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Introduction: Mouse prolactin-inducible protein (mPIP) is secreted in mouse saliva and has been found to bind oral bacteria, showing the highest affinity for streptococci. Comparisons between the oral flora of mPIP knockout mice and their wild-type controls showed differences in the genera colonizing the two groups of mice. These findings suggested a role for mPIP in the colonization of the mouse oral cavity, possibly modulating the oral flora. In this in vitro study, we focused on the contribution of this protein to aggregation of oral bacteria, a process thought to promote the clearance of bacteria from the oral cavity, and one that could influence the composition of the oral bacterial community., Methods: The aggregation of selected human and mouse oral streptococci was measured spectrophotometrically. The aggregation of oral bacteria by saliva from mPIP knockout mice, which lack mPIP, was compared with that of saliva from wild-type mice., Results: Both wild-type saliva and mPIP knockout mouse saliva induced aggregation of human strain Streptococcus gordonii SK120 and mouse streptococci strains M105/6 and M106/2. Bacterial aggregation induced by the saliva of wild-type mice was significantly higher than the aggregation induced by saliva from mPIP knockout mice for all the bacterial strains., Conclusion: In this study it was confirmed that mPIP plays a role in the aggregation of oral bacteria. The salivary components promoting aggregation of oral bacteria are considered to be part of the oral defense mechanisms so these findings provide insight into a possible function of mPIP in host defense by promoting aggregation of oral bacteria.

Introduction: Microbial interactions are considered to be important for bacterial colonization. Interactions that inhibit colonization of pathogens could possibly be used as a new treatment approach for periodontitis. The aim of this study was to test this hypothesis on soft surfaces in vitro, taking into account the hydrodynamic forces continuously present in vivo., Methods: Cultured epithelial cells were precolonized with Streptococcus sanguinis KTH-4, Streptococcus cristatus CC5A, Streptococcus salivarius TOVE and Streptococcus mitis BMS before Aggregatibacter actinomycetemcomitans colonization. Experiments were performed in a modified Robbins-device-type flow cell. Bacterial colonization and the number of epithelial cells were evaluated by microbial culturing and quantitative polymerase chain reaction., Results: The streptococci were able to inhibit A. actinomycetemcomitans colonization on soft tissue surfaces under flow conditions. Statistically significant differences were found between streptococcal pretreatments and the controls, with the most pronounced effect caused by S. sanguinis., Conclusion: These data confirm the possibility of applying beneficial bacteria in periodontal treatment.

Aim: The aim of the study was to evaluate the adhesion of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus rhamnosus strain GG to saliva-coated surfaces in vitro., Methods: Fifteen radiolabeled dairy L. delbrueckii subsp. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated hydroxyapatite beads and polystyrene microtiter plates and the radioactivity was measured by liquid scintillation counter. The effects of lysozyme on the adhesion of lactobacilli and of pretreatment with lactobacilli on the adhesion of Streptococcus sanguinis were also assessed., Results: All strains tested adhered to saliva-coated surfaces but with significantly different binding frequencies. The adhesion of the L. delbrueckii subsp. bulgaricus strains remained lower in comparison to L. rhamnosus strain GG. One L. delbrueckii subsp. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment of the samples significantly increased lactobacillus adhesion to saliva-coated surfaces., Conclusion: The present results showed significant variations in the adhesion capacity of the Lactobacillus strains studied. Adhesion to oral surfaces is of primary importance for bacterial colonization in the mouth. Only one of the L. delbrueckii subsp. bulgaricus dairy starter culture strains investigated had a high adhesion percentage. This strain might then be considered for further investigations in the oral environment.

Introduction: The effects of Streptococcus salivarius on the competence-stimulating peptide (CSP)-dependent biofilm formation by Streptococcus mutans were investigated., Methods: Biofilms were grown on 96-well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm., Results: S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC-3 and FSC-3DeltaglrA in separate dual-species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP-binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene-dependent quorum sensing systems., Conclusion: It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell-to-cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.

Background/aim: We investigated the mechanisms of adherence of salivary and serum proteins, which mimic gingival crevicular fluid (GCF), to Porphyromonas gingivalis, and the effects of these adhered proteins on coaggregation and hemagglutination properties., Methods: The amounts of salivary and serum proteins adhering to P. gingivalis were determined using (3)H-labeled and non-labeled proteins. The coaggregation between P. gingivalis and Streptococcus oralis or Streptococcus gordonii was observed. Hemagglutination was evaluated using sheep erythrocytes. Proteins that interacted with zinc or copper in saliva and serum and on P. gingivalis were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis., Results: The amount of salivary or serum proteins that adhered to the surface of P. gingivalis strains was increased by cations, especially zinc and copper ions. The pretreatment of bacterial cells with salivary or serum proteins before the assay inhibited coaggregation with gram-positive bacteria and hemagglutination. These phenomena were enhanced by the presence of zinc or copper ions during the pretreatment of P. gingivalis with proteins. We detected protein bands that were related to these cations in saliva and serum and on P. gingivalis., Conclusions: Our findings suggest that zinc and copper ions markedly enhanced the adhesion and accumulation of salivary and serum proteins on cells of P. gingivalis and inhibited the coaggregation and hemagglutination of P. gingivalis. These cations might be useful for limiting the settlement of P. gingivalis in the gingival sulcus with the goal of preventing periodontal disease.

Introduction: Microbial interactions are considered important in the adhesion process of pathogenic bacteria in the oral cavity. This study addressed the hypothesis that a streptococcal biofilm influences the hard tissue colonization by the periodontopathogen Aggregatibacter actinomycetemcomitans under hydrodynamic conditions., Methods: The colonization of a green-fluorescent-protein-labelled A. actinomycetemcomitans strain on surfaces coated with a streptococcal biofilm, was monitored in real time using a confocal laser scanning microscope-mounted flow cell. Culture and quantitative polymerase chain reaction data were obtained in parallel from a Modified Robbins Device., Results: Colonization of A. actinomycetemcomitans was inhibited by the four tested streptococci (Streptococcus sanguinis, Streptococcus cristatus, Streptococcus salivarius, and Streptococcus mitis). The most inhibiting species was S. sanguinis., Conclusion: These results confirmed the hypothesis that some bacterial species influence A. actinomycetemcomitans colonization of hard surfaces in vitro under hydrodynamic conditions.

Background/aims: Streptococcus parasanguinis is a primary colonizer of the tooth surface. Its adhesion is mediated by the long fimbriae, which are composed of multiple subunits of a serine-rich glycoprotein, Fap1. Previous studies revealed that a chromosomal region located downstream of fap1 is involved in the secretion and glycosylation of Fap1. In this study, we investigated the role of a glycosylation-associated gene, gap3, in Fap1 biogenesis., Methods: A gap3 non-polar mutant was constructed by insertional inactivation. The phenotype of the mutant and the subcellular distribution of its products were investigated. The binding ability of the mutant was tested with saliva-coated hydroxyapatite (SHA). Electron microscopy was used to observe the morphological changes on the mutant cell surface. Confocal microscopy was utilized to determine biofilm formation ability., Results: The gap3 mutant produced a partially glycosylated Fap1 precursor, that was less stable than mature Fap1. The Fap1 precursor was distributed in all subcellular fractions including the cell surface and culture medium although in decreased amounts. These data suggest a role for Gap3 in Fap1 glycosylation as well as a link between glycosylation and secretion of Fap1. The gap3 mutant had reduced binding to saliva-coated hydroxyapatite. Electron microscopy revealed that the gap3 mutant had lost its long fimbriae. Biofilm formation was also inhibited by the gap3 mutation. Fewer gap3 mutant cells adhered to the biofilm surface and microcolony formation was decreased., Conclusion: Gap3 is required for the complete glycosylation and secretion of Fap1, which is important for fimbrial assembly, bacterial adhesion, and in vitro biofilm formation.

Introduction: Periodontitis results from a shift in the subgingival microflora into a more pathogenic direction with Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans considered as periodontopathogens. In many cases, treatment procures only a temporary shift towards a less pathogenic microflora. An alternative treatment could be the deliberate colonization of pockets with antagonistic microorganisms to control the adhesion of periodontopathogens. The aim of this study was to identify bacterial strains that reduce adhesion of periodontopathogens to surfaces., Methods: Streptococcus sanguinis, Streptococcus crista, Streptococcus salivarius, Streptococcus mitis, Actinomyces naeslundii, and Haemophilus parainfluenzae were evaluated as potential antagonists against P. gingivalis ATCC 33277, P. intermedia ATCC 49046, and A. actinomycetemcomitans ATCC 43718 as periodontopathogens. Adhesion of periodontopathogens to the bottom plate of a parallel plate flow chamber was studied in the absence (control) and the presence of pre-adhering antagonistic strains up to a surface coverage of 5%., Results: The largest reduction caused by antagonistic strains was observed for P. gingivalis. All antagonistic strains except S. crista ATCC 49999 inhibited the adhesion of P. gingivalis by at least 1.6 cells per adhering antagonist, with the largest significant reduction observed for A. naeslundii ATCC 51655 (3.8 cells per adhering antagonist). Adhering antagonists had a minimal effect on the adhesion of A. actinomycetemcomitans ATCC 43718. Intermediate but significant reductions were perceived for P. intermedia, most notably caused by S. mitis BMS., Conclusion: The adhesion of P. gingivalis was inhibited best by antagonistic strains, while S. mitis BMS appeared to be the most successful antagonist.

Introduction: Autoinducer-2 (AI-2) is a widespread communication-signal molecule that allows bacteria to sense and react to environmental factors. In some streptococci AI-2 is reported to be involved in virulence expression and biofilm formation. It has earlier been shown that the alga Delisea pulchra produces bromated furanones, which prevent bacterial colonization of the algae., Methods and Results: We have previously published a novel and simple synthesis of (Z)-5-bromomethylene-2(5H)-furanone. In this study we showed that our synthesized furanone inhibited biofilm formation and bioluminescence induction by Streptococcus anginosus, Streptococcus intermedius, and Streptococcus mutans, as well as bioluminescence induction by Vibrio harveyi BB152., Conclusion: We suggest that the effect is linked to interference with the AI-2 signaling pathway because adding furanone to the medium had no effect on the ability of the AI-2-defective S. anginosus luxS and S. intermedius luxS mutants to form biofilms.

The surface of the oral plaque bacterium Streptococcus cristatus is decorated with a lateral tuft of fibrils. The fibrillar tuft functions in the adhesion of S. cristatus to heterologous bacterial species in the plaque biofilm. The tuft typically consists of a densely packed fringe of shorter fibrils 238 +/- 19 nm long with longer, less abundant fibrils 403 +/- 66 nm long projecting through the fringe of short fibrils. The two types of fibrils in the tufts of S. cristatus have been refractory to biochemical separation, complicating their characterization. A hexadecane partition assay was used to enrich for subpopulations of S. cristatus CR311 (type strain NCTC 12479) having distinct fibrillar morphotypes. Negative staining in the TEM revealed that cells of a hydrophobic subpopulation of S. cristatus (CR311var1) carried only the long fibrils (395 +/- 32 nm). A hydrophilic subpopulation of S. cristatus (CR311var3) consisted of mixed morphotypes having no fibrils or remnant short fibrils (223 +/- 49 nm). No long fibrils were observed on any cells in the CR311var3 subpopulation. The CR311var3 morphotype, unlike the wild-type strain and CR311var1, was not able to form corncobs with either Corynebacterium matruchotii or Fusobacterium nucleatum. Variant CR311var3 did not express the novel gene srpA, which encodes a high molecular weight (321,882 Da) serine-rich protein, SrpA. The SrpA protein contains two extensive repeat motifs of 17 and 71 amino acids and a gram-positive cell wall anchor consensus sequence (LPNTG). The unusual properties of SrpA most closely resemble those of Fap1, the fimbrial-associated adhesin protein of Streptococcus parasanguis. The association of long fibrils, high surface hydrophobicity, ability to form corncob formations, and expression of the srpA gene suggest that SrpA is a long fibril protein in S. cristatus.

Dental plaque is composed of a biofilm community of microorganisms on teeth that coats the oral cavity, including attaching to the teeth, and provides a protective reservoir for oral microbial pathogens, which are the primary cause of persistent and chronic infectious diseases. Oral streptococci are pioneering organisms that play an important role in biofilm formation on tooth surfaces as well as being primary causative agents of dental caries. The purpose of this study was to clarify the role of the E2f1 gene in susceptibility to dry mouth and bacterial adherence of oral streptococci to tooth surfaces in animal model experiments. A mutation of the E2f1 gene in mice is known to cause enhanced T-lymphocyte proliferation, leading to testicular atrophy, splenomegaly, salivary gland dysplasia, and other systemic and organ-specific autoimmunity. We found a decreased volume of saliva production and protein production rate, along with increased amylase activity, IgA concentration, and mucin 1 concentration in E2F-1(-/-) mice as compared with the control C57BL/6 mice. Further, we quantified the recolonization of oral streptococci in E2F-1(-/-) mice and found that a higher number of some oral streptococci were colonized on the teeth of these mice. In particular, following oral ingestion of 1% sucrose in water, the colonization of Streptococcus mutans increased in comparison with other streptococci. Our results suggest that the E2f1 gene may affect susceptibility for oral biofilm formation by streptococci in humans with dry mouth.

Cranberry juice is known to inhibit bacterial adhesion. We examined the inhibitory effect of cranberry juice on the adhesion of oral streptococci strains labeled with [3H]-thymidine to saliva-coated hydroxyapatite beads (s-HA). When the bacterial cells were momentarily exposed to cranberry juice, their adherence to s-HA decreased significantly compared with the control (P < 0.01). Their hydrophobicity also decreased dependently with the concentration of cranberry juice. We also evaluated the inhibitory effect of cranberry juice on biofilm formation. By using a microplate system, we found that the high molecular mass constituents of cranberry juice inhibited the biofilm formation of the tested streptococci. The inhibitory activity was related to the reduction of the hydrophobicity. The present findings suggest that cranberry juice component(s) can inhibit colonization by oral streptococci to the tooth surface and can thus slow development of dental plaque., (Copyright Blackwell Munksgaard, 2004.)

Zinc is a known inhibitor of acid production by mutans streptococci. Our primary objective was to extend current knowledge of the physiologic bases for this inhibition and also for zinc inhibition of alkali production by Streptococcus rattus FA-1 and Streptococcus salivarius ATCC 13419. Zinc at concentrations as low as 0.01-0.1 mm not only inhibited acid production by cells of Streptococcus mutans GS-5 in suspensions or in biofilms but also sensitized glycolysis by intact cells to acidification. Zinc reversibly inhibited the F-ATPase of permeabilized cells of S. mutans with a 50% inhibitory concentration of about 1 mm for cells in suspensions. Zinc reversibly inhibited the phosphoenolpyruvate: sugar phosphotransferase system with 50% inhibition at about 0.3 mm ZnSO4, or about half that concentration when the zinc-citrate chelate was used. The reversibility of these inhibitory actions of zinc correlates with findings that it is mainly bacteriostatic rather than bactericidal. Zinc inhibited alkali production from arginine or urea and was a potent enzyme inhibitor for arginine deiminase of S. rattus FA-1 and for urease of S. salivarius. In addition, zinc citrate at high levels of 10-20 mm was weakly bactericidal.

Streptococcus salivarius is divided into two serological subgroups that carry either fibrils or fimbriae. Although fimbriae have been observed on up to 50% of S. salivarius strains in the human oral cavity, no function has yet been assigned to them. To determine whether S. salivarius fimbriae have a role in adhesion, we examined the ability of S. salivarius to coaggregate with selected microorganisms involved in periodontal diseases. Our results show that S. salivarius coaggregated with Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia. However, only fimbriated S. salivarius cells were able to coaggregate with P. intermedia, suggesting a specific role for these structures in the interaction. Heat treatment, sensitivity to sugars, amino acids, and EDTA, as well as protease treatment were also used to further characterize coaggregation between S. salivarius and periodontopathogens.

Interactions between bacterial oral flora and Candida albicans are important in denture plaque formation. This study therefore first aimed to quantify the coadherence of C. albicans and bacteria by the use of a bioluminescent adenosine triphosphate (ATP) assay based on the firefly luciferase-luciferin system. The second aim was to examine the effect of i) dietary sugars (used for preculture) and ii) enzymatic digestion of fungi on the coadherence. When yeast was preincubated in yeast nitrogen base medium (YNB) supplemented with 250 mM glucose, the yeast coadhered with all isolates of Streptoccus mutans and Streptococcus sanguis, and no significant coadhesion was observed with the isolates of Streptococcus sobrinus, Streptococcus salivarius, Lactobacillus and Actinomyces. However, when the yeast was precultured in YNB supplemented with 500 mM galactose, the yeast coadhered with S. salivarius and Actinomyces, which was not observed when the yeast was grown in YNB with glucose. In addition, the coadherence of the yeast with the isolates of S. sanguis was significantly reduced. Enzymatic digestion of yeast and a reverse transcription polymerase chain reaction assay revealed that expression of at least two types of proteinaceous adhesins are involved in these phenomena.

A total of eight bacterial isolates belonging to six species, and a select group of 12 oral Candida albicans isolates, were used to study the effect of bacteria on germ-tube formation. Briefly, each bacterial suspension (10(5-6) cells/ml) was mixed with a C. albicans suspension (10(7) cells/ml) and incubated at 37 degrees C for 90 min with bovine serum, and the percentage germ-tube-positive Candida cells was quantified using a haemocytometer, under light microscopy. In general, out of eight bacteria, Streptococcus sanguis SK21A, Streptococcus salivarius SK56, Escherichia coli ATCC 25922, and S. salivarius OBU3 suppressed germ-tube formation to varying degrees, with different C albicans isolates. Porphyromonas gingivalis Pg 50, Lactobacillus casei ATCC 7469 and Prevotella intermedia OBU4 elicited significant enhancement of germ-tube formation, whereas S. sanguis OBU 2 had no effect. E. coli ATCC 25922 was the only organism to show statistically significant suppression of germ-tube formation (p=0.0312). A significant increase in the germ tube production of C. albicans isolated from HIV-infected compared with HIV-free individuals was also noted. The current results tend to suggest that commensal and transient oral bacterial populations may selectively influence the differential expression of germ-tube-forming ability of C. albicans isolates.

Acid tolerance and acidogenicity of non-mutans streptococci and their capacity of acid adaptation were studied. The cells of non-mutans streptococci (Streptococcus sanguis [Streptococcus sanguinis], Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis) grown at pH 7.0 showed 0.0088% to 71% viability after acidification at pH 4.0 for 60 min, whereas the cells of mutans streptococci (Streptococcus mutans) were not killed by the acidification. Washed cells of non-mutans streptococci lowered pH to 4.04-4.33 in the presence of glucose, while mutans streptococci cells lowered pH to 3.70. When the growth pH was shifted to 5.5 for 30-90 min, the viability of non-mutans streptococci after the acidification at pH 4.0 for 60 min increased (0.25% to 91%) and the minimum pH values of the cells in the presence of glucose decreased (3.90 4.19). Along with the increase in acid tolerance and acidogenicity, non-mutans streptococci increased activities of H(+)-ATPase and arginine deiminase and amounts of stress proteins cross-reacting with 60 kDa and 70 kDa heat shock proteins (Hsp60 and Hsp70). These results indicate that non-mutans streptococci were capable of increasing their acid tolerance and acidogenicity in response to environmental acidification. Furthermore, it is suggested that the acid adaptation observed in non-mutans streptococci cells could involve the induction of H(+)-ATPase, arginine deiminase and stress protein syntheses. The strains of non-mutans streptococci, which are pioneer bacteria for dental plaque formation and predominant in plaque microbial flora, may play a significant role in shifting the dental plaque environment toward acidic and consequently promoting the colonization of more acid-tolerant and acidogenic bacteria such as mutans streptococci and lactobacilli.

The activity of glucosyltransferases (GTF), a group of enzymes that synthesize water-soluble and -insoluble glucans from sucrose, significantly contributes to the cariogenicity of mutans streptococci. Streptococcus downei produces four glucosyltransferases, GTFI, which produces insoluble glucan, and GTFS, GTFT, and GTFU, which synthesize soluble glucans. We have previously reported that inactivation of gtfS results in altered adherence and have now examined its interaction with other enzymes by constructing mutants which were gtfS, gtfS/gtfT, gtfS/gtfI and gtfI. The mutants were tested for their ability to accumulate on wires and on plastic microtiter trays in the presence of sucrose. The gtfS mutant displayed a reduced ability to adhere compared to the wild type but there was no further reduction of adherence in a gtfS/gtfT mutant. In contrast, the gtfS/gtfI double mutant showed a drastic reduction in adherence and when gtfI alone was inactivated, bacteria were unable to adhere to a hard surface. The results confirmed that insoluble glucan is required for strong adherence to a smooth surface but that the amount and structure of this glucan is dependent upon the availability of soluble glucans to act as primer molecules.

Our previous research has demonstrated that with the more aciduric oral bacteria, an acid shock to sub-lethal pH values results in the induction of an acid tolerance response that protects the cells at extremely low pH (pH 3.0-4.0) that kills unadapted control cells maintained at pH 7.5 (Oral Microbiol Immunol 1997: 12: 266-273). In this study, we were interested in comparing the protein profiles of acid-shocked and control cells of nine organisms from three acid-ogenic genera that could be categorized as strong, weak and non-acid responders in an attempt to identify proteins that could be classified as acid-regulated proteins and which may be important in the process of survival at very low pH. For this, log-phase cultures were rapidly acidified from pH 7.5 to 5.5 in the presence of [14C]-amino acids for varying periods up to 2 h, the period previously shown to be required for maximum induction of the acid response. The cells were extracted for total protein and subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide chromatography with comparable control and acid-shocked protein profiles compared by scanning and computer analysis. Of particular interest were the proteins in the acid-shocked cells that showed enhanced labeling (i.e., synthesis) over the control cells, since these were considered acid-regulated proteins of importance in pH homeostasis. Streptococcus mutans LT11 generated the most rapid and complex pattern: a total of 36 acid-regulated proteins showing enhanced synthesis, with 25 appearing within the first 30 min of acid shock. The enhanced synthesis was transient with all proteins, with the exception of two with molecular weights of 50/49 and 33/32 kDa. Within the acid-regulated proteins were proteins having molecular weights comparable to the heat shock proteins and the various subunits of the membrane H+/ATPase. By comparison, the strong responder, Lactobacillus casei 151, showed the enhanced formation of only nine proteins within the first 30 min of the acid shock, with a total of 11 acid-regulated proteins formed during the 2-h adaptation period with enhanced synthesis transient for seven of these proteins. Streptococcus salivarius AT2 and Streptococcus gordonii TH12 had the formation of 6 and 8 proteins enhanced, while the weakly responding organisms, Streptococcus sanguis ATCC 10,556 and Streptococcus oralis ATCC 10,557, exhibited 8 and 6 such proteins, respectively. Even non-responding strains unable to survive at very low pH, such as Streptococcus sobrinus CH125/43, Streptococcus mitis ATCC 12,261 and Actinomyces naeslundii 301-13 showed the initial formation of 3-9 acid-regulated proteins, but protein synthesis was not sustained over the entire adaptation period. Clearly, the survival of oral bacteria at very low pH is related, not to the total number of the acid-regulated proteins induced per se but to the formation of key proteins that function to augment normal pH homeostasis.

It was previously shown that a low-molecular-weight chitosan and its derivatives N-carboxymethyl chitosan and imidazolyl chitosan inhibit Streptococcus mutans adsorption to hydroxyapatite. The ability of the same molecules to interfere with adhesive properties of other oral streptococci (Streptococcus sanguis, Streptococcus gordonii, Streptococcus constellatus, Streptococcus anginosus, Streptococcus intermedius, Streptococcus oralis, Streptococcus salivarius, Streptococcus vestibularis) was tested. When saliva-coated or -uncoated hydroxyapatite beads were treated with N-carboxymethyl chitosan, a reduction varying from 60% to 98% depending on strains was observed. Low-molecular-weight chitosans and imidazolyl chitosan did not have any effect. Growth in N-carboxymethyl chitosan-supplemented medium (final concentrations ranging from 20 to 500 micrograms.ml-1) caused a dose related reduction in the adsorption of all strains to hydroxyapatite and in their affinity towards xylene. No effect was observed with low-molecular-weight chitosans and imidazolyl chitosan. In contrast to what observed with S. mutans, the three polysaccharides did not affect detachment from hydroxyapatite beads and adherence to cheek epithelial cells of the other streptococci. These results suggest that low-molecular-weight chitosans and/or imidazolyl chitosan, selectively affecting S. mutans adsorption to hydroxyapatite, may be very interesting as potential anti-dental caries agents.

Using 21 species of oral bacteria, representing six acidogenic genera, we undertook to determine whether the pH-limiting exponential growth is related to the ability of the organisms to generate an acid-tolerance response that results in enhanced survival at low pH. The lower pH limit of exponential growth varied by more than two units with that of Neisseria A182 at pH 6.34; growth of Lactobacillus casei RB1014 stopped at pH 3.81, with species of Actinomyces, Enterococcus, Prevotella and Streptococcus falling between these limits. The working hypothesis was that the organisms with the higher pH limits for growth are unable to respond to acidic environments in order to survive, whereas the more aciduric organisms would possess or acquire acid tolerance. Adaptation to acid tolerance was tested by determining whether the prior exposure of exponential-phase cells to a low, sub-lethal pH would trigger the induction of a mechanism that would enhance survival at a pH killing pH 7.5 control cells. The killing pH varied from pH 4.5 for Prevotella intermedia ATCC 25611 to pH 2.3 for the three Lactobacillus casei strains in the study, with the three Streptococcus mutans strains killed at pH 3.0 for 3 h. The adaptation experiments revealed three groups of organisms: non-acid-responders, generally representing strains with the highest terminal pH values; weak acid-responders in the middle of the pH list, generating low numbers of survivors at one or two pH values, and the aciduric, strong responders generating a high number of survivors at pH values in the range 6.0 to 3.5, but not at pH 7.5. Predominant among the latter group were the S. mutans and Lactobacilli casei strains, with the most significant adaptive response exhibited by S. mutans LT11 and S. mutans Ingbritt, involving a process that required protein synthesis. Time course experiments with the latter organisms indicated that 90-120 min was required after exposure to the triggering pH before the acid response was fully functional. These results indicate that the sudden exposure of strains of oral streptococci and lactobacilli, as well as Enterococcus faecalis, to pH values between 6.0 and 3.5 results in the induction of an acid tolerance response that enhances the survival of these strains at or below pH 3.5.

Twenty-one oral Streptococcus isolates of known interbacterial coaggregation groups were tested against one another (as both producers and indicators) to detect bacteriocin-like inhibitory activity. In agar-based antagonism tests, seven strains produced small inhibitory zones (< or = 3 mm diameter) but in liquid medium, only strain Streptococcus gordonii DL1 (Challis) produced a detectable antibacterial action (bacteriocin STH1). Five strains were sensitive to bacteriocin STH1, but neither the production of nor the sensitivity to any of the antagonistic agents correlated with coaggregation groupings. Four strains (C219, 903, 118 and Wicky) developed stable resistance in response to the bacteriocin, whereas one isolate (strain 34) remained sensitive following repeated bacteriocin exposure. With one exception (strain 903), bacteriocin STH1-sensitive strains were competent for genetic transformation, but not all competent strains were bacteriocin-sensitive. Bacteriocin-resistant derivatives of transformable strains exhibited decreased competence (80-90% reduction) compared with their parent strains.

Strong interspecies adherence was demonstrated among the periodontal pathogens Treponema denticola, Bacteroides forsythus and Porphyromonas gingivalis, and between these pathogens and the commensal plaque organism Streptococcus crista. Adherence showed specificity and demonstrated saturation binding kinetics. Binding between B. forsythus and P. gingivalis appeared to be a unimodal protein-protein interaction. Binding between the other organisms was at least bimodal involving interactions between combinations of proteins and carbohydrates with a variety of sugar specificities. Salivary molecules prevented adherence between T. denticola and S. crista, and serum eliminated B. forsythus binding to P. gingivalis. All other interactions occurred to some degree in the presence of serum and saliva. Such interbacterial binding interactions may be important in the establishment of periodontopathic plaque.

Strains of Candida albicans, Candida krusei, Candida kefyr, Candida tropicalis, Candida parapsilosis and Candida guilliermondii were grown in the presence or absence of concentrations of sodium hypochlorite below the minimal inhibitory concentration and tested for a range of characteristics that may be associated with pathogenicity. Sodium hypochlorite is used routinely in hospitals in Australia for disinfection procedures, and these experiments were designed to assess the efficacy of hypochlorite as a sterilizing agent for acrylic dentures. Candida showed varying abilities to adhere to surfaces that may be present in the oral cavity. Sodium hypochlorite reduced the adhesion of all C. albicans strains and most other Candida species to both polystyrene and buccal epithelial cells. A biofilm of Streptococcus gordonii reduced the adhesion of most C. albicans strains and most other Candida species to polystyrene. However, Candida species were able to coaggregate with S. gordonii in suspension, with one strain of C. albicans, GDH 2346, showing greater coaggregating ability than the other strains or species. Sodium hypochlorite increased coaggregation of all C. albicans strains and most other Candida species. Examination of cell wall proteins from strains of C. albicans and a strain of C. parapsilosis showed that growth in hypochlorite increased the amount of protein in some existing bands and, in one strain of C. albicans, increased the number of detectable protein bands ranging from 56 to 26 kDa. Only 4 strains of C. albicans were able to produce hyphae, and 3 of these same strains and C. parapsilosis were able to produce proteinase. Hypochlorite increased the rate of blastospore to hyphal transition but had no effect on proteinase production or activity. It is concluded that hypochlorite at a concentration below the minimal inhibitory concentration may function as an anti-adhesin for Candida species but may not affect their more pathogenic characteristics.

While studying the oral bacterial biota of mice, we observed an unidentified streptococcus (TG) that eventually became the dominant species of the oral cavities of all other mice in our animal facility. We found that the strain is indigenous to Jackson Laboratory mice but is absent in animals from Charles River Laboratories. TG was also transmitted from artificially contaminated BALB/c mice to the oral cavities of 4 other mouse strains. Streptococcus sp. TG stimulated the secretory and systemic immune systems of artificially contaminated Charles River BALB/c mice but did not provoke clinical symptoms. The increase in antibody level to TG did not prevent its colonization and persistence in these mice. In mice from Jackson Laboratory, the secretory and systemic immune response to TG was significantly lower. In vitro, Streptococcus sp. TG inhibited murine oral lactobacilli and staphylococci, probably due to the production of hydrogen peroxide.

The adhesion and accumulation of oral streptococci on saliva-coated hydroxyapatite was examined in strains representing species that appear in initial plaque (Streptococcus sanguise FC1 and Streptococcus oralis C5) and in more mature plaque (Streptococcus gordonii G9B). Washed cells of strains FC1 and C5 did not attach better to saliva-coated hydroxyapatite than did strain G9B, suggesting that the degree of initial adhesiveness does not alone account for the temporal appearance of these bacteria in dental plaque. Growing cells of each strain were also examined for their ability to accumulate on saliva-coated hydroxyapatite. The addition of sucrose to the medium promoted the accumulation of strain G9B more than it promoted the accumulation of strains FC1 and C5. Sucrose also enhanced the accumulation of adhesion-defective mutants of each strain to levels similar to those of the respective parent strains. These results suggest that sucrose-dependent accumulation may facilitate the colonization of the tooth surface by these species of oral streptococci when adhesion is limited by reduced bacterial adhesiveness or limited pellicle-binding sites.

Fibrinogen inhibits the coaggregation between Porphyromonas gingivalis and Streptococcus oralis. In this study, we determined which bacterium interacts with fibrinogen in this inhibitory process. Although preincubation of each bacterium with fibrinogen did not inhibit coaggregation, its activity was completely eliminated by the addition of protease inhibitors such as N-ethylmaleimide (NEM), p-chloromercuriphenyl sulfonate and N alpha-p-tosyl-L-lysine chloromethyl ketone to the preincubation mixture with fibrinogen and P. gingivalis. However, the inhibition of coaggregation was not found after preincubation of S. oralis with fibrinogen in the presence or absence of the protease inhibitors. Labelled materials were recovered from the extract of P. gingivalis cells incubated with radioiodinated fibrinogen in the presence of NEM but not in the absence of NEM. In the binding experiment, P. gingivalis showed a much higher binding activity to fibrinogen than S. oralis. These findings suggest that fibrinogen and its fragment(s) may mask directly or indirectly the aggregation site with S. oralis on the P. gingivalis cells in its inhibitory process of coaggregation.

Most investigations of mechanisms accounting for intergeneric coaggregation have emphasized stereospecific rather than nonspecific interactions. The purpose of this investigation was to determine the relative importance of lectin-carbohydrate and nonspecific hydrophobic and ionic interactions, using a model based on strains with one of the most well understood specific coaggregation mechanisms, the lactose-reversible coaggregation of Actinomyces naeslundii and Streptococcus oralis. The kinetics of coadhesion and desorption of coadherent bacteria were studied using S. oralis 34 bound to hexadecane droplets as an affinity support for the adhesion of A. naeslundii WVU 398A. Light, confocal microscopy and transmission electron microscopy confirmed that A. naeslundii cells adhered only to the S. oralis cells, not to exposed hexadecane between the streptococci. Coadhesion was inhibited by lactose concentrations as low as 2.0 mM. The rate of coadhesion was halved at 60 mM lactose. The hydrophobicity inhibitors bovine serum albumin and defatted bovine serum albumin and the salts LiCl and KCl failed to inhibit coadhesion in the hexadecane assay, and bovine serum albumin also failed to inhibit coaggregation in a bacterial aggregation assay on glass slides. High concentrations of the salts achieved a 50% rate decrease in A. naeslundii adhesion to the S. oralis-coated droplets only when they were combined with > 20 mM lactose. Sodium dodecyl sulfate (SDS) and Tween 20 inhibition was tested by the slide coaggregation assay because they tended to emulsify the droplets; SDS was inhibitory. Lactose selectively desorbed A. naeslundii from S. oralis-coated droplets at low concentrations equivalent to those that inhibited coadhesion. Neither LiCl nor KCl desorbed A. naeslundii from the droplets, even at 500 mM. At low concentrations, SDS but not Tween 20 eluted both A. naeslundii and S. oralis from the droplets. Although the SDS results might suggest a degree of cooperative charge interactions, the results support the hypothesis that stereospecific, beta-galactoside-sensitive interactions have a much greater impact than nonspecific interactions on the coadhesion of A. naeslundii and S. oralis.

Bovine caseinate, derivatives of its glycosylated moiety [caseinoglycomacropeptide (CGP)], and caseinophosphopeptides were evaluated as inhibitors of adhesion of oral bacteria to saliva-coated hydroxyapatite beads (S-HA). All milk casein-derived components behaved as potent inhibitors of Streptococcus sanguis OMZ 9 and Streptococcus sobrinus OMZ 176 adhesion to S-HA, whereas neither bovine serum albumin nor polyethyleneglycol were able to interfere with the adhesion of these strains. By contrast, none of the molecular species tested was able to inhibit the attachment of Actinomyces viscosus Ny 1 to S-HA. On the other hand, casein derivatives were shown to displace human serum albumin from S-HA beads. They were also able to bind to the bacterial cell surface of all strains examined. Collectively, these findings suggest that interactions between acidic casein-derived milk components and the biological surfaces involved in bacterial adhesion to S-HA result in an inhibitory effect that is selective for the oral streptococci examined.

Despite the numerous investigations on the adhesion of microorganisms to buccal epithelial cells, it is difficult to correlate different results, as disparate adherence values have been reported for a given organism. As one reason for this disparity may be the indigenous or natural bacterial populations on human buccal epithelial cells, the effect of the latter on subsequent microbial adherence in vitro was examined. There was a highly significant correlation between the degree of natural bacterial colonization on pooled buccal epithelial cells from 8 healthy donors and the adhesion of a single isolate each of Streptococcus mitis, Escherichia coli and Actinomyces naeslundii. However, no such relationship could be established for Candida albicans, Streptococcus milleri and another isolate of Streptococcus mitis. As in previous studies, variation in adherence values was found, both between samples from different donors, and from the same donor over time, but to a far lesser degree in pooled samples from different donors. These results imply that natural bacterial populations on buccal epithelial cells may affect the adhesion values derived from laboratory experimentation, and hence such data should be interpreted with caution.

The mechanism of bacterial adherence to the mat of bacteria in preformed dental plaque is not well defined. This study measured the influence of saliva on the adherence of bacteria in suspension to a continuous bacterial surface in vitro. Twenty different pairs of bacteria were tested, consisting of Streptococcus spp., Haemophilus spp. and Actinomyces spp. The species were chosen based on the parameters of coaggregation, and salivary agglutination. The results were expressed as bacteria that adhered per mm2 of bacterial surface. When both the surface bacteria and the bacteria in suspension agglutinated in saliva, interbacterial adherence was increased 2.5-fold when a salivary coating was placed on the surface. When one or both of the bacteria did not agglutinate in saliva, interbacterial adherence was increased only slightly when the surface was saliva-coated. The results suggested that salivary-mediated adherence is significant to plaque formation once the tooth surface becomes covered with bacteria. Thus bacteria that are capable of agglutinating in saliva may have a distinct advantage in colonization of the plaque surface.

Strains of Streptococcus, Actinomyces and Lactobacillus were grown on glass surfaces in semi-defined medium (pH 7.0) with mucin, at a dilution rate of D = 0.1 h-1, in a modified chemostat. The accumulation of cells followed four phases. In phase 1 (0-1 h), cells did not divide on the surfaces and adhesion accounted for rapid accumulation. Phase 2 (1-4 h) comprised adhesion and cell division, and accumulation slowed, cell number doubling times (Cdt) Streptococcus, 2.7 h to 8.6 h, Actinomyces, 2.3 h to 7.5 h and Lactobacillus, 3.6 h to 3.8 h. Cell division on surfaces accounted for accumulation in phase 3 (4 h to 12 h): Cdt Streptococcus, 1.7 h to 5.2 h, Actinomyces, 2.4 h to 7.5 h and Lactobacillus, 2.2 h to 7.2 h. The biofilm stabilized in Phase 4, Cdt 18.5 h to 90.2 h. The numbers (10(6) colony-forming units per cm2) of cells in stable biofilms were Streptococcus, 4.02 to 5.12, Actinomyces, 12.5 and 34.0 and Lactobacillus, 2.77. Accumulation increased (Cdt 0.9 h-2.7 h) when cells were exposed to glucose excess or high dilution rates and phase 2 of accumulation did not occur.

The ability of oral lactobacilli to coaggregate with streptococci and actinomycetes was investigated. Of the 7 species of lactobacilli studied, only two were capable of coaggregation and the coaggregation was restricted to streptococci. Lactobacillus salivarius strains (2/4) coaggregated with Streptococcus salivarius, Streptococcus gordonii, Streptococcus crista and tufted Streptococcus sanguis II strains. Lactobacillus fermentum (2/3) coaggregated with S. gordonii and S. sanguis. The coaggregation between L. salivarius and S. salivarius, S. gordonii or tufted S. sanguis II strains was mediated by a protein on the surface of the lactobacilli and was not inhibited by lactose. The coaggregation between L. fermentum and the streptococci was mediated by protein on the surface of the streptococci and was inhibited by lactose.

Adherence of Porphyromonas gingivalis to strains of Streptococcus sanguis and Streptococcus mitis deposited on nitrocellulose paper was investigated. A variety of laboratory strains and clinical isolates of P. gingivalis bound to both S. sanguis and S. mitis. Binding of P. gingivalis to all but one of the streptococci was not inhibited by salivary molecules. Pretreatment of P. gingivalis with periodate and pretreatment of S. sanguis and S. mitis with pronase decreased binding, suggesting that adherence may be mediated by a protein on the streptococci interacting with a carbohydrate on P. gingivalis. Binding was not inhibited by a selection of simple sugars. The ability to adhere to early plaque bacteria such as S. sanguis and S. mitis may be important in the colonization of the mouth by P. gingivalis.

Streptococcus mitis ATCC 903 aggregated when suspended in salt solutions containing the ions zinc, aluminium, lanthanum and cerium. This aggregation was very rapid as compared to spontaneous aggregation occurring in this strain. It was not inhibited by alkaline pH. Washed bacteria treated previously with zinc sulphate recovered and retained their ability to aggregate spontaneously at a slow rate. No such effect was observed with lanthanum-induced aggregation. The aggregates caused by lanthanum chloride were stable in sodium chloride up to 5 M concentrations. Magnesium sulphate dissociated these aggregates at 250 mM. Aggregation induced by zinc sulphate was less stable in these salts. The spontaneously aggregated cells were dissociated completely at 10 mM magnesium sulphate or 100 mM sodium chloride. Bacteria which had lost their ability to aggregate, owing to trypsin or beta-galactosidase treatment, were re-aggregated after addition of zinc, lanthanum or aluminium ions. Galactosamine inhibited the spontaneous aggregation and aggregation induced by zinc but not the aggregation induced by lanthanum or aluminium ions. In conclusion, the results provide a molecular model of induced and spontaneous aggregations where the two phenomena are qualitatively different.

To demonstrate the specificity of cellular interaction in the spontaneous aggregation of Streptococcus mitis ATCC 903 the inhibitory effect of various sugars was studied. More than 90% inhibition was recorded in 0.1 M concentrations of D-glucosamine, D-galactosamine and D-mannosamine. A reduction of the inhibition by more than 50% was observed when the N-acetylated derivatives of the hexosamines were tested. Polymers containing hexosamines such as hyaluronic acid, heparin and fetuin were potent inhibitors, in contrast to dextrans of different molecular weights. Inhibition was less than 20% in lactose, melibiose, cellobiose, methyl beta-D-galactopyranoside and a number of other sugars tested in 0.1 M concentration. The bacteria retained their ability to aggregate after removal of the inhibitory sugars by washing in phosphate buffer. These findings support the hypothesis that the specific nature of the spontaneous aggregation of S.mitis ATCC 903 depends on a lectin-ligand type interaction.

The aggregation properties of Streptococcus mitis ATCC 903 cells modified by treatment with heat or different enzymes was investigated. Bacteria that had the ability to aggregate spontaneously lost this capacity by treatment with proteolytic enzymes, beta-galactosidase or heat. Cells subjected to different types of modification were mixed in various proportions and their aggregation properties were recorded. To discriminate between the two kinds of cells in the suspension, one partner in the aggregation reaction was labelled with 14C-palmitic acid. Bacteria treated with beta-galactosidase co-aggregated with spontaneously aggregating cells (not modified) and with cells treated with heat. Heat-treated cells co-aggregated with spontaneously aggregating cells and with cells treated with beta galactosidase. Cells treated with trypsin did not co-aggregate either with spontaneously aggregating cells or cells treated with heat or beta-galactosidase. These findings are consistent with the hypothesis that two surface components are required for specific aggregation of S. mitis cells. We suggest that both components are degraded or released from the bacterial surface by treatment with trypsin (and other proteolytic enzymes) as shown by the inability of these cells to take part in any co-aggregation with spontaneously aggregating cells. Treatment with beta-galactosidase degrades a carbohydrate receptor constituting the terminal part of a glycoprotein. Heat treatment inactivates a protein lectin. The fact that heat-treated bacteria and bacteria treated with beta-galactosidase aggregate when mixed supports the assumption that two components take part in the aggregation reaction.

Spontaneous aggregation of Streptococcus mitis ATCC 903 was highly dependent on various electrolytes such as sodium chloride, sodium phosphate or potassium phosphate with an optimum at 10 mM concentration. No aggregation was obtained in distilled water. Addition of a number of mono- or divalent ions to bacteria incubated in 10 mM phosphate or tris-maleate buffers resulted in an inhibition of aggregation. A 50% inhibition of aggregation was obtained at low concentrations of calcium, magnesium, sulphate and carbonate ions. Other mono- and divalent ions tested were needed in significantly higher concentrations in order to obtain the same inhibition. Aggregation inhibition by calcium or magnesium salts was reversed by chelation with EDTA. Washing bacteria previously treated with magnesium or calcium salts resulted in a complete recovery of aggregating ability. Spontaneously aggregated cells were completely dissociated at 10 mM magnesium sulphate or 100 mM sodium chloride. The results indicate that electrostatic interactions are of major importance for aggregation of S. mitis ATCC 903 whereas hydrophobic interaction plays a minor role.

Outer membrane vesicles purified from Bacteroides gingivalis culture supernatant bound to serum coated hydroxyapatite (SeHA). The immobilized vesicles served as receptors for a number of species of oral streptococci. The binding of Streptococcus sanguis 12 to SeHA was increased 10 times by the vesicles. Vesicle-associated binding increased proportionally with an increase in the number of bound vesicles. Arginine and lactose both partially reduced binding of S. sanguis. Heating the vesicles destroyed their binding ability whereas heating S. sanguis reduced but did not eliminate vesicle-mediated binding.

Previous studies have shown that the mechanism of spontaneous aggregation of Streptococcus mitis ATCC 903 depends on a lectin-ligand type interaction. To study the specificity of the ligand, the binding of a number of lectins of different sugar specificities to the surface of untreated, trypsin and beta-galactosidase-treated bacteria was studied by assessing aggregation. Untreated bacteria were rapidly aggregated by concanavalin A (Con A), wheat-germ agglutination (WGA) and helix pomatia lectin (HPL). Other lectins tested, e.g. peanut agglutinin and soy bean lectin, did not induce aggregation. Lectin-induced aggregation was distinguished from the spontaneous one by recording the course of aggregation and inhibition of lectins by specific sugars. Trypsin-treated bacteria lost their ability for both spontaneous and lectin-induced aggregation. beta-galactosidase-treated bacteria were aggregated only in the presence of Con A and HPL. The bacteria retained their ability for spontaneous aggregation after removal of lectins and inhibitory sugars. These findings suggest that ligand is of glycoprotein nature, since it was removed from the bacterial surface by treatment with trypsin, as shown by the inability of treated cells for both spontaneous and lectin-induced aggregation. Partial degradation of the carbohydrate part of the ligand is indicated by the ability of beta-galactosidase-treated bacteria to aggregate in the presence of Con A and HPL.

Endothelial cells were prepared from rat heart, and the ability of various bacterial species to adhere to these cells was investigated, with special regard to alpha-hemolytic streptococci. The endothelial cells were identified by morphology and by the presence of F VIII antigen. Seventeen bacterial strains representing various species were used in the in vitro adherence tests; 10 strains represented various strains of alpha-hemolytic streptococci, 5 of which were isolated from patients with infective endocarditis and 5 from the throats of healthy individuals; 2 were Neisseria meningitidis, 2 N. gonorrhoeae, 2 S. aureus and 1 E. coli. The highest adherence rate was found for alpha-hemolytic streptococci isolated from patients with infective endocarditis. The difference compared to carrier strains of alpha-hemolytic streptococci and to the other tested strains was statistically highly significant.