D.C. Ellwood

The influence of growth rate and nutrient limitation on the microbial composition and biochemical properties of a mixed culture of oral bacteria grown in a chemostat.

A sample of human dental plaque was homogenized in transport fluid and inoculated simultaneously into a glucose-limited and a glucose-excess chemostat maintained at pH 7.0 and a dilution rate (D) of 0.05 h-1. In an attempt to ensure the establishment of slow-growing bacterial populations, two further inoculations of each chemostat with fresh samples of dental plaque took place before a steady-state was attained at this dilution rate. The dilution rate was increased step-wise to D = 0.6 h-1, and then returned directly to D = 0.05 h-1. Contrary to chemostat theory, microbial communities with a high species diversity were maintained under all of the experimental conditions employed, although not all of the bacterial populations present in the inocula established successfully in the chemostat. At each steady-state the bacteriological composition and biochemical properties (fermentation products, enzyme assays and acid production) of the communities of each chemostat was determined. Higher cell yields and a slightly more diverse community were obtained from the glucose-limited chemostat at all dilution rates. A complex mixture of end products of metabolism was obtained from the glucose-limited chemostat, suggesting amino acid catabolism, while lactate was the predominant acid of the glucose-excess culture. In washed-cell experiments, communities from the glucose-excess chemostat produced the lower terminal pH values following a pulse of glucose, with the lowest pH values occurring at the higher dilution rates. A film of micro-organisms, which accumulated around the neck of the chemostat, was sampled at the end of the experiment. The microbial composition of the films from each chemostat differed markedly, and both were different to the community of the bulk fluid of the respective chemostat. Spirochaetes and a population of yeasts were detected in the films from the glucose-limited and glucose-excess chemostats, respectively. No invertase or glucosyltransferase activity, and little glucoamylase-specific glycogen was detected in the communities from either chemostat, although significant endogenous activity, particularly at high dilution rates, was obtained with washed-cells from the glucose-excess chemostat. The results suggest that the chemostat could make a valuable contribution to the study of the ecology of dental plaque.

Inhibition by the antimicrobial agent chlorhexidine of acid production and sugar transport in oral streptococcal bacteria

Oral streptococci transport sugars via the phosphoenolpyruvate-phosphotransferase (PEP-PTS) system. In a specific assay of this system, low concentrations of chlorhexidine abolished the activity of the glucose and sucrose PTS in batch-grown cells of Streptococcus mutans Ingbritt and B13, Strep. sanguis NCTC 7865, Strep. mitis ATCC 903, Strep. milleri NCTC 10709 and Strep. salivarius NCTC 8606. Intact cells and cells made permeable to the assay reagents with toluene were used. Toluenized cells were more sensitive to chlorhexidine than intact cells (0.09 and 0.25 mM, respectively). This PTS-inhibitory concentration of chlorhexidine reduced acid production from glucose in pH fall experiments to values higher than are obtained solely from endogenous metabolism. The effect of chlorhexidine on rates of acid production was determined at pH 7.0 using cells washed with either 135 mM NaCl or 135 mM KCl. In general, faster rates of acid production from the metabolism of glucose and sucrose were obtained with potassium-treated cells. Addition of the PTS-inhibitory concentration of chlorhexidine markedly reduced or totally abolished acid production by NaCl-treated cells; a greater residual-activity was detected in the same cells washed with KCl (except with Strep. mutans B13 and Strep. mitis ATCC 903). The PTS-inhibitory concentration of chlorhexidine had little or no effect on the viability of cells. The results confirm the existence of sugar uptake systems in oral streptococci additional to the PTS and provide an explanation for the additive anti-caries effect of mouth-rinses containing both fluoride and chlorhexidine.

Evidence that glucose and sucrose uptake in oral streptococcal bacteria involves independent phosphotransferase and proton-motive force-mediated mechanisms

Sugar transport and glycolysis in Streptococcus sanguis NCTC 7865, Streptococcus mitis ATCC 903, Streptococcus salivarius NCTC 8606 and several strains of Streptococcus mutans were investigated by following the rate of acid production by washed bacteria at a constant pH of 7.0. The phosphoenolpyruvate-phosphotransferase system (PTS) was inhibited by low concentrations of chlorhexidine. When this PTS-inhibitory concentration of chlorhexidine was added to cells washed and re-suspended in KCl, glucose uptake and glycolysis continued at a greatly-reduced rate. Chlorhexidine abolished glucose and sucrose uptake and metabolism in bacteria washed and incubated in saline. The Na+-inhibition was reproduced in KCl-washed bacteria using the cyclic peptide ionophores, valinomycin and gramicidin, to dissipate K+ and H+ gradients across the cell membrane. Glucose metabolism by Strep. mutans B13 was more resistant to chlorhexidine than that of Strep. mutans NCTC 10449 or Strep. sanguis but was more sensitive to the ionophores. Valinomycin had a greater inhibitory effect on strain B13 than the other two. That ion gradients are important in the chlorhexidine-resistant glucose-uptake mechanism was confirmed using the classical uncoupling agents, carbonylcyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol and KSCN. Glucose metabolism was inhibited in the presence of both the uncouplers and the PTS-inhibitory concentration of chlorhexidine and significant inhibition was also observed in the absence of the PTS inhibitor. Lactate or the ATPase inhibitor, dicyclohexyl carbodiimide (DCCD), had similar inhibitory effects on the non-PTS uptake system.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of Bioenergetic Processes to the Pathogenic Properties of Oral Bacteria

The energized membrane has been shown to affect properties (sugar transport, acid production, intracellular polysaccharide formation, and glycosyltransferase secretion) related to the pathogenicity of oral bacteria. The activity of the energized membrane was susceptible to modulation by environmental conditions likely to be encountered by bacteria in dental plaque.

Growth of Streptococcus mutans in a chemostat

A strain of Streptococcus mutans isolated from a carious lesion of a patient and known to cause caries in hamsters and monkeys were grown in a complex medium in a chemostat. There were changes in the ability of the organism to stick to surfaces, glucose utilization, and acid production of the organism when grown at different dilution rates.

Additive Inhibitory Effects of Combinations of Fluoride and Chlorhexidine on Acid Production by Streptococcus mutans and Streptococcus sanguis

The effect of 0.07 or 0.15 mM chlorhexidine (CHX) and 4.0 or 8.0 mM potassium fluoride (F), added singly and in combinations, on acid production by Streptococcus mutans and Streptococcus sanguis was studied. Cells were grown in a chemostat under different environmental conditions and acid production from glucose or sucrose was measured as a rate at pH 7.0 and by pH-fall experiments. CHX had a greater inhibitory action on S. mutans while S. sanguis was more sensitive to F. Growth conditions affected the sensitivity of both strains to the two inhibitors and, in general, cells grown glucose-limited were the most sensitive. Combinations of F and CHX showed additive inhibitory effects on acid production, irrespective of the method of measurement.

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen, Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (3H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (Δp) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference (ΔΨ) dissipator, KSCN. The membrane ATPase inhibitor, N,N1-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (ΔpH) and ΔΨ. ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to ΔpH.

Environmental Regulation of Carbohydrate Metabolism by Streptococcus sanguis NCTC 7865 Grown in a Chemostat

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.

Variations in surface polymers of Streptococcus mutans.

The cell wall composition of strains of S mutans with respect to sugars and proteins appears to be correlated to the serological grouping although groups c and E are rather similar. There also appear to be similarities in the structure of the polysaccharide formed by the glycosyltransferases from organisms of serological groups b and d. However, the activity of these enzymes appears to be variable in these groups. The most noteworthy difference found was that between the three Ingbritt strains. All three strains gave identical results with regard to their cell wall composition, and presumably this would mean that they were identical serologically. However, Ingbritt LH differed considerably from both the others in the types of polysaccharide formed by their glycosyltransferases from sucrose. Ingbritt B was a reisolate from monkeys, whereas Ingbritt LH was maintained in laboratory culture, and this may explain the difference. Clearly, more work will be required to explain this difference and as c strains are commonly isolated from plaque, it would seem desirable to clear up this point.

Effect of Environmental Conditions on the Fluoride Sensitivity of Acid Production by S. sanguis NCTC 7865

Growth and environmental conditions affected the fluoride (F) sensitivity of acid production by Streptococcus sanguis NCTC 7865. Cells grown glucose-limited in a chemostat were generally more sensitive than those harvested from cultures in which there was an excess of glucose (amino acid-limited). There was no consistent relationship between the growth rate of cells and their F sensitivity. Slower-growing cells (mean generation time = 14 hr) were more sensitive than those growing quickly when glucose was the limiting nutrient, whereas the faster growing cells from the glucose-excess culture were most susceptible. The pH of the environment markedly affected the F sensitivity of cells: 2 mM F- was sufficient to abolish acid production by cells incubated at pH 5.0, whereas 24 mM F-did not totally inhibit glycolysis at pH 7.0 or 8.0. Regardless of pH and growth conditions, the cationic composition of the environment had the most pronounced effect on acid production and fluoride sensitivity. Cells washed and re-suspended in KCl were more acidogenic and more sensitive to F than the same cells treated with saline. At pH 7.0 and 8.0, saline-washed cells were comparatively unaffected by F, while glycolysis by the same cells at the same pH but washed in KCl could be inhibited by up to 80%. These results suggested that F inhibition could not be explained merely on the basis of HF uptake at low pH values. Since it has been shown previously that the activity of the energized membrane is maintained by K+ and dissipated in the presence of Na+, it was proposed that proton motive force (pmf) might be involved in the uptake of F-.