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.

Intravitreal bevacizumab (Avastin) treatment of choroidal neovascularisation in patients with angioid streaks.

OBJECTIVE: To evaluate the safety and efficacy of intravitreal bevacizumab (Avastin) as treatment for choroidal neovascularisation (CNV) associated with angioid streaks METHODS: A non-randomised, interventional case series conducted on eyes with subfoveal CNV associated with angioid streaks. Intravitreal bevacizumab (1.25 mg in 0.05 ml) was injected into nine eyes of six patients between August 2005 and December 2007. Treatment efficacy was assessed based on pre- and post-treatment visual acuity and optical coherence tomography (OCT). RESULTS: With a mean follow-up of 19 months (range 10 to 28 months), the best corrected visual acuity improved by three or more lines in four eyes (44.4%), remained within two lines of baseline in four eyes (44.4%) and decreased by three or more lines in one eye (11.1%). Central foveal thickness (CFT) measured by OCT decreased an average of 67.7 microm (range +11 to -175 microm) with an average improvement in standardised change in macular thickening of 46.6% (range -12% to +84.5%). No injection-related complications or drug-related side effects were observed. CONCLUSIONS: Intravitreal bevacizumab for the treatment of subfoveal CNV secondary to angioid streaks mildly reduced central foveal thickness with a trend toward stabilisation of visual acuity. Additional follow-up and a larger patient cohort are needed to evaluate the long-term effects of this treatment.

Why be down in the mouth? Three decades of research in oral microbiology.

This paper describes some of the work done in the author's laboratory over the past 35 years. The research covers the following areas: the physiology of oral streptococci and their interactions; the physiology of some Gram-negative anaerobes and their interactions in relation to periodontal diseases; preventing the major dental diseases; and the future of oral microbiology.

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.

The effect of oxygen on the growth and physiology of Porphyromonas gingivalis.

Oxygen constitutes a constant challenge for the survival of strict anaerobes in the oral environment. The aim of this study was to investigate the effect of oxygen on the physiology and growth of Porphyromonas gingivalis in a continuous culture system when grown under conditions of hemin limitation and excess. Results showed that, when grown in the presence of hemin at 0.5 mg/l, P. gingivalis could tolerate low levels of oxygen, being able to reach steady-state when 6% oxygen was present in the incoming gas mixture. When the hemin concentration was increased to 5 mg/l, the culture tolerated 10% oxygen. Anaerobically-grown cells were coccoid in shape, whereas those grown in the presence of oxygen were bacillary. Acetate was the predominant end-product in cultures grown in the presence of oxygen or in cultures hemin-limited. Despite some changes in the activity of Arg- and Lys-gingipain, most of the proteolytic activity was retained in the presence of oxygen. Activity of each of the three anti-oxidant enzymes tested (NADH oxidase, NADH peroxidase and SOD) was detected under all conditions and usually increased under oxygenated environments. Higher activities were also seen in the hemin-limited cultures. These results show some of the changes that occur in the physiology of P. gingivalis as a result of oxidative stress and confirm that hemin has a protective effect on the growth of the microorganism in the presence of oxygen.

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.

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.

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.

Transpupillary thermotherapy of subfoveal occult choroidal neovascularization.

Choroidal neovascularization secondary to age-related macular degeneration is a leading cause of vision loss in adults. Although most patients present with occult CNV, treatment has focused on the small percentage of eyes with well-delineated, classic CNV. Transpupillary thermotherapy is a recent advancement in the management of occult CNV. Transpupillary thermotherapy acts in a subthreshold manner by slightly raising the choroidal temperature. A recent pilot study demonstrated that 56% of treated eyes remained stable one year after treatment with only 25% losing two lines of visual acuity. The TTT4CNV study will further evaluate the effectiveness of transpupillary thermotherapy in a randomized, double-blind trial.

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.

Pseudotumor cerebri in children receiving recombinant human growth hormone.

PURPOSE: This article represents the first report in the ophthalmology literature of an association between pseudotumor cerebri (PTC) and recombinant human growth hormone (rhGH). DESIGN: Noncomparative case series. PARTICIPANTS: Three children receiving rhGH for short stature with Turner syndrome, Jeune syndrome, or Down syndrome. METHODS: Children underwent full ocular examination. After papilledema was identified, patients underwent lumbar puncture and imaging with either magnetic resonance imaging or computerized tomography. Treatment was under the guidance of the primary physician or neurosurgeon. The rhGH was discontinued in all children. MAIN OUTCOME MEASURES: Visual acuity and evaluation of the optic nerve for resolution of papilledema were followed at each examination. RESULTS: In all three cases, papilledema resolved with the cessation of rhGH, and treatment with acetazolamide or prednisone. Visual acuity was unchanged in case 1, decreased by two to three lines in case 2, and was inconsistent in case 3. One child (case 2) required a ventriculoperitoneal shunt for persistent elevation of intracranial pressure. CONCLUSION: There appears to be a causal relationship between the initiation of rhGH with the development of PTC. Children should have a complete ophthalmic evaluation if they report headache or visual disturbances. Baseline examination with routine follow-up should be instituted when children cannot adequately communicate.

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.

Studies on fusobacteria associated with periodontal diseases.

The physiological and metabolic characteristics of representative isolates of the various subspecies of Fusobacterium nucleatum were investigated by growing them in continuous culture in chemically-defined, media. Behaving almost identically, these organisms were found to obtain energy from the fermentation of simple carbohydrates such as glucose or fructose or from the fermentation of certain amino acids, free or in the form of small peptides. The latter can be attacked by aminopeptidase activity which was shown to be essential for the growth of the organism in an environment lacking fermentable carbohydrate and free amino acids but replete with small peptides. This metabolic versatility may explain the presence of F. nucleatum in both supra- and sub-gingival dental plaque and why it is often found together with organisms such as Porphyromonas gingivalis which display powerful endopeptidase activities. Using the technique of allozyme electrophoresis, the current subspeciation of F. nucleatum was shown to be of doubtful validity and evidence, based upon physiological and metabolic properties, for differences in pathogenicity between isolates was not detected. While this organism is a member of various bacterial consortia associated with periodontal diseases, its contribution to the disease process remains unclear.

ADRF Trebitsch Scholarship. The microbial contamination of toothbrushes. A pilot study.

Ten individuals were each supplied with a new toothbrush of the same type and brand, together with identical tubes of fluoridated toothpaste. After a three-week period, during which subjects were asked to follow their usual oral hygiene practices, the toothbrushes were collected and assayed for microbial contamination using a range of selective growth media. The total microbial load per toothbrush was found to be 10(4) to 10(5) colony forming units. Staphylococci were found on all toothbrushes and streptococci on all but one. These two genera were also quantitatively dominant. Candida, corynebacteria, pseudomonads and coliforms were identified in 70, 60, 50 and 30 per cent of toothbrushes, respectively. However, mutans streptococci, lactobacilli and black-pigmented Gram-negative anaerobic rods were not detected on any of the toothbrushes. For each individual, information on variables such as toothbrush rinsing practices and post-brushing storage methods and environment was collected. No obvious relationship between such variables and microbial load was apparent but it is suggested that more extensive studies are needed, taking into account additional parameters such as age and degree of toothbrush wear and the use of pre-brushing mouthwashes.

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.

Investigations of the taxonomy and systematics of Fusobacterium nucleatum using allozyme electrophoresis.

Fusobacterium nucleatum forms part of the resident microbiota in both oral and extraoral sites in humans and animals. It is also involved in infections in such sites. Despite the genetic heterogeneity within the species, it has been divided into five subspecies, the validities of which have been questioned. In the present study, 44 F. nucleatum isolates were examined at 21 enzyme loci by using the allozyme electrophoretic technique to establish an accurate genetic framework for taxonomic purposes. Three distinct genetic clusters were identified; one cluster consisted exclusively of extraoral isolates, another cluster consisted predominantly of human oral isolates, and the third cluster consisted of a single human oral isolate. Our results highlight the urgent need for extensive biochemical, immunological, and epidemiological studies to accurately define the systematics of the genus fusobacterium based on the framework derived in this study by using 21 independent genetic characteristics.

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.