Oral bacterial attachment to titanium surfaces: a scanning electron microscopy study.

Despite the wide use of dental implants, the understanding of the mechanism(s) of bacterial attachment to implant surfaces and of the factors that affect such attachment is limited. In this study, the attachment of oral bacteria--including Streptococcus sanguis, Actinomyces viscosus, and Porphyromonas gingivalis--to titanium (Ti) discs with different surface morphology (smooth, grooved, or rough) was examined by scanning electron microscopy (SEM). The most bacterial attachment was observed on the rough BSA-coated Ti surfaces. The smooth surfaces promoted poor attachment for S. sanguis and A. viscosus. However, P. gingivalis attached equally well to both the smooth and grooved coated Ti surfaces, based on direct cell quantitation and examination with SEM. Cell-surface fimbriae (which may play a role in adhesion) of both A. viscosus and P. gingivalis observed were associated with the Ti surfaces. Ti implant surface characteristics appeared to influence oral bacterial attachment in vitro. The in vitro attachment system has proven its usefulness for future bacterial attachment studies with model implant surfaces.

Glucan-binding proteins of Streptococcus sobrinus B13 grown in high glucose media.

To improve the yield of an 87-kDa glucan-binding protein (GBP) of Streptococcus sobrinus B13 (serogroup d), trypticase-yeast extract (TYE) medium supplemented with higher (1 and 2%) than the usual amount (0.2%) of glucose was used for growth. The production of this GBP extracellularly in 1.0 and 2.0% glucose-TYE media was examined and compared with the control (0.2% glucose). Upon analysis using SDS-PAGE, extracellular culture concentrates of 1.0 and 2.0% glucose-TYE cultures revealed similar protein profiles as the control. Higher glucose concentrations did not inhibit the synthesis of the 87-kDa GBP. Cells grown in 1.0 or 2.0% glucose-supplemented media aggregated rapidly compared to those observed in the control cells (0.2% glucose grown). Higher cell yield and higher extracellular protein content were obtainable in both 1.0 and 2% glucose-TYE cultures, thus improving the yield of the 87 kDa GBP.

An 87-kilodalton glucan-binding protein of Streptococcus sobrinus B13.

An 87-kDa glucan-binding protein (GBP) of Streptococcus sobrinus B13 (serotype d) was isolated and purified from extracellular culture supernatant by using affinity chromatography on Sephadex G-50 and elution with a guanidine HCl gradient. Western blot (immunoblot) analysis showed it to be antigenically related, but not completely identical, to the 74-kDa GBP of Streptococcus mutans Ingbritt. The 87-kDa GBP has no glucosyltransferase activity. A possible role for this GBP in the cariogenicity of S. sobrinus B13 is suggested.

Synergistic effect of pyrophosphate and sodium dodecyl sulfate on periodontal pathogens.

Our previous studies have shown that pyrophosphate (PPi), the anticalculus component of tartar-control dentifrices, inhibits the growth of organisms associated with coronal and root surface caries. The purposes of this investigation were to: 1) determine if periodontal pathogens are similarly susceptible to the growth-inhibitory properties of PPi; and 2) determine if combinations of pyrophosphate-sodium dodecyl sulfate (PPi-SDS) inhibit growth synergistically. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Eikenella corrodens, and Campylobacter rectus (formerly Wolinella recta) were cultured in appropriate enriched media under anaerobic conditions. Inhibition assays were performed in tubes containing media supplemented with PPi and/or SDS. A range of concentrations of PPi and SDS in 2-fold increments was employed, with each concentration assayed in triplicate. Minimal inhibitory concentration (MIC) analyses revealed all of the bacteria were susceptible to PPi and SDS, with MICs of 0.67% (25 mM) and 0.01% w/v respectively. Combination studies with PPi-SDS showed much greater growth inhibition against P. gingivalis and A. actinomycetemcomitans than achieved with the agents individually. Determination of fractional inhibitory concentration indices indicated a synergistic growth-inhibitory effect. Under the constraints of the conditions employed, these studies demonstrate the efficacy of PPi-SDS combinations in inhibiting the growth of periodontal pathogens. It is conceivable that these compounds may have clinical benefit as a subgingival irrigant.

Growth inhibition of selected food-borne bacteria, particularly Listeria monocytogenes, by plant extracts.

Six extracts from Chinese medicinal plants: Tin Men Chu, Sey Lau Pai, Siu Mao Heung, Bak Tao Yung, Kam Chin Chiu and Liao Ya, were tested for their inhibitory effect on selected food-borne bacteria by the well assay technique. Among them, Tin Men Chu, Siu Mao Heung and Sey Lau Pai inhibited the growth of Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli, Shigella flexneri, Streptococcus faecalis, Salmonella paratyphi, Salm. enteritidis, Enterobacter aerogenes, Pseudomonas fluorescens, Proteus vulgaris, Alcaligenes faecalis, and three strains of Listeria monocytogenes. Two of these three extracts, Tin Men Chu and Siu Mao Heung, suppressed the growth of L. monocytogenes Scott A in cabbage juice. This inhibition was prevented by the addition of protein but not sodium chloride. Plant extracts show potential to control the growth of food-borne bacteria.

In vitro screening of Chinese medicinal toothpastes: their effects on growth and plaque formation of mutans streptococci.

Many plant extracts or derivatives have been incorporated into commercial toothpastes to treat oral diseases related to caries or periodontal diseases in China. However, no information is available concerning their in vitro effects on oral bacteria. Thirty-one Chinese medicinal toothpastes were selected for this study. Their ability to inhibit growth, in vitro plaque formation and glucosyltransferase (GTF) activity of Streptococcus sobrinus B13 and Streptococcus mutans 3209 were examined. Eighty-seven percent of the tested toothpastes inhibited the growth of the mutans streptococci, with zones ranging from 0.8 to 2.5 cm. At 10 mg/ml, 74% of the toothpastes inhibited in vitro plaque formation by S. mutans. Among these, 60% completely suppressed water-insoluble glucan synthesis from sucrose by GTF. Based on data obtained from our study, the incorporation of natural plant products or their derivatives into dentifrices seems a feasible means of promoting oral health and controlling dental diseases.

Gallotannins inhibit growth, water-insoluble glucan synthesis, and aggregation of mutans streptococci.

During screening for anti-plaque agents of plant origin, ethanolic extracts from Melaphis chinensis (Bell), the Chinese Nutgall, exhibited strong inhibition of glucosyltransferase (GTF), in vitro adherence and glucan-induced agglutination of Streptococcus mutans 3209 and S. sobrinus B13. More than 91% inhibition of water-insoluble glucan synthesis from sucrose by GTF was noted at a concentration as low as 7.8 micrograms/mL. Bactericidal effects on other mutans streptococci, S. salivarius, and Actinomyces viscosus were also evident. Through chemical fractionation and analyses, along with bioassays, the active components were identified as gallotannins.

Cytoplasmic proteins of Streptococcus mutans (serotype c) and their interaction with fluoride.

The protein profile of the cytoplasmic proteins of Streptococcus mutans GS-5 was determined by two-dimensional gel electrophoresis. Use of this recently developed, high-resolution analytical tool showed in excess of 140 cytoplasmic proteins. The profile consisted of mostly acidic components with pI values between 3.70 and 5.30 and relative molecular weights mainly in the 13,000 to 90,000 range. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were resolved into 40 to 45 components. The binding of fluoride by the proteins reached a maximum value in 15 min, and it was linear with exogenous F- doses of up to 60 to 80 ppm per mg of protein (60 to 80 micrograms/g). The proteins bound 22 to 138 times more F- from assay mixtures containing 1 mM CaCl2 than from assay mixtures containing such ions as HgCl2, ZnCl2, CuCl2, MgCl2, MnCl2, or SnCl2. When NaF, SnF2, NH4F, CsF, (CH3)4NF, and Na2PO3F were used as sources of F- (adjusted to 10 ppm of F- in all cases), the proteins bound 2.1, 1.8, 1.6, 1.4, and 0.3 ppm of F- per mg of protein, respectively. Initial fractionation of the plasma proteins by preparative column isoelectric focusing indicated that proteins with pI values of 4.1 to 4.5 as well as those with pI values of 5.0 to 5.3 bound twice as much F- as did the proteins outside these pI values.

The action of selected agents on the accumulation of 18F by Streptococcus mutans.

The action of certain substances known to induce cellular alterations, or encounted in the oral cavity, on the accumulation of 18F by Streptococcus mutans GS-5 has been investigated. A 62-67% inhibition in the number of 18F atoms bound per mg dry weight of cells could be induced by a 15 min pretreatment with 2.7 X 10(-4) M cetyltrimethylammoniumbromide, 1 X 10(-1) M acetic anhydride, or 7 X 10(-2) M HCl. Plate counts indicated that alteration of the cellular composition rather than viability was responsible for this diminution in 18F accumulation. Prior exposure for 15 min of this organism to 1 M HCHO or 0.1 M NaOH did not alter 18F accumulation. Of the common salts encountered in the oral cavity, CaCl2 enhanced 18F binding. Pretreatment of the assay cells for 15-160 min with 0.1-10 mg/ml of trypsin, pronase, protease, alpha-glucosidase, dextranase, or lactoferrin had no significant effect on the accumulation of 18F. However, pre-exposure of cells for 60 min to 1-10 mg/ml of either amylase or lipase induced a 40-67% inhibition in the binding of 18F, while lysozyme enhanced the binding of 18F by the cells. It would appear then that the binding of 18F by S. mutans may be altered by certain substances encountered in the oral cavity.

Properties of Streptococcus mutans grown in a synthetic medium: binding of glucosyltransferase and in vitro adherence, and binding of dextran/glucan and glycoprotein and agglutination.

The influence of culture media on various properties of Streptococcus mutans was investigated. Strains of S. mutans (serotypes c, d, f, and g) were grown in a complex medium (Todd-Hewitt broth [THB]) or a synthetic medium (SYN). The SYN cells, in contrast to THB cells, did not bind extracellular glucosyltransferase and did not produce in vitro adherence. Both types of cells possessed constitutive levels of glucosyltransferase. B13 cells grown in SYN plus invertase-treated glucose possessed the same level of constitutive enzyme as THB cells. In contrast to THB cells, the SYN cells of seven serotype strains did not agglutinate upon the addition of high-molecular-weight dextran/glucan. Significant quantities of lower-molecular-weight (2 x 10(4) or 7 x 10(4)) dextran and B13 glucan were bound by SYN cells. SYN cells agglutinated weakly in anti-glucan serum (titers, 0 to 16), whereas THB cells possessed titers of 32 to 256. Evidence for the existence of a second binding site in agglutination which does not possess a glucan-like polymer has been obtained. B13 cells grown in invertase-treated THB agglutinated to the same degree as normal THB cells. The nature of this site is unknown. SYN cells possess the type-specific polysaccharide antigen. B13 cells did not bind from THB a glycoprotein which reacts with antisera to the A, B, or T blood group antigens or which allows agglutination upon the addition of dextran. The results demonstrate that S. mutans grown in a chemically defined medium possesse markedly different biochemical and biological activities than cells grown in a complex organic medium.

Dextran/glucan binding by Streptococcus mutans: the role of molecular size and binding site in agglutination.

1) S. mutans strains of serotypes a, d and g were strongly agglutinated with soluble glucans and dextran T2000. Homologous glucan did not in all cases produce agglutination. 2) The quantity of low molecular weight dextrans bound (T20 and T70) does not correspond to the agglutination induced by glucan or T2000. 3) The agglutination and binding of high molecular weight glucan by B13 cells was sensitive to heat, trypsin, dextranase, EDTA, SDS and urea, whereas no inhibition of binding of T20 and T70 was seen. 4) Pretreatment of B13 cells with anti-d, or anti-glucan sera, or Con A, RCA I, or RCA II completely inhibited agglutination by T2000 and caused a significant reduction of the binding of glucan. No reduction in the binding of T20 and T70 occurred. 5) An agglutination-negative mutant was agglutinated by sucrose but not by T2000 or high molecular weight glucan. It bound normal levels of T20 and T70. 6) The results indicate that B13 cells possess multiple glucan binding sites and that the site responsible for agglutination consists of both polysaccharide and protein. 7) Inhibition studies on agglutination and adherence using B13 cells indicate that the two processes involve different mechanisms.

Architecture and chemistry of microconidial walls of Trichophyton mentagrophytes.

The ultrastructure and chemical composition of the walls of Trichophyton mentagrophytes microconidia were investigated with particular emphasis on the localization of the major structural components within the walls. The walls consisted of carbohydrate (56.1% neutral polysaccharide, and 16.0% chitin), protein (22.6%), lipid (6.5%), ash (1.7%), and trace amounts of melanin (0.2%) and phosphorus (0.2%). in thin sections, three distince layers were recognized. The electron-transparent pellicle (15 to 20 nm thick) covering the outermost surface of the wall consisted of a glycoprotein-lipid complex and was mostly extracted by sodium phosphate buffer (0.1 M, pH 6.5) containing 8 M urea, 1% (vol/vol) mercaptoethanol, and 1% (wt/vol) sodium dodecyl sulfate. The middle electron-dense layer (30 to 50 nm thick) represented the proteinaceous rodlet layer embedded in polysaccharides and could be completely solubilized by hot alkali extraction (1 N NaOH, 100 DEGREES C, 1 h). The thick inner layer (200 to 300 nm thick) was relatively resistant to the above treatments and was found to consist of amorphous glucans and microfibrillar chitin. Approximately half of the inner wall glucans was susceptible to (1 leads to 3)-beta-glucanase.

Isolation and characterization of the rodlet layer of Trichophyton mentagrophytes microconidial wall.

The rodlet layer of the microconidial wall of Trichophyton mentagrophytes was isolated and partially characterized. The purified microconidial walls were first extracted with urea (8M), mercaptoethanol (1%), and sodium dodecyl sulfate (1%) followed by enzymatic digestion with glusulase (snail intestinal enzymes) and purified (1 leads to 3)-beta-D-glucanase and chitinase. The purified rodlet layer was 15 to 30 nm thick and accounted for approximately 10% of the original wall weight. The pattern of rodlet patches, as revealed by electron microscopy of freeze-etched preparations of the isolated layer, was essentially the same as that observed on the intact microconidial wall. The rodlet layer was found to be resistant to most of the common organic solvents, cell wall lytic enzymes, mild acid treatments, and surface-active agents, but was solubilized in boiling 1 N NaOH with concomitant disorientation of the rodlet patterns. A melanin or melanin-like pigment appeared to be intimately associated with this rodlet layer and was solubilized during a hot-alkali treatment. Protein (80 to 85%) and glucomannan (7 to 10%) were the major components of the rodlet layer. The rodlet layer did not contain any appreciable amounts of lipid or phosphorus.