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.

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.

Orica's groundwater treatment plant in Botany, NSW, Australia, was designed to remove and destroy volatile organic compounds from polluted groundwater and to treat the water for reuse on the Botany Industrial Park. The initial steps in this process involved acidification of the groundwater and air stripping. During this operation, very large quantities of a biofilm formed within the air stripper, necessitating weekly clean-outs. We investigated the composition of this biofilm using molecular methods. Total DNA extracted from biofilm material was used as a template for amplification of both bacterial 16 S ribosomal DNA (rDNA) and the eukaryotic rDNA internal transcribed spacer region. Cloning and sequencing of these products showed that the biofilm was composed primarily of a bacterium belonging to the genus Acidocella, a filamentous fungus (Trichoderma asperellum), and the ascomycetous yeasts Pichia, Candida and Geotrichum. This unusual biofilm was composed of acidophiles that were capable of rapidly generating large amounts of biomass under these conditions. When acidification of the groundwater ceased, the biofilm no longer formed.

Interactions between sulfur and metals were studied in aerobic and anaerobic biofilms grown on domestic waste water at 15°C. The dominant metals in the waste water were iron, zinc and copper, which were present in average concentrations of 0.5mg/l, 0.6mg/l and 0.1m/l, respectively. Copper and zinc were found to accumulate in the anaerobic biofilm owing to precipitation of metal sulfides. Iron supplementation (approximately 5.5mg Fe/l) to the anaerobic waste water reduced the zinc and copper precipitation due to sulfide precipitation with iron. However, even at these elevated iron concentrations in the waste water, sulfide precipitation in the biofilm was controlled largely by zinc and copper. In the aerobic biofilm, addition of iron resulted in accumulation of iron and phosphate, probably owing to precipitation of iron phosphates and iron (oxy)hydroxides. The potential importance of extracellular polymeric substances (EPS) on metal sorption in sewer biofilms was studied. EPS consisted mainly of proteins (13–260mg/(g volatile solids)) and to a lesser extent of carbohydrates (8–26mg/(g volatile solids)). The EPS composition remained relatively constant during experimental runs, but differed significantly between them. No relationships between the metal content of the biofilm and the amount of extracted EPS were found, which suggests that EPS did not play a major role in the metal accumulation.

The susceptibility of Pseudomonas aeruginosa (PA01) bacteria to ultraviolet-A (UV-A) light in photocatalytic and non-photocatalytic systems was investigated. Thin films of TiO2 were deposited on glass slides using a dip-coating method. Disinfection of both planktonic and biofilm bacterial cells was studied over both coated and uncoated slides exposed to low intensity (1.0 mW/cm2) UV-A irradiation. Photocatalytic treatment of planktonic PA01 cultures pro-vided a 4-log reduction in the number of viable cells in 3 h whereas UV light alone produced a 1-log reduction. For biofilm bacteria, photocatalytic treatment did not enhance killing relative to UV treatment alone; however, the UV illumination resulted in a 2- to 3-log reduction in viable cells as compared with non-illuminated controls. Possible explanations for the discrepancy between susceptibility of planktonic and biofilm bacteria to photocatalytic oxidation are discussed.

A site polluted for many years with polychlorinated biphenyls (PCB) was used to elucidate the metabolic adaptation of microbial communities to these xenobiotics. Soil samples taken along a gradient of PCB-pollution at this site were used to grow biofilm communities on PCB oil. The biofilm communities originating from the non-polluted soil formed rather uniform and thin bacterial layers on PCB oil, while the biofilms originating from contaminated soil samples formed agglomerated structures on the PCB droplets. Biofilm communities were very diverse but those from highly polluted soil were dominated by Burkholderia species, a genus known for degrading several PCBs. All biofilm communities could transform low to medium chlorinated PCB congeners but a strong increase in the rate and degree of PCB transformation in communities from heavily polluted soil was observed. Notably, pentachlorinated congeners were transformed only by biofilms derived from the highly polluted soil but at the same time the content of trichlorinated congeners did not decrease. It is assumed that biofilms from the highly contaminated soil reductively dechlorinated PCB, converting pentachlorinated congeners to trichlorinated congeners in the spherical biofilm aggregates by diffusing to the surface of the aggregates, where aerobic transformation took place.

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.

This review details the current literature on relevant aspects of biofilm formation and microbial control in dental practice. To date, there is no published evidence of a serious public health risk from biofilm-contaminated dental waterlines. However, there remain few effective methods of decontamination of such waterlines.

Actinomycetes have been used with enormous success in industrial processes; however, little is known about biofilm formation by these filamentous microbes, or community development on insoluble substrates such as cellulose. We hypothesized that biofilm formation is a general strategy used by actinomycetes in the degradation of cellulose, and that it may serve as a means for these microbes to secure nutrients and persist in their environments. The objective of this study was to examine biofilm production by Thermobifida fusca, an actinomycete that rapidly degrades cellulose by means of a well-characterized extracellular cellulase system. Thermobifida fusca cells grew as biofilms attached to both nutritive (e.g. dialysis tubing membrane) and non-nutritive surfaces. Dialysis tubing was colonized by T. fusca aleuriospores but not by mycelial pellets, except when mycelial pellets were disrupted by sonication. Microscopic examination of surface-attached growth revealed structures characteristic of biofilms, with cells embedded in fibrous material suggestive of an extracellular polymeric matrix. Concanavalin A bound to the extracellular polymeric substance of biofilms and mycelial pellets, indicating alpha-linked d-mannosyl and/or alpha-linked d-glucosyl residues. The carbohydrate content of both biofilms and mycelial pellets increased during growth. Also, DNase I inhibited biofilm production, suggesting a role for extracellular DNA in T. fusca biofilm development. Cellulose degradation and expression of celE (encoding endoglucanase E5) was similar for T. fusca biofilms and mycelial pellets. Results of this study indicate that, in the life cycle of this actinomycete, cellulose is specifically colonized by aleuriospores, which germinate, grow and degrade cellulose, ultimately developing into biofilms encased in a carbohydrate-containing exopolymeric matrix, a hallmark of biofilm production.

In previous work, we described a protocol for measuring biofilm formation on opaque and non-opaque surfaces using reflected light. In the present work, we used the reflectance assay to assess biofilm formation for 14 strains of Enterococcus and compared our results with those obtained using a Crystal Violet (CV) assay. In general, the results for the two assays were in agreement: both identified the same Enterococcus strain as the highest biofilm-forming strain and the same two strains as the lowest biofilm-forming strains. However, two Enterococcus strains identified as high biofilm formers by the CV assay were not identified as such by the reflectance assay. A single numerical value was obtained for each of the reflectance assay results that corresponded well with the confluence of a biofilm (and presumably its depth) using phase contrast microscopy.

The aim of this research was to study the effect of varying incubation times and sucrose exposures on lesion development and biofilm composition using a multi-species biofilm caries model. Two studies were conducted. In study 1, enamel specimens were divided into four groups, inoculated with a mixed overnight culture of Streptococcus mutans, Lactobacillus casei, Actinomycesnaeslundii, Streptococcusparasanguis and Streptococcussalivarius, and exposed to circulating trypticase soy broth + 5% (w/v) sucrose (TSBS; 30 min, three times per day (3 × /day)) and a mineral washing solution containing 0.25 p.p.m. fluoride (22.5 h/day) for 2, 5, 6 or 8 days. In study 2, additional enamel specimens were divided into four groups and exposed to the same biofilm for 7 days, but with variations in the feeding schedule: TSBS 3×/day for 5 min, TSBS 3×/day for 15 min, TSBS 3 × /day for 30 min, or TSBS 3×/day for 30 min + 1×/day for 15 min. At the end of each study, bacterial colonization counts and lesion size were determined. In study 1, specimens developed significantly deeper carious lesions with longer demineralization time (average lesion depth was 52.16 μm, 67.86 μm, 84.91 μm, and 99.97 μm, respectively, for 2, 5, 6, and 8 days). In study 2, there was no significant difference in size between the lesions developed at feeding schedules of 3 × /day for 5 or 15 min. Lesions exposed to longer (30 min) and more frequent feeding schedules (4×/day) were significantly larger than the other groups. In both studies, all five bacterial species were able to colonize the enamel and were present in all groups at the end of the experiments, with predominance of lactobacilli over S. mutans. In conclusion, larger lesions were observed with increased incubation time and more frequent feeding schedules, with small variations in biofilm composition.

Proteus mirabilis can persist in biofilms, with the bacteria in this state tending to resist antibiotic therapy. Until now, the relationship between the action of ciprofloxacin and the production of reactive oxygen species (ROS) has not been studied in planktonic and biofilmic P. mirabilis. Our results show that ciprofloxacin stimulates the production of ROS in planktonic P. mirabilis, but that the increase in ROS was observed in sensitive strains (n = 4) only in the absence of the extracellular matrix (ECM). This augmentation of ROS was principally intracellular, invoking an increase in intracellular superoxide dismutase (SOD). ROS were assayed by chemiluminescence (CL) and SOD by inhibition of reduction of nitroblue tetrazolium in the presence of methionine, riboflavin and light. The antibiotic-resistant strains (n = 4) did not suffer oxidative stress and exhibited a higher antioxidant capacity than antibiotic-sensitive ones, as indicated by tripyridyltriazine assay. Both types of bacterial strain showed a reduction in antioxidant capacity in the presence of ciprofloxacin, and only the resistant bacteria returned to normal count levels within 5 min of introduction of antibiotic. Ciprofloxacin stimulated ROS more than it did nitric oxide (NO) in planktonic bacteria, as determined by Griess's reaction. Proteus mirabilis biofilms treated with ciprofloxacin did not suffer any increase in ROS but there was an increase in NO and the ratio of intracellular ROS:NO decreased to 25%. Biofilms of P. mirabilis were neither stressed nor inhibited by 40 µg ciprofloxacin/ml, a dose higher than the minimum inhibitory concentration (i.e. supra MIC). Both resistant and sensitive strains maintained the number of viable bacteria in biofilms incubated with supra MIC ciprofloxacin at concentrations that stressed and reduced substantially the number of colony-forming units of planktonic bacteria per millilitre. These results contribute to understanding of the differences between biofilmic and planktonic bacteria, with respect to susceptibility to oxidative stress caused by ciprofloxacin and also the antioxidant effect of ECM.

Titanium has proven to be the heat exchanger material of choice for sea-water-cooled power plants owing to its outstanding resistance to pitting and crevice corrosion in a wide range of aggressive media. However, the inertness of the titanium surface makes it highly susceptible to biofilm formation and subsequent biofouling. This can hinder the heat transfer properties and flow of water. Fouling control strategies in condensers include a combination of mechanical, chemical and thermal treatments. However, reports from various industrial situations suggest that mechanical treatment may not have long-term effects. This study aimed to find out whether mechanical cleaning eventually enhances biofilm formation and increases the adherence of biofilm. In our studies epifluorescence micrographs of biofilms on control and mechanically treated titanium surfaces clearly showed accelerated biofilm formation as well as increased adherence on the mechanically cleaned surface. Total counts of viable bacteria acquired by culturing technique, and biofilm thickness measurements made using microscopic techniques, confirmed this observation. Surface profilometry showed increased roughness of the titanium surface, facilitating adherence of biofilm. The number of microbial species was higher on mechanically cleaned and re-exposed surfaces than on fresh titanium. Thus we concluded that mechanical cleaning can increase biofilm formation and adherence of biofilm, thereby increasing the potential of biofouling in the long term.

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.

Biofilm-formation in Staphylococcus epidermidis depends on the expression of the icaADBC operon encoding the enzymes required for the synthesis of polysaccharide intercellular adhesin (PIA). Different S. epidermidis strains vary widely in the degree of PIA and biofilm that they produce. In 11 clinical S. epidermidis strains we analyzed the biofilm-forming capacity in relation to the amount of ica expressed in static biofilm cultures. In mid-exponential growth phase no correlation could be detected between the level of ica transcription and the biofilm-forming phenotype. When the different strains were grown under conditions leading to a biofilm-negative phenotype, ica-expression was highly upregulated. Sequence analysis demonstrated that the observed differences were not due to major mutations in the ica promoter region but apparently to other strain-specific regulators.

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.

The aim of this work was to study the community level physiological profile of a developing oral biofilm. Mixed-species biofilms, derived from human saliva, were grown in a constant-depth film fermentor for up to 240 h. Biolog GP2 microplates were inoculated with saliva or homogenized biofilms and incubated at 37 °C. The results showed that a biofilm community could utilize a range of substrates more diverse than those of their planktonic counterparts. Following the construction of dendrograms, a temporal relationship was demonstrated between the inoculum and biofilms, and the difference in communities over time was also resolved by using an eight-digit code of positive carbon sources. The diversity of the populations could be determined through the use of the community level physiological profile and the treatment of the data may augment the interpretation of the results. Methods such as community level physiological profiling may provide a valuable insight into the composition of biofilm communities associated with oral health and disease.

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.

Study of natural microbial ecosystems is hampered by our inability to closely duplicate the development of a community in the laboratory environment. The lack of such systems is particularly acute when endolithic microbes are examined. In this study, we have developed a system that enables laboratory growth of cryptoendolithic microbial communities resembling those found in the porous sandstones making up the Colorado Plateau in southeastern Utah. Microscopic examination of natural and in vitro cryptoendolithic biofilms shows that, on a gross level, the laboratory biofilm is developing in a fashion that is structurally similar to that of the native community. Further confirmation that the laboratory system resembles the native community was obtained by using denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis to examine the biofilms for the presence of cyanobacteria and the Geobacteriaceae. We found that the molecular profiles for these two groups of bacteria were nearly identical in the native and in vitro biofilms. These data show that our system for growing cryptoendolithic bacteria in the laboratory is suitable for the study of these ecosystems and will allow for experimental manipulations that are not possible in the field.

This study sought to understand the cell surface characteristics, viability and biofilm-forming potential of Enterococcus faecalis cells sequentially exposed to EDTA and calcium hydroxide, as in endodontic treatment. Bacterial cells exposed to EDTA and calcium hydroxide were assayed for cell viability, membrane integrity, cell surface hydrophobicity and surface charge, while alteration in the surface topography of E. faecalis cells was examined using atomic force microscopy (AFM). The bacterial adherence potential to type I collagen was also examined to assess the biofilm-forming capacity of E. faecalis cells exposed to EDTA and calcium hydroxide. It was found that calcium hydroxide treatment reduced the viability of E. faecalis. However, prior exposure to EDTA significantly reduced the antibacterial effect of calcium hydroxide (P < 0.05). Calcium hydroxide treatment resulted in impaired cell wall morphology, observed as increased surface roughness and pore formation under AFM. However, these topographical changes induced by calcium hydroxide were significantly reduced in EDTA pretreated cells (P < 0.05). Calcium hydroxide treatment caused reduction in hydrophobicity and adherence of E. faecalis to type I collagen. These effects due to calcium hydroxide were also significantly altered in EDTA-pretreated cells (P < 0.001). The findings from this study showed that the antibacterial and anti-adherence effect of calcium hydroxide was diminished by prior exposure of E. faecalis cells to EDTA.