C.W. Keevil

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.

Coaggregation amongst aquatic biofilm bacteria

Aquatic autochthonous flora isolated directly from a water biofilm model was tested for itscapacity to take part in coaggregation. Two assay methods were compared and a visualsemi‐quantitative method was found to be the most consistent and easy to use. All isolatesdemonstrated coaggregation with at least one other isolate to some extent, giving rise to visibleflocs. One isolate, identified as Micrococcus luteus, was found to coaggregate with all ofthe other isolates to different degrees, highlighting a possible role as a bridging organism in thebiofilm consortium. The addition of certain simple sugars reversed coaggregation betweenparticular isolates suggesting that lectin‐like adhesins were operating between these members ofthe aquatic autochthonous flora by a mechanism similar to that reported to occur between dentalplaque bacteria. The time taken for coaggregation to begin varied considerably between differentpairs of isolates (1–16 h), suggesting that this may play a role in determining the successionalorder of isolates during biofilm development.

Mycobacterium fortuitum and Mycobacterium chelonae biofilm formation under high and low nutrient conditions

The rapidly growing mycobacteria (RGM) are broadly disbursed in the environment. They have been recovered from freshwater, seawater, wastewater and even potable water samples and are increasingly associated with non‐tuberculous mycobacterial disease. There is scant evidence that non‐tuberculous mycobacteria (NTM) and RGM form biofilms. Therefore, an experimental system was designed to assess the ability of RGM to form biofilms under controlled laboratory conditions. A flat plate reactor flow cell was attached to either a high or low nutrient reservoir and monitored by image analysis over time. Two surfaces were chosen for assessment of biofilm growth: silastic which is commonly used in medical settings and high density polyethylene (HDPE) which is prevalent in water distribution systems. The results show that Mycobacterium fortuitum and M. chelonae formed biofilms under both high and low nutrient conditions on both surfaces studied. These results suggest that RGM may form biofilms under a variety of conditions in industrial and medical environments.

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.

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen‐limited and anaerobic conditions. The growth and metabolic efficiency of oxygen‐limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2–3 μm long, and were encapsulated by a layer of extracellular material. The majority of cells were non‐flagellated and fimbriae were not observed. Chemostat‐grown cells were significantly more adhesive for HEp‐2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen‐limited and anaerobic cultures were significantly more adhesive for Hep‐2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro‐organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.

Inhibition of the synthesis and secretion of extracellular glucosyl- and fructosyltransferase in Streptococcus sanguis by sodium ions.

The influence of Na+ and K+ on the synthesis and secretion of extracellular glucosyltransferase (GTF; EC 2.4.1.5) and fructosyltransferase (FTF; EC 2.4.1.10) by Streptococcus sanguis NCTC 7865 and Streptococcus sanguis Challis NCTC 7868 has been determined. No FTF and little or no mutansucrase (GTF-I) activities were detectable during growth on glucose or sucrose unless the Na+/K+ ratio of the cultures was kept low. Increasing K+ concentrations stimulated the production of FTF and dextransucrase (GTF-S), but all glycosyltransferase activities decreased in high K+ media when the growth pH was maintained with NaOH instead of KOH, indicating that the Na+/K+ ratio effect was due principally to Na+ inhibition. Significant GTF and FTF activities were detected in a putative GTF- mutant of strain Challis grown in high K+ medium but not in high Na+ medium, suggesting that the mutant might be defective in a regulatory gene.

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.