The concept that bacteria live preferentially in matrix-enclosed communities attached to surfaces has emerged gradually from scientific observations over an extended period of time, but the pace at which this concept has advanced has accelerated sharply during the past two decades. Because Antonie van Leuwenhoek examined his own teeth scrapings with his primitive microscope, he probably saw more biofilm fragments than planktonic cells, and dental microbiologists and waste-water engineers have had a lengthy association with biofilms without using that term. Early microscopic observations of marine systems showed that most bacteria adhered actively to surfaces, and the role of surfaces in the migration and maturation of myxobacterial communities was noted very early in the study of these fascinating organisms. The new concept that was crystallized in a Scientific American article in February 1978 (Costerton, J. W., Geesey, G. G. & Cheng, K. J. (1978) How bacteria stick. Scientific American238, 86–95) was that these surface associations were the rule (and not the exception) in all nutrient-sufficient microbial ecosystems, and that most bacteria in the biosphere grow in biofilms.

Oral bacteria form mixed-species biofilms known as dental plaque. Growth of these complex microbial communities is often controlled with the use of antimicrobial mouthrinses. Novel laboratory methods for testing the efficacy of antimicrobials in situ are necessary to complement current clinical testing protocols. In this study, we examined the effects of antimicrobial agents on a streptococcal biofilm grown in a saliva-conditioned flowcell. The flowcell coupled with confocal laser scanning microscopy enabled examination of growing oral biofilms in situ without disruption of the microbial community. Biofilms composed of Streptococcus gordonii DL1 were grown in an in vitro flowcell and treated with several commercially available antimicrobial mouthrinses containing essential oils, triclosan, cetylpyridinium chloride/domiphen or chlorhexidine. The results of this study revealed varying abilities of the antimicrobial agents to cause cellular damage on the growing biofilm in situ. This study therefore demonstrated the usefulness of the flowcell in the rapid assessment of antimicrobial efficacy.

A fermentation system was designed to model the human colonic microflora in vitro. The system provided a framework of mucin beads to encourage the adhesion of bacteria, which was encased within a dialysis membrane. The void between the beads was inoculated with faeces from human donors. Water and metabolites were removed from the fermentation by osmosis using a solution of polyethylene glycol (PEG). The system was concomitantly inoculated alongside a conventional single-stage chemostat. Three fermentations were carried out using inocula from three healthy human donors.

Bacterial populations from the chemostat and biofilm system were enumerated using fluorescence in situ hybridization. The culture fluid was also analysed for its short-chain fatty acid (SCFA) content. A higher cell density was achieved in the biofilm fermentation system (taking into account the contribution made by the bead-associated bacteria) as compared with the chemostat, owing to the removal of water and metabolites. Evaluation of the bacterial populations revealed that the biofilm system was able to support two distinct groups of bacteria: bacteria growing in association with the mucin beads and planktonic bacteria in the culture fluid. Furthermore, distinct differences were observed between populations in the biofilm fermenter system and the chemostat, with the former supporting higher populations of clostridia and Escherichia coli. SCFA levels were lower in the biofilm system than in the chemostat, as in the former they were removed via the osmotic effect of the PEG. These experiments demonstrated the potential usefulness of the biofilm system for investigating the complexity of the human colonic microflora and the contribution made by sessile bacterial populations.

A natural microphytobenthic assemblage from the Eden Estuary, Scotland, was used to study the effect of temperature and irradiance on the sustainability and species composition of a natural transient biofilm. Three tidal tanks were maintained: tank 1 at 10 °C; tank 2 at 18 °C; and tank 3 at 26 °C. Within each tank, five cores were unshaded (350 μmol/m2 per s), five cores were semi-shaded (175 μmol/m2 per s), and five cores were shaded (70 μmol/m2 per s). Chlorophyll a increased in all treatments, but accumulated slower with increasing temperature. Surface diatom biomass of biofilms (as determined from measurements of minimum fluorescence, Fo15), grown at 10 °C and 18 °C was sustained above initial levels after 21 days, whilst biofilms grown at 26 °C suffered a severe loss of diatoms after 14 days, probably owing to nutrient limitation. Species richness and diversity of diatom assemblages illustrated a variety of responses to the temperature and light conditions. Diatoms at 10 °C acclimated to the different light levels by varying the ratios of diadinoxanthin: chlorophyll a, whilst at 18 °C the diatom species composition changed dramatically in response to shading. Ratios of zeaxanthin: chlorophyll a increased with increasing temperature, indicating an increase in cyanobacterial biomass at 26 °C after 21 days. Increased temperature significantly increased the maximum theoretical electron transport rate (rETRmax) in the short term (days), although light and temperature treatments did not affect the maximum light utilization coefficient (αrETR). Limitations of the fluorescence methodology used to study the resultant mixed community of benthic phototrophs is discussed. Diatom vertical migration in response to light, and an alteration of the main functional taxonomic group, has implications for the interpretation and value of fluorescence data.

Tolerance of Staphylococcus epidermidis in biofilms to killing by rifampin was correlated with limitation of bacterial growth in the biofilm state. Intact biofilm experienced a 0.62 log reduction when treated with 0.1 μg rifampin/ml for 4 h whereas the same treatment of exponential-phase planktonic cells produced a log reduction of 4.48. Stationary-phase planktonic cells were nearly as tolerant as intact biofilm cells, experiencing a 1.11 log reduction. Biofilm bacteria grew at only 10% of the maximum rate at which they grew on the same medium in planktonic culture. Killing was localized near the surface of the biofilm adjacent to the nutrient source, as revealed by staining with a respiratory dye. Increased nutrient concentration during antibiotic treatment enhanced killing of biofilm cells. Changing the oxygen tension in the gas phase above the biofilm during antibiotic treatment barely affected killing. It was hypothesized that the biofilm harbors significant numbers of stationary-phase-like cells in the nutrient-limited depths of the biofilm, and that these inactive cells are the ones that survive antibiotic challenge.

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.

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.

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.

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.

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.