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.

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.

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.

Bacterial invasion of pulpal dentin wall in vitro.

Instrumented root canals of extracted human teeth were inoculated with known pulpal bacterial isolates. The inoculated teeth were immersed in the appropriate culture media and incubated at 37 degrees C for varying periods. Streptococci multiplied in the root canals and invaded the radicular dentinal tubules. The extent of bacterial invasion was time-dependent. This experimental model of bacterial invasion was time-dependent. This experimental model may be useful in investigating the effect of intra-canal medicaments on microorganisms lodged in the pulpal dentin wall.

Competitive binding among oral strptococci to hydroxyapatite.

The relative affinity of various oral streptococci for hydroxyapatite (HA) differed widely. Streptococcus mutans affinity for dextran-coated HA was the highest of all the streptococci to any HA coating. S salivarius had the lowest affinity, and S sanguis affnity was higher then S miteor for saliva-coated HA. Competition for binding sites on saliva-dextran-coated, dextran-coated HA. Hhwever, competition between this pair was not for the same binding site but for closely associated sites.

Experimental formation of "corn cob" in vitro.

Organisms isolated from corn cob were examined for aggregation with Bacterionema matruchotti. Such aggregations, interpreted as corn cob formation, in vitro, occurred with five isolated identified as Streptococcus sanguis. A cell-surface component of Streptococcus sanguis or a phospholipid-related substance of Bacterionema matruchotii appeared to be involved.

Reduction of dental decay in rampant caries individuals following short-term kanamycin treatment.

A week of kanamycin gel treatment before and after the placement of dental restorations, compared to a placebo gel treatment, significantly reduced the levels of cultivable bacteria, S mutans and S sanguis, in the plaque samples collected immediately after the completion of the gel treatments, and was associated with a 46% reduction in new carious surfaces in the 14- to 37-month period following the gel treatment.

Microbial characterization of an experimental cariogenic plaque in man.

Experimentally induced plaque seemed to originate by direct contact inoculation from the vestibular mucosa and saliva. During the next seven days, this plaque developed its own characteristics. Populations of Streptococcus mutans usually less than 2% of total streptococci population in plaques that were less than three days old, increased between days 3 and 7. Proportions of S sanguis, high in early samples, decreased after day 3. Populations of S salivarius, which usually outnumbered other streptococci, fluctuated widely through day 3, and then increased in proportion in subjects who were more productive of experimental caries, but decreased in subjects who were less productive. Proportions of plaque flora comprising lactobacilli paralleled those of S salivarius.

Compounds which affect the adherence of Streptococcus sanguis and Streptococcus mutans to hydroxyapatite.

Several compounds were evaluated in an in vitro assay system for their ability to block the adherence of Streptococcus sanguis to saliva-coated hydroxyapatite and Streptococcus mutans to dextran-coated hydroxyapatitite. Fatty acids, ranging from C-12 to C-20, the enzyme amylase, chlorhexidine, human sera, and several serum proteins blocked S sanguis adherence to saliva-coated hydroxyapatite. Chlorhexidine blocked S mutans adherence to dextran-coated hydroxyapatite, but human sera and serum proteins did not. The effects of these compounds on the adherence of these organisms to hydroxyapatite may help in the development of specific plaque control methods for use in human populations.

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)

Comparison of antiplaque agents using an in vitro assay reflecting oral conditions.

An in vitro assay is described using saliva-treated bovine enamel slabs for determining the potential of chemotherapeutic agents to adsorb to tooth surfaces and act against plaque-forming bacteria. Chlorhexidine was found to inhibit the formation of in vitro plaque by Actinomyces viscosus, A naeslundii, Streptococcus mutans and S sanguis. Actinobolin was found to have marked antibacterial properties but limited adsorptive qualities.

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.

The viability of microorganisms in carious lesions five years after covering with a fissure sealant.

Carious lesions that were covered with a pit and fissure sealant for five years yielded bacterial cultures that were predominantly negative. Sixteen of 18 test sites judged to have active caries in 1972 were found inactive in 1977; ten of 12 sites suspected to have caries in 1972 were deemed to have inactive caries in 1977. Sealant treatment resulted in an apparent 89% reversal from a caries-active to a caries-inactive state. These data confirm and extend previous observations that a limited number of cultivable organisms persist in some lesions but their numbers are few, and they do not appear capable of continuing the destruction of tooth structure.

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.