There is growing evidence to suggest that non-typeable Hemophilus influenzae (NTHi), an important cause of otitis media in children, is able to grow as a biofilm in the middle ear. This observation may help to explain bacterial persistence in chronic infections. We evaluate the usefulness of rapid freezing and freeze substitution as a means of preparing biofilms for ultrastructural examination by comparing the morphology of cryofixed specimens with the morphology produced using more conventional chemical fixation and dehydration methods. Chemical fixation and dehydration methods produced substantial ultrastructural damage to individual NTHi in the biofilm and loss of extracellular matrix, even in the presence of ruthenium red. In comparison, cryofixed and freeze-substituted NTHi biofilms showed significantly improved preservation of bacterial ultrastructure and biofilm organization. The intracellular contents of NTHi prepared using the cryomethods showed little evidence of aggregation, and bacteria within the biofilm were closely packed and surrounded by an abundant extracellular matrix. Although high-pressure freezing of NTHi biofilms followed by freeze substitution was highly effective for preserving ultrastructure when examined by transmission electron microscopy, immersion in liquid propane offered an alternative, “less technical”, freezing method. Immersion in liquid propane followed by freeze substitution and critical point drying was most effective for preserving ultrastructural details in specimens examined by scanning electron microscopy.

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.

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.

Bacteria often choose a sessile biofilm lifestyle as a strategy to overcome environmental stress. In this study, we describe biofilm formation by Helicobacter pylori on a polystyrene surface, evaluating the viability and the morphological dynamics of bacterial cells during multicellular aggregate development. Moreover, we investigate expression of the luxS and rpoD genes, which are involved in biofilm formation.

Two clinically susceptible and resistant strains of H. pylori were analyzed, as well as H. pylori ATCC 43629 for reference. The dominant form of expression, clustered bacterial cells arranged in an abundant matrix, was recorded after 2 days of incubation without shaking. Coccoid (spherical) cells with a “wrinkled” aspect presented the prevalent morphology (59.26%) among cells in the biofilm structure as observed by scanning electron microscopy. In aged H. pylori cultures, death occurred in single cells or cells grouped into microcolonies in which degenerated bacteria were localized inside the aggregates. The expression of luxS and rpoD genes among the sessile bacterial population reached a maximum after 2 days, with a significant reduction at subsequent time-points. No differences in gene expression and biofilm formation were recorded in the three evaluated strains.

The morphological fickleness expressed in the life cycle by H. pylori strains emphasizes the bacterium's ability to overcome environmental stress, balancing its spread both outside and inside the host.

We have investigated the role of smectite clay particles in biofilm formation by several different species of bacteria (Pseudomonas syringae, Escherichia coli, Staphylococcus aureus and Bacillus subtilis). We observed that the presence of clay particles enhances the formation of biofilms and, after 24 h, the bacterial populations in the clay mixtures were greater than the respective populations in media without clay. Smectite-bearing clay slurries uniformly develop bacteria–clay aggregates with a substantial biofilm component within 24 h, while the exclusively bacterial suspensions do not develop any observable biofilm component. The biofilm–clay aggregates vary in size from tens of micrometers to several millimeters. Biofilm formation was evaluated by phase contrast microscopy and fluorescence staining. Biofilm promotion by smectite clays may indicate the importance of transport of bacteria by aerosol dust particles.

Candida albicans forms part of the normal human flora whose growth is usually restricted by the normal flora bacteria and the host's immune system. It is an opportunistic fungal pathogen that causes infections in immunocompromised individuals, mechanical trauma victims and iatrogenic patients. Candida albicans can ingress the human host by adhering to a plastic surface (i.e. prosthetic devices, catheters, artificial organs, etc.) that is subsequently implanted, and forms a protective biofilm that provides a continuous reservoir of yeast to be hematogenously dispersed. In order for the medical profession to battle device-related infections, initial adhesion and biofilm formation of C. albicans needs to be better understood. There has been some skepticism as to whether the initial adhesion events bear any relationship to subsequent biofilm formation. Thus, to better comprehend the relationship between the initial adhesion rates and growth rate and biofilm formation, these events were studied on two different, well-defined culture surfaces, native polystyrene and Pluronic F127-conditioned polystyrene. The adhesion studies determined that Pluronic F127 adsorption dramatically reduced the adhesion of C. albicans to polystyrene. The biofilm growth studies, analyzed by confocal scanning laser microscopy, revealed that Pluronic F127 decreased the biofilm surface coverage, cluster group size, thickness and the presence of hyphal elements over the untreated polystyrene. These findings indicate that the effect of a material's surface chemistry on the initial adhesion process has a direct influence on subsequent biofilm formation.

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.

We have compared the outer membrane protein (OMP) pattern of sessile Pseudomonas aeruginosa cells with that of its stationary-phase free-floating counterparts. Two-dimensional electrophoretic gels were compared using computing scanning densitometry. The level of 103 OMPs (i.e. about 50% of the spots that fulfilled our statistical selection threshold) was significantly and reproducibly different in biofilm organisms as compared with their planktonic counterparts. Some OMPs that specifically either accumulated or decreased were identified by mass spectrometry and the role of these proteins in the biofilm phenotype is discussed.

Infrared spectra of Pseudomonas aeruginosa PAO1 biofilms grown on 40° ZnSe and 40° AMTIR (amorphous material transmitting infrared radiation, Ge33As12Se55) internal reflection elements (IRE) at 26 °C in a flow cell were collected. The depth of penetration of the evanescent wave was sufficient to identify four biofilm stages during the experiment: (1) cell/substratum attachment, (2) lag phase, (3) later-stage growth, and (4) restructuring (e.g. rearrangement, detachment or “erosion”). Our experimental results provide the first detection via a non-perturbative technique of an excess of protein in the neighbourhood of the surface. Our mathematical analysis applied to the data supports the conclusion that our observations cannot be completely accounted for by the changing characteristics of bacterial proteins but, rather, shows that an accumulation of excess protein near the attachment surface is taking place. On the assumption that biofilm cells are approximately of the same size as those of the planktonic culture, this suggests the presence of extracellular proteins. Our data do not support the presence of extracellular nucleic acids near the attachment surface in these biofilms. This conclusion is based on our observation that, during the initial 20 h of development (stage 2 and the beginning of stage 3), biofilms had a higher proportion of protein to nucleic acid than in the latter part of stage 3 and in stage 4. In the restructuring phase this ratio became equal to the ratio determined for planktonic cultures. The decrease of biomass during stage 4 is inconsistent with the physical removal or sloughing-off of cellular aggregates from biofilms within a few micrometres of the substrate surface but, instead, supports a model of gradual cell migration away from it. During this cell migration there is also a gradual loss of extracellular protein. Alternatively, therefore, the restructuring may involve cell lysis and the observed loss of biomass may be due to soluble macromolecules being removed from the neighbourhood of the surface.

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.

Microbial adhesion and biofilm formation on surfaces pose major problems and risks to human health. One way to circumvent this problem is to coat surfaces (in this report stainless steel) with a non-toxic fish extract that generates an abiotic surface with less bacterial attachment than uncoated surfaces or surfaces coated with, for example, tryptone soy broth. The bacteria grow well in the fish extract; hence a general bacteriocidal effect is not the reason for the antifouling effect. Bacterial attachment was quantified by different methods including (a) direct fluorescence microscopy, (b) removal by ultrasound and subsequent quantification of the adhered bacteria, and (c) regrowth of the adhered bacteria measured by indirect conductometry. Surprisingly, the bacterial counts on surfaces coated with aqueous fish extract were 10–100 times lower than on surfaces coated with laboratory broths when surfaces were submerged in bacterial suspensions. The effect was seen for Pseudomonas fluorescens AH2, Pseudomonas aeruginosa PAO1, Escherichia coli MG1655, Vibrio anguillarum 90-11-287 and Aeromonas salmonicida Jno 3175/88. It lasted for at least 7 days. Atomic force microscopy showed that steel surfaces conditioned with fish extract were covered by a thin layer of spherical, nanosized particles. Chemical analysis of the surfaces coated with adsorbed fish extract using X-ray photoelectron spectroscopy revealed that the layer was proteinaceous and had a thickness less than 2 nm. Numerous protein bands/peaks were also detected by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry techniques. We conclude that coating the stainless steel surface with fish extract results in a thin protein layer that reduces bacterial adhesion significantly.

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.

Biofilm growth within a water distribution system could lead to operational problems such as pipe corrosion, water quality deterioration and other undesirable impacts in water distribution systems. With the high ambient temperatures experienced in Singapore, the operating environment in water distribution systems is expected to be more conducive to biofilm development. We have recently conducted a survey on biofilms potentially present in a local water distribution system.

The survey results indicated that residual chlorine (±standard deviation) decreased from 1.49±0.61 mg/l (water plant outlets) to 0.82±0.21 mg/l (block pipes) or 0.18±0.06 mg/l (unit pipes), respectively. Consumed chlorine, instead of residual chlorine, was found to be correlated with biofilm bacterial population. Assimilable organic carbon (AOC) level was 160±66 μg acetate C/l, and AOC:PO4-P:NO3-N was about 8:13:1. Carbon source seemed to be the limiting nutrient for bacterial growth. The concentration of iron increased from <0.04 mg/l (water plant outlets) to 0.22±0.10 mg/l (all sites). All samples showed negative results in a coliform test. The average heterotrophic plate count (HPC) for the suspended bacteria was 20 colony-forming units (c.f.u.)/ml (2 days, 35 °C) or 290 c.f.u./ml (8 days, 35 °C). The average HPC for the biofilm bacteria was 6500 c.f.u./cm2 (2 days, 35 °C) or 29000 c.f.u./cm2 (8 days, 35 °C). High HPC values in samples B2a, B2b and B3a (representing biofilm samples at site 2 from block/unit pipes and biofilm samples at site 3 from block pipes, respectively) illustrated that the relevant sample sites had a higher probaboility of biofilm growth.

The effects of oxygen availability on in vitro biofilm formation by an Escherichia coli K-12 strain and 13 clinical E. coli strains were compared. All E. coli strains were capable of forming monospecies biofilm on polystyrene in aerobic media. The K-12 strain produced biofilm in both aerobic glucose minimal medium (ABTG), and aerobic trypticase soy broth (TSB) whereas the majority of the clinical strains produced significant biofilm only in aerobic TSB (9 of 13). In anaerobic media, E. coli K-12 and 9 of the 13 clinical strains were capable of forming biofilm in vitro. Only three clinical strains formed biofilm in anaerobic TSB whereas six clinical strains produced detectable biofilm in anaerobic ABTG. None of the strains tested were capable of forming biofilm in both anaerobic ABTG and anaerobic TSB. Strains that were good biofilm formers in aerobic ABTG also produced the highest amount of biofilm in anaerobic ABTG (R2 = 0.90). Image analysis revealed notable differences in architecture for biofilms grown in the presence and in the absence of oxygen. In aerobic ABTG, the biofilm was dominated by tall, mushroom-shaped microcolonies with pores and channels whereas biofilm in anaerobic ABTG was thinner and less heterogeneous, resulting in reduced maximum thickness and biovolume. Analysis of phospholipid fatty acid (PLFA) profiles from E. coli K-12 and three clinical strains did not reveal a specific pattern associated with the biofilm phenotypes. Interestingly, the clinical E. coli strains adjusted their PLFA composition much more than did E. coli K-12 in response to changes in growth regimens. Collectively, the results indicate that oxygen availability may affect E. coli biofilm formation in minimal and complex media. The results confirm that E. coli K-12 and some clinical E. coli strains are capable of forming in vitro biofilm under anaerobic conditions. However, the data also suggest that this attribute is highly strain dependent and may vary significantly among clinical isolates.

Four bacterial isolates (Chryseobacterium sp., Pseudomonas fluorescens and two Stenotrophomonas maltophilia isolates) originally isolated from the water system aboard the Mir Space Station were grown in two concentrations of nutrient broth under modeled reduced gravity using clinorotation. Sampling was performed over a 7 day period and planktonic cells were enumerated using 4′,6-diamidino-2-phenylindole (DAPI), while those attached to stainless steel were enumerated using the LIVE/DEAD® BacLight™ kit and DAPI. On some of the sampling days for all the isolates, planktonic cell counts were higher under modeled reduced gravity as compared with the normal gravity controls. In contrast, the number of cells of P. fluorescens and one S. maltophilia isolate attached to the stainless steel disks was higher under modeled reduced gravity as compared with normal gravity, whereas no such differences were observed for Chryseobacterium sp. and the other S. maltophilia isolate. Differences in motility among isolates appeared to influence the growth of planktonic cells under modeled reduced gravity but did not appear to be related to biofilm formation.

Pseudomonas aeruginosa PAO1 was grown on 40° ZnSe and 40° AMTIR® (Amorphous Material Transmitting Infrared Radiation, Ge33As12Se55) internal reflection elements in a thermostatted flow cell. Information about the chemistry of the biofilm development was obtained using Fourier transform infrared spectroscopy. The weakly absorbing C–H stretching region of the infrared spectrum was measured for the first time and revealed qualitative and quantitative changes not evident in the amide region studied by previous investigators. There is a chemical difference between the early growth period and subsequent development of the biofilm.

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.

We have made a comparative analysis of the extracellular phospholipid composition of biofilms of Gram-negative and Gram-positive bacteria. The surface film of a growing bacterial community contains small membrane vesicles and a bilipid layer covering the entire surface of that community. These supracommunity films containing the bilipid layer can cover the entire surface of a Petri dish and form a boundary between bacterial communities and the environment. A mixed bacterial lawn, formed by unrelated bacteria, also becomes covered with a single film containing a lipid bilayer. The phospholipid compositions of the bacterial cell and surface film bilipid layer reflect the nature of the bacterial strains forming the community, but have some specific differences.

The aim of this study was to investigate bacterial survival and biofilm formation on toothbrushes. Fifteen healthy volunteers each used a normal toothbrush and an antibacterial toothbrush of the same design for two separate 5 week periods. Bacteria were removed from the brush head by swabbing and mechanical agitation in 10ml of tryptone soya broth, cultured aerobically on selective and non-selective media, and classified by Gram staining, catalase and oxidase tests. Survival of Staphylococcus epidermidis and Pseudomonas aeruginosa was monitored in the laboratory on both types of brush over 8 days. Scanning electron microscopy was used to observe biofilm formation on antibacterial and conventional brushes used for various times. Numbers of bacteria isolated from conventional and antibacterial brushes from different individuals ranged from 8.3×103 to 4.7×106 and from 1×102 to 1.2×106 colony-forming units/ml, respectively. A larger number of bacteria were isolated from conventional brushes than from antibacterial brushes used by the same individuals but no statistically significant difference was demonstrated. No differences in the relative proportions of Gram-negative and Gram-positive rods or cocci were seen. Staphylococci, presumptive coliforms and pseudomonads were isolated from 48%, 28% and 16% of brushes, respectively. Pseudomonas aeruginosa was viable for at least 4 days on conventional, and 2–3 days on antibacterial, brushes, whilst S. epidermidis survived for 6–8 days on antibacterial and more than 8 days on conventional brushes. Biofilms formed on the heads and bristles of both conventional and antibacterial brushes. Extensive, mixed community biofilms developed after several months of use. We conclude that toothbrushes may be a reservoir of opportunistic pathogens including staphylococci and pseudomonad-like organisms and must be considered as a potential source of haematogenous infections and cross-infection.

Epilithic biofilms play key roles in rocky shore ecosystems. The diversity and biomass of epilithic biofilms were determined along a subtropical intertidal rocky shore at Xpicob, Southern Gulf of Mexico (Campeche, Mexico). Biofilm diversity was assessed by polymerase chain reaction (PCR) amplification of partial 16 S rRNA genes using single-strand conformation polymorphism (SSCP). Triplicate 2 cm×2 cm biofilm samples per site were taken randomly from 0.6 m×0.6 m quadrats located in two sampling sites, established 20 m apart within a homogeneous calcareous intertidal platform. Twenty-two partial rRNA sequences, belonging to four bacterial divisions (Cyanobacteria, Bacteroidetes, Actinobacteria and Proteobacteria), were recovered from these biofilms; of these, cyanobacteria were the most abundant (41%). The occurrence of cyanobacterial sequences in most samples, along with the detection of high levels of chlorophyll a and phycobiliprotein, indicates that these organisms are dominant within the biofilms. Consistent with previous reports, thick-sheathed cyanobacteria such as those found in this study (Xenococcus, Myxosarcina and Chroococcidiopsis) are typical in habitats in intertidal zones. In addition, most of the detected organisms from other bacterial lineages had closest relatives displaying biofilm phenotypes that suggested stressful conditions (i.e. desiccation) prevailed in the intertidal shores; this was selecting for biofilm-forming or thick-sheathed organisms as an ecological adaptation to withstand the conditions. Unweighted pair-group method with arithmetic mean (UPGMA) analysis of SSCP profiles indicated that there was significant spatial heterogeneity in biofilm community composition across different scales of analysis and that this heterogeneity was related to the distance at which samples were taken. This analysis showed that certain organisms appeared to be station specific and that the biofilm community structure varied even on a small scale (1 cm). Since no significant differences in bulk sea water or substratum physicochemistry were observed, these results suggest that small-scale variability of environmental conditions, surface properties and/or biological interactions (i.e. allelopathy) may be important factors structuring these biofilm communities.