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Immunization with dextransucrases, levansucrases, and glycosidic hydrolases from oral streptococci. II. Immunization with glucosyltransferases, fructosyltransferases, and glycosidic hydrolases from oral streptococci in monkeys.

The feasibility of immunizing monkeys with enzymes from oral streptococci in an attempt to reduce dental caries was investigated. Forty rhesus monkeys, Macaca mulatta, were used. Cariogenic streptococci, S mutans, were implanted into all the monkeys' mouths. There was no pathological effect resulting from immunization. Of the 40 animals, 30 retained the implanted flora throughout the experiment; the remaining 10 were reimplanted until the streptococci remained. In six months, gross carious lesions were evident with plaque. Inhibitiors present in the monkey sera after immunization inhibited glucosyltransferase, fructosyltransferase, and neuraminidase activities. It was presumed the inhibitors were antibodies. There was a reduction of 68.6% in the total carious lesions in the animals immunized intraorally with glucosyltransferase, 62.4% reduction in those injected with fructosyltransferase, and 57.4% reduction in total lesions in those immunized with glycosidic hydrolases after 19 months, as compared to the control group. There were no gross lesions apparent in the group immunized with glycosidic hydrolases. It appears that immunization with enzymes significantly reduces carries and is feasible in a primate model.

Animals

Surface potential and adherence of oral streptococci to solid surfaces.

Strains of oral streptococci belonging to the same species but with different zeta-potential adhere with different strengths or become desorbed at different rates. However, strains of various species but with approximately the same zeta-potential also adhere differently. The adherence was found to be affected by the presence of polyvalent of counter ions in the microorganisms studied.

Adhesiveness

Effect of inorganic ions and surface active organic compounds on the adherence of oral streptococci.

A reduction in the adherence of oral streptococci was observed after topical application of aluminum ions to standardized dentin test pieces whereas pyrophosphate and tripolyphosphate seemed to inhibit the adherence after incorporation in the incubation solution. A pronounced effect was recorded after topical application of surfactants with the positively charged primary amino group as end group. Hydrofluorides of several such aliphatic amines effectively reduced the number of colony-forming units washed off from the test pieces. Also the free base of hexadecylamine showed a similar effect. Inhibition of adherence was also obtained with long chain esters of lysine, with an optimum at 16 carbon atoms in the alcohol group. These compounds have the advantage of being built up from molecules known to the body.

Adhesiveness

Interactions between human serum proteins and oral streptococci reveal occurrence of receptors for aggregated beta 2-microglobulin.

A total of 31 strains of oral streptococci representing Streptococcus mutans, Streptococcus sanguis, Streptococcus mitior, Streptococcus salivarius, and Streptococcus milleri were tested for possible binding of human immunoglobulins G, G1, G2, G3, G4, A1, A2, M1, and M2 and haptoglobin, hemoglobin, fibrinogen, and aggregated beta 2-microglobulin. Radiolabeled beta 2-microglobulin in aggregated form showed affinity for 20 of the 31 strains tested. Binding activity for the protein was found in strains belonging to all five species. The bacterial receptor was resistant to trypsin. Monomeric, unlabeled beta 2-microglobulin did not interfere with the binding of the aggregated form. Of the other proteins tested, only the immunoglobulin A1 protein showed positive binding, and that was only with a single strain of S. milleri. beta 2-Microglobulin is present on all nucleated cell membranes in vivo. The reaction between aggregated beta 2-microglobulin and oral streptococci is a new type of human-bacterium interaction which should be considered in studies of bacterial adherence.

Beta-Globulins

More sensitive test agar for detection of dextranase-producing oral streptococci and identification of two glucan synthesis-defective dextranase mutants of Streptococcus mutans 6715.

A more sensitive test agar was developed to detect oral microbes with relatively low dextranase activity and to identify dextranase-negative mutants. Several oral streptococci that had previously been scored as dextranase negative readily decolorized the new, blue dextran-containing medium. To assess whether dextranase plays a role in glucan synthesis by oral streptococci, various glucan synthesis-defective mutants were tested for dextranase activity on the new medium. Mutants 4 and 27, which do not cause smooth-surface caries and which synthesize more soluble glucan than their parent, Streptococcus mutans 6715-13, were markedly deficient in these dextranase activity tests.

Culture Media

Experimental endocarditis induced by dental manipulation and oral streptococci.

Circumstantial evidence has strongly implicated dental manipulation as an etiologic factor in the development of infective endocarditis. The introduction of human oral streptococci via the oral cavity in rabbits with vegetative cardiac lesions yields a 94 per cent incidence of infective endocarditis, if the number of inoculated organisms is above a threshold level of 10(7).

Animals

A study of the interaction between oral streptococci and hard surfaces.

A rotating disc method was used to compare the tendencies of two oral streptococci to deposit on to glass and polystyrene surfaces from electrolyte solutions of varying ionic strength. Streptococcus salivarius had a greater tendency to deposit than had Streptococcus mitior under these condition. In addition to the balance of van der Waals' forces of attraction and electrostatic forces of repulsion, it is suggested that the adsorption to the glass and polystyrene surfaces of material present in the outer layers of the cell wall could play a significant part in the deposition of S. salivarius.

Adsorption

Occurrence and distribution of sucrose-metabolizing enzymes in oral streptococci.

Specific growth rates, growth yields, and the level and cellular distribution of three sucrose-metabolizing enzyme activities were determined for seven oral streptococci (Streptococcus mutans strains E49, BHT, 10449, SL-1, and LM-7, S. sanguis 10558, and S. salivarius 25975). Cultures were grown in a fermentor at pH 6 with either 20 mM glucose or 10 mM sucrose. Generation times varied between 21 and 70 min. Whereas some strains grew 10 to 50% more slowly with sucrose than with glucose, others did not. Growth was always logarithmic, and the growth yields were similar. Glcosyl transferase (EC 2.4.1.5) was largely extracellular; in sucrose cultures it was appreciably lower, but no major shift to a cell-associated form was found. In glucose cultures, the activity varied between 4 and 140 IU per 6-liter culture. The glucan formed was mostly or exclusively water insoluble. Glcosyl transferase was stimulated weakly (60% or less) by various dextrans. Fructosyl transferase (EC 2.4.1.10) was primarily extracellular (except in glucose cultures of S. salivarius) and varied between 0 and 337 IU/culture. In S. salivarius, the extracellular fructosyl transferase was induced by sucrose. In all S. Mutans cultures, the total fructosyl transferase activity was lower after growth with sucrose. All strains had extra- and intracellular invertase (EC 3.2.1.26) activity. Total levels varied between 210 and 3,500 IU/culture. Less extracellular activity was present in sucrose cultures. Only S. salivarius had appreciable activity in the cellular particulate fraction. Invertase activity was significantly higher than the combined glucosyl and fructosyl transferase activities in all cultures.

Fructose

Bacteriocins as factors in the in vitro interaction between oral streptococci in plaque.

The effect of bacteriocins on the composition of dental plaque flora was studied in vitro with bacterial plaque formed by oral streptococci on glass rods suspended in broth medium. Cell-free preparations containing mutacin SW31, a bacteriocin produced by Streptococcus mutans, killed sensitive cells present in the plaque selectively, but did not affect resistant cells. Similar preparations from a non-bacteriocinogenic mutant exerted only a slight effect. Mixed growth of bacteriocin-producing S. mutans SW31 with the sensitive S. sanguis Ny101 resulted in a nearly single-strain plaque of the former. Mutacin could be detected in the plaque substance as well as in the surrounding medium. A non-bacteriocinogenic mutant, which was shown not to be altered in functions affecting growth in plaque, allowed substantial growth of S. sanguis Ny101. Sequential inoculation of the bacteriocinogenic plaque former S. sanguis P3A3 and the sensitive S. mutans OMZ61 showed that the cells of the latter are killed rapidly in established plaques when inoculated with the bacteriocinogenic strain. A trypsin-sensitive antagonistic substance could be detected in the plaque, but not in the surrounding medium. The results indicate that bacteriocins can be active in plaque in vitro and suggest that bacteriocins could play a role in determining the composition of plaque in vivo.

Bacteriocins

New method for the isolation of Streptococcus mutans and its differentiation from other oral streptococci.

A new, improved agar medium for the isolation of Streptococcus mutans, the etiological agent of dental caries, was developed. In contrast to mitis-salivarius agar, this medium not only recovers a greater number of S. mutans strains from most oral specimens but, because of its mannitol and sorbitol content, it also facilitates the differentiation of S. mutans from other oral streptococci, e.g., S. salivarius, S. mitis, and S. sanguis, which do not grow or produce scanty growth only after 10 days of incubation. The medium is easy to prepare because of its simple and unique composition, is characterized by the presence of an acid indicator, and can be utilized under aerobic and anaerobic conditions as well. The medium cannot be used to distinguish among the eight serotypes, a to g and SL-1, of S. mutans. Mannitol-utilizing bacteria such as streptococci (e.g., S. faecalis) and other microorganisms (e.g., Staphylococcus aureus) are able to grow on this medium and can be distinguished from S. mutans by their unique colony morphology.

Aerobiosis

In vitro studies of dental plaque formation: adsorption of oral streptococci to hydroxyaptite.

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 (Ka) 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 10(9) 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.

Binding Sites

Metabolism of the polysaccharides of human dental plaque. Part II. Purification and properties of Cladosporium resinae (1 leads to 3)-alpha-D-glucanase, and the enzymic hydrolysis of glucans synthesised by extracellular D-glucosyltransferases of oral streptococci.

Cladosporium resinae (1 leads to 3)-alpha-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1 leads to 3)-alpha-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1 leads to 3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1 leads to 3)-alpha-D-glucosidic linkages that occur in mycodextran, isolichein, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucan synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1 leads to 6)-alpha-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1 leads to 3)-alpha-D-glucanase to determine the relative proportions of (1 leads to 6)-linked to (1 leads to 3)-linked sequences of D-glucose residues in the insoluble glucans produce by oral streptococci. The simultaneous action of the two D-glucanoses was highly effective in solubilizing the glucans.

Cladosporium

Specificity of coaggregation reactions between human oral streptococci and strains of Actinomyces viscosus or Actinomyces naeslundii.

Coaggregation reactions between actinomycete and streptococcal cells occurred frequently when human strains of Actinomyces viscosus or A. naeslundii were mixed with human isolates of Streptococcus sanguis or S. mitis, but were infrequent with other oral actinomycetes and streptococci. Two groups of actinomycetes and four groups of streptococci were defined by the patterns of their coaggregation reactions and by the ability of beta-linked galactosides (i.e., lactose) to reverse these reactions. Coaggregations occurred by one of the following three kinds to cell-cell interactions: (i) coaggregation that was blocked by heating the streptococcus but not the actinomycete and was not reversed by lactose; (ii) coaggregation that was blocked by heating the actinomycete but not the streptococcus and was reversed by lactose; and (iii) coaggregation that was blocked only by heating both cell types. The latter reaction was a combination of the first two since lactose reversed coaggregation between heated streptococci and unheated actinomycetes but did not reverse coaggregations between unheated streptococci and heated actinomycetes. Cells that could be heat inactivated also were inactivated by amino group acetylation or protease digestion, whereas cells that were unaffected by heat were not inactivated by these treatments. Coaggregation reactions of each kind were Ca2+ dependent and insensitive to dextranase treatment. These findings are consistent with the hypothesis that human strains of A. viscosus and A. naeslundii coaggregate with strains of S. sanguis and S. mitis by a system of specific cell surface interactions between protein or glycoprotein receptors on one cell type and carbohydrates on the other type.

Acetylation

Changing agglutination activities of salivary immunoglobulin A preparations against oral streptococci.

Immunoglobulin A (IgA) was prepared from samples of parotid saliva collected from two individuals over a 150-day period. Standardized solutions of the IgA were tested for agglutinating antibody activity against strains of streptococcus salivarius, S. sanguis, S. mitis, and S. mutans that had been isolated from a homologous individual on day 30 and preserved in the lyophilized state. The agglutinating activity of the standardized IgA preparations against these strains was found to change over time, whereas the agglutination patterns of IgA prepared from saliva samples collected 3 days apart were generally similar. Differences in agglutination titers were also observed between IgA samples prepared from saliva collected on day 0 and day 150. The data indicate that the oral cavity may harbor streptococci of different antigenic composition representing the same species at a given time and that parotid IgA agglutinating antibody against these serotypes changes over time.

Adult

Acid production from Lycasin, maltitol, sorbitol and xylitol by oral streptococci and lactobacilli.

The acid production from maltitol was compared with the acid production from hydrogenated starch hydrolysate (Lycasin), sorbitol and xylitol by a number of oral strains and reference strains of Streptococcus mutans, S. sanguis, S. salivarius, S. mitior, S. milleri, S. faecalis, S. faecium, S. avium, Lactobacillus casei and L. salivarius. The polyols were added to a final concentration of 1.0% to two different basal media. Incubation was performed at 37 degrees C for 7 days after which the pH was recorded. Maltitol was fermented only by the lactobacilli (about two thirds of the strains). Lycasin was fermented by all strains of S. faecalis, more than 90% of the lactobacilli, about half of the S. sanguis strains, about one third of the S. mutans strains, and by a few other streptococcal strains. Acid production from sorbitol was observed among more than 80% of the S. mutans strains and the S. faecalis strains and most of the lactobacilli strains. Sorbitol-fermenting strains of S. sanguis and of S. mitior, all isolated from sorbitol-consumers, were observed. No other sorbitol-fermenting streptococci were found. Only the reference strains L. salivarius subsp. salivarius ATCC 11741 and S. avium ATCC 14025 fermented xylitol.

Acids