rodents are increased if diets containing large found in mixed dental plaque, though total elimination

Transcription

1 INFECTION AND IMMUNITY, Nov. 1977, p Copyright ) 1977 American Society for Microbiology Vol. 18, No. 2 Printed in U.S.A. Influence of Salivary Components and Extracellular Polysaccharide Synthesis from Sucrose on the Attachment of Streptococcus mutans 6715 to Hydroxyapatite Surfaces W. B. CLARK AND R. J. GIBBONS* Forsyth Dental Center, Harvard School of Dental Medicine, Boston, Massachusetts Received for publication 11 April 1977 The adsorption of 3H-labeled Streptococcus mutans 6715 cells to disks of hydroxyapatite (HA) was studied. The number of streptococci that adsorbed was logarithmically related to the concentration of cells available up to at least 2 x 108 per ml; equilibrium occurred within 45 min. Assay reliability was verified by direct scanning electron microscopic counts. Untreated HA disks exposed to buffered saline (PBS)-suspended streptococci at a concentration of 1.1 x 108 per ml absorbed 3.2 x 106 cells per cm2; approximately 3% of the surface area was, therefore, occupied by adsorbed organisms. The presence of adsorbed salivary components on HA reduced the number of attaching S. mutans cells by half. When S. mutans cells were suspended in saliva to mimic conditions existing in the mouth, the number of streptococci adsorbing to saliva-treated HA was reduced more than 30-fold compared to untreated HA. Approximately one-half of the streptococci adsorbed to untreated or to saliva-treated HA disks could be desorbed over a 4-h period with M phosphate buffer. S. mutans cells exposed to sucrose to permit extracellular polysaccharide synthesis before or during adsorption attached in fewer numbers to both saliva-treated and untreated HA than PBS-treated organisms. When S. mutans cells adsorbed on untreated HA were exposed to sucrose, fewer organisms could be desorbed; thus, in situ polysaccharide synthesis promoted their more firm attachment to untreated HA. However, when saliva-suspended streptococci were adsorbed to saliva-treated HA surfaces, exposure to sucrose before or subsequent to adsorption did not promote more firm attachment. Evidently, the powerful adherence-inhibiting and desorptive effects of salivary components overshadowed any promoting effects attributable to glucan synthesis from sucrose. Similarly, no differences were noted in the desorption of S. mutans cells from human teeth after exposure to sucrose, glucose, or PBS relative to a strain of Streptococcus mitis (S. mitior). Thus, no evidence was obtained to support the hypothesis that glucan synthesis from sucrose was essential for, or promoted, the attachment of S. mutans cells to HA surfaces exposed to saliva or to the smooth surfaces of human teeth. Extracellular glucans synthesized from sucrose are known to be involved in dental plaque formation by Streptococcus mutans (for recent reviews, see references 13, 15). These polysaccharides are also associated with the ability of S. mutans to form adherent deposits on solid surfaces in vitro. For dental plaque formation to occur, bacterial cells must first attach to the tooth surface, and then, while proliferating, the organisms must adhere to each other to resist removal by oral cleansing forces. Bacterial accumulations many micrometers in thickness thereby develop (13). Several observations suggest that extracellular glucans synthesized from sucrose are involved in and promote the accumulation of S. mutans cells on teeth under natural conditions. For example, the proportions 514 and the numbers of S. mutans cells that can be recovered from the teeth of both humans and rodents are increased if diets containing large quantities of sucrose are consumed (3, 39). Substituting glucose for sucrose in the diet results in a reduction in the proportions of S. mutans found in mixed dental plaque, though total elimination of the organism does not occur (3, 39). That glucans can promote cell-to-cell adhesion, thereby facilitating accumulation of S. mutans cells, is further supported by the finding that high-molecular-weight glucans induce aggregation of washed S. mutans cells (9). The surface of this organism appears to contain at least two types of receptors that specifically bind glucan molecules, and these receptors probably account for the ability of S. mutans cells to be held

2 Voi 18, 1977 together by glucans, allowing accumulation (6, 34) İn in vitro models, strains of S. mutans have been observed to form large adherent deposits on surfaces of glass, wire, and extracted teeth incubated in broth cultures containing sucrose but not glucose (7, 23). Thick deposits also form on glass when heat-killed S. mutans cells are incubated with active glucosyltransferase preparations and sucrose (26, 27). These observations have been interpreted as suggesting that glucan synthesis from sucrose played an important role in natural attachment of S. mutans cells to teeth. However, these in vitro models based upon the formation of thick streptococcal deposits cannot necessarily distinguish between adherent interactions involved in cell attachment to the surface and those required for cell-to-cell accumulation. Early studies by Krasse showed that dietary sucrose increased the extent of implantation of S. mutans on teeth of humans and hamsters (19, 20); this has also been interpreted as suggesting that glucan synthesis from sucrose either enhanced or was required for the initial attachment of this organism to teeth. However, recent studies by van Houte et al. (35) have shown that all of 11 strains of S. mutans, representative of the five serogroups delineated by Bratthall (1), could establish and persistently colonize teeth of rats fed diets with no detectable sucrose. In addition, the minimum infective dose for 9 of 11 strains studied was similar in rats fed diets containing either glucose or sucrose (35). Implantation of the two divergent strains was enhanced by sucrose, though these strains clearly could colonize in its absence (39). Colonization in these experiments mainly occurred in occlusal fissures (35, 39). These observations suggest that S. mutans cells can attach to at least some areas of rodent teeth firmly enough to promote persistent colonization in the absence of significant glucan synthesis for sucrose. Under natural conditions, teeth are covered by a membranous film, termed the acquired pellicle, which is thought to consist of selectively adsorbed salivary components (13). The initial attachment of S. mutans cells to teeth presumably involves adhesive interactions between surface molecules of the bacterium and constituents of the acquired salivary pellicle (13, 15). The present investigation studied effects of salivary components on attachment of S. mutans cells to hydroxyapatite (HA) surfaces and assessed the role of glucan synthesis from sucrose on this process. MATERIALS AND METHODS Cultures and cultural conditions. S. mutans 6715 and Streptococcus mitis (S. mitior) 26R were ATTACHMENT OF S. MUTANS TO HYDROXYAPATITE 515 maintained by weekly transfer in Trypticase soy broth (BBL) or on Trypticase soy (BBL) blood agar plates. Tritium-labeled cells of S. mutans 6715 were prepared by growing the organism in Trypticase broth (9) containing a solution of 0.2% glucose, 0.01% sucrose (TGB+S), and 10,uCi of [ 3H]thymidine per ml (New England Nuclear). The glucose and sucrose were autoclaved separately and added to complete the medium. S. mutans 6715 cells cultured in this medium possess cell-bound glucosyltransferase activity (34). Cells from overnight cultures were harvested by centrifugation, washed twice, and suspended in saline at a concentration of 1.1 x 108 cells per ml. Samples of such suspensions, which had specific activities of 4 x 105 to 6 x 105 cpm/108 streptococci, were lyophilized and stored under vacuum. Preliminary experiments indicated that when such lyophilized streptococci were suspended in 0.01 M phosphate-buffered saline (PBS) at ph 7.0, less than 1% of the radioactivity of the suspension passed through 0.45-,im membrane filters, and only negligible quantities of this soluble material adsorbed to HA. To remove this small quantity of soluble 3H-labeled material, lyophilized cells of S. mutans were suspended in 10 ml of PBS and washed once by centrifugation before use. The streptococci were then suspended in either PBS, clarified whole saliva (CWS), or in an ultrafiltrate of saliva at a concentration of 1.1 x 108 cells per ml. Clumps of cells and long chains were broken up by passing the suspensions through a syringe with a 21-gauge needle 15 to 20 times. Microscopic examination of the suspensions indicated that most cells were single or paired; chains containing more than three cells were sparse. Preparation of saliva. Whole paraffin-stimulated saliva from one adult donor, age 28, was collected in a container chilled over ice. The saliva was clarified by centrifugation at 12,000 x g for 10 min and heated at 60 C for 30 min to inactivate degradative enzymes (12, 16). Salivary ultrafiltrate was prepared by first centrifuging clarified, heat-inactivated saliva for 2 h at 110,000 x g to remove mucinous components; then the supernatant was filtered through membranes with a molecular exclusion limit of <1,000 (Diaflo Filter Corp.) before use as a suspending fluid. Preparation and treatment of HA disks. HA disks, 1 cm in diameter, were prepared by compressing 150 mg of HA powder (Monsanto Chemicals) for 15 min at 8,000 lb of pressure (ca. 3,558.4 N). Disk surfaces appeared smooth and shiny, suggesting their intact nature. Each disk was equilibrated with PBS overnight before use. In some experiments, equilibrated HA disks were exposed to 2 ml of CWS or to human serum for 1 to 24 h at room temperature to form a film of selectively adsorbed components. Disks exposed to PBS served as controls. Sodium azide was added to the CWS, serum, or PBS at a final concentration of 0.04% to inhibit microbial growth during HA pretreatment periods. Disks were then rinsed, before use, in PBS. Bacterial adsorption to HA disks. Equilibrated HA disks were suspended vertically, using stainlesssteel wire holders in 10 ml of 3H-labeled S. mutans 6715 cells suspended in PBS or in CWS at 37 C for 90 min, unless otherwise noted. Bacterial suspensions were stirred continuously throughout the incubation period by a small magnetic stirrer. After incubation,

3 516 CLARK AND GIBBONS the disks were rinsed by immersion in a large volume of saline to remove unattached streptococcal cells. The counts per minute of cells adsorbed to disks was determined by scintillation counting with a Packard Tri-Carb scintillation counter. Known numbers of 3Hlabeled streptococci contained in 10-,ul samples were placed on HA disks and counted in a similar manner. The number of streptococci adsorbed to experimental disks was calculated from these values. Assay reliability was ascertained by comparing the number of cells that adsorbed to duplicate HA disks, as determined by scintillation counting, with the number of cells counted by direct microscopic examination of disks using scanning electron photomicrographs of known magnification. Desorption of S. mutans from HA. Studies of streptococcal desorption were performed with appropriately treated HA disks that were exposed to 3Hlabeled S. mutans 6715 cells for 10 min. The disks were rinsed, and desorption of organisms was accomplished by incubating disks in 6 ml of either M phosphate buffer (ph 7.0) or CWS (ph 7.0). Desorption fluid was changed hourly for 4 successive h. Cells desorbed after each 1-h period were collected by filtering the desorption fluid through 0.45-t2m membrane filters (Millipore Corp., Bedford, Mass.). The filters were then counted for radioactivity. Known numbers of 3H-labeled cells collected on filters were counted in a similar manner, so that counts per minute could be related to the number of streptococci. The number of cells remaining adsorbed to HA disks was determined as described above. Effect of extracellular polysaccharide synthesis on adsorption and desorption. To determine the influence of previous extracellular polysaccharide synthesis on adsorption and desorption of S. mutans 6715 cells, washed streptococcal suspensions were incubated with 0.05 M sucrose in PBS for 30 min at 37 C. The organisms were centrifuged and suspended in PBS or CWS; aggregated streptococci were dispersed by rapid and repeated passage through a 21- gauge syringe needle. The suspensions were then incubated with untreated or with CWS-treated HA disks as described above. The effect of polysaccharide synthesis by previously adsorbed S. mutans cells was also studied. Streptococci were adsorbed to untreated or to saliva-treated HA disks, and the disks were rinsed to remove unattached cells. They were then incubated in either PBS or CWS containing 0.05 M sucrose or 0.05 M glucose for 30 min at 37 C. Disks incubated in CWS or PBS alone served as controls. After incubation, the streptococci were desorbed with either M phosphate buffer or CWS as described. To confirm synthesis of polymeric material by adsorbed S. mutans cells exposed to sucrose, HA disks were examined by scanning electron microscopy as previously described (8). In vivo experiments. To determine if extracellular polysaccharide synthesis from sucrose promoted firmer attachment of S. mutans 6715 cells on human INFECT. IMMUN. teeth in vivo, desorption of this organism was compared to S. mitis (S. mitior) 26, a strain that does not synthesize extracellular glucans (21). A streptomycinresistant mutant of strain 26 was isolated as previously described (37). S. mutans 6715 was already streptomycin resistant. Both streptococci were cultured in TGB+S overnight. The organisms were harvested by centrifugation, washed twice, and suspended in saline containing 1% Trypticase (BBL) at a concentration of 2.6 x 108 cells per ml. Equal volumes of each cell suspension were mixed together, and dilutions of the mixture were plated in duplicate on mitis salivarius agar (Difco) plates containing 200,ug of streptomycin per ml. These were incubated in an atmosphere of 80% N2, 10% H2, and 10% CO2 for 48 h at 35 C. The actual proportions of colony-forming units of each species in the mixture was determined by their distinctive colonial morphologies. The six maxillary anterior teeth of three adult volunteers were cleaned by careful tooth brushing, and 1 ml of the streptococcal mixture was placed into the mouth of each subject. After 5 min, the mixture was expectorated, and the subjects thoroughly rinsed their mouths with water. The buccal surfaces of three individual teeth on the right side were sampled by forceful rubbing with Calgiswabs 15 min later (37, 38). Immediately after base line samples were taken, each subject rinsed five times with a total of 25 ml of either saline, 25% glucose, or 25% sucrose periodically over a 30-min period. The three anterior teeth on the left side were sampled with Calgiswabs (Inolex Corp., Glenwood, Ill.) after 4 h. All swab samples were immediately placed in 2 ml of saline containing 1% Trypticase, and the streptococci were dispersed for 1 min by a Vortex mixer. Dilutions of the resulting suspensions were plated in duplicate on mitis salivarius agar (Difco) plates containing streptomycin as described above. The relative proportions of colony-forming units of S. mutans to S. mitis (S. mitior) recovered from teeth were multiplied by the reciprocal of their proportions in the mixture introduced into the mouth to reflect equal opportunity for attachment (21, 37). RESULTS Adsorption assay. The reliability of enumerating 3H-labeled streptococci on HA disks by scintillation counting was verified by direct scanning electron microscopic counting. With duplicate HA disks, the number of adsorbed cells determined by direct microscopic count of 20 random fields on each disk was 9.1 ± 0.4 x 105 per cm2, whereas the number of adsorbed streptococci on duplicate HA disks estimated by radioactive assay was 8.9 ± 0.1 x 105 per cm2. The small standard errors observed in the radioactive assay indicate its reproducibility; they further suggest the reproducible nature of these disks. At an initial concentration of 1.1 x 108 cells per ml, the number of S. mutans 6715 cells adsorbing to uncoated HA disks increased with time up to 45 min, and equilibrium appeared to be reached thereafter (Fig. 1). The log of the number of streptococci that became adsorbed was directly related to the concentration of cells available up to at least 2.24 x 10' per ml at

4 Voi,. 18, x 0-. o 0 6n a /6 x MINUTES INCUBATION FIG. 1. Adsorption of PBS-suspended S. mutans 6715 cells to untreated HA disks over time. conditions of equilibrium (Fig. 2). Thus, the radioactive assay seems a reliable and sensitive procedure for studying adsorption of S. mutans to HA surfaces. Influence of saliva on initial sorption. Untreated HA disks exposed to PBS suspensions of S. mutans 6715 at a concentration of 1.1 x 108 per ml adsorbed 3.18 x 10' streptococci per cm). Prior treatment of HA disks with saliva for 1 or 24 h to form a film of selectively adsorbed salivary components reduced the number of S. mutans 6715 cells that adsorbed (Table 1). Precoating HA disks with human serum for 24 h also reduced streptococcal adsorption (Table 1). When CWS was used as a suspending fluid, the number of S. mutans 6715 cells adsorbing to HA disks treated with saliva for 24 h was reduced by approximately 15-fold compared with organisms suspended in PBS (Table 1). TABLE 1. ATTACHMENT OF S. MUTANS TO HYDROXYAPATITE This marked inhibition of streptococcal adsorption was apparently due to high-molecularweight components of saliva, for when the organisms were suspended in a salivary ultrafiltrate consisting of low-molecular-weight components (<1,000), including unbound salivary cations and anions, the number of cells that adsorbed was similar to the number adsorbed when organisms were suspended in PBS (Table 1). S. mutans cells were also preincubated in CWS for 1 h and then washed twice and suspended in PBS for assay. Fewer such salivatreated streptococci adsorbed to either un- o 0 co 0 0 o 0L 6.60r I 2 3 NO. STREPTOCOCCI AVAI LABLE/ML x 10-8 FIG. 2. Relation between concentration of streptococci available and number that adsorb to untreated HA disks at equilibrium. Effect of salivary components on adsorption of S. mutans 6715 to HA disks Total no. cells -± SEa Adsorption Prior treatment of Suspension fluid Prior HA treatment (h) adsorbedper +- S relative to streptococci eor - untreated HA (%) None PBS PBS 3.18 ± None PBS Saliva (1) 2.43 ± None PBS Saliva (24) 1.51 ± None PBS Serum (24) 0.67 ± None Saliva Saliva (24) 0.11 ± None Saliva-ultrafiltrate Saliva (24) 1.79 ± None Saliva Serum (24) 0.18 ± CWS PBS PBS 2.64 ± CWS PBS Saliva (24) 0.80 ± None 0.05 M sucrose in PBS PBS 1.81 ± None 0.05 M sucrose in PBS Saliva (1) 0.16 ± M sucrose PBS PBS 1.32 ± M sucrose PBS Saliva (24) 1.33 ± M sucrose Saliva Saliva (24) 0.13 ± a SE, Standard error of the mean. 517

5 518 CLARK AND GIBBONS treated or saliva-treated HA disks compared to untreated organisms (Table 1). This finding suggests that adsorption-inhibiting salivary components became firmly bound to the streptococcal cell surface and could not be removed by washing. The binding of salivary components to S. mutans cells has been previously noted (5, 11, 12, 30). Desorption of S. mutans. Nearly half of PBS-suspended streptococci that adsorbed to untreated HA disks could be desorbed over a 4- h period with M phosphate buffer (Table 2), suggesting they were reversibly attached. Increasing the molarity of phosphate buffer to 1 M did not appreciably increase the percentage of cells that could be removed, nor did extending the desorption period to 8 h. Thus, a proportion of S. mutans 6715 cells apparently can bind tenaciously to HA or saliva-treated HA without synthesizing extracellular glucans from sucrose. These observations raised the possibility that HA may contain two types of binding sites for S. mutans 6715; alternatively, there could be different populations of S. mutans cells present. However, the percentage of cells that could be desorbed from HA surfaces exposed to streptococcal concentrations over a 50-fold range did not differ significantly (data not shown). Hence, the HA surfaces probably do not contain a limited number of sites that bind S. mutans cells more firmly than other sites. In addition, S. mutans cells desorbed from HA surfaces were found to readsorb to HA comparably to cells that had not been previously adsorbed; the proportion of such cells capable of subsequent desorbtion was also similar. Thus, there does not appear to be two populations of S. mutans cells in the suspensions differing in their adsorptive properties to HA. Similar percentages of streptococci were also desorbed from disks exposed to streptococcal suspensions for 5 and for 45 min. This similarity suggests that the strength of streptococcal adsorption bonds to untreated HA did not appreciably change over this time. Evidently, a fixed proportion of S. mutans cells INFECT. IMMIJN. adsorbing to untreated HA become firmly attached and resist desorption. The percentage of S. mutans cells that could be desorbed with M phosphate buffer from HA disks treated with saliva for 1 or 24 h was generally similar to the percentage desorbed from untreated HA disks (Table 2). However, substantially more cells were desorbed from HA disks that had been pretreated with whole human serum for 24 h. When saliva was used as a desorptive fluid in place of phosphate buffer, almost 80% of the streptococci became desorbed from saliva-treated HA disks (Table 2). A similar percentage of cells was desorbed when salivasuspended streptococci were adsorbed onto saliva-treated HA (Table 2), even though markedly fewer cells attached because of the inhibition caused by use of saliva as a suspension fluid (see Table 1). Thus, components of saliva not only strongly inhibited the adsorption of S. mutans 6715 cells to saliva-treated HA disks, but they also effectively fostered their desorption. Influence of extracellular polysaccharide synthesis on adsorption. Addition of 0.05 M sucrose to PBS-suspended S. mutans 6715 resulted in obvious aggregation of the organisms, and significantly fewer streptococci attached to both untreated and saliva-treated HA (Table 1). Similarly, S. mutans 6715 exposed to sucrose, to permit extracellular polysaccharide synthesis, and subsequently washed and dispersed also tended to adsorb in fewer numbers than untreated organisms (Table 1). Use of saliva as a suspending fluid also strongly inhibited adsorption of such cells to saliva-treated HA. These observations, therefore, indicate that synthesis of extracellular polysaccharides from sucrose by S. mutans cells, before or during adsorption, does not promote their attachment to HA or to saliva-treated HA surfaces. Influence of extracellular polysaccharide synthesis on desorption. S. mutans cells exposed to sucrose before or during adsorption to untreated HA were less firmly bound than PBStreated streptococci, as judged by the percentage TABLE 2. Effect of salivary components on desorption of S. mutans 6715 from HA disks Streptococci suspen- Prior treatment of HA (h) Desorption fluid Cells ± SE" Desorbed sion fluid i PBS None Phosphate buffer 45 ± 3 PBS Saliva (1) Phosphate buffer 43 ± 2 PBS Saliva (24) Phosphate buffer 56 ± 4 PBS Serum (24) Phosphate buffer 75 ± 1 PBS Saliva (24) Saliva 79 ± 3 Saliva Saliva (24) Phosphate buffer 70 ± 2 Saliva Saliva (24) Saliva 71 ± 1 Saliva Serum (24) Saliva 70 ± 1 SE, Standard error of the mean.

6 VOL. 18, 1977 of organisms that could be desorbed with phosphate buffer (Table 3). Similarly, streptococci exposed to glucose after adsorption on HA disks were desorbed to an extent comparable to organisms with PBS exposure. However, when previously adsorbed S. mutans 6715 cells were incubated with 0.05 M sucrose, an increased percentage of organisms resisted desorption (Table 3). Synthesis of extracellular polymeric material by such organisms was confirmed by scanning electron microscopy (Fig. 3A and B). Thus, TABLE 3. Effect of extracellular polysaccharide synthesis from sucrose on desorption of S. mutans 6715 from untreated HA by phosphate buffera Cells desorbed Streptococci exposed to: + SEb in 4 h (%) 0.05 M sucrose during adsorption 76 ± 2 PBS before adsorption ± M sucrose before adsorption ± 6 PBS after adsorption ± M glucose after adsorption 47 ± M sucrose after adsorption ± 3 " Cells were adsorbed onto HA disks from PBS suspension. ' SE, Standard error of the mean. ATTACHMENT OF S. MUTANS TO HYDROXYAPATITE 519 synthesis of extracellular polysaccharides from sucrose in situ by adsorbed S. mutans 6715 cells appeared to promote their more firm attachment to untreated HA surfaces. In contrast to the enhancing effect of sucrose noted above, exposure of the organisms to sucrose either before or after streptococcal adsorption did not reduce the percentage of cells that could be desorbed from saliva-treated HA disks when CWS was used as the desorption fluid (Table 4). Evidently, the potent desorptive effects of free salivary components on salivatreated HA surfaces overshadowed any adherence-promoting effect of polymer synthesis. To rule out the possibility that antibodies or other substances in the saliva might have completely inhibited polysaccharide synthesis, CWS- and PBS-suspended S. mutans 6715 cells were incubated alone and with 0.5 M sucrose for 30 min. Marked streptococcal agglutination was observed in suspensions exposed to sucrose, which is indicative of extracellular glucan synthesis (9), whereas little or no agglutination occurred in control suspensions. Influence of sucrose, glucose, or saline rinses on desorption of S. mutans 6715 rel- Downloaded from on November 12, 2018 by guest - lmrsa%3;1 e.p, +. sj*>7,s, *n '. FIG. 3. Scanning electron photomicrograph of S. mutans 6715 cells adsorbed to untreated HA disks. (A) Adsorbed streptococci were exposed to PBS for 30 min. (B) Adsorbed streptococci were exposed to 0.05M sucrose for 30 min. Note the large quantities of extracellular polysaccharide synthesized. Bars, I Ian.

7 520 CLARK AND GIBBONS TABI,E 4. Effect of extracellular polysaccharide synthesis from sucrose on desorption of S. mutans 6715 cells from saliva-treated HA by saliva"a Cells desorbed Streptococci exposed to: ± SE in 4 h (%) 0.05 M sucrose before adsorption 71 ± 4 Saliva after adsorption 71 ± M glucose after adsorption 79 ± M sucrose after adsorption 72 ± 2 a Cells were adsorbed onto 24-h saliva-treated HA disks from saliva suspensions. 'SE, Standard error of the mean. ative to S. mitis (S. mitior) 26R from human teeth. The apparent ineffectiveness of sucrose in promoting firmer attachment of S. mutans cells to saliva-treated HA surfaces was further studied in human mouths. No appreciable differences were noted in clearance of S. mutans 6715 from teeth of individuals who had rinsed with either 25% sucrose, 25% glucose, or saline, as judged by the ratio of S. mutans cells to cells of S. mitis (S. mitior) 26R recovered after 4 h (Table 5). Thus, exposure of S. mutans 6715 cells to sucrose did not appear to promote more firm attachment to smooth tooth surfaces under the in vivo conditions studied. DISCUSSION The abilities of S. mutans cells to attach to tooth surfaces and to accumulate during proliferation are considered important parameters contributing to the cariogenic potential of these organisms (13, 14). This potential prompted development of various in vitro models for studying adherence of S. mutans to solid surfaces (32). Strains of S. mutans will form adherent microbial deposits on surfaces of wire, glass, and extracted teeth when grown in the presence of sucrose, which specifically promotes extracellular polysaccharide synthesis (7, 18, 23). Heatkilled S. mutans cells also form thick deposits on glass when they are statically incubated with glucosyltransferase preparations and sucrose (26, 27). Such studies have frequently been interpreted as suggesting that extracellular glucan synthesis is required for attachment of S. mutans cells to solid surfaces. However, models based on formation of thick streptococcal masses cannot discriminate between parameters involved in attachment of S. mutans cells to the surface and those involved in cell-to-cell accumulation of S. mutans cells. It is, therefore, possible that attachment of S. mutans cells to solid surfaces could entail different surface components than those involved in accumulation. Because of this possibility, adsorption of washed S. mutans and other oral bacterial cells to untreated or saliva-treated human enamel powder (17), to synthetic hydroxyapatite (10, 22, 24), to slices of bovine enamel (29), and to slices of whale dentin (28) has been studied. Bacterial accumulation does not occur in these systems, and they show that S. mutans cells can attach in the absence of glucan synthesis (17, 22, 29). Such studies further show that the presence of salivary components that selectively adsorb to hydroxyapatite can significantly influence the number of cells of Streptococcus sanguis and S. mitis (S. mitior) that attach (10, 17, 29). The presence of a salivary coating generally has less effect upon the numbers of S. mutans cells that adsorb (17, 22, 24), but this does not necessarily imply that salivary components are unimportant, as suggested by others (32). In our investigation, parameters affecting adsorption and desorption of S. mutans cells were studied using 3H-labeled streptococci and untreated and saliva-treated disks of compressed hydroxyapatite. This system is not complicated by bacterial accumulation, and the surface area of HA exposed to saliva or bacterial suspensions more closely simulates the surface area-to-volume ratios involved in in vivo exposure of teeth to oral fluids. Under the conditions used, adsorption of S. mutans cells reached equilibrium within 45 min, and the number of streptococci adsorbing was logarithmically related to the number of cells available at the concentrations studied. Bacterial quantitation was performed by scintillation counting, and the reliability of this procedure was verified by direct scanning electron microscopic counts. In stirred reaction mixtures containing 10i PBS-suspended S. mutans cells, approximately 3 x 106 streptococci adsorbed per cm2 of untreated HA under conditions of equilibrium. Thus, only a small percentage of the available streptococci became attached, and adsorbed cells occupied approximately 3% of the available surface area. Hydroxyapatite disks pretreated with saliva for 1 or 24 h adsorbed fewer TABLE 5. INFE,CT. IMMIJN. Effect of rinsing with sucrose, glucose, and saline on relative ratio of S. mutans 6715 to S. mitis (S. mitior) 26 remaining on teeth Time Relative proportions of S. mutans to S. mitisa Subject Tie h Saline Glucose Sucrose ± ± ± ± ± ± ± ± ± ± ± ± ± 0.04 a Mean values of three teeth sampled in each subject + the standard error of the mean.

8 VOI,. 18, 1977 ATTACHMENT OF S. MUTANS TO HYDROXYAPATITE 521 streptococcal cells, and when the streptococci were suspended in CWS, their overall net adsorption was reduced more than 30-fold. Clearly, salivary components can exert important influences on adsorption of S. mutans cells to HA surfaces. High-molecular-weight salivary components appear primarily responsible for inhibition of adsorption observed, since streptococci suspended in an ultrafiltrate of saliva adsorbed comparably to buffer-suspended streptococci. At least part of this inhibitory effect may be due to binding of salivary components to surfaces of S. mutans cells, for organisms pretreated with CWS and then washed and suspended in buffer adhered in fewer numbers than untreated streptococci. Since the attachment of S. mutans cells to saliva-treated HA disks probably entails interactions of surface components of the organism with adsorbed salivary molecules, it seems likely that similar components free in the saliva competitively inhibit these interactions. Free salivary components would also be expected to partially saturate receptors on the streptococcal surface. The nature of the salivary component(s) responsible for the marked inhibition of streptococcal adsorption has not been elucidated. S. mutans cells have been reported to bind bloodgroup-reactive mucins (11), antibodies (2), lysozyme (30), and certain agglutinating factors (5, 12) when exposed to saliva. Blood-group-reactive mucinous glycoproteins (41), as well as salivary antibodies of the immunoglobulin A type (40), have been shown to inhibit adsorption of oral streptococci to buccal epithelial cells, and data are available suggesting that blood-groupreactive glycoproteins are either identical to, or associated with, the receptors on epithelial cells to which these streptococci attach (41). Bloodgroup-reactive mucins are among those salivary components that selectively adsorb to HA, and they comprise part of the acquired pellicle that naturally develops on human teeth (4, 31, 33). It is, therefore, reasonable to suspect that adsorption of S. mutans cells to saliva-treated HA surfaces and its inhibition by free salivary components at least partially entails interactions of this organism with blood-group-reactive salivary glycoproteins (11). The colonization of teeth in vivo initiates by adsorption of saliva-suspended bacteria to the acquired pellicle covering teeth (15). In individuals naturally infected with S. mutans, van Houte and Green (38) demonstrated that approximately 104 cells of S. mutans per ml of saliva were required before it became probable that a few cells would adsorb to a cleaned tooth surface. In the present study, approximately 1 x 105 S. mutans cells adsorbed per cm2 of salivatreated HA when the streptococci were suspended in saliva at a concentration of 108 organisms per ml. Since the number of streptococci adsorbed was proportional to the number of cells available in the in vitro system utilized, then, by extrapolation, a concentration of 104 saliva-suspended S. mutans cells would be expected to result in approximately 10 S. mutans cells becoming adsorbed per cm2 of salivatreated HA. Since the surface area of a given tooth sampled by van Houte and Green was less than 1 cm2, it would appear that the adsorptive behavior of S. mutans cells in the in vitro model used closely mimics that of natural S. mutans cells in human mouths. Formation of large deposits of S. mutans on solid supports, such as wire or glass, in vitro depends on glucan synthesis from sucrose (7, 18, 23, 26). However, cells of S. mutans attached readily to untreated or saliva-treated HA in the absence of glucan synthesis. Moreover, streptococci exposed to sucrose before or during adsorption did not attach in higher numbers than PBS-treated streptococci to either untreated or saliva-treated HA surfaces. In fact, they tended to adsorb less well. Thus, the glucans formed by S. mutans do not seem to be innately sticky. Exposure of S. mutans cells already adsorbed on untreated HA disks to sucrose significantly reduced the percentage of streptococci desorbed. This indicates that in situ glucan synthesis can promote more firm attachment of streptococci to untreated HA. This observation is consistent with the suggestion of Marshall and co-workers (25) that synthesis of bacterial polymers capable of binding to a surface can increase the strength of bacterial adsorption to that surface. In the case of S. mutans, these polymers evidently must be formed while the streptococci are associated with the surface; apparently glucan molecules on the surface of free streptococcal cells are unable to get into the proper steric arrangement to strongly interact with the surface and promote streptococcal adsorption. This explains why deposits of S. mutans cells formed on glass or wire surfaces in vitro do not readily reattach once dislodged. This similarly explains the observation by Mukasa and Slade that addition of preformed glucans to S. mutans cells does not result in formation of adherent streptococcal masses on glass surfaces (26); these investigators postulated that an "active" form of glucan was required. It is important to note that incubation with sucrose did not promote firm attachment of S. mutans cells when they were adsorbed on salivatreated HA and subsequently exposed to CWS. This phenomenon could be because the powerful

9 522 CLARK AND GIBBONS desorptive effects of salivary components overshadowed any adherence-promoting effects that glucan synthesis from sucrose may have exerted, or it may indicate that glucans bind less well to saliva-treated HA than to untreated HA. That glucan synthesis from sucrose did take place under the conditions used was indicated when saliva-suspended S. mutans cells agglutinated shortly after sucrose addition. Thus, if there were antibodies to S. mutans glycosyltransferases in the saliva, they did not totally inhibit glucan synthesis. Exposure of S. mutans cells adsorbed onto smooth surfaces of human teeth in vivo to sucrose also did not significantly promote more firm attachment compared with cells of S. mitis (S. mitior), which are unable to synthesize large quantities of glucan from sucrose, and compared with exposure to glucose or PBS. Thus, observations based on both the in vitro model and on in vivo studies in humans do not support the notion that glucan synthesis from sucrose is essential for, or necessarily enhances, attachment of S. mutans cells to teeth. These observations are, therefore, consistent with and extend the recent report of van Houte and coworkers (35) that S. mutans strains can implant and persistently colonize fissures of rodent molar teeth in the absence of sucrose. The data obtained are also in accord with reports that teeth of humans who consume little or no sucrose may, nevertheless, be persistently infected by S. mutans (36). Clearly these studies show both that cells of S. mutans can attach to teeth by interacting with components of the enamel pellicle and that glucan synthesis from sucrose is not required for this process. 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