Genetic studies on reference strains of mutans streptococci

Twenty four reference strains (serotype a-h) belonging to the mutans group of streptococci were compared for DNA fragment patterns of rDNA after treatment with Hind III. It was shown that Streptococcus cricetus (serotype a), S. rattus (serotype b), and S. downei (serotype h) reveals comparatively homogeneous patterns while S. mutans (serotype c, e and f) exhibits differences between the different serotypes as well as within single serotypes. S. sobrinus had an intermediary diversity. These data support the previous findings that S. mutans is heterogeneous at the serological, biochemical and genetical level.     

Dental Caries Induction in Experimental Animals by Clinical Strains of Streptococcus mutans Isolated from Japanese Children

Oral implantation and the cariogenic activity of clinical strains of Streptococcus mutans which had been isolated from Japanese children and labeled with streptomycin-resistance were examined in specific pathogen-free Sprague-Dawley rats. All the seven strains tested were easily implanted and persisted during the experimental period. Extensive carious lesions were produced in rats inoculated with clinical strains of S. mutans belonging to serotypes c, d, e, and f, and maintained on caries-inducing diet #2000. Noninfected rats did not develop dental caries when fed diet #2000. Type d S. mutans preferentially induced smooth surface caries in the rats. Strains of other serotypes primarily developed caries of pit and fissure origin. Caries also developed in rats inoculated with reference S. mutans strains BH-TR and FAIR (type b) that had been maintained in the laboratories for many years. However, the cariogenicity of the laboratory strains was found to have decreased markedly. All three S. sanguis strains could be implanted, but only one strain induced definite fissure caries. Two S. salivarius strains could not be implanted well in the rats and therefore they were not cariogenic. Four different species of lactobacilli also failed to induce dental caries in rats subjected to similar caries test regimen on diet #2000. S. mutans strain MT6R (type c) also induce caries in golden hamsters and ICR mice, but of variable degrees.     

Activity of fructanase in batch cultures of oral streptococci

Several strains of oral streptococci produced fructanase when grown in the absence of d-fructan in a complex medium supplemented with d-glucose. The major part of the activity was extracellular, and only 1–5% was associated with the cells. Release of fructanase began early in the exponential phase and the enzyme was stable in the stationary phase for several h if the pH did not fall below 6. Among the strains of Streptococcus mutans, serotypes a, d, and g released the highest amount of fructanase, and the low level of enzyme produced by strains of serotype c was increased when d-fructose replaced d-glucose as carbon source for growth. Fructanase of S. mutans readily hydrolysed (2 → 6)-β-d-fructans, but (2 → 1)-β-d-fructans and inulin were more resistant. Adsorption of fructanase to (2 → 6)-β-d-fructan, or inhibition with Tris buffer, provided effective means of eliminating fructanase activity from culture filtrates. This procedure should permit a more accurate determination of fructosyltransferase activity of S. mutans strains.     

Investigating the candidacy of the serotype specific rhamnan polysaccharide based glycoconjugates to prevent disease caused by the dental pathogen <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.     

Characterization of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> diversity by determining restriction fragment-length polymorphisms of the <Emphasis Type="Italic">gtfB</Emphasis> gene of isolates from 5-year-old children and their mothers

The diversity between Streptococcus mutans clinical isolates from 5-year-old children and their mothers in two South African ethnic groups was investigated. The gtfB gene encoding for glucosyltransferase (EC 2.4.1.5), an enzyme responsible for the synthesis of extracellular polysaccharides was characterized by PCR-RFLP with HaeIII restriction enzyme digestion. Forty-seven children were examined for dental caries and 128 S. mutans clinical isolates cultured from samples of their saliva and plaque and from the saliva of their mothers. Thirty-three children had active caries (70%) and the remainder (n = 14) were caries-free. Caries prevalence was significantly different (p = 0.02) between black African and coloured children, but no differences were found between gtfB amplitypes by caries or ethnic grouping. Thirty-four (27%) of the S. mutans clinical isolates investigated did not ferment melibiose. Melibiose-negative phenotypes (n = 10) isolated from four families showed gtfB RFLP patterns identical to each other. Mothers and children harboured between one and three amplitypes. GtfB amplitypes were identical in 17 families (17/47), of which nine only were identical to S. mutans reference strains. The percentage match between S. mutans amplitype from mothers and their children was low (13%) in the caries-free group compared to children with caries (44%). RFLP analysis of the gtfB gene showed the diversity of S. mutans genotypes within two South African populations that were acquired from mothers and other sources.     

A Role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans

Streptococcus mutans is a major etiological agent in dental caries. Salivary agglutinin is one of the main salivary components binding to S.mutans. To learn more about the interaction of salivary agglutinin with S.mutans, parotid, submandibular, sublingual and palatal saliva samples were incubated with S. mutans suspension. Both depleted saliva samples and bacterial extracts were analyzed by SDS-PAGE and immunoblotting. Salivary agglutinin was present in all types of glandular saliva and in all cases bound to S.mutans, also to PC337C, a P1^– mutant of S.mutans. Agglutinin was separated by SDS-PAGE under reducing and non-reducing conditions and then transferred to nitrocellulose. Non-reduced agglutinin bound S.mutans, but reduced agglutinin did not. Adhesion of S.mutans to agglutinin-coated microplates was inhibited by amine-containing components, 1 M NaCl or KCl and EDTA. Adhesion decreased with decreasing pH with no adhesion below pH 5.0. These data suggest that calcium-dependent electrostatic interactions play a role in binding. By immunoblotting was demonstrated that blood group antigens and Lewis antigens were present on agglutinin. Synthetic blood group antigens and Lewis antigens covalently coupled to polyacrylamide were tested for binding to S.mutans. Only Le^a(Gal1,3(Fuc1,4)GlcNAc) bound to S.mutans, whereas the blood group antigens Le^b, Le^x, Le^y, H1, H2, A, B and sialylated Le^a did not. Le^a without galactose (Fuc1,4GlcNAc) still bound to S. mutans, but Le^a without fucose (Gal1,3GlcNAc) did not. Binding of agglutinin to S. mutans was not inhibited by Le^a. In conclusion, S. mutans can bind to Le^a carbohydrate epitopes in which the fucose is an essential residue. Le^a carbohydrate epitopes are present on salivary agglutinin but play no major role in binding.     

Inhibition of Rat Dental Caries by Dextranase from a Strain of Spicaria violacea

Dextranase AD17 obtained from a culture liquor of a strain of Spicaria violacea was assessed for its ability to inhibit the development of dental caries in conventional Sprague-Dawley rats which had been infected with one of the Streptococcus mutans strains, MT6R (serotype c), OMZ 176R (d), or MT-703R (e). These experiments showed that caries was significantly inhibited when rats were given cariogenic diet # 2000 and drinking water containing AD17 at a concentration of 10 units/g, as compared to control rats not given dextranase. The inhibitory effects of AD17 were more prominent in smooth surface caries than in total caries. AD17 had a tendency to retard both the establishment of inoculated S. mutans and plaque deposition on tooth surfaces. However, S. mutans could be implanted in the rat oral cavity after repeated inoculation of the bacteria, even in the presence of AD17. These results suggest that the anticaries activity of AD17 is due to not only inhibition of adherence of S. mutans cells on tooth surfaces but also to physicochemical changes of dental plaque formed under the enzymatic action of AD17. Preliminary histopathological examination showed that AD17 had no significant toxicity in rats.     

Effective Immunity to Dental Caries: Protection of Gnotobiotic Rats by Local,Immunization with a Ribosomal Preparation from Streptococcus mutans

A ribosomal preparation from Streptococcus mutans 6715 was characterized for its ability to induce an immune response in gnotobiotic rats which was protective against S. mutans-induced dental caries. Animals injected in the salivary gland region with the S. mutans ribosomal vaccine developed significantly higher (P < 0.01) salivary IgA and serum IgG antibody activities against whole S. mutans cells and ribosomal preparations than nonimmunized rats. Vaccinated animals had significantly lower (67%; P < 0.01) levels of S. mutans adherent to their molar surfaces than the control rats after infection with the homologous, cariogenic S. mutans. The immunized animals had significantly fewer (P < 0.01) carious lesions on their buccal, sulcal, and proximal molar surfaces than the nonimmunized rats following challenge with the virulent organism. Animals injected with the ribosomal preparation developed salivary IgA and serum IgG antibodies with specificities to various cell surface-associated antigens such as lipoteichoic acid and glucosyltransferase, suggesting that the observed protection may be due to antibodies against cell surface contaminants of the ribosomal vaccine. These results are the first demonstration that a ribosomal preparation from S. mutans protected rats from caries formation after challenge with the homologous, virulent S. mutans.     

Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram-Positive Bacteria

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.     

Streptococcus mutans and dental caries in humans: a bacteriological and immunological study

Plaque samples from caries-active subjects showed a higher incidence of S. mutans than plaque samples from caries-free subjects. This was especially evident in approximal incisor plaque. S. mutans serotype d was almost exclusively present in approximal plaque obtained from caries-active subjects. Tooth surfaces infected with S. mutans still harbored this micro-organism 10 months later, while uninfected tooth surfaces remained free of S. mutans.Caries development predominantly occurs on those tooth surfaces which harbor relatively high percentages of S. mutans (> 5%). It is unlikely that serum or saliva antibodies against S. mutans play a major role in the protection against dental caries in these caries-free subjects since subjects with the greatest number of decayed surfaces showed the highest antibody titre as measured by haemagglutination or by the enzyme-linked immuno sorbent assay (ELISA).The present investigation shows that serum or salivary antibodies against S. mutans do not seem to play a role in caries resistance in young adults. However, this does not preclude a role of antibodies, prior to infection with S. mutans, in man.     

Isolation and Characterization of the Serotype g Carbohydrate Moiety from an Enzyme Lysate of Streptococcus mutans 6715 Cell Walls

The serotype-specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell-wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo-N-acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C-25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G-100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan—polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A-25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re-chromatographed on a Bio-Gel P-100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti-serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4 : 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti-serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an α-linked galactose-glucose terminal linkage.     

Therapeutic effect of llama derived VHH fragments against Streptococcus mutans on the development of dental caries

Streptococcus mutans is the main cause of dental caries. We evaluated the therapeutic effect of variable regions of a llama heavy chain antibody fragments directed against S. mutans named S36-VHH (S for Streptococcus) alone or fused with glucose oxidase (GOx) from Aspergillus niger. Western blot analysis and ELISA revealed binding of the S36-VHH to the streptococcal antigen I/II adhesin molecule of S. mutans serotype C. In a rat-desalivated caries model, daily administration of S36-VHH significantly reduced the development of smooth surface caries. No additional therapeutic effect of GOx was observed. Our results suggest that llama VHH antibodies may be a potential benefit as prophylaxis against dental caries.     

Single-Plex Quantitative Assays for the Detection and Quantification of Most Pneumococcal Serotypes

Streptococcus pneumoniae globally kills more children than any other infectious disease every year. A prerequisite for pneumococcal disease and transmission is colonization of the nasopharynx. While the introduction of pneumococcal conjugate vaccines has reduced the burden of pneumococcal disease, understanding the impact of vaccination on nasopharyngeal colonization has been hampered by the lack of sensitive quantitative methods for the detection of >90 known S. pneumoniae serotypes. In this work, we developed 27 new quantitative (q)PCR reactions and optimized 26 for a total of 53 qPCR reactions targeting pneumococcal serotypes or serogroups, including all vaccine types. Reactions proved to be target-specific with a limit of detection of 2 genome equivalents per reaction. Given the number of probes required for these assays and their unknown shelf-life, the stability of cryopreserved reagents was evaluated. Our studies demonstrate that two-year cryopreserved probes had similar limit of detection as freshly-diluted probes. Moreover, efficiency and limit of detection of 1-month cryopreserved, ready-to-use, qPCR reaction mixtures were similar to those of freshly prepared mixtures. Using these reactions, our proof-of-concept studies utilizing nasopharyngeal samples (N=30) collected from young children detected samples containing ≥2 serotypes/serogroups. Samples colonized by multiple serotypes/serogroups always had a serotype that contributes at least 50% of the pneumococcal load. In addition, a molecular approach called S6-q(PCR)² was developed and proven to individually detect and quantify epidemiologically-important serogroup 6 strains including 6A, 6B, 6C and 6D. This technology will be useful for epidemiological studies, diagnostic platforms and to study the pneumobiome.     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.     

Electrophoretic studies of extracellular glucosyltransferases and fructosyltransferases from seventeen strains of Streptococcus mutans

Streptococcus mutans was classified by the electrophoretic properties of glucosyltransferases (GTases) and fructosyltransferases (FTases). The cells of serotypes a, d and g did not release extracellular FTases, although those from other serotypes did. The enzymes from cells of serotypes d and g synthesized a good deal of insoluble polysaccharide compared with other serotypes. The enzymes were applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polyacrylamide gel-isoelectric focussing (PAG-IEF). Gels were stained for their activity and protein content. Enzymes belonging to the same serotype gave the same specific pattern on both gels. The seven serotypes could be classified into the following four groups: serotypes d and g, serotype a, serotypes c, e and f, and serotype b. The results agree well with some previous reports based on other methods. The molecular weights of three GTase bands were 156K, 146K and 135K, and of four kinds of FTase bands were 108K, 95K, 80K and 76K. The isoelectric points of main enzymes were 4.25, 4.60, 5.00, 5.55 and 5.70. Those of FTases were 4.25 and 4.60.Abbreviations GTase glucosyltransferase - FTase fructosyltransferase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PAG-IEF polyacrylamide gel-isoelectric focussing - PAS periodic acid-Schiff     

Sterilization effects of a TiO<Subscript>2</Subscript> photocatalytic film against a<Emphasis Type="Italic">Streptococcus mutans</Emphasis> culture

Streptococcus mutans is one of the more significant pathogens involved in the development of dental caries in humans. The purpose of this research was to design a TiO₂-coated dental instrument and to determine the bactericidal effects of the instrument onS. mutants. TiO₂ photocatalytic films were prepared by the low-pressure metal-organic chemical vapor deposition (LPMOCVD) method using titanium tetraisopropoxide (TTIP) as precursor. The photocatalytic reaction was carried out on a TiO₂-coated pyrex petri dish with an ultraviolet (UV) light emitting diode (LED) illuminator or a fluorescent lamp light source. Our data indicates that the relative survival ratio ofS. mutans when plated onto TiO₂ photocatalytic films and under exposure to UV-A light for 15 min was 0.01%. In addition, a fluorescent lamp light source also had bactericidal effects on theS. mutans plated TiO₂ photocatalytic films. These results indicate that TiO₂-coated dental materials or devices may be useful in dental treatments for the prevention of carious or enamel demineralization.     

Effect of neovestitol–vestitol containing Brazilian red propolis on accumulation of biofilm in vitro and development of dental caries in vivo

The present study examined the influences of the neovestitol–vestitol (NV) containing fraction isolated from Brazilian red propolis on the development of biofilm and expression of virulence factors by Streptococcus mutans using saliva-coated surfaces of hydroxyapatite. In addition, NV was tested in a rodent model of dental caries to assess its potential effectiveness in vivo. Topical applications of NV (800 μg ml^?1) significantly impaired the accumulation of biofilms of S. mutans by largely disrupting the synthesis of glucosyltransferase-derived exopolysaccharides and the expression of genes associated with the adaptive stress response, such as copYAZ and sloA. Of even greater impact, NV was as effective as fluoride (positive control) in reducing the development of carious lesions in vivo. NV is a promising natural anti-biofilm agent that targets essential virulence traits in S. mutans, which are associated with the formation of cariogenic biofilm and the subsequent onset of dental caries disease.     

Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand

One hundred and eighty-seven strains of Cryptococcus neoformans isolated from patients in Thailand were charcterized by biochemical varieties relating to serogroups. Canavanine-glycine-bromothymol blue (CGB) agar was used for differentiating the varieties of C. neoformans. Slide agglutination tests were performed with Crypto Check (Iatron, Inc., Tokyo) to determine their serotypes. Fifty-five percent (10 out of 18) of the pre-AIDS isolates were serotype B, 28% were serotype A, 5% were serotype D, and an unexpected 11% (2 out of 18) were serotype C. These are the first to be recorded in Asia. In contrast, among the 169 clinical isolates obtained between January 1993 and March 1995 (AIDS epidemic), serotype A was outstandingly predominant-93% (157 out of 169), serotype B was relatively low (3.6%) and both serotypes D and AD were 1.8%. The pattern of serotypes of the 59 isolates from known HIV-positive patients was closely similar to the total isolates during the AIDS epidemic. In determining the varieties of C. neoformans by CGB, only 1 of the 187 isolates gave a false reaction. On the basis of our findings, we believe that in the pre-AIDS era either C. neoformans var. gattii serorype B or serotype C were the common causative agents of cryptococcosis in Thailand. The advent of AIDS changed the pattern of serotypes with serotype A becoming predominant as has been reported world wide.     

Survey of plasmid profiles of <Emphasis Type="Italic">Shigella</Emphasis> species isolated in Malaysia during 1994–2000

Summary Plasmid profiling was used to characterize 219 strains of Shigellaspecies isolated from sporadic cases of shigellosis in Malaysia during the period 1994–2000. Heterogeneous plasmid patterns were observed in all Shigella spp. There was a correlation between plasmid patterns and serotypes of S. flexneri, S. dysenteriaeand S. sonnei. Five common small plasmids (>20.0 kb) were observed in S. flexneri1b and 2a, whereas six common small plasmids were found in serotype 3a. Some of these plasmids appeared to maintain their existence stably in each individual serotype. Plasmids of size 11.40 and 4.20 kb were present only in S. flexneri2a isolates, whereas the 4.40 kb plasmid was unique for serotype 3a. Large (>150 kb) or mid-range plasmid (20.0–150 kb) was not observed from any S. flexneri1b isolates. Eighty-nine percent of S. flexneriof various serotypes harboured the plasmid of 3.20 kb. All S. dysenteriaetype 2 isolates harboured the 9.00 kb plasmid, while four common small plasmids were found in S. sonneiisolates. The 2.10 kb plasmid was only seen in S. sonnei. Streptomycin resistance in S. dysenteriaetype 2 and multi-drug resistance in S. sonneimay be associated with the 9.00 and 14.8 kb plasmids, respectively. Plasmid profiling provided a further discrimination beyond serotyping and a useful alternative genotypic marker for differentiation of Shigellaspecies. To the best of our knowledge, this is the first report on the plasmid prevalence of the Malaysian Shigellaspecies.     

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

An inhibitor of $\text{Streptococcus}$,$\text{mutans}$ endodextranase was detected in proteins prepared from batch cultures of $\text{S.}$,$\text{mutans}$ strains representing serotypes $\text{a}$ through $\text{g}$. Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of $\text{S.}$,$\text{mutans}$ known to produce this enzyme.