Acid tolerance properties of dental biofilms in vivo.

BACKGROUND: The ecological plaque hypothesis explains caries development as the result of the enrichment of acid tolerant bacteria in dental biofilms in response to prolonged periods of low pH. Acid production by an acid tolerant microflora causes demineralisation of tooth enamel and thus, individuals with a greater proportion of acid tolerant bacteria would be expected to be more prone to caries development. Biofilm acid tolerance could therefore be a possible biomarker for caries prediction. However, little is known about the stability of biofilm acid tolerance over time in vivo or the distribution throughout the oral cavity. Therefore the aim of this study was to assess intra-individual differences in biofilm acid-tolerance between different tooth surfaces and inter-individual variation as well as stability of acid tolerance over time. RESULTS: The majority of the adolescents showed low scores for biofilm acid tolerance. In 14 of 20 individuals no differences were seen between the three tooth sites examined. In the remaining six, acid-tolerance at the premolar site differed from one of the other sites. At 51 of 60 tooth sites, acid-tolerance at baseline was unchanged after 1 month. However, acid tolerance values changed over a 1-year period in 50% of the individuals. CONCLUSIONS: Biofilm acid tolerance showed short-term stability and low variation between different sites in the same individual suggesting that the acid tolerance could be a promising biological biomarker candidate for caries prediction. Further evaluation is however needed and prospective clinical trials are called for to evaluate the diagnostic accuracy.

Bacterial profiles and proteolytic activity in peri-implantitis versus healthy sites.

Peri-implantitis is a biofilm-induced destructive inflammatory process that, over time, results in loss of supporting bone around an osseointegrated dental implant. Biofilms at peri-implantitis sites have been reported to be dominated by Gram-negative anaerobic rods with a proteolytic metabolism such as, Fusobacterium, Porphyromonas, Prevotella and Tannerella, as well as anaerobic Gram-positive cocci. In this study, we hypothesized that protease activity is instrumental in driving bone destruction and we therefore compared the microbial composition and level of protease activity in samples of peri-implant biofluid (PIBF) from 25 healthy subjects (H group) and 25 subjects with peri-implantitis (PI group). Microbial composition was investigated using culture techniques and protease activity was determined using a FITC-labelled casein substrate. The microbial composition was highly variable in subjects both in the H and PI groups but one prominent difference was the prevalence of Porphyromonas/Prevotella and anaerobic Gram positive cocci which was significantly higher in the PI than in the H group. A subgroup of subjects with peri-implantitis displayed a high level of protease activity in the PIBF compared to healthy subjects. However, this activity could not be related to the presence of specific bacterial species. We propose that a high level of protease activity may be a predictive factor for disease progression in peri-implantitis. Further longitudinal studies are however required to determine whether assessment of protease activity could serve as a useful method to identify patients at risk for progressive tissue destruction.

Observed changes in the alertness and communicative involvement of students with multiple and severe disability following in-class mentor modelling for staff in segregated and general education classrooms.

BACKGROUND: The improvement of engagement and involvement in communicative and socially centred exchanges for individuals with multiple and severe disability (MSD) presents complex and urgent challenges to educators. This paper reports the findings of an intervention study designed to enhance the interactive skills of students with MSD using an in-class mentor model of staff development to improve the skills and strategies of their communication partners in two distinct educational settings. METHODS: Observational data were collected on eight students with MSD and their 16 teachers and teachers' aides (paraprofessionals), using a multiple baseline across students design, replicated across special and general school setting types. RESULTS: Results indicated variable improvements in student alertness and increased communicative interactions. In some cases significant differences in communicative involvement and awake-active-alert activity were observed. CONCLUSIONS: These findings underline the complexity of variables involved in delivering educational and communicative interventions for staff working with this population. Implications for further research and application to daily practices in classrooms are discussed.

Fluoride-supplemented milk inhibits acid tolerance in root caries biofilms.

In this study we investigated the effect of fluoride on plaque acid tolerance. The test group consumed 200 ml of milk supplemented with 5 mg F/l as NaF once a day, the milk control group drank 200 ml of unsupplemented milk, and the no-milk control group did not consume milk in this manner. Plaque samples were taken at baseline and after 15 months. The proportion of acid-tolerant bacteria in plaque was estimated using LIVE/DEAD® BacLight™ staining after exposure to pH 3.5 for 2 h. The fluoride group showed a statistically significant decrease in plaque acid tolerance compared to baseline. This study shows that daily intake of fluoride in milk reduces plaque acid tolerance.

Chitosan nanoparticles affect the acid tolerance response in adhered cells of Streptococcus mutans.

In this study we evaluated the effect of chitosan nanoparticles on the acid tolerance response (ATR) of adhered Streptococcus mutans. An ATR was induced by exposing S. mutans to pH 5.5 for 2 h and confirmed by exposing the acid-adapted cells to pH 3.5 for 30 min, with the majority of cells appearing viable according to the LIVE/DEAD® technique. However, when chitosan nanoparticles were present during the exposure to pH 5.5, no ATR occurred as most cells appeared dead after the pH 3.5 shock. We conclude that the chitosan nanoparticles tested had the ability to hinder ATR induction in adhered S. mutans.

A new generation of antibodies.

Antibodies, proteins produced in animals that bind with high specificity and affinity to a seemingly limitless variety of biomolecules, have an essential role in clinical medicine and basic biological research. Methods to produce antibody fragments on the surface of bacterial viruses were surveyed for their ability to replace animal-dependent antibodies. The use of filamentous phage to display antibody fragments derived from semisynthetic antibody genes was found to produce proteins that bind to antigens with a variety, specificity, and affinity similar to those produced in animal systems.

Siderophores: structure and function of microbial iron transport compounds.

Siderophores are common products of aerobic and facultative anaerobic bacteria and of fungi. Elucidation of the molecular genetics of siderophore synthesis, and the regulation of this process by iron, has been facilitated by the fact that E. coli uses its own siderophores as well as those derived from other species, including fungi. Overproduction of the siderophore and its transport system at low iron is in this species well established to be the result of negative transcriptional repression, but the detailed mechanism may be positive in other organisms. Siderophores are transported across the double membrane envelope of E. coli via a gating mechanism linking the inner and outer membranes.

Site-directed mutagenesis of the ferric uptake regulation gene of Escherichia coli.

The 12 histidine and four cysteine residues of the Fur repressor of Escherichia coli were changed, respectively, to leucine and serine by site-directed mutagenesis of the fur gene. The affects of these mutations were measured in vivo by ligation of the mutated genes to a wild-type fur promoter followed by measurement of the ability of these plasmids to regulate expression of a lacZ fusion in the aerobactin operon. In vitro affects were assayed by insertion of the mutated genes in the expression vector pMON2064 attended by isolation of the altered Fur proteins and appraisal of their capacity to bind to operator DNA. The results suggest that cysteine residues at positions 92 and 95 are important for the activity of the Fur protein.

An electron spin resonance study of the Mn(II) and Cu(II) complexes of the Fur repressor protein.

EPR spectra of Mn(II) Fur complex suggested the presence of Mn(II) in one site per Fur monomer in which Mn(II) is present in a low symmetry environment. The binding of the Mn(II) Fur complex to a DNA fragment "iron box" has a slight broadening effect on the Mn(II) signal and hence it altered the symmetry of the Mn(II) environment. We also report EPR spectra of Cu(II) Fur and Cu(II) C92S C95S mutant Fur complexes as models for Fe(II) complexes; the anisotropic g values and A values observed indicate the presence of Cu(II) in two different environments in the protein; a major axially distorted Cu(II) site bound to nitrogen and a minor distorted tetrahedral sulfur bound to the Cu(II) site. The effect of metal ion on Fur DNA binding is also discussed.

Siderophores.

Siderophores, defined as low-iron-induced virtually ferric-specific ligands, are widely distributed in microbial species where they function in the sequestration and transport of iron. In Escherichia coli biosynthesis of siderophores is negatively regulated at the transcriptional level by ferrous ion in conjunction with a repressor protein, Fur (ferric uptake regulation). Siderophores are a component of the virulence armamentarium of clinical isolates of E. coli and of certain other microorganisms pathogenic to man, animals, and plants. The mechanism whereby cells accumulate sufficient iron for activation of essential enzymes while, at the same time, avoiding excess of a potentially toxic element remains one of the most challenging problems in biochemistry.

Identification of 2,3-dihydroxybenzoic acid as a Brucella abortus siderophore.

Brucella abortus grown in low-iron medium or in the presence of iron chelators [ethylenediamine-di(o-hydroxyphenylacetic acid) and 2,2-dipyridyl] showed reduced cell yields and released a material positive in chemical and biological assays for catechols. This material was purified from culture fluids of B. abortus 2308 by chromatography on agarose-iminodiacetic acid-Fe3+ and identified as 2,3-dihydroxybenzoic acid (2,3-DHBA) by thin-layer chromatography, paper electrophoresis, and UV-visible nuclear magnetic resonance and mass spectroscopy. No other major catechols were observed at different stages of growth, and 2,3-DHBA was also produced upon iron limitation by representative strains of B. abortus biotypes 1, 5, 6, and 9. Both synthetic 2,3-DHBA and the natural catechol relieved the growth inhibition of B. abortus 2308 by ethylenediamine-di(o-hydroxyphenylacetic acid), and 2,3-DHBA promoted 55Fe uptake by B. abortus 2308 by an energy-dependent mechanism. Two other monocatechols tested, 2,3-dihydroxybenzoyl-Ser and 2,3-dihydroxybenzoyl-Gly, also promoted 55Fe uptake. More complex catechol siderophores (agrobactin and enterobactin), hydroxamate siderophores (aerobactin, ferrichrome, and deferriferrioxamine mesylate [Desferal]), and an EDTA-related siderophore (rhizobactin) failed to mediate 55Fe uptake. B. abortus cells grown in low-iron medium or in medium with iron had similar rates of iron uptake when supplied with 55Fe-2,3-DHBA, and the release of 2,3-DHBA under iron starvation was not associated with the expression of new outer membrane proteins. These results suggest an uptake system in which only the synthesis of the siderophore is regulated by the iron available for growth.

Negative transcriptional control of iron transport in Erwinia chrysanthemi involves an iron-responsive two-factor system.

Systemic virulence of the phytopathogen Erwinia chrysanthemi 3937 requires a functional iron assimilation system which, in this enterobacterium, is mediated by the siderophore chrysobactin and the outer membrane transport protein Fct. We investigated the regulation of this system by iron. No direct similarity with the Escherichia coli fur gene was found. Insertional mutagenesis allowed isolation of a regulatory mutant which expressed chrysobactin and two other high-affinity iron transport systems previously characterized in strain 3937, regardless of the iron level. RNA/DNA hybridization analysis established that regulation of chrysobactin by iron occurs at the transcriptional level. From a wild-type gene library, a recombinant cosmid able to restore normal regulation in the mutant strain was isolated. By generating a series of subclones and mini-Mulac insertions, we identified a regulatory locus (cbr) extending beyond c. 2.5kb which encodes two polypeptides, CbrA and CbrB, with molecular weights of 34,000 and 55,000 respectively. Functional analysis of the locus suggests that the cognate genes cbrA and cbrB are clustered within an operon. Their expression was studied through chromosomal lac gene fusions, in the presence of plasmid-borne wild-type constructions, under high- and low-iron conditions. In summary, the data show that in the presence of iron, cbr negatively regulates the chrysobactin biosynthetic and transport genes, while under conditions of depletion, cbr is subject to negative autogeneous regulation.

Ferric iron uptake in Erwinia chrysanthemi mediated by chrysobactin and related catechol-type compounds.

Erwinia chrysanthemi 3937 possesses a saturable, high-affinity transport system for the ferric complex of its native siderophore chrysobactin, [N-alpha-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine]. Uptake of 55Fe-labeled chrysobactin was completely inhibited by respiratory poison or low temperature and was significantly reduced in rich medium. The kinetics of chrysobactin-mediated iron transport were determined to have apparent Km and Vmax values of about 30 nM and of 90 pmol/mg.min, respectively. Isomers of chrysobactin and analogs with progressively shorter side chains mediated ferric iron transport as efficiently as the native siderophore, which indicates that the chrysobactin receptor primarily recognizes the catechol-iron center. Free ligand in excess only moderately reduced the accumulation of 55Fe. Chrysobactin may therefore be regarded as a true siderophore for E. chrysanthemi.

Mechanism and regulation of synthesis of aerobactin in Escherichia coli K12 (pColV-K30).

The aerobactin operon of the virulence plasmid pColV-K30 of Escherichia coli K12 consists of four genes for biosynthesis and one for transport of the siderophore. Regulation by iron occurs at the transcriptional level and is mediated by a ferrous iron binding protein designated Fur (ferric uptake regulation). The metallated Fur repressor binds at a palindromic dyad, the "iron box" operator, situated in the vicinity of the RNA polymerase attachment site of the promoter. Evidence suggests that the ferrous iron enters the C-terminal domain of Fur to cause a conformational change in the N-terminal part of the protein. This results in greatly enhanced affinity of the repressor for the operator.

Iron(III) complexes of chrysobactin, the siderophore of Erwinia chrysanthemi.

The phytopathogenic bacterium Erwinia chrysanthemi produces the monocatecholate siderophore chrysobactin under conditions of iron deprivation. Only the catecholate hydroxyl groups participate in metal coordination, and chrysobactin is therefore unable to provide full 1:1 coordination of Fe(III). The stoichiometry in aqueous solution is a variable dependent on pH and metal/ligand ratio, in addition to being concentration dependent. At neutral pH and concentrations of about 0.1 mM, ferric chrysobactin exists as a mixture of bis and tris complexes. Chrysobactin and its isomers form optically active tris complexes. The dominant configuration depends on the chirality of the amino acid to which the catecholate moiety is attached.

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.

Structural dynamics and functional domains of the fur protein.

Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12. Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin. Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding. Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.