crose (Todd Hewitt broth, Difco), and 1% sucrose ... grown in Todd-Hewitt broth at 37°C for 18 h, and the ..... Appelbaum, B., E. Golub, S. C. Holt, and B. Rosan.
INFECTION AND IMMUNITY, Apr. 1981, p. 364-372 0019-9567/81/040364-09$02.00/0
Vol. 32, No. 1
Adherence of Streptococcus sanguis Clinical Isolates to Smooth Surfaces and Interaction of the Isolates with Streptococcus mutans Glucosyltransferase SHIGEYUKI HAMADA,' * MITSUO TORII,' SHOZO KOTANI,' AND YASUHIKO TSUCHITANI2
Department of Dental Research, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo, 141 Japan,' and Departments of Oral Microbiology and Operative Dentistry, Osaka University Dental School, Kita-ku, Osaka, 530 Japan2
Streptococcus sanguis isolated from human dental plaque were grown in ToddHewitt broth. Cells were collected by centrifugation and lyophilized after extensive washing with water. The cell-associated glucosyltransferase (GTase) activities of S. sanguis strains were assayed with ['4C]sucrose. Strain differences in GTase activity were significant within the same serotype or biotype or both. The ability of S. sanguis cells to adhere to smooth glass surfaces was generally weak, irrespective of significant cell-associated GTase activity synthesizing water-insoluble, gel-like glucans. Resting cells of most S. sanguis strains bound extracellular GTase from Streptococcus mutans strain B13 (serotype d), resulting in the strong adherence of the S. sanguis cells to smooth glass surfaces in the presence of sucrose. Conversely, S. mutans B13 cells also could bind extracellular GTase from some strains of S. sanguis examined. The sucrose-dependent adherence of S. mutans cells was not altered, although S. sanguis strains from which the extracellular GTases were obtained did not produce significant adherence in the presence of sucrose. In view of these findings, it was suggested that S. mutans GTase could affect the adherence of S. sanguis to smooth tooth surfaces in the oral cavity. It has been shown that bacterial adherence to tooth surfaces is a primary requirement in the initiation of dental plaque formation and caries development. Among the many oral bacterial species examined, Streptococcus mutans has been suggested to be a major causative agent of dental caries. The ability of S. mutans to colonize tooth surfaces could be ascribed to its ability to synthesize water-insoluble, adherent glucans from sucrose. These glucans appear to be an important factor in the stabilization of initial attachment of bacterial cells to the tooth surfaces (10). However, several in vivo studies revealed that Streptococcus sanguis is one of the earliest colonizers on the tooth surfaces when teeth erupt (2) or new tooth surfaces are exposed by mechanical cleansing (3, 29-31). In vitro studies also indicate that the adherence of S. sanguis to tooth slabs or hydroxyapatite is greater than that of S. mutans and other oral streptococci (1, 5, 24). On the other hand, uptake of sucrose results in an increased proportion of S. mutans compared with other bacterial species, including glucan-producing S. sanguis, in dental plaque (13, 26). S. sanguis has a high affinity for hydroxyapa-
tite and an ability to synthesize water-soluble and water-insoluble glucans from sucrose. However, most strains of this species develop no significant dental plaque (12) in animal model systems. Therefore, they are generally noncariogenic in experimental animals fed a caries-inducing diet (6, 17). In a preceding study, we isolated more than 100 isolates of S. sanguis from human mouths and classified them by their biological abilities and immunological specificities (12). The present study examined the ability of recent clinical isolates of S. sanguis to adhere to glass surfaces under various experimental conditions and compared the adherence ability of S. sanguis with that of S. mutans. MATERIALS AND METHODS Bacterial strains and cultural condition. S. sanguis (110 strains) were isolated from human dental plaque as described in our previous study (12). For comparison, clinically isolated S. mutans strains MT9001 (serotype c), MT6367 (serotype d), MT9007 (serotype e), MT3051 (serotype f), and MT6219 (serotype g); S. salivarius strain HT9; and S. mitior were employed. S. milleri strains were kindly provided by 364
VOL. 32, 1981 S. Edwardsson (22). Organisms were grown in ToddHewitt broth (Difco) supplemented with 0.5% glucose at 37°C for 18 h unless otherwise stated. Adherence of growing cells to a glass surface. To assess the adherence of growing cells of oral streptococci to a glass surface, organisms were grown at 37°C at a 300 angle for 18 h in a clean test tube (10 by 75 mm). Media (3.0 ml) containing no sucrose (tryptose phosphate broth, Difco), a trace amount of sucrose (Todd Hewitt broth, Difco), and 1% sucrose (Tryptose phosphate broth supplemented with 1% sucrose) were used to examine the effect of sucrose on the adherence of growing cells. After incubation, the tubes were gently rotated, and the detached cells were transferred to a second tube. After addition of 3 ml of potassium phosphate buffer (0.05 M, pH 6.8; KPB) to the side of the first tube, the tube was rotated again. The released cells were transferred to a third tube. The second and third tubes were centrifuged, and the supernatants were discarded to remove the brown culture media. Then KPB (3.0 ml) was added to all tubes, and the cells were suspended extensively with an ultrasonic oscillator. The percent adherence was determined by reading the optical density at 550 nm as described previously (11). Adherence of resting cells to a glass surface. In a 10- by 75-mm glass tube, 2.0 ml of cell suspension (0.5 mg [dry weight] per ml of KPB) and 0.4 ml of 5% sucrose in KPB were added, mixed well in a Vortex mixer, and incubated at 37°C at a 300 angle to the horizontal for 18 h. A single lot of lyophilized cells was used in this experiment. Merthiolate (0.01% final concentration) was included in the reaction mixture as a preservative. The percent adherence was determined as above. Assay of cell-associated, water-insoluble glucan synthesis. Cell-bound, water-insoluble glucan synthesis due to cell-associated glucosyltransferase (GTase) of whole cells of S. sanguis and S. mutans was assessed essentially as described previously (11). In brief, lyophilized cells (2.0 mg [dry weight]) suspended in 900 pJ of KPB containing 0.02% merthiolate were mixed with 100 Ld of ['4C]sucrose solution (110,000 dpm) which contained 0.05 ,uCi of [U-'4C]sucrose (New England Nuclear, Boston; specific activity, 396 mCi/mmol, 100 ,Ci/ml) and 12.5 umol of unlabeled sucrose (reagent grade). After incubation for 18 h at 37°C, the cells were washed twice with KPB (2.0 ml each) to remove free [14C]sucrose and its degradative products, and the radioactivity of the precipitated cells was counted in an Aloka liquid scintillation counter (model LSC-673; Aloka Ltd., Tokyo, Japan). Preparation of extracellular GTase. Extracellular GTase preparations were obtained from S. sanguis strains ST3, ST160, and ST205 which belonged to type IA (serotype/biotype), IIB, and IVA (12), and S. mutans B13 (serotype d). S. sanguis strains were grown in Todd-Hewitt broth at 37°C for 18 h, and the culture supernatants were precipitated by 50% saturated ammonium sulfate. The resulting precipitates were dissolved in water and dialyzed against excess KPB. The GTase activities of the preparations from strains ST3, ST160, and ST205 were 1.2, 0.9, and 4.5 dextransucrase units per mg of protein, respectively,
ADHERENCE OF S. SANGUIS CELLS
365
when assayed by the description of Koepsell and Tsuchiya (15). S. mutans B13 GTase was that employed in our previous study, and the enzyme activity was 4.1 dextransucrase units per mg of protein (11). Binding of extracellular GTase to streptococcal cells. Lyophilized cells (2 mg) of clinical isolates of S. sanguis suspended in KPB (2 ml) were mixed with 20 pd of B13 GTase. After standing for 10 min at 37°C, the cells were washed twice with KPB (2 ml each) to remove unbound GTase. Cell-associated, water-insoluble glucan synthesis due to bound GTase was assessed with [14C]sucrose as described above. "Background" glucan synthesis due to intrinsic cellassociated GTase of S. sanguis strains was also assayed with the cells which had not been treated with B13 GTase. The net increase in glucan synthesis was due to exogeneous GTase bound to the cell surface. The adherence of S. sanguis cells with or without GTase treatment to a glass surface was determined in the presence of sucrose, as described above. Binding of S. sanguis GTase (20 pi each) or S. mutans B13 GTase (10 ptl) to nontreated and heat-treated cells (2 mg) of S. mutans B13 was similarly assessed. Cellassociated, water-insoluble glucan synthesis from [14C]sucrose and cell adherence to a glass surface were measured (see above). Effect of various treatments on the binding of GTase to whole cells. Lyophilized cells (15 mg) of S. sanguis strains ST3, ST160, ST173, and ST205 were pretreated at 37°C for 60 min in 3 ml of the following agents: 0.2% dextranase AD17 (9) dissolved in acetate buffer (pH 5.8, 0.1 M); 0.2% crystalline trypsin in KPB (0.05 M, pH 7.1); 0.2% egg white lysozyme in KPB; 1 N NaOH; 1 N HCl; or 1% Brij 35. Cells (15 mg) were also heated in boiling water (3 ml) for 10 min. After treatment, the cell suspension was centrifuged and washed three times with KPB. The binding of GTase from S. mutans to the cells which had been treated enzymatically, chemically, or physically was assessed as described in the above sections. Percentage binding to the control without pretreatment was calculated in terms of cell-associated glucan synthesis and cell adherence to a glass surface. Scanning electron microscopic observation of streptococcal cells adherent to a glass surface. Samples of cell adherence of S. sanguis ST3 and S. mutans B13 with or without pretreatment by S. mutans B13 GTase to a cover glass were made for scanning electron microscopic observations as described above. These samples were fixed for 1 h in freshly prepared 2.5% glutaraldehyde in KPB (0.05 M, pH 7.0) at 0°C, washed in excess saline and water, and dried as previously described (9). The extensively dried surfaces of the samples were coated with gold to a thickness of ca. 10 nm, and the samples were observed and photographed in a scanning electron microscope (model MSM-4T, Akashi Works, Tokyo, Japan).
RESULTS Adherence of growing cells of oral streptococci. Growing cells of S. sanguis and other oral streptococcal species besides S. mutans did not signiflcantly adhere to a glass surface when cultured in broth media irrespective of their
366
HAMADA ET AL.
sucrose content (Table 1). S. mutans, however, produced marked adherence to a glass surface in a 1% sucrose-containing broth. Almost all of the growing cells of fresh S. mutans isolates belonging to a different serotype adhered to the glass surface in the sucrose broth. Trace amounts of sucrose (ca. 0.01 to 0.05%) in a commercial ToddHewitt broth (11) were found to be sufflcient to promote an increased adherence to glass (Table
1).
Cell-associated glucan synthesis by S. and cell adherence. The adherence of S. mutans cells to smooth surfaces has been shown to depend on de novo synthesis of cellassociated, water-insoluble glucan from sucrose. Thus, to relate glucan-producing ability and cellular adherence of S. sanguis, it is important to survey the ability of the bacteria to synthesize cell-associated, water-insoluble glucans with fresh clinical isolates of S. sanguis. As shown in Table 2, the glucan synthesis by lyophilized whole cells of clinical strains belonging to types IA and IIIA varied markedly, from less than 200 sanguis
INFECT. IMMUN. TABLE 2. Cell-associated, water-insoluble glucan synthesis by cells of clinically isolated strains of S. sanguisa Sero-
No. of strains examined
B
type
I II III IV
A B A A
22 15 37 15
% of strains synthesizing
glucan at (dpm): 1 \ >10,000 1' 00-
0
20.000 10.000 Glucan synthesized (dDm)
30.000
FIG. 1. Cell-associated, water-insoluble glucan synthesis by cells of clinical isolates of S. sanguis with or without pretreatment with extracellular GTase of S. mutans B13. Symbols: -, cell-associated glucan synthesis due to intrinsic cell-associated GTase of S. sanguis cells; c, cell-associated glucan synthesis after binding of S. mutans B13 GTase onto S. sanguis cells.
367
binding. When cells of S. sanguis strains were pretreated with dextranase, trypsin, or NaOH, subsequent binding of S. mutans GTase to the cells and sucrose-dependent adherence to a glass surface markedly decreased (Table 3). On the other hand, HCI or heat treatment of cells resulted in a decreased binding of GTase, but the cell adherence remained unchanged or rather increased (Table 3). Treatment with lysozyme or Brij 35 increased GTase binding and cell adherence. Morphology of S. sanguis cells adhered to a glass surface. Binding of S. mutans B13 GTase to S. sanguis cells resulted in not only an increased adherence to a glass surface (Fig. 2) but also a significant change in macroscopic appearance of the adhered S. sanguis cells in the presence of sucrose. Scanning electron microscopy revealed that cells of S. sanguis ST3 synthesized large quantities of gelatinous polysaccharide from sucrose. The cells were covered with amorphous material and embedded in the
Tvpe
IA
STrI68 P
1 J-! 3
185
_ !
____
173 !
209 _ == 154 _ 187 166 _ 177 _
144-
GTase and synthesized relatively large amounts Type II B === ST202 of cell-associated glucan. As described in the section above, S. sanguis 197 7_-= cells did not significantly adhere to a glass sur216 204 e face in the presence of sucrose by the enzymatic action of intrinsic cell-associated GTase. 'How- Type lIIA ever, the binding of S. mutans GTase to S. ST181 140I =W. sanguis cells resulted in marked increases in 183, 134 ! sucrose-dependent cell adherence (Fig. 2). Most -1 195!: = strains, irrespective of their serotype and bio186 ! 175 type, produced strong adherence in which more s 109 7777 than 60% of the applied cells adhered to glass 111_ (Fig. 2). Type IVA The effects of the concentration of S. mutans ST143 6 _ GTase on subsequent cell-associated glucan syn=~~~~~~~~~~~~~~~~~~~~~~~ 152 thesis and cell adherence were then investigated. 193 205 A rapid increase and gradual saturation of the 207 GTase binding to the cells of S. sanguis strains 7ST3, ST160, and ST205 were observed, whereas 0 100 50 the GTase binding to the cells of strains ST173, and ST204 was not significant when increased Adherence (%) quantities (0 to 50 pl) of the GTase were added FIG. 2. Adherence of resting cells of clinical iso(Fig. 3A). However, maximum cell adherence lates of S. sanguis to a glass surface in the presence was obtained in most strains when small quan- of exogenous sucrose. Symbols: -, cell adherence due tities (ca. 10 ,ul or less) of the GTase were added to intrinsic GTase activity of S. sanguis cells; o, cell to the reaction mixture (Fig. 3B). adherence of S. sanguis after binding of S. mutans Treatments of S. sanguis cells and GTase GTase onto S. sanguis cells. .1 Ao
-
368
HAMADA ET AL.
INFECT. IMMUN.
not so prominent as compared with that in nonheated cells. However, GTases from strains ST3 and ST205 moderately promoted adherence of heated cells of S. mutans B13. Under the same conditions, B13 GTase bound to heat-treated B13 cells and produced a marked adherence to a glass surface (Table 4).
b. 100
I-
av
:O
B13 GTase ( ul)
FIG. 3. Binding of S. mutans B13 GTase to the cells of S. sanguis strains and subsequent cell-associated water-insoluble glucan synthesis (a) and cellular adherence (b) in the presence of sucrose. Cells (2 mg, dry weight) were incubated with varying quantities of GTase in KPB and washed with KPB; then, ["4C]sucrose (a) and 1% sucrose (b) were added. After incubation at 37°C for 18 h, cell-associated [14C]glucan synthesis and percent cell adherence were quantitated as described in the text. Symbols: 0, ST3; A, ST160; E, ST173; 0, ST204; A, ST205; U, B13.
thick, voluminous accumulation of polysaccharide (Fig. 4A). However, if the S. sanguis cells were pretreated with S. mutans B13 GTase, individual organisms became clearly distinguishable, but adhered tightly to the glass surface (Fig. 4B). The shape of the organisms was very similar to that of S. mutans B13 cells (Fig. 4C). Binding of S. sanguis GTase to S. mutans B13 cells. As summarized in Table 4, extracellular GTase prepared from three strains of S. sanguis which belonged to different serotypes was bound to nonheated and heated cells of S. mutans B13. Nonheated B13 cells bound S. sanguis GTase, accompanying variable but significant increases in cell adherence to a glass surface in the presence of sucrose. B13 extracellular GTase also bound to the nonheated B13 cells, resulting in a further increase in the adherence ability. On the other hand, the binding of S. sanguis GTases to heat-treated B13 cells was
DISCUSSION S. sanguis have been known to possess a high affinity for enamel surfaces both in vivo and in vitro (2, 23, 24, 31). It has also been shown that S. sanguis and S. mitior attach to saliva-coated surfaces more effectively than do strains of S. mutans and S. salivarius (1, 5, 20, 25). Salivary glycoproteins are most likely to participate in the binding of S. sanguis to dental plaque or interbacterial aggregation, in which terminal sialic acids play a key role (19, 21). The wide variety of reactivity of S. sanguis may explain the earliest colonization of this species in teeth among various oral streptococci. However, no extensive survey on the sucrose-mediated adherence of S. sanguis cells to surfaces has been carried out. Both growing and resting cells of S. sanguis showed weak ability to adhere to glass surfaces in the presence of sucrose, whereas the adherence of those of S. mutans was markedly enhanced upon addition of sucrose (Table 1; Fig. 1 and 2). However, cell-associated GTase activity of S. sanguis cells was found to differ significantly from strain to strain. Some strains such as ST168 and ST181 synthesized large quantities of glucan due to intrinsic cell-associated GTase, but they did not show significant adherence to a glass surface in the presence of sucrose (Fig. 1 and 2). Therefore, these results indicate that cell-associated glucan synthesis by S. sanguis from sucrose does not necessarily result in significant cellular adherence to smooth surfaces. Related to this, certain noncariogenic mutants of serotype g S. mutans strains have been reported to possess properties similar to those of S. sanguis in terms of glucan synthesis and cell adherence to glass surfaces (7, 14, 16, 28). The chemical structure of glucans produced by S. sanguis should differ from that of S. mutans glucans in spite of the water insolubility of both glucans. It is of interest to note that glucans synthesized by S. sanguis cells are more voluminous than those of S. mutans, and the appearance of S. sanguis glucans is gel-like, reminiscent of soft agar (Fig. 4A). In our experiences, sucrose-grown cells of S. mutans which adhered tightly to a glass surface could be dispersed by extensive Vortex mixing or sonic oscillation, whereas those of S. sanguis which are easily detached from a glass surface tended to form a
ADHERENCE OF S. SANGUIS CELLS
VOL. 32, 1981
369
TABLE 3. Effect of various pretreatments of S. sanguis cells on the binding of S. mutans B13 GTase and subsequent cell adherence to a glass surface' ST3 Treatment of S. sanguis cells
Relative Relative GTase aherbmdmg binding eneM (^ ne()(%)
ST160 Relative GTase bmdmg binding ence (%)
ST173
Relative
alher-
ence
M
ST205
Relative Relative Relative GTase GTase adherbmdmg bidn mdmg(%) ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ence ~ ~ ~enc,,o ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ bidig ~ ~ ~ ~ ~(%) ~ ~ ~ ~ ~ ~ ~ idngi g
Relative eav Rlte adherec ec % ec
100 100 100 100 100 100 100 None 100 11 24 6 23 19 36 Dextranase (0.2%)b 3 23 11 23 5 20 10 17 1 22 Trypsin (0.2%) 72 91 10 54 17 NaOH (1.ON) 36 11 37 41 46 110 107 HCI (1.0N) 108 108 48 99 127 124 100 125 257 131 93 Brij 35 (1.0%) 78 107 136 201 151 Lysozyme (0.2%) 468 104 105 107 29 104 17 155 44 Heat (100°C, 10 min) 122 19 105 a S. sanguis cells were pretreated by various procedures followed by washing with KPB. The cell suspension was reconstituted, and the binding of S. mutans B13 GTase and cell adherence were assessed as described in the text. The rate of relative GTase binding and cell adherence is expressed as a percentage of that of nontreated control cells. b Final concentration.
lump of cell-glucan complex after Vortex mixing. This result suggests that sucrose-grown cells of S. sanguis are apt to form homotypic cell-to-cell aggregates through cell-associated, water-insoluble, gel-like glucans, whereas newly synthesized glucans from sucrose by S. mutans have a tendency to bind bacterial cells to various surfaces. Many strains of S. sanguis have been found to bind GTase from S. mutans, which resulted in markedly enhanced adherence to a glass surface and increased synthesis of variable amounts of cell-associated, water-insoluble glucans (Fig. 1 and 2). The glucan synthesized by S. mutans GTase bound to the cell surface of S. sanguis prescribed the adherence ability and cellular morphology of S. sanguis in the presence of sucrose, overcoming the effect of intrinsic GTase activity of S. sanguis (Fig. 4). Certain strains of S. sanguis have been known to produce heterotypic cell-to-cell aggregates with many strains of Actinomyces species (4). These properties of S. sanguis may contribute to growth or enlargement of dental plaque mass, although S. sanguis per se possess weak adherence ability in general. In this connection, it was reported by other investigators (18) that the adsorption of extracellular GTase from S. mutans strain GS5 (serotype c) to the cells of S. sanguis ST3, which we had provided them, resulted in low levels of adherence to a glass surface in the presence of sucrose. This finding is contradictory to our results (Fig. 2) (8). The discrepancy may be ascribed to the differences in the properties of S. mutans GTases, variations in the assay systems for cellular adherence to glass surfaces, or changes in surface structures of S. sanguis cells during laboratory transfers. The salivary concentrations of S. mutans and
S. sanguis are important determinants in the colonization of smooth surfaces (31). S. sanguis appears to be concentrated from saliva onto tooth surfaces (1, 5). It has been reported that S. sanguis averaged about 106 colony-forming units per ml in adults (27). However, Carlsson et al. (2, 3) reported that S. sanguis as well as S. mutans could not be recovered from the mouths of infants before tooth eruption or from edentulous persons without dentures. Therefore, solid surfaces such as teeth and dentures appear to favor the colonization of S. sanguis and S. mutans. Furthermore, it is expected from the results presented in Fig. 2 that the interaction of S. sanguis with extracellular GTase from S. mutans would result in an enhanced cellular adherence to tooth surfaces. However, these findings do not readily support the significance of S. mutans GTase for S. sanguis in vivo, because when sucrose ingestion increased in humans and monkeys, the proportions of S. mutans increased, whereas those of S. sanguis decreased (6, 13, 26). Both streptococcal species might benefit from glucan synthesis, but S. mutans more so. Therefore, although proportions of S. sanguis could decrease as compared with those of S. mutans, its actual number on the tooth surface could be increased. But direct data have yet to be obtained. The GTase binding site(s) of S. sanguis appears to be glucan or protein entities or both located on the surface layer of the cells, based on the results shown in Table 4. In S. mutans, surface glucan on the cells was considered to be the most important component in the binding of GTase and subsequent cellular adherence to smooth surfaces (11). Pretreatment of S. mutans cells with a protease resulted in an almost com-
i. 5pl
P:-.
A A
s~~~~~~~~~A
FIG. 4. Adherence of S. sanguis cells on the glass surface in the presence of sucrose: scanning electron micrograph. (A) S. sanguis ST3 cells; (B) S. sanguis ST3 cells pretreated with S. mutans B13 GTase; (C) S. mutans B13 cells.
370
VOL. 32, 1981
ADHERENCE OF S. SANGUIS CELLS
A'7
.4
.""
J.-
TABLE 4. Binding of S. sanguis GTase to S. mutans B13 cells and subsequent cell-associated glucan synthesis and adherence to a glass surface a Nonheated cells
GTase from:
S. sanguis ST3 ST160 ST205 S. mutans B13
Glucan
371
Heat-treated cells Glucan
synthesized (dpm)
Adher ence
synthe-
(%)
sized (dpm)
Adher erence (%)
21,630 9,835 26,652
81 70 62
3,204 1,505 4,692
55 10 43
5,652
81
4,616
84
-
sanguis cells (Table 4), whereas no significant effect was found by the treatments of S. mutans cells with HCl or heat (100°C, 10 min) (11). These results indicate that the GTase binding entity of S. sanguis cells is different from that of S. mutans cells; however, it is not clearly elucidated at this point. LITERATURE CITED
611 7 53 None 2,262 a S. mutans B13 cells (2 mg [dry weight]) were treated with GTase from S. sanguis (20 p1l) or S. mutans B13 (10 pl). After removing unbound GTase by centrifugation, cell-associated, water-insoluble glucan synthesis and cellular adherence were quantitated as described in the text.
plete loss of subsequent sucrose-dependent adherence in spite of sufficient binding of exogenous GTase (11). In S. sanguis, however, binding ability of S. mutans GTase onto cells was destroyed by trypsin treatment. HCl and heat treatments of S. sanguis cells also diminished the binding ability of S. mutans GTase to S.
1. Appelbaum, B., E. Golub, S. C. Holt, and B. Rosan. 1979. In vitro studies of dental plaque formation: ad-. sorption of oral streptococci to hydroxyapatite. Infect. Immun. 25:717-728. 2. Carlsson, J., H. Grahnen, G. Jonsson, and S. Wikner. 1970. Establishment of Streptococcus sanguis in the mouths of infants. Arch. Oral Biol. 15:1143-1148. 3. Carlsson, J., G. Soderholm, and L. Almfeldt. 1969. Prevalence of Streptococcus sanguis and Streptococcus mutans in the mouth of persons wearing full-dentures. Arch. Oral Biol. 14:243-249. 4. Cisar, J. O., P. E. Kolenbrander, and F. C. McIntire. 1979. Specificity of coaggregation reactions between human oral streptococci and strains of Actinomyces viscosus orActinomyces naeslundii. Infect. Immun. 24: 742-752. 5. Clark, W. B., L. L. Bammann, and R. J. Gibbons. 1978. Comparative estimates of bacterial affinities and adsorption sites on hydroxyapatite surfaces. Infect. Immun. 19:846-853. 6. de Stoppelaar, J. D., J. van Houte, and 0. Backer Dirks. 1970. The effect of carbohydrate restriction on
372
7.
8.
9.
10.
11.
12.
13. 14.
15. 16.
17.
18.
19.
HAMADA ET AL.
the presence of Streptococcus mutans, Streptococcus sanguis and iodophilic polysaccharide-producing bacteria in human dental plaque. Caries Res. 4:114-123. Freedman, M. L., and J. M. Tanzer. 1974. Dissociation of plaque formation from glucan-induced agglutination in mutants of Streptococcus mutans. Infect. Immun. 10:189-196. Hamada, S., Y. Kobayashi, and H. D. Slade. 1978. Cell-bound glucan synthesis and subsequent adherence of oral streptococci due to the binding of extracellular glucosyltransferase to the streptococcal cell surface. Microbiol. Immunol. 22:279-282. Hamada, S., J. Mizuno, Y. Murayama, T. Ooshima, N. Masuda, and S. Sobue. 1975. Effect of dextranase on the extracellular polysaccharide synthesis of Streptococcus mutans: chemical and scanning electron microscopy studies. Infect. Immun. 12:1415-1425. Hamada, S., and H. D. Slade. 1980. Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiol. Rev. 44:331-384. Hamada, S., and M. Torii. 1978. Effect of sucrose in culture media on the location of glucosyltransferase of Streptococcus mutans and cell adherence to glass surfaces. Infect. Immun. 20:592-599. Hamada, S., M. Torii, Y. Tsuchitani, and S. Kotani. 1980. Isolation and immunobiological classification of Streptococcus sanguis from human tooth surfaces. J. Clin. Microbiol. 12:243-249. Kilian, M., and G. RIola. 1976. Initial colonization of teeth in monkeys as related to diet. Infect. Immun. 14: 1022-1027. Kishimoto, E., T. Koga, and M. Inoue. 1979. Cariogenic virulence of Streptococcus mutans AHT mutants with altered in vitro adherence abllities in hamsters. Microbiol. Immunol. 23:849-857. Koepsell, H. J., and H. M. Tsuchiya. 1952. Enzymatic synthesis of dextran. J. Bacteriol. 63:293-295. Koga, T., and M. Inoue. 1978. Cellular adherence, glucosyltransferase adsorption, and glucan synthesis of Streptococcus mutans AHT mutants. Infect. Immun. 19:402-410. Krasse, B., and J. Carlsson. 1970. Various types of streptococci and experimental caries in hamsters. Arch. Oral Biol. 15:25-32. Kuramitsu, H., and L. Ingersoll. 1977. Molecular basis for the different sucrose-dependent adherence properties of Streptococcus mutans and Streptococcus sanguis. Infect. Immun. 17:330-337. Levine, M. J., M. C. Herzberg, M. S. Levine, S. A.
INFECT. IMMUN.
20.
21.
22. 23. 24.
25.
26.
27.
28.
29.
30.
31.
Ellison, M. W. Stinson, H. C. iA, and T. van Dyke. 1978. Specificity of salivary-bacterial interactions: Role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans. Infect. Immun. 19:107-115. Liljemark, W. F., S. V. Schauer, and C. G. Bloomquist. 1978. Compounds which affect the adherence of Streptococcus sanguis and Streptococcus mutans to hydroxyapatite. J. Dent. Res. 57:373-379. McBride, B. C., and M. T. Gisslow. 1977. Role of sialic acid in saliva-induced aggregation of Streptococcus sanguis. Infect. Immun. 18:35-40. Mejare, B., and S. Edwardsson. 1975. Streptococcus milleri (Guthof); an indigenous organism of the human oral cavity. Arch. Oral Biol. 20:757-762. Olsson, J., and B. Krasse. 1976. A method for studying adherence of oral streptococci to solid surfaces. Scand. J. Dent. Res. 84:20-28. 0rstavik, D., F. W. Kraus, and L. Carolyn Henshaw. 1974. In vitro attachment of streptococci to the tooth surface. Infect. Immun. 9:794-800. R6nstrom, A., S. Edwardsson, and R. Attstrom. 1977. Streptococcus sanguis and Streptococcus salivalius in early plaque formation on plastic films. J. Periodont. Res. 12:331-339. Staat, R. H., T. H. Gawronski, D. E. Cressey, R. S. Harris, and L. E. A. Folke. 1975. Effects of dietary sucrose levels on the quantity and microbial composition of human dental plaque. J. Dent. Res. 54:872-880. Svanberg, M., and W. J. Loesche. 1977. The salivary concentration of Streptococci mutans and Streptococci sanguis and their colonization of artificial tooth fissures in man. Arch. Oral Biol. 22:441-447. Tanzer, J. M., M. L. Freedman, R. J. Fitzgerald, and R. H. Larson. 1974. Diminished virulence of glucan synthesis-defective mutants of Streptococcus mutans. Infect. Immun. 10:197-203. Van Houte, J., R. J. Gibbons, and S. B. Banghart. 1970. Adherence as a determinant of the presence of Streptococcus salivarius and Streptococcus sanguis on the human tooth surface. Arch. Oral Biol. 15:1025-1034. Van Houte, J., R. J. Gibbons, and A. J. Pulkkinen. 1971. Adherence as an ecological determinant for streptococci in the human mouth. Arch. Oral Biol. 16:11311141. Van Houte, J., and D. B. Green. 1974. Relationship between the concentration of bacteria in saliva and the colonization of teeth in humans. Infect. Immun. 9:624630.
