D.J. Bradshaw

Dental plaque as a biofilm

Dental plaque is the diverse microbial community found on the tooth surface embedded in a matrix of polymers of bacterial and salivary origin. Once a tooth surface is cleaned, a conditioning film of proteins and glycoproteins is adsorbed rapidly to the tooth surface. Plaque formation involves the interaction between early bacterial colonisers and this film (the acquired enamel pellicle). To facilitate colonisation of the tooth surface, some receptors on salivary molecules are only exposed to bacteria once the molecule is adsorbed to a surface. Subsequently, secondary colonisers adhere to the already attached early colonisers (co-aggregation) through specific molecular interactions. These can involve protein-protein or carbohydrate-protein (lectin) interactions, and this process contributes to determining the pattern of bacterial succession. As the biofilm develops, gradients in biologically significant factors develop, and these permit the co-existence of species that would be incompatible with each other in a homogeneous environment. Dental plaque develops naturally, but it is also associated with two of the most prevalent diseases affecting industrialised societies (caries and periodontal diseases). Future strategies to control dental plaque will be targeted to interfering with the formation, structure and pattern of development of this biofilm.

Microbial Biofilm Formation and Contamination of Dental-Unit Water Systems in General Dental Practice

ABSTRACT Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 105CFU · ml−1; in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by BacLight staining, but only 5% of this “viable by BacLight” fraction produced colonies on agar plates. Legionella pneumophila,Mycobacterium spp., Candida spp., andPseudomonas spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci andFusobacterium spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases, P < 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered “cleaner” than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.

Effects of Carbohydrate Pulses and pH on Population Shifts within Oral Microbial Communities in vitro

A mixed culture chemostat system was used to distinguish between the effects of carbohydrate availability per se and the low pH generated from carbohydrate metabolism on the proportions of bacteria within microbial communities. Nine oral bacteria were grown at pH 7 and pulsed with glucose on ten consecutive days. In one chemostat, the pH was maintained automatically at 7 throughout the experimental period, while in the other, pH control was discontinued for six hours after each pulse. Glucose pulses at neutral pH had little effect on the composition of the microflora. Only the proportions of A. viscosus and V. dispar increased; L. casei and S. mutans remained at low levels (0.2% and 1.0%, respectively). Acetate and propionate were the predominant end-products of metabolism; lactate levels were low. In contrast, when pH was allowed to fall after each glucose pulse, the composition of the microflora altered dramatically. The amounts of L. casei and S. mutans increased both as a proportion of the total count and in absolute numbers, as did V. dispar, whereas the amounts of the other Gram-negative organisms (B. intermedius, F. nucleatum, and N. subflava) and S. sanguis were considerably reduced. Lactate formed a major portion of the metabolic end-products. Successive glucose pulses resulted in both amplified changes in the microflora and a steadily greater rate and final extent of acid production. This is in agreement with the reported shifts in the oral microflora in vivo in response to frequent carbohydrate intake. Analysis of the data strongly suggests that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, is responsible for the widely reported shifts in composition and metabolism of the oral microflora in vivo.

Analysis of pH–Driven Disruption of Oral Microbial Communities in vitro

Previously, a mixed culture chemostat system was used to demonstrate that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, was responsible for the shifts observed in the oral microflora which are associated with high carbohydrate diets and the development of dental caries. The aim of this study was to determine more accurately the microbially generated pH at which such shifts occurred. Nine oral bacteria were grown in three independent chemostats, and pulsed with glucose on 10 consecutive days. In one chemostat, pH control was discontinued for 6 h, and the pH fall was restricted to a minimum value of pH 5.5; the pH fall was arrested in the other two chemostats at either pH 5.0, or at pH 4.5. When the pH was allowed to fall, the numbers and proportions of Streptococcus mutans and Lactobacillus rhamnosus increased; this increase was directly related to the magnitude of the pH fall. Veillonella dispar was the most numerous organism following all glucose pulsing regimes, especially at low pH. The increase in proportions of acidogenic bacteria was accompanied by a fall in the proportions of acid–sensitive species (Fusobacterium nucleatum, Prevotella nigrescens, Streptococcus gordonii and Streptococcus oralis). The counts of these species were relatively stable between pH 5.5 and 4.5, but were markedly reduced when the pH fell below pH 4.5; Neisseria subflava could not persist in the culture at pH 4.5 or below. The data suggest that the disruption of communities associated with glucose metabolism and low pH can be explained in terms of a two–stage process. A fall in pH to a value between pH 5.5 and 4.5 may allow the enrichment of potentially cariogenic species, whilst permitting species associated with health to remain relatively unaffected. A further reduction in pH (<pH 4.5) may not only enhance the competitiveness of odontopathogens, but inhibit the growth and metabolism of non–caries–associated species. The results also indicate that species other that mutans streptococci or lactobacilli are competitive at pH values low enough to demineralise enamel, and thus suggest that a broader range of micro–organisms may be associated with caries initiation.

Physiological Approaches to the Control of Oral Biofilms

Evidence that physiological strategies may be potential routes for oral biofilm control has come from (i) observations of the variations in the intra-oral distribution of members of the resident oral microflora, (ii) changes in plaque composition in health and disease, and (iii) data from laboratory model systems. Key physiological factors that were identified as significant in modulating the microflora included the local pH, redox potential (Eh), and nutrient availability. Increases in mutans streptococci and lactobacilli occur at sites with caries; growth of these species is selectively enhanced at low pH. In contrast, periodontal diseases are associated with plaque accumulation, followed by an inflammatory host response. The increases in Gram-negative, proteolytic, and obligately anaerobic bacteria reflect a low redox potential and a change in nutrient status due to the increased flow of gingival crevicular fluid (GCF). Consequently, physiological strategies for oral biofilm control should focus on reducing the frequency of low pH in plaque by (i) inhibiting acid production, (ii) using sugar substitutes, and (iii) promoting alkali generation from arginine or urea supplements. Similarly, strategies to make the pocket environment less favorable to periodontopathogens include (i) anti-inflammatory agents to reduce the flow of (and hence nutrient supply by) GCF, (ii) bacterial protease inhibitors, and (iii) redox agents to raise the Ehh locally. Most laboratory and clinical findings support the concept of physiological control. However, some data suggest that the ordered structure and metabolically interactive organization of mature dental plaque could generate a community with a high level of homeostasis that is relatively resistant to deliberate external manipulation.

Effect of oxygen, inoculum composition and flow rate on development of mixed-culture oral biofilms

The effect of aeration on the development of a defined biofilm consortium of oral bacteria was investigated in a two-stage chemostat system. An inoculum comprising 10 species, including both facultatively anaerobic and obligately anaerobic bacteria, and species associated with oral health and disease, was inoculated into an anaerobic first-stage chemostat vessel. The effluent from this chemostat was linked to an aerated [200 ml CO2/air (5:95, v/v) min-1] second-stage vessel, in which removable hydroxyapatite discs were inserted to allow biofilm formation. Comparisons were made of planktonic and biofilm communities in the aerated second-stage vessel by means of viable counts. Both planktonic and early biofilm communities were dominated by Neisseria subflava, comprising > 40% of total c.f.u. in the fluid phase, and > 80% of c.f.u. in 2 h biofilms. Obligate anaerobes persisted in this mixed culture, and succession in biofilms led them to predominate only after 7 d. Despite the continuous addition of air, the dissolved oxygen tension (dO2) within the culture remained low (< 5% of air saturation), and the redox potential (Eh) was -275 mV. In order to assess the significance of the presence of N. subflava in community development, a subsequent experiment omitted this aerobe from the inoculum, to produce a nine-species culture. The planktonic phase was predominated by three streptococcal species, Prevotella nigrescens and Fusobacterium nucleatum. Biofilms again underwent successional changes, with anaerobes increasing in proportion with time. In contrast to the culture including N. subflava, dO2 was 50-60% of air saturation, and the Eh was +50 mV. In the final experiment, the rate of addition of first-stage culture was reduced to 1/10 of that in the previous experiment, in order to determine whether anaerobes were growing, rather than merely persisting in the aerated culture. The data for the planktonic phase indicated that the anaerobes were growing in aerated (dO2 40-50%, Eh +100 mV) conditions. Once again, anaerobes increased in proportion in older biofilms. The study indicates that mixed cultures can protect obligate anaerobes from the toxic effects of oxygen, both in the biofilm and planktonic modes of growth.

Effects of Glucose and Fluoride on Competition and Metabolism within in vitro Dental Bacterial Communities and Biofilms

Antimicrobial effects of fluoride in vivo remain contentious. Previous studies suggested that 1 mM NaF reduced acid production from glucose, and prevented the enrichment of bacteria associated with caries in a chemostat model. The present study examines the effects of a lower fluoride concentration (0.53 mM, 10 ppm NaF) in both biofilm and planktonic microbial communities. Nine oral species were grown at pH 7.0 and pulsed on 10 successive days with glucose; bacterial metabolism was allowed to reduce the pH for 6 h before being returned to neutrality, either in the presence or absence of NaF. In addition, 10-day-old mixed culture biofilms were overlaid with glucose, with or without NaF, and the pH change followed by microelectrode. After 10 days, chemostat pH dropped to ca. pH 4.5 following glucose pulses, and the community was dominated by Streptococcus mutans (rising from 4 to 23% of total CFU) and Veillonella dispar (16 to 73%). In comparison, after 10 days pulsing with glucose + fluoride, the final pH was significantly higher (ca. pH 4.9) (paired t test, p < 0.0001). The culture was predominated by V. dispar (70%) and Actinomyces naeslundii (13%), whereas S. mutans proportions were significantly lower (t test, p = 0.04), remaining <3% of the total flora, compared to the culture without fluoride. Biofilm pH fell to only pH 5.55 1 h after glucose/fluoride overlay, compared to 4.55 with glucose alone (paired t test, p < 0.000001). Analysis of the data suggests that fluoride exerts dual antimicrobial modes of action. Fluoride prevents enrichment of S. mutans by inhibiting critical metabolic processes (direct effect) and, in an inter-related way, by reducing environmental acidification (indirect effect) in biofilms.

The Effects of Triclosan and Zinc Citrate, Alone and in Combination, on a Community of Oral Bacteria Grown in vitro

A mixed-culture chemostat system has been used as a more stringent laboratory system for evaluation of the antimicrobial effects of Triclosan and zinc citrate. The inhibitors were added alone, and in combination, as a pulse (a high initial inhibitor concentration which decreased with time) or as a dose (concentration of inhibitor increased with time) to give maximum concentrations of 34.5 μmol/L Triclosan and 39.8 μmol/L zinc citrate. When dosed, Triclosan inhibited A. viscosus and all five Gram-negative species, whereas zinc citrate had less effect, probably due to complexation by media components. Similar effects were seen when Triclosan was pulsed, except that S. mutans was the most sensitive Gram-positive species and V. dispar was unaffected. However, when the inhibitors were dosed or pulsed in combination, marked complementary and additive inhibitory effects were observed, particularly against Gram-negative species, although S. gordonii and S. oralis were relatively unaffected. The data confirm that increased effects can be obtained with suitable combinations of antimicrobial agents and suggest that, under certain conditions, apparently broad-spectrum antimicrobial agents may be acting more selectively than hitherto suspected.

Prevention of Population Shifts in Oral Microbial Communities in vitro by Low Fluoride Concentrations

A continuous culture system has been used to study the effects of low (sub-MIC) levels of sodium fluoride on the stability and metabolism of a defined oral microbial community. The microflora was also subjected to glucose pulses at pH 7.0, with and without subsequent pH control. At pH 7.0, a continuous supply of 1 mmol/L NaF reduced slightly the viable counts of the oral microflora, although their proportions were relatively unaffected. At pH 7.0, during glucose pulsing, 1 mmol/L NaF prevented the rise in proportions of A. viscosus and reduced the levels of B. intermedius. Glucose pulsing without pH control and in the absence of fluoride markedly inhibited the growth of many species, and L. casei, V. dispar, and S. mutans predominated in the culture. Fluoride (1 mmol/L), either pulsed with the glucose or provided continuously, reduced both the rate of change and the degree of fall in pH, and in doing so prevented the enrichment of S. mutans in the culture. Fluoride also reduced the pH-mediated inhibition of other members of the oral community, although S. sanguis was inhibited even further. Thus, even sub-MIC levels of fluoride may have a beneficial anti-bacterial effect on dental plaque by interfering with acid production. This would reduce the pH-mediated disruption to the balance of the microflora and suppress the selection of S. mutans.

Oral anaerobes cannot survive oxygen stress without interacting with facuItative/aerobic species as a microbial commmunity

D.J. Bradshaw, P.D. Marsh, G.K. Watson and C. Allison. 1997. Anaerobic bacteria are found commonly as components of mixed culture biofilms in many aerated habitats, including the mouth. Previous studies showed that anaerobes could survive in planktonic and biofilm communities in aerated conditions when part of a community including facultative and/or aerobic species, and the numbers and proportions of anaerobic species increased as biofilms aged. When the obligate anaerobes were grown in the absence of aerobic/facultative species, however, they were unable to grow in either the planktonic or biofilm culture. The mean survival times of organisms in the aerated culture containing four anaerobic species varied from around 5 min for Fusobacterium nucleatum and Veillonella dispar, to less than 4 min for Porphyromonas gingivalis and Prevotella nigrescens. In addition, in this culture, the biofilm mode of growth did not provide a haven for these bacteria in the absence of oxygen‐consuming species.