Architecture of a typical biofilm. Three-dimensional reconstruction of V. cholerae biofilms. Bacteria carrying a plasmid constitutively expressing the green fluorescent protein (GFP) were incubated in chambers containing borosilicate glass. At 6 h, the wells were emptied, washed, and examined with a CSLM using 488- and 510-nm excitation and emission wavelengths, respectively. The top and center panels show horizontal (xy or top-down view) projected images at low and high magnification, respectively. Islands of bacterial aggregates are visible on the surface. The bottom panel is a sagittal (xz or side view) view of the same biofilm. The relative intensity of the pseudo-colored images is shown at the lower right corner and correlates with cell density. Bars, 50 μm (top panel) and 10 μm (center and bottom panels). This image was kindly provided by Fitnat Yildiz and Gary Schoolnik. A diagrammatic representation of various biofilms is also shown in Fig. 5. Reproduced with permission from reference 282.

Biofilm on a plant root. Biofilm of GFP-labeled Pseudomonas fluorescens WCS365 on the root of a tomato plant. A large microcolony of bacteria is apparent on the root surface and is indicated by the yellow arrow. The white arrows highlight three smaller colonies that have formed at plant root cell boundaries, which may be the site of release of root exudates used by bacteria as nutrient sources. The diffuse appearance of some bacterial cells in the large microcolonies suggests that these bacteria are covered by an EPS. EPS may play a role in formation of these microcolonies (see text), suggesting that these communities have many of the characteristics of typical bacterial biofilms. This image is kindly provided by Guido Bloemberg.

Mixed-species oral biofilm. Confocal image using live-dead stain (Molecular Probes, Inc.) of a mixed-species dental biofilm formed overnight in a flow cell. The inoculum used was saliva, and the chamber was incubated at 37°C with a flow of saliva at 0.2 ml/min. This image is a 0.5-μm slice through the 20-μm biofilm; the slice is between 1.0 and 1.5 μm from the substratum (saliva-coated glass). The green staining indicates live cells, while red bacteria either are dead or have a compromised membrane. The inset in the upper left corner of the figure is a higher magnification of the boxed area in the center of the image. The red arrow points to individual dead or damaged cells, and the green arrow points to a microcolony of live cells. This biofilm comprises a variety of oral microbes that have been reconstituted in an in vitro system. The image is kindly provided by Paul Kolenbrander and Rob Palmer.

Ecology of microbial communities. Top-down view of an idealized surface-attached microbial community, illustrating some of the major concepts pertaining to the ecology of biofilms discussed in the text. The four microcolonies at the center of the figure represent organisms that both generate and consume hydrogen and comprise two organisms that participate in syntrophism (see text). Fermenting organisms produce organic acids used by the hydrogen producers, and these fermenting organisms gain their carbon and energy by utilizing various sugars. In addition to potential metabolic interactions between organisms, signaling molecules may aid in inter- and intraspecies communication. The factors described above (as well as environmental influences) may all contribute to the spatial organization of the biofilm. As shown here, microcolonies in natural communities can comprise either a single or multiple bacterial species. The proximity of different microbes allows the possibility of physical interactions in addition to communication via diffusible factors.

Syntrophism in a sludge granule. Photomicrograph of in situ hybridization of a sludge granule obtained from a methanogenic reactor to illustrate biofilm organisms participating in a metabolic interaction. Fluorescein-labeled fluorescent probes were used to identify organisms specific to the order Methanomicrobiales (green and green arrow), and rhodamine-labeled probes were used to localize syntrophic propionate-oxidizing bacteria related to the genus Syntrophus (red and red arrow). The double (red and green) labeling results in yellow fluorescence. The results indicate that the syntrophic microcolonies are intertwined with chains of methanogens (yellow and yellow arrow). The metabolic interactions between these two organisms speed the anaerobic degradation of certain compounds (see text for details). Bar, 20 μm. This figure was modified from Harmsen et al. (92a), with permission to use this image kindly provided by Willem de Vos.

Biofilm development in gram-negative organisms. This figure outlines the current models for the early stages in biofilm formation in three of the best-studied model organisms, P. aeruginosa, E. coli, and V. cholerae. (A) In P. aeruginosa, flagella are required to bring the bacterium into proximity with the surface, and LPS mediates early interactions, with an additional possible role for outer membrane proteins (OMPs). Once bacteria are on the surface in a monolayer, type IV pilus-mediated twitching motility is required for the cells to aggregate into microcolonies. The production of pili is regulated at least in part by nutritional signals via Crc. Documented changes in gene expression at this early stage include upregulation of the alginate biosynthesis genes and downregulation of flagellar synthesis. The production of cell-to-cell signaling molecules (acyl-HSLs) is required for formation of the mature biofilm. Alginate may also play a structural role in this process. (B.) In E. coli, flagellum-mediated swimming is required for both approaching and moving across the surface. Organism-surface interactions require type I pili and the outer membrane protein Ag43. Finally, the EPS known as colanic acid is required for development of the normal E. coli biofilm architecture. (C) V. cholerae, like E. coli, utilizes the flagella to approach and spread across the surface. The MshA pili, and possibly one or more unidentified outer membrane proteins, are required for attachment to the surface. This initial surface attachment appears to be stabilized by EPS. Formation of the mature biofilm, with its associated three-dimensional structure, also requires production of EPS. Vps refers to the EPS produced by V. cholerae.