Ann S. McDermid

The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50

Porphyromonas gingivalis strain W50 was grown under haemin-limitation and haemin-excess conditions in a chemostat at pH 7.5. The maximum specific growth rate (mumax) was determined at both haemin concentrations (mumax = 0.236 +/- 0.052 and 0.271 +/- 0.039 h-1, respectively). This enabled dilution rates to be adjusted so that the virulence and enzyme activity of haemin-limited and haemin-replete cells could be compared at identical relative growth rates (murel) of 0.25, 0.50 and 0.75 of their respective mumax. The data showed that the fastest growing cells were significantly more virulent than those grown more slowly, irrespective of haemin concentration. However, at each growth rate tested, cells grown under haemin-excess conditions were always more virulent than haemin-limited cells. Trypsin-like enzyme activity of whole cultures was also greater at each growth rate under haemin-excess conditions while, conversely, collagenolytic activity was generally higher in haemin-limited cultures. Thus, although growth rate had an effect on the virulence and enzyme activity of P. gingivalis, the availability of haemin for growth was the most significant factor.

Isolation of colonial variants of Bacteroides gingivalis W50 with a reduced virulence

The spontaneous appearance of unusual colony forms was observed during prolonged growth of Bacteroides gingivalis W50 in a chemostat. Two variants were selected for further study which could be distinguished from the parent strain by the rate and intensity of pigmentation of their colonies. For example, after anaerobic incubation for 14 days, variant W50/BR1 produced brown colonies whereas those of the parent strain were black; in contrast, variant W50/BE1 did not show signs of pigmentation until incubation had continued for 21 days. In subsequent studies in the chemostat, variant W50/BE1 bred true even after prolonged growth whereas other colony forms appeared after incubation of variant W50/BR1 for 14 days. The relatedness of W50/BR1 and W50/BE1 to the parent strain was confirmed by comparisons of the whole-cell fatty-acid profiles, the patterns of pre-formed enzymes and by the metabolic end products after growth. However, the variants did differ from the parent strain in their virulence in a mouse pathogenicity model. The parent strain killed all mice given infective doses greater than 5 x 10(8) cfu whereas W50/BR1 was much less virulent (2 out of 10 mice killed and higher infective doses needed for higher mortality rates) and W50/BE1 was avirulent at all infective doses tested.

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.

A Mixed-culture Chemostat System to Predict the Effect of Anti-microbial Agents on the Oral Flora: Preliminary Studies using Chlorhexidine

A mixed-culture chemostat system, composed of nine bacterial species representative of plaque in health and disease, has been assessed as an improved laboratory method of evaluating the likely in vivo effects of antimicrobial agents used in dentistry. The advantages of the system include reproducibility, the long-term stable cultivation of bacteria under controllable conditions, and repeated sampling, for bacteriological and biochemical studies, without disrupting the stability of the community. The effects of (i) the continuous provision of chlorhexidine (CHX) and (ii) three pulses of CHX (final concentration in both experiments = 0.24 mmol/L) on the composition of the chemostat communities were monitored. Only L. casei survived the continuous provision of CHX; the other bacteria were killed and were lost at different rates which generally corresponded to their known sensitivities to CHX. After each CHX pulse, the numbers of bacteria fell markedly. Again, L. casei was least affected, while A. viscosus, B. intermedius, and F. nucleatum were temporarily undetectable but returned to their original levels within 2-4 generation times. Counts of S. mutans were affected more by CHX than those of S. sanguis or S. mitior. The effect of successive pulses of CHX on the viability of some bacteria and on acid production (as measured by pH-fall experiments) decreased, suggesting that adaptation to CHX had occurred. The fact that the in vitro observations paralleled previous clinical findings suggests that the mixed-culture system could be used as a predictive model of the probable effect on the oral flora of new anti-microbial agents prior to expensive trials in animals or human volunteers.

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.

The effect of environmental pH on the physiology and surface structures of Salmonella serotype Enteritidis phage type 4

The incidence of food-poisoning caused by Salmonella serotype Enteritidis PT4 has increased. Implicated food products display pH levels between 4 and 9. Accordingly, the effect of growth at extremes of pH on the presence of surface structures and the carriage of a 38-MDa plasmid was determined by growing a clinical isolate of Enteritidis PT4 in a chemostat. Steady-state growth was possible over the pH range 4.35-9.45, corresponding to the pH extremes associated with key reservoirs implicated in outbreaks. Without pH control, cultures stabilised at pH 7.10. Growth at extremes of pH had significant effects on the distribution of cell surface structures; at pH 9.45, only 3% of cells were fimbriate compared with 52% at pH 7.10 and 20% at pH 4.35. The proportion of motile cells and the presence of flagella was also reduced at extremes of pH. A 38-MDa plasmid was present in cells grown in the chemostat at pH 7.10, but not in cells grown at pH 4.35 or pH 9.45. Thus, environmental pH may have a significant impact on the virulence potential of Enteritidis PT4.

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.

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-.