Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

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Bacterial biofilms : A common cause of persistent infections

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.

The biofilm matrix

Biofilms--matrix-enclosed microbial accretions that adhere to biological or non-biological surfaces--represent a significant and incompletely understood mode of growth for bacteria. Biofilm formation appears early in the fossil record (approximately 3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages, including the 'living fossils' in the most deeply dividing branches of the phylogenetic tree. It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments. Recent advances show that biofilms are structurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments and also disperse to colonize new niches. The implications of these survival and propagative mechanisms in the context of both the natural environment and infectious diseases are discussed in this review.

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Bacterial biofilms: from the natural environment to infectious diseases.

Though biofilms were first described by Antonie van Leeuwenhoek, the theory describing the biofilm process was not developed until 1978. We now understand that biofilms are universal, occurring in aquatic and industrial water systems as well as a large number of environments and medical devices relevant for public health. Using tools such as the scanning electron microscope and, more recently, the confocal laser scanning microscope, biofilm researchers now understand that biofilms are not unstructured, homogeneous deposits of cells and accumulated slime, but complex communities of surface-associated cells enclosed in a polymer matrix containing open water channels. Further studies have shown that the biofilm phenotype can be described in terms of the genes expressed by biofilm-associated cells. Microorganisms growing in a biofilm are highly resistant to antimicrobial agents by one or more mechanisms. Biofilm-associated microorganisms have been shown to be associated with several human diseases, such as native valve endocarditis and cystic fibrosis, and to colonize a wide variety of medical devices. Though epidemiologic evidence points to biofilms as a source of several infectious diseases, the exact mechanisms by which biofilm-associated microorganisms elicit disease are poorly understood. Detachment of cells or cell aggregates, production of endotoxin, increased resistance to the host immune system, and provision of a niche for the generation of resistant organisms are all biofilm processes which could initiate the disease process. Effective strategies to prevent or control biofilms on medical devices must take into consideration the unique and tenacious nature of biofilms. Current intervention strategies are designed to prevent initial device colonization, minimize microbial cell attachment to the device, penetrate the biofilm matrix and kill the associated cells, or remove the device from the patient. In the future, treatments may be based on inhibition of genes involved in cell attachment and biofilm formation.

Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

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Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

https://www.cell.com/trends/microbiology/pdf/S0966-842X(00)01913-2.pdf

Mechanisms of biofilm resistance to antimicrobial agents

▪ Abstract Biofilms can be defined as communities of microorganisms attached to a surface. It is clear that microorganisms undergo profound changes during their transition from planktonic (free-swimming) organisms to cells that are part of a complex, surface-attached community. These changes are reflected in the new phenotypic characteristics developed by biofilm bacteria and occur in response to a variety of environmental signals. Recent genetic and molecular approaches used to study bacterial and fungal biofilms have identified genes and regulatory circuits important for initial cell-surface interactions, biofilm maturation, and the return of biofilm microorganisms to a planktonic mode of growth. Studies to date suggest that the planktonic-biofilm transition is a complex and highly regulated process. The results reviewed in this article indicate that the formation of biofilms serves as a new model system for the study of microbial development.

Biofilm Formation as Microbial Development

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.

Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development

Populations of surface-attached microorganisms comprising either single or multiple species are commonly referred to as biofilms. Using a simple assay for the initiation of biofilm formation (e.g. attachment to an abiotic surface) by Pseudomonas fluorescens strain WCS365, we have shown that: (i) P. fluorescens can form biofilms on an abiotic surface when grown on a range of nutrients; (ii) protein synthesis is required for the early events of biofilm formation; (iii) one (or more) extracytoplasmic protein plays a role in interactions with an abiotic surface; (iv) the osmolarity of the medium affects the ability of the cell to form biofilms. We have isolated transposon mutants defective for the initiation of biofilm formation, which we term surface attachment defective (sad). Molecular analysis of the sad mutants revealed that the ClpP protein (a component of the cytoplasmic Clp protease) participates in biofilm formation in this organism. Our genetic analyses suggest that biofilm formation can proceed via multiple, convergent signalling pathways, which are regulated by various environmental signals. Finally, of the 24 sad mutants analysed in this study, only three had defects in genes of known function. This result suggests that our screen is uncovering novel aspects of bacterial physiology.

George A. O'Toole

Papers

Mechanisms of biofilm resistance to antimicrobial agents

Biofilm Formation as Microbial Development

Microbial Biofilms: from Ecology to Molecular Genetics

Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development

Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis