Competition for dietary carbohydrates between streptococci in dental plaque

Abstract The predominant microorganisms in dental plaque, i.e., streptococci and actinomycetes, are carbohydrate fermenters. In the natural environment these organisms experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. Recently, we have studied the competition between pairs of oral Streptococcus species in the chemostat under continuous glucose or sucrose limitation and under glucose pulsed conditions.
In the present study we have investigated the competition for dietary carbohydrates in dental plaque of gnotobiotic rats infected with the same combinations of streptococci. The rats were exposed to similar regimes of carbohydrate administration as in the chemostat by incorporation of low molecular carbohydrates in the diet and drinking water. Addition of glucose to the diet favoured the organism with the highest q _max for glucose. This result parallels the outcome of earlier competition experiments in glucose-pulsed chemostats, which showed that the organism with the highest q _max glucose became dominant in the cultures. Previous observations that bacteriocin production and extracellular glucan synthesis are major ecological factors for oral streptococci were also confirmed in this experiment.     

Effect of utilization of substrates on the composition of dental plaque

Abstract The microbiota in the mouth is subjected to substrate limitations. In this study we have evaluated the role of competition for carbon and energy substrates on the proportions of 2 microbial species in a simplified plaque ecosystem in gnotobiotic rats. Germ-free rats were inoculated with a combination of Streptococcus sanguis and Streptococcus mutans , or with a combination of Streptococcus milleri and S. mutans . The available carbon and energy sources were varied through the host's diet. 3 Experimental diets were tested: (i) a basal diet low in soluble carbohydrates; (ii) an arginine-supplemented diet; (iii) a sucrose-supplemented diet. Arginine is used for growth by S. sanguis and S. milleri , but not by S. mutans . Sucrose is rapidly fermented by all 3 species.
The total number of viable organisms on the dentition increased when arginine or sucrose were supplied in the diet. With the arginine-supplemented diet, S. sanguis and S. milleri increased while S. mutans decreased. With the sucrose-supplemented diet, S. mutans increased while S. sanguis and S. milleri decreased. These results were explained by assuming that the organism with the highest growth rate on the supplementary substrate competes most favourably. Changes in the environmental pH, due to breakdown of sucrose and arginine, might also have affected the competition between the streptococci. In addition, production of extracellular glucans from sucrose could be a competitive advantage for S. mutans .     

Surface free energies of oral streptococci

Abstract The surface free energies ( γ _b) of a variety of oral streptococci were determined from contact angle measurements on bacterial deposits, using the concept of dispersion and polar components. At least four strains of each species were tested. Strains of Streptococcus mutans, S. sanguis and S. salivarius possessed relatively high surface free energies (103 ± 12 mJ · m⁻²) and at the species level no significant difference was found. In contrast, the strains of S. mitis had remarkably low surface free energies (45 ± 14 mJ · m⁻²). S. milleri appeared to be a heterogeneous species, showing surface free energies over a range of 32–119 mJ · m⁻². No significant differences were observed between laboratory strains and strains freshly isolated from the oral cavity.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture.

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Surface free energies of oral streptococci and their adhesion to solids

Abstract The adhesion of 3 strains of oral streptococci from a buffered suspension onto 3 different solid substrata was studied. Representative strains of streptococci were selected on the basis of their surface free energy ( γ _b), namely Streptococcus mitis L1 ( γ _b= 37 mJ·m⁻²), Streptococcus sanguis CH3 (95 mJ·m⁻²) and Streptococcus mutans NS (117 mJ·m⁻²). Solid substrata were also selected on basis of their surface free energy ( γ _s), and included polytetrafluorethylene ( γ _s= 20 mJ·m⁻²), polymethylmethacrylate (53 mJ·m⁻²) and glass (109 mJ·m⁻²). Bacterial adhesion was measured as the number of bacteria adhering per cm² at equilibrium. Equilibrium was usually obtained within 20 min. S. sanguis CH3, having an intermediate surface free energy did not show a clear preference for any of the 3 solids. S. mitis L1, however, the lowest surface free energy strain, adhered in highest numbers to the low energy solid PTFE, whereas the highest γ _b strain, S. mutans NS, adhered in highest numbers to the highest γ _s solid, glass. Calculation of the interfacial free energy of adhesion ( ΔF _adh) for each bacterial strain showed that this parameter was predictive of bacterial adhesion to solid substrata.     

Accumulation of carbohydrate by Escherichia coli B/r/1 during growth at low water activity

The carbohydrate content of Escherichia coli B/r/1, grown in a glucose or arabinose-limited salts medium in a chemostat, increased by a factor of 2–4 when the water activity (a_w) of the medium was reduced to 0.986 by addition of NaCl, KCl or sucrose. The biomass decreased by 30–45%. The sucrose system resulted in the lowest biomass and carbohydrate content. The monosaccharide part of the accumulated carbohydrate consisted of glucose or glucose and arabinose in the cultures fed glucose and arabinose, respectively, and accounted for 50% or more of the total carbohydrate in the NaCl and KCl systems and 16.79% in the sucrose system. In addition, the K⁺ content depended on the solute and related inversely to the monosaccharide content, being highest in the sucrose system. The combined molarity of the monosaccharide and K⁺ was deduced to be far in excess of that required for osmotic equilibration of the cultures, especially in the sucrose system. These observations are discussed in the context of osmoregulation, the effects of solutes on glucose metabolism and the morphological changes that occur in cultures at low a_w.     

A method for evaluating the cariogenicity of oral bacteria using radio-labelled synthetic hydroxyapatite

R. YAMAGUCHI, M. SATO, H. TSUCHIYA, K. YAMAMOTO, Y. DOI AND F. IWAKU. 1996. Radioactive hydroxyapatite was synthesized using ⁴⁵Ca to evaluate the cariogenicity of oral streptococci. Discs prepared from it were suspended in media containing sucrose, then inoculated with Streptococcus mutans or Streptococcus sanguis . The radioactivity in the supernatant fluid was measured at specified time intervals. Released ⁴⁵Ca in the supernatant fluids markedly increased in both species during the experimental period, while Strep, mutans showed much higher decalcification than Strep, sanguis . The present method would be useful for semi-quantitative evaluation of bacterial decalcification ability.     

Molecular basis for the association of glucosyltransferases with the cell surface of oral streptococci

The expression of Streptococcus mutans mutant glucosyltransferase-I enzymes in S. sanguis and S. milleri suggests that cell-associated glucosyltransferase activity is dependent upon both glucan synthesis and glucan binding by the carboxyl-terminal repeating units of the enzyme. Mutant enzymes lacking these repeating units were only present in the extracellular fluids of these transformed streptococcal strains.     

Environmental pH as a factor in the competition between strains of the oral streptococci Streptococcus mutans, S. sanguis, and "S. mitior" growing in continuous culture

Strains of Streptococcus mutans (biotype 1), Streptococcus sanguis, and Streptococcus mitior have been grown in mixed continuous culture in a semidefined medium under glucose limitation at a growth rate of D = 0.1 h-1. The effect of varying the environmental pH on the proportions of the different populations within the community has been determined. Initially the populations were allowed to reach steady state at pH 7.0 when S. sanguis was dominant with S. mutans and "S. mitior" maintaining similar populations. The medium pH was then lowered in steps of 0.5 pH units from pH 7.0 to 4.5, and the community was grown at each step for at least 15 generations. Viable counts of each species were made at 24-h intervals. The population ratios established at pH 7.0 remained relatively stable when the environmental pH was set at pH 6.5. However, after the medium pH was lowered to 6.0 (days 18-27), the population of S. mutans began to increase and the S. mitior population began to decline. A further change was seen at pH 5.5 (days 27-34) when S. mutans became dominant, S. sanguis declined, and S. mitior was not detectable. At pH 4.5, both S. mutans and S. sanguis were reduced in numbers, but survived until the experimental run was terminated (44 days). Samples of culture fluid were taken throughout the experiment and analyzed for the presence of the acid products of glucose metabolism. The amounts of lactic acid produced by the community increased as the environmental pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 Growing in Pure or Mixed Culture on Human Teeth in an Artificial Mouth

Streptococcus mutans and Streptococcus mitior were both capable of colonizing the surfaces of human teeth in an artificial mouth. Although fermentable carbohydrate was not essential for colonization, highest numbers were recovered after 5 d if 5–0°_***0 (w/v) sucrose had been available intermittently. When grown together in mixed culture the interaction of the two species was affected profoundly by the available nutrients. In the presence of synthetic saliva alone, Strep. mitior was strongly antagonistic to Strep. mutans. When a nutrient broth containing sucrose was also provided intermittently there was slight inhibition of Strep. mutans accompanied by an increase in the proportion of Strep. mitior in the plaque, although the former was the dominant organism under these conditions. When 0–5% (w/v) glucose replaced the sucrose, mutual antagonism occurred and fewer organisms were recovered than if only synthetic saliva had been available. One reason why a high-sucrose diet encourages colonization by Strep. mutans may be that insoluble extracellular polysaccharide confers a competitive advantage upon it in the face of antagonistic agents such as the hydrogen peroxide produced by Strep. mitior.     

Genetic relationships among the oral streptococci.

Genetic relationships and species limits among the oral streptococci were determined by an analysis of electrophoretically demonstrable variation in 16 metabolic enzymes. Fifty isolates represented 40 electrophoretic types, among which the mean genetic diversity per locus was 0.857. Mannitol-1-phosphate dehydrogenase was not detected in isolates of the sanguis species complex, and glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were absent in species of the mutans complex. Clustering from a matrix of Gower's coefficient of genetic similarity placed the 40 electrophoretic types in 10 well-defined groups corresponding to the Streptococcus species S. mutans, S. sobrinus, S. cricetus, S. rattus, S. ferus, S. oralis (mitior), two distinct assemblages of S. sanguis strains, and two subdivisions of "S. milleri." The assignments of isolates to these groups were the same as those indicated by DNA hybridization experiments, and the coefficient of correlation between genetic distance estimated by multilocus enzyme electrophoresis and genetic similarity indexed by DNA hybridization was -0.897 (P less than 0.001) for 50 pairwise combinations of isolates. S. ferus, which is widely believed to be a member of the mutans complex, was shown to be phylogenetically closer to species of the sanguis complex.     

Effects of dietary carbohydrates on mutacin production and activity

Although mutacins (bacteriocins produced by Streptococcus mutans) were shown to be active in vivo, their ecological role in the oral cavity is still controversial. In the present paper, the effect of dietary carbohydrates, one of the ecological parameters which influences oral bacterial populations, on the activity and the production of mutacins from four S. mutans strains (C67-1, Ny257, Ny266 and T8) is described. Results obtained by the deferred antagonism test in solid media and by the mixed cultivation of the mutacinogenic strains with a sensitive indicator strain in liquid batch cultures, indicate that a minimal fermentable sugar concentration is needed for mutacin production. Among all the fermentable carbohydrates tested (fructose, glucose, lactose, mannitol and sucrose), none significantly affected the production and the activity of the four mutacinogenic strains used, in concentrations up to 5%. Although the results do not discount the possibility of mutacin inactivation in vivo, they indicate that they are not affected by dietary carbohydrates.     

Metabolism of nitric oxide in soil and denitrifying bacteria

Abstract Production and consumption of NO was measured under anaerobic conditions in a slightly alkaline and an acidic soil as well as in pure cultures of denitrifying Pseudomonas aeruginosa, P. stutzeri, P. fluorescens, Paracoccus denitrificans, Azospirillum brasilense , and A. lipoferum . Growing bacterial cultures reduced nitrate and intermediately accumulated nitrite, NO, N₂O, but not NO₂. Addition of formaldehyde inhibited NO production and NO consumption. In the presence of acetylene NO was reduced to N₂O. Net NO release rates in denitrifying bacterial suspensions and in soil samples decreased hyperbolically with increasing NO up to mixing ratios of about 5 ppmv NO. This behaviour could be modelled by assuming a constant rate of NO production simultaneously with a NO consumption activity that increased with NO until V _max was reached. The data allowed calculation of the gross rates ( P ) of NO production, of the rate constants ( k ), V _max and K _m of NO consumption, and of the NO compensation mixing ratio ( m _c). In soil, P was larger than V _max resulting in net NO release even at high NO mixing ratios unless P was selectively inhibited by chlorate + chlorite or by aerobic incubation conditions. In bacteria, V _max was somewhat larger than P resulting in net NO uptake at high NO mixing ratios. Both P and V _max were dependent on the supply of electron donor (e.g. glucose). Both in soil (aerobic or anaerobic) and in pure culture, the K _m values of NO consumption were in a similar low range of about 0.5–6.0 nM. Anaerobic soil and denitrifying bacteria exhibited m _c values of 1.6–2.1 ppmv NO and 0.2–4.0 ppmv NO, respectively.     

The effect of oxygen on the growth and acid production of Streptococcus mutans and Streptococcus sanguis

Abstract Pure cultures of Streptococcus mutans NCTC 10499 and Streptococcus sanguis ATCC10556 were grown in a glucose-limited chemostat under varying concentrations of oxygen in the gas phase. Both streptococci consumed large amounts of oxygen by the partial oxidation of sugars, thus maintaining an anaerobic environment. With increasing oxygen concentrations the degradation products from glucose become more oxidized. Ethanol gradually disappeared from the culture fluid while the acetate concentration increased. In the case of S. sanguis , the products became even more oxidized at higher oxygen concentrations, and carbon dioxide was formed instead of formate. Sudden increase in the oxygen concentration in the gas phase caused elevated oxygen tensions in the cultures, which led to a decrease in the growth rate of the streptococci.     

Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates

Abstract From chemostat enrichments conducted at dilution rates of 0.025, 0.12 and 0.25 h⁻¹ glutamate- and aspartate-fermenting bacteria were isolated. The dominant aspartate-fermenting strains in all these enrichments belonged to the genus Campylobacter , whereas 3 dissimilar types of glutamate-fermenting bacteria predominated at the different dilution rates. One of these strains was identified as Clostridium cochlearium . The remaining two were designated as strain DKglu16 (glutamate → acetate + propionate + ammonium + carbon dioxide) and DKglu21 (glutamate → acetate + formate + ammonium + carbon dioxide). Grown in continuous culture under glutamate limitation, strain DKglu16 (μ_max= 0.13 h⁻¹; K _s= 1.9 μM) outcompeted C. cochlearium (μ_max= 0.36 h⁻¹; K _s= 7 μM) at low dilution rates, but was outgrown at higher rates of dilution (0.044 h⁻¹). In glutamate-limited continuous culture the competitiveness of strain DKglu16 increased considerably when lactate was added to the feed in addition to glutamate.     

Effect of carbohydrates on fructosyltransferase expression and distribution in Streptococcus mutans GS-5 biofilms

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.     

Sucrose phosphorylase of the rumen bacterium Pseudobutyrivibrio ruminis strain A

Aims:  To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose.
Methods and Results:  Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis . Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The K _m for glucose-1-P formation and fructose release were 3·88 × 10⁻³ and 5·56 × 10⁻³ mol l⁻¹ sucrose, respectively – while the V _max of the reactions were −0·579 and 0·9  μ mol mg protein⁻¹ min⁻¹. The enzyme also released free glucose from glucose phosphate.
Conclusion:  Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage.
Significance and Impact of the Study:  Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.     

Soil microbial carbon uptake characteristics in relation to soil management

Abstract The kinetics of glucose uptake by soil microbial communities in 16 different soild (7 under monocultures and 9 under crop rotations) differing in microbial biomass content, % C_org, pH and clay content were investigated at 22°C. The V _max value of microbial bimasses under monoculture, was o.27 μg C_gluc · μg⁻¹ C_mic · h⁻¹ (range 0.18–0.44), twice as high as the mean value of V _max of microbial biomasses under rotations (0.13 μg C_gluc, range 0.07–0.19). Mean values of K _m were 714 μg C_gluc and 290 μg C_gluc · g⁻¹ soil, respectively.
These differences were highly significant ( P =0.001, based on SE) and could not be relate to particle size distribution of the soils, pH or C_org. A Michaelis-Menten type uptake response was apparent over the total range of glucose concentrations used (45.4–1453.3 μg C_gluc · g⁻¹ soil) for microbial biomasses under rotation while the majority of microbial biomasses under monocultures showed a similar response only at low glucose concentrations. A different uptake mechanism appeared to be involved at higher glucose concentrations (similar to diffusion) in monoculture soils.     

Detection of the response regulator AgrA in the cytosolic fraction of Staphylococcus aureus by monoclonal antibodies

Abstract The interaction of salivary lysozyme with the surface protein antigen (PAc) of Streptococcus mutans and the interaction of lysozyme with the pathogen were examined by ELISA using S. mutans MT8148 (PAc⁺) and the PAc-defective mutant EM-2 (PAc⁻). The lysozyme clearly bound to the S. mutans wild type but not to the S. mutans mutant. Furthermore, lysozyme bound directly in the fluid phase to the rPAc, of which the binding kinetics were determined ( K _on= 3.63 ± 0.04 × 10³M⁻¹ s ⁻¹, K _off= 1.72 ± 0.04 × 10⁻⁵s⁻¹ and K_on / K_off= 2.11 × 10⁸M⁻¹) using surface plasmon resonance. The kinetics of both association and dissociation were relatively slow. In addition, anti-lysozyme antibody significantly inhibited the binding of salivary components to the rPAc. The present findings indicate that lysozyme is one of the major salivary components interacting with S. mutans PAc.