Comparative study of two variants of the mouth Streptococcus sanguis with different colonial morphologies and abilities to adhere.

A rough (R) variant of Streptococcus sanguis strain G26 showing increased in-vitro adherence and a slower growth rate compared with its smooth (S) wild-type strain. Serological comparison revealed no difference in antigenic composition. The R-form was less sensitive to NaCl, NaF and NaN₃ but more sensitive to chlorhexidine. The R-form was more readily transformed than the smooth wild-type. Cells of both phenotypes synthesized α-(1 → 6)-rich, dextran-like glucans and did not appear to produce α-(1 → 3)-rich water insoluble glucans. Furthermore, no significant difference was observed in the caries-producing potential in rats. Tests for cell-bound lipoteichoic acid (LTA) showed that hot phenol extracts of R-cells contained more LTA than did extracts of S-cells. The significance of these findings for increased in vitro adherence of the R-form is discussed.     

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.     

Neuraminidase activity: a biochemical marker to distinguish Streptococcus mitis from Streptococcus sanguis

Selected reference and freshly isolated strains of Streptococcus mitis (mitior) and Streptococcus sanguis were assayed for cell-associated neuraminidase activity by their ability to hydrolyze [3H-] sialyllactitol. A cell-associated neuraminidase was detected with S. mitis and S. sanguis serotype II (reclassified as S. mitis) but not with S. sanguis serotypes I and III. Neuraminidase activity of S. mitis correlated with this organism's inability to hydrolyze arginine, aesculin, and few, if any, sugars. The findings indicate that the presence of cell-associated neuraminidase activity is useful for the taxonomic classification of S. mitis.     

Fluoride accumulation by a strain of human oral Streptococcus sanguis.

Cells of Streptococcus sanguis strain H7PR3 accumulated fluoride when incubated with sodium fluoride at pH 5.5. In media containing 1 ppm of added F^? (5 × 10^?5 M NaF) the intracellular concentration of the anion at equilibrium was about 6 ppm. Incubation of the cells in the presence of iodoacetamide, or in the absence of added glucose, or at different temperatures between 22–37 °C, had very little effect on the amount of fluoride taken up by the cells so that the accumulation system appeared to be independent of energy-producing metabolic processes. Part of the fluoride taken up by the cells was irreversibly bound and maximum binding was observed at 1 ppm of added fluoride. The apparent affinity constant for the system was 0.3 ppm (1.5 × 10^?5 M). Fluoride was shown to bind to cytoplasmic components within the cells. A second fraction, the loosely bound fluoride, was concentrated two-fold within the cells and could be removed from the cells by washing with buffer. Uptake and binding were observed also at pH 7.0. Accumulative processes such as these probably contribute to the reported uptake of fluoride by dental plaque, and may be important in determining the metabolic susceptibilities of oral microorganisms to the anion.     

Studies on the bacterial components which bind Streptococcus sanguis and Streptococcus mutans to hydroxyapatite.

Experiments utilizing radio labelled oral streptococci in an in-vitro assay system have demonstrated the selective ability of certain species of these bacteria preferentially to adhere to saliva-coated and dextran-coated hydroxyapatite (HA). Pretreatment of Streptococcus sanguis and Streptococcus mutans with the proteolyic enzymes trypsin and pronase greatly reduced their ability to adhere to saliva or dextran-coated HA respectively. Pretreatment of Strep. sanguis with lipase increased its adsorption to saliva-coated HA and stearic acid-blocked adsorption. Cell walls isolated from Strep. sanguis proved effective in blocking Strep. sanguis adsorption to salivacoated apatite. Cells of Strep. mutans after treatment with dextranase or low molecular weight dextrans did not adhere well to dextran-coated HA.     

Kinetic analysis of Streptococcus sanguis adhesion to artificial pellicle

Studies of equilibria between Streptococcus sanguis and artificial pellicle have suggested that there are multiple binding sites for the organism. In the present study, adhesion of S. sanguis to saliva-coated hydroxylapatite was examined by means of kinetic methods. Cell-pellicle complex formation was measured from initiation of binding to equilibrium. Rate constants were calculated for forward reactions (adsorption) and reverse reactions (desorption). Initial binding obeyed reversible, first-order kinetics, whereas desorption of bound cells followed biphasic kinetics. Initial desorption proceeded approximately ten times faster than the slower second rate. The results are consistent with the mechanism C + P reversible CP* in equilibrium with CP in which CP* represents the reversible equilibrium that shifts at a discrete rate to the high-affinity CP state. Thus, the biphasic binding behavior that has been previously deduced from equilibrium studies may be attributed to a time-dependent shift from close apposition to pellicle, stabilized by low-specificity forces, to a higher-affinity binding.     

影响血链球菌粘附因素的研究

目的 探讨ＰＨ值、钙离子对血链球菌粘附至血清,包被羟磷灰石的影响。方法 采用Ｃｌａｒｋ创立的体外粘附实验模型,以「＾３Ｈ」胸腺嘧核标记对血锭球菌的粘附进行定量观察。结果不同ＰＨ值组测得的放射性测量计数率差异有显著性;其中ＰＨ值为６．０－７．５时ｅｐｍ值最高,ＰＨ值小于５．５时ｅｐｍ则明显降低。不同钙离子浓度的实验组ｃｐｍ值均显著高于对照组,当钙离子浓度为１．２５３．７５ｍｍｏｌ／Ｌ时ｅｐｍ了高。结     

Identification of a Streptococcus sanguis receptor for salivary agglutinins

The objective of this study was to characterize a fraction from oral streptococci containing receptor activity for salivary agglutinin molecules. Several species and strains of streptococci were disrupted in a Ribi press. The supernatant was nuclease-treated and subjected to differential centrifugation. Receptor activity in the fractions was measured by the inhibition of saliva-mediated bacterial aggregation. In addition, bacterial strains were tested for their ability to aggregate and to deplete saliva of agglutinin activity. Three patterns of activity were observed: Streptococcus sanguis M5 depleted saliva of agglutinin activity and aggregated well; Streptococcus sanguis CC5A depleted saliva of agglutinin but did not aggregate well; and Streptococcus faecalis S-161 neither depleted saliva of agglutinin nor did it aggregate. The 105,000 g supernatant fractions derived from Ribi-disrupted Streptococcus sanguis M5 and CC5A, but not from Streptococcus faecalis, showed dose-dependent inhibition of saliva-mediated aggregation. This inhibitory activity was non-dialyzable, had the same heat and trypsin sensitivity as that seen with intact bacteria, and was not due to enzymatic digestion of the salivary agglutinin. Iso-electric focusing revealed a single active region with a pI of 5.5 which was clearly separated from the bulk of the bacterial proteins.     

Evidence for an ecto-ATPase on the cell wall of Streptococcus sanguis

Certain strains of viridans streptococci bind platelets, which release ATP from dense granules and then aggregate. By hydrolyzing the released ATP to the platelet agonist, ADP, cell wall-associated ATPase activity of Streptococcus sanguis may amplify the aggregation of platelets. To identify and characterize this ecto-ATPase activity, whole cells were incubated with [¹⁴C]-ATP. The cell-free nucleotides were separated by thin-layer chromatography and quantified by liquid scintillation counting. Whole-cell activity showed temperature and pH optima in the physiological range. To isolate a soluble fraction with ATPase activity from the cell wall, whole cells were digested under osmotically stable conditions to produce protoplasts. Protoplasts and cells were separated from soluble cell wall materials by centrifugation. ATPase activity in cell fractions was identified by zymograms of native 8% polycrylamide gels after electrophoresis. The ecto-ATPase preparation, membrane and cytoplasmic ATPase in lysed protoplasts showed different zymograms and sensitivity to inhibition by DCCD, ouabain vanadate, azide and NEM. In electron micrographs of ultrathin sections of cells of S. sanguis , ATPase activity was localized to the cell wall. Since the pattern of localization to the wall changed with the phase of growth, the ecto-ATPase of S. sanguis may be associated with the development and maintenance of the cell wall.     

Characteristics of a hydrogen peroxide-forming pyruvate oxidase from Streptococcus sanguis

A cytoplasmic pyruvate oxidase was partially purified from Streptococcus sanguis ATCC 10556. The enzyme used pyruvate, inorganic phosphate and oxygen as substrates, and formed acetyl phosphate and hydrogen peroxide. In this reaction carbon dioxide can also be expected, but this product was not looked for. The enzyme was dependent on thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and magnesium ions for activity. Its relative molecular weight (Afr) was 260,000 as calculated from its migration distance in polyacrylamide gradient gel. The enzyme was hysteretic and its activity was not influenced by various low molecular-weight substances known to be present in the cytoplasm. The pH optimum of the enzyme was 6.7 to 7.5 and its activity was not inhibited by 1 mM hydrogen peroxide or iodoacetamide, or by 10 mM potassium cyanide, sodium azide, iodoacetate, sodium fluoride, zinc chloride, EDTA, or dithiothreitol.     

Cell surface mutants of Streptococcus sanguis with altered adherence properties

Two mutants of Streptococcus sanguis (Challis) with altered cell-surface hydrophobicities were isolated after mutagenesis with ethyl methanesulphonate. Cells of one mutant were more hydrophobic than those of the parent strain and they adhered better to hexadecane, octyl-Sepharose, hydroxylapatite, saliva-treated hydaroxylapatite, and to Actinomyces viscosus. The second mutant had reduced hydrophobicity, and cells adhered less well to all the above surfaces except the actinomycetes, adsorption to which was similar to that of the wild-type strain. The differences in hydrophobicity of the mutants were associated with alterations in amount and exposure of surface proteins, in particular two envelope proteins ( M _r 43,000 and 45,000). The properties of the mutants support the contention that hydrophobic forces are determinants in S. sanguis adherence.     

Dental caries in gnotobiotic rats immunized with purified glucosyltransferase from Streptococcus sanguis.

A highly significant decrease in smooth-surface carious lesions (p < 0.001) was produced in gnotobiotic Sprague-Dawley rats, monocontaminated with a cariogenic Streptococcus sanguis and immunized with a purified glucosyltransferase from the same organism. No significant difference was observed in the number of fissure-caries lesions.     

Effect of neuraminidase on the adherence to salivary pellicle of Streptococcus sanguis and Streptococcus mitis

Neuraminidase-sensitive adherence to experimental salivary pellicles was studied using eight strains of Streptococcus sanguis and five strains of Streptococcus mitis. Approximately 60% of the strains of each species showed significantly lower adherence to neuraminidase-treated versus untreated saliva-coated hydroxyapatite. In addition, the adherence of several of these streptococcal strains to saliva-coated hydroxyapatite and neuraminidase-treated saliva-coated hydroxyapatite was inhibited using galactose and N-acetyl-D-galactosamine. Results from these studies suggested that several salivary receptors mediate adherence of these species.     

Chemiluminescence of neutrophils from patients with Beh?et's disease and its correlation with an increased proportion of uncommon serotypes of Streptococcus sanguis in the oral flora

Zymosan-induced chemiluminescence was investigated in whole blood and in neutrophils: in both, the peak count was frequently elevated in Beh?et's disease, and was significantly higher than in healthy controls; similarly the peak time was shorter. There were more uncommon serotypes of Streptococcus sanguis in the oral flora of patients with Beh?et's disease. Common serotypes were present in the flora of healthy controls, but not in patients with the disease. The percentage of Strep. sanguis in the oral flora was significantly correlated with the level of chemiluminescence response. Thus infection with uncommon serotypes of Strep. sanguis may play a role in the aetiology of Beh?et's disease.     

The role of transamination in ammonium assimilation in Streptococcus sanguis.

Synthesis of aspartate, alanine, valine, leucine and isoleucine by the addition of ammonium to suspensions of Strep. sanguis was clearly detected and no glutamine nor asparagine was found. Increase of glutamate was not predominant, although glutamate dehydrogenase is considered the primary enzyme for ammonium assimilation. High aspartate aminotransferase activity found in cell-free extract can account for the low metabolic pools of glutamate and 2-oxoglutarate and the high metabolic pool of aspartate. Since ammonium-stimulated ¹⁴CO₂ formation from [1-¹⁴C]-D-glucose, most NADPH necessary for ammonium fixation might be regenerated from the hexose-monophosphate shunt.     

Some aspects of protease production by a strain of Streptococcus sanguis

Our previous studies indicated that Arginine (Arg) plays a key nutritional role in Streptococcus sanguis P4A7 and that this organism can grow on whole casein as the sole nitrogen source. Its protease activities were therefore studied after glucose-limited continuous culture in a chemically-defined medium with either free amino acids or casein as the nitrogen source. Both culture supernatant and cell-associated endopeptidase (EP) and exopeptidase (amino-AP and carboxy-CP) activities were determined. Growth rate (mu) had little effect on EP, 75% of which was consistently in culture supernatants; AP and CP both decreased as mu was increased and both were predominantly cell-associated. At high growth pH, EP was substantially increased while AP and CP activities were optimal at pH 7. The most striking nutritional effect occurred under nitrogen limitation (glucose excess) when EP and AP were greatly increased and CP greatly decreased. It was concluded that S. sanguis is well equipped to scavenge its environment for Arg under a wide range of growth conditions.     

Lysis of Streptococcus sanguis by an extracellular enzyme from the bacterium Streptococcus mutans from human dental plaque

The ability of crude extracellular enzyme produced by the oral bacterium Streptococcus mutans AL7-1 to lyse living cells of Streptococcus sanguis ATCC 10556, 10557 and 10558 was examined. This enzyme showed lytic activity of living cells and cell walls of only Strep. sanguis ATCC 10558 strain and severed at random the long chains of this strain of living cells. Early log phase cells of this strain were more sensitive to this lytic enzyme than were late-log phase cells. In view of these results, the relationship between this lytic enzyme from Strep. mutans and a decrease in the number of serotype III strains of Strep. sanguis in dental plaque is discussed.     

不同分子量的壳聚糖对血链菌生物膜的脱落作用

目的:观察不同分子量的壳聚糖促进血链菌生物膜表面的脱落作用。方法:在模拟人口腔环境的人工口腔内,形成4h血链菌生物膜,分别放入黏度5cps(小分子量)、80cps(中分子量)和600cps(大分子量)的2%壳聚糖溶液中,作用时间为3、10、30min,然后应用激光共聚焦扫描显微镜和死菌/活菌荧光染色技术相结合,比较其促进血链菌生物膜表面脱落的作用,对所得数据进行方差分析。结果:壳聚糖应用于4h血链菌生物膜,可以有效促进细菌脱落(P<0.05),黏度5cps(小分子量)的壳聚糖的作用效果最好(P<0.01)。结论:黏度5cps(小分子量)的壳聚糖是一种有效促进菌斑生物膜脱落的药物。     

Susceptibility of biofilms of Streptococcus sanguis to chlorhexidine gluconate and cetylpyridinium chloride

Biofilms of Streptococcus sanguis and planktonic cells of the organism were exposed to chlorhexidine gluconate and cetylpyridinium chloride, at concentrations used clinically, and survivors enumerated. S. sanguis exhibited a lower susceptibility to both antiseptics when it comprised a biofilm than when the organism was in the planktonic form. No viable bacteria were detectable after 5 min of exposure of planktonic cells to 0.2% (w/v) chlorhexidine gluconate or 0.05% (w/v) cetylpyridinium chloride, whereas viable bacteria survived in biofilms of S. sanguis even after exposure to these agents for 4 h. Older biofilms (7 days old) had similar susceptibilities to the antiseptics as younger biofilms (4 days old). Chlorhexidine achieved kills corresponding to approximately a 2 log₁₀ reduction in the viable count of biofilms, containing approximately 10⁷ colony-forming units after 5 min of incubation, whereas the corresponding kills achieved by cetylpyridinium chloride amounted to approximately a 1 log₁₀ reduction. However, on a molar basis, cetylpyridinium chloride was the more effective of the two antiseptics. Minimum inhibitory concentration determinations showed chlorhexidine gluconate to be more effective against S. sanguis than cetylpyridinium chloride. The results of this study have revealed that the minimum inhibitory concentration is not a reliable predictor of the relative effectiveness of antimicrobial agents against. S. sanguis biofilms.     

Effect of chlorhexidine on the relative proportions of Streptococcus mutans and Streptococcus sanguis in hamster plaque.

The effect of chlorhexidine on the proportions of Streptococcus mutans and Streptococcus sanguis in plaque was studied in hamsters fed a diet containing 28% sucrose. In animals given chlorhexidine in their drinking water for 10 d a decrease in the population of S. mutans and an increase of S. sanguis occurred in the plaque. Following the removal of chlorhexidine the population of S. mutans increased again in the presence of sucrose and the number of S. sanguis returned to initial values. When animals were given a sucrose-free diet the low proportion of S. mutans observed following the short-term chlorhexidine period persisted. These data indicate that there is an inverse relationship between the number of S. sanguis and S. mutans in plaque and that the sensitivity in vivo of S. mutans to chlorheximide can be used to suppress the population of S. mutans with a concomitant rise in the proportion of S. sanguis.