Efficacy of low concentrations of sodium hypochlorite and low-powered Er,Cr:YSGG laser activated irrigation against an Enterococcus faecalis biofilm.

AIM: To establish the antibacterial efficacy of low concentrations of sodium hypochlorite with and without Er,Cr:YSGG laser activation on Enterococcus faecalis biofilms in extracted teeth. METHODOLOGY: The root canals of 96 decoronated single-rooted extracted human teeth were prepared to a size 40, 0.06 taper 1 mm beyond the apex. They were mounted within a flow cell, which was sterilized before pumping a nutrient media through the root canals. The flow cell was inoculated with E. faecalis (ATCC 700802) and cultivated for 4 weeks. The root-ends were sealed, and the roots were then subjected to one of six treatment groups: group 1: syringe irrigation (SI) with saline (control) using a 27 -gauge Monoject needle 1 mm from the apex for 2 min; group 2: as for group 1 but with 1% NaOCl; group 3: as for group 1 but with 4% NaOCl; group 4: 0.5% NaOCl irrigation for 15 s followed by laser-activated irrigation (LAI) with four 15-s cycles replenishing the irrigant between cycles; group 5: as for group 4 but with 1% NaOCl as the irrigant; group 6: as for group 4 but with 4% NaOCl as the irrigant. Following treatment, teeth were crushed and viable bacteria were quantitated by serial dilution and plating. The colony-forming unit values were compared between groups using one-way anova and Tukey-adjusted post hoc tests. A two-tailed P value of <0.05 was considered statistically significant. RESULTS: The mean number of cells recovered from the 1% NaOCl SI group was significantly higher than that from the 4% NaOCl LAI group (P = 0.02). CONCLUSION: Within the limitations of this laboratory study, low-powered (0.5 W) Er,Cr:YSGG laser activation did not improve the antibacterial effect of low concentrations of sodium hypochlorite.

Clonal diversity in biofilm formation by Enterococcus faecalis in response to environmental stress associated with endodontic irrigants and medicaments.

AIM: To determine whether clonal diversity within E. faecalis affects biofilm formation when exposed to antimicrobial compounds found in endodontic medicaments and irrigants. METHODOLOGY: Five human isolates of E. faecalis were compared; biofilms were grown in microtitre trays in the presence of sodium hypochlorite, calcium hydroxide, chlorhexidine, tetracycline or clindamycin. Biofilms were quantified by staining with crystal violet and optical density determined with a microplate reader. Slime production (an amorphous extracellular matrix comprising polysaccharides, glycoproteins and glycolipids loosely attached to the cell surface) was determined qualitatively by growth on Congo red agar plates. Linear mixed models were used to examine whether medicaments affected biofilm growth of the isolates in the presence of the medicaments or irrigants. RESULTS: Overall, different endodontic antimicrobials significantly altered biofilm growth in E. faecalis isolates. Two E. faecalis isolates significantly (P < 0.0001) increased biofilm formation in the presence of tetracycline and one in the presence of NaOCl (P = 0.018). Qualitatively, slime production also varied between isolates and correlated with biofilm production. CONCLUSIONS: When subjected to sub-minimum inhibitory concentration (MIC) levels of antimicrobial compounds found in endodontic medicaments, E. faecalis isolates demonstrated significant clonal variation in their capacity to form biofilms. Interestingly, there was a correlation between slime production and the ability of isolates to form a biofilm in the presence of antimicrobials. The results indicate that isolates of E. faecalis that form biofilms in response to endodontic medicaments may be more likely to survive endodontic treatment.

Proteomic identification of proteinase inhibitors in the porcine enamel matrix derivative, EMD(®).

BACKGROUND AND OBJECTIVE: The porcine enamel matrix derivative, EMD(®), which is the active component of Emdogain(®), is used widely in periodontics because of its ability to promote the regeneration of soft and hard tissues and to reduce inflammation. Previous studies have used indirect methods to explain its angiogenic and proliferative effects on cells associated with wound healing. In this study we used proteomic techniques to identify proteins in EMD other than amelogenins. MATERIAL AND METHODS: Proteins in EMD were separated by two-dimensional gel electrophoresis and were identified using mass spectrometry. Proteomic results were validated by western blot analysis of Emdogain. RESULTS: Fourteen proteins of porcine origin were identified and included the serine and cysteine proteinase inhibitors alpha1-antichymotrypsin and fetuin A, respectively. Alpha1-antichymotrypsin is an acute-phase factor that has been reported to indirectly down-regulate the expression of the gelatinase MMP-9. Fetuin A, a major glycoprotein component of bone and teeth, is a potent inhibitor of ectopic calcification of vascular and soft tissues and has been implicated in both osteogenesis and bone resorption. It also facilitates plasma membrane repair in damaged fibroblasts. CONCLUSION: EMD contains a number of high-molecular-weight compounds which include the proteinase inhibitors, fetuin A and alpha1-antichymotrypsin.

The use of live-animal micro-computed tomography to determine the effect of a novel phospholipase A2 inhibitor on alveolar bone loss in an in vivo mouse model of periodontitis.

BACKGROUND AND OBJECTIVE: Live-animal micro-computed tomography is a new and promising technique that can be used to quantify changes in bone volume for periodontal disease models. The major aim of this study was to develop the methodology of live-animal micro-computed tomography and to determine the effect of a novel secretory phospholipase A2 inhibitor on alveolar bone loss. MATERIAL AND METHODS: Periodontitis was induced in mice by oral infection with Porphyromonas gingivalis over a period of 13 wk, and live-animal micro-computed tomography scans were taken at different time-points to determine bone volume changes with disease progression. This enabled conclusions to be made as to when treatment was most likely to be effective. In addition, the model was used to investigate a novel drug, the secretory phospholipase A2 inhibitor, KHO64, and its potential ability to inhibit osteoclast bone resorption and treat periodontitis. RESULTS: The results from live-animal micro-computed tomography scans revealed greater, statistically significant, bone volume loss in diseased mice compared with normal mice (p < 0.05). This corresponded to a larger area from the cemento-enamel junction to the alveolar bone crest, as assessed by stereo imaging (p < 0.001). These techniques can therefore detect and quantify alveolar bone loss. Both methods revealed that KHO64 had no significant effect on the volume of bone resorption. CONCLUSION: Live-animal micro-computed tomography is a robust, reproducible technique that clearly demonstrates significant time-dependent changes in alveolar bone volume in a small-animal model of periodontitis.

Effect of dietary omega-3 polyunsaturated fatty acids on experimental periodontitis in the mouse.

BACKGROUND AND OBJECTIVE: Periodontitis is an infective disease caused predominantly by gram-negative anerobes. The host inflammatory response to these bacteria causes alveolar bone loss, which characterizes periodontitis. Omega-3 polyunsaturated fatty acids have recognized anti-inflammatory effects; their oxygenated derivatives are key mediators in reducing inflammation. In this study we tested the hypothesis that dietary supplementation with tuna fish oil rich in the n-3 polyunsaturated fatty acid, docosahexaenoic acid, would reduce alveolar bone loss in mice inoculated with periodontopathic bacteria. MATERIAL AND METHODS: Adult mice were fed experimental diets containing either 10% tuna oil or Sunola oil for 57 d. After 14 d, 35 mice on each diet were inoculated orally with Porphyromonas gingivalis, with a mixture of P. gingivalis and Fusobacterium nucleatum, with carboxymethylcellulose or remained untreated. The mice were killed, and soft tissue biopsies from the oral cavity of treated mice were used to determine the polyunsaturated fatty acid concentrations. The maxilla was removed, stained and digitally imaged to assess bone loss around the upper molars. RESULTS: n-3 polyunsaturated fatty acid levels were significantly higher in oral soft tissues of mice fed tuna oil compared with the control group. Mice fed tuna oil and inoculated with P. gingivalis or with the combination of F. nucleatum and P. gingivalis exhibited 72% and 54% less alveolar bone loss respectively, compared with the treatment control group. CONCLUSION: Alveolar bone loss was inversely related to n-3 polyunsaturated fatty acid tissue levels. In conclusion, fish oil dietary supplementation may have potential benefits as a host modulatory agent in the prevention and/or adjunctive management of periodontitis.

The inability of Streptococcus mutans and Lactobacillus acidophilus to form a biofilm in vitro on dentine pretreated with ozone.

BACKGROUND: The use of ozone therapy in the treatment of dental caries is equivocal. The aim of this study was to use an in vitro model to determine the effects of prior ozone application to dentine on biofilm formation and to measure any associated reduction in bacteria viability. METHODS: Twenty dentine discs were bonded to the bases of 5 mL polycarbonate screw top vials. Ten dentine discs were infused with ozone for 40 seconds, 10 samples remained untreated as a control. The vials were filled with nutrient medium, sterilized and placed into the outflow from a continuous chemostat culture of Streptococcus mutans and Lactobacillus acidophilus for four weeks. At the conclusion of the experiment bacterial growth was monitored by taking optical density readings of the growth medium in each vial and the outer surface of the dentine specimens were examined by scanning electron microscopy as shown by SEM analysis. RESULTS: Ozone infusion prevented biofilm formation on all the treated samples while there was substantial biofilm present on the control specimens. While the average optical density of the control specimens was almost twice that of the ozone infused dentine (0.710 for the control with a SD of 0.288 and 0.446 for the ozonated samples with a SD of 0.371), the results were not significant (p > 0.05). CONCLUSIONS: This preliminary study has shown that the infusion of ozone into non-carious dentine prevented biofilm formation in vitro from S. mutans and L. acidophilus over a four-week period. The possibility exists that ozone treatment may alter the surface wettability of dentine through reaction with organic constituents.

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.

Microbiological evaluation of endodontic files after cleaning and steam sterilization procedures.

BACKGROUND: Infection control procedures are essential for modern dental practice and they are continually evolving to meet the dental profession's high standards. The present study evaluated the efficacy of two cleaning procedures to reduce bacterial numbers on endodontic files, and evaluated the effect of biological debris on the subsequent sterilization of files. METHODS: Stainless steel and nickel-titanium (NiTi) files were examined upon removal from the manufacturer's packaging, after instrumentation in root canals of human teeth inoculated with a broth containing two anaerobic species and one facultative anaerobic species of bacteria, and after instrumentation and cleaning with either an ultrasonic bath or a thermal disinfector. For each file, the bacterial numbers were quantified using routine microbiological techniques in an anaerobic chamber. RESULTS: No bacteria were detected from files direct from their packets. The size, taper and type of file did not affect the ability of either of the cleaning procedures to reduce bacterial numbers. However, an absence of bacteria was more likely when files were cleaned in the thermal disinfector. No bacteria were detected from files that were-subjected to steam sterilization irrespective of the type of prior cleaning procedure. CONCLUSIONS: Steam sterilization eliminated all bacteria from the endodontic files irrespective of the presence of biological debris. The majority of bacteria were eliminated from endodontic files after either ultrasonic cleaning or using a thermal disinfector.

A SEM evaluation of debris removal from endodontic files after cleaning and steam sterilization procedures.

BACKGROUND: In recent times, it has been proposed to classify endodontic files as single-use items due to a perceived inability to adequately clean the instruments. The purpose of the present study was to quantify the surface debris on files removed from the manufacturer's packaging, and after cleaning using an ultrasonic bath or a thermal disinfector. METHODS: Stainless steel and rotary nickel-titanium files were examined after removal from the manufacturer's packaging, after instrumentation in broth-contaminated human teeth, and after various cleaning procedures. The cleaning procedures consisted of either a thermal disinfector cycle, ultrasonication with the files placed in a perforated container or ultrasonication with the files loosely placed in a beaker. The presence of manufacturing debris and biological debris was evaluated using scanning electron microscopy and quantified using image analysis software. RESULTS: The effectiveness of cleaning was not affected by variation in the size or taper of the files when an effective cleaning procedure was used. Cleaning the files in a thermal disinfector or by ultrasonication within a container did not consistently achieve complete removal of biological debris. Placing the files loosely in the ultrasonic bath achieved the most effective cleaning, an average of 98.33 per cent of the file surface area was freed of any biological debris. CONCLUSIONS: A conventional cleaning method is capable of effectively removing biological debris from endodontic files. The efficacy of ultrasonic cleaning was impaired when the files were placed within a perforated container.

Studies on NADH oxidase and alkyl hydroperoxide reductase produced by Porphyromonas gingivalis.

Enzymes that detoxify oxygen or oxygen radicals are important to anaerobic microorganisms that inhabit oxygenated environments. In previous studies we have determined that Porphyromonas gingivalis W50 cell extracts possess NADH oxidase-like activity, which increases slightly under oxygenated conditions. The aim of this study was to characterize the protein responsible for this activity in order to establish whether it protects the microorganism from oxidative stress. Protein purification based on NADH oxidase activity did not isolate a conventional NADH oxidase. Instead, the NADH oxidase activity was found to be associated with a FAD-dependent enzyme identified as 4-hydroxybutyryl-CoA dehydratase (AbfD). The biological significance of this activity with respect to protection against oxidative stress is not clear; hydrogen peroxide (H2O2) was present after completion of the NADH oxidase assay with the purified protein. Northern blot analysis, examining the expression of other proteins likely to function as NADH oxidases/peroxidases in P. gingivalis, revealed the transcription of a protein similar to alkyl-hydroperoxide reductase (AhpF-C), which could serve as an NADH oxidase and H2O2-detoxification system. AhpF is transcribed in a polycystronic way with its neighboring gene, which encodes for the coupling protein AhpC. No transcript could be detected for the closest match to an NADH oxidase identified in the P. gingivalis genome sequence. In conclusion, P. gingivalis seems to lack a protective NADH oxidase but AhpF-C could contribute to its moderate tolerance to reactive oxygen species by metabolizing H2O2.

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.

Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments.

The authors compared the differences in tolerance to oxygen of the anaerobic periodontopathic bacteria Fusobacterium nucleatum and Porphyromonas gingivalis, and explored the possibility that F. nucleatum might be able to support the growth of P. gingivalis in aerated and CO2-depleted environments. Both micro-organisms were grown as monocultures and in co-culture in the presence and absence of CO2 and under different aerated conditions using a continuous culture system. At steady state, viable counts were performed and the activities of the enzymes superoxide dismutase and NADH oxidase/peroxidase were assayed in P. gingivalis. In co-culture, F. nucleatum was able to support the growth of P. gingivalis in aerated and CO2-depleted environments in which P. gingivalis, as a monoculture, was not able to survive. F. nucleatum not only appeared to have a much higher tolerance to oxygen than P. gingivalis, but a significant increase in its numbers occurred under moderately oxygenated conditions. F. nucleatum might have an additional indirect role in dental plaque maturation, contributing to the reducing conditions necessary for the survival of P. gingivalis and possibly other anaerobes less tolerant to oxygen. Additionally, F. nucleatum is able to generate a capnophilic environment essential for the growth of P. gingivalis.

Sodium ion-driven serine/threonine transport in Porphyromonas gingivalis.

Porphyromonas gingivalis is an asaccharolytic, gram-negative bacterium that relies on the fermentation of amino acids for metabolic energy. When grown in continuous culture in complex medium containing 4 mM (each) free serine, threonine, and arginine, P. gingivalis assimilated mainly glutamate/glutamine, serine, threonine, aspartate/asparagine, and leucine in free and/or peptide form. Serine and threonine were assimilated in approximately equal amounts in free and peptide form. We characterized serine transport in this bacterium by measuring uptake of the radiolabeled amino acid in washed cells of P. gingivalis energized with a tetrapeptide not containing serine. Serine was transported by a single system with an affinity constant for transport (K(t)) of 24 microM that was competitively inhibited by threonine. Serine transport was dependent on sodium ion concentration in the suspending buffer, and the addition of the ionophore gramicidin caused the inhibition of serine uptake. Together these data indicate that serine transport was sodium ion-motive force driven. A P. gingivalis gene potentially encoding a serine transporter was identified by sequence similarity to an Escherichia coli serine transporter (SstT). This P. gingivalis gene, designated sstT, was inactivated by insertion of a Bacteroides tetQ gene, producing the mutant W50ST. The mutant was unable to transport serine, confirming the presence of a single serine transporter in this bacterium under these growth conditions. The transport of serine by P. gingivalis was dependent on the presence of free cysteine in the suspension buffer. Other reducing agents were unable to stimulate serine uptake. These data show that P. gingivalis assimilates free serine and threonine from culture media via a cysteine-activated, sodium ion-motive force-driven serine/threonine transporter.

The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture.

Fusobacterium nucleatum ATCC 10953 was grown in continuous culture and the atmosphere changed stepwise from nitrogen (anaerobiosis) to a mixture of air: oxygen (40:60). No significant differences in biomass were observed and the baseline low level of superoxide dismutase increased only slightly; catalase and peroxidase activities were never detected but the level of NADH oxidase increased more than three-fold when oxygen was introduced into the system. In relation to acidic end-products, the relative proportion of acetate increased while that of butyrate decreased. Due mainly, it would seem, to NADH oxidase activity, the culture maintained a low redox potential (E(h)=-274 mV) even under an atmosphere of 40% oxygen in air and dissolved oxygen was not detected. This may, in part, explain the protective role of F. nucleatum for anaerobes in both biofilm and planktonic phases of aerated, mixed cultures of oral bacteria.

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.

The influence of intracellular polyglucose and prior growth rate on the survival of Fusobacterium nucleatum under starvation conditions.

Grown in a chemically defined medium containing glucose at a dilution rate of D = 0.065 h-1, Fusobacterium nucleatum D212B-2 produced large amounts of intracellular polyglucose. Aliquots of this culture were starved by anaerobic incubation at 37 degrees C and at various times, assayed for intracellular polyglucose content and viability. This protocol was repeated using cells grown under the same conditions in a chemically defined medium, a medium lacking carbohydrate and in which the organism produced no intracellular polyglucose. Both cultures had 50% survival time values of about 1.5 h and were not eliminated even after 32 h of starvation. It was, therefore concluded that starvation-survival is not influenced by intracellular polyglucose. Starvation-survival was also determined for cells grown in a chemically defined medium at D = 0.048 h-1 and D = 0.12 h-1. The faster-grown cells had a 50% survival time of 3.8 h but were completely eliminated after 8-16 h of starvation. In contrast, slower-grown cells had a 50% survival time of 1.5 h but were not completely eliminated after 32 h of starvation. This illustrates the importance of cell history and technique standardization in comparing the starvation-survival of different organisms.

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.