Antonie van Leeuwenhoek 45 (1979) 25 33
25
Streptococcus mutans and dental caries in humans: a bacteriological and immunological study J. H. J. Hu~s IN 'T VELD, W. H. VAN PALENSTE1N HELDERMAN AND O. BACKER DIRKS Department o f Preventive Dentistry, UniversiO' o f Utrecht, Sorbonnelaan 16, Utrecht, The Netherlands
HuIs 1N 'T VELD, J. H. J., VAN PALENSTEINHELDERMAN,W. H. and BACKER D1RKs,O. 1979. Streptococcus mutans and dental caries in humans: a bacteriological and immunological study. Antonie van Leeuwenhoek 45: 25--33. Plaque samples from caries-active subjects showed a higher incidence of S. mutans than plaque samples from caries-free subjects. This was especially evident in approximal incisor plaque. S. mutans serotype d was almost exclusively
present in approximal plaque obtained from caries-active subjects. Tooth surfaces infected with S. mutans still harbored this micro-organism 10 months later, while uninfected tooth surfaces remained free of S. mutans. Caries development predominantly occurs on those tooth surfaces which harbor relatively high percentages ofS. mutans ( > 5~). It is unlikely that serum or saliva antibodies against S. mutans play a major role in the protection against dental caries in these caries-free subjects since subjects with the greatest number of decayed surfaces showed the highest antibody titre as measured by haemagglutination or by the enzyme-linked immuno sorbent assay (ELISA).
INTRODUCTION
Dental caries is our most wide-spread disease, affecting more than 99 percent of the adult population. It is widely accepted that this is the result of faulty eating habits, especially the frequent ingestion of sucrose-containing food. The disease is initiated by the action of a bacterial plaque located on the tooth surface. The cariogenicity of dental plaque arises from the production of lactic acid by many plaque bacteria, resulting in a plaque pH which is low enough to dissolve the mineral of the tooth. At the same time, the structural nature of the dental plaque restricts the inward diffusion of potential neutralizing agents from the saliva whilst favouring the retention of bacterial products formed within the plaque (Winkler and Backer Dirks, 1958). Therefore, the dental plaque and its bacterial composition are generally considered to be of predominant significance in the
26
J. H. J. HUIS IN 'T VELD ET AL.
etiology of dental caries. Amongst these bacteria, Streptococcus mutans seems to be the most strongly associated with dental caries. The cariogenicity of this organism is due to a strong acid formation and to the ability to adhere and accumulate on tooth surfaces (Mc CABEet al., 1967). In addition to acid production from exogenous carbohydrates, S. mutans like many other oral streptococci produces acid from intracellular stored glycogen-like polysaccharides (IPS). It has been postulated (Gibbons and Nygaard, 1968) that IPS synthesizing bacteria produce more acid per unit of time when exogenous carbohydrate is present, prolong acid production when exogenous carbohydrate is depleted and have a better chance of survival than bacteria incapable of synthesizing IPS. The unique cariogenic potential of S. rnutans is, however, associated with its ability to initiate dental plaque formation. This is a two step process. First, S. mutans adheres to the so-called acquired pellicle, a structural entity separate from dental plaque. This layer consists of salivary glycoproteins which have become selectively adsorbed to the tooth surface. At a second stage, particularly in the presence of sucrose, an irreversible adhesive interaction will occur between the bacterial cells, mainly due to the synthesis of large amounts of extracellular polysaccharides (glucan, EPS) (van Houte, Winkler and Jansen, 1969). In contrast to IPS, glucan is only formed when sucrose is offered as the substrate, because the enzymes involved (glucosyltransferases) require the energy contained in the disaccharide bond for the reaction. Unlike other oral bacteria, such as Streptococcus sanguis, S. rnutans synthesises in addition to a water soluble 0~(1-6)-linked glucan, a cell-associated waterinsoluble glucan. The latter type of glucan is rich in 0~(1-3) linkages which explains its different physico-chemical character. De Stoppelaar et al. (1971) have demonstrated that a mutant of S. mutans which lacks the ability to synthesise the insoluble cell-associated glucans, produced less plaque and consequently induced significantly less caries in animals. The ability to synthesise glucan molecules which bind to specific sites on the surface of the S. mutans cell, thereby linking the organisms together, is a unique property of S. mutans. Other types of plaque bacteria, including S. sanguis, do not possess this characteristic. S. rnutans has therefore been used by several investigators (Bowen et al., 1975; Lehner et al., 1975; Mc Ghee et al., 1975) to immunise animals and reduction in caries was observed. Protection against caries coincided with raised levels of haemagglutinating antibodies in serum and saliva against S. mutans. However, the mechanism of this protection is still unknown. Both salivary and serum immunoglobulins may contribute to this immunity, since saliva continuously bathes the teeth and serum immunoglobulins are found in the gingival fluid. It is conceivable that antibodies present in serum or oral fluids may interfere with bacterial adherence and thereby prevent caries. (Evans and Genco, 1973; Linzer and Slade, 1976). Studying the relationship between immunoglobulins and caries in man may help to elucidate a possible role of immunity.
THE WINKLER SYMPOSIUM
27 RESULTS
Occurrence o f Streptococcus mutans in plaque T h e subjects used in this s t u d y were 18-20 years old m i l i t a r y recruits free f r o m p a s t o r present c a r i o u s lesions ( " c a r i e s - f l e e subjects"). T w o c o n t r o l groups, a r a n d o m l y selected g r o u p a n d a g r o u p with high caries experience, were also selected. O n e o u t o f a p p r o x i m a t e l y 2000 m i l i t a r y recruits a p p e a r e d to be absolutely free o f caries experience. P l a q u e samples were collected f r o m three clinically i n t a c t t o o t h surfaces in each i n d i v i d u a l a n d the p r e v a l e n c e o f S. rnutans in each s a m p l e was d e t e r m i n e d . (Huis in 't Veld et al., 1978). S e r o t y p i n g o f the different S. mutans strains was p e r f o r m e d b y the r i n g p r e c i p i t a t i o n test using specific sera. T a b l e 1 shows the f r e q u e n c y o f i s o l a t i o n a n d the d i s t r i b u t i o n o f S. mutans s e r o t y p e s in v a r i o u s p l a q u e s a m p l e s f r o m a r m y recruits with differing caries experience. In all three groups, S. mutans was isolated least in the a p p r o x i m a l
Table 1. Frequency of isolation and distribution of serotypes of Streptococcus mutans isolated from three different sites of individuals with various caries experiences Caries-free Frequency of detection
Randomly selected
2/20
Caries-active
13/20
9/12
At Serotypes
1c
7c
1 e/c
4e
7c 1 e/c 1 c/d
2f
B2
Frequency of detection Serotypes
10/20 5c le 3f
16/20 8c le ld
1 c/d
10/12 3c le ld 5 c/d
1 f/d
5 c/d Frequency of detection
12/20
C3 Serotypes
7c 2e 2f 1 c/f
1A : approximal incisor plaque sample z B: approximal molar plaque sample 3C : fissure molar plaque sample
17/20 10c 2e 3f 2 c/d
12/12 11 c 1 c/d
28
J. H. J. HUIS IN 'T VELD ET AL.
incisor sample (A) while an increasing number of positive isolates were found in the approximal molar plaque (B) and in plaque samples from the fissures of the molars (C). Out of all recruits studied, only 4 belonging to the caries-free group did not harbor S. rnutans in any of the three plaque samples. Serotypes c, e and f were present in the plaque of most of the subjects selected. In Dutch military recruits, serotype a and b could not be detected. Serotype d, in combination with other serotypes, was particularly present in approximal molar samples of people belonging to the randomly selected and caries-active group. Only one approximal plaque sample of a caries-free recruit contained a serotype d strain. A sequential bacteriological study, 10 months later, revealed that the serotype d strain had disappeared from the site in the caries-free individual. Surfaces infected with S. mutans still harbored this organism after 10 months, while uninfected surfaces remained free of S. mutans. Some exceptions occurred but always near the level of detection of S. mutans (0.5 ~o). The sequential study also showed that the composition of serotypes in plaque remained rather constant during this time period. Surfaces infected with mixtures of serotypes generally harbored the same serotypes 10 months later. Even in the mouths of individuals where a great variation in serotypes existed at different surfaces, the same serotypes persisted in the individual sites. However, fluctuation in the percentage of S. mutans, as compared with total bacterial counts, was observed. All four caries-free recruits,who did not have S. mutans in the plaque at any site, remained S. mutans free after 10 months. S. mutans and caries progression
When a careful radiographic examination is carried out on clinically intact approximal surfaces, different stages of caries lesion formation can be observed. For example, 2/3 of the randomly selected recruits appeared to have either enamel lesions or dentinal lesions. We have evaluated the progression of caries lesions on standardised X-rays over a 10-month period. Caries progression occurred at 14 tooth surfaces. (Table 2) 71~ of these surfaces harboured S. mutans percentages higher than 5~ , while S. mutans could not be detected at 51~o of the tooth surfaces without caries progression. Of Table 2. Prevalence of S. mutans at approximal tooth surfaces with and without caries-progression I Streptococcus rnutans
Caries progression No caries progression
N
0%
0-5%
14 41
1 21
3 17
1Figures represent numbers of tooth surfaces.
/> 5% 10 3
29
THE W1NKLER SYMPOSIUM
Table 3. Mean serum log2 titre + standard deviation against Rantz and Randall extracts of reference S. m u t a n s strains of different serotypes in people with differing caries experience Serotypes
a
b
c
d
e
Caries-free group n-20
6.0_+1.5
4.9_+1.6
6.5_+ l.l
6.3_+ 1.1
5.0_+1.4
At random group n = 20
6.2 + 1.3
5.3 + 1.2
6.8 _+ 1.0
6.7 + 0.9
5.6 + 1.8
Caries-active group n = 12
7.6 -+ 0.7
6.0 -+ 1.2
7.9 -+ 0.7
7.0 -+ 0.6
6. l -+ 1.2
Table 4. Mean serum IgG and parotid IgA antibody levels against S. m u t a n s C67-1 as measured with ELISA Caries free n = 15
Randomly selected n - 15
Caries active n - 10
Serum IgG
0.94 + 0.54
I. 18 + 0.51
1.64 + 0.61
Parotid IgA
0.87 _+0.53
0.96 + 0.48
1.1 _+ 0.54
1Figures represent absorption values at 449 nm. Serum dilution : 1/ 1000. Parotid saliva dilution : !/4.
course Table 2 also shows that there is no 100~ correlation: relative high S. mutans percentages could be detected at tooth surfaces without any caries progression. Serum and salivary antibodies against S. mutans
Haemagglutination and the enzyme-linked immunosorbent assay (ELISA) were used to determine antibodies against S. mutans. The ELISA was a modification of the method described by Ruitenberg et al., 1967. Haem agglu t in a t ion
In parotid saliva haemagglutinating antibody titers were low in all three groups (up to l : 32) and did not show any correlation with caries experience. In serum, antibody titers could be detected up to a maximum of 1:512. Serum haemagglutination titers against serotypes a, b, c, d and e were higher in those people with the highest caries scores (Table 3). Caries-free individuals showed the lowest titers when tested against extracts of the different serotypes. Statistically significant differences in antibody titers to serotype c were observed between the caries-free and the caries-active group (p < 0.01). Extracts of freshly isolated S. mutans strains, tested against serum from the same individual showed about the same titer as reference strains.
30
J. H. J. HUIS IN 'T VELD ET AL.
ELISA
In serum, the predominant specific antibody type appeared to be IgG, where ELISA titers up to 1 : 5000 could be detected. In some sera, low levels of IgA antibodies against S. rnutans were found. With this techniques no serum IgM antibodies against S. mutans could be detected. The ELISA also showed that the recruits with the highest caries experience possessed the highest concentration of specific IgG (Table 4). Significant differences between the caries-free and the caries-active group were found (p < 0.025). The sequential study indicated that serum IgG levels stayed constant over a 10 month period. Even subjects who developed caries during this period showed no significant increase in serum IgG levels. In parotid saliva low IgA antibodies against S. mutans were detectable. No significant differences between the cariesfree, the randomly selected and the caries-active group were observed (Table 3). Strong fluctuations in specific IgA levels over the 10 month period were observed.
DISCUSSION
Animal experiments have revealed that antibodies against cell wall antigens of S. mutans are protective against dental caries. (Bowen et al., 1975; Lehner et al.,
1975). The purpose of the present study was to elucidate any role of antibodies against S. mutans cell wall antigens in the protection against dental caries in humans. Since one might expect that the antibody titer against S. mutans is a reflection of past or present stimulation of the immune system by that organism, a bacteriological study to detect S. mutans in these individuals was also undertaken. S. mutans was present in about 9 5 ~ of the people selected. A difference in S. rnutans incidence between the caries-free and the other two groups was found and this difference was especially evident in approximal incisor plaque samples (Table 1). Until now little information on the distribution of the different serotypes in human dental plaque has been available. Serotypes c, d and e are found widespread (Bratthall, 1972; Duany et al., 1972) while serotypes a, b, f and g have been isolated occasionally (Perch et al., 1974). Our results confirm these studies, but in addition show that serotype d is predominantly present in subjects with active caries. Conclusive evidence about the relationship between S. mutans and the caries process can only be obtained from sequential analyses. Unfortunately few of those studies have been carried out sofar (de Stoppelaar, 1971; Ikeda et al., 1973). Although in these investigations different age groups and different tooth surfaces were studied, our results are in good agreement with the above mentioned reports: progressing caries lesions mainly occurs at those tooth surfaces with a relatively high percentage of S. mutans (> 5~o, Table 2). From these
THE WINKLER SYMPOSIUM
31
studies the conclusion seems to be justified that sofar, S. mutans is the only oral microorganism positively correlated with the initiation of dental caries. But at the same time, Table 2 shows that S. mutans does not appear to be the only microorganism associated with the onset of caries. Caries also develops without the presence of detectable levels of S. mutans. The last part of this investigation deals with the possible role of antibodies against S. mutans in the protection against dental caries. Two different techniques were applied to detect antibodies in serum and saliva. The high degree of S. mutans infection in caries-active individuals is reflected by raised serum antibody levels. In our study, subjects with the greatest number of decayed surfaces showed the highest haemagglutinating antibody titers against S. mutans serotype c, the most commonly occurring serotype (Table 3). Our caries-free group harbored low levels of S. mutans, which were constant over a period of time and it is therefore likely to assume that the low haemagglutinating antibody titer in this group is consequence of the low degree of infection. Although serotypes a and b could never be detected in our plaque samples, titers against serotypes a were relatively high while those against serotype b were the lowest. Cross-reacting antigens between serotypes a and b (Bratthall, 1972) might be responsible for this. The principle of haemagglutination and ELISA is different, but both techniques lead to the same conclusion; subjects with the highest caries experience showed the highest antibody titers against S. mutans. ELISA has advantages compared to conventional techniques such as haemagglutination and complement fixation. It has been applied for the detection of low levels of antibodies in various body fluids. Besides, it provides better information about the nature of the antibodies and facilitates quantitation of antibody levels. For example, in our assay system an absorption value of 0.3 corresponds to a serum ELISA titer of about 1 : 500. An absorption value of 1.6 with a titer of about 1 : 5000. The ELISA technique has also certain limitations. In the presence of excess IgG, low levels of other antibody classes might not be detected because of masking of the antigen by the excess IgG. The failure to detect any specific IgM with ELISA might be a consequence of the masking effect by IgG. Therefore, the presence of low levels of specific IgM in serum, the most efficient haemagglutinating antibody, can not be excluded. However, high levels of specific IgM antibodies are not to be expected in a chronic disease like dental caries. In contrast with the constant levels of specific IgG in serum, the specific salivary IgA content varied with time. Care must be taken when interpreting these results, since daily fluctuations in specific IgA titers within one person were observed (Huis in 't Veld, unpublished results). At times, no specific serum IgA could be detected in individuals with a relatively high specific salivary IgA titer. Preliminary observations on the specificity of the ELISA test revealed that crossreactivity between the different serotypes of S. mutans occurred. Cross-reactivity was the weakest with serotype b strains, a serotype rarely found in human dental
32
J. H. J. HUIS IN 'T VELD ET AL.
plaque. Since we have used whole cells of S. mutans as the antigen, antibodies against all cell wall components, cell wall carbohydrates as well as cell-associated GTF, lipoteichoic acids or other cell wall components may have been measured. Furthermore, the presence of naturally occurring human serum antibodies to teichoic acids (Duany et al., 1972) and the low antigenic specificity of these components have been well established (Knox and Wicken, 1972). It is therefore not surprising that cross-reactivity was observed. The present investigation shows that serum or salivary antibodies against S. mutans do not seem to play a role in caries resistance in young adults. However, this does not preclude a role of antibodies, prior to infection with S. mutans, in
man.
REFERENCES BOWEN, W. H., COHEN, B., COLE,M. F. and COLMAN,G. 1975. Immunisation against dental caries. --Brit. Dent. J. 139: 45-58. BRATTHALL, D. 1972. Demonstration of Streptococcus mutans strains in some selected areas of the world. - - Odontol. Revy 23: 401-410. DUANY, F., JABLON, J. M. and ZINNER, D. D. 1972. Epidemiologic studies of caries-free and cariesactive students, - - J. Dent. Res. 51 : 723-726. EVANS, R. T. and GENCO, R. J. 1973, Inhibition of glucosyltransferase activity by antisera to known serotypes of Streptococcus mutans. - - Infect. Immun. 7: 237-241. G1BBONS, R. J. and NYGAARD, M. 1968. Synthesis of insoluble dextran and its significance in the formation of gelatinous deposits by plaque-forming streptococci. - - Arch. Oral. Biol. 13: 1249-1262. GUGGENHEIM, B. and SCHROEDER,H. E. 1967. Biochemical and morphological aspects of extracellular polysaccharides produced by cariogenic streptococci. - - Helv. Odontol. Acta l l : 131-152. VAN HOUTE, J., WINKLER, K. C. and JANSEN, H. M. 1969. Iodophilic polysaccharide synthesis, acid production and growth in oral streptococci. - - A r c h . Oral. Biol. 14:45 61. HuIs 1N 'T VELD, J., BANNET, D., VAN PALENSTEINHELDERMAN,W. U., SAMPAIOCAMARGO, P. and BACKER DIRKS, O. 1978. Antibodies against Streptococcus mutans and glucosyltransferases in caries-free and caries-active military recruits. Adv. Exp. Med. Biol. 107:369 383. IKEDA. T., SANDHAM, H. J. and BRADLEY, E. L. 1973. Changes in Streptococcus mutans and lactobacilli in plaque in relation of dental caries in negro children. - - Arch. Oral. Biol. 18 : 555-566. KNox, K. W. and WlCKEN, A. J. 1972. Reaction of dextrans with antisera to teichoic acids. Arch. Oral. Biol. 17: 1491-1494. LEHNER,T., CHALLACOMBE,S. J. and CALDWELL,J. 1975. An immunological investigation into the prevention of caries in deciduous teeth of Rhesus monkeys. - - Arch. Oral. Biol. 20:305-310. LINZER, R. VAN SLADE, H, D. 1976. Characterization of an anti-glucosyltransferase serum specific Infect. Immun. 13:494 500. for insoluble glucan synthesis by Streptococcus rnutans. MCCABE, R, M., KEYES, P. M. and HOWEL, A. 1967. An in vitro method for assessing the plaque forming ability of oral bacteria. - - Arch. Oral, Biol. 12: 1653 1656. PERCH, B., KJEMS, E. and RAVIN, T. 1974. Biochemical and serological properties Streptococcus mutans from various h u m a n and animal sources, - - Acta. Pathol. Microbiol. Scand. B 82: 357 370.
THE WINKLER SYMPOSIUM
33
RUITENBERG, E. J., STEERENBERG,P. A., BROSE, B. J. M. and Buys, J. 1967. Reliability of the ELISA for the serodiagnosis of Trichinella spira/is infections in conventionally raised pigs. - - J . Immunol. Methods 10: 67-83. DE STOPPELAAR,J. D. 1971. p. 222-228. In R. W. Fearnhead and M. V. Stack (eds). Tooth enamel II Symposium. - - J o h n Wright and Sons Ltd., Bristol. DE STOPPELAAR, J. D., KONIG, K. G., PLASSCHAERT~A. J. M. and VAN DER HOEVEN, J. S. 1971. Decreased cariogenicity of a m u t a n t of Streptococcus mutans. - - Arch. Oral. Biol. 16:971 975. WINKLER. K. C. and BACKER DIRKS. O. 1958. The mechanism of the dental plaque. Int. Dent. J. 8:561 585.
25
Streptococcus mutans and dental caries in humans: a bacteriological and immunological study J. H. J. Hu~s IN 'T VELD, W. H. VAN PALENSTE1N HELDERMAN AND O. BACKER DIRKS Department o f Preventive Dentistry, UniversiO' o f Utrecht, Sorbonnelaan 16, Utrecht, The Netherlands
HuIs 1N 'T VELD, J. H. J., VAN PALENSTEINHELDERMAN,W. H. and BACKER D1RKs,O. 1979. Streptococcus mutans and dental caries in humans: a bacteriological and immunological study. Antonie van Leeuwenhoek 45: 25--33. Plaque samples from caries-active subjects showed a higher incidence of S. mutans than plaque samples from caries-free subjects. This was especially evident in approximal incisor plaque. S. mutans serotype d was almost exclusively
present in approximal plaque obtained from caries-active subjects. Tooth surfaces infected with S. mutans still harbored this micro-organism 10 months later, while uninfected tooth surfaces remained free of S. mutans. Caries development predominantly occurs on those tooth surfaces which harbor relatively high percentages ofS. mutans ( > 5~). It is unlikely that serum or saliva antibodies against S. mutans play a major role in the protection against dental caries in these caries-free subjects since subjects with the greatest number of decayed surfaces showed the highest antibody titre as measured by haemagglutination or by the enzyme-linked immuno sorbent assay (ELISA).
INTRODUCTION
Dental caries is our most wide-spread disease, affecting more than 99 percent of the adult population. It is widely accepted that this is the result of faulty eating habits, especially the frequent ingestion of sucrose-containing food. The disease is initiated by the action of a bacterial plaque located on the tooth surface. The cariogenicity of dental plaque arises from the production of lactic acid by many plaque bacteria, resulting in a plaque pH which is low enough to dissolve the mineral of the tooth. At the same time, the structural nature of the dental plaque restricts the inward diffusion of potential neutralizing agents from the saliva whilst favouring the retention of bacterial products formed within the plaque (Winkler and Backer Dirks, 1958). Therefore, the dental plaque and its bacterial composition are generally considered to be of predominant significance in the
26
J. H. J. HUIS IN 'T VELD ET AL.
etiology of dental caries. Amongst these bacteria, Streptococcus mutans seems to be the most strongly associated with dental caries. The cariogenicity of this organism is due to a strong acid formation and to the ability to adhere and accumulate on tooth surfaces (Mc CABEet al., 1967). In addition to acid production from exogenous carbohydrates, S. mutans like many other oral streptococci produces acid from intracellular stored glycogen-like polysaccharides (IPS). It has been postulated (Gibbons and Nygaard, 1968) that IPS synthesizing bacteria produce more acid per unit of time when exogenous carbohydrate is present, prolong acid production when exogenous carbohydrate is depleted and have a better chance of survival than bacteria incapable of synthesizing IPS. The unique cariogenic potential of S. rnutans is, however, associated with its ability to initiate dental plaque formation. This is a two step process. First, S. mutans adheres to the so-called acquired pellicle, a structural entity separate from dental plaque. This layer consists of salivary glycoproteins which have become selectively adsorbed to the tooth surface. At a second stage, particularly in the presence of sucrose, an irreversible adhesive interaction will occur between the bacterial cells, mainly due to the synthesis of large amounts of extracellular polysaccharides (glucan, EPS) (van Houte, Winkler and Jansen, 1969). In contrast to IPS, glucan is only formed when sucrose is offered as the substrate, because the enzymes involved (glucosyltransferases) require the energy contained in the disaccharide bond for the reaction. Unlike other oral bacteria, such as Streptococcus sanguis, S. rnutans synthesises in addition to a water soluble 0~(1-6)-linked glucan, a cell-associated waterinsoluble glucan. The latter type of glucan is rich in 0~(1-3) linkages which explains its different physico-chemical character. De Stoppelaar et al. (1971) have demonstrated that a mutant of S. mutans which lacks the ability to synthesise the insoluble cell-associated glucans, produced less plaque and consequently induced significantly less caries in animals. The ability to synthesise glucan molecules which bind to specific sites on the surface of the S. mutans cell, thereby linking the organisms together, is a unique property of S. mutans. Other types of plaque bacteria, including S. sanguis, do not possess this characteristic. S. rnutans has therefore been used by several investigators (Bowen et al., 1975; Lehner et al., 1975; Mc Ghee et al., 1975) to immunise animals and reduction in caries was observed. Protection against caries coincided with raised levels of haemagglutinating antibodies in serum and saliva against S. mutans. However, the mechanism of this protection is still unknown. Both salivary and serum immunoglobulins may contribute to this immunity, since saliva continuously bathes the teeth and serum immunoglobulins are found in the gingival fluid. It is conceivable that antibodies present in serum or oral fluids may interfere with bacterial adherence and thereby prevent caries. (Evans and Genco, 1973; Linzer and Slade, 1976). Studying the relationship between immunoglobulins and caries in man may help to elucidate a possible role of immunity.
THE WINKLER SYMPOSIUM
27 RESULTS
Occurrence o f Streptococcus mutans in plaque T h e subjects used in this s t u d y were 18-20 years old m i l i t a r y recruits free f r o m p a s t o r present c a r i o u s lesions ( " c a r i e s - f l e e subjects"). T w o c o n t r o l groups, a r a n d o m l y selected g r o u p a n d a g r o u p with high caries experience, were also selected. O n e o u t o f a p p r o x i m a t e l y 2000 m i l i t a r y recruits a p p e a r e d to be absolutely free o f caries experience. P l a q u e samples were collected f r o m three clinically i n t a c t t o o t h surfaces in each i n d i v i d u a l a n d the p r e v a l e n c e o f S. rnutans in each s a m p l e was d e t e r m i n e d . (Huis in 't Veld et al., 1978). S e r o t y p i n g o f the different S. mutans strains was p e r f o r m e d b y the r i n g p r e c i p i t a t i o n test using specific sera. T a b l e 1 shows the f r e q u e n c y o f i s o l a t i o n a n d the d i s t r i b u t i o n o f S. mutans s e r o t y p e s in v a r i o u s p l a q u e s a m p l e s f r o m a r m y recruits with differing caries experience. In all three groups, S. mutans was isolated least in the a p p r o x i m a l
Table 1. Frequency of isolation and distribution of serotypes of Streptococcus mutans isolated from three different sites of individuals with various caries experiences Caries-free Frequency of detection
Randomly selected
2/20
Caries-active
13/20
9/12
At Serotypes
1c
7c
1 e/c
4e
7c 1 e/c 1 c/d
2f
B2
Frequency of detection Serotypes
10/20 5c le 3f
16/20 8c le ld
1 c/d
10/12 3c le ld 5 c/d
1 f/d
5 c/d Frequency of detection
12/20
C3 Serotypes
7c 2e 2f 1 c/f
1A : approximal incisor plaque sample z B: approximal molar plaque sample 3C : fissure molar plaque sample
17/20 10c 2e 3f 2 c/d
12/12 11 c 1 c/d
28
J. H. J. HUIS IN 'T VELD ET AL.
incisor sample (A) while an increasing number of positive isolates were found in the approximal molar plaque (B) and in plaque samples from the fissures of the molars (C). Out of all recruits studied, only 4 belonging to the caries-free group did not harbor S. rnutans in any of the three plaque samples. Serotypes c, e and f were present in the plaque of most of the subjects selected. In Dutch military recruits, serotype a and b could not be detected. Serotype d, in combination with other serotypes, was particularly present in approximal molar samples of people belonging to the randomly selected and caries-active group. Only one approximal plaque sample of a caries-free recruit contained a serotype d strain. A sequential bacteriological study, 10 months later, revealed that the serotype d strain had disappeared from the site in the caries-free individual. Surfaces infected with S. mutans still harbored this organism after 10 months, while uninfected surfaces remained free of S. mutans. Some exceptions occurred but always near the level of detection of S. mutans (0.5 ~o). The sequential study also showed that the composition of serotypes in plaque remained rather constant during this time period. Surfaces infected with mixtures of serotypes generally harbored the same serotypes 10 months later. Even in the mouths of individuals where a great variation in serotypes existed at different surfaces, the same serotypes persisted in the individual sites. However, fluctuation in the percentage of S. mutans, as compared with total bacterial counts, was observed. All four caries-free recruits,who did not have S. mutans in the plaque at any site, remained S. mutans free after 10 months. S. mutans and caries progression
When a careful radiographic examination is carried out on clinically intact approximal surfaces, different stages of caries lesion formation can be observed. For example, 2/3 of the randomly selected recruits appeared to have either enamel lesions or dentinal lesions. We have evaluated the progression of caries lesions on standardised X-rays over a 10-month period. Caries progression occurred at 14 tooth surfaces. (Table 2) 71~ of these surfaces harboured S. mutans percentages higher than 5~ , while S. mutans could not be detected at 51~o of the tooth surfaces without caries progression. Of Table 2. Prevalence of S. mutans at approximal tooth surfaces with and without caries-progression I Streptococcus rnutans
Caries progression No caries progression
N
0%
0-5%
14 41
1 21
3 17
1Figures represent numbers of tooth surfaces.
/> 5% 10 3
29
THE W1NKLER SYMPOSIUM
Table 3. Mean serum log2 titre + standard deviation against Rantz and Randall extracts of reference S. m u t a n s strains of different serotypes in people with differing caries experience Serotypes
a
b
c
d
e
Caries-free group n-20
6.0_+1.5
4.9_+1.6
6.5_+ l.l
6.3_+ 1.1
5.0_+1.4
At random group n = 20
6.2 + 1.3
5.3 + 1.2
6.8 _+ 1.0
6.7 + 0.9
5.6 + 1.8
Caries-active group n = 12
7.6 -+ 0.7
6.0 -+ 1.2
7.9 -+ 0.7
7.0 -+ 0.6
6. l -+ 1.2
Table 4. Mean serum IgG and parotid IgA antibody levels against S. m u t a n s C67-1 as measured with ELISA Caries free n = 15
Randomly selected n - 15
Caries active n - 10
Serum IgG
0.94 + 0.54
I. 18 + 0.51
1.64 + 0.61
Parotid IgA
0.87 _+0.53
0.96 + 0.48
1.1 _+ 0.54
1Figures represent absorption values at 449 nm. Serum dilution : 1/ 1000. Parotid saliva dilution : !/4.
course Table 2 also shows that there is no 100~ correlation: relative high S. mutans percentages could be detected at tooth surfaces without any caries progression. Serum and salivary antibodies against S. mutans
Haemagglutination and the enzyme-linked immunosorbent assay (ELISA) were used to determine antibodies against S. mutans. The ELISA was a modification of the method described by Ruitenberg et al., 1967. Haem agglu t in a t ion
In parotid saliva haemagglutinating antibody titers were low in all three groups (up to l : 32) and did not show any correlation with caries experience. In serum, antibody titers could be detected up to a maximum of 1:512. Serum haemagglutination titers against serotypes a, b, c, d and e were higher in those people with the highest caries scores (Table 3). Caries-free individuals showed the lowest titers when tested against extracts of the different serotypes. Statistically significant differences in antibody titers to serotype c were observed between the caries-free and the caries-active group (p < 0.01). Extracts of freshly isolated S. mutans strains, tested against serum from the same individual showed about the same titer as reference strains.
30
J. H. J. HUIS IN 'T VELD ET AL.
ELISA
In serum, the predominant specific antibody type appeared to be IgG, where ELISA titers up to 1 : 5000 could be detected. In some sera, low levels of IgA antibodies against S. rnutans were found. With this techniques no serum IgM antibodies against S. mutans could be detected. The ELISA also showed that the recruits with the highest caries experience possessed the highest concentration of specific IgG (Table 4). Significant differences between the caries-free and the caries-active group were found (p < 0.025). The sequential study indicated that serum IgG levels stayed constant over a 10 month period. Even subjects who developed caries during this period showed no significant increase in serum IgG levels. In parotid saliva low IgA antibodies against S. mutans were detectable. No significant differences between the cariesfree, the randomly selected and the caries-active group were observed (Table 3). Strong fluctuations in specific IgA levels over the 10 month period were observed.
DISCUSSION
Animal experiments have revealed that antibodies against cell wall antigens of S. mutans are protective against dental caries. (Bowen et al., 1975; Lehner et al.,
1975). The purpose of the present study was to elucidate any role of antibodies against S. mutans cell wall antigens in the protection against dental caries in humans. Since one might expect that the antibody titer against S. mutans is a reflection of past or present stimulation of the immune system by that organism, a bacteriological study to detect S. mutans in these individuals was also undertaken. S. mutans was present in about 9 5 ~ of the people selected. A difference in S. rnutans incidence between the caries-free and the other two groups was found and this difference was especially evident in approximal incisor plaque samples (Table 1). Until now little information on the distribution of the different serotypes in human dental plaque has been available. Serotypes c, d and e are found widespread (Bratthall, 1972; Duany et al., 1972) while serotypes a, b, f and g have been isolated occasionally (Perch et al., 1974). Our results confirm these studies, but in addition show that serotype d is predominantly present in subjects with active caries. Conclusive evidence about the relationship between S. mutans and the caries process can only be obtained from sequential analyses. Unfortunately few of those studies have been carried out sofar (de Stoppelaar, 1971; Ikeda et al., 1973). Although in these investigations different age groups and different tooth surfaces were studied, our results are in good agreement with the above mentioned reports: progressing caries lesions mainly occurs at those tooth surfaces with a relatively high percentage of S. mutans (> 5~o, Table 2). From these
THE WINKLER SYMPOSIUM
31
studies the conclusion seems to be justified that sofar, S. mutans is the only oral microorganism positively correlated with the initiation of dental caries. But at the same time, Table 2 shows that S. mutans does not appear to be the only microorganism associated with the onset of caries. Caries also develops without the presence of detectable levels of S. mutans. The last part of this investigation deals with the possible role of antibodies against S. mutans in the protection against dental caries. Two different techniques were applied to detect antibodies in serum and saliva. The high degree of S. mutans infection in caries-active individuals is reflected by raised serum antibody levels. In our study, subjects with the greatest number of decayed surfaces showed the highest haemagglutinating antibody titers against S. mutans serotype c, the most commonly occurring serotype (Table 3). Our caries-free group harbored low levels of S. mutans, which were constant over a period of time and it is therefore likely to assume that the low haemagglutinating antibody titer in this group is consequence of the low degree of infection. Although serotypes a and b could never be detected in our plaque samples, titers against serotypes a were relatively high while those against serotype b were the lowest. Cross-reacting antigens between serotypes a and b (Bratthall, 1972) might be responsible for this. The principle of haemagglutination and ELISA is different, but both techniques lead to the same conclusion; subjects with the highest caries experience showed the highest antibody titers against S. mutans. ELISA has advantages compared to conventional techniques such as haemagglutination and complement fixation. It has been applied for the detection of low levels of antibodies in various body fluids. Besides, it provides better information about the nature of the antibodies and facilitates quantitation of antibody levels. For example, in our assay system an absorption value of 0.3 corresponds to a serum ELISA titer of about 1 : 500. An absorption value of 1.6 with a titer of about 1 : 5000. The ELISA technique has also certain limitations. In the presence of excess IgG, low levels of other antibody classes might not be detected because of masking of the antigen by the excess IgG. The failure to detect any specific IgM with ELISA might be a consequence of the masking effect by IgG. Therefore, the presence of low levels of specific IgM in serum, the most efficient haemagglutinating antibody, can not be excluded. However, high levels of specific IgM antibodies are not to be expected in a chronic disease like dental caries. In contrast with the constant levels of specific IgG in serum, the specific salivary IgA content varied with time. Care must be taken when interpreting these results, since daily fluctuations in specific IgA titers within one person were observed (Huis in 't Veld, unpublished results). At times, no specific serum IgA could be detected in individuals with a relatively high specific salivary IgA titer. Preliminary observations on the specificity of the ELISA test revealed that crossreactivity between the different serotypes of S. mutans occurred. Cross-reactivity was the weakest with serotype b strains, a serotype rarely found in human dental
32
J. H. J. HUIS IN 'T VELD ET AL.
plaque. Since we have used whole cells of S. mutans as the antigen, antibodies against all cell wall components, cell wall carbohydrates as well as cell-associated GTF, lipoteichoic acids or other cell wall components may have been measured. Furthermore, the presence of naturally occurring human serum antibodies to teichoic acids (Duany et al., 1972) and the low antigenic specificity of these components have been well established (Knox and Wicken, 1972). It is therefore not surprising that cross-reactivity was observed. The present investigation shows that serum or salivary antibodies against S. mutans do not seem to play a role in caries resistance in young adults. However, this does not preclude a role of antibodies, prior to infection with S. mutans, in
man.
REFERENCES BOWEN, W. H., COHEN, B., COLE,M. F. and COLMAN,G. 1975. Immunisation against dental caries. --Brit. Dent. J. 139: 45-58. BRATTHALL, D. 1972. Demonstration of Streptococcus mutans strains in some selected areas of the world. - - Odontol. Revy 23: 401-410. DUANY, F., JABLON, J. M. and ZINNER, D. D. 1972. Epidemiologic studies of caries-free and cariesactive students, - - J. Dent. Res. 51 : 723-726. EVANS, R. T. and GENCO, R. J. 1973, Inhibition of glucosyltransferase activity by antisera to known serotypes of Streptococcus mutans. - - Infect. Immun. 7: 237-241. G1BBONS, R. J. and NYGAARD, M. 1968. Synthesis of insoluble dextran and its significance in the formation of gelatinous deposits by plaque-forming streptococci. - - Arch. Oral. Biol. 13: 1249-1262. GUGGENHEIM, B. and SCHROEDER,H. E. 1967. Biochemical and morphological aspects of extracellular polysaccharides produced by cariogenic streptococci. - - Helv. Odontol. Acta l l : 131-152. VAN HOUTE, J., WINKLER, K. C. and JANSEN, H. M. 1969. Iodophilic polysaccharide synthesis, acid production and growth in oral streptococci. - - A r c h . Oral. Biol. 14:45 61. HuIs 1N 'T VELD, J., BANNET, D., VAN PALENSTEINHELDERMAN,W. U., SAMPAIOCAMARGO, P. and BACKER DIRKS, O. 1978. Antibodies against Streptococcus mutans and glucosyltransferases in caries-free and caries-active military recruits. Adv. Exp. Med. Biol. 107:369 383. IKEDA. T., SANDHAM, H. J. and BRADLEY, E. L. 1973. Changes in Streptococcus mutans and lactobacilli in plaque in relation of dental caries in negro children. - - Arch. Oral. Biol. 18 : 555-566. KNox, K. W. and WlCKEN, A. J. 1972. Reaction of dextrans with antisera to teichoic acids. Arch. Oral. Biol. 17: 1491-1494. LEHNER,T., CHALLACOMBE,S. J. and CALDWELL,J. 1975. An immunological investigation into the prevention of caries in deciduous teeth of Rhesus monkeys. - - Arch. Oral. Biol. 20:305-310. LINZER, R. VAN SLADE, H, D. 1976. Characterization of an anti-glucosyltransferase serum specific Infect. Immun. 13:494 500. for insoluble glucan synthesis by Streptococcus rnutans. MCCABE, R, M., KEYES, P. M. and HOWEL, A. 1967. An in vitro method for assessing the plaque forming ability of oral bacteria. - - Arch. Oral, Biol. 12: 1653 1656. PERCH, B., KJEMS, E. and RAVIN, T. 1974. Biochemical and serological properties Streptococcus mutans from various h u m a n and animal sources, - - Acta. Pathol. Microbiol. Scand. B 82: 357 370.
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RUITENBERG, E. J., STEERENBERG,P. A., BROSE, B. J. M. and Buys, J. 1967. Reliability of the ELISA for the serodiagnosis of Trichinella spira/is infections in conventionally raised pigs. - - J . Immunol. Methods 10: 67-83. DE STOPPELAAR,J. D. 1971. p. 222-228. In R. W. Fearnhead and M. V. Stack (eds). Tooth enamel II Symposium. - - J o h n Wright and Sons Ltd., Bristol. DE STOPPELAAR, J. D., KONIG, K. G., PLASSCHAERT~A. J. M. and VAN DER HOEVEN, J. S. 1971. Decreased cariogenicity of a m u t a n t of Streptococcus mutans. - - Arch. Oral. Biol. 16:971 975. WINKLER. K. C. and BACKER DIRKS. O. 1958. The mechanism of the dental plaque. Int. Dent. J. 8:561 585.
