Effect of various rinsing protocols after use of amine fluoride/stannous fluoride toothpaste on the bacterial composition of dental plaque.

This clinical study evaluated the effect of different oral hygiene protocols on the bacterial composition of dental plaque. After a 2-week period of using fluoride-free toothpaste, 30 participants followed three 1-week experimental protocols, each followed by 2-week fluoride-free washout periods in a randomized crossover examiner-blind controlled trial. The 1-week experimental protocols comprised the use of AmF/SnF(2) toothpaste twice daily, after which participants either (1) rinsed with tap water, (2) did not rinse but only spat out the toothpaste, or (3) rinsed with an AmF/SnF(2) mouthwash. In the fluoride-free washout periods, the participants brushed their teeth with fluoride-free toothpaste without further instructions. Six hours after the last brushing (+/- rinsing) of each period, buccal plaque samples in the upper molar region were taken. The microbiota composition of the plaque samples was analyzed by checkerboard DNA:DNA hybridization. A statistically significant reduction was found in the total amount of DNA of the 39 major plaque species measured, and in the proportions of some acid-producing bacterial strains after the period having used the AmF/SnF(2) toothpaste + AmF/SnF(2) mouthrinsing. The results indicate that using the AmF/SnF(2) toothpaste and rinse combination could result in plaque of lower cariogenicity.

Microbiota of plaque microcosm biofilms: effect of three times daily sucrose pulses in different simulated oral environments.

AIM: To explore the Ecological Plaque Hypothesis for dental caries. To test modification of the microbiota of dental plaque microcosm biofilms by sucrose pulsing during growth in two different simulated oral fluids, and with a urea-induced plaque pH elevation. METHODS: Plaque microcosm biofilms were cultured in an 'artificial mouth' with and without 6-min 5% w/v sucrose pulses every 8 h in an environment of continuously supplied saliva-like defined medium with mucin (DMM), or basal medium mucin (BMM, a high-peptone-yeast extract oral fluid analogue), and also in DMM + 10 mmol/l urea, with sucrose pulsing. Forty plaque species were quantified by checkerboard DNA:DNA hybridization analysis. RESULTS: Sucrose pulsing extended rapid plaque growth in DMM and BMM, inducing major microbiota changes in DMM but not in BMM. In DMM, some streptococci and lactobacilli were unaffected while others implicated in caries, together with Candida albicans and Capnocytophaga gingivalis, increased. Aerobic, microaerophilic and major anaerobic species decreased. Elevation of the pH(max) from 6.4 to 7.0 had almost no effect on the microbiota. BMM plaques were distinct from DMM plaques with particularly low levels of Candida albicans and Actinomyces. CONCLUSIONS: Modest sucrose exposure in a saliva-like environment causes profound changes in the developmental self-organization of plaque microcosms, supporting the Ecological Plaque Hypothesis. Nevertheless, there is significant stability in microbial composition with varying pH near neutrality. Increases in levels of specific bacteria in response to sucrose could be characteristic of organisms particularly important in caries.

The concomitant deposition of strontium and fluoride in dental plaque.

We have investigated the feasibility of incorporating Sr into dental plaque by means of an enzyme-dependent system known to increase Ca, P, and F levels in plaque. A solution containing Ca (20 mmol/L), P (12 mmol/L), MFP (4.7 mmol/L), F (0.3 mmol/L), and urea (500 mmol/L) was modified by equimolar replacement of Ca with 1, 2, 5, and 10 mmol/L Sr. Thin films of human salivary sediment incubated in these solutions showed increasing levels of acid-extractable Sr as the solution Sr increased. When the concentration exceeded 2 mmol/L, deposition of Ca, P, and F was reduced. In artificial plaque, grown on bovine enamel, from mixed human salivary organisms and treated with the solution containing 2 mmol Sr/L, there was a slightly smaller uptake of Ca, P, and Sr, but a greater uptake of F than in sediment treated with the same solution. Natural human plaque treated 12 times in vivo over three days with this solution (in the form of a mouthrinse) also showed substantial increases (from five- to 26-fold) in the concentrations of all four ions. Absolute levels of Ca, P, F, and especially Sr were, however, lower than those in the artificial plaque samples. (Ca + Sr)/P ratios suggested apatite deposition, and the correlation between amounts of Ca and Sr deposited in natural plaque samples suggested that Sr, like F, is structurally incorporated into this apatite. Fluctuations in the pH of natural plaque may promote apatite crystal maturation, causing a slow loss of Sr.(ABSTRACT TRUNCATED AT 250 WORDS)

Therapeutic mineral enrichment of dental plaque visualized by transmission electron microscopy.

The form, location, and distribution of fluorhydroxyapatite deposited in dental plaque by a urease-mediated mineral enrichment process have been studied by transmission electron microscopy. Artificial plaque was formed in terylene gauze in the mouth of one subject and immersed for five min four times per day in a mineral-enriching solution. Contralateral control plaque remained untreated. The effect on natural plaque was studied in two subjects who withheld oral hygiene for four days and mouthrinsed with this solution for two min four times per day during the last two days. Mineral deposits were seen in all plaque samples exposed to the test solution. None was detected in any control sample. The deposits were scattered in the interbacterial matrix as needle-shaped crystals, the size and shape of apatite, together with amorphous material. The crystals appeared larger and more perfect, and the amorphous material less conspicuous, with longer in vivo rinsing periods. Platelet-shaped crystals of octacalcium phosphate were never seen. Mineral was also seen within the remnants of dead bacterial cells and within degenerating epithelial cells. Crystals were never seen within intact bacterial cells, as in calculus formation. The presence of a single crystal type and the relative absence of densely-mineralized foci are other differences between this mineral-enrichment process and supra-gingival calculus formation. A longer-term study is necessary to determine whether the solution promotes calculus by providing nucleation seeds.

A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH, and mineralization.

A plaque growth chamber was developed for long-term growth of five separate plaques from the same plaque or saliva sample under identical conditions of temperature and gas phase. Reagent addition and growth conditions for each plaque could be independently controlled, and each was accessible for sequential sampling and electrode insertion. Plaques were cultured for over six weeks on pellicle-coated Lux (TM) 25-mm diameter cover-slips at 35 degrees C under 5% CO2 in N2, and supplied with a medium containing 0.25% mucin (BMM) at 3.6 mL/h, and with periodic 5% sucrose. Electron microscopy and flora analysis of microcosm plaques showed that they had close similarities to reported characteristics of natural dental plaques. Diverse motile bacteria were present. Sucrose-induced Stephan pH curves and urea-induced pH rises were also similar to those reported for natural plaques. Changes in plaque urease, calcium, phosphate concentrations, and the flora were followed over five weeks in a plaque supplied with BMM containing additional 2.5 mmol/L calcium and 7.5 mmol/L phosphate. Despite this high environmental calcium phosphate concentration, there was no continuing increase in calcium levels, although plaque phosphate doubled. Urease levels fluctuated. Changes in the cultivable flora were minor. A urea-containing calcium phosphate/mono-fluorophosphate pH 5 solution, applied for six min every two h for seven days, increased plaque calcium, phosphate, and fluoride to high levels. Thus, plaques grown over several weeks in the multi-station artificial mouth exhibited metabolic and pH behavior typical of natural plaques, could be analyzed during development, and the system allowed manipulation of environmental variables important in plaque pH control and calcification.

pH regulation of urease levels in Streptococcus salivarius.

Potential mechanisms for regulation of urease levels in Streptococcus salivarius were examined, including: induction by urea, nitrogen or carbon source repression, and effects of pH and CO2 (because CO2 enrichment enhanced urease detection on urea agar plates). Regulation by either pH or CO2 was confirmed by comparison of the urease accumulation pattern during anaerobic growth under CO2 with that under N2. Under CO2, there was an initial buffering plateau at pH 6.2 and a rate of Streptococcus salivarius urease accumulation three-fold that under N2, with a pH 7.6 plateau. With both gas phases there was also an increase in the rate of urease appearance coincident with the decrease in medium pH following the pH plateau. The effects of pH, CO2, and HCO3- on urease levels and on growth were separately assessed by culture in media containing 0, 25, 100 mmol/L KHCO3 buffered at different pH levels. There was an inverse relationship between the logarithm of the urease level after 24-hour growth and the pH during growth-the urease specific activity was 100-fold higher at pH 5.5, compared with pH 7.0 and above. HCO3-/CO2 (100 mmol/L) had little effect on urease levels, but was essential for growth at pH 5.5. There was no significant urease induction by urea, or repression by ammonia or glucose. There was also evidence of pH regulation of urease levels in some staphylococci, Klebsiella pneumonia, and Corynebacterium renale, but not in Actinomyces naeslundii and several other species. We conclude that the external pH is a major factor regulating urease levels in S. salivarius and possibly some other species-a mechanism equivalent to urease repression by OH-.

Checkerboard DNA-DNA hybridisation technology focused on the analysis of Gram-positive cariogenic bacteria.

Checkerboard DNA-DNA hybridisation enabled the quantitative analysis of plaque samples against 40 microbial species simultaneously, using digoxygenin-labelled, whole-genome DNA probes. This technique was initially developed to study the predominantly Gram-negative sub-gingival plaque microbiota. The aim of this study was to apply it to a suite of predominantly Gram-positive microorganisms, such as those implicated in cariogenesis. To specifically target Gram-positive species (and Candida albicans) required optimisation and modification of DNA extraction, prehybridisation, hybridisation, and antibody detection conditions. The suitability of the revised technique for clinical and epidemiological studies was confirmed using interproximal plaque from small groups of 5- to 6-year-old children of high (decayed, missing, or filled teeth (dmft)> or =5, n=8) and zero (n=5) caries rates.

In-vitro urea-dependent pH-changes by human salivary bacteria and dispersed, artificial-mouth, bacterial plaques.

The pH effects of urea metabolism were studied in washed salivary-sediment bacteria from subjects that had up to 10-fold variation in oral ureolytic activity, and in dispersed artificial-mouth plaques. Adequate evaluation required analysis of the [OH-] as well as the pH curve. An initial constant rate of pH-change, lasting until pH 7.8, was derived from the pH curve; this gave the best correlation (r = 0.95) with the ureolysis rate. From the [OH-]-curve, between pH 7.8 and 8.3 (approx.), a constant and maximal rate of change in [OH-] was determined. Although theoretically this was directly related to the rate of ammonia release, it was 10(-2) to 10(-3) times its value and correlated less well (r = 0.83) with ureolysis. Together with the initial and final pH, these two rates largely described urea-induced pH changes. After 12.5-fold dilution of the cells, changes in the pH curve were minor. Although the rate of ureolytic ammonia release was proportional to cell-protein concentration, the reduction in ureolytic activity was compensated by a corresponding reduction in cell pH-buffering. Consequently, in order to relate pH and [OH-] changes to ureolysis, it was necessary to control, or correct for, variations in the cell mass present. Buffering capacity in plaques was greater than in sediments. The 10-fold range in oral ureolytic activity by salivary bacteria gave a 10-20-fold range in base changes.

pH changes during simultaneous metabolism of urea and carbohydrate by human salivary bacteria in vitro.

The effect of the wide natural variation in oral ureolysis rates on the pH changes resulting from simultaneous metabolism of 25 mM urea and 2.8 mM glucose in salivary-sediment bacteria were investigated. The pH curves were complex, and included distinctive plateaux indicative of balanced acid and base production. These neutralization plateaux occurred at different pHs, which were a function (r2 = 0.98) of the ureolytic rate as measured by the log of the initial pH-change rate in the urea-only reaction. In the simplest case, the pH curve was characterized by a rise or fall to the neutralization plateau, a variable period of time at the plateau (up to 1 h), then a pH rise. The pattern of pH changes induced by glucose alone varied between different sediments: in some cases, the pH decreased smoothly to an end-point; in others, the curve was more complex, and these features became superimposed on the urea/glucose curve. The rate of ureolytic ammonia release was almost constant and unaffected by simultaneous carbohydrate metabolism. Concomitant metabolism of endogenous carbohydrate present in sediments prepared 1-2 h following a meal was of sufficient magnitude to affect ureolytic pH curves. If the ureolytic activity was high, this effect was negligible; if it was low, metabolism of the endogenous carbohydrates could completely suppress the ureolytic pH rise. Soluble salivary components had little effect on ureolysis but pH changes were modified by buffering, and the presence of urea, ammonia, N-catabolic and acidogenic substrates in the saliva.

Urease activity in Streptococcus salivarius at low pH.

Arginine metabolism to alkali by the arginine deiminase system in oral bacteria increases their acid tolerance. The potential of urease activity in Streptococcus salivarius to fulfil a similar role was examined. In cell extracts between pH 5.0 and 8.0, urease activity was over 80% the maximal rate. The urease rate was zero at pH 4.3, and at pH 3.6 the enzyme was rapidly inactivated (t 1/2 of 0.6 min). The pH range of intact cells was broader. In Strep. salivarius cells acidified to pH 2.6 for 5 min, urease was completely retained and the ureolytic pH rise was rapid. There was no urease activity after acidification to pH 2. In cells acidified to maintain the pH between 3.3 and 4, viability was maintained for a short period (extrapolation indicated 20 min) and then decreased. This acidification induced alkali generation or acid removal that decreased in parallel to loss of viability. A small fraction (10%) of the urease was rapidly inactivated, after which both the remaining urease and pH response decreased at a similar rate to cell viability (t 1/2 of 15-20 min), but for at least 1 h following acidification, a rapid ureolysis induced rise in pH to above 7. In cells held at pH 3.6 and treated to compromise their membranes by freeze-thawing or transient acidification to pH 2.3, 70-80% of the urease was lost rapidly and the remainder inactivated at a rate similar to that in intact cells. Therefore, although at pH below 4, S. salivarius urease is outside its pH activity range and the free enzyme is rapidly inactivated, intact cells the urease is protected and ureolytic generation of ammonia is capable of substantially raising the pH for at least 1 h while the cell population is being progressively killed by acid.

Factors affecting the resting pH of in vitro human microcosm dental plaque and Streptococcus mutans biofilms.

The aim was to examine factors that potentially control the resting pH, defined as the pH unaffected by meals, of microcosm dental plaques and Streptococcus mutans biofilms under standard conditions, and to examine the effect of supplying urea at concentrations found intraorally. Microcosm plaques were cultured from plaque bacteria-enriched saliva in an 'artificial mouth' with a continuous supply of a medium including 0.25% mucin [Basal Medium Mucin, (BMM), 3.6 ml/hr per plaque] and a periodic supply of sucrose. The steady-state resting pH was 6.4 (range +/- 0.1) in BMM containing no urea and supplied at the standard flowrate. This is a robust property of the ecosystem. In one experiment with a replicated (n = 9) set of measurements, the resting pH was approx. pH 6.3, 6.4, 6.7 and 7.3 with 0, 1, 5 and 20 mmol/l urea in the BMM. The magnitude of sucrose- and urea-induced pH responses was unaffected by elevating the resting pH to produce parallel pH curves. The sucrose-induced pH curves were analogous to those classically reported by Stephan that showed an association between caries activity and increasingly acidic plaque pH responses to glucose. Stopping the BMM flow caused a pH rise, indicating continuing net alkali generation from BMM components in the absence of a fluid flow. Step. mutans monoculture biofilms had an acidic resting pH of 5.0 to 5.3, which increased to 6.8 following an adventitious superinfection by Bacillus cereus. It was concluded that the resting pH in plaque results from a delicate balance between alkali and acid generation, which is in turn dependent both on the bacterial composition of the plaque and on the supply of substrates and buffers from, and metabolite clearance into, flowing oral fluid. In vivo the resting pH will vary with site-specific changing saliva flows. Urea continuously supplied at concentrations normal for saliva and gingival crevicular fluid can raise the resting pH of microcosm plaque by an amount tat in vivo would probably be significant in reducing dental caries.

Inhibition by ethanol of the growth of biofilm and dispersed microcosm dental plaques.

Inhibition of microcosm plaque biofilm growth by periodic application of ethanol was compared with the minimum inhibitory concentration (MIC) and bactericidal effects of ethanol on liquid cultures of dispersed plaque bacteria. Microcosm plaques were cultured from saliva in a multiplaque 'artificial mouth' and their growth in wet weight measured daily. Nutrient conditions included: a continuous supply of a medium containing 0.25 percent mucin, and 8-hourly 5 percent (w/v) sucrose (1.5 ml over 6 min). Plaque biofilm growth was strongly inhibited by exposure to 40 percent (v/v) ethanol applied in volumes of 3.75 ml over 15 min, six times daily. Application of 1.5 ml over 6 min inhibited much less or not at all. Ethanol concentrations lower than 40 percent caused less inhibition, with 10 percent having almost no effect. The pH response to sucrose was unchanged by prior application of 40 percent ethanol for 30 min. Some evidence was obtained for either bacterial adaptation to ethanol or selection of ethanol-resistant bacteria. The MIC and bactericidal effects of ethanol were assessed by growth of dispersed plaque in liquid culture; the bactericidal effect was measured as the induced delay in growth. The aerobic and anaerobic MIC of ethanol for growth was 10 percent and 8 percent; 50 percent inhibition of growth rate occurred at 3.7 percent and 2.8 percent. Ethanol (40 percent) was bactericidal within 1-2 min, but 10 percent had almost no effect. It was concluded that, despite the well-known high ethanol sensitivity of dispersed plaque bacteria, prolonged application of ethanol concentrations in the order of 40 percent are necessary to inhibit growth of plaque biofilms.

The source of variation in ureolysis in artificial plaques cultured from human salivary bacteria.

Artificial-mouth plaques were cultured for 7 days from the saliva of two individuals, one with high and one with low salivary ureolytic activity. There was a 7.5-fold range in the resulting plaque ureolysis rates (per mg of protein), and the composition of the flora varied widely. The average rate of ureolysis of artificial plaque was similar to that in natural plaques but higher than in salivary sediment (after correction of sediment rates for the presence of 50 per cent non-bacterial protein). The average rate of ureolysis per ureolytic bacterium was 2.5 times higher in the artificial plaques than in saliva. Although the saliva inocula were from subjects with a 3-fold difference in salivary ureolysis rate, this difference was not reflected in the ureolytic activity of the artificial plaques. Neither was this difference evident in the ureolytic activity of the corresponding natural plaques. The established hypothesis that plaque ureolysis is derived mainly from an unidentified active segment of the total ureolytic flora was tested in the artificial plaques by analysis of variance to determine the contribution of the known ureolytic bacteria. Plaque ureolysis rates were almost entirely explained (r2 = 86 per cent) by the percentage of total detectable ureolytic bacteria in the plaque flora. The plaque bacteria giving strong ureolytic reactions on agar plates were all Gram-positive cocci and in 6 of the 9 plaques were streptococci only. Therefore, in artificial plaques the physiologically significant bacteria comprise a high proportion of the total ureolytic flora and are Gram-positive cocci, mainly streptococci.

Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system.

The aim was to establish defined-species consortium plaque biofilms to investigate enamel and root caries in an artificial mouth. Strains of the putative enamel and root caries pathogens, Streptococcus mutans, Strep. sobrinus, Actinomyces naeslundii and Lactobacillus rhamnosus, were screened in batch culture for potential cariogenic properties: a low terminal pH, ability to aggregate, and catabolic diversity. The strains selected were grown as monoculture biofilms and as consortium plaque biofilms in a multiplaque artificial mouth. The biofilms were supplied with a constant flow of a simulated oral fluid and were given periodic sucrose (and in some instances glucose) to simulate meals. All the bacteria except L. rhamnosus formed large, monospecies biofilms with resting pH in the range 5.3-5.8. The consortia biofilms were larger and had a resting pH of 4.9-5.3. The consortia biofilms supplied with 8-hourly carbohydrate comprised mainly 'mutans' streptococci (58, SD 5.5%) and L. rhamnosus (42, SD 5.7%). A. naeslundii characteristically was absent or present in a low percentage (up to 4% colony-forming units). All biofilms demineralized polished bovine enamel and dentine blocks, as assessed by microradiography and enamel-surface microhardness measurement. The consortia also demineralized intact enamel and tooth roots; they were more cariogenic to enamel than any of the monoculture biofilms, as measured by enamel-surface softening, but variation in lesion depth was proportional to biofilm wet weight irrespective of acidogen composition (r = 0.93, p < 0.05). Enamel lesions had a well-mineralized intact surface and a zone of subsurface demineralization, typical of early natural lesions. Dentine and root lesions showed extensive demineralization but lacked a pronounced surface mineralized zone. Substitution of glucose for sucrose had no effect on the cariogenicity of the consortium to bovine enamel or human roots and had no major effect on the plaque composition. Continuously supplied fluoride (19 parts/10(6)) resulted in a substantially reduced enamel surface softening and subsurface demineralization of intact roots. It was concluded that consortia biofilms of selected caries pathogens generate realistic caries lesions in all tooth hard tissues under controlled growth conditions in the artificial mouth. This in vitro caries experimental model may prove useful for the study of interrelations between the plaque biofilm, tooth tissues and the oral environment, and for the development of procedures to modify the course of caries development.

The pH response to urea and the effect of liquid flow in 'artificial mouth' microcosm plaques.

This study examined in detailed the pH response of microcosm plaque biofilms to the application of 500 mmol/l urea, and the effect of modifying the flow rate of BMM (a basal medium containing 0.25% mucin). Microcosm plaques were cultured from the mixed salivary bacteria in a multi-plaque 'artificial mouth' supplied continuously with BMM at 3.6 ml/h per plaque, and periodically with sucrose (5 or 10%). Urea (500 mmol/l) induced a pH response that was the inverse of the Stephan pH curve induced by sucrose. In thicker plaques the ureolytic pH response was delayed and slower. With no BMM flow, the urea-induced pH curve reached a maximum and then slowly decreased indicating loss of ammonia. A flow of BMM reduced the magnitude of the pH response. Urea dilution explained (r2 = 0.97) the reduction in the maximum rate of pH rise caused by an increasing BMM flow. There were, however, additional flow-rate effects on the magnitude of the pH rise, the curve areas and the maximum rate of pH decrease back to the resting pH. These effects were greatest at low BMM flow rates, indicating that ammonia clearance may be limited at higher flow rates by the rate of intraplaque diffusion and metabolism. Application of 50 instead of 500 mmol/l urea reduced the rate of pH rise about 10-fold, and the area of the curve about seven fold. Metabolism of arginine (50 mmol/l) generated only about half the pH response of the same amount of urea.(ABSTRACT TRUNCATED AT 250 WORDS)

pH gradients induced by urea metabolism in 'artificial mouth' microcosm plaques.

Evidence was sought for urea-induced pH gradients in dental plaque microcosm biofilms cultured from the mixed salivary bacteria in a multi plaque 'artificial mouth'. Application of 500 mmol/l urea for short periods (6 min) to 5-8 mm maximum-thickness plaques induced intraplaque pH gradients of up to 0.7 pH units with the surface alkaline relative to the inner plaque. These pH gradients persisted for more than 5 h in the absence of a flow of fluid. With 30-min urea applications and a flow of a basal medium containing mucin (BMM, pH 7.0), the pH of the inner (deeper) plaque regions also increased. Although the pH gradient initially formed was alkaline at the plaque surface, the BMM flow lowered the surface pH to neutrality whilst the inner layers were still alkaline, thereby reversing the pH gradient. In thick microcosm dental plaques, urea-induced pH gradients can therefore form and last many hours. They probably result from the significant time taken for urea to penetrate to the inner layers of plaque, its rapid metabolism by the outer plaque layers, and a rate-limiting clearance of ammonia. Even a slow BMM flow over the plaque greatly increased the rate of return to the resting pH, causing the gradients to change polarity.

Kinetics and product stoichiometry of ureolysis by human salivary bacteria and artificial mouth plaques.

Ureolysis was investigated in salivary bacteria from persons with widely-differing oral ureolytic activities. Rate curves and product stoichiometry were established for urea disappearance, ammonia appearance and conversion of [14C]-urea to 14CO2. Ammonia, released stoichiometrically from urea, was best measured by a direct phenate-hypochlorite reaction. About 80 per cent of the urea-C was liberated as free CO2. Slight deviations from ammonia stoichiometry and most of the CO2 loss occurred in the first 5-10 min of reaction, when the rate of urea disappearance was constant and up to 2-fold higher than subsequently. This rate-change suggests that flux in the ureolysis pathway may be under feedback control. Ureolysis by salivary-sediment bacteria followed Michaelis-Menten kinetics with a Km of 2.5 mM; rates of end-product formation were independent of urea concentration between 25 and 500 mM. Ureolysis was inhibited 98 per cent by 5 mM acetohydroxamic acid, a urease inhibitor, and could be partly solubilized by sonication to give an enzyme preparation which, without cofactor supplementation, quantitatively hydrolysed urea. Thus urea metabolism by oral bacteria may principally involve urease-catalysed hydrolysis, rather than non-urease pathways.

pH responses to sucrose and the formation of pH gradients in thick 'artificial mouth' microcosm plaques.

Artificial microcosm plaques were grown in a five-plaque culture system for up to 6 weeks, reaching a maximum depth of several mm. Procedures for long-term pH measurement with glass electrodes were established; they showed that the application of 5 or 10% sucrose for 6 min with a slow continuous flow of a basal medium containing mucin (BMM) generated the pH changes characteristic of in vivo Stephan curves. These pH responses were reproducible between plaques. Plaque mass and thickness were critical variables. Successive, sucrose-induced pH curves in plaques up to 4 mm thickness showed minor reductions only in the amplitude and rates of pH change. In plaques over 4 mm thick there was a pronounced reduction in pH response to successive sucrose applications, indicating increased diffusion limitations--a result of plaque growth to seal in the freshly-inserted pH electrode. In plaques of 6 mm maximum thickness, 10% sucrose induced a decrease to below pH 5.5 lasting 24 h, compared to the pH response in 2 mm thick plaque, which returned to the resting pH in 2 h. Differences in pH of up to 0.9 units were identified in thick plaques between inner and outer layers. The BMM flow rate was a critical determinant of the amplitude of the pH response to sucrose and subsequent return to resting pH. These results confirm, for microcosm plaque, the importance of clearance dynamics and diffusion-limited gradients in regulating plaque pH.

Inhibitory effect of ZnCl(2) on glycolysis in human oral microbes.

Although the inhibition of bacterial glycolysis by zinc ions might be expected to moderate dental caries, there has not been a comparison of the effect of Zn on different organisms under both fixed pH and free-fall conditions. Here, the effect of ZnCl(2) on Streptococcus salivarius, Strep. mutans, Strep. sobrinus, Actinomyces naeslundii and Lactobacillus casei, as well as on mixtures of oral organisms outgrown from human dental plaque and saliva, was surveyed. pH-stat experiments were performed at pH 7, 6 or 5 in a solution containing 5% glucose and a suspension of the test organism; pH-fall experiments started at pH 7. In both cases, acid production was monitored for 60 min, when samples were taken for Zn and lactate determinations. Under pH-stat conditions, acid production was inhibited by Zn most strongly in Strep. sobrinus and Strep. salivarius. In terms of total acid production averted, however, the effect of Zn under both pH-stat and pH-fall conditions was clearly greatest with Strep. salivarius. A. naeslundii was inhibited the least strongly under pH-stat conditions. Cultured oral organism mixtures were more sensitive to moderate concentrations of zinc (0.2-0.3mM initial concentration) than were the single species to higher concentrations (1mM). Packed cell layers responded to Zn quite differently from suspensions, the pH often falling in the presence of 1mM Zn at a rate similar to the no Zn control. As streptococci had the highest acidogenesis rates in both pH-stat and pH-fall experiments, it seems likely that inhibition of acid production with these organisms would be of more value in moderating caries than the inhibition of less acidogenic organisms such as A. naeslundii.

The bacteria responsible for ureolysis in artificial dental plaque.

The origin of ureolytic activity in artificial-mouth plaques was established by assessing the contribution to plaque ureolytic activity of the isolated bacteria. To overcome losses of ureolytic activity caused by the unstable presence of urease in oral bacteria, ureolytic bacteria were isolated from an exceptionally active plaque (1 mumol NH3/min per mg protein) in which 63 per cent of the flora was ureolytic. After their ability to metabolize urea was stabilized, 13 ureolytic bacteria remained: seven strains of Streptococcus salivarius, one Streptococcus bovis, two Staphylococcus epidermidis and three Staphylococcus haemolyticus. Their urease activity, measured after growth into stationary phase, was reproducible and strain specific with a 20-fold range within each genus. The mean ureolytic activity of each species, when weighted by its calculated incidence in the original plaque, accounted for 40 per cent of the total plaque ureolytic activity. However, these values for urease levels were only a small fraction of the bacterial ureolytic potential. Urease per mg cell protein measured during the growth cycle of a selected Strep. salivarius, and Staph. epidermidis, varied 10-fold, and reached much higher activities (i.e. 6-8 mumol NH3/min per mg of cell protein) than under the growth conditions that were used to assess the contribution of these species to total plaque ureolysis. Thus urea metabolism in artificial plaque was due mainly to Strep. salivarius, with a small contribution from Staph. epidermidis. The presence of further unidentified species of ureolytic oral bacteria need not be invoked.