Effect of crevicular fluid and lysosomal enzymes on the adherence of streptococci and bacteroides to hydroxyapatite.

Samples of hydroxyapatite (HA) beads smaller than 1 mg were coated with 10 microliter of either saliva, serum, or human crevicular fluid before being added to a suspension of Streptococcus sanguis or Bacteroides gingivalis. In some assays, preparations of a granular fraction, elastase, or cathepsin G from human leukocytes were used to coat HA or to treat saliva-coated HA (SHA) before mixing with bacteria. The number of cells adhering to the beads was then counted under a scanning electron microscope by a standardized procedure. More cells were found to adhere to SHA in this assay than in the conventional large-scale assay. Human crevicular fluid, even when diluted up to three times, completely inhibited the adherence of S. sanguis to HA. A 100% inhibition of S. sanguis adherence was also observed when HA was coated with the granular fraction of leukocytes, and a 65% inhibition observed when SHA was treated with the enzyme preparation. When used to coat HA, elastase and cathepsin G reduced the adherence of S. sanguis by 30 and 50%, respectively. The binding of S. sanguis to elastase- or cathepsin G-treated SHA was also reduced. B. gingivalis 33277 was found to adhere in high numbers to SHA. Coating HA with crevicular fluid or with the lysosomal enzyme preparation had a limited negative effect. We postulate that crevicular fluid prevents the adherence of S. sanguis by virtue of either its enzyme content or its albumin content or both.     

In Vitro Attachment of Streptococci to the Tooth Surface

The ability of Streptococcus strains to adhere to the tooth surface in vitro was investigated. Polished enamel slabs, with and without acquired pellicles, were incubated with buffer suspensions of oral streptococci, and attached bacteria were counted under a microscope using incident light. Low numbers of bacteria adhered to uncoated enamel; the presence of an acquired pellicle significantly enhanced the attachment of all strains tested. The adherence of Streptococcus sanguis was significantly greater than that of Streptococcus salivarius, and both of these strains adhered in greater numbers than did Streptococcus mutans. When bacteria were suspended in whole saliva, the adherence of S. salivarius and S. mutans was inhibited, whereas the adherence of S. sanguis was enhanced in some experiments and inhibited in others. The adherence of S. sanguis and S. salivarius was consistently inhibited by parotid fluid; this inhibitory effect persisted after thorough washing and resonication of the bacterial cells. Incubation in oral fluids was associated with the attachment of bacterial clumps to the pellicle, and parallel investigation revealed agglutination of S. sanguis and S. salivarius by whole saliva and, in particular, parotid fluid. The results are discussed in terms of surface microecology, and are related to the development of dental plaque.     

Aggregation and Adherence of Streptococcus sanguis: Role of Human Salivary Immunoglobulin A

Fourteen freshly isolated strains of Streptococcus sanguis were obtained from the dental plaque of five healthy adults. Whole saliva was collected concomitant with the plaque isolates from the five subjects, and a second whole saliva sample was collected 10 weeks later. All possible combinations of the first five saliva samples, the second five saliva samples, and 14 strains of bacteria were tested for aggregation. Of the 140 combinations examined, 108 of 140 (77%) of the strains aggregated with the first saliva samples and 95 of 140 (68%) aggregated with the second saliva samples. Overall, 72% of the strains aggregated with both the first and second saliva samples. Removal of immunoglobulin A (IgA) from these same salivas resulted in 38 of 108 (35%) of the aggregates decreasing in intensity with the first saliva samples and 27 of 95 (29%) of the aggregates decreasing in intensity with the second saliva samples. No aggregates increased in intensity with saliva samples when IgA had been removed. Removal of IgA from saliva also resulted in a mean decrease of 46% in adherence of S. sanguis to hydroxyapatite coated with the IgA-deficient saliva. Several strains of S. sanguis were shown to aggregate strongly with human salivary and colostral IgA. In addition, S. sanguis strain S7 showed a 31% stimulation of adherence to hydroxyapatite precoated with human salivary IgA over the uncoated controls. Stepwise removal of IgA from saliva resulted in a decrease in aggregation intensity from strong (4+) to weak (1+ to 2+). Similarly, the adherence of S. sanguis to hydroxyapatite coated with these saliva samples decreased linearly as the salivary IgA was depleted. Alternatively, the addition of a small quantity of salivary IgA (20 mug/ml) to progressively diluted saliva maintained a high level of adherence and strong aggregation until the saliva dilutions reached between 1:8 in the adherence experiments and 1:32 for the aggregations. These data indicate that salivary IgA may play an important role in the microbial ecology of human dental plaque formation.     

Interference of secretory immunoglobulin A with sorption of oral bacteria to hydroxyapatite.

The potential of secretory immunoglobulin A (S-IgA) to interfere with the initial phase of dental plaque formation was studied by using an in vitro method which permits the quantitative determination of the sorption of radiolabeled oral bacterial cells to hydroxyapatite (HA) beads. The importance of specific S-IgA antibodies was evaluated by a comparison of the effect of pure preparations of colostral S-IgA, polymeric myeloma IgA, or preabsorbed S-IgA. Specific antibody molecules bound at the HA surface significantly enhanced the sorption of two Streptococcus sanguis strains. In contrast, HA-bound S-IgA antibodies inhibited the sorption of Streptococcus mitior and Streptococcus salivarius. The same was true for Streptococcus mutans cells, but only when they were propagated in the absence of sucrose. Suspended in saliva, cells of all streptococcal species adhered in significantly lower numbers to HA. Comparative experiments with bacteria suspended in solutions of various preparations of IgA or immunoglobulin-deficient salivas with S-IgA or myeloma IgA added indicated that the adherence inhibition seen with S. Sanguis, S. mitior, S. salivarius, and glucose-grown S. mutans was partly attributable to functions of S-IgA antibodies. Under the in vitro conditions of the study, S-IgA antibodies had no effect on the sorption of sucrose-grown S. mutans, Actinomyces viscosus, and Actinomyces naeslundii to HA. The results indicated that S-IgA can interfere with the sorption of some oral bacteria to HA by several different functions.     

Effect of bacterial aggregation on the adherence of oral streptococci to hydroxyapatite.

Several in vitro assay systems to measure the adherence of human dental plaque bacteria to solid surfaces such as teeth, glass, and hydroxyapatite have been published. In many studies a variety of macromolecular solutes have been used to study the adherence process. Often these solutes are able to aggregate the test bacterial and thus may alter the outcome of adherence experiments. In this study, the effects of the aggregation of Streptococcus sanguis on adherence to spheroidal hydroxyapatite is described. Adherence of preformed aggregates and of bacteria which were aggregating during the adherence reaction was examined. Bacteria were aggregated with whole saliva, concanavalin A, and wheat germ lectin. Further effects of the coaggregation of S. mitis and Actinomyces viscosus to saliva-coated spheroidal hydroxyapatite are presented. These studies suggest that formation of large aggregates resulted in a decrease in the numbers of organisms which adhered. In contrast, the formation of small aggregates actually increased the numbers of bacteria that adhered. All increases in adherent bacteria occurred at low concentrations of aggregating substance in which visible bacterial aggregation was not evident. The data indicate that adequate dose-response experiments must be performed to ensure that solutes used as probes to study adherence mechanisms do not affect the adherence simply as a result of aggregation of the test microorganisms.     

Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis.

Fimbriae are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. It has been demonstrated that purified fimbriae bind to whole human saliva adsorbed to hydroxyapatite (HAP) beads, and the binding appears to be mediated by specific protein-protein interactions. Recently, we expressed the recombinant fimbrillin protein (r-Fim) of P. gingivalis corresponding to amino acid residues 10 to 337 of the native fimbrillin (A. Sharma, H.T. Sojar, J.-Y. Lee, and R.J. Genco, Infect. Immun. 61:3570-3573, 1993). We examined the ability of individual salivary components to promote the direct attachment of r-Fim to HAP beads. Purified r-Fim was radiolabeled with 125I and incubated with HAP beads which were coated with saliva or purified individual salivary components. Whole, parotid, and submandibular-sublingual salivas increased the binding of 125I-r-Fim to HAP beads. Submandibular-sublingual saliva was most effective in increasing the binding of 125I-r-Fim to HAP beads (1.8 times greater than that to uncoated HAP beads). The binding of 125I-r-Fim to HAP beads coated with acidic proline-rich protein 1 (PRP1) or statherin was four and two times greater, respectively, than that to uncoated HAP beads. PRP1 and statherin molecules were also found to bind 125I-r-Fim in an overlay assay. The binding of intact P. gingivalis cells to HAP beads coated with PRP1 or statherin was also enhanced, by 5.4 and 4.3 times, respectively, over that to uncoated HAP beads. The interactions of PRP1 and statherin with 125I-r-Fim were not inhibited by the addition of carbohydrates or amino acids. PRP1 and statherin in solution did not show inhibitory activity on 125I-r-Fim binding to HAP beads coated with PRP1 or statherin. These results suggest that P. gingivalis fimbriae bind strongly through protein-protein interactions to acidic proline-rich protein and statherin molecules which coat surfaces.     

Salivary-agglutinin-mediated adherence of Streptococcus mutans to early plaque bacteria.

Interspecies binding is important in the colonization of the oral cavity by bacteria. Streptococcus mutans can adhere to other plaque bacteria, such as Streptococcus sanguis and Actinomyces viscosus, and this adherence is enhanced by saliva. The salivary and bacterial molecules that mediate this interaction were investigated. Salivary agglutinin, a mucinlike glycoprotein known to mediate the aggregation of many oral streptococci in vitro, was found to mediate the adherence of S. mutans to S. sanguis or A. viscosus. Adherence of S. mutans to saliva- or agglutinin-coated S. sanguis and A. viscosus was inhibited by antibodies to the bacterial agglutinin receptor. Expression of the S. sanguis receptor (SSP-5) gene in Enterococcus faecalis increased adhesion of this organism to saliva- or agglutinin-coated S. sanguis and A. viscosus. This interaction could be inhibited by antibodies to the agglutinin receptor. The results suggest that salivary agglutinin can promote adherence of S. mutans to S. sanguis and A. viscosus through interactions with the agglutinin receptor on S. mutans.     

Cell-to-cell interaction of Streptococcus sanguis and Propionibacterium acnes on saliva-coated hydroxyapatite.

Cell-to-cell interaction (coaggregation) between Propionibacterium acnes PK93 and Streptococcus sanguis DL1 was measured on saliva-coated hydroxyapatite beads (SHA) at bacterial concentrations between 1.3 X 10(6) and 6.7 X 10(8) cells per ml. Four hundredfold more DL1 than PK93 cells adhered to the saliva-coated beads, and the adherence of S. sanguis was proportional to cell input. SHA precoated with 3 X 10(8) DL1 cells bound 75 to 80% of available PK93 cells at all input amounts tested, up to an input of 8 X 10(7) cells. Adherence of PK93 to DL1-coated SHA approached saturation at an input of approximately 10(9) PK93 cells, when 1.5 X 10(8) bound. The coaggregation on SHA occurred either in buffer or saliva and was inhibited by N-acetylgalactosamine and by lactose; the attachment of DL1 to SHA was not inhibited by these sugars. S. sanguis 34 and heat-treated DL1 cells, neither of which form coaggregates with PK93, attached to SHA, but such cells did not bind PK93 cells. The findings of this study indicate that bacteria unable to attach to saliva-coated hydroxyapatite can indeed adhere to such a surface by strong lectin-mediated cell-to-cell interactions with bacteria already attached to the surface.     

Adherence of Streptococcus sanguis to conformationally specific determinants in fibronectin.

The adherence of Streptococcus sanguis to specific receptors exposed or deposited at the site of endothelial damage may play an important role in the development of infective endocarditis. Adherence of the Challis strain of S. sanguis to gelatin (or collagen) and gelatin-binding components of plasma was examined with an enzyme-linked immunosorbent assay. S. sanguis adhered poorly to immobilized gelatin and to molecular or fibrillar collagen. However, in the presence of fresh human plasma, the adherence of S. sanguis to all three substrates increased as much as eightfold. Removal of gelatin-binding proteins eliminates the ability of plasma to enhance adherence of S. sanguis to the substrates. Addition of purified human plasma fibronectin (Fn) to the absorbed plasma restored the adherence-promoting ability in a dose-dependent manner. A similar dose-dependent increase in S. sanguis adherence was observed when increasing concentrations of Fn alone were added to the gelatin-coated assay wells. S. sanguis adherence to immobilized fibronectin could not be inhibited by preincubating either the bacteria or the gelatin-coated assay wells with Fn or by including excess soluble Fn in the assay mixture. Studies with peptides purified from trypsin digests of Fn indicated that the 160- to 180-kilodalton (kDa) fragments which retain both the gelatin-binding and the cell-binding regions of the intact molecule support adherence of S. sanguis to gelatin. The 160- to 180-kDa fragments inhibited the interaction of S. sanguis with immobilized Fn. In contrast, intact Fn and the 31-kDa amino-terminal fragment were unable to inhibit the adherence when used in equivalent or greater molar amounts. These in vitro results suggest that in the presence of whole plasma, S. sanguis binds to immobilized gelatin or collagen via Fn bound to the immobilized substrates. Our finding that adherence of S. sanguis to immobilized Fn can occur in the presence of large concentrations of Fn, whether in plasma or purified, indicates that a S. sanguis-binding domain is cryptic in the Fn molecule while in solution and is exposed by a conformational change when the Fn becomes bound to gelatin-coated plastic. The ability of peptide fragments of Fn to inhibit S. sanguis adherence is consistent with this hypothesis.     

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.     

Inhibition of the interaction of Streptococcus sanguis with hexadecane droplets by 55- and 60-kilodalton hydrophobic proteins of human saliva.

The effect of salivary secretions on the hydrophobicity of Streptococcus sanguis was investigated. Pretreatment of the bacteria with paraffin-stimulated whole saliva resulted in a 79% inhibition of adhesion to hexadecane droplets. Column chromatography on Sepharose 4B and sodium dodecyl sulfate gel electrophoretic analysis indicated that the inhibitory activity of saliva resided in a fraction containing material of approximately 60,000 molecular weight. The active components, which we have termed the hydrophobic components (HC), bind to octyl-Sepharose beads. Pretreatment of S. sanguis with HC resulted in a dose-dependent inhibition of the streptococcus-hexadecane interaction that reached a maximum of 85%. Furthermore, HC effectively blocked the ability of S. sanguis to adhere to hydroxyapatite beads coated with either whole saliva or HC. Sodium dodecyl sulfate-polyacrylamide gel analysis indicated that HC eluted from octyl-Sepharose consisted primarily of two proteins (60 kDa and 55 kilodaltons) which could be resolved by high-pressure liquid chromatography. Both of these proteins were able to inhibit the binding of S. sanguis to hexadecane in a dose-dependent manner; however, the 60-kilodalton molecule was slightly more effective in this assay. Amino acid analysis of these proteins showed that both proteins contained a high percentage of nonpolar amino acids. These findings suggest that certain components of saliva influence the interaction of S. sanguis with hydrophobic surfaces.     

Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay.

There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of 3H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.     

Inhibition of Porphyromonas gingivalis adhesion to Streptococcus gordonii by human submandibular-sublingual saliva.

Porphyromonas gingivalis W50 adheres in vitro to biofilms of Streptococcus gordonii G9B. This phenomenon is believed to facilitate the initial colonization of the oral cavity by P. gingivalis and to contribute to the maturation of dental plaque. In this report, we describe the modulating effects of human submandibular-sublingual saliva (HSMSL) on this in vitro model of intergeneric bacterial adhesion (coaggregation). HSMSL inhibited P. gingivalis adhesion to S. gordonii by 50% at a concentration of 57 micrograms of protein per ml. Maximum inhibitory activity was associated with a 43-kDa protein obtained by sequential Sephadex G200 gel filtration and CM52 ion-exchange chromatography of HSMSL. Pools of other column fractions of HSMSL showed no effect or were slightly stimulatory for bacterial adhesion. The binding of radioiodinated column fractions containing the 43-kDa protein by P. gingivalis was accompanied by their rapid enzymatic degradation. Treating P. gingivalis at 60 degrees C for 30 min or with protease inhibitors (phenylmethylsulfonyl fluoride and sodium iodoacetate) reduced adherence to streptococcal biofilms. These treatments did not prevent P. gingivalis from binding soluble HSMSL saliva components, although subsequent proteolysis was nearly eliminated. These observations indicate that surface-associated proteases of P. gingivalis, either independently or in concert with adjacent surface adhesins, interact with surfaces of oral streptococci to facilitate interbacterial adhesion. The adhesion-blocking properties of HSMSL, particularly the 43-kDa protein, may represent an important host defense mechanism in the oral cavity.     

Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis.

Fimbriae are important in the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas (Bacteroides) gingivalis fimbriae appear to mediate adherence to oral epithelial cells and the pellicle-coated tooth surface. The role and contribution of fimbriae in the binding of P. gingivalis to hydroxyapatite (HAP) coated with saliva as a model for the pellicle-coated tooth surface were investigated. 3H-labeled P. gingivalis or the radioiodinated purified fimbriae were incubated with 2 mg of HAP beads coated with whole human saliva (sHAP) and layered on 100% Percoll to separate unbound from sHAP-bound components. The radioactivity of the washed beads was a measure of the bound components. The binding of P. gingivalis 2561 (381) cells and that of purified fimbriae were concentration dependent and saturable at approximately 10(8) cells and 40 micrograms of fimbriae added, respectively. The addition of fimbriae inhibited binding of P. gingivalis to sHAP beads by 65%, while the 75-kDa protein, which is another major surface component of P. gingivalis 2561, did not show significant inhibition, suggesting that the fimbriae are important in adherence. Encapsulated and sparsely fimbriated P. gingivalis W50 did not bind to sHAP beads. On the basis of the predicted sequence of the fimbrillin, a structural subunit of fimbriae, a series of peptides were synthesized and used to localize the active fimbrillin domains involved in P. gingivalis adherence to sHAP beads. Peptides from the carboxyl-terminal one-third of the fimbrillin strongly inhibited P. gingivalis binding to sHAP beads. Active residues within the sequence of inhibitory peptide 226-245 (peptide containing residues 226 to 245) and peptide 293-306 were identified by using smaller fragments prepared either by trypsin cleavage of the peptide 226-245 or by synthesis of smaller segments of peptide 293-306. Hemagglutinin activity, lectinlike binding, and ionic interaction did not seem to be involved in this binding since lysine, arginine, carbohydrates, and calcium ions failed to affect the binding of P. gingivalis. The observation that poly-L-lysine, bovine serum albumin, and defatted bovine serum albumin, even at high concentrations, only partially blocked the binding of P. gingivalis indicates that hydrophobic interactions are not the major forces involved in P. gingivalis binding to sHAP beads. Protease inhibitors such as EDTA, leupeptin, pepstatin, 1,10-phenanthroline, and phenylmethylsulfonyl fluoride did not interfere with the binding of P. gingivalis. However, the binding of P. gingivalis to trypsin- or chymotrypsin-pretreated sHAP beads was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of salivary alpha-amylase binding to Streptococcus sanguis.

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.     

Bacterial and Candida albicans adhesion on rapid prototyping-produced 3D-scaffolds manufactured as bone replacement materials

Rapid prototyping (RP)-produced scaffolds are gaining increasing importance in scaffold-guided tissue engineering. Microbial adhesion on the surface of replacement materials has a strong influence on healing and long-term outcome. Consequently, it is important to examine the adherence of microorganisms on RP-produced scaffolds. This research focussed on manufacturing of scaffolds by 3D-bioplotting and examination of their microbial adhesion characteristics. Tricalciumphosphate (TCP), calcium/sodium alginate, and poly(lactide-co-glycolic acid) (PLGA) constructs were produced and used to study the adhesion of dental pathogens. Six oral bacterial strains, one Candida strain and human saliva were used for the adhesion studies. The number of colony forming units (CFU) were determined and scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were performed. Microorganisms adhered to all scaffolds. All strains, except for Streptococcus oralis, adhered best to PLGA scaffolds. Streptococcus oralis adhered to each of the biomaterials equally. Streptococcus mutans and Enterococcus faecalis adhered best to PLGA scaffolds, followed by alginate and TCP. Prevotella nigrescens, Porphyromonas gingivalis, Streptococcus sanguis, and Candida albicans showed the highest adherence to PLGA, followed by TCP and alginate. In contrast, the microorganisms of saliva adhered significantly better to TCP, followed by PLGA and alginate. SEM observations correlated with the results of the CFU determinations. CLSM detected bacteria within deeper sheets of alginate. In conclusion, because of the high adherence rate of oral pathogens to the scaffolds, the application of these biomaterials for bone replacement in oral surgery could result in biomaterial-related infections. Strategies to decrease microbial adherence and to prevent infections due to oral pathogens are discussed.     

Adherence of Streptococcus mutans and Streptococcus sanguis to salivary components bound to glass.

Adherence of radiolabeled Streptococcus mutans and Streptococcus sanguis to saliva-treated glass surfaces was studied under conditions which minimized bacteria-glass interactions. Treatment of glass with an alkylsilane solution decreased nonspecific bacterial adherence and enhanced adsorption of radiolabeled salivary components to these surfaces. Addition of Triton X-100 to the bacterial suspensions also reduced nonspecific adherence to siliconized glass, but did not affect adherence to salivary components attached to siliconized glass. Calcium stimulated S. mutans adherence to saliva-free glass, but inhibited adherence to saliva-treated glass. S. sanguis adherence to either saliva-free or saliva-treated glass was inhibited slightly at high calcium ion concentrations. Adherence of streptococci to saliva-treated glass exhibited saturation kinetics, and the numbers of binding sites on the experimental salivary pellicle and the affinity constants for bacteria-saliva attachment were determined. Preincubation of the streptococci with whole saliva decreased their capacity to adhere to saliva-treated glass, but not to saliva-free glass. Bacteria adherent to saliva-treated glass surfaces were readily desorbed by washing with saliva. The addition of homologous antisera, ammonium sulfate-precipitated immunoglobulins, or Fab fragments to the bacterial suspensions inhibited cell adherence to saliva-treated glass.     

Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to Oral Surfaces and Its Possible Role in Colonization of the Mouth and of Periodontal Pockets

This investigation examined the ability of cells of Bacteroides melaninogenicus subsp. asaccharolyticus 381 to adhere to surfaces that might be important for its initial colonization of the mouth and its subsequent colonization in periodontal pockets. Of 48 asaccharolytic strains of B. melaninogenicus, 47 agglutinated human erythrocytes, whereas none of 20 fermentative strains, which included reference cultures of the subspecies intermedius and melaninogenicus, were active. Electron microscopy indicated that both asaccharolytic and fermentative strains possessed pili; hence, the presence of pili did not correlate with the hemagglutinating activities of B. melaninogenicus strains. Both asaccharolytic and fermentative B. melaninogenicus strains suspended in phosphate-buffered saline adhered in high numbers to buccal epithelial cells and to the surfaces of several gram-positive bacteria tested, including Actinomyces viscosus, A. naeslundii, A. israelii, Streptococcus sanguis, and S. mitis. B. melaninogenicus subsp. asaccharolyticus 381 also attached, but in comparatively low numbers, to untreated and to saliva-treated hydroxyapatite. Addition of clarified whole saliva to suspensions of strain 381 almost completely eliminated adherence to buccal epithelial cells and to hydroxyapatite surfaces, but saliva had no detectable effect on attachment to gram-positive plaque bacteria. Both fermentative and nonfermentative strains of B. melaninogenicus also attached in high numbers to crevicular epithelial cells derived from human periodontal pockets, but normal human serum strongly inhibited attachment. Serum also inhibited attachment of strain 381 to saliva- and serum-treated hydroxyapatite, but it had little effect upon attachment to gram-positive bacteria. These observations suggested that salivary and serum components would strongly inhibit the attachment of B. melaninogenicus cells to several oral surfaces, but not to the surfaces of certain gram-positive bacteria commonly present in human dental plaque. This was confirmed by an in vivo experiment in which streptomycin-labeled cells of B. melaninogenicus 381-R were introduced into the mouths of two volunteers. After 10 min, several hundred-fold higher numbers of the organism were recovered from preformed bacterial plaque present on teeth than from clean tooth surfaces or from the buccal mucosa and tongue dorsum. High numbers of B. melaninogenicus cells were also recovered from preformed plaque after 150 min, but virtually no cells of the organism were recovered from the other surfaces studied. These data suggest that the presence of dental plaque containing Actinomyces and other gram-positive bacteria may be essential for the attachment and colonization of B. melaninogenicus cells after their initial introduction into the mouth. Similarly, the presence of subgingival plaque containing gram-positive bacteria may be necessary for its secondary colonization in periodontal pockets.     

Adherence of mutans streptococci to other oral bacteria.

Adherence of mutans streptococci to strains of Actinomyces viscosus, Streptococcus sanguis, and Streptococcus mitis immobilized on a nitrocellulose membrane was measured. Strains of Streptococcus mutans, S. sobrinus, and S. rattus bound in a lactose-independent manner to a variety of the actinomyces and streptococci. Most of these reactions could proceed in the presence of whole saliva although adherence of S. rattus BHT to the streptococci was inhibited by salivary molecules. In contrast, adherence of S. mutans 10449 and KPSK2 to A. viscosus, S. sanguis, and S. mitis was enhanced by salivary molecules. S. mutans KPSK2, S. sobrinus OMZ 176, and S. rattus FA-1 binding to A. viscosus NC3 and S. sanguis G9B exhibited saturation kinetics. Adherence to A. viscosus NC3 was of a higher avidity than adherence to S. sanguis G9B. Attachment of S. mutans KPSK2 to S. sanguis G9B and of S. mutans OMZ 176 to A. viscosus NC3 and S. sanguis G9B was inhibited by heat treatment of the mutans streptococci. Attachment of S. mutans KPSK2 to A. viscosus NC3 and of S. rattus FA-1 to A. viscosus NC3 and S. sanguis G9B was unaffected by heat. These observations suggest that the mutans streptococci can adhere to a variety of early plaque bacteria by several distinct mechanisms. Such interactions may be important in the colonization of tooth surfaces by the mutans streptococci.     

Ability of Veillonella and Neisseria Species to Attach to Oral Surfaces and Their Proportions Present Indigenously

The present study describes the distribution of Veillonella and Neisseria species in the human oral cavity and indicates that their ability to attach to oral surfaces correlates with their proportions found in various sites of the mouth. The mean percentages of Veillonella and Neisseria of the total flora cultivable on anaerobic blood-agar plates was found to be: plaque, 0.75 and <0.13, respectively; lip, 0.38 and <0.05; cheek, 0.66 and <0.14; tongue dorsum, 9.4 and <0.12; saliva, 5.0 and <0.9. The ability of Veillonella and Neisseria species to attach to tooth surfaces was studied by cleaning the labial surfaces of incisors to render them relatively free of viable bacteria. Samples taken 1 hr later contained <0.27% Veillonella and <0.4% Neisseria, whereas saliva to which these teeth were exposed contained 20-fold higher proportions of Veillonella. These data indicate that Veillonella and Neisseria species possess a feeble ability to attach to cleaned teeth. The ability of these organisms to adhere to other oral surfaces was determined by introducing mixtures of streptomycin-resistant strains into the mouths of volunteers for 5 min. Labeled strains of Streptococcus sanguis and S. salivarius were included for comparative purposes. Analysis of samples obtained from oral surfaces after 45 min indicated that Veillonella and Neisseria adhere very poorly to preformed dental plaque as compared to S. sanguis. In contrast, Veillonella adhered to the tongue dorsum markedly better than Neisseria, S. sanguis, and S. salivarius. The greater ability of Veillonella to adhere to the tongue in relation to the other organisms studied correlates with the high proportions of Veillonella found on this site. The feeble ability of Neisseria to attach to surfaces in the oral cavity is reflected by their low proportions found on these surfaces.