental area. The subjects were instructed to use the Bass technique and dental floss, and their teeth were polished weekly with a rubber cup and prophylaxis paste (Buffalo prophylaxis paste, Buffalo Dental Manufacturing G. Inc., NY, USA), until the gingival index scores (12) approached zero. Individual soft acrylic onlays were made that covered the teeth participating in the experiment, Material and methods the neighboring teeth, and the surrounding gingiExperimental teeth - Two class V fillings, with their va in all subjects. The onlays did not interfere with cervical rnargins placed subgingivally, were placed the cervical part of the teeth or with the marginal in two o f three neighboring teeth in the anterior gingiva. The subjects used l.he onlays every time or in ihe canine-premolar region of 16 patients. they performed oral hygiene during the 14-day The teeth to be filled had cervical abrasion/erosion experimental period in order to prevent, them from defects. One defect was filled with a composite disturbing the plaque and the development of ginresin (Silux 3M Dental Products, St. Paul, MN, givitis (13). On day 14 stimulated whole saliva USA, o r Prismafil, DeTrey/DentSply, Zurich, was sampled and both on day 0 and day 14 plaque Switzerland). The second defect was restored with samples were obtained as described earlier. a glass ionomer cement (Chemfil II, DeTrey/ Bacteriologic procedures - The bacterial deposit DenlSply, Zurich, Switzerland). The third of the on the paper strips were dispersed by treating the three experimental teeth showed a non-abrasive vial in a mixer (Vortex-Genie, Scientific Industries and non-filled enamel surface. The evaluations Inc., Springfield, MA, USA) at the maximal setwere macie about 1 yr after insertioti ofthe fillings. ting for 30 s. Saliva samples were treated in the The oral hygiene regime of the involved patients same way. A spiral plater (Spiral systems. Inc., was relatively good. Toothbrushing with dentifrice Gincinatti, OH) was used to spread aliquots on was used twice a day by all patietits, while proxi- Mitis Salivarius Bacitracin medium (GOLD et al. mal hygiene was practiced every day by 68.6% 1973), Mitis Salivarius Agar (Difco Laboratories, and irregularly by 41.9"/|, ofthe padents. Detroit, MI, USA) and Rogosa SL agar (Difco). Cross-sectional sttidy The patients were not informed about the registrations in advance of the visit in order to relate the effect of their customary oral hygiene procedures to the occurrence of number of bacteria on the different experiment
The inoculated agar plates were incubated al 37°G in an atmosphere of 5% GO, in air for 48 h. The number of lactobacilli, total streptococci (14) and S. mutans (15) per ml saliva and in the plaque samples were determined.
15
BACTERIA ON GLASS IONOMER CEMENT
unit; range: 10^-1.4 x 10*), five glass ionomer cement surfaces (median: 5x 10^ CFU/surface unit; range: lO'-Ll x 10**) and seven enamel surfaces (median: 2.3 x 10' CFU/surface unit; range: 7x 10^-1.2 x 10^). The intraindividual differences of number of lactobacilli, S. mutans, and total streptococci between the test surfaces were not statistically significant.
Statistical analyses - Within each subject the number of 5. mutans, total streptococci, and lactobacilli were ranked in the plaque samples of the cross-sectional study and of day 0 and day 14 of the experimental plaque formation study. The sums of the intraindividual ranks for each dental material and the enamel were then compared using Friedman's two-way analysis of variance (13). In this way each subject served as a statistical unit. The numbers of microorganisms in saliva were related to their numbers on the test surfaces at the cross-sectional visit and at day 14 in the experimental plaque formation study.
Experimental
Results Cross-sectional study - The numbers of micro-
organisms per ml of saliva are shown in Table 1. One subject had a number of lactobacilli higher than 10^/ml saliva, while seven subjects showed lactobacilli levels between lO^-lO-'/ml saliva. Lactobacilli could not be isolated from the test surfaces of any of the subjects. The salivary levels of 6". mutans were 4.5 X lO'/ml or less in 10 of the 16 subjects. This should be the salivary content of S. mutans, which supports the establishment of this microorganism on tooth surfaces (9). S. mutans were recovered from enamel surfaces of only two subjects (2-10- CFU/surface unit). The sahvary levels of total streptococci in 14 of the 16 subjects was lO'/ml or more. Total streptococci were found on six composite surfaces (median: 2.4 x 10'' CFU/surface
plaque formation
study -
The
numbers of streptococci, lactobacilli, and S. mutans per ml of saliva at day 14 are given in Table 1. Table 2 shows the number of microorganisms recovered from the test surfaces at day 0 and day 14. The differences between the test surfaces were not statistically different. The salivary level of S. mutans and total streptococci at day 14 and their proportions on the test surfaces are shown in Figs. 1 and 2. When the number of 5. mutans in saliva was below 10*/ml no S. mutans was recovered from the test surfaces. There was a similar distribution pattern for the test surfaces in each salivary interval of S. mutans. At day 0 lactobacilh could not be isolated from the test surfaces. At day 14 lactobacilli were found on one enamel surface (3 x 10" CFU/surface unit), two composite surfaces (10- CFU/surface unit), and two glass ionomer cement surfaces (10" CFU/surface unit). Because of the low numbers of lactobacilli on the test surfaces no comparisons could be made with the salivary levels.
Table 1 .Vumber of mtcroorganiwis per ml of .saliva. CRS, cro.s.s-.seclumal .study. EPF, day 14 of experimental plaque formation study
No. of subjects
CRS Median (range)
EPF Median (range)
Laclobacilli
16
3.0 X 10(
Dijken JWV van, Persson S, Sjostrom S: Presence oi Streptococcus mutans and lactobacilli in saliva and on enamel, glass ionomer cement, and composite resin surfaces. Scand J Dent Res 1991; 99: 13-9. Abstract - The quantity of 6". mutans, total streptococci, and lactobacilli on sound enamel surfaces and 1-yr-old glass ionomer cement and composite resin fillings with the cervical margins placed subgingivally was compared intraindividually. The amount of bacteria was compared to their number in saliva. The evaluation was done in a cross sectional study, where the patients continued to use their customary oral hygiene procedures and during a 14-day period of experimental plaque formation. The number of lactobacilli and S. tnutans recovered from the test surfaces indicated that the critical salivary concentrations necessary for the isolation of 5. mutans and lactobacilli from glass ionomer cement and composite resin surfaces are the same as for the enamel surfaces. The fluoride levels in plaque adjacent to gla.ss iononner cement will not become high enough to inhibit the accumulation of the investigated bacteria. Key words: composite resin; glass ionomer cement; lactobacilli; S. mulans. Jan van Dijken, Department ofCariology, Faculty of Odontology, University of Umea, Umea, 90187 Sweden. Accepted for publication 9 June 1990.
The composition of dental plaque can be affected by the chemical properties of different restorative materials. SKJORLAND showed both in vitro and in vivo that composite resins harbor high proportions of S. mutans, but they are scarcely demonstrable on enamel, amalgam and silicate cement (1-3). The caries preventive effects of fluoride have stimulated the inclusion of fluoride m a host of dental materials (4). One expla-
nation of the cariostatic effect of fluoride is the interference of fluoride on the adherence of bacteria to the enamel pellicle or with the subsequent multiplication and aggregation of plaque bacteria (5). However, several studies suggest that the normal resistance of most of the oral organisms is such that their growth would not be inhibited by the levels of fluoride normafly found in the plaque (6, 7). Plaque fluoride levels adjacent to a fluoride
14
VAN DIJKEN ET AL.
releasiag material like glass ionomer cement can be c^ansiderably higher due to the constantly d^isolubilizing surface of the cement. Plaque formation may be hampered because of the fluoride release and absorption to enamel (^8). Earlier studies have shown a relationship between the salivary concentration of ^S". mutans and lactobacilli and their colonisation on enamel surfaces (9, 10). Both bacterial affinity and the number of bacteria in saliva available for attachment explain differences in plaque formation on different surfaces jn the oral cavity. The purpose of this in vivo study was to compare intraindividually the occurrence of 5. mutans and lactobacilli oti glass ionomer cement, composite resin, a n d sound enamel surfaces, and to relate it t o salivary levels of these bacteria. The evaluation was done 1) in patients who continued to use their customary oral hygiene procedures and 2) during a 14-day period o f experimental plaque formation.
surfaces. Samples of stimulated whole saliva was obtained by parafiin wax chewing. One ml was transferred to 4 ml of a buffered salt solution, which contained per liter 4.3 g NaGl, 0.42 g KCl, 1.0 g NajHPO^, 10 g (3-glycerophosphate, 0.24 g GaCl,, and 0.1 g MgGl,-6H,O (11). A bacterial plaque sample was taken from the experimental teeth after isolation with cotton rolls and a suction device. All tooth surfaces were sprayed with water and carefully dried with compressed air. Plaque samples were obtained by pressing a strip of filter paper of standardized area for 30 s against the test surface. The paper strips were then transferred to 4 ml of the buffered salt solution. Saliva and plaque samples were brought to the laboratory and cultured within 3 h. Experimental plaque formation study - All subjects
were started on an oral hygiene regime in order . to eliminate all marginal inflammation in the experimental area. The subjects were instructed to use the Bass technique and dental floss, and their teeth were polished weekly with a rubber cup and prophylaxis paste (Buffalo prophylaxis paste, Buffalo Dental Manufacturing G. Inc., NY, USA), until the gingival index scores (12) approached zero. Individual soft acrylic onlays were made that covered the teeth participating in the experiment, Material and methods the neighboring teeth, and the surrounding gingiExperimental teeth - Two class V fillings, with their va in all subjects. The onlays did not interfere with cervical rnargins placed subgingivally, were placed the cervical part of the teeth or with the marginal in two o f three neighboring teeth in the anterior gingiva. The subjects used l.he onlays every time or in ihe canine-premolar region of 16 patients. they performed oral hygiene during the 14-day The teeth to be filled had cervical abrasion/erosion experimental period in order to prevent, them from defects. One defect was filled with a composite disturbing the plaque and the development of ginresin (Silux 3M Dental Products, St. Paul, MN, givitis (13). On day 14 stimulated whole saliva USA, o r Prismafil, DeTrey/DentSply, Zurich, was sampled and both on day 0 and day 14 plaque Switzerland). The second defect was restored with samples were obtained as described earlier. a glass ionomer cement (Chemfil II, DeTrey/ Bacteriologic procedures - The bacterial deposit DenlSply, Zurich, Switzerland). The third of the on the paper strips were dispersed by treating the three experimental teeth showed a non-abrasive vial in a mixer (Vortex-Genie, Scientific Industries and non-filled enamel surface. The evaluations Inc., Springfield, MA, USA) at the maximal setwere macie about 1 yr after insertioti ofthe fillings. ting for 30 s. Saliva samples were treated in the The oral hygiene regime of the involved patients same way. A spiral plater (Spiral systems. Inc., was relatively good. Toothbrushing with dentifrice Gincinatti, OH) was used to spread aliquots on was used twice a day by all patietits, while proxi- Mitis Salivarius Bacitracin medium (GOLD et al. mal hygiene was practiced every day by 68.6% 1973), Mitis Salivarius Agar (Difco Laboratories, and irregularly by 41.9"/|, ofthe padents. Detroit, MI, USA) and Rogosa SL agar (Difco). Cross-sectional sttidy The patients were not informed about the registrations in advance of the visit in order to relate the effect of their customary oral hygiene procedures to the occurrence of number of bacteria on the different experiment
The inoculated agar plates were incubated al 37°G in an atmosphere of 5% GO, in air for 48 h. The number of lactobacilli, total streptococci (14) and S. mutans (15) per ml saliva and in the plaque samples were determined.
15
BACTERIA ON GLASS IONOMER CEMENT
unit; range: 10^-1.4 x 10*), five glass ionomer cement surfaces (median: 5x 10^ CFU/surface unit; range: lO'-Ll x 10**) and seven enamel surfaces (median: 2.3 x 10' CFU/surface unit; range: 7x 10^-1.2 x 10^). The intraindividual differences of number of lactobacilli, S. mutans, and total streptococci between the test surfaces were not statistically significant.
Statistical analyses - Within each subject the number of 5. mutans, total streptococci, and lactobacilli were ranked in the plaque samples of the cross-sectional study and of day 0 and day 14 of the experimental plaque formation study. The sums of the intraindividual ranks for each dental material and the enamel were then compared using Friedman's two-way analysis of variance (13). In this way each subject served as a statistical unit. The numbers of microorganisms in saliva were related to their numbers on the test surfaces at the cross-sectional visit and at day 14 in the experimental plaque formation study.
Experimental
Results Cross-sectional study - The numbers of micro-
organisms per ml of saliva are shown in Table 1. One subject had a number of lactobacilli higher than 10^/ml saliva, while seven subjects showed lactobacilli levels between lO^-lO-'/ml saliva. Lactobacilli could not be isolated from the test surfaces of any of the subjects. The salivary levels of 6". mutans were 4.5 X lO'/ml or less in 10 of the 16 subjects. This should be the salivary content of S. mutans, which supports the establishment of this microorganism on tooth surfaces (9). S. mutans were recovered from enamel surfaces of only two subjects (2-10- CFU/surface unit). The sahvary levels of total streptococci in 14 of the 16 subjects was lO'/ml or more. Total streptococci were found on six composite surfaces (median: 2.4 x 10'' CFU/surface
plaque formation
study -
The
numbers of streptococci, lactobacilli, and S. mutans per ml of saliva at day 14 are given in Table 1. Table 2 shows the number of microorganisms recovered from the test surfaces at day 0 and day 14. The differences between the test surfaces were not statistically different. The salivary level of S. mutans and total streptococci at day 14 and their proportions on the test surfaces are shown in Figs. 1 and 2. When the number of 5. mutans in saliva was below 10*/ml no S. mutans was recovered from the test surfaces. There was a similar distribution pattern for the test surfaces in each salivary interval of S. mutans. At day 0 lactobacilh could not be isolated from the test surfaces. At day 14 lactobacilli were found on one enamel surface (3 x 10" CFU/surface unit), two composite surfaces (10- CFU/surface unit), and two glass ionomer cement surfaces (10" CFU/surface unit). Because of the low numbers of lactobacilli on the test surfaces no comparisons could be made with the salivary levels.
Table 1 .Vumber of mtcroorganiwis per ml of .saliva. CRS, cro.s.s-.seclumal .study. EPF, day 14 of experimental plaque formation study
No. of subjects
CRS Median (range)
EPF Median (range)
Laclobacilli
16
3.0 X 10(
