Komei Fukui

Detection of Streptococcus mutans by PCR amplification of spaP gene

Synthetic oligonucleotide primers were used in the polymerase chain reaction (PCR) to amplify a sequence of the spaP gene, which encodes the surface protein antigen I/II of Streptococcus mutans. A DNA fragment of c. 192 bp was amplified from lysed S. mutans cells or isolated DNA. With S. mutans cells, the lower limit of detection was 4-40 cfu. With these primers, 13 reference and 50 clinical strains of S. mutans were identified. Amplification of the 192-bp product was not demonstrated when 41 strains of other streptococcal and non-streptococcal species were tested. The spaP gene PCR has potential for the rapid diagnosis of S. mutans infections.

Serological typing of reference strains and clinical isolates of Streptococcus mutans by agglutination reactions and/or radioimmunoassay.

Rabbit antisera were prepared against eight different serotypes of Streptococcus mutans. After absorption with heterologous serotype strains the antisera were able to distinguish 21 reference strains of different serotypes of S. mutans by simple agglutination reactions and radioimmunoassay, both using intact whole cells. The reference serotypes a, b, d and g were differentiated by specific agglutination using the corresponding monospecific antiserum. Strains of serotypes c, e, f and h were discriminated by differential agglutination using anti-c, anti-e, anti-f or anti-h serum respectively, but the antisera cross-reacted slightly with other serotype strains. Using these antisera a total of 261 clinical isolates of S. mutans from human dental plaques were serotyped by agglutination reactions, in addition to which radioimmunoassay and the classical immunodiffusion tests in agar were also used in some cases. The results were quite consistent in the three methods. The predominant strain was of serotype c (187), followed by serotype f (28), g (10), d (5), e (4) and b (1). 26 strains were untypable and there were no serotype a or h strains.

A comparison of competitive enzyme immunoassay and precipitin inhibition tests in the analysis of polysaccharide antigenic determinants of oral streptococci.

A rapid and convenient method of competitive enzyme immunoassay (EIA) was explored for the immunological analysis by MAbs of polysaccharide antigens prepared from the mutans group of streptococci, and optimal conditions for it were established. These included the concentrations of antigen, monoclonal antibodies (MAbs) and labelled antibody, temperature, pH and reaction time. The results were compared with those obtained by precipitin inhibition tests using the classical quantitative precipitin reaction, and they were found to be consistent with the latter method. It was concluded that the EIA system can be extended to the immunological study of polysaccharide antigens of oral streptococci using MAbs which perform poorly in precipitation reactions.

Streptococcus mutans serotype b strain (St. rattus, Coykendall): first isolation in Japan from human dental plaque.

Streptococcus mutans has been implicated for many years as one of the major microorganisms responsible for dental caries in man and animals (1, 6, 14, 20, 21). The bacterial species has been divided into groups on the basis of biochemical (1, 29, 31) or serological (1, 3, 4, 27) reactions, and DNA homology (8, 9, 12). So far eight different serological types (a through h) have been recognized (1, 20, 27). However, the predominant clinical isolates from human material have been of serotype c, followed by serotypes d, g, e, and f (7, 19, 20, 25, 29). In particular serotype b has never been isolated in Japan from human dental plaque whereas it was previously found in some other countries (2, 5, 13, 15, 16, 34). This communication deals with the first isolation in Japan of a serotype b strain of S. mutans (Streptococcus rattus nom. rev. after Coykendall) from human dental plaque and describes its biochemical and serological characteristics. Nineteen strains of various known serotypes (a through h) of S. mutans, HSI, E49, FA1, BHT, Ingbritt, MT6R, OMZ176, MT615R, LM7, P4, MT703, SE17, OMZ175, MT557, OMZ65, 6715, KIR, MF25, BFe12, were used as reference strains. They were generous gifts of Dr. Shigeyuki Hamada (National Institute of Health, Tokyo) and of Dr. R.R.B. Russell (Dental Research Unit, Royal College of Surgeons of England, Surrey, England). Dental plaques were sampled from the first and second molar teeth of 261 female students ranging from 18 to 21 years old and suspended in sterile saline. A portion of the suspension was cultured on MitisSalivarius (MS, Difco) agar plates containing bacitracin or potassium tellurite in a CO2 GasPak system (BBL). All reference strains and isolates suspected as S. mutans on MS agar plates were examined for biochemical and other properties following essentially the previously described methods (1, 29, 31) ; morphology in a gram-stained preparation, acid from various substrates added to a basal medium, Phenol Red Broth Base (Difco), ability to hydrolyze arginine and esculin, growth in brain heart infusion broth (BHI, Difco) at 45 C or 10 C or containing 6.5% NaC1 or adjusted to pH 9.6, hydrogen peroxide production, sensitivity to bacitracin,

Type-specific antigen of Streptococcus rattus strains (KAY1, FA1 and BHT). II: Ultrastructural localization

Streptococcus rattus strains (KAY1, FA1 and BHT) were examined by electron microscopy in whole cell-mounted samples and ultrathin sections for the localization of the type-specific antigen, using specific antiserum and ferritin-labelled anti-rabbit IgG as secondary antibody. In thin sections as well as whole cell-mounted samples the antigen was observed as irregular masses over the entire surface of the cells, in particular in the septal region between cells. The localization of the antigen was also examined in thin sections of a cell wall fraction prepared by treating the whole cells with glass beads in a disintegrator. Ferritin particles were observed around the outer surface, but not as many in number as those in whole cell-mounted or sectioned samples and rarely on the inner surface of the cell wall.

Type-Specific Antigen of Streptococcus rattus Strains (KAY1 and FA1). I. Characterization

A serotype-specific antigen was extracted from Streptococcus rattus KAY1 strain isolated first in Japan from human dental plaque and purified on an ion exchange column to compare it chemically and immunologically with that of FA1 strain which had been examined extensively by previous workers. Antigens of both strains reacted in a double diffusion test specifically with anti-FA1 serum which had previously been demonstrated specific for the strains in the same test, agglutination reactions and/or radioimmunoassay using whole cells. After separation on a DEAE-Sephadex A-25 ion exchange column the antigen was found to be resistant to various enzymatic treatments with pronases, lipase and nucleases and produce a single precipitin band against absorbed anti-FA1 serum in immunoelectrophoresis. Chemical analysis of this antigen revealed that it composed of carbohydrate, protein and a few percentages of glycerol and phosphorus. Hapten inhibition tests between antigen and antibody showed that galactose as well as glucose were the most potent inhibitors, suggesting their involvement in the antigenic determinant. Involvement of the sugars was also supported by gas chromatographic analysis and abolishment of reactivity with antiserum after the treatment of antigens with NaIO4. Moreover, protein does not seem to be involved since after SDS-PAGE analysis an enzyme immunoassay gave a negative reaction with immunoblotted antigens.

Directional immobilization of sodium- and potassium-activated ATPase to expose its cytoplasmic part to the liquid phase on microtiter plates by wheat germ agglutinin.

A convenient method for highly efficient and directional immobilization of intact sodium- and potassium-activated ATPase (Na,K-ATPase) using wheat germ agglutinin linked on microtiter plates was developed. Wheat germ agglutinin, which bound tightly to the beta-subunit of Na,K-ATPase and had no effect on the Na,K-ATPase activity, the potassium-activated p-nitrophenylphosphatase activity, or the inhibitory action of ouabain, was covalently linked to microtiter plates and used as an immobilizer of the enzyme. The amount of Na,K-ATPase coupled to microtiter plates in this immobilizing system was more than 10-fold greater than that used in the direct immobilizing system (O. Urayama, M. Nakao, H. Nagamune, and H. Sugiyama, (1984) Anal. Biochem. 141, 194-198). Also in this system, the cytoplasmic domain of Na,K-ATPase was exposed to the liquid phase. This technique was useful for investigating the reactivities of monoclonal antibody specific for the cytoplasmic domain of the enzyme. Moreover, because this technique was used successfully in the immobilization of periodic acid--Schiff positive staining glycoprotein 1 prepared from human erythrocytes and human alpha 2-macroglobulin, the technique should also be useful for other membrane or secreted proteins that possess N-linked sugar chains containing bisecting N-acetylglucosamine or a high amount of sialic acid.

Lysis of Streptococcus mutans with Achromopeptidase and Release of Osmotically Fragile Bodies

Until recently research on the genetics, metabolism and membrane structure of Streptococcus mutans has been hampered by the resistance of this bacterium to enzymatic hydrolysis (3). The difficulty, however, was partially overcome by the development of several techniques involving addition of high concentrations of salt (4), high pH (14), or detergent to cause lysis of lysozyme-treated cells (2). On the other hand, the search for enzymes capable of hydrolyzing cell walls or intact cells of S. mutans indicated that the L-ll enzyme of a Flavobacterium species (9), the M-l enzyme of Streptomyces globisporus (16) and the H-402 enzyme from Streptomyces griseus (17) could cause lysis. Only a few of these enzymes enabled a few investigators to succeed in the formation of protop lasts or osmotically fragile bodies from S. mutans (13, 15) opening opportunities for many types of studies. The present paper describes a new enzyme preparation, achromopeptidase (TBL-l, Wako Pure Chemical Industries, Osaka, Japan) capable of hydrolyzing intact cells as well as cell walls of S. mutans, its serotype-dependent sensitivity to the enzyme and successful formation of osmotically fragile bodies from this bacterium. Strains of Streptococcus mutans used were HSI (serotype a), FAI (serotype b), Ingbritt (serotype c), OMZ 176 (serotype d), LM 7 (serotype e), SE 17 (serotype f), and OMZ 65 (serotype g). All strains were gifts from Dr. Shigeyuki Hamada (National Institute of Health of Japan, Tokyo). Each strain of these bacteria was cultivated in brain heart infusion broth (BHI, Difco) at 37 C for 15 hr. Bacterial cells were harvested and washed three times by centrifugation in a 0.01 M Tris-HCl buffer solution (pH 8.0) containing 0.01 M NaCl. The cells were resuspended in the buffer solution to give an optical density (O.D.) reading of about 0.5 at 660 nm in a Bausch and Lomb spectrophotometer (Shimadzu Seisakusho, Ltd., Kyoto, Japan). The reaction was started by incubating the cell suspension at 37 C in a total volume of 5.0 ml with achromopeptidase at a concentration of 100 to 1,600 ftg per ml. At intervals, a decrease in turbidity was followed by measurement of O.D. at the same wavelength. To obtain purified cell walls of each serotype, cells were treated for 12 min in a glass bottle with glass beads (0.1 mm in diameter) in a B. Braun disintegrator (Melsungen, West Germany). The cell wall fraction was purified by differential centrifugation of the treated cells in a solution of 1.0 M NaCl containing 1.0% Triton X-lOO. The fraction was further subjected to repeated

Cross-reactive Polysaccharide Antigens (Types a, d, and h) of the Mutans Group of Streptococci: Different Molecular Forms of the Type as Distinguishable by Monoclonal Antibodies

As compared to the previous precipitin inhibition tests differences were found in the reactivities of monoclonal antibodies (MAbs), a-4 and a-84 with Streptococcus cricetus (serotype a) in an enzyme immunoassay using whole cells, purified cell wall antigen and haptenic sugars coated onto microtitre wells. Investigation into the differences led to the finding that the purified antigen from S. cricetus cells consisted mainly of three forms with different molecular weights and sugar contents. MAb a-4 reacted with a high molecular weight form (AgI, molecular weight of 46,000) and low molecular weight forms (AgII and AgIII, molecular weights of 9,800 and 20,000, respectively) whereas MAb a-84 reacted only with the high-molecular form. Gas chromatographic analysis revealed that all antigens contained rhamnose, galactose and glucose but in different ratios of the sugars. Although the binding site of AgII/AgIII with MAb a-4 seemed to be slightly different from that of AgI with MAb a-84, the predominant immunodeterminant of the antigens was considered to be the same. On the basis of these results, the chemical structures of the antigenic determinants are suggested. The nature of the antigen-antibody reactions is discussed.

Scanning electron microscopic studies of the extracellular polysaccharides (EP) synthesized in colonies of Streptococcus mutans: development of EP and the effect of dextranase on them.

It has been known for years that certain species of streptococci often isolated from dental plaque of humans produce extracellular polysaccharides (3). It has been suggested that among the extracellular polysaccharides (EP) the water-insoluble forms play an important role in the initiation of dental caries (3-5). Whittaker and Drucker (8) first reported the colonial morphology of Streptococcus mutans as revealed by scanning electron microscopy but mentioned little about the chemical nature and morphology of the EP. Guggenheim and Schroeder ( 1) examined S. mutans electron microscopically and reported that globular forms of EP were sensitive to a-1 ,6-dextranase while Hamada et a! (3) showed that the fibrillar material was sensitive to their dextranase preparation and concluded that the material was mainly composed of a-1 ,6-glucosidic linkages. However, there is little information about the development of the morphological structures of EP or the developmental relationship between the different morphological forms of EP. The present paper describes in detail the initial observation by scanning electron microscopy of the extracellular polysaccharides synthesized in the colonies of S. mutans and shows the morphological relationship between the different structures of EP as well as the effects of dextranase on them. Streptococcus mutans, HS-1 (serotype a), FA-I (serotype b), LM (serotype c), SEll (serotype f), and OMZ 65 (serotype g) were used. All the strains were kindly provided by Dr. Shigeyuki Hamada (National Institute of Health of Japan, Tokyo). They were grown on Mitis-Salivarius agar (Difco) plates for 48 hr at 37 C under an atomosphere of 5% C02 and 95% air, as reported previously (7). Strains of each serotype were also grown similarly except that agar plates lacking sucrose but otherwise having the same formula as that of the Mitis-Salivarius agar were used. Blocks of agar (2 mm X 2 mm X 2 mm) containing colonies were cut, washed three times in 0.01 M phosphate buffer (pH 7.2) and fixed with 2% glutaraldehyde in the buffer for 2 hr at 4 C. After being washed again in the buffer solution the agar blocks were treated in micro titer plates with dextranase ( a-1 ,6glucan, 6-glucanohydrolase, Sigma Chemical Co., St. Louis, Mo., U.S.A.) for 48 hr at 37 C. Reaction was carried out in a total volume of 0.5 ml with a final con-