The structural organization of four microbial communities was analysed by a novel computer program, COMSTAT, which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria, Pseudomonas putida, P. aureofaciens, P. fluorescens and P. aeruginosa, tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure -mean thickness, roughness, substratum coverage and surface to volume ratioshowed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum, and formed flat, uniform biofilms. P. aureofaciens resembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 003 mM, 01 mM or 05 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover, biofilm roughness increased with lower citrate concentrations, whereas surface to volume ratio increased with higher citrate concentrations.

SummaryBiofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and COMSTAT image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.

Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfpbased reporter technology has been applied for non-destructive, single-celllevel detection of quorum sensing in laboratory-based P. aeruginosa biofilms. It is reported that a synthetic halogenated furanone compound, which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra, is capable of interfering with AHL-mediated quorum sensing in P. aeruginosa. It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis. In addition, it reduces the production of important virulence factors, indicating a general effect on target genes of the las quorum sensing circuit. The furanone was applied to P. aeruginosa biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does, however, affect the architecture of the biofilm and enhances the process of bacterial detachment, leading to a loss of bacterial biomass from the substratum.

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans -produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis . Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.

During the course of chronic cystic fibrosis (CF) infections, Pseudomonas aeruginosa undergoes a conversion to a mucoid phenotype, which is characterized by overproduction of the exopolysaccharide alginate. Chronic P. aeruginosa infections involve surface-attached, highly antibiotic-resistant communities of microorganisms organized in biofilms. Although biofilm formation and the conversion to mucoidy are both important aspects of CF pathogenesis, the relationship between them is at the present unclear. In this study, we report that the overproduction of alginate affects biofilm development on an abiotic surface. Biofilms formed by an alginateoverproducing strain exhibit a highly structured architecture and are significantly more resistant to the antibiotic tobramycin than a biofilm formed by an isogenic nonmucoid strain. These results suggest that an important consequence of the conversion to mucoidy is an altered biofilm architecture that shows increasing resistance to antimicrobial treatments.

Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces N-acylhomoserine lactones (AHLs) due to an inactivation of the cepR gene. cepR and the cepI AHL synthase gene together constitute the cep quorum-sensing system of B. cepacia. By using a gene replacement method, two defined mutants, H111-I and H111-R, were constructed in which cepI and cepR, respectively, had been inactivated. These mutants were used to demonstrate that biofilm formation by B. cepacia H111 requires a functional cep quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment, but rather controls the maturation of the biofilm. Furthermore, it is shown that B. cepacia is capable of swarming motility, a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of B. cepacia is quorum-sensing-regulated, possibly through the control of biosurfactant production. Complementation of the cepR mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility per se is not essential for biofilm formation on abiotic surfaces.

SummaryReversible phase variation between the rugose and smooth colony variants is predicted to be important for the survival of Vibrio cholerae in natural aquatic habitats. Microarray expression profiling studies of the rugose and smooth variants of the same strain led to the identification of 124 differentially regulated genes. Further expression profiling experiments showed how these genes are regulated by the VpsR and HapR transcription factors, which, respectively, positively and negatively regulate production of VPS El Tor , a rugose-associated extracellular polysaccharide. The study of mutants of rpoN and rpoS demonstrated the effects of these alternative sigma factors on phase variation-specific gene expression. Bioinformatics analysis of these expression data shows that 'rugosity' and 'smoothness' are determined by a complex hierarchy of positive and negative regulators, which also affect the biofilm, surface hydrophobicity and motility phenotypes of the organism.

In this study, stratified patterns of protein synthesis and growth were demonstrated in Pseudomonas aeruginosa biofilms. Spatial patterns of protein synthetic activity inside biofilms were characterized by the use of two green fluorescent protein (GFP) reporter gene constructs. One construct carried an isopropyl-␤-Dthiogalactopyranoside (IPTG)-inducible gfpmut2 gene encoding a stable GFP. The second construct carried a GFP derivative, gfp-AGA, encoding an unstable GFP under the control of the growth-rate-dependent rrnBp 1 promoter. Both GFP reporters indicated that active protein synthesis was restricted to a narrow band in the part of the biofilm adjacent to the source of oxygen. The zone of active GFP expression was approximately 60 m wide in colony biofilms and 30 m wide in flow cell biofilms. The region of the biofilm in which cells were capable of elongation was mapped by treating colony biofilms with carbenicillin, which blocks cell division, and then measuring individual cell lengths by transmission electron microscopy. Cell elongation was localized at the air interface of the biofilm. The heterogeneous anabolic patterns measured inside these biofilms were likely a result of oxygen limitation in the biofilm. Oxygen microelectrode measurements showed that oxygen only penetrated approximately 50 m into the biofilm. P. aeruginosa was incapable of anaerobic growth in the medium used for this investigation. These results show that while mature P. aeruginosa biofilms contain active, growing cells, they can also harbor large numbers of cells that are inactive and not growing.Biofilms are communities of microorganisms that are associated with surfaces. Cell clusters in biofilms are known to be characterized by gradients in the concentrations of oxygen, nutrients, and metabolic waste products. It is widely recognized that such chemical heterogeneity in microbial biofilms can lead to microorganisms in the biofilm exhibiting different rates of growth and metabolic activity. Even pure cultures of microorganisms growing in biofilms experience these gradients and may exist in a range of metabolic states. The variety of growth states that can be represented in a biofilm, even for a single species, surely contributes to the special ecology and antimicrobial tolerance manifested by biofilms. Given the fundamental significance of growth status, it is surprising that there have been few investigations in which the growth patterns in biofilms have been visualized.Several studies have investigated spatial patterns of cellular activity inside biofilms, using approaches such as staining with nucleic acid dyes that differentially indicate DNA and RNA (17), hybridization to 16S rRNA with fluorescently tagged oligonucleotide probes (9), the induction of alkaline phosphatase followed by staining with a fluorogenic phosphatase substrate (8,19), and green fluorescent protein (GFP) expression from a growth-rate-dependent promoter (11). These previous investigations have revealed gradients in metabolic activity in biofilms.The purpose ...