In vitro inhibition of adherence of Streptococcus mutans strains by nonadherent mutants of S. mutans 6715.

Four nonadherent mutants from Streptococcus mutans 6715 mutant UAB66 (serotype g) with similar phenotypes were shown to inhibit the adherence of adherence-proficient S. mutans serotypes c and g strains. One mutant, UAB108, was shown to inhibit adherence by wild-type strains representing serotypes a, d, and e as well. This inhibition of adherence was seen with pairs of strains grown in partially defined (PD) medium supplemented with 1% sucrose in both microtiter plates and glass tubes. The inhibiting factor was present in culture supernatant fluids of inhibiting strains grown in PD medium plus 1% sucrose and was heat stable. Ethanol precipitation of culture supernatant fluids of these strains yielded a water-soluble polymer which effectively inhibited the adherence of UAB66. This polymer, isolated from UAB108, was also shown to inhibit the adherence of UAB66 at lower concentrations than that needed to inhibit adherence with dextran T10. Partially purified glucosyltransferase, isolated from the culture supernatant fluids of glucose-grown UAB108, produced a water-soluble glucan which was shown to inhibit the adherence of UAB66 as well. The methods developed permit rapid screening for strains or mutants of strains or both that inhibit adherence or plaque formation or both by wild-type strains of S. mutans.     

Decreased oral colonization of Streptococcus mutans during aging of Sprague-Dawley rats.

The colonization by streptomycin-resistant Streptococcus mutans strains of the teeth of conventional and ex-germfree Sprague-Dawley rats of various ages fed either a high-sucrose or a high-glucose diet was studied. Bacterial colonization occurred with increasingly greater difficulty as the rats became older. This was observed in studies of the implantation of the test organism after oral inoculation with different cell numbers as well as its transmission between infected and uninfected rats. With rat fed sucrose diet, the effect of age could not be demonstrated until they were age 3 months or older; the results from rats fed a glucose diet suggest that changes may already have occurred early after weaning. Changes in susceptibility to colonization during aging manifested themselves as a decrease in the proportions of rats which became infected as well as lower population levels in infected rats. The possible mechanism(s) involved as well as the possible significance of the findings was discussed.     

Preemption of Streptococcus mutans 10449S colonization by its mutant 805.

Oral infection of rats with Streptococcus mutans mutant 805 was used to prevent the establishment of its highly virulent wild-type progenitor NCTC 10449S. The dose of wild-type cells required to colonize 100% of the specific pathogen-free Osborne-Mendel rats (21 to 43 days old) consuming caries test diet 2000 was greater than 4 x 10(5) but less than 4 x 10(6) cells. Therefore, the latter dose was used to challenge rats which had already been colonized by an oral dose of about 6 x 10(8) cells of mutant 805. This prior infection by 805 either completely protected rats from subsequent colonization by wild-type cells or greatly delayed and diminished their emergence. Rats in which wild-type cells became established showed much lower percentages of wild-type cells in their total recoverable floras than did rats that were not first infected by the mutant. Large doses of mutant 805, however, did not displace wild-type cells from rats once it became established. There was no evidence of reversion of the mutant, which is defective in intracellular polysaccharide synthesis and hence is less virulent than wild-type cells. The data indicate that the S. mutans cell which first colonizes rats gains the strongest ecological position and is difficult to displace. Also, they suggest the possible prophylactic utility of infection by this mutant of S. mutans.     

Effects of local immunization with glucosyltransferase on colonization of hamsters by Streptococcus mutans.

Experiments were performed to study the effect of antibody to Streptococcus mutans glucosyltransferase (GTF) on the implantation of these organisms in hamsters. Salivary (immunoglobulin A) and serum (immunoglobulin G) antibodies to GTF and GTF-inhibiting activity were elicited by injection of GTF in Freund complete adjuvant in the salivary gland region. Sham-immunized and GTF-immunized groups were then orally challenged with approximately 10(7), 10(8), or 10(9) colony-forming units of cariogenic S. mutans 6715. The results were evaluated by systematically swabbing molars 4 days and approximately 4 weeks after challenge. In general, fewer GTF-immunized hamsters became infected with S. mutans after challenge with 10(7) or 10(8) organisms than did identically challenged sham-immunized hamsters. Of the animals that did become infected, fewer S. mutans colony-forming units were recovered from GTF-immunized hamsters. These results indicate that the presence of antibody to GTF can diminish the ability of S. mutans to implant in the oral cavity of immunized hamsters.     

In vitro and in vivo complementation of Streptococcus mutans mutants defective in adherence

Previous studies have shown that adherence-defective mutants of Streptococcus mutans PS14, serotype c, can be grouped into several different phenotypic groups. In this study a method was developed to test for complementation between pairs of nonadhering mutants which possess different genotypic defects. Mutant strains UAB95 and a spectinomycin-resistant derivative of UAB95 (UAB516) were found to exhibit increased levels of adherence when grown together with UAB230 in media containing sucrose as compared to the adherence of each strain grown separately. An increase in caries was also observed in gnotobiotic rats mixedly infected with the two mutants as compared to either strain alone. Tests revealed that UAB95 produced more water-insoluble glucan than its parent strain but had a defect in glucan binding. UAB230 was found to produce levels of a defective glucan that could not be bound by mutant or wild-type cells. Our results suggest that UAB95 produces a water-insoluble glucan which is bound by UAB230, thus allowing complementation for adherence and caries production.     

Impaired colonization of gnotobiotic and conventional rats by streptomycin-resistant strains of Streptococcus mutans.

Colonization of streptomycin-resistant mutants derived from Streptococcus mutans strain LB1, a human isolate, and strain FA-1, a rodent isolate, was studied in gnotobiotic and conventional rats. Mutants resistent to 2.0 mg of streptomycin per ml were isolated by using both stepwise (suffix "R"M) and one-step (suffix "R"1) selections. Rats were infected with mixtures of parental and streptomycin-resistant strains, and the proportions of each strain present in samples from the intestinal canal, tongue dorsum, teeth, and fissure plaque were determined. Combinations of strains investigated were LB1 and FA-1"R"M; FA-1 and LB1"R"M; LB1 and LB1"R"1; FA-1 and FA-1"R"1. In gnotobiotic rats, nonresistant strains predominated in every oral sample studied at 7 and 21 days after infection. Similarly, when conventional exgermfree rats were infected with FA-1 and FA-1"R"1, FA-1 dominated in all samples. Streptomycin-sensitive revertants were not detected in rats monoinfected with strains LB1"R"1 and FA-1"R"1 for 21 days. No antagonistic interactions were observed between the strains in in vitro experiments. Streptomycin-resistent mutants attached to hydroxyapatite treated with rat or human saliva in equal or higher numbers than did parental strains. However, parental strains appeared to grow faster in Trypticase soy broth then streptomycin-resistant mutants. These observations indicate that induction of streptomycin resistance frequently impairs the colonization properties of S. mutans strains, possibly by altering their rate of growth.     

Differential inhibition of Streptococcus mutans in vitro adherence by anti-glucosyltransferase antibodies.

Antibodies prepared against an insoluble-soluble glucan-synthesizing fraction significantly inhibited in vitro adherence of Streptococcus mutans, whereas antibodies directed against a soluble glucan-synthesizing fraction were much less inhibitory.     

Binding of a Streptococcus mutans cationic protein to kidney in vitro.

An 8-kDa protein, with binding activity for heparin and heparan sulfate of basal laminae of animal tissues, was isolated from Streptococcus mutans MT703 and purified to homogeneity. Binding of radioiodinated 8-kDa protein to rabbit kidney tissue in vitro showed a high degree of specificity, as indicated by saturation kinetics, time dependence, and competitive inhibition by unlabeled protein. Binding activity for kidney tissue was competitively inhibited by selected glycosaminoglycans and polyanions in the following order: heparin greater than dextran sulfate greater than heparan sulfate greater than chondroitin sulfate greater than lipoteichoic acid greater than keratan sulfate greater than hyaluronic acid. Binding of the streptococcal protein to rabbit kidney tissue was also strongly inhibited by protamine sulfate, polylysine, and a random copolymer of lysine and alanine. Among the monosaccharides tested at 50 mM, glucosamine 2,3- or 2,6-disulfate, glucuronic acid, glucose 6-phosphate, and glucose 6-sulfate inhibited 50% or more of the binding activity, whereas N-acetylglucosamine 3-sulfate, glucosamine 6-sulfate, N-acetyl-glucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and a selection of neutral sugars were not inhibitory. The heparin-binding protein was detected on the cell wall of S. mutans and in the culture medium following growth. Several other species of streptococci produce an immunologically related protein of similar size.     

Antigens of Streptococcus mutans I. Characterization of a Serotype-Specific Determinant from Streptococcus mutans

A membrane-associated glycerol teichoic acid antigen has been isolated from Streptococcus mutans AHT and a similar antigen has been demonstrated to be present in each of the other Bratthall serotype a organisms studied. Trichloroacetic acid-extracted material was resolved into two phosphorus-containing antigenic fractions (B and C) by agarose chromatography. Fraction B was preliminarily identified as a phospholipid moiety with a glycerol-to-phosphorus ratio of 2:1, and fraction C showed a ratio of 1:1 indicative of a glycerol teichoic acid. This latter fraction also was associated with glucose, galactose, alanine, and fatty acids. Diglycerol triphosphate, the compound characteristically released from 1-3 phosphodiester-linked glycerol teichoic acids by alkaline hydrolysis, was isolated and characterized. Alanine was identified as its alkaline-labile, ester-linked D-isomer. A glyceride was isolated containing a disaccharide of glucose and galactose attached to the 2-hydroxyl group of glycerol. Hapten inhibition analysis demonstrated that beta-galactosides were the greatest inhibitors of the precipitin reaction (>75%), whereas glucose and its derivatives inhibited to a much lesser extent (<30%). Comparative immunodiffusion and immuno-electrophoresis analyses demonstrated that all six Bratthall serotype a organisms tested contained this antigenic determinant and that it was absent in serotypes b, c, and d. It is suggested that the common antigenic determinant of this serotype within S. mutans may be a beta-galactoside associated with a glycerol teichoic acid and possibly other polymers.     

Plasmid-mediated transformation of Streptococcus mutans.

Streptococcus mutans GS-5 was transformed to erythromycin resistance with streptococcal plasmid pVA736. Transformation frequencies were higher with plasmids reisolated from transformed GS-5 cells relative to plasmid originally derived from S. sanguis Challis.     

Expression of Streptococcus mutans gtf genes in Streptococcus milleri.

The Streptococcus mutans glucosyltransferase (GTF) genes gtfB and gtfC were ligated into Escherichia coli-streptococcus shuttle plasmids and introduced into Streptococcus milleri. gtfB transformant KSB8 formed an S. mutans-like rough colony on mitis salivarius agar and expressed an extracellular GTF-I, of 158 kDa, and two cell-bound GTF-Is, of 158 and 135 kDa. gtfC transformant KSC43 formed a semirough colony on mitis salivarius agar and expressed primarily an extracellular GTF-SI, of 146 kDa, and two cell-bound GTF-SIs, of 146 and 152 kDa. The extracellular GTFs from KSB8 and KSC43 were purified and characterized. The two types of GTF also reacted specifically with monoclonal antibodies directed against each enzyme. Both enzymes synthesized significant amounts of oligosaccharides, consisting primarily of alpha-1,6-glucosidic linkages, as well as water-insoluble glucans, containing alpha-1,3-glucosidic linkages. Insoluble-glucan-synthesizing activities of both enzymes were stimulated (three- to sixfold) by the addition of dextran T10 and were inhibited in the presence of 1.5 M ammonium sulfate. The Km(s) for sucrose and the optimal pHs were also similar for both enzymes. However, when the transformants were grown in Todd-Hewitt broth supplemented with sucrose, KSC43 cells, expressing GTF-SI activity, adhered to glass surfaces in vitro, while KSB8 cells, expressing GTF-I activity, did not. These results are discussed relative to the potential role of the gtfB and gftC genes in S. mutans cariogenicity.     

Use of monoclonal antibodies in local passive immunization to prevent colonization of human teeth by Streptococcus mutans.

Local passive immunization with monoclonal antibodies (MAbs) raised against streptococcal antigen (SA) I/II protects monkeys against colonization of teeth by Streptococcus mutans and the subsequent development of dental caries. In this study we extended the preclinical experiments to human subjects. In the first study of eight healthy subjects, four had anti-SA I/II MAb (immunoglobulin G2a [IgG2a]) and four had saline applied to their teeth on three occasions. A streptomycin-resistant S. mutans strain (Guy K2 strain, serotype c) was then implanted onto the teeth, and the organism was cultured sequentially from dental plaque and saliva up to 100 days after the first treatment with MAb. Decreased colonization by S. mutans was found in the dental plaque collected from smooth surfaces and fissures and in saliva of subjects whose teeth were treated with the MAb, as compared with the saline-treated control subjects. The experiment was then repeated on seven new subjects, and the effect of anti-SA I/II MAb was compared with that of an unrelated MAb to Campylobacter jejuni. The results again showed a consistently lower level of colonization of teeth in the anti-SA I/II MAb-treated subjects as compared with those sham immunized with the unrelated MAb. There was little difference in serum IgG, IgM, or IgA, gingival fluid IgG, or salivary IgA anti-SA I/II antibodies between the immunized and sham-immunized subjects, before and after the investigation. No side effects were observed, and the gingival and plaque indices remained unchanged. A sensitive radioimmunoassay failed to detect changes in anti-MAb (IgG2a) antibodies in any of the three fluids examined. We suggest that local passive immunization by means of MAb might be an alternative approach in the prevention of colonization of teeth by S. mutans and the development of dental caries.     

Growth of Streptococcus mutans on various selective media.

The ability of Streptococcus mutans to grow on mitis-salivarius (MS) agar, MC agar, mitis-sucrose-bacitracin (MSB), BCY agar, and MM10 sucrose agar was studied. Batch cultures of S. mutans serotype a demonstrated no growth on MSB agar. Certain serotype d and g strains did not grow on MC agar. The yield for most strains of other serotypes on these selective media was lower compared with that on MS agar. The number of total colony-forming units on BCY and MM10 sucrose agar was similar to the blood agar results. Similar data were obtained when fermenter-grown strains, harvested in the middle or the end of the logarithmic growth phase, were used for inoculation of the various media. Enumeration of S. mutans from plaque samples plated on MC and MSB agar yielded about 75% of the counts obtained on MS or the nonselective medium. When the proportions of S. mutans were expressed as a percentage of the total cultivable flora, the selective media (MC and MSB agar) showed approximately 10% lower values than the MS, BCY, and MM10 sucrose agar.     

Growth rates of Actinomyces viscosus and Streptococcus mutans during early colonization of tooth surfaces in gnotobiotic rats.

Germfree Osborne-Mendel rats were monoassociated with Actinomyces viscosus or Streptococcus mutans. The adherence and subsequent growth of these organisms on the tooth surface was studied by means of total viable cell counts. Both A. viscosus and S. mutans showed a lag phase and an exponential growth phase, similar to logarithmic growth in batch cultures. The exponential growth rates of S. mutans and A. viscosus were 0.63 h-1 (doubling time [td] = 1.1 h) and 0.24 h-1 (td = 2.9 h), respectively. After a period of rapid growth, the rate declined and the populations approached a steady state. The presence of a sucrose-containing diet did not significantly influence the exponential growth rates of A. viscosus and S. mutans, but had a slight negative effect on the initial adherence of S. mutans at the tooth surface.     

Inhibitory Effects of MoAbs against a Surface Protein Antigen in Real-Time Adherence In vitro and Recolonization In vivo of Streptococcus mutans

A surface protein antigen (PAc) of Streptococcus mutans, particularly the A-region of the molecule, has been reported to interact with salivary components on the tooth surface. It might be a candidate antigen inducing the production of antibodies against the adherence of S. mutans to the tooth surface. We investigated the effects of monoclonal antibodies (MoAbs) obtained by immunization of synthetic PAc peptides that completely correspond to the amino acid sequence of part of the A-region. These MoAbs recognize several core B-cell epitopes in the sequence. Two (KH5 and SH2) of these antibodies reacted with both S. mutans and Streptococcus sobrinus, but not with Streptococcus sanguis, Streptococcus salivarius, Porphyromonas gingivalis or Lactobacillus casei. They clearly inhibited the real-time adherence of S. mutans to salivary components in a biosensor. KH5, which showed a real-time inhibition (71%), also significantly prevented the recolonization of S. mutans on the tooth surface in rats. These results suggested that the core B-cell epitope (-Y---L--Y----) recognized by KH5 was the essential sequence in the antigenic epitopes of PAc protein recognized specifically by the inhibitory antibody. Therefore, the amino acid residues were found to be important in the initial attachment of S. mutans to the tooth surface. These results provide for the mechanism of PAc molecule in the initial attachment of S. mutans on the tooth surface and more effective designs for the removal of S. mutans and S. sobrinus from the oral cavity.     

Effect of microbial interaction on the colonization rate of Actinomyces viscosus or Streptococcus mutans in the dental plaque of rats.

The resident oral microflora of conventional Osborne-Mendel rats was challenged with Actinomyces viscosus or Streptococcus mutans strains. The adherence of the inoculated organism to the tooth surface and the subsequent growth were studied by means of viable counts determination. The initial growth rate of S. mutans in conventional rats was lower than in mono-associated gnotobiotic rats (doubling time, td = 5 h versus td = 1.1 h). The delayed start of growth and the low initial growth rate indicated that a competitive interaction between S. mutans and the resident microflora occurred. The initial growth rate of A. viscosus in conventional rats (td = 3.1 h) was approximately the same as that in gnotobiotic rats (td = 2.8 h). The start of growth of A. viscosus was only slightly delayed compared with the start in gnotobiotic rats. These results suggest a neutralistic relationship between A. viscosus and the resident microflora. A. viscosus reached a stationary level about 7 days after inoculation, whereas the S. mutans strains did not reach stationary levels until 2 weeks after inoculation.     

Pyruvate dehydrogenase activity in Streptococcus mutans.

Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.     

Fluoride uptake by Streptococcus mutans 6715.

The short-term kinetics of fluoride uptake by cells from 20- to 22-h cultures of Streptococcus mutans strain 6715 were studied using rapid filtration and centrifugation techniques. Saline-suspended organisms were diluted with fluoride-containing solutions buffered at four different pH values (2.0, 4.0, 5.5, and 8.2). Fluoride disappearance from the medium was inversely related to pH and to the duration of the exposure at any given pH. The uptake was rapid and extensive at the lower pH values and decreased as the pH increased. Media fluoride concentrations subsequently increased; i.e., fluoride was released from the cells. The presence of glucose, cyanide, or iodoacetate did not influence the results. However, preincubation of the cells in fluoride-free buffers, followed by the addition of fluoride, reduced fluoride uptake markedly. Cell-to-media pH gradients were determined by the distribution of 14C-labeled 5,5-dimethyl-2,4-oxazolidinedione. Fluoride uptake was found to be a function of the magnitude of the pH gradient (P less than 0.001). It is hypothesized that fluoride uptake occurs by the diffusion of hydrogen fluoride and the subsequent trapping of ionic fluoride.     

Effects of oxygen on glucose-limited growth of Streptococcus mutans.

Aerotolerance in the growth of Streptococcus mutans and related streptococci was examined under glucose-limited conditions. The growth rate of all strains tested was more or less retarded when they were transferred from anaerobic to aerobic conditions. As for growth yield, however, some strains (group 1) showed reduced values for the increment of cellular dry weight change (in grams per mole of glucose), whereas others showed either unaltered (group 2) or increased (group 3) yields. The characteristic feature of strains in groups 2 and 3 was their high activity of both glucose- and pyruvate-dependent oxygen uptake when strains were grown under aerobic conditions. By contrast, the activity of pyruvate-dependent oxygen uptake by group 1 strains was negligibly low, whether grown under aerobic or anaerobic conditions. Some strains (group 1a) consumed oxygen in the presence of glucose at a much faster rate than others did (group 1b). There seems to be a good correspondence of these aerotolerance groupings to those based on serotypes of S. mutans. Thus, the groups 1a, 1b, 2, and 3 correspond to serotypes g, a+d, c+f, and b, respectively.     

Comparative recovery of Streptococcus mutans on ten isolation media.

The ability of Streptococcus mutans (Bratthall serotypes a through e) to grow on 10 isolation media was examined. The number and morphology of the colonies were observed to vary on different media. The use of blood-sucrose media consistently produced the highest recoveries. Mitis salivarius agar (MS) and higher recovery values than modified medium 10 (MM10SB), Trypticase-yeast extract-cystine medium (TYC), or MS with 1% tellurite (MST). MST with 40% sucrose (MS40S), MST with 20% sucrose and 0.2 U of bacitracin per ml (MSB), and Carlsson medium with 1% sulfasoxazole (MC), media formulated for the selection of S. mutans, were the most inhibitory for all serotypes. The morphology of several S. mutans strains was atypical on MC and MS40S, making positive identification difficult. Absence of growth of serotype a strains on MSB and serotype d strains on MC were the two major differences observed among the serotypes. Results are discussed in terms of the difficulties in making quantitative determinations from cultural data.