Jost Wingender

The biofilm matrix

The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.

What are Bacterial Extracellular Polymeric Substances

The vast majority of microorganisms live and grow in aggregated forms such as biofilms and flocs (“planktonic biofilms”). This mode of existence is lumped in the somewhat inexact but generally accepted expression “biofilm”. The common feature of all these phenomena is that the microorganisms are embedded in a matrix of extracellular polymeric substances (EPS). The production of EPS is a general property of microorganisms in natural environments and has been shown to occur both in prokaryotic (Bacteria, Archaea) and in eukaryotic (algae, fungi) microorganisms. Biofilms containing mixed populations of these organisms are ubiquitously distributed in natural soil and aquatic environments, on tissues of plants, animals and man as well as in technical systems such as filters and other porous materials, reservoirs, plumbing systems, pipelines, ship hulls, heat exchangers, separation membranes, etc. (Costerton et al. 1987; 1995; Flemming and Schaule 1996). Biofilms develop adherent to a solid surface (substratum) at solid-water interfaces, but can also be found at water-oil, water-air and solid-air interfaces. Biofilms are accumulations of microorganisms (prokaryotic and eukaryotic unicellular organisms), EPS, multivalent cations, biogenic and inorganic particles as well as colloidal and dissolved compounds. EPS are mainly responsible for the structural and functional integrity of biofilms and are considered as the key components that determine the physicochemical and biological properties of biofilms.

The role of intermolecular interactions: studies on model systems for bacterial biofilms.

The mechanical stability of biofilms and other microbial aggregates is of great importance for both the maintenance of biofilm processes and the removal of undesired biofilms. The binding forces are weak interactions such as London dispersion forces, electrostatic interactions and hydrogen bonds. In a first attempt to rank their contribution, the viscosity of solutions of extracellular polymeric substances (EPS) from a mucoid strain of Pseudomonas aeruginosa is measured. In order to distinguish the binding forces, substances are chosen which individually address the different types of bonds. Polyacrylic acid is identified as a suitable model system for EPS when molecular interactions are studied. Electrostatic interactions and hydrogen bonds are found to be the dominating forces among macromolecules within the biofilm.

Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa.

Fluorescently labelled lectins were used in combination with epifluorescence microscopy and confocal laser scanning microscopy to allow the visualization and characterization of carbohydrate-containing extracellular polymeric substances (EPS) in biofilms of Pseudomonas aeruginosa. A mucoid strain characterized by an overproduction of the exopolysaccharide alginate, and an isogenic, non-mucoid strain were used. Model biofilms grown on polycarbonate filters were treated with lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) that were fluorescently labelled with fluorescein isothiocyanate or tetramethyl rhodamine isothiocyanate. Fluorescently labelled ConA yielded cloud-like regions that were heterogeneously distributed within mucoid biofilms, whereas these structures were only rarely present in biofilms of the non-mucoid strain. The bacteria visualized with the fluorochrome SYTO 9 were localized both within and between the ConA-stained regions. In WGA-treated biofilms, the lectin was predominantly associated with bacterial cells. Alginate seemed to be involved in the interaction of ConA with the EPS matrix, since (i) pre-treatment of biofilms with an alginate lyase resulted in a loss of ConA biofilm staining, and (ii) using an enzyme-linked lectinsorbent assay (ELLA), ConA was shown to bind to purified alginate, but not to alginate that was degraded by alginate lyase. The application of fluorescently labelled lectins in combination with ELLA was found to be useful for the visualization and characterization of extracellular polysaccharide structures in P. aeruginosa biofilms.

Metagenome survey of biofilms in drinking-water networks

ABSTRACT Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria. Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia. Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria. The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.

Uniaxial compression measurement device for investigation of the mechanical stability of biofilms.

The mechanical stability of biofilms is important for biotechnology, as sloughing of the biomass due to mechanical failure of the biofilm matrix can lead to severe interferences with biofilm processes. In cases of biofouling, biofilms have to be removed, in which case their mechanical stability must be overcome. The apparent modulus of elasticity and the yield strength as obtained from uniaxial compression experiments can be taken as parameters indicative for the mechanical stability of a biofilm. A film rheometer is presented which allows for the determination of these quantities, using model biofilms of Pseudomonas aeruginosa grown on membrane filters. The compressive stress-strain behaviour up to the point of failure is recorded at a compression speed of 1 microm s(-1). In accordance with the stress-strain curve, the investigated biofilm can be described as viscoelastic material, which demonstrates plastic flow properties. The extracellular polymeric substances (EPS), which keep biofilms together, form a temporary network of fluctuating junction points. Above the yield point, the gel structure fails and the system behaves as a highly viscous fluid. The apparent modulus of elasticity and the yield point are considered to be useful parameters for characterizing the mechanical properties of biofilms.

Community structure and co-operation in biofilms: Cohesiveness in biofilm matrix polymers

A review with 60 refs. Topics discussed include microbial aggregates, the role of extracellular polymeric substances (EPS) as microbial aggregate construction material, the cohesiveness of matrix polymers, and the role of EPS in microbial aggregation.

Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa.

Publisher Summary This chapter discusses that Biofilms of Pseudomonas aeruginosa have been intensively studied during the last decade. Pseudomonas aeruginosa offers several advantages as a model organism in biofilm research. This gram-negative bacterium is well characterized with respect to its molecular genetics, biochemistry, and physiology; it is also of hygienic relevance as an opportunistic pathogen, because biofilms harboring this bacterium in technical water systems and on medical devices may be a source of human infections. The extracellular polymeric substances (EPS) of P. aeruginosa predominantly consist of different polysaccharides and proteins. Mucoid variants of P. aeruginosa are characterized by an overproduction of the viscous exopolysaccharide alginate, resulting in the production of large slimy colonies, when the bacteria are cultivated overnight on common agar-based media. Alginates from P. aeruginosa are high molecular weight unbranched copolymers consisting of (1→ 4)-linked uronic acid residues of β- D -mannuronate and α- L -guluronate. The formation of biofilms is regarded as part of a natural “life cycle” of mucoid P. aeruginosa. Alginates represent major components of the EPS of mucoid P. aeruginosa and have been implicated in the development as well as the maintenance of the mechanical stability of biofilms formed by P aeruginosa on living and abiotic surfaces. In this chapter, methods of isolation and biochemical characterization are described for both whole EPS and alginate from mucoid strains of P. aeruginosa grown as a confluent bacterial lawn on agar media as simple in vitro model systems for bacterial biofilms.

Contamination of drinking water by coliforms from biofilms grown on rubber-coated valves

In water samples from drinking water distribution systems, coliform bacteria (predominantly Citrobacter species) were repeatedly detected. Disinfection and flushing of the systems did not erase the problem. The pattern of the coliform occurrences indicated contamination originating from biofilms. After inspection of internal surfaces of the systems, no significant biofilm growth was observed on pipe surfaces, but in a number of cases, visible biofilms were detected on rubber-coated valves which harboured the same coliform species as those found in the drinking water samples. In these cases, the rubber-coated valves seemed to act as point sources for the contamination of water.

Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in the nutrition of the bacterium. As most studies concerning extracellular enzymes have been performed on planktonic cultures of non-mucoid P. aeruginosa strains, knowledge of the potential role of these enzymes in biofilm formation in mucoid (alginate-producing) P. aeruginosa remains limited. Here we show that mucoid P. aeruginosa produces extracellular hydrolases during biofilm growth. Overexpression of the extracellular lipases LipA and LipC, the esterase EstA and the proteolytic elastase LasB from plasmids revealed that some of these hydrolases affected the composition and physicochemical properties of the extracellular polymeric substances (EPS). While no influence of LipA was observed, the overexpression of estA and lasB led to increased concentrations of extracellular rhamnolipids with enhanced levels of mono-rhamnolipids, elevated amounts of total carbohydrates and decreased alginate concentrations, resulting in increased EPS hydrophobicity and viscosity. Moreover, we observed an influence of the enzymes on cellular motility. Overexpression of estA resulted in a loss of twitching motility, although it enhanced the ability to swim and swarm. The lasB-overexpression strain showed an overall enhanced motility compared with the parent strain. Moreover, the EstA- and LasB-overproduction strains completely lost the ability to form 3D biofilms, whereas the overproduction of LipC increased cell aggregation and the heterogeneity of the biofilms formed. Overall, these findings indicate that directly or indirectly, the secreted enzymes EstA, LasB and LipC can influence the formation and architecture of mucoid P. aeruginosa biofilms as a result of changes in EPS composition and properties, as well as the motility of the cells.