Antimicrobial and ultrastructural properties of root canal filling materials exposed to bacterial challenge.

INTRODUCTION: Chemo-mechanical preparation of the root canal leaves behind viable bacteria which can lead to treatment failure. Materials used inside the root canal should possess antimicrobial properties and also resist disintegration in the presence of biofilm. METHODS: Gutta-percha, three root canal sealers (Pulp Canal Sealer, AH Plus and BioRoot RCS) and materials used to make posts (a metal and a resin) were evaluated. Their antimicrobial activity against Enterococcus faecalis in direct contact was assessed by scanning electron microscopy and live-dead staining using confocal microscopy over a period of eight weeks. The materials' structural integrity was assessed by scanning electron microscopy. RESULTS: The antimicrobial activity of the materials varied. The metal alloy posts as well as BioRoot RCS sealer did not allow any biofilm accumulation; but gutta-percha, Pulp Canal Sealer and resin from fibre-reinforced posts encouraged thick biofilm accumulation. Microstructural changes were observed in AH Plus (washout) and BioRoot (crystal deposition) in contact with biofilm. The Pulp Canal and BioRoot RCS sealers exhibited a modified ion leaching pattern in contact with microbially loaded media. CONCLUSIONS: The microbial challenge affected the material microstructure in some of the materials tested and allowed biofilm accumulation. Although clinical success depends on a number of factors, materials that are structurally sound and exhibit antimicrobial properties are preferable for endodontic therapy and tooth restoration involving entry in the root canal.

Microbial motility involvement in biofilm structure formation--a 3D modelling study.

A computational model explaining formation of mushroom-like biofilm colonies is proposed in this study. The biofilm model combines for the first time cell growth with twitching motility in a three-dimensional individual-based approach. Model simulations describe the tendency of motile cells to form flat biofilms spreading out on the substratum, in contrast with the immotile variants that form only round colonies. These computational results are in good qualitative agreement with the experimental data obtained from Pseudomonas aeruginosa biofilms grown in flowcells. Simulations reveal that motile cells can possess a serious ecological advantage by becoming less affected by mass transfer limitations. Twitching motility alone appears to be insufficient to generate mushroom-like biofilm structures with caps on stalks. Rather, a substrate limitation-induced detachment of motile cells followed by reattachment could explain this intriguing effect leading to higher-level biofilm structure.

Cell division theory and individual-based modeling of microbial lag: part II. Modeling lag phenomena induced by temperature shifts.

This paper is the second in a series of two, and studies microbial lag in cell number and/or biomass measurements caused by temperature changes with an individual-based modeling approach. For this purpose, the theory of cell division, as discussed in the first part of this series of research papers, was implemented in the individual-based modeling framework BacSim. Simulations of this model are compared with experimental data of Escherichia coli, growing in an aerated, glucose-rich medium and subjected to sudden temperature shifts. The premise of a constant cell volume under changing temperature conditions predicts no lag in cell numbers after the shift, in contrast to the experimental observations. Based on literature research, two biological mechanisms that could be responsible for the observed lag phenomena are proposed. The first assumes that the average cell volume depends on temperature while the second assumes that a lag in biomass growth occurs after the temperature shift. For a lag in cell number caused by an increased average cell volume, the cell biomass always increases at the maximal rate. Therefore, cells are evidently not stressed and do not have to adapt to the new conditions, as opposed to a lag in biomass growth. Implementation and simulation of both mechanisms are found to describe the experimental observations equally well. Therefore, further research is needed to distinguish between the two mechanisms. This can be done by observing, in addition to cell numbers, a measure for the average cell volumes. In conclusion, the individual-based modeling approach is a good methodology to investigate and test biological theories and assumptions. Also, based on the simulations, suggestions for further experimental observations can be made.

Individual-based modelling of growth and migration of Salmonella enteritidis in hens' eggs.

An individual-based model (IbM) was developed to describe the growth and migration of Salmonella enteritidis in hens' eggs. The Bacteria Simulator (BacSim) environment was used to implement the model; the bacteria are represented by spheres that grow by nutrient uptake and divide in two daughter cells upon exceeding a certain threshold volume. Motility of the Salmonella bacteria was described by a run and tumble mechanism. For the sake of simplicity, the bacteria were assumed to grow exponentially, an appropriate assumption for the initial phase of growth relevant for shelf-life predictions. Both albumen and yolk were assumed to be homogeneous. The impact of several model parameters (chemotaxis, growth rate, initial contamination numbers and bacterial swimming speed) was assessed by a sensitivity analysis. The results show that chemotaxis towards the yolk would have a strong effect on the time needed to reach the vitelline membrane, an aspect that future research should focus on. The contamination position had less impact on the time to reach the vitelline membrane. The simulation results illustrate the need for more detailed knowledge on the subject of bacterial migration in hens' eggs. Our model can easily incorporate this knowledge when it becomes available.

Individual-based modelling of biofilms.

Understanding the emergence of the complex organization of biofilms from the interactions of its parts, individual cells and their environment, is the aim of the individual-based modelling (IbM) approach. This IbM is version 2 of BacSim, a model of Escherichia coli colony growth, which was developed into a two-dimensional multi-substrate, multi-species model of nitrifying biofilms. It was compared with the established biomass-based model (BbM) of Picioreanu and others. Both models assume that biofilm growth is due to the processes of diffusion, reaction and growth (including biomass growth, division and spreading). In the IbM, each bacterium was a spherical cell in continuous space and had variable growth parameters. Spreading of biomass occurred by shoving of cells to minimize overlap between cells. In the BbM, biomass was distributed in a discrete grid and each species had uniform growth parameters. Spreading of biomass occurred by cellular automata rules. In the IbM, the effect of random variation of growth parameters of individual bacteria was negligible in contrast to the E. coli colony model, because the heterogeneity of substrate concentrations in the biofilm was more important. The growth of a single cell into a clone, and therefore also the growth of the less abundant species, depended on the randomly chosen site of attachment, owing to the heterogeneity of substrate concentrations in the biofilm. The IbM agreed with the BbM regarding the overall growth of the biofilm, due to the same diffusion-reaction processes. However, the biofilm shape was different due to the different biomass spreading mechanisms. The IbM biofilm was more confluent and rounded due to the steady, deterministic and directionally unconstrained spreading of the bacteria. Since the biofilm shape is influenced by the spreading mechanism, it is partially independent of growth, which is driven by diffusion-reaction. Chance in initial attachment events modifies the biofilm shape and the growth of single cells because of the high heterogeneity of substrate concentrations in the biofilm, which again results from the interaction of diffusion-reaction with spreading. This stresses the primary importance of spreading and chance in addition to diffusion-reaction in the emergence of the complexity of the biofilm community.

Effect of EPS on biofilm structure and function as revealed by an individual-based model of biofilm growth.

We have simulated a nitrifying biofilm with one ammonia and one nitrite oxidising species in order to elucidate the effect of various extracellular polymeric substance (EPS) production scenarios on biofilm structure and function. The individual-based model (IbM) BacSim simulates diffusion of all substrates on a two-dimensional lattice. Each bacterium is individually simulated as a sphere of given size in a continuous, three-dimensional space. EPS production kinetics was described by a growth rate dependent and an independent term (Leudeking-Piret equation). The structure of the biofilm was dramatically influenced by EPS production or capsule formation. EPS production decreased growth of producers and stimulated growth of non-producers because of the energy cost involved. For the same reason, EPS accumulation can fall as its rate of production increases. The patchiness and roughness of the biofilm decreased and the porosity increased due to EPS production. EPS density was maximal in the middle of the vertical profile. Introduction of binding forces between like cells increased clustering.

O-demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(I)alamin.

The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates: electrochemically produced cob(I) alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isobestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (delta epsilon 540 = 4.40 nM-1.cm-1. The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of synringate-grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme-bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho-hydroxyl or para-carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol.min-1.(mg protein)-1 [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: Km = 1.2 mM, Vmax = 220 nmol min-1.(mg protein)-1, and Vmax/Km = 180 nmol.min-1.(mg protein) 1.mM-1 3,5-Dihydroxyanisole demethylation by extracts of 3,5-dihydroxyanisole-grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower.

Holophaga foetida gen. nov., sp. nov., a new, homoacetogenic bacterium degrading methoxylated aromatic compounds.

A polyphasic approach was used in which genotypic and phenotypic properties of a gram-negative, obligately anaerobic, rod-shaped bacterium isolated from a black anoxic freshwater mud sample were determined. Based on these results, the name Holophaga foetida gen. nov., sp. nov. is proposed. This microorganism produced dimethylsulfide and methanethiol during growth on trimethoxybenzoate or syringate. The only other compounds utilized were pyruvate and trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol. The aromatic compounds were degraded to acetate. Although comparison of the signature nucleotide pattern of the five established subclasses of Proteobacteria with the 16S rDNA sequence of Holophaga foetida revealed a relationship to members of the delta-subclass, the phylogenetic position within the radiation of this class is so deep and dependent upon the number and selection of reference sequences that its affiliation to the Proteobacteria must be considered tentative. The type strain is H. foetida strain TMBS4 (DSM 6591).