No abstract

The extracellular matrix is a complex and extremely important component of all biofilms, providing architectural structure and mechanical stability to the attached population. The matrix is composed of cells, water and secreted/released extracellular macromolecules. In addition, a range of enzymic and regulatory activities can be found within the matrix. Together, these different components and activities are likely to interact and in so doing create a series of local environments within the matrix which co-exist as a functional consortium. The matrix architecture is also subject to a number of extrinsic factors, including fluctuations in nutrient and gaseous levels and fluid shear. Together, these intrinsic and extrinsic factors combine to produce a dynamic, heterogeneous microenvironment for the attached and enveloped cells.

Cultures of two strains of freshwater bacterial isolates adhered readily to inert glass surfaces exposed in the growth medium. The process of microbial film formation could be followed by a new staining technique based on congo red, a dye specific for carbohydrate material. In conjunction with a chemical assay for total carbohydrate, the association of extracellular polysaccharides with attached cells was demonstrated. Under optimal growth conditions, the involvement of exopolysaccharide in the adhesion process appeared to follow the initial attachment of bacterial cells, leading to the formation of microcolonies enmeshed in polysaccharides. A non-polysaccharide-producing mutant attached to glass slides in numbers similar to the wild-type bacteria, but did not form microcolonies. Growth conditions such as glucose or Ca2+ limitation which affected polysaccharide synthesis in the wild-type prevented microcolony formation, but not cell attachment. It is proposed that exopolysaccharide production is involved in the development of the surface films, but possibly not in the initial adhesion process. In those strains which do produce polysaccharide, the cells which attach develop into microcolonies.

Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21³C. Biofilm formation was maximal ca. 20^50 h after inoculation of the liquid medium and, as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spent culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C T ) exogenous homoserines can trigger such responses in P. fluorescens, its own signal substance is likely to possess a longer ( s C V ) fatty acyl chain. z

The effect of concentrated cell-free extracellular material from stationary-phase cultures of Burkholderia cepacia 10661 and Pseudomonas aeruginosa PAO1 on virulence factor production in B. cepacia was assessed. While increasing concentrations of the B. cepacia exoproduct caused a slight increase in siderophore, lipase, and protease production in the producing organism, a significant increase in productivity was observed for all three virulence factors with the addition of the PAO1 exoproduct. Moreover, the addition of the exoproduct from a strain of P. aeruginosa producing reduced amounts of autoinducer caused only a slightly greater response than that of the control. Both B. cepacia 10661 and P. aeruginosa PAO1, along with two matched clinical isolates of both organisms obtained from a cystic fibrotic patient, were shown to produce variable amounts of three different types of autoinducer. The potential for interspecies signalling in microbial pathogenicity is discussed.Burkholderia cepacia, formerly known as Pseudomonas cepacia, is now recognized as an important opportunistic agent of human disease (6,8). In particular, it has received a great deal of attention owing to its increasing association with fatal pulmonary infections in patients with cystic fibrotic (CF) lung disease (9, 13). Clinically, B. cepacia colonization of CF patients can result in an asymptomatic carriage, a slow and continuous decline in lung function, or third, a rapid deterioration of the lung accompanied by fever, necrotizing pneumonia, and in some cases, bacteremia (6, 9). This third syndrome is not observed with other CF pathogens. Generally, it has been noted that B. cepacia colonizes the lung after infection by other microorganisms (24,25). Indeed, in a recent survey of the population with CF lung disease in Greater Manchester, England, only 3.3% of patients were colonized exclusively with B. cepacia (11). Instead, over 80% of the patients were cocolonized with P. aeruginosa. Whereas P. aeruginosa produces a panoply of virulence factors which play an active role in the organism's pathogenicity (7,26), little is known about the pathophysiology of B. cepacia (5,14). Isolated strains of B. cepacia are variable in their abilities to produce hemolysins, lipase and protease (5), exopolysaccharide (2, 17), and ironchelating siderophores (12, 21) in vitro. Consequently, there has been no direct correlation with any of these virulence factors to the organism's pathogenic status in vivo. However, a novel possibility might be the active coaggregation of B. cepacia and P. aeruginosa in the lungs, whereby one species synergistically enhances the virulence determinants of the other. To this effect, the enhancement of B. cepacia attachment to different surfaces by P. aeruginosa exoproducts has been demonstrated (1, 18).It is now recognized that individual cells within a multicellular system can both generate signals and respond to those produced by the surrounding cells, providing a basis for cells to change in response to prevailing environmental...