INFECTION AND IMMUNITY, Aug. 1979, p. 717-728 0019-9567/79/08-0717/12$02.00/0

Vol. 25, No. 2

In Vitro Studies of Dental Plaque Formation: Adsorption of Oral Streptococci to Hydroxyapatite BENJAMIN APPELBAUM,' ELLIS GOLUB,2 STANLEY C. HOLT,3 AND BURTON ROSAN'* Departments of Microbiology' and Biochemistry,2 School of Dental Medicine and Center for Oral Health Research, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Microbiology, The University of Massachusetts, Amherst, Massachusetts 010033 Received for publication 19 April 1979

A mixture of saliva-coated hydroxyapatite beads and radioactively labeled bacteria has been employed as an in vitro model for the initial phase of dental plaque formation. Adsorption in this model can be expressed by the Langmuir adsorption isotherm, and the adherence of oral streptococci can be expressed as the product of the affinity constant (K.) and the number of binding sites (N), KaN. With this approach, Streptococcus sanguis serotype 1 strains adhered better (KaN = [187 ± 72] X 10-2) than serotype 2 strains (KaN = [97 + 84] X 10-2); a t test showed this difference to be statistically significant to the 99.99% confidence level. Strains of S. mitis, S. mutans, and S. salivarius did not appear to adhere as well. To analyze the bacterial receptors involved in adherence, competition studies in which increasing quantities of unlabeled bacteria were added to a fixed quantity (4 x 109 cells per ml) of 3H-labeled serotype 1, reference strain S. sanguis G9B, were performed. These studies indicated that the type 1 strains competed for the same, or closely related, binding sites. Competition studies using serotype 2 S. sanguis strains resulted in an increased binding of reference strain G9B to hydroxyapatite. Scanning electron microscopy indicated this effect was due to the formation of localized aggregations of bacteria, presumably representing the two bacterial types. The results of competition studies with S. mitis were variable, and several strains of other oral bacteria showed little or no competition.

Several studies suggest that the initial phase of many bacterial diseases involves the attachment of the microorganisms to the target tissue (8). The epitome of this process is seen in dental caries and periodontal disease, where adherent plaque appears to be a direct precursor of disease (41). Thus, plaque formation can be considered the initial phase in these disease processes, and therefore, understanding the mechanisms involved in plaque formation is important not only to understanding these diseases but also to the development of rational control measures for caries and periodontal diseases. Streptococcus sanguis appear to be among the first organisms to colonize plaque (4, 5, 42). This species also interacts with salivary glycoproteins, some of which are responsible for the formation of the pellicle on tooth surfaces (21, 27). In addition, S. sanguis appear to interact specifically with Actinomyces viscosus (2, 9, 18), S. mutans (24), and Bacterionema matruchotti (28, 39), organisms which are found in increasing numbers as plaque matures. These observations suggested that S. sanguis could play an important role in plaque formation and thus would be

a good model for the study of the mechanisms of adherence, particularly the initial attachment of bacteria to the tooth surface. The hypothesis forming the basis of these studies is that specific bacterial receptors are involved in adherence to tooth surfaces. This concept is supported not only by reports suggesting differences in the affinity of various streptococci for tooth surfaces and other oral tissues (15, 22), but by studies at the ultrastructural level indicating differences in surfaces among streptococci (12, 13). Clark et al. (6) and Slots and Gibbons (35) have suggested recently that hydroxyapatite (HA) beads can be used as a suitable in vitro model of adherence to tooth surfaces. Initial attachment could be expressed quantitatively in terms of the Langmuir adsorption isotherm (1, 6, 14, 26), which is a mathematical expression of adsorption derived by considering the process as a reversible reaction which follows the law of mass action. We have employed this analysis to estimate the relative adherence of strains representing the various serotypes of S. sanguis and other oral streptococci. These experiments are a prelude to studies in which attempts will 717

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be made to define the specific surface receptors involved in the initial attachment of oral bacteria to tooth surfaces during the formation of dental plaque. MATERIALS AND METHODS Organisms. The strains used in this study are listed in Table 1. Those strains designated as recent plaque isolates were isolated in the summer of 1977 from human dental plaque. Plaque samples were streaked onto Mitis-Salivarius agar, containing 0.001% potassium tellurite (MST agar) and incubated for 48 h at 370C. Colonies showing the typical appearance of S. sanguis were picked and restreaked on MST three times to insure purity of the culture. After Gram staining, stock cultures were made and immediately lyophilized and frozen at -700C as a source of cultures for future studies. The physiological characteristics of the fresh isolates were determined by using the tests described by Carlsson (3) and Facklam (10). Cell growth and labeling conditions. Streptococcal strains were grown in 5 ml of brain heart infuTABLE 1. Strains used in this study Strain

S. sanguis serotype'

B-4 G9B 38 23A 32A 47A2 72x42 72x36 5913 CC5A

1 1 1 1 1 1 1 1 1 1

Enole 72x38 Blackburn M-5 72x34 Challis 10556 10558 SS34 10557 9811 6249

2 2 2 2 2 2 2 2

9652 S161 6715

S. mitis S. mitis S. mitis S. mitis S. salivarius S. faecalis S. mutans

Sourceb

Plaque Plaque Recent human isolate Recent human isolate Recent human isolate Recent human isolate

C. Moutonc; plaque isolate

Plaque

R. J. Gibbonsd

R. J. Gibbons

R. J. Gibbons Capnocytophaga sp. Serotype based upon data of Rosan (31). b Unless specifically indicated, sources of these strains and any alternate designations they may have, have been published (32). 'C. Mouton, S.U.N.Y. School of Dentistry, Buffalo, N.Y. dR. J. Gibbons, Forsyth Dental Center, Boston, Mass. a

sion broth (BHI; Difco Laboratories) for 24 h at 37°C. A 0.5% inoculum was used to inoculate larger quantities (15 to 100 ml) of BHI. [methyl-1',2'-3H]thymidine (specific activity, 80 to 100 Ci/mmol, New England Nuclear Corp., Boston, Mass.) was used at a final concentration of 2 ,uCi/ml for radioactive labeling of cells. After incubation for 20 h, the cells were harvested by centrifugation (16,300 X g, 15 min, 4°C) in plastic centrifuge bottles, washed three times with buffered KCl (2 mM potassium phosphate buffer, pH 6.8, 1 mM CaCl2, and 0.05 M KCl), and suspended in buffered KCl to a turbidity of 300 Klett units (6 x 109 cells per ml; see below), using a Klett-Summerson colorimeter with a blue no. 47 filter. The bacterial stock cell suspension was diluted in buffered KCl to give a series of cell suspensions ranging from 50 to 300 Klett units (1 x 109 to 6 x 109 cells per ml). A 50-jl amount of each 3H-labeled cell suspension was counted in Aquasol-2 (New England Nuclear) to determine its specific activity, which was approximately 450,000 cpm/6 x 109 cells per ml for most S. sanguis strains tested. Strains of Capnocytophaga sp. were grown in BHI broth, supplemented with hemin (5 /g/ml) and sodium succinate (1%, wt/vol), and incubated in an atmosphere of 4% carbon dioxide. Tritiated thymidine at a final concentration of 2 ,Ci/ml was used to label these strains, which were then treated in the same manner as the streptococci. Diluted streptococcal suspensions were counted in a Petroff-Hausser chamber. The preparations contained primarily single cells and pairs of cocci; chains of three cells were also present, but not as numerous, and longer chains were not common. Scanning electron microscopy (SEM) examination of cells bound to HA further confirmed that the distribution of bound cells was predominantly singles and pairs; only occasionally was a longer chain observed (see, for example, Fig. 5A). Thus, no special steps were found necessary to reduce chain length. Direct microscopic count of the bacterial suspensions indicated that 1 Klett unit corresponded to (2 ± 0.10) x 107 streptococci per milliliter (5.6% counting error, based upon a total of 60 samples for 6 different strains). Preparation of saliva. Whole, paraffin-stimulated saliva was collected into ice-chilled tubes and clarified by centrifugation (16,300 x g, 15 min, 4°C). The supernatant fluids were heated for 30 min at 60°C, and sodium azide was then added to a final concentration of 0.004%. If not used immediately, saliva was stored at 40C. Adherence assay. The assay used was based upon that described by Clark et al. (6); it consists, essentially, of measuring the adherence of radioisotopically labeled bacteria to saliva-coated, spheroidal HA beads. A 40-mg amount of HA beads (see below) in 2-ml plastic tubes (Provial; Cooke Engineering Co. [Dynatech Corp.], Alexandria, Va.) were washed once with 1 ml of buffered KCl, mixed with 1 ml of clarified whole saliva, and incubated for 2 h at ambient temperature with rotation (20 rpm). Spectrophotometric assays of the amounts of protein (25) and carbohydrate (29) present in whole saliva, on washed saliva-coated beads, and in the resulting "adsorbed" saliva (i.e.,

VOL. 25, 1979 saliva removed from the HA beads after 2 h of incubation with HA) indicated that the beads were saturated with salivary glycoproteins under these conditions. Unadsorbed saliva was removed, and the beads were washed three times with 1 ml of buffered KCl before the addition of 1 ml of a labeled cell suspension; each assay was done in triplicate. The cells were equilibrated with the saliva-coated beads (by rotation, 20 rpm) for 2 h at 37°C. The beads were allowed to settle, and 200 IL of the supernatant fluids was removed and counted to determine c, the total number of free cells per milliliter at equilibrium in supernatant fluids. The remaining unbound cells were removed, and the beads were then washed three times with buffered KCl, transferred to glass scintillation vials by using 0.4 ml of buffered KCl, and counted to determine q, the total number of cells bound at equilibrium to 40 mg of HA beads. Controls indicated that no significant quenching of counts could be attributed to the beads. Langmuir adsorption isotherm and the determination of adherence parameters. One linearization of the Langmuir adsorption isotherm is c/q = Kd/N + (1/N x c), where Kd is the dissociation constant (cells per milliliter), the reciprocal of the Kd is the affinity constant, Ka, and N is the total number of available binding sites. If adsorption follows the Langmuir isotherm, then a plot of c versus c/q results in a straight line of positive slope equal to 1/N, and in x and y intercepts of -Kd and Kd/N, respectively (see, for example, Fig. 1B). The data can also be plotted as c (abscissa) versus q (ordinate), in which case a curvilinear relationship is obtained (Fig. 1A). The plateau in this curve indicates that the number of bacteria bound to HA is approaching saturation. For several strains, a c-versusq plot gave a straight line of positive slope, suggesting that the system does not become saturated at conditions that result in saturation when other strains are used. The values for Kd and N were determined by regression analysis by using the method of least squares from the experimentally determined values of c and q. The product KaN was chosen as an index of the binding of a given strain to HA, since this product combines the affinity (Ka) and the number of sites (N) into a single factor. It can be shown that KaN = q(1/ c + Ka); therefore, KaN is a linear function of q (the number of cells bound), is modulated by the relationship between the free cell concentration and the affinity constant Ka, and is independent of N, regardless of the range of cell concentrations employed in the adsorption experiments. Since KaN represents the reciprocal of the y-intercept, its units represent the number of cells bound per 40 mg, divided by the number of cells free per milliliter. It should also be noted that all assays were carried out in triplicate, such that each point on any of the curves represents the average of three determinations for that particular value; the statistics of the assay will be discussed in further detail below. Precision of the assay. From adsorption studies with two serotype 1 (31) S. sanguis strains, we have examined the precision of this assay. For strain G9B (data from 26 separate experiments), the values of Kd, N, and KaN + standard deviations are (6.1 + 3.27) x

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108 cells per ml, (10.3 ± 4.34) x 108 sites, and (165 ± 64) x 10-2, respectively; for strain 38, N equals (15.3 ± 4.72) X 10' sites, and KaN is (167 ± 70) X 10-2, representing data from 16 different experiments. Competition experiments. To determine what effect the presence of another (unlabeled, competing) strain would have on the adherence of the reference strain, 3H-labeled G9B, increasing concentrations of competing cells were added to a fixed quantity of 3Hlabeled G9B, and the adherence of G9B was measured. The rationale behind this approach was that if two cells bind to the same site as increasing concentrations of competing cells are added, the adherence of the reference cell [3H]G9B should decrease as the available binding sites become occupied by the competitor. If the competing strain does not bind to saliva-coated HA, or does not bind to the same site as G9B (thereby having a different receptor), barring interfering factors such as steric hindrance, it should have little or no effect upon the binding of 3H-labeled G9B, and the adherence 3H-labeled G9B should remain constant. In carrying out these experiments, individual stock suspensions of both competitor and control (G9B) strains, at a concentration of 6 x 109 cells per ml, were prepared. Reference strain G9B was suspended to a concentration of 1.2 X 1010 cells per ml, and the competing strain was suspended to a concentration of 2.4 x 10"° cells per ml. These suspensions were then mixed, and buffered KCl was added to give a final concentration of 4 x 109 cells per ml of 3H-labeled G9B; increasing concentrations of competing cells, ranging from 2 x 109 to 1.2 x 1010 cells per ml, were added to the fixed concentration of 3H-labeled G9B. A 1-ml amount of each concentration of the mixed suspension was added to the saliva-coated beads, and after 2 h of equilibration at 370C, the adherence of 3Hlabeled G9B was determined. Controls containing (i) no competing cells and (ii) homologous, unlabeled G9B were done for each experiment. All points were done in triplicate. In these experiments, adherence was expressed as the percentage of control counts per minute bound. The latter was determined from the percentage of G9B cells bound to the beads at the input cell concentration of 4 x 109 cells per ml (usually 2.25 X 105 cpm). The percent G9B bound under these conditions varied from 13 to 18%; this value was arbitrarily called 100% adherence. In the competition experiments, the percentage of control counts per minute bound was normalized as follows: (percent G9B bound in mixed suspension/percent G9B bound in control) x 100. This percentage was plotted against the ratio of competing cells to control [3H]G9B cells in the mixture. SEM. HA beads with and without attached cells were treated identically for SEM. Samples were fixed for 1 h in freshly prepared 2% (vol/vol) glutaraldehyde in 0.2 M potassium phosphate buffer (pH 7.2). The fixative was washed out of the samples with several changes of the buffer, and the samples were dehydrated through a graded (20, 50, 70, 100%) acetone series. All samples were critical-point dried in a Polaron 3000 critical point dryer. The resulting dried surfaces were coated with gold to a thickness of approximately 30 nm in a Polaron E5000 sputter coater. The

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specimen stage was cooled with cold water to reduce surface artifacts as a result of heating. A JEOL-JSM35 scanning electron microscope was used, with the resultant images recorded on Polaroid type 665P/N

film. Surface area of the HA beads. The spheroidal HA beads ranged in size from 75 to 185 im in diameter (size as specified by Gallard-Schlessinger Chemical Corp., Carle Place, N.Y.) and have a density of 0.7 g/ ml. Clark et al. (6) reported that these beads have an approximate surface area of 0.27 cm2/mg. We have calculated the surface area of the beads, assuming an average diameter of 135 ,m per bead (G. R. Hill, B.D.H. Chemicals, Ltd., Poole, England, personal communication), and find that the approximate surface area is 0.63 cm2/mg or 25.2 cm2/40 mg of HA beads. This represents a minimum calculation, since it does not include the numerous pores present in the beads (see, for example, Fig. 6a). Scintillation counting. All samples were -counted in 5 ml of Aquasol-2 in an Intertechnique model SL-30 liquid scintillation spectrometer.

RESULTS Effects of saliva. The adsorption of strain 34 to saliva-coated and noncoated HA is shown in Fig. 1. Adsorption to noncoated HA beads follows the Langmuir isotherm (Fig. 1A and B). However, when saliva-coated beads are utilized, adsorption no longer appears to follow the Langmuir model (Fig. 1A), and the number of cells bound appears to be directly proportional to the number of cells added to the system. It was assumed that such adsorption was not specific, since the sites could not be saturated, despite the large number of cells added. Although this strain was received as S. sanguis, it does not produce ammonia from arginine and does not contain a glycerol teichoic acid, indicating that it is more closely related to S. mitis (33). Inhibitory effects of saliva on the adherence of S. salivarius 9652 were also observed (data not shown). This is in agreement with the results obtained by other investigators (30). The effects of saliva upon a representative S. 5

sanguis strain G9B are shown in Fig. 2; the difference between adherence to saliva-coated and uncoated HA beads was within the limits of error and thus was not considered significant; other S. sanguis strains examined (data not shown) showed similar adsorption isotherms in the presence and absence of saliva. Even though there were no significant differences in the affinity constant or the number of binding sites of the S. sanguis strains, the data from strain 34 and S. salivarius suggested that coating HA beads with saliva must have some effect on the nature of the binding sites. Since, in vivo, enamel is constantly in contact with saliva, we chose to use saliva-coated beads for the assay to mimic in vivo conditions as closely as possible. Effects of cell concentration. The number of organisms used in our assays was predicated on the assumption that the closer the experimental numbers of bacteria were to the theoretical number of binding sites, the more accurate the assay would be. Thus, input cell concentrations ranging from 1 x 109 to 6 x 109 cells per ml were employed in our assay; the results shown in Fig. 2 indicate the validity of our assumption. However, the adherence parameters reported by Clark et al. (6) were calculated from experiments in which significantly lower concentrations of S. sanguis (1 X 107 to 6.5 x 107 cells per ml) were used. To determine whether differences in the input cell numbers had an effect upon the adherence parameters, S. sanguis strain G9B was assayed at input cell concentrations ranging from 1 x 107 through 6 x 109 cells per ml. The results (Fig. 3A) show a typical Langmuir adsorption curve, with saturation occurring at high cell concentrations. In Fig. 3B, the data from the lower portion of the curve in Fig. 3A has been expanded to show the curve at input cell concentrations ranging from 1 x 107 to 1 x 109 cells per ml. The smooth curve suggests that multiple binding sites, if present, cannot be distinguished

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FIG. 2. Adsorption of 3H-labeled S. sanguis strain G9B cells to saliva-coated and uncoated HA. Symbols: 0, saliva-coated HA; 0. noncoated HA.

ADHERENCE OF ORAL STREPTOCOCCI

VOL. 25, 1979

721

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in this assay. Thus, for the purposes of our assay it appears that we are dealing with a binding site which becomes saturated when approximately 4 109 to 5 x 109 cells per ml are added to 40 mg of saliva-coated HA beads. Adherence of oral bacteria. The strains listed in Table 1 were all examined for their ability to adhere to saliva-coated HA beads. Initial examinations of Ka and N of several S. sanguis strains showed that although the constants were similar for some strains, no clear-cut relationship between either parameter separately was found. However, it was reasoned that the ability of an organism to adhere at a fixed input of bacteria would be dependent upon two parameters: the number of binding sites available (N) and the affinity (Ka) of the organism for these sites. Thus the product of these parameters, KaN, was chosen to describe the adherence of bacteria to HA (see above). The results, expressed as KaN (Fig. 4), indicate that the type 1 S. sanguis strains adhered better than did the type 2 strains. The average KaN for nine strains X

of S. sanguis type 1 was (178 + 66)

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x

10-2,

excluding strains 38 and G9B. Since the latter two strains have been used as controls for nearly all experiments, it was thought that their inclusion would bias the data; however, even if included, the KaN is (187 ± 72) x 10-2 for 54 determinations. The KaN for serotype 2 strains of S. sanguis was (97 + 84) x 10-2. The differences between the KaN of serotype 1 and serotype 2 S. sanguis strains are significant (by the t test, t = 3.39, significant to 0.01 level). In the calculation of KaN for serotype 2 strains, the data for strains 10558 and 71x22 have been omitted, since under the conditions employed these strains showed the same type of nonspecific adsorption to saliva-coated beads as did strain 34. Other strains of oral bacteria examined

50

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S.rvtis

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S.foecolis s- -

-

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adhered relatively poorly, including strains of S. mutans, S. salivarius, S. faecalis, and Capnocytophaga. S. salivarius and S. mutans strains appear to have relatively low adherence values, as has been reported by other investigators, albeit differences in numerical value between our studies and theirs (6) were noted. S. faecalis (16) and Capnocytophaga represent strains that are not ordinarily associated with attached plaque (36) and thus should act as negative controls. As expected, they did not adhere well. Strains of S. mitis, on the other hand, adhered to approximately the same extent as did serotype 2 S. sanguis strains; KaN for the S. mitis strains tested (six determinations) is (95 + 56) x 10-2. Since only four S. mitis strains have been examined, and in view of the known heterogeneity of S. mitis, no conclusions regarding the relative adherence of this group can be drawn at this time. The heterogeneous behavior of these organisms in adherence assays was also observed in the competition experiments. Competition experiments. The results of the adsorption experiments suggested at least three possible groupings of surface receptors: those in serotype 1, serotype 2, and S. mitis. If these groupings were valid, it was assumed that cells having the same surface receptor should compete for the same binding site. Experiments designed to test this hypothesis used strain G9B as the radioactively labeled reference strain. Attempts were made to perform competition experiments and analyze the data by using the Langmuir isotherm, but as the results below show, interactions between the competing cells occurred which made this type of analysis unworkable (20). Therefore, competition at a fixed concentration of labeled cells, as described above, was chosen. The results of these competition studies are shown in Fig. 5 and 7; Fig. 6 shows scanning electron micrographs of cells actually bound to HA. Figure 6A indicates that control [3H]G9B cells, in the absence of compet-

722

INFECT. IMMUN.

APPELBAUM ET AL.

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Competing cells 1 R[ti R 3H S.sanguis G9BJ FIG. 5. Results of competition assays, where increasing quantities of unlabeled S. sanguis serotypes 1 and 2 cells were added to a fixed quantity (4 x 109 streptococci per ml) of 3H-labeled serotype 1 S. sanguis G9B, and the adherence of 3H-labeled G9B was determined. (A and B) S. sanguis serotype I as competitors. (C) Competition with representative S. sanguis serotype 2 strains.

ing cells, were bound, primarily as singlets and doublets, in the matrix of the HA beads. A mixture of unlabeled and labeled G9B cells at a 3:1 ratio but at a fourfold higher concentration, i.e., a total of 1.6 x 1010 cells per ml, showed the same total number of cells bound when any given field was examined (compare Fig. 6A and 6B). The results suggested that these cells must be competing with each other for the same, limited number of discrete binding sites on the surface of the HA and that the reduced adherence of the labeled cells, in the presence of unlabeled, competing cells, does in fact correspond to competition. Serotype 1 S. sanguis strains, as a group, clearly inhibited the adherence of 3H-labeled G9B cells (Fig. 5A and B). However, two types of inhibition were seen. The first, observed with strains G9B, 5913, or 47A2 as competing cells, was a direct inhibition (Fig. 5A). In contrast, when strains 38, 32A, or 23A were used as com-

petitors (Fig. 5B), there was an initial increase in the number of 3H-labeled G9B cells bound; then, as more competing cells were added, competition appeared to occur. These data suggest that either an interaction between the competing strains and the reference strain took place or that adsorption to two slightly different sites occurred. SEM analysis of cells adherent to the beads from the mixture containing a 1:1 ratio of unlabeled 38 and 3H-labeled G9B suggested that the cells appeared to be covering more of the surface of the beads (Fig. 6C) than observed on beads saturated with control G9B cells (Fig. 6B). The competition of 38 and 3H-labeled G9B indicated that the cells were more closely spaced, suggesting attachment to similar or closely related binding sites, rather than interacting with one another or accumulating as distinct foci on the surface of the HA. Type 2 S. sanguis strains, on the other hand, competed very slightly, if at all, with 3H-labeled G9B. On the contrary (Fig. 5C), at a low ratio of unlabeled cells to labeled cells, the type 2 strains all show an enhancing effect upon adherence of 3H-labeled G9B. This enhancing effect appears to be associated with an interaction between these two serotypes and, as suggested by SEM examination (Fig. 6D) of a mixture containing strain Challis and 3H-labeled G9B, localized foci of bacteria can be observed. Variable results were seen when strains of S. mitis were used as competitor; some strains, such as 6249 (Fig. 7A) were strong competitors of 3H-labeled G9B, whereas other strains, such as strain 34 (not shown) and ATCC 10557 (Fig. 7A) competed in a manner similar to that of type 2 S. sanguis (Fig. 5C). When S. mutans strain 6715 cells were used as the competitor, strain 3H-labeled G9B showed a marked increase in adherence, in proportion to the numbers of S. mutans cells added (Fig. 7B), again, presumably the result of cell-to-cell interaction. However, there was no significant difference in the number of cells attached, nor were any localized aggregates or foci of bacteria seen, when beads from this experiment were examined by SEM (data not shown). Additional controls, in which the label was placed on S. mutans (i.e., 3H-labeled 6715), showed only a minimal (19% of total input counts per minute) binding of strain 6715 to the saliva-coated HA beads, either in the presence or absence of 4 x 109 G9B cells per ml. Other investigators have noted in SEM

FIG. 6. SEM examination of bacterial cells bound to HA beads. (a) Control, containing 3H-labeled G9B at an input of 4 x 109 cells per ml. (b) G9B homologous competition assay, containing G9B cells at an input of 1.6 X 1010 cells per ml. (c) Mixture of unlabeled 38 and 3H-labeled G9B in a 1:1 ratio. (d) Mixture of unlabeled

Challis and 3H-labeled G9B cells at a ratio of 1:2.

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INFECT. IMMUN.

APPELBAUM ET AL.

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examination of S. mutans 6715 attached to HA beads that only a minimal number of cells are bound (R. H. Staat, personal communication). These results suggest that the two strains, S. sanguis G9B and S. mutans 6715, are probably binding to two separate and distinct sites on the saliva-coated HA surface. The data do not, as yet, suggest the nature of this effect. Strains that show little or no binding to saliva-coated HA beads, such as S. salivarius 9652 and Capnocytophaga, had no effect upon the adherence of G9B (Fig. 7B). S. faecalis strain S161, which also binds poorly to saliva-coated HA, only slightly inhibited the adherence of G9B (Fig. 7B). All strains listed in Table 1 were used in the competition experiments; the results in Fig. 5 and 7 are representative results. Additional controls for the competition experiments were obtained from a dual-label experiment. A culture of G9B was labeled with ["4C]thymidine (0.2 MCi/ml). The cells were washed and assayed, first, for adsorption by using the Langmuir isotherm, and second, as a competitor of 3H-labeled G9B cells by using increasing concentrations of

"C-labeled cells. In this experiment, calculated on the basis of Langmuir adsorption, 8.9 x 108 binding sites were available for binding strain G9B to the saliva-coated beads (Table 2). In the mixtures containing equal proportions of 3Hand "C-labeled cells, the 3H-labeled cells were shown to occupy 53%, and the "C-labeled cells occupied 47% of the total sites available; thus, the total number of sites available and the number of sites occupied by each type of strain are in agreement with the predictions of the model. DISCUSSION The experiments performed in this study confirm that the use of the Langmuir adsorption isotherm (1, 26) for studying the initial attachment of bacteria to HA, as suggested by Clark et al. (6) and Gibbons et al. (14), is valid, and thus is a useful model for studies of in vitro plaque formation. However, the system does have several limitations which must be understood to avoid misinterpretation of the data obtained. First, it is necessary to use large numbers of cells to insure saturation of the binding

ADHERENCE OF ORAL STREPTOCOCCI

VOL. 25, 1979

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TABLE 2. Determination of adherence parameters of strain G9B by a dual-label experiment No. of binding % of sites ocExperimental conditions total cupied

Control (single label) Dual-label mixture Sites occupied by 3H cells Sites occupied by 14C cells aTotal number of binding sites each case was 8.6 x 108.

(X108') 8.6

100

4.6 53 3.5 47 available (N) in

sites and to obtain reliable predictions of N in the adsorption equation; the numbers of cells used in these studies generally ranged from 1 x 109 to 6 x 109 cells per ml. This contrasts sharply with the low numbers of cells (3 x 105 to 6 x 107 per ml, depending upon the strain) used by Clark et al. (6) in essentially the same assay system.

Indeed, these investigators suggest that the model requires the use of relatively low numbers

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of cells, so that the proportion of cells adhering to the beads is high relative to the cells added. Since the adsorption curve is curvilinear, it is true that at a low cell input a higher proportion of cells will adhere; however, a low cell concentration requires a larger extrapolation to estimate the number of binding sites derived from the isotherm equation. The basis for the use of relatively low cell concentrations was the possibility that interactions between cells may occur. However, the results of our studies suggest that in most instances interbacterial interactions do not appear to interfere with the attachment of cells to the HA. Certainly the data obtained with most of the strains in this study do fit the Langmuir isotherm, and comparisons of experimental curves with theoretical curves predicted by the equations were excellent (data not shown); thus, on an experimental basis the use of high cell concentrations to estimate N appears to be justified. The differences in numbers of cells used in our experiments compared with those used by Clark et al. (6) accounts for the differences in affinity and number of binding sites predicted for S. mutans and S. sanguis observed in the two studies. At the lower cell concentrations (1 x 107 to 6.5 x 107 cells per ml) of strain G9B, the HA beads are not saturated (Fig. 3B). To determine what the adherence parameters would be for data obtained with these cell concentrations, a trial calculation was performed, and N was calculated to be 8.6 x 106 sites. However, based upon the saturating cell concentrations shown in Fig. 3A, N equals 7.8 x 108 sites, a 100-fold difference. The fact that the curves in Fig. 3A and 3B are smooth indicates that for practical purposes we are dealing with a single type of binding site on the HA surface. This site(s) requires high cell numbers to be saturated. If two grossly different types of binding sites with different affinities were involved, a biphasic curve (26) would have been expected over the range of cell concentrations used. Although the differences between the results of our study and that of Clark et al. (6) may be due to the use of different growth media or strains or both, it is clear that the results obtained do support the use of high cell concentrations in this assay. Thus, the differences in adherence parameters observed in our studies compared to those of Clark et al. appear to be directly related to the difference in input cells used rather than difference in affinity or number of detectable binding sites. However, it is of interest that the patterns of adherence were similar. Thus, S. mutans did not adhere well, the adherence of S. salivarius decreased with the addition of saliva to the beads, and S. san-

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guis generally adhered much better than the other species. Thus, although the data disagree quantitatively, they are nearly in complete agreement in qualitative terms. Since our eventual goal is to define specific bacterial receptor sites, it is obvious that the quantitative aspects and the proper experimental methods to achieve quantitation must be considered. The coefficient of variation for the parameters determined from binding experiments was found to be approximately 40%. This could be the result of both biological and analytical errors, and we have attempted to analyze the source of this variation. Analytical errors in the determination of free and bound cells could be ruled out, since each value is the mean of triplicate determinations, with a coefficient of variation near 10%. Uncertainties in the calculated parameter values could arise from curve-fitting errors, and although it was not determined, this probably accounted for an additional 10 to 20% variation. The residual error was probably the result of biological variations, such as alterations in saliva, slight variation in the proportions of pairs and single cocci which were not detected in cell count, variations in the amount of DNA per cell, or variations in the quantity of surface receptors present on the bacteria. Studies in other adherence assay systems also suggest that although good precision is obtained among replicate samples on a single day, considerable variation in the attachment of bacteria to epithelial cells in replicate experiments has been observed (11, 17, 19, 37, 38, 40). The major source of this error (20% experimental error for Escherichia coli to uroepithelial cells [37]) has been attributed to differences among the epithelial cells, i.e., differences in the quantity of receptor present, or differences in the overall population of cells, which, consequently, may affect their ability to bind bacteria. However, there are also other bacterial factors, i.e., the degree of piliation of, for example, E. coli strains (17), which may be related to the nutrition of the organism, that may also contribute to the overall variation. This variation probably accounts for the differences observed in the effects of saliva on the adherence of S. sanguis to HA in our studies compared with those of other investigators (6, 23, 24, 30). The studies of Liljemark and Schauer (23, 24) employed only one point and measured the percent adherence at a single concentration of cells. Since the isotherm is essentially a curvilinear relationship, the conclusions from this approach must be looked upon with some degree of caution. Moreover, only two of the strains studied by these investigators showed increased adherence, one strain showed a decrease, and one strain showed no significant difference in

INFECT. IMMUN.

adherence in the presence of saliva (24). The more recent studies of Clark et al. (6), employing the same model (except for cell numbers) used in this study, concluded that saliva increased the number of binding sites but decreased the affinity of S. sanguis. Although our studies also indicated changes in these parameters for strains G9B (Fig. 2) and 38 (not shown), these changes were within the limits of experimental error and thus were not considered significant. The salivacoated beads in the experiment of Clark et al. (6) had a KaN of 0.22 x 10-2 as compared with the figure of 0.28 x 102 for the uncoated beads, and as suggested by our studies such differences may not be significant. However, it is clear that saliva does affect the attachment of other organisms, e.g., S. salivarius (30) and S. mutans (6, 7). Thus it cannot be assumed that, because no differences in the numerical expression of adherence are noted, the same binding sites are involved in the adherence of S. sanguis to coated and uncoated beads. Indeed, the studies of salivary aggregation of S. sanguis (21, 27) strongly suggest that specific salivary binding sites are involved in interactions with specific receptors on the organisms. Since it is these receptors we eventually hope to define, saliva remains a vital component of the assay, despite the coincidence that no significant numerical difference was detected between the coated and uncoated beads. The differences in the quantitative estimates of adherence among the different serotypes and species could be due to differences in the amount of receptors or the types of receptors present on the cell surface. These possibilities might be distinguished in competition experiments in which reduction in adherence of control labeled cells, when mixed with unlabeled cold cells, could be used not only to determine similarity among the receptors but also, perhaps, to roughly estimate the quantity of receptor by noting how effectively one strain competed with the control, labeled strain. The results of studies suggested at least four different patterns of adherence, which it is assumed reflects differences in the receptors on the surfaces of the streptococci tested. In general, this pattern reflected the differences already observed in the direct (Langmuir adsorption) adherence assay. The first pattern was illustrated among some serotype 1 strains, such as 47A2 and 5913, which appeared to compete directly for the same binding site as the control strain (G9B), suggesting a closer relationship between the surfaces of these cells and that of the control. However, differences in the efficiency of competition might indicate quantitative differences in the amount of receptors. The direct competition between these serotypes was confirmed in the SEM photo-

VOL. 25, 1979

graphs which show essentially the same number of cells binding as in the control of 3H-labeled G9B cells alone or where cold G9B cells compete with labeled G9B cells (Fig. 6a). Another type of competition was manifested with other serotype 1 strains, e.g., 38, 23A, and 32A, in which there appears to be an initial enhancement of the adherence of G9B followed by an inhibition. An electron micrograph of this type of interaction (Fig. 6c) shows an increase in the total number of cells adhering, but few if any aggregates appear to form. It would appear that mixing the cells together alters the vicinal apatite such as to make it more receptive. A point then appears to be reached, and the two strains begin to compete for the remaining sites. The idea that one cell could influence the adherence of another cell in the vicinity is reminiscent of ultrastructural studies of early dental plaque where different streptococci appear to form adjacent microcolonies (34). A third pattern of adherence was observed when unlabeled serotype 2 cells were mixed with labeled G9B. Rather than competition, there appeared to be an enhancement of adherence of G9B. SEM examination (Fig. 6d) confirmed that a greater number of bacteria adhered compared with the number adhering to controls. The distribution of the organisms indicated a tendency of the organisms to aggregate in certain areas of the beads, suggesting that some interbacterial interaction had occurred between these serotypes. A pattern similar to this was observed for mixtures of G9B and S. mitis strains 34 and 10557 (Fig. 7A). In contrast, two other S. mitis strains, 6249 and 9811, competed as effectively as homologous G9B cells for the binding sites. These differences among S. mitis strains may reflect the known heterogeneity of the surfaces of these organisms (33). Mixtures of S. mutans strain 6715 and labeled G9B also followed the same pattern as mixtures of serotype 2 strains with G9B; i.e., they appeared to enhance the adherence of G9B. However, if the labeled cells were reversed (3H-labeled S. mutans; unlabeled G9B), there was no increase in the adherence of S. mutans cells alone. This study suggests that S. mutans cells can enhance the adherence of S. sanguis but not vice versa; the SEM of this interaction was inconclusive and gave no indication of the type of interaction which might be occurring between these two species. In any event, only one strain of S. mutans was examined, and these results may not necessarily be applicable to other strains of S. mutans. These experiments suggest that several kinds of bacterial surface receptors may be involved in adherence. Interestingly, similar patterns of specificity have been observed by Isaacson et al.

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(17) for the competitive inhibition of E. coli by various preparations of purified pili. In a sense, the results parallel the studies of the salivary aggregation of bacteria in which several different glycoproteins appear to be involved (21, 27). Although it is by no means clear that the same glycoproteins are involved in adherence and aggregation, it is clear that the two phenomena are complex. This is not really surprising when one compares the complexity of bacterial forms found even in the earliest stages of plaque formation. Particularly interesting is the idea that, in addition to different surface receptors reacting with different salivary glycoproteins, the organisms themselves may exert some influence on the surrounding beads. However, it is apparent that in the absence of any real information concerning the surface receptors involved in adherence, further speculation would be fruitless. The key answers should come when we can isolate specific polymers which can compete successfully for the binding sites. Definition of such molecules will be a first step in unraveling the complex interactions which appear to be involved in plaque formation. ACKNOWLEDGMENTS We thank Mary Dennis and Erika Musante for technical assistance, Lois Argenbright for saliva donations, and Stuart Schiffman for writing computer programs. This research was supported by Public Health Service grants I-F32-DE-05164 (to B.A.), DE-05123 (to S.C.H.), and DE-02623 and DE-03180 (to B.R.) from the National Institute of Dental Research. LITERATURE CITED 1. Adamson, A. A. 1967. Physical chemistry of surfaces, 2nd ed., p. 397-402. Interscience Publishers, Inc., New York. 2. Bourgeau, G., and B. C. McBride. 1976. Dextran-mediated interbacterial aggregation between dextran-synthesizing streptococci and Actinomyces viscosus. Infect. Immun. 13:1228-1234. 3. Carlsson, J. 1968. A numerical taxonomic study of human oral streptococci. Odontol. Revy 19:137-160. 4. Carlsson, J., H. Grahnen, and G. Jonsson. 1975. Lactobacilli and streptococci in the mouth of children. Caries Res. 9:333-339. 5. Carlsson, J., H. Grahnen, G. Jonsson, and S. Wickner. 1970. Establishment of Streptococcus sanguis in the mouth of infants. Arch. Oral Biol. 15:1143-1148. 6. Clark, W. B., L. L. Bammann, and R. J. Gibbons. 1977. Comparative estimates of bacterial affinities and adsorption sites on hydroxyapatite surfaces. Irfect. Immun. 19:846-853. 7. Clark, W. B., and R. J. Gibbons. 1977. Influence of salivary components and extracellular polysaccharide synthesis from sucrose on the attachment of Streptococcus mutans 6715 to hydroxyapatite surfaces. Infect. Immun. 18:514-523. 8. Costerton, J. W., G. G. Geesey, and K. G. Cheng. 1978. How bacteria stick. Sci. Am. 238:86-95. 9. Ellen, R. P., and I. B. Balcerzak-Raczkowski. 1977. Interbacterial aggregation of Actinomyces naeslundii and dental plaque streptococci. J. Periodontal Res. 12: 11-20.

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10. Facklam, R. R. 1977. Physiological differentiation of viridans streptococci. J. Clin. Microbiol. 5:184-201. 11. Frost, A. J., D. D. Wanasinghe, and J. B. Woolcock. 1977. Some factors affecting selective adherence of microorganisms in the bovine mammary gland. Infect. Immun. 15:245-253. 12. Gibbons, R. J., and J. van Houte. 1973. On the formation of dental plaques. J. Periodontol. 44:347-360. 13. Gibbons, R. J., and J. van Houte. 1975. Bacterial adherence in oral microbial ecology. Annu. Rev. Microbiol. 27:19-44. 14. Gibbons, R. J., E. C. Moreno, and D. M. Spinell. 1976. Model delineating the effects of a salivary pellicle on the adsorption of Streptococcus miteor onto hydroxyapatite. Infect. Immun. 14:1109-1112. 15. Gibbons, R. J., D. M. Spinell, and Z. Skobe. 1976. Selective adherence as a determinant of the host tropisms of certain indigenous and pathogenic bacteria. Infect. Immun. 13:238-246. 16. Gold, 0. G., H. V. Jordon, and J. van Houte. 1975. The prevalence of enterococci in the human mouth and their pathogenicity in animal models. Arch. Oral Biol. 20:473-479. 17. Isaacson, R. E., P. C. Fusco, C. C. Brinton, and H. W. Moon. 1978. In vitro adhesion of Escherichia coli to porcine small intestine epithelial cells: pili as adhesive factors. Infect. Immun. 21:392-397. 18. Kelstrup, J., and T. D. Funder-Nielson. 1974. Aggregation of oral streptococci with fusobacterium and actinomyces. J. Biol. Buccale 2:347-362. 19. Kimura, L. H., and N. N. Pearsall. 1978. Adherence of Candida albicans to human buccal epithelial cells. Infect. Immun. 21:64-68. 20. Kresak, M., E. C. Moreno, R. T. Zahradnik, and D. I. Hay. 1977. Adsorption of amino acids onto hydroxyapatite. J. Colloid Interface Sci. 59:283-292. 21. Levine, M. G., M. C. Herzberg, M. S. Levine, S. A. Ellison, M. W. Stinson, H. C. Li, 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. 22. Liljemark, W. F., and R. J. Gibbons. 1972. Proportional distribution and relative adherence of Streptococcus miteor (mitis) on various surfaces in the human oral cavity. Infect. Immun. 6:852-859. 23. Liljemark, W. F., and S. V. Schauer. 1975. Studies on the bacterial components which bind Streptococcus sanguis and Streptoccus mutans to hydroxyapatite. Arch. Oral Biol. 20:609-615. 24. Liljemark, W. F., and S. V. Schauer. 1977. Competitive binding among oral streptococci to hydroxyapatite. J. Dent. Res. 56:157-165. 25. Lowry, 0. H., N. G. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

INFECT. IMMUN. 26. Marshall, K. 1976. Interfaces in microbial ecology, p. 2735. Harvard University Press, Cambridge, Mass. 27. McBride, B. C., and M. T. Gisslow. 1977. Role of sialic acid in saliva-induced aggregation of Streptococcus sanguis. Infect. Immun. 18:35-40. 28. Mouton, C., H. Reynolds, and R. J. Genco. 1978. Combined micromanipulation, culture and immunofluorescent techniques for isolation of the coccal organisms comprising the "corn cob" configuration of human dental plaque. J. Biol. Buccale. 5:321-332. 29. Nowotny, A. 1969. Basic exercises in immunochemistry, p. 102-104. Springer-Verlag, New York. 30. 0rstavik, D., F. W. Kraus, and L. C. Henshaw. 1974. In vitro attachment of streptococci to the tooth surface. Infect. Immun. 9:794-800. 31. Rosan, B. 1973. Antigens of Streptococcus sanguis. Infect. Immun. 7:205-211. 32. Rosan, B. 1976. Relationship of the cell wall composition of group H streptococci and Streptococcus sanguis to their serological properties. Infect. Immun. 13:11441153. 33. Rosan, B. 1978. Absence of glycerol teichoic acids in certain oral streptococci. Science 201:918-920. 34. Rosan, B., C. H. Lai, and M. A. Listgarten. 1976. Streptococcus sanguis: a model in the application of immunochemical analysis for the in situ localization of bacteria in dental plaque. J. Dent. Res. 55(A):124-141. 35. Slots, J., and R. J. Gibbons. 1978. Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets. Infect. Immun. 19: 254-264. 36. Socransky, S. S. 1977. Microbiology of periodontal disease-present status and future considerations. J. Periodontol. 48:497-504. 37. Svanborg Eden, C., B. Eriksson, and L. A. Hanson. 1977. Adhesion of Escherichia coli to human uroepithelial cells in vitro. Infect. Immun. 18:767-774. 38. Svanborg Eden, C., and H. A. Hansson. 1978. Escherichia coli pili as possible mediators of attachment to human urinary tract epithelial cells. Infect. Immun. 21: 229-237. 39. Takazoe, I., T. Matsukubo, and T. Katow. 1978. Experimental formation of "corn cob" in vitro. J. Dent. Res. 57:384-387. 40. Tramont, E. C., and C. Wilson. 1977. Variations in buccal cell adhesion of Neisseria gonorrhoeae. Infect. Immun. 16:709-711. 41. van Houte, J. 1977. Oral bacteria colonization: mechanisms and implications, p. 3-32. In H. M. Stiles, W. J. Loesche, and T. C. O'Brien (ed.), Microbial aspects of dental caries. Information Retrieval, Washington, D.C. 42. 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.