Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis.

Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

Systematic study of genes influencing cellular chain length in Streptococcus sanguinis.

Streptococcus sanguinis is a Gram-positive bacterium that is indigenous to the oral cavity. S. sanguinis, a primary colonizer of the oral cavity, serves as a tether for the attachment of other oral pathogens. The colonization of microbes on the tooth surface forms dental plaque, which can lead to the onset of periodontal disease. We examined a comprehensive mutant library to identify genes related to cellular chain length and morphology using phase-contrast microscopy. A number of hypothetical genes related to the cellular chain length were identified in this study. Genes related to the cellular chain length were analysed along with clusters of orthologous groups (COG) for gene functions. It was discovered that the highest proportion of COG functions related to cellular chain length was 'cell division and chromosome separation'. However, different COG functions were also found to be related with altered cellular chain length. This suggested that different genes related with multiple mechanisms contribute to the cellular chain length in S. sanguinis SK36.

TLR2 sensing of F. nucleatum and S. sanguinis distinctly triggered gingival innate response.

Gingival tissue faces constant exposure to micro-organisms. It functions as part of the host response, an anti-microbial barrier that recognizes and discriminates between commensal and pathogenic bacteria. This study aimed to evaluate and compare the effects of cell wall extracts from different periodontal bacteria, commensals Streptococcus sanguinis and Fusobacterium nucleatum and the pathogen Porphyromonas gingivalis, on the innate immune response of gingival keratinocytes and the role of TLR2 in regulating this. We assayed mRNA levels to determine the expression of human beta-defensins (hbetaD2, hbetaD3), interleukin-1alpha, -1beta, 6 and 8 and matrix metalloproteinase-9. F. nucleatum extracts induced beta-defensin and inflammatory marker mRNA expression at higher levels than P. gingivalis. Extracts from the Gram-positive commensal S. sanguinis did not upregulate the host response. TLR2 extinction inhibited the upregulation of beta-defensin and cytokine transcripts by F. nucleatum extracts but, in contrast, led to a weak induction of hbetaD3 after challenge with S. sanguinis extracts. Although F. nucleatum strongly induces innate immune and inflammatory mediators, S. sanguinis limits their expression through TLR2. Together, our data demonstrate that gingival keratinocytes recognize and discriminate between Gram-positive and Gram-negative commensal extracts, in part through TLR2, to activate different signaling pathways of the innate immune host response.

Adhesion of Streptococcus sanguinis to glass surfaces measured by isothermal microcalorimetry (IMC).

Bacterial adhesion is the first step in the development of the oral biofilm, called dental plaque. Plaque is the cause of caries, periodontal diseases, and periimplantitis. Investigations of dental plaque, including bacterial adhesion, employ various in vivo and in vitro models using microscopic methods. Microcalorimetry offers another direct approach. The model organism Streptococcus sanguinis is one of the first colonizers adhering to the saliva-coated human tooth surfaces or dental materials within minutes after tooth cleaning. TAM III thermostats, equipped with microcalorimeters, were used for isothermal microcalorimetric (IMC) measurements of heat production as a function of time, expressed by power-time (p-t) curves. Continuous measurements of heat production of growing S. sanguinis cells showed their overall metabolic activity and were highly reproducible. For the adhesion experiments the bacteria were allowed to adhere to different amounts of glass beads. Growing S. sanguinis cells produced a characteristic p-t curve with a maximum of 500 microW at 4.5 h when reaching 10(9) cells ml(-1). The same number of stationary S. sanguinis cells, suspended in PBS produced only approximately 30 microW at 0.5 h due to adhesion. But the amount of heat increased with available glass surface area, indicating that a portion of the heat of adhesion was measured. Similar results were obtained with stationary S. sanguinis cells suspended in human saliva. This study shows that microcalorimetric evaluation of initial bacterial adhesion is indeed possible and may become a rapid, reproducible screening method to study adhesion of different bacteria to different dental materials or to modified surfaces.

Cytochemical differences in bacterial glycocalyx.

To examine new cytochemical aspects of the bacterial adhesion, a strain 41452/01 of the oral commensal Streptococcus sanguis and a wild strain of Staphylococcus aureus were grown with and without sucrose supplementation for 6 days. Osmiumtetraoxyde (OsO4), uranyl acetate (UA), ruthenium red (RR), cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs), and the tannic acid-metal salt technique (TAMST) were applied for electron microscopy. Cytochemically, only RR-positive fimbriae in S. sanguis were visualized. By contrast, some types of fimbriae staining were observed in S. aureus glycocalyx: RR-positive, OsO4-positive, tannophilic and CB-positive with ceasing point at 0.3 M MgCl2. The CB staining with CEC, used for the first time for visualization of glycoproteins of bacterial glycocalyx, also reveals intacellular CB-positive substances-probably the monomeric molecules, that is, subunits forming the fimbriae via extracellular assembly. Thus, glycosylated components of the biofilm matrix can be reliably related to single cells. The visualization of intracellular components by CB with CEC enables clear distinction between S. aureus and other bacteria, which do not produce CB-positive substances. The small quantities of tannophilic substances found in S. aureus makes the use of TAMST for the same purpose difficult. The present work protocol enables, for the first time, a partial cytochemical differentiation of the bacterial glycocalyx.

Distribution of cells between solid/liquid and liquid/liquid interfaces.

The use of aqueous two-phase systems (ATPSs) and each system's individual phase-forming species to prevent Streptococcus sanguis attachment onto hydroxyapatite discs was explored. The strategy that we followed was to attach the cells to a solid surface in the presence of an additional interface. Conditions under which, simultaneously, the phase-forming species form two phases and the cells proliferate were identified. Growth curves were constructed in the presence of various polymers and salts commonly used to prepare ATPSs. Several aqueous two-phase systems were selected such that bacterial growth was comparable to that observed in pure medium. Cells were allowed to attach to hydroxyapatite discs for 7 days in the presence of varying concentrations of media, media with polymer, media with salt, and media with ATPS. Streptococcus sanguis attachment to the disks was evaluated by scanning electron microscopy. The addition of a PEG/Na(2)SO(4) ATPS to high concentrations of yeast-tryptone (YT) media (>65%) and of a PEG/MgSO(4) ATPS to nutrient-limited media reduces surface coverage of S. sanguis to less than 10%. Comparison of the attachment levels for the systems containing PEG/Na(2)SO(4) to media containing the individual phase-forming species and to the YT reference systems indicated that nutrient availability did not affect attachment.

A role for glycoprotein Ib in Streptococcus sanguis-induced platelet aggregation.

Numerous studies have implicated bacteria in cardiovascular disease, but there is a paucity of information on the mechanism involved. In this study we show how the common oral bacterium Streptococcus sanguis can directly interact with platelets, resulting in activation and aggregate formation. Platelet aggregation was dependent on glycoprotein IIb/IIIa (GPIIb/IIIa) and thromboxane. Platelets could also directly bind to S sanguis, but this interaction was not inhibited by GPIIb/IIIa antagonists. Antibodies to GPIb could inhibit both platelet aggregation and platelet adhesion to bacteria. This suggested a direct interaction between GPIb and S sanguis; however, this interaction did not require von Willebrand factor, the normal ligand for GPIb. By use of a range of monoclonal antibodies to GPIb and the enzyme mocharagin, which cleaves GPIb at amino acid 282, the interaction was localized to a region within the N-terminal 1-225 portion of GPIbalpha. Furthermore S sanguis failed to induce aggregation of platelets from a patient with Bernard-Soulier disease, the organism bound to Chinese hamster ovary cells transfected with the GPIbalpha gene but did not bind to mock-transfected cells and biotin-labeled S sanguis cells bound to purified GPIb in ligand blots. It is suggested that the interaction between S sanguis and GPIb is important in the pathogenesis of infective endocarditis and may also play a contributory role in some cases of myocardial infarction.

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.

Involvement of human mucous saliva and salivary mucins in the aggregation of the oral bacteria Streptococcus sanguis, Streptococcus oralis, and Streptococcus rattus.

The contribution of human parotid (Par) and submandibular/sublingual (SM/SL) saliva and of the human whole salivary mucin fraction (HWSM) to saliva-induced bacterial aggregation was studied for S. sanguis C476, S. oralis I581, and S. rattus HG 59. The mucous SM/SL saliva showed a much higher aggregation potency towards the S. sanguis and S. oralis strain than did the serous Par saliva. The SM/SL saliva-induced aggregation was observed after 30 min, at 60 min followed by the Par saliva-induced aggregation, and showed a 4-fold higher aggregation titer of 128 for S. sanguis, and an 8-fold higher titer of 516 for S. oralis. In contrast, the Par saliva showed a slightly higher aggregation activity than the SM/SL saliva towards S. rattus as judged by a twofold higher titer of 64. Morphologically, however, the SM/SL saliva-induced aggregation of S. rattus was far more pronounced as was also found for S. sanguis. Finally, the HWSM-induced aggregation showed a 4 to 8-fold higher titer than the originating salivary source, measuring 2048 for S. oralis and 128 for S. rattus. Moreover, no difference was observed in aggregation activity between the HWSM from whole saliva of a blood group O donor and the HWSM from SM/SL saliva of a blood group A donor. All the data point to an important, though not exclusive role of the human salivary mucin fraction in the saliva-induced aggregation of these strains.

Spontaneous switching of the sucrose-promoted colony phenotype in Streptococcus sanguis.

Streptococcus sanguis on media containing 3% sucrose gives rise to characteristic hard cohesive colonies (designated Spp+). Populations of Spp+ bacteria (strain Challis) on sucrose media switch to a soft noncohesive phenotype (designated Spp-) at a frequency of 10(-4) to 10(-3). Spp- bacteria switch back to Spp+ bacteria at a similar frequency. Successive rounds of Spp variation were observed. The Spp phenotypic switch was associated with changes in extracellular glucosyltransferase activity.

Intergeneric rosettes: sequestered surface recognition among human periodontal bacteria.

The human oral bacteria, Streptococcus sanguis and Bacteroides loescheii, when mixed in equal numbers in vitro, formed large settling coaggregates. As the relative number of each cell type was changed, coaggregates became smaller until at cell-type ratios of 10 to 1, rosettes formed. Rosettes consisting of a streptococcal cell in the center surrounded by bacteroides cells exhibited surface recognition properties of only the bacteroides, which coaggregated with many other cell types such as Actinomyces naeslundii, and formed large settling multigeneric aggregates. The ecological significance of these results derives from the following: (i) the direct demonstration that intergeneric coaggregates can protect the central cell from or prevent its access to other cells in the environment, and (ii) the potential for these effects to occur during bacterial succession of various cell types observed in progressively more severe stages of human periodontal disease.

The surface free energy of oral streptococci after being coated with saliva and its relation to adhesion in the mouth.

Contact angle measurements on layers of bacteria were used to determine the bacterial surface free energy (gamma b) of a variety of oral streptococcal strains, both without and after being coated with human whole saliva. At least four isolates of each species, either freshly isolated or laboratory strains, were used. The species Streptococcus mutans, S. sanguis, and S. salivarius were homogeneous, having high surface free energies, and were not affected by saliva treatment (gamma b = 106 +/- 12 and 107 +/- 10 erg X cm-2 in the absence and presence of saliva coating, respectively; n = 20). S. mitis had a very low surface free energy (46 +/- 15; n = 5), which was significantly increased after salivary adsorption (71 +/- 14 erg X cm-2; p less than 0.002). The species S. milleri contained strains with both high and low gamma b. Calculation of the interfacial free energy of adhesion (delta F adh) for bacteria from a saliva suspension to solid surfaces with various arbitrary surface free energies (gamma s) showed that, theoretically, most strains will encounter thermodynamically favorable conditions for adhesion to surfaces with a gamma s above 62 erg X cm-2. However, S. mitis strains not coated with saliva would only be able to adhere to surfaces with gamma s lower than this value. Saliva-coating reverses the calculated relationship with gamma s for these strains. The results indicate that an enamel surface with a low gamma s value would be thermodynamically unfavorable for adhesion of most oral streptococci.

A conceptual model for the co-existence of Streptococcus spp. and Actinomyces spp. in dental plaque.

One of the most important questions in ecology is how to explain the co-existence of the variety of physiologically related organisms in the same habitat. A model is presented for the co-existence of Streptococcus species and Actinomyces species in dental plaque. The hypothesis is that these organisms co-exist because they simultaneously utilize several carbon and energy substrates. The hypothesis follows from the observation that the growth yield of oral streptococci and actinomyces in saliva is limited by carbohydrate. Preliminary experiments were undertaken to test the hypothesis using mixed chemostat cultures and gnotobiotic rats. Competition between S. mutans K1R and A. viscosus Ut2 in mixed chemostat cultures on glucose and asparagine was hampered by the early appearance of high-glucose-affinity variants of A. viscosus. From the physiological characteristics of S. sanguis and S. milleri, it might be predicted that simultaneous utilization of carbohydrate and arginine would enable these organisms to co-exist with S. mutans in an ecosystem. To test this mechanism under natural conditions, germ-free rats were inoculated with a combination of S. mutans K1R and S. sanguis P4A7 or the combination S. mutans K1R and S. milleri B448. The rats were fed on three different diets: (1) 58% cornstarch; (2) 48% cornstarch and 10% sucrose; and (3) 53% cornstarch and 5% arginine. The results of this experiment demonstrated that dietary arginine caused a significant decrease of the ratios K1R/P4A7 and K1R/B448 in dental plaque.(ABSTRACT TRUNCATED AT 250 WORDS)