Strong genetic control of emergence of human primary incisors.

Our understanding of tooth eruption in humans remains incomplete. We hypothesized that genetic factors contribute significantly to phenotypic variation in the emergence of primary incisors. We applied model-fitting to data from Australian twins to quantify contributions of genetic and environmental factors to variation in timing of the emergence of human primary incisors. There were no significant differences in incisor emergence times between zygosity groups or sexes. Emergence times of maxillary central incisors and mandibular lateral incisors were less variable than those of maxillary lateral incisors and mandibular central incisors. Maxillary lateral incisors displayed significant directional asymmetry, the left side emerging earlier than the right. Variation in timing of the emergence of the primary incisors was under strong genetic control, with a small but significant contribution from the external environment. Estimates of narrow-sense heritability ranged from 82 to 94% in males and 71 to 96% in females.

An in vitro investigation of marginal dentine caries abutting composite resin and glass ionomer cement restorations.

BACKGROUND: There are a number of studies citing the primary reason for replacing auto cure glass ionomer cements was due to recurrent caries. The purpose of this study was to use an in vitro model to measure caries at the dentine restoration interface of bonded composite resin and auto cure glass ionomer cement restorations and to measure the amount of surface degradation occurring in the restorative materials. METHODS: Specimens of auto cure glass ionomer cements (Riva Fast, Fuji IX Fast, Ketac Molar Quick and Fuji VII) and bonded composite resin restorations (Ice, SDI) were placed separately at the dentino-enamel junction of 10 recently extracted human third molar teeth, disinfected and placed into the overflow from a continuous culture of S. mutans for two weeks. Restorations were sectioned and prepared for scanning electron microscopy (SEM) and electron probe microanalysis (EPMA). Restoration tooth interfaces were photographed and the distance from the surface of the teeth to the surface of the restorations measured. EPMA of percentage weights of calcium, phosphorous and fluoride were made outwards from the restoration surface 130pm at a depth of 10 microm below the surface of the dentine. RESULTS: There were significant differences between the surface heights of composite resin, auto cure glass ionomer cements compared to teeth surfaces. Percentage weights of calcium and phosphorus levels were similar to non-demineralized dentine in the auto cure glass ionomer cement samples but there were significant reductions in mineral content of dentine adjacent to bonded composite resin restorations. Fluoride levels were mixed. CONCLUSIONS: This study shows that placing a bonded composite resin restoration into dentine affords little protection to the surrounding tooth from caries attack although insignificant degradation of the restorative surface occurs. Placing a glass ionomer cement restoration into dentine protects the surrounding tooth from caries but degradation of the restoration surface occurs.

Differences between normal and demineralized dentine pretreated with silver fluoride and potassium iodide after an in vitro challenge by Streptococcus mutans.

BACKGROUND: The application of diamine silver fluoride (Ag(NH3)2F) and potassium iodide (KI) to demineralized dentine has been shown to inhibit the growth of Streptococcus mutans. The purpose of this study was to observe the differences between demineralized and non-demineralized dentine treated with AgF/KI. METHODS: Thirty-five dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials which were filled with nutrient medium, sterilized and placed into the overflow from a continuous culture of S. mutans. Samples were divided as follows: 10 samples of demineralized dentine; 10 samples of demineralized dentine treated with AgF/KI; 5 samples of non-demineralized dentine; and 10 samples of non-demineralized dentine treated with AgF/KI. Following two weeks connected to the Chemostat, an electron probe microanalysis (EPMA) of percentage weights and penetration depths of calcium, phosphorous silver and fluoride was conducted. Bacterial growth was monitored by taking optical density readings of the growth medium in each vial and outer surfaces of the specimens were examined by scanning electron microscopy (SEM). RESULTS: AgF/KI treatment of demineralized and non-demineralized dentine prevented biofilm formation and reduced further demineralization by S. mutans. AgF/KI treatment of demineralized dentine was more effective in reducing dentine breakdown and the growth of S. mutans. Significantly higher levels of silver and fluoride were deposited within demineralized dentine. CONCLUSIONS: A topical treatment with AgF/KI on dentine reduced in vitro caries development and inhibited surface biofilm formation. Reduction of in vitro caries development and viability of S. mutans was more pronounced on the dentine samples that had been demineralized prior to the application of

An in vitro model to measure the effect of a silver fluoride and potassium iodide treatment on the permeability of demineralized dentine to Streptococcus mutans.

BACKGROUND: Diamine silver fluoride (Ag(NH3)2F), referred to as AgF, has been used to reduce the incidence of caries in primary dentitions but has been limited by the associated staining of both teeth and restorative materials. The application of potassium iodide (KI), following AgF prevents staining but its effects on the ability of AgF to reduce caries are not known. The aim of this study was to develop an in vitro model that would provide an indication of the permeability of demineralized dentine to Streptococcus mutans after treatment of the dentine with AgF followed by KI. METHODS: Forty dentine discs were bonded to the base of forty 5mL polycarbonate screw top vials (that had had their bases removed), filled with nutrient medium, sterilized and placed into a continuous culture of S. mutans. Samples were divided into four groups as follows: 10 samples of demineralized dentine as a control, 10 samples of demineralized dentine treated with AgF/KI, 10 samples of demineralized dentine treated with KI and 10 samples of demineralized dentine treated with AgF. After two weeks the optical density of the growth medium chambers was measured to determine bacterial penetration and growth. Cultures were plated out to determine migration through the discs by S. mutans. RESULTS: S. mutans migrated through all dentine discs. However, the samples treated with AgF and AgF/KI had significantly lower optical densities than the corresponding controls. The range of optical densities was least amongst demineralized samples treated with AgF/KI. CONCLUSIONS: Under the conditions of this study, treatment of demineralized dentine discs with AgF followed by KI allowed the penetration of S. mutans. Based on optical density measurements, the treatment resulted in significantly fewer microorganisms being present subjacent to the discs treated with AgF and KI than the control discs at the end of the experimental period.

Changes in growth and polyglucose synthesis in response to fructose metabolism by Fusobacterium nucleatum grown in continuous culture.

Fusobacterium nucleatum, grown in a chemically defined medium at micro(rel) = 0.5, produced greater cell yields and undetectable levels of intracellular polyglucose (IP) when fructose was substituted for glucose. The utilisation and metabolism of fructose by growing cells was studied and the effect of the energy-yielding amino acids, glutamate, serine, histidine and lysine on cell yield, IP synthesis and acidic end-products was investigated. When F. nucleatum was grown on elevated amino acid levels, IP was synthesised from fructose and amino acids were metabolised to lactate, acetate, butyrate and formate. Under these conditions, IP synthesis was associated with the cells being replete with amino acid-derived energy; an observation supported by the absence of IP when the levels of (energy yielding) amino acids were reduced. Compared with fructose, glucose was less efficiently removed from the growth medium and produced less biomass and markedly lower levels of IP during energy-limited growth.

The characterization of a (nutritionally important) proline iminopeptidase from Eikenella corrodens.

Eikenella corrodens generates energy primarily through the oxidative deamination of specific amino acids, a process that is coupled to dissimilatory nitrate reduction to nitrite. Cell yields resulting from chemostat-growth of the organism in simple, chemically defined media containing varying amounts of proline confirm that this amino acid is a likely source of energy for E. corrodens in the oral environment. The importance of proline in ATP generation by the organism is reflected in molar growth yields, which showed that biomass production per mole of this amino acid was significantly higher than that for other amino acids. The organism was found to express, constitutively, the enzyme proline iminopeptidase, which releases proline from the N-terminus of small peptides. The enzyme was partially purified and characterized and found to exist in the cytoplasm as a 35 kDa monomer. Inhibition studies showed that the enzyme, although classified as a serine protease, also appears to require thiol groups for activity, a finding which is consistent with previous reports. The enzyme obeyed Michaelis-Menten kinetics and was found to have a Km value of 0.223 mM for the substrate proline-p-nitroanilide.

An aminopeptidase nutritionally important to Fusobacterium nucleatum.

The properties of an aminopeptidase (AP) from Fusobacterium nucleatum were studied in view of the fact that this organism, along with other Gram-negative anaerobes involved in periodontal diseases, survives in the subgingival environment by obtaining energy via the fermentation of a small number of peptide-derived amino acids. The AP was found to be cell-associated and was isolated from disrupted chemostat-grown cells. It was purified by (NH4)2SO4 fractionation, two column chromatographic steps and IEF. The enzyme was found to have a molecular mass of 54 kDa, a pI of 5.1, a pH optimum between 7.5 and 8.0 and, using Leu-Ala as substrate, it gave K(m) and Vmax values of 0.66 mM and 0.12 mumol min-1 mg-1, respectively. No complete homology was found between the N-terminal sequence of the first 20 amino acids (MDXKXYVDLKERFLRYVKFN...) and any other published sequence, but residues 8-20 gave a 62% match with residues 9-21 of an AP from Haemophilus influenzae. The enzyme was inactivated by chelating agents, bestatin, p-hydroxymercuribenzoate and some heavy metals. Cobalt ions restored EDTA-inactivated activity but did not reverse inhibition by 1,10-phenanthroline. In addition, bestatin and 1,10-phenanthroline had an inhibitory effect on the batch growth of F. nucleatum in a complex medium in which peptidase activities would be nutritionally essential. No such inhibition was observed in a chemically defined medium in which growth was not dependent upon peptidase activities. The peptidase described in this paper therefore appears to be a cobalt-activated metallo-AP which, together with other peptidases, is considered to be important in the survival of F. nucleatum in the subgingival environment of the mouth.

The behaviour of Fusobacterium nucleatum chemostat-grown in glucose- and amino acid-based chemically defined media.

Fusobacterium nucleatum is a Gram-negative anaerobe, found in a number of different areas of human and animal bodies as part of the resident microbiota. However, it also appears to be involved in polymicrobial infections in such sites. It occurs in the oral cavity where it is a prominent member of various bacterial consortia associated with periodontal diseases. Like most fusobacteria, it derives energy via the fermentation of amino acids which it can obtain through the dissimilation of small peptides. However, the role of simple carbohydrates, such as glucose, in its growth and metabolism are not well understood. Accordingly, the aim of the present study was to study the behaviour ofF. nucleatum grown anaerobically in continuous culture in two different chemically-defined media (CDM); one containing only amino acids as the energy source, the other containing glucose as the predominant energy provider. At various dilution rates the culture were assayed for dry weight, intracellular polyglucose (IP) content, residual amino acids and glucose and acidic metabolic end-products. In the carbohydrate-free CDM the acidic end-products were a constant acetate : butyrate : formate of 1.5 : 1 : 0.4. The values of Y(max)amino acids, maximum yield of bacteria per mol of amino acids consumed, for two strains were estimated to be 15.2 and 18.6 g dry wt/mol, respectively. Them(amino acids), maintenance energy requirement for growth on amino acids, for the two strains was 0.81 and 0.94 mmol/g dry wt/h, respectively. Growth of one strain in the glucose-based CDM gave an estimated Y(max)glucose of 67.2 and an m(glucose) of 0.38; the acidic end-products were a fairly constant acetate : butyrate : formate : lactate of 0.7 : 1 : 0.3 : 2.5. Only at low growth rates, and then only in small amount, was IP produced in this medium. Overall, it was concluded that the occurrence of F. nucleatum in widely-differing oral niches may be explained, at least in part, by its metabolic versatility.

Energy production and peptidase activity in Eikenella corrodens.

Eikenella corrodens 33EK(L), a clinical isolate, was assayed for its ability to utilise amino acids as substrates in the reduction of nitrate to nitrite. The metabolism of proline, glutamate, serine and glutamine was found to result in relatively high rates of nitrate reduction. The ability of cells to metabolise these amino acids from a variety of small peptides was also determined. E. corrodens was found to possess a relatively specific proline aminopeptidase as well as a putative carboxypeptidase activity for glutamate. Energy production in this organism appears to be via oxidative deamination of these key amino acids linked to a respiratory chain, with nitrate acting as the ultimate electron acceptor.

Some observations on the nutritional requirements of Eikenella corrodens ATCC 23834T grown in continuous culture.

The type strain, ATCC 23834, of Eikenella corrodens was grown anaerobically in continuous culture in chemically defined media. Initial experiments showed that glucose was not utilized and it was subsequently omitted from all media. The initial chemically defined medium contained varying levels of 14 amino acids and 20 mM potassium nitrate, an essential nutrient for growth under these conditions. In this medium, the doubling time was 2.1 h, the optimum growth temperature 34 degrees C and the pH 7.2. The key growth-promoting amino acids were glutamate and serine, and the culture appeared to be nitrate limited. By growing the organism in a series of chemically defined media containing 40 mM nitrate and varying levels of these amino acids, glutamate was found to be the major contributor to biomass increase. Its catabolism may be linked to a respiratory chain involving nitrate as the ultimate electron acceptor.

The breakdown and utilization of peptides by strains of Fusobacterium nucleatum.

It has been claimed that most strains of Fusobacterium nucleatum require peptides rather than free amino acids for growth. In contrast, we have shown that, under continuous culture conditions, all strains tested grow in a chemically defined medium (CDM). The purposes of this study were to determine whether resting cells of F. nucleatum could attack unsubstituted peptides and whether growing cells could utilize a peptide fraction prepared from a commercial peptone. Resting cells cleaved all 19 peptides, containing 3-6 residues, and the 4 key energy-yielding amino acids--Glu, His, Ser and Lys--were rapidly taken up. A hydrophilic Casitone fraction, rich in Glu, promoted growth and peptides < 1 kDa were rapidly utilized. The cleaved residues metabolized were those previously shown to limit growth in CDM: Glu, Ser, His and Lys. The endopeptidase activities of Porphyromonas gingivalis would provide the necessary peptides for the growth of F. nucleatum, which may partly explain why these two organisms frequently coexist in periodontally diseased sites.

Aspects of the growth and metabolism of Fusobacterium nucleatum ATCC 10953 in continuous culture.

Fusobacterium nucleatum ATCC 10953, a type strain of one of the newly proposed subspecies of this group of organisms, was grown anaerobically in continuous culture in a chemically defined medium. Its response to conditions of varying pH, nutritional environment, and imposed growth rate were then examined. The organism failed to grow at pH 7.8 but grew at pH 5.8, although the cell yield was greatly reduced. At pH 6.8 the cell yield was halved and less than 50% of available glucose was consumed. The optimum growth pH was around 7.4 when the culture appeared to be limited for both glucose and the amino acids glutamate, histidine and serine. Some intracellular polyglucose (IP) was produced and acetate, butyrate and ammonia were the major fermentation end-products, as they were under all growth conditions tested. Increasing the available glucose or amino acids did not alter cell numbers but the amount of IP was greatly increased. When glucose was omitted from the medium, the cell yield was halved and the culture then became limited for lysine as well as for glutamate, histidine and serine. Growth rate had little overall effect on the organism's physiology and the maximum growth rate at pH 7.4 was 0.20 h-1, a doubling time of 3.5 h. Glucose was thus channelled into stable IP synthesis only when the growth limitation imposed by lack of fermentable amino acids was relieved. The metabolism of IP and the ability to obtain carbon and energy from a variety of substrates may explain why F. nucleatum is one of the most commonly detected organisms in subgingival dental plaque.

Identification of components in Fusobacterium nucleatum chemostat-culture supernatants that are potent inhibitors of human gingival fibroblast proliferation.

The present investigation concerned the effect of chemostat-culture cell-free supernatants of Fusobacterium nucleatum on the growth and synthetic activity of human gingival fibroblasts in vitro. Human gingival fibroblasts were cultured in fetal calf serum supplemented Dulbecco-Vogt medium containing various dilutions of conditioned or unconditioned bacterial culture medium. Cell proliferation was monitored by assessing cell growth over 5 days or incorporation of [3H]-thymidine into DNA. Protein and proteoglycan synthesis were monitored by the incorporation of [3H]-proline and [35S]-sulfate, respectively, into macromolecules. While the conditioned culture medium caused a complete inhibition of cell growth and incorporation of [3H]-thymidine DNA, there was no discernible effect on protein or proteoglycan synthesis. This indicated that the cells remained viable yet unable to divide. Such a view was supported by the observation that the inhibitory effect was reversible upon removal of the conditioned medium. This activity had a molecular size less than 30,000, was heat-stable and nonvolatile. Chemical analysis of the conditioned bacterial culture supernatants indicated that high proportions of butyrate, ammonium, and acetate were present. When these components were added to unconditioned medium and tested, most of the inhibitory activity could be attributed to ammonium and butyrate. Since many bacteria which constitute the subgingival microflora release ammonium and butyrate, a very high concentration of these metabolites may well accumulate. Clearly, the potential for inhibition of fibroblast proliferation has ramifications related to diminished tissue repair following bacterially-induced periodontal destruction.

Effects of pulsing with xylitol on mixed continuous cultures of oral streptococci.

Continuous culture is a means whereby organisms can be grown at rates approaching those occurring naturally. Moreover, the effect of adding transient excesses of various nutrients to the culture vessel ('pulsing') simulates the effect of dietary challenge on dental plaque organisms. Mixed cultures of Streptococcus mutans T8 and Streptococcus milleri B448 were grown glucose-limited in a chemically defined medium under an atmosphere of 5 per cent carbon dioxide in nitrogen, at a dilution rate of D = 0.1 h-1 and controlled pH of 7.0. The level of arginine in the medium reservoir was adjusted so that Strep. milleri predominated over Strep. mutans in a stable coexistence. After equilibration, the culture vessel was pulsed with various carbohydrates to a final concentration of 5 x 10(-2)mol/L. Samples were then taken at regular intervals and differential viable counts of Strep. mutans and Strep. milleri were done on mitissalivarius agar. Results demonstrated that pulsing with glucose, fructose, 'coupling sugar', lactose, xylose and sorbitol gave Strep. mutans a clear ecological advantage. In direct contrast, pulsing with xylitol resulted in a marked antimicrobial effect on Strep. mutans while Strep. milleri was essentially unaffected. This supports recent findings by other workers that uptake of this pentitol by Strep. mutans in batch culture sets up a 'futile cycle', leading to depressed growth or even cell death.

Factors affecting peptide catabolism by oral streptococci.

The binding of a number of unsubstituted peptides to Streptococcus sanguis and Streptococcus mutans and their subsequent degradation by such cells were examined. Peptides were added to cell suspensions prepared from glucose-limited growth in a chemostat and, at appropriate time intervals, cell-free filtrates were analyzed for peptides and their constituent amino acid residues by high-pressure liquid chromatography techniques. The results indicated that peptide hydrophobicity plays a limited role in peptide binding, but that charge and chain-length are probably important. In S. sanguis, carboxypeptidase activity rapidly released C-terminal arginine (Arg); this amino acid was less rapidly released from the N-terminus but a number of other residues were also released by aminopeptidase activity. When Arg is buried in the peptide, the rate of its release depends upon the number and type of residues between it and the N-terminus. In contrast, S. mutans possessed only weak peptidase activities. The nature of its peptidase activities indicates that S. sanguis can obtain the metabolically important Arg from a variety of peptides.

Some aspects of protease production by a strain of Streptococcus sanguis.

Our previous studies indicated that Arginine (Arg) plays a key nutritional role in Streptococcus sanguis P4A7 and that this organism can grow on whole casein as the sole nitrogen source. Its protease activities were therefore studied after glucose-limited continuous culture in a chemically-defined medium with either free amino acids or casein as the nitrogen source. Both culture supernatant and cell-associated endopeptidase (EP) and exopeptidase (amino-AP and carboxy-CP) activities were determined. Growth rate (mu) had little effect on EP, 75% of which was consistently in culture supernatants; AP and CP both decreased as mu was increased and both were predominantly cell-associated. At high growth pH, EP was substantially increased while AP and CP activities were optimal at pH 7. The most striking nutritional effect occurred under nitrogen limitation (glucose excess) when EP and AP were greatly increased and CP greatly decreased. It was concluded that S. sanguis is well equipped to scavenge its environment for Arg under a wide range of growth conditions.

Response of a Streptococcus sanguis strain to arginine-containing peptides.

For dental plaque organisms such as Streptococcus sanguis, the ecological importance of the ability to utilize arginine as an energy source has been established in previous studies. The present investigation was undertaken to determine the ability of a strain of S. sanguis to process unsubstituted arginine-containing peptides. The organism was grown under glucose-limited conditions in a chemically defined medium, and peptide was added to washed, resting cells in a pH-stat at pH 7.0. Filtrates taken at appropriate time intervals were assayed for peptide, free amino acids, and metabolites. Irrespective of the position of the arginine residue, all peptides tested were attacked, although those that possessed a C-terminal arginine (including a tetrapeptide) were processed at a faster rate than were those in which arginine was N terminal. However, C-terminal arginine was cleaved only slowly from a peptide containing 24 residues. In each case, most of the released arginine was converted to ornithine via the arginine deiminase pathway. Such peptidase activities appeared to occur at or near the cell surface and were probably constitutive. It was found that the organism grew in chemically defined medium containing arginine that was present solely in the form of a tripeptide, and also that a strain of S. mutans possessed only a limited ability to attack arginine-containing peptides and was unable to utilize the released arginine.

Influence of arginine on the coexistence ofStreptococcus mutans andS. milleri in glucose-limited mixed continuous culture.

Dental plaque is a complex community of bacteria coexisting in an environment frequently limited by carbon and energy sources. UnlikeStreptococcus mutans, other oral streptococci such asS. milleri andS. sanguis have an absolute requirement for and actually consume all available arginine when grown glucose limited in a chemically defined medium. The conditions, particularly in terms of arginine concentration, under which the dental plaque bacteriaS. mutans andS. milleri would coexist under glucose-limiting conditions were investigated. The minimum level of arginine supporting optimal growth ofS. milleri was found to be ca. 50µM, and above this level these strains outcompetedS. mutans. However, coexistence withS. mutans could be achieved at arginine levels of 14-40µM, depending upon theS. milleri andS. mutans strains used. Under such dual limitation,S. milleri was unable to respond to glucose pulses but did respond to pulses of arginine and arginine plus glucose. One of the twoS. milleri strains did not tolerate low pH. In contrast,S. mutans did not tolerate high pH whereasS. milleri was unaffected. This is relevant to dental plaque where arginine catabolism produces a pH rise. Additionally, arginine is an important nutrient since it can be used as an energy source by some oral streptococci.