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Caries Res 1991:25:146-152
Dental Location of Streptococcus mutans and Streptococcus sobrinus in Humans Harboring Both Species1 Birgitta Lindquist, Claes-Göran Emilson Department of Cariology, Faculty of Odontology, University of Göteborg, Sweden
Key Words. Dental plaque • Streptococcus mutans ■Streptococcus sobrinus Abstract. The distribution and prevalence of Streptococcus mutans and Streptococcus sobrinus were deter mined in plaque samples from the cervical areas of all buccal, lingual and approximal tooth surfaces and fron the fissures of all occlusal sites in 40 subjects harboring both species. S. mutans was detected more often and it higher numbers than S. sobrinus. There were more teeth detected with S. mutans only than with S. sobrinu only. Most teeth harbored both of these mutans streptococci species, indicating a positive association. Th highest numbers of CFU for both species were detected on the molars, with the lowest incidence on the anterioi teeth. The presence of S. mutans was relatively similar on all teeth tested, while the presence of S. sobrinus wa relatively higher on the molars compared to the anterior teeth. S. mutans and S. sobrinus were found to colo nize the buccal surfaces in almost equal numbers. On all other surfaces, S. mutans was detected more fre quently or in higher numbers compared to S. sobrinus. No significant differences could be found in the relative proportions of S. mutans and S. sobrinus between sound, decayed or filled tooth surfaces.
1 This study was supported by the Swedish Medical Research Council (project No. 4548).
1981; Kristoffersson et al., 1984; Lindquist and Emil son, 1990]. Moreover, it is not known whether S. mu tans and S. sobrinus colonize the same or differeni tooth surfaces. Since strains of S. mutans and S. sobri nus appear to attach to different receptors of salivary pellicles [Gibbons et al., 1986], it is likely that the pat tern of their colonization of the teeth will exhibit dif ferences. The purpose of this study was to examine the distribution and prevalence of S. mutans and S. sobri nus in subjects harboring both species.
Materials and Methods Subjects. Saliva and plaque samples were collected from 40 sub jects, who harbored both S. mutans and S. sobrinus. They ranged in age from 13 to 71 years (mean 29.8 years). Clinical Examination. All subjects were examined clinically and radiographically as specified by Koch [1967], No frank carious le sions were detected but some subjects had incipient caries not re-
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The prevalence of the mutans streptococcal species Streptococcus mutans and Streptococcus sobrinus in humans has been examined mainly in saliva samples, and little information is available about their simul taneous prevalence and distribution in the dentition. S. mutans is often found alone in humans, whereas S. sobrinus is generally detected in subjects who also harbor S. mutans [Walter and Shklair, 1982; Emilson, 1983; Beighton et al., 1987], S. sobrinus has been re ported to be the sole species isolated in only a few sub jects [Huis i n' t Veld et al., 1979; Keene et al., 1977; Emilson and Thorselius, 1988], The mutans streptococci do not colonize the teeth uniformly but seem to be located in the dentition in a particular distribution pattern with a decrease in pre valence from molars to anterior teeth [Keene et al.,
S. mutans and S. sobrinus in the Dentition
Table 1. Mean percentage of different teeth colonized by S. mutans (S.m.) and S. sobrinus (S.sob.) in subjects harboring both spe cies of mutans streptococci (MS) Teeth
% of teeth colonized by MS S.m. S.sob.
Molars 73 59 Premolars 54 43 Anterior teeth 48 37
53 34 29
% of teeth colonized by S.m. S.m.> S.sob. > S.sob. only only S.sob.1 S.m. 19 16 13
23 20 19
18 11 11
13 7 5
1 Denotes that S. mutans predominated.
Statistical Analysis. In the analysis of microbial associations, each individual was considered as the basic unit since the level of mutans streptococci within each subject must be considered to in fluence the prevalence of S. mutans and S. sobrinus on individual tooth surfaces. The mean differences in the distribution of S. mu tans and S. sobrinus on groups of teeth and tooth surfaces within each subject were evaluated. For the whole group of subjects the statistical differences between the mean occurrence of these two species were calculated by an analysis of variance using a paired t test.
Results The mean number of mutans streptococci in saliva was 1.04xl06 CFU/ml ranging from 8.4x 103 to 10.1 x 106 CFU (median 0.49 x 106). The mean sali vary number of S. mutans was 0.8 x 106 and S. sobri nus was 0.3 x 106, while the relative proportions of these mutans species were 53.2 and 46.8% of the total mutans streptococci, respectively. A total of 5,082 available tooth surfaces, of which 2,951 were colonized by mutans streptococci (mean 58%), were sampled from 40 subjects. The mean per centage of molar teeth which harbored mutans strepto cocci was 73%, whereas 54% of the premolars and 48% of the anterior teeth were colonized (table 1). Levels of S. mutans predominated over S. sobrinus, both with respect to the proportion of teeth with both species present or with only one species. S. mutans was isolated more frequently from all available tooth surfaces than was S. sobrinus (2,529 and 2,155 positive sites, respectively). In table 2, the mean percentage of different tooth surfaces colonized by S. mutans and/or S. sobrinus is presented for 40
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quiring restorative treatment. The presence of all restorations in cluding fillings and crowns was recorded for each subject. A mean DMF surfaces value of 39 (range 10-82) was recorded. Sample Collection. All subjects refrained from tooth brushing during the preceding 24 h and from eating and drinking I h before sampling. Paraffin-stimulated saliva was collected and I ml was transferred to VMG1I transport medium [Möller, 1966] and trans ported to the laboratory. The teeth were cleaned with water and then isolated with cotton rolls and gently dried with compressed air before plaque samples were obtained from all individual tooth sur faces. Separate samples were collected using the tip of a wooden toothpick (TePe Röd, Munhygienprodukter AB, Malmö, Sweden) by scraping the cervical area of buccal and lingual tooth surfaces and along the entire fissure or margins of restorations in occlusal surfaces. The tips with plaque were cut off and dropped into wells in a microtiter plate containing 0.3 ml reduced transport fluid [Syed and Loesche, 1972]. Approximal anterior and posterior tooth sur faces were sampled by pressing a toothpick into each interproximal space until firm contact was obtained. Both sides of the toothpick were then immediately pressed against the surface of mitis salivarius bacitracin (MSB) agar [Gold et al., 1973], which is selective for mutans streptococci. The distal surfaces of the third molars were excluded because of the risk of saliva contamination during sam pling. Good reproducibility in repeated sampling has been shown previously using toothpicks for the estimation of mutans strepto cocci [Kristoffersson et al., 1984], with a low variation in recovery over long periods [Emilson et al., 1987]. More surfaces were found to be positive for mutans streptococci when sampling with a tooth pick than with a carver, needle or unwaxed floss [Wennerholm et al., 1987]. All saliva and plaque samples were transferred to the lab oratory and cultured within 2 h of collection. Bacteriological Procedures. The saliva samples were dispersed on a Whirlimixer for 30 s, serially diluted in 0.05 M phosphate buf fer (pH 7.3), and 25-jj.I portions spotted in duplicate onto MSB agar. Each plaque sample was sonically dispersed in the microtiter wells for 10 s at 60 W power using a Branson sonifier (W185). Por tions of 25 pi were then plated in duplicate on MSB agar. All agar plates were incubated in 5% CO, and 95% N, for 48 h at 37 °C. The colonial characteristics of S. mutans [Krasse, 1966] and S. sobrinus [Emilson, 1983] were used for colony counting. Representative and doubtful colonies of mutans streptococci were tested by specific antisera [Bratthall, 1972]. The number of colony-forming units per milliliter saliva (CFU/ml) for S. mutans and S. sobrinus in saliva was determined. The prevalence of the mutans streptococci in each plaque sample was expressed by the following score: score 0= 400 CFU. The lower limit of 5 CFU for positive detection was used in order to avoid or limit the possibility that the undiluted samples would be scored as positive due to contamination with sa liva containing mutans streptococci. The reason for this limitation was based on observations that sites with more than 4 CFU of mutans streptococci have shown low variations in recovery over long periods and good reproducibility in repeated samples [Emilson et al., 1987]. Plaque samples yielding mutans score of 4 (>400 CFU) were not countable and therefore colonies on the agar surface were further subcultured on MSB agar. The percentage of each species in relation to each other was then determined. For each subject, the mean proportion of S. mutans and/or S. sobrinus in relation to the total number of mutans streptococci was calculated for different tooth surfaces and teeth and expressed as a percentage.
147
148
Lindquist/Emilson
Table 2. Mean percentage of tooth surfaces colonized by S. mutans (S.m.) and S. sobrinus (S.sob.) in subjects harboring both spe cies of mutans streptococci (MS) Tooth surface % of surfaces colonized by
35 61 76 49
31 51 64 45
■
1
^
2
□
3
E 3
4
% of surfaces colonized by --------------------------------------
MS S.m. S.sob. Occlusal Approximal Buccal Lingual
S co re
18 43 61 37
S.m. S.m.> S.sob. > S.sob. only S.sob1 S.m. only 17 20 15 12
9 19 27 22
4 10 12 4
5 12 22 11
Denotes that S. mutans predominated.
S.sob. (100%)
bucc.
>
appr.
>
ling.
>
occl.
S.sob. > S.m.
bucc.
>
appr.
>
ling.
>
occl.
S.sob. < S.m.
bucc.
>
ling.
>
appr.
>
occl.
S.m. ( 100%)
appr.
>
occl.
>
bucc.
>
ling.
Tooth surfaces: bucc. = buccal; appr. = approximal; ling. = lingual; occl. = occlusal. Surfaces connected by an interrupted line arc statistically equivalent using paired t test.
subjects. S. mutans was detected on 31% of all oc clusal surfaces whereas S. sobrinus was present only on 18%. S. mutans alone was detected on 17% of the occlusal sites whereas S. sobrinus alone was present on 4%. For buccal surfaces, the proportions of S. mu tans and S. sobrinus were similar (64 versus 61%, re spectively). Fifteen percent of the buccal sites showed S. mutans alone and 12% only S. sobrinus. S. sobrinus was detected more often on approximal and buccal surfaces than on occlusal and lingual surfaces. Table 3 shows the statistical comparison (paired t test) of the prevalence of S. sobrinus and S. mutans on the different tooth surfaces presented in table 2. Of the tooth surfaces harboring S. sobrinus alone or to gether with S. mutans. the buccal sites showed the highest isolation frequencies while the occlusal tooth surfaces showed the lowest (p 50%) by S. sobrinus (dark columns) or S. mutans (light columns).
Table 4. Computation of the odds ratio for the detection of S. sobrinus in the absence ( - ) or presence ( + ) of S. mutans S. sobrinus + S. mutans + Odds ratio-
2,206 1,168
624 1,084
1,084/1,168 = 3.32 624/2,206
in samples with S. mutans was 1,084/1,168 (0.93) and the odds of detecting S. sobrinus in the absence of S. mutans was 624/2,206 (0.28). The odds ratio for the detection of S. sobrinus in the presence of S. mutans, as opposed to in the absence of this species, was thus 0.93/0.28 = 3.32. In general, different tooth surfaces, either filled, decayed or sound, showed, with some exceptions, sim ilar distribution values when the mean proportions of
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Figure 3 is a three-dimensional bar chart illustrat ing the detection of and association between S. mutans and S. sobrinus in plaque samples from all avail able tooth surfaces with mutans scores 0-3. Sites with the mutans scon' of 4 (737 surfaces) were not included due to uncountable numbers of CFU of the individual species. There were 2,13 1 sites in which no mutans streptococci were detected and another 75 sites posi tive for mutans streptococci (score l) but where S. mutans and S. sobrinus each occurred in numbers less than 5 CFU, thus resulting in a total sum of 2,206 sites with these species scored as absent. The majority of surfaces colonized by mutans streptococci har bored only one species with S. mutans predominat ing. Flowever, both species were detected together in a large number of sites, thus showing a positive associa tion. The positive association was further revealed by using the odds ratio as described by Socransky et al. [1988]; the basic data for computing the odds ratio are presented in table 4. The odds of detecting S. sobrinus
S. mutans and S. sobrinus in relation to the total mutans streptococci were compared. Thus there was a tendency for higher percentages of S. mutans rather than S. sobrinus on the surfaces filled with composite. Buccal surfaces with amalgam restorations showed an especially higher relative proportion of S. sobrinus. However, these differences were not statistically sig nificant.
Discussion S. sobrinus is not as prevalent as S. mutans in hu mans. In Sweden, S. sobrinus has been detected in 34-44% of teenagers, adults and elderly people [Emilson, 1983; Emilson and Thorselius, 1988; Rask et al., in press]. Since S. mutans and S. sobrinus show many fundamental differences [Gibbons, 1986] which could influence the adherence to the tooth surface and the colonization pattern of the teeth, only subjects har boring both species were selected in the present study. In this group of subjects, the levels of mutans strepto cocci in saliva ranged from low to high counts, colo nized a mean of 58% of all tooth surfaces, and showed different relative proportions of S. mutans and S. sob rinus on different tooth surfaces. Molar surfaces were colonized by S. mutans and S. sobrinus to a higher degree than those of premolars and anterior teeth. This colonization pattern is in agreement with earlier observations for mutans streptococci [Keene et al., 1981; Kristoffersson et al., 1984; Lindquist and Emilson, 1990]. However, the de creasing colonization gradient from molars to ante rior teeth was not as apparent for S. mutans as it was for S. sobrinus when either of these species predomi nated. This observation may be interpreted as indica tive of environmental factors which are more favor able for colonization and accumulation of 5. sobrinus on posterior teeth than on anterior teeth. It has been suggested that the colonization of S. sobrinus is more sucrose-dependent than S. mutans [Gibbons et al., 1986] so that it might accumulate on surfaces more ef fectively through sucrose-induced glucan binding [Rolla et al., 1983; Gibbons et al., 1986]. The retention and elimination of sucrose seem to differ between var ious parts of the mouth. Recently it was shown that sugar clearance was slower from molar surfaces and upper incisors than from surfaces of the lower inci sors [Britse and Lagerlof, 1986]. This finding, together with the glucan binding potential, may explain the
higher detection frequency of S. sobrinus in the molar region, where the third molars were highly colonized. The results suggest a positive association between S. mutans and S. sobrinus in plaque. The odds ratio revealed that the probability of detecting S. sobrinus at a site in the presence of S. mutans was about 3 times higher than finding S. sobrinus in the absence of S. mutans. The large numbers of sites where both spe cies were detected show that S. mutans and S. sobrinus are capable of coexisting. This observation is in agree ment with results from other studies showing that, in saliva, S. sobrinus is generally found in mouths, which also harbor S. mutans, and that only in a few subjects has it been reported to be the only mutans streptococci isolated [Huis in ’t Veld et al., 1979; Keene et al., 1977; Emilson and Thorselius, 1988]. An interesting observation in this study, in which only subjects with both species present were selected, was that each of the species could be detected in sub stantial numbers of sites in the absence of the other species. This illustrates that the two species might prefer a different habitat and/or that their ability to colonize differs at many sites in the dentition. In an environment with a large number of microbial species the different colonization pattern could be due to any one of several factors such as competition for nu trients and binding sites, or to the production of inhib itory bacteriocin substances. The higher prevalence in saliva of S. mutans com pared with S. sobrinus was also reflected in a similar difference in the total numbers of tooth surfaces col onized by these two species. This is in accordance with earlier observations showing a highly significant correlation between the population of mutans strepto cocci in saliva and the proportion of colonized tooth surfaces [Dutchin and Van Houte, 1978; Emilson, 1983; Togelius et al., 1984; Lindquist et al., 1989]. However, on different tooth surfaces the prevalence of S. mutans and S. sobrinus was different, which could have affected their proportions in saliva. Thus, occlusal sites had lower detection levels of S. sobrinus than 5. mutans, while for other tooth surfaces and es pecially for buccal sites the proportions of surfaces harboring S. mutans and S. sobrinus were more simi lar. The differences in colonization were also evident in the distribution of mutans streptococci scores, which showed that a high number of the buccal sur faces positive for 5. sobrinus were also highly colo nized by this species. For occlusal, lingual and approximal tooth surfaces, S. sobrinus was detected
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150
mainly in low numbers, i.e. mutans streptococci scores of 1 or 2. S. sobrinus has been shown to be more acidogenic than the other species of mutans streptococci [De Soet et al., 1989]. In humans there are indications that the caries prevalence is higher in subjects harboring both S. mutans and S. sobrinus than in subjects with only S. mutans [Köhler and Bjarnason, 1987], The finding in this study that S. sobrinus showed a higher prefer ence to colonize posterior rather than anterior teeth and the possible association between S. sobrinus and smooth surface caries [Huis in 't Veld et al., 1979] seem to coincide with the general pattern of tooth sur faces most susceptible to and affected by dental caries [Ainamo, 1970; Lindquist and Emilson, 1990]. These observations also imply that it has become important to discriminate between the different species of mutans streptococci when analyzing the prevalence of these cariogenic microorganisms in humans. In earlier studies, a higher prevalence of mutans streptococci has been noted for tooth-colored fillings than from sound surfaces [Keene and Brown, 1983; Lundin and Emilson, 1989; Lindquist and Emilson, 1990], but in these studies no speciation of mutans streptococci was performed. Comparing the preval ence of S. mutans and S. sobrinus in the present study, it was found that tooth surfaces restored by tooth-col ored fillings were preferentially colonized by S. mutans. while amalgam-filled buccal sites showed a high relative proportion of S. sobrinus. The reason for this is unclear but it is possible that this is only a reflection of the predominance of tooth-colored fillings on ante rior teeth with their relatively higher proportion of S. mutans, whereas the amalgam fillings were present in the posterior sites with high numbers of S. sobrinus. References Ainamo J: Concomitant periodontal disease and dental caries in young males. Suom Hammaslääk Toim 1970;66:303-366. Beighton D, Rippon H R, Thomas H EC: The distribution of Strepto coccus mutans serotypes and dental caries in a group of 5to 8-year-old Hampshire schoolchildren. Br Dent J 1987; 162: 103-106. Bratthall D: Immunofluorescent identification of Streptococcus mutans. Odontol Revy 1972:23:181-196. Britse A, Lagerlöf F: Clearance of sucrose from different parts of the oral cavity (abstract 1064). J Dent Res (special issue) 1986: 65:844. De Soet JJ, Toors FA, De Graaff J: Acidogenesis by oral strepto cocci at different pH values. Caries Res 1989;23:14-17. Dutchin S, Van Houte J: Colonization of teeth in humans by Strept
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ococcus mutans as related to its concentration in saliva and hos tage. Infect Immun 1978:20:120-125. Emilson CG: Prevalence of Streptococcus mutans with different co lonial morphologies in human plaque and saliva. Scand J Dent Res 1983;91:26-32. Emilson CG, Lindquist B, Wennerholm K: Recolonization of hu man tooth surfaces by Streptococcus mutans after suppression by Chlorhexidine treatment. J Dent Res 1987;66:1503-1508. Emilson CG, Thorselius I: Prevalence of mutans streptococci and lactobacilli in elderly Swedish individuals. Scand J Dent Res 1988;96:14-21. Gibbons RJ: Concluding remarks and areas for research: in Hamada S, Michalek SM, Kinyino H, Menaker L, McGhee JL (eds): Molecular Microbiology and Immunobiology of Streptococcus mutans. Amsterdam, Elsevier, 1986, pp 433-445. Gibbons RJ, Cohen L, Hay DI: Strains of Streptococcus mutans and Streptococcus sobrinus attach to different pellicle receptors. Infect Immun 1986:52:555-561. Gold OG, Jordan HV, Van Houte J: A selective medium for Streptococcus mutans. Arch Oral Biol 1973;18:1357-1364. Huis in ’t Veld JHJ, Van Palenstein Helderman WH, Backer-Dirks O: Streptococcus mutans and dental caries in humans: A bacteri ological and immunological study. Antonie van Leeuwenhoek 1979:45:25-33. Keene HJ, Brown CK: Colonization of metallic and nonmetallic restorations by Streptococcus mutans in vivo. Clin Prev Dent 1983;5:3-7. Keene HJ, Horton IM, Handler SF: Streptococcus mutansapproximal plaque index as a new epidemiologic tool for defining the parameters of Streptococcus mutans infection in human popula tions. Arch Oral Biol 1981;26:345-355. Keene HJ, Shklair IL, Mickel GJ, Wirthlin MR: Distribution of Streptococcus mutans biotypes in five human populations. J Dent Res 1977;56:5-10. Koch G: Effect of sodium fluoride in dentifrice and mouthwash on incidence of dental caries in school-children. Odontol Revy 1967;18(suppl 12). Köhler B, Bjarnason S: Mutans streptococci, lactobacilli and caries prevalence in 11- and 12-year-old Icelandic children. Commu nity Dent Oral Epidemiol 1987;15:332-335. Krasse B: Human streptococci and experimental caries in hamsters. Arch Oral Biol 1966:11:429-436. Kristoffersson K, Axelsson P, Bratthall D: Effect of professional tooth cleaning program on interdentally localized Streptococcus mutans. Caries Res 1984;18:385-390. Lindquist B, Emilson CG, Wennerholm K: Relationship between mutans streptococci in saliva and their colonization of the tooth surfaces. Oral Microbiol Immunol 1989;4:71-76. Lindquist B, Emilson CG: Distribution and prevalence of mutans streptococci in the human dentition. J Dent Res 1990:69: 1160-1166. Lundin SÄ, Emilson CG: Microflora in plaque from approximal posterior composite resin restorations. Quintessence Int 1989; 20:413-416. Möller AJR: Microbiological examination of root canals and peri apical tissues of human teeth; thesis. Odontol Tidskr (special is sue) 1966;74:1-380. Rask PI, Emilson CG, Krasse B, Sundberg H: Dental caries and salivary and microbial conditions in 50-60 years old persons. Community Dent Oral Epidemiol, in press.
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Wennerholm K, Lindquist B, Emilson CG: Mutans streptococci on different tooth surfaces: A comparison between various sam pling methods (abstract 26). Swed Dent J (special issue) 1987; 11:281-282.
Received: June 15, 1990 Accepted after revision: September 23, 1990 B. Lindquist Department of Cariology Faculty of Odontology University of Göteborg Box 33070 S-400 33 Göteborg (Sweden)
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Rolla G, Ciardi JE, Schultz SA: Adsorption of glucosyltransferase to saliva coated hydroxyapatite. Possible mechanism for su crose dependent bacterial colonization of teeth. Scand J Dent Res 1983;91:112-117. Socransky SS, Haffajee AD, Dzink JL, Hillman JD: Associations between microbial species in subgingival plaque samples. Oral Microbiol Immunol 1988;3:1-7. Syed SA, Loesche WJ: Survival of human dental plaque flora in various transport media. Appl Microbiol 1972;24:638-644. Togelius J, Kristoffersson K, Anderson H, Bratthall D: Strepto coccus mutans in saliva: Intraindividual variations and relation to the number of colonized sites. Acta Odontol Scand 1984:42: 157-163. Walter RG, Shklair IL: Streptococcus mutans in caries-free and car ies-active naval recruits. J Dent Res 1982;61:1229-1232.
Caries Res 1991:25:146-152
Dental Location of Streptococcus mutans and Streptococcus sobrinus in Humans Harboring Both Species1 Birgitta Lindquist, Claes-Göran Emilson Department of Cariology, Faculty of Odontology, University of Göteborg, Sweden
Key Words. Dental plaque • Streptococcus mutans ■Streptococcus sobrinus Abstract. The distribution and prevalence of Streptococcus mutans and Streptococcus sobrinus were deter mined in plaque samples from the cervical areas of all buccal, lingual and approximal tooth surfaces and fron the fissures of all occlusal sites in 40 subjects harboring both species. S. mutans was detected more often and it higher numbers than S. sobrinus. There were more teeth detected with S. mutans only than with S. sobrinu only. Most teeth harbored both of these mutans streptococci species, indicating a positive association. Th highest numbers of CFU for both species were detected on the molars, with the lowest incidence on the anterioi teeth. The presence of S. mutans was relatively similar on all teeth tested, while the presence of S. sobrinus wa relatively higher on the molars compared to the anterior teeth. S. mutans and S. sobrinus were found to colo nize the buccal surfaces in almost equal numbers. On all other surfaces, S. mutans was detected more fre quently or in higher numbers compared to S. sobrinus. No significant differences could be found in the relative proportions of S. mutans and S. sobrinus between sound, decayed or filled tooth surfaces.
1 This study was supported by the Swedish Medical Research Council (project No. 4548).
1981; Kristoffersson et al., 1984; Lindquist and Emil son, 1990]. Moreover, it is not known whether S. mu tans and S. sobrinus colonize the same or differeni tooth surfaces. Since strains of S. mutans and S. sobri nus appear to attach to different receptors of salivary pellicles [Gibbons et al., 1986], it is likely that the pat tern of their colonization of the teeth will exhibit dif ferences. The purpose of this study was to examine the distribution and prevalence of S. mutans and S. sobri nus in subjects harboring both species.
Materials and Methods Subjects. Saliva and plaque samples were collected from 40 sub jects, who harbored both S. mutans and S. sobrinus. They ranged in age from 13 to 71 years (mean 29.8 years). Clinical Examination. All subjects were examined clinically and radiographically as specified by Koch [1967], No frank carious le sions were detected but some subjects had incipient caries not re-
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The prevalence of the mutans streptococcal species Streptococcus mutans and Streptococcus sobrinus in humans has been examined mainly in saliva samples, and little information is available about their simul taneous prevalence and distribution in the dentition. S. mutans is often found alone in humans, whereas S. sobrinus is generally detected in subjects who also harbor S. mutans [Walter and Shklair, 1982; Emilson, 1983; Beighton et al., 1987], S. sobrinus has been re ported to be the sole species isolated in only a few sub jects [Huis i n' t Veld et al., 1979; Keene et al., 1977; Emilson and Thorselius, 1988], The mutans streptococci do not colonize the teeth uniformly but seem to be located in the dentition in a particular distribution pattern with a decrease in pre valence from molars to anterior teeth [Keene et al.,
S. mutans and S. sobrinus in the Dentition
Table 1. Mean percentage of different teeth colonized by S. mutans (S.m.) and S. sobrinus (S.sob.) in subjects harboring both spe cies of mutans streptococci (MS) Teeth
% of teeth colonized by MS S.m. S.sob.
Molars 73 59 Premolars 54 43 Anterior teeth 48 37
53 34 29
% of teeth colonized by S.m. S.m.> S.sob. > S.sob. only only S.sob.1 S.m. 19 16 13
23 20 19
18 11 11
13 7 5
1 Denotes that S. mutans predominated.
Statistical Analysis. In the analysis of microbial associations, each individual was considered as the basic unit since the level of mutans streptococci within each subject must be considered to in fluence the prevalence of S. mutans and S. sobrinus on individual tooth surfaces. The mean differences in the distribution of S. mu tans and S. sobrinus on groups of teeth and tooth surfaces within each subject were evaluated. For the whole group of subjects the statistical differences between the mean occurrence of these two species were calculated by an analysis of variance using a paired t test.
Results The mean number of mutans streptococci in saliva was 1.04xl06 CFU/ml ranging from 8.4x 103 to 10.1 x 106 CFU (median 0.49 x 106). The mean sali vary number of S. mutans was 0.8 x 106 and S. sobri nus was 0.3 x 106, while the relative proportions of these mutans species were 53.2 and 46.8% of the total mutans streptococci, respectively. A total of 5,082 available tooth surfaces, of which 2,951 were colonized by mutans streptococci (mean 58%), were sampled from 40 subjects. The mean per centage of molar teeth which harbored mutans strepto cocci was 73%, whereas 54% of the premolars and 48% of the anterior teeth were colonized (table 1). Levels of S. mutans predominated over S. sobrinus, both with respect to the proportion of teeth with both species present or with only one species. S. mutans was isolated more frequently from all available tooth surfaces than was S. sobrinus (2,529 and 2,155 positive sites, respectively). In table 2, the mean percentage of different tooth surfaces colonized by S. mutans and/or S. sobrinus is presented for 40
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quiring restorative treatment. The presence of all restorations in cluding fillings and crowns was recorded for each subject. A mean DMF surfaces value of 39 (range 10-82) was recorded. Sample Collection. All subjects refrained from tooth brushing during the preceding 24 h and from eating and drinking I h before sampling. Paraffin-stimulated saliva was collected and I ml was transferred to VMG1I transport medium [Möller, 1966] and trans ported to the laboratory. The teeth were cleaned with water and then isolated with cotton rolls and gently dried with compressed air before plaque samples were obtained from all individual tooth sur faces. Separate samples were collected using the tip of a wooden toothpick (TePe Röd, Munhygienprodukter AB, Malmö, Sweden) by scraping the cervical area of buccal and lingual tooth surfaces and along the entire fissure or margins of restorations in occlusal surfaces. The tips with plaque were cut off and dropped into wells in a microtiter plate containing 0.3 ml reduced transport fluid [Syed and Loesche, 1972]. Approximal anterior and posterior tooth sur faces were sampled by pressing a toothpick into each interproximal space until firm contact was obtained. Both sides of the toothpick were then immediately pressed against the surface of mitis salivarius bacitracin (MSB) agar [Gold et al., 1973], which is selective for mutans streptococci. The distal surfaces of the third molars were excluded because of the risk of saliva contamination during sam pling. Good reproducibility in repeated sampling has been shown previously using toothpicks for the estimation of mutans strepto cocci [Kristoffersson et al., 1984], with a low variation in recovery over long periods [Emilson et al., 1987]. More surfaces were found to be positive for mutans streptococci when sampling with a tooth pick than with a carver, needle or unwaxed floss [Wennerholm et al., 1987]. All saliva and plaque samples were transferred to the lab oratory and cultured within 2 h of collection. Bacteriological Procedures. The saliva samples were dispersed on a Whirlimixer for 30 s, serially diluted in 0.05 M phosphate buf fer (pH 7.3), and 25-jj.I portions spotted in duplicate onto MSB agar. Each plaque sample was sonically dispersed in the microtiter wells for 10 s at 60 W power using a Branson sonifier (W185). Por tions of 25 pi were then plated in duplicate on MSB agar. All agar plates were incubated in 5% CO, and 95% N, for 48 h at 37 °C. The colonial characteristics of S. mutans [Krasse, 1966] and S. sobrinus [Emilson, 1983] were used for colony counting. Representative and doubtful colonies of mutans streptococci were tested by specific antisera [Bratthall, 1972]. The number of colony-forming units per milliliter saliva (CFU/ml) for S. mutans and S. sobrinus in saliva was determined. The prevalence of the mutans streptococci in each plaque sample was expressed by the following score: score 0= 400 CFU. The lower limit of 5 CFU for positive detection was used in order to avoid or limit the possibility that the undiluted samples would be scored as positive due to contamination with sa liva containing mutans streptococci. The reason for this limitation was based on observations that sites with more than 4 CFU of mutans streptococci have shown low variations in recovery over long periods and good reproducibility in repeated samples [Emilson et al., 1987]. Plaque samples yielding mutans score of 4 (>400 CFU) were not countable and therefore colonies on the agar surface were further subcultured on MSB agar. The percentage of each species in relation to each other was then determined. For each subject, the mean proportion of S. mutans and/or S. sobrinus in relation to the total number of mutans streptococci was calculated for different tooth surfaces and teeth and expressed as a percentage.
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Table 2. Mean percentage of tooth surfaces colonized by S. mutans (S.m.) and S. sobrinus (S.sob.) in subjects harboring both spe cies of mutans streptococci (MS) Tooth surface % of surfaces colonized by
35 61 76 49
31 51 64 45
■
1
^
2
□
3
E 3
4
% of surfaces colonized by --------------------------------------
MS S.m. S.sob. Occlusal Approximal Buccal Lingual
S co re
18 43 61 37
S.m. S.m.> S.sob. > S.sob. only S.sob1 S.m. only 17 20 15 12
9 19 27 22
4 10 12 4
5 12 22 11
Denotes that S. mutans predominated.
S.sob. (100%)
bucc.
>
appr.
>
ling.
>
occl.
S.sob. > S.m.
bucc.
>
appr.
>
ling.
>
occl.
S.sob. < S.m.
bucc.
>
ling.
>
appr.
>
occl.
S.m. ( 100%)
appr.
>
occl.
>
bucc.
>
ling.
Tooth surfaces: bucc. = buccal; appr. = approximal; ling. = lingual; occl. = occlusal. Surfaces connected by an interrupted line arc statistically equivalent using paired t test.
subjects. S. mutans was detected on 31% of all oc clusal surfaces whereas S. sobrinus was present only on 18%. S. mutans alone was detected on 17% of the occlusal sites whereas S. sobrinus alone was present on 4%. For buccal surfaces, the proportions of S. mu tans and S. sobrinus were similar (64 versus 61%, re spectively). Fifteen percent of the buccal sites showed S. mutans alone and 12% only S. sobrinus. S. sobrinus was detected more often on approximal and buccal surfaces than on occlusal and lingual surfaces. Table 3 shows the statistical comparison (paired t test) of the prevalence of S. sobrinus and S. mutans on the different tooth surfaces presented in table 2. Of the tooth surfaces harboring S. sobrinus alone or to gether with S. mutans. the buccal sites showed the highest isolation frequencies while the occlusal tooth surfaces showed the lowest (p 50%) by S. sobrinus (dark columns) or S. mutans (light columns).
Table 4. Computation of the odds ratio for the detection of S. sobrinus in the absence ( - ) or presence ( + ) of S. mutans S. sobrinus + S. mutans + Odds ratio-
2,206 1,168
624 1,084
1,084/1,168 = 3.32 624/2,206
in samples with S. mutans was 1,084/1,168 (0.93) and the odds of detecting S. sobrinus in the absence of S. mutans was 624/2,206 (0.28). The odds ratio for the detection of S. sobrinus in the presence of S. mutans, as opposed to in the absence of this species, was thus 0.93/0.28 = 3.32. In general, different tooth surfaces, either filled, decayed or sound, showed, with some exceptions, sim ilar distribution values when the mean proportions of
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Figure 3 is a three-dimensional bar chart illustrat ing the detection of and association between S. mutans and S. sobrinus in plaque samples from all avail able tooth surfaces with mutans scores 0-3. Sites with the mutans scon' of 4 (737 surfaces) were not included due to uncountable numbers of CFU of the individual species. There were 2,13 1 sites in which no mutans streptococci were detected and another 75 sites posi tive for mutans streptococci (score l) but where S. mutans and S. sobrinus each occurred in numbers less than 5 CFU, thus resulting in a total sum of 2,206 sites with these species scored as absent. The majority of surfaces colonized by mutans streptococci har bored only one species with S. mutans predominat ing. Flowever, both species were detected together in a large number of sites, thus showing a positive associa tion. The positive association was further revealed by using the odds ratio as described by Socransky et al. [1988]; the basic data for computing the odds ratio are presented in table 4. The odds of detecting S. sobrinus
S. mutans and S. sobrinus in relation to the total mutans streptococci were compared. Thus there was a tendency for higher percentages of S. mutans rather than S. sobrinus on the surfaces filled with composite. Buccal surfaces with amalgam restorations showed an especially higher relative proportion of S. sobrinus. However, these differences were not statistically sig nificant.
Discussion S. sobrinus is not as prevalent as S. mutans in hu mans. In Sweden, S. sobrinus has been detected in 34-44% of teenagers, adults and elderly people [Emilson, 1983; Emilson and Thorselius, 1988; Rask et al., in press]. Since S. mutans and S. sobrinus show many fundamental differences [Gibbons, 1986] which could influence the adherence to the tooth surface and the colonization pattern of the teeth, only subjects har boring both species were selected in the present study. In this group of subjects, the levels of mutans strepto cocci in saliva ranged from low to high counts, colo nized a mean of 58% of all tooth surfaces, and showed different relative proportions of S. mutans and S. sob rinus on different tooth surfaces. Molar surfaces were colonized by S. mutans and S. sobrinus to a higher degree than those of premolars and anterior teeth. This colonization pattern is in agreement with earlier observations for mutans streptococci [Keene et al., 1981; Kristoffersson et al., 1984; Lindquist and Emilson, 1990]. However, the de creasing colonization gradient from molars to ante rior teeth was not as apparent for S. mutans as it was for S. sobrinus when either of these species predomi nated. This observation may be interpreted as indica tive of environmental factors which are more favor able for colonization and accumulation of 5. sobrinus on posterior teeth than on anterior teeth. It has been suggested that the colonization of S. sobrinus is more sucrose-dependent than S. mutans [Gibbons et al., 1986] so that it might accumulate on surfaces more ef fectively through sucrose-induced glucan binding [Rolla et al., 1983; Gibbons et al., 1986]. The retention and elimination of sucrose seem to differ between var ious parts of the mouth. Recently it was shown that sugar clearance was slower from molar surfaces and upper incisors than from surfaces of the lower inci sors [Britse and Lagerlof, 1986]. This finding, together with the glucan binding potential, may explain the
higher detection frequency of S. sobrinus in the molar region, where the third molars were highly colonized. The results suggest a positive association between S. mutans and S. sobrinus in plaque. The odds ratio revealed that the probability of detecting S. sobrinus at a site in the presence of S. mutans was about 3 times higher than finding S. sobrinus in the absence of S. mutans. The large numbers of sites where both spe cies were detected show that S. mutans and S. sobrinus are capable of coexisting. This observation is in agree ment with results from other studies showing that, in saliva, S. sobrinus is generally found in mouths, which also harbor S. mutans, and that only in a few subjects has it been reported to be the only mutans streptococci isolated [Huis in ’t Veld et al., 1979; Keene et al., 1977; Emilson and Thorselius, 1988]. An interesting observation in this study, in which only subjects with both species present were selected, was that each of the species could be detected in sub stantial numbers of sites in the absence of the other species. This illustrates that the two species might prefer a different habitat and/or that their ability to colonize differs at many sites in the dentition. In an environment with a large number of microbial species the different colonization pattern could be due to any one of several factors such as competition for nu trients and binding sites, or to the production of inhib itory bacteriocin substances. The higher prevalence in saliva of S. mutans com pared with S. sobrinus was also reflected in a similar difference in the total numbers of tooth surfaces col onized by these two species. This is in accordance with earlier observations showing a highly significant correlation between the population of mutans strepto cocci in saliva and the proportion of colonized tooth surfaces [Dutchin and Van Houte, 1978; Emilson, 1983; Togelius et al., 1984; Lindquist et al., 1989]. However, on different tooth surfaces the prevalence of S. mutans and S. sobrinus was different, which could have affected their proportions in saliva. Thus, occlusal sites had lower detection levels of S. sobrinus than 5. mutans, while for other tooth surfaces and es pecially for buccal sites the proportions of surfaces harboring S. mutans and S. sobrinus were more simi lar. The differences in colonization were also evident in the distribution of mutans streptococci scores, which showed that a high number of the buccal sur faces positive for 5. sobrinus were also highly colo nized by this species. For occlusal, lingual and approximal tooth surfaces, S. sobrinus was detected
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mainly in low numbers, i.e. mutans streptococci scores of 1 or 2. S. sobrinus has been shown to be more acidogenic than the other species of mutans streptococci [De Soet et al., 1989]. In humans there are indications that the caries prevalence is higher in subjects harboring both S. mutans and S. sobrinus than in subjects with only S. mutans [Köhler and Bjarnason, 1987], The finding in this study that S. sobrinus showed a higher prefer ence to colonize posterior rather than anterior teeth and the possible association between S. sobrinus and smooth surface caries [Huis in 't Veld et al., 1979] seem to coincide with the general pattern of tooth sur faces most susceptible to and affected by dental caries [Ainamo, 1970; Lindquist and Emilson, 1990]. These observations also imply that it has become important to discriminate between the different species of mutans streptococci when analyzing the prevalence of these cariogenic microorganisms in humans. In earlier studies, a higher prevalence of mutans streptococci has been noted for tooth-colored fillings than from sound surfaces [Keene and Brown, 1983; Lundin and Emilson, 1989; Lindquist and Emilson, 1990], but in these studies no speciation of mutans streptococci was performed. Comparing the preval ence of S. mutans and S. sobrinus in the present study, it was found that tooth surfaces restored by tooth-col ored fillings were preferentially colonized by S. mutans. while amalgam-filled buccal sites showed a high relative proportion of S. sobrinus. The reason for this is unclear but it is possible that this is only a reflection of the predominance of tooth-colored fillings on ante rior teeth with their relatively higher proportion of S. mutans, whereas the amalgam fillings were present in the posterior sites with high numbers of S. sobrinus. References Ainamo J: Concomitant periodontal disease and dental caries in young males. Suom Hammaslääk Toim 1970;66:303-366. Beighton D, Rippon H R, Thomas H EC: The distribution of Strepto coccus mutans serotypes and dental caries in a group of 5to 8-year-old Hampshire schoolchildren. Br Dent J 1987; 162: 103-106. Bratthall D: Immunofluorescent identification of Streptococcus mutans. Odontol Revy 1972:23:181-196. Britse A, Lagerlöf F: Clearance of sucrose from different parts of the oral cavity (abstract 1064). J Dent Res (special issue) 1986: 65:844. De Soet JJ, Toors FA, De Graaff J: Acidogenesis by oral strepto cocci at different pH values. Caries Res 1989;23:14-17. Dutchin S, Van Houte J: Colonization of teeth in humans by Strept
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ococcus mutans as related to its concentration in saliva and hos tage. Infect Immun 1978:20:120-125. Emilson CG: Prevalence of Streptococcus mutans with different co lonial morphologies in human plaque and saliva. Scand J Dent Res 1983;91:26-32. Emilson CG, Lindquist B, Wennerholm K: Recolonization of hu man tooth surfaces by Streptococcus mutans after suppression by Chlorhexidine treatment. J Dent Res 1987;66:1503-1508. Emilson CG, Thorselius I: Prevalence of mutans streptococci and lactobacilli in elderly Swedish individuals. Scand J Dent Res 1988;96:14-21. Gibbons RJ: Concluding remarks and areas for research: in Hamada S, Michalek SM, Kinyino H, Menaker L, McGhee JL (eds): Molecular Microbiology and Immunobiology of Streptococcus mutans. Amsterdam, Elsevier, 1986, pp 433-445. Gibbons RJ, Cohen L, Hay DI: Strains of Streptococcus mutans and Streptococcus sobrinus attach to different pellicle receptors. Infect Immun 1986:52:555-561. Gold OG, Jordan HV, Van Houte J: A selective medium for Streptococcus mutans. Arch Oral Biol 1973;18:1357-1364. Huis in ’t Veld JHJ, Van Palenstein Helderman WH, Backer-Dirks O: Streptococcus mutans and dental caries in humans: A bacteri ological and immunological study. Antonie van Leeuwenhoek 1979:45:25-33. Keene HJ, Brown CK: Colonization of metallic and nonmetallic restorations by Streptococcus mutans in vivo. Clin Prev Dent 1983;5:3-7. Keene HJ, Horton IM, Handler SF: Streptococcus mutansapproximal plaque index as a new epidemiologic tool for defining the parameters of Streptococcus mutans infection in human popula tions. Arch Oral Biol 1981;26:345-355. Keene HJ, Shklair IL, Mickel GJ, Wirthlin MR: Distribution of Streptococcus mutans biotypes in five human populations. J Dent Res 1977;56:5-10. Koch G: Effect of sodium fluoride in dentifrice and mouthwash on incidence of dental caries in school-children. Odontol Revy 1967;18(suppl 12). Köhler B, Bjarnason S: Mutans streptococci, lactobacilli and caries prevalence in 11- and 12-year-old Icelandic children. Commu nity Dent Oral Epidemiol 1987;15:332-335. Krasse B: Human streptococci and experimental caries in hamsters. Arch Oral Biol 1966:11:429-436. Kristoffersson K, Axelsson P, Bratthall D: Effect of professional tooth cleaning program on interdentally localized Streptococcus mutans. Caries Res 1984;18:385-390. Lindquist B, Emilson CG, Wennerholm K: Relationship between mutans streptococci in saliva and their colonization of the tooth surfaces. Oral Microbiol Immunol 1989;4:71-76. Lindquist B, Emilson CG: Distribution and prevalence of mutans streptococci in the human dentition. J Dent Res 1990:69: 1160-1166. Lundin SÄ, Emilson CG: Microflora in plaque from approximal posterior composite resin restorations. Quintessence Int 1989; 20:413-416. Möller AJR: Microbiological examination of root canals and peri apical tissues of human teeth; thesis. Odontol Tidskr (special is sue) 1966;74:1-380. Rask PI, Emilson CG, Krasse B, Sundberg H: Dental caries and salivary and microbial conditions in 50-60 years old persons. Community Dent Oral Epidemiol, in press.
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Wennerholm K, Lindquist B, Emilson CG: Mutans streptococci on different tooth surfaces: A comparison between various sam pling methods (abstract 26). Swed Dent J (special issue) 1987; 11:281-282.
Received: June 15, 1990 Accepted after revision: September 23, 1990 B. Lindquist Department of Cariology Faculty of Odontology University of Göteborg Box 33070 S-400 33 Göteborg (Sweden)
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Rolla G, Ciardi JE, Schultz SA: Adsorption of glucosyltransferase to saliva coated hydroxyapatite. Possible mechanism for su crose dependent bacterial colonization of teeth. Scand J Dent Res 1983;91:112-117. Socransky SS, Haffajee AD, Dzink JL, Hillman JD: Associations between microbial species in subgingival plaque samples. Oral Microbiol Immunol 1988;3:1-7. Syed SA, Loesche WJ: Survival of human dental plaque flora in various transport media. Appl Microbiol 1972;24:638-644. Togelius J, Kristoffersson K, Anderson H, Bratthall D: Strepto coccus mutans in saliva: Intraindividual variations and relation to the number of colonized sites. Acta Odontol Scand 1984:42: 157-163. Walter RG, Shklair IL: Streptococcus mutans in caries-free and car ies-active naval recruits. J Dent Res 1982;61:1229-1232.
