Enterococcus faecalis is known to produce biofilms on biomaterials, but the manner in which this occurs is unknown. Herein we report that adhesion of E. faecalis in biofilms appeared to be mediated by cell wall surface projections attaching cells to the substratum. Biofilm formation was observed on the polystyrene surface of 96-well plates and also on the surface of cellulose kidney dialysis tubing used as a model for biofilm formation on catheters. Qualitative differences involved the packing of E. faecalis cells in biofilms, with greater intercellular spacing detected in the 96-well plate, whereas bacteria were tightly packed on the surface of cellulose catheters. Distribution of adherent bacterial cells accumulating on the two surfaces revealed obvious differences, with most of the bacteria attaching to the polystyrene surface as single cells or diplococci separated from neighboring organisms by intervals of uncolonized surface. In contrast, enterococci on the cellulose surface were found as multi-layer cellular aggregates or microcolonies, even when much of the total surface was free from attached bacteria. Microcolonies stained intensely for neutral hexose sugars using the periodic acid–Schiff (PAS) stain. Surface projections, presumably exopolysaccharide, anchored bacteria to the substratum and appeared to elevate the cells above the surface. These slender surface projections could be seen over the entire enterococcal cell wall, with the exception of areas adjacent to septal regions where new cell wall formation was occurring. Rod-like interconnections were also observed between adjacent diplococci. These results suggested that biofilm formation varies on different substrates and that enterococcal surface projections may be involved in E. faecalis colonization and adhesion within biofilms.

Bacteria in nature grow mostly as biofilms, surface-associated cells enveloped by hydrated extracellular polymeric substances (EPS) of bacterial origin. The composite of EPS and biofilm cells is measured when quantifying bacterial biomass from natural samples. However, little is known regarding the relative magnitude of the EPS and cellular fractions of biofilm biomass, particularly in unsaturated systems such as soil and food surfaces. In this study, we examined the cellular and extracellular fractions of Pseudomonas aeruginosa biofilms for DNA, protein and carbohydrate content. Biofilms were cultured in a model laboratory system that simulates the nutrient gradients and poorly mixed nature of unsaturated systems. We found that unsaturated biofilms exhibited two growth phases – an initial rapid phase and a second phase of slower or negligible development. However, no lag phase was observed for either carbon source. Cellular DNA accumulated linearly with biomass whereas cellular protein and carbohydrates accumulated exponentially with biomass. High levels of carbohydrate, protein and DNA were observed in the EPS of all samples, representing as much as 50% of these macromolecules in the biofilm. Most EPS accumulated during the second phase of growth, when cellular DNA increased only slightly. However, for biofilms cultured on a poorly bioavailable carbon source, EPS DNA decreased during the second phase, suggesting that EPS may affect bacterial survival under nutrient-limited conditions. Whether a product of overflow metabolism or cell lysis, EPS is a significant component of unsaturated biofilm biomass that probably impacts on bacterial ecology.

We present an overview of all the features of the unified multi-component cellular automaton (UMCCA) model. The UMCCA model describes quantitatively the complexity of biofilms for all biofilm components: active bacteria, inert biomass and extracellular polymeric substances (EPS). It also includes original donor substrate, two types of soluble microbial products (SMP) and oxygen. All mass balances and parameters used in UMCCA are based on our unified theory, which reconciles the apparently disparate findings about active and inert biomass, EPS and SMP. The UMCCA model captures all trends observed experimentally regarding biofilm density, namely large density differences within individual biofilm samples, with bottom layers (closest to the substratum) being as much as 5 to 10 times denser than top layers, which appear to be “fluffy” and porous. To capture this effect, the UMCCA model employs the novel idea of biofilm consolidation: the fluid over the biofilm creates pressures and vibrations that cause the biofilm to consolidate, or pack itself to a higher density over time. As a result, each biofilm compartment in the model output consolidates to a different degree that depends on the age of its biomass, which is higher for the bottom layers of the biofilm. The UMCCA model can also be used to describe biofilm mechanical properties variable in time and space, and it can be linked to finite-element software to study the behavior of biofilms under tensile or compressive stress and to perform a stress analysis of the deformed biofilm, possibly indicating where it is likely to fail or detach.

Here we describe an experimental study of the mechanical properties of bacterial biofilms formed from the early dental plaque colonizer Streptococcus mutans. The S. mutans biofilms demonstrated the behavior of rheological fluids, with properties similar to those of organic polymers and other biological fluids. The time-dependent response of the biofilms was modeled on the basis of principles of viscoelasticity theory. The static and dynamic responses were defined in terms of the creep compliance, storage and loss moduli, and viscosity. The creep compliance and stress relaxation functions of S. mutans biofilms were characterized using the Burger model. Implications for developing more effective mechanical removal strategies of dental plaque biofilms are discussed.

Principles of antimicrobial biomaterials: why they do not (usually) work

Characterization of bacterial adhesion onto surfaces of stainless steel by micromanipulation

Yersinia spp./C. elegans infection model: biofilms on a biotic surface

Periodontal pathogens in dental unit waterlines

Biofilms as reservoirs of antibiotic resistance

Systemic antimicrobials and periodontitis – a classic biofilm disease

Prion disease in the dental setting

Reviewing the role of biofilms in infection control

Infection control in dental technology laboratories

Review of the treatment aspects of dental unit water systems

Myxococcus xanthus is a motile soil bacterium with complex social behaviors. Upon starvation, a developmental program is initiated that results in cellular aggregation and fruiting body formation. This process requires the exopolysaccharide (EPS) component of the extracellular matrix. With prolonged starvation, a part of the cells within a fruiting body die, while the other cells differentiate into spores. Extensive genetic and biochemical information has been generated that elucidates this interesting developmental process. Little is known, however, about the detailed three-dimensional structural features of native fruiting bodies or the EPS and distribution of live/dead cells (spores) within these structures. In this study, changes in the three-dimensional architecture of fruiting bodies and the distribution of the extracellular matrix within the fruiting bodies during the developmental process were investigated using a gfp-expressing M. xanthus strain and carbohydrate-specific lectins or monoclonal antibodies in combination with confocal laser scanning microscopy. The extracellular matrix was found to form a scaffold within the fruiting body structure. Furthermore, using a bacterial viability staining assay, the distribution of live/dead cells within fruiting bodies was examined at different times. The majority of live cells were found to localize at the outer layer of a mature fruiting body, with dead cells underneath.

We present experimental results of colony formation in bacteria as an example of pattern formation resulting from reproduction and movement in biological populations. The bacterium Bacillus subtilis is known to exhibit at least five distinct types of colony pattern, depending on the substrate softness and nutrient concentration: diffusion-limited aggregation (DLA), compact Eden-like, dense branching morphology (DBM), concentric ring-like, and disk. We established a morphological diagram of the colony patterns, and then examined and characterized both macroscopically and microscopically how the the colonies grow. There seem to be two kinds of bacterial cells – active and inactive – and the active form drives the colony interfaces outwards. The active cells may be clearly distinguished from the inactive ones as they form the characteristic fingernail-like structure at the tips of growing branches of the DBM colony. The concentric ring-like colony is formed as a consequence of repeated alternate migration and resting of the growing interface, the cycle time for which seems to be independent of the substrate softness and nutrient concentration. So far there have been several phenomenological models proposed to qualitatively explain or reproduce the patterns observed in bacterial colonies. A few of them are reviewed here, systematically and critically, in light of our experimental results.

Extracellular polysaccharide (EPS) from Klebsiella oxytoca was chemically modified to increase net negative charge in order to expand the efficiency of metal binding. Chlorosulfonic acid (CSA) or a complex of dimethylformamide and sulfur trioxide (DMF–SO3) was used to introduce sulfate substituents onto the native polymer. Additionally, sequential treatment with divinylsulfone (DVS) and glycine or DVS and iminodiacetic acid (IDA) was used to introduce carboxyl residues. Native EPS exhibited a molecular mass distribution of 1500–1700 kDa and removed 38% and 19%, respectively, of lead and cadmium ions. Modification with CSA resulted in a reduction of molecular mass to 450–600 kDa and a removal from solution of lead and cadmium of 75% and 66%, respectively. Modification of EPS with the DMF–SO3 complex, while it did not affect the molecular mass of the polymer, resulted in decreased levels of lead and cadmium removal. Covalent substitution of EPS with DVS–glycine resulted in an increase of molecular mass to 3000 kDa and lead and cadmium removals of 46% and 47%, respectively. Modification of EPS with DVS–IDA increased molecular mass to 2200 kDa and demonstrated lead and cadmium removals of 57% each. Immobilization of native EPS and of the CSA and DVS–IDA modifications to oxirane–acrylic beads resulted in significant increases in metal binding per gram of bound polymer. This suggests a method for metal ion recovery using chelate desorption.

This list includes conferences that had been announced at the time of going to press. We are happy to include details of other biofilm-related conferences – please send details of these to the editor-in-chief.

Two ultrasonic devices – flat (T1) and curved (T2) ultrasonic transducers – were developed to remove biofilms from opened and closed surfaces, respectively. The aim is to standardize biofilm removal for in situ sanitary control in the food industry. The biofilms studied in this work were model biofilms made with milk on stainless steel sheets. We have shown in a previous study that sonication could be employed to remove and resuspend biofilm consistently, with a good recovery rate, from opened surfaces. Plate counting was used to assess the efficiency of each treatment. A total removal of Escherichia coli and Staphylococcus aureus from model biofilms was obtained with T1: 10 s at 40 kHz. However, ultrasound applied with T2 (a patented curved transducer developed for closed surfaces: 10 s at 40 kHz) failed to completely remove these model biofilms: 30±7% and 66±10% for E. coli and S. aureus biofilms, respectively. In order to improve the biofilm removal from closed surfaces with T2, the effect of the application of ultrasound in combination with chelating agent preparations was investigated. The application of ultrasound with T2 in 0.05 mol EDTA or EGTA per litre dislodged the E. coli milk model biofilm, with 100±10% and 100±5% recovery yields, respectively. These results showed a synergism between ultrasonic waves and chelator preparations, i.e. the combination achieved three times the recovery rate of sonication alone (30%). However, when the same treatment was applied to the S. aureus milk model biofilm, the combined treatment with EDTA or EGTA did not significantly improve the recovery of the biofilm cells: 74±26% with EDTA at 0.025 mol/l and 41–47% with EGTA at 0.025 mol/l and 0.05 mol/l, respectively, compared with 66±10% for sonication alone. The combined treatment was in agreement with an industrial control, i.e. a good reproducible recovery of the biofilm in a few seconds (10 s) for E. coli milk biofilms but not for S. aureus biofilms.

A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a chemical signalling molecule, or microbial hormone, that triggers aerial mycelium formation and secondary metabolism in Streptomyces griseus. A-factor pro- duced in a growth-dependent manner switches on the transcription of adpA, encoding a transcriptional activator, by binding to ArpA, the A-factor receptor protein, which has bound to the adpA promoter, and dissociating the bound ArpA from the DNA. AdpA then activates a number of genes of various functions required for morphological development and secondary metabolism, forming an AdpA regulon. ArpA, which belongs to the TetR family, contains a helix–turn–helix DNA-binding motif in its N-terminal portion and an A-factor-binding pocket (5 Å (0.5 nm) diameter and 20 Å (2 nm) long) in its C-terminal portion, as implied by X-ray crystallography of CprB, an ArpA homologue. The ligand pocket, which can accommodate an entire A-factor-type molecule of γ-butyrolactone, is completely embedded in the C-terminal portion. Upon binding A-factor, a long helix connecting the A-factor-binding and ligand-binding domains is relocated, as a result of which the DNA-binding helix moves outside, resulting in dissociation from DNA. AdpA, which belongs to the AraC/XylS family, contains a ThiJ/PfpI/DJ-1-like dimerization domain in its N-terminal portion and an AraC/XylS-type DNA-binding domain in its C-terminal portion. For transcriptional activation, AdpA can bind to various positions with respect to the transcriptional start points of the target genes and sometimes to multiple sites. We show here how A-factor triggers secondary metabolism and morphological development in S. griseus, with emphasis on the two key transcriptional factors, ArpA and AdpA, in the A-factor regulatory cascade.

The human oral microbial ecosystem is one of the best characterized and highly complex bacterial communities known. It is estimated that about 600 bacterial species exist in the mouth and that 85% of those are currently known at the molecular (16 S rRNA) level. The major bacterial physiologies occurring in the oral cavity have been known for many years, and knowledge exists of the distribution of organisms in time (as plaque accumulates) and in space (different environments within the mouth). However, only rudimentary data are available on interactions between the bacterial species. It is precisely these interactions that, along with the interactions of the developing community with its human host, must drive the succession of genera that is observed to occur. In the reductionist scientific approach to studying such interactions, disrupted plaque is used to isolate single organisms, and interactions between these organisms are examined by recombining the organisms ex situ. Lessons learned from these in vitro studies can be applied to understand empirical observations made in vivo. However, this approach begins with the primary assumption that the chosen interaction does in fact occur in vivo. The accessibility and the well-characterized nature of the oral ecosystem presents an opportunity for approaching the problem from the opposite direction; one can capture a community very early in development in vivo, then apply in vitro methods to sort out the interactions within that community. This latter approach begins with a set of organisms known to interact in vivo. A combination of both approaches should yield robust microbiological data suitable for in silico modeling and analyses.

This list includes conferences that had been announced at the time of going to press. We are happy to include details of other biofilm-related conferences– please send details of these to the editor-in-chief.

Mathematical modeling of spatial biofilm structure has been in development for the past 10 years, its main goal being to derive the dynamics of biofilm structure from first-principle descriptions of the various physical, chemical and biological processes involved in biofilm formation. Early efforts described development of unrestricted monospecies consortia, often considering diffusion and reaction of a single solute species. Multi-dimensional modeling of biofilms has presently reached a stage where multi-species systems with any number of bacterial and solute species, reactions and arbitrary detachment scenarios may be readily implemented using a general-purpose software framework introduced recently. The present work presents motivations for the mathematical modeling of biofilm structure and provides an overview on major contributions to this field from pioneering efforts using cellular automata (CA) to more recent methods using the preferred individual-based modeling (IbM). Recent examples illustrate how biofilm models can be used to study the microbial ecology in: (a) development of multi-species nitrifying biofilms with anammox bacteria, (b) interspecies hydrogen transfer in anaerobic digestion methanogenic consortia, (c) competition between flock-formers and filamentous bacteria influenced by environmental conditions and its effect on morphology of activated sludge flocs, and (d) a two-species biofilm system with structured biomass describing extracellular polymeric substances (EPS) and internal storage compounds. As recent efforts from direct comparison of structure predicted by three-dimensional modeling with that observed by confocal laser scanning microscopy imaging of biofilms grown in laboratory flow cells show a good agreement of predicted structures, multi-dimensional modeling approaches presently constitute a mature and established methodology to enhance our understanding of biofilm systems.

Biofilm initiation and development is a complex process that includes several major stages. This study examined the process of Listeria monocytogenes biofilm initiation and consequent spatial and physiological patterns formed in the biofilm as the result of bacterial response to a changing environment. This is a first attempt at establishing a link between the spatial organization of biofilm-associated bacteria and major stress factors such as surface bioavailability and nutrient content. Developed linear models allow evaluation of some important parameters of the surface population, such as the critical colony size and the intercolony distance in the case of diffusion-limited nutrient access. Variations in bacterial physiological patterns during biofilm initiation as a function of bacterial population density during surface colonization are reported.

The desiccation tolerance of Pseudomonas putida strain mt-2 cells within intact unsaturated biofilms and cells cultivated in liquid media was examined. Since as soils dry there can be an increase in the solute potential and reductions in nutrient availability, we also assessed the effect of the solute and matric (low-water-content) components of the total water potential, and nutrient availability on cell survival. Biofilms were cultivated in model laboratory systems that can simulate the matric and solute components of the total water potential and the poorly mixed nature of unsaturated habitats. Desiccation stress was imposed by exposing biofilm and planktonic cells to environments with controlled relative humidities. Our results show that biofilm cells are more tolerant to air drying than are planktonic cells, and tolerance is enhanced when there is an adequate supply of nutrients available to the cells. We provide evidence suggesting that a major factor contributing to desiccation sensitivity is the matric stress itself rather than solute stress and that matric stress is inherently more stressful to P. putida than is the thermodynamically equivalent solute stress.

Aggregation is a common phenomenon in microbiology. It manifests itself in different forms ranging from loose irregular structures such as effluent floc and marine snow to the ordered aggregates that show the reproducible patterns seen in many microbial colonies. Aggregation conveys advantages to microorganisms. These include transfer of chemical signals, exchange of genetic information, protection from adverse environmental conditions, metabolic cooperation between different species, as well as cell differentiation in some populations. Coaggregation is now recognized as a mechanism for allowing specific association between collaborating bacterial species. We describe a simple cellular automaton that illustrates structures that could form when different species interact. In particular the importance of neighbours is investigated.

In spite of the large range of morphologies observed for biofilms, there is strong experimental and theoretical evidence that the complex nature of biofilm structure dynamics is primarily a consequence of the effect of environmental conditions on biofilm development. It has been observed from the operation of industrial and laboratory-scale biofilm reactors that the structure of biofilms results from a balance of the detachment forces and the regimen of transport of a growth-limiting substrate. The overall performance of biofilm reactors is intrinsically dependent on biofilm morphology. The spatial distribution of the diverse dissolved and particulate components through the biofilm matrix and the shape of its external surface influence the rates of the occurring bioconversions, and structure also influences the stability of the biofilm in terms of resistance to mechanical stress. Individual-based modelling (IbM) of biofilms structure dynamics is used here to unify observations from the operation of biofilm reactors by simulating biofilm growth under variable detachment forces and mass transport regimens for a growth-limiting substrate. The IbM is a bottom-up approach, where the global system behaviour is derived from the local interactions of multiple elements acting independently. Transport and reaction of a solute species, local microbial growth rates and the effect of external detachment forces applied to the biofilm are modelled using differential approaches. Simulations carried out in two-dimensional space using this model illustrate a range of biofilm morphologies that emerge from different reactor operation parameters, reproducing trends observed experimentally. Comparison of multi-dimensional modelling results with those obtained using one-dimensional approaches enforces the need to use multi-dimensional modelling to predict properties that derive from the spatial biofilm structure.