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

▪ Abstract Quorum sensing is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological activities. These processes include symbiosis, virulence, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. In general, Gram-negative bacteria use acylated homoserine lactones as autoinducers, and Gram-positive bacteria use processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between bacterial species. Furthermore, there is mounting data suggesting that ba...

Quorum Sensing in Bacteria

Bacteria in nature often exist as sessile communities called biofilms. These communities develop structures that are morphologically and physiologically differentiated from free-living bacteria. A cell-to-cell signal is involved in the development of Pseudomonas aeruginosa biofilms. A specific signaling mutant, a lasI mutant, forms flat, undifferentiated biofilms that unlike wild-type biofilms are sensitive to the biocide sodium dodecyl sulfate. Mutant biofilms appeared normal when grown in the presence of a synthetic signal molecule. The involvement of an intercellular signal molecule in the development of P. aeruginosa biofilms suggests possible targets to control biofilm growth on catheters, in cystic fibrosis, and in other environments where P. aeruginosa biofilms are a persistent problem.

https://science.sciencemag.org/content/sci/280/5361/295.full.pdf

The involvement of cell-to-cell signals in the development of a bacterial biofilm

Prokaryotic biofilms that predominate in a diverse range of ecosystems are often composed of highly structured multispecies communities. Within these communities metabolic activities are integrated, and developmental sequences, not unlike those of multicellular organisms, can be detected. These structural adaptations and interrelationships are made possible by the expression of sets of genes that result in phenotypes that differ profoundly from those of planktonically grown cells of the same species. Molecular and microscopic evidence suggest the existence of a succession of de facto biofilm phenotypes. We submit that complex cell-cell interactions within prokaryotic communities are an ancient characteristic, the development of which was facilitated by the localization of cells at surfaces. In addition to spatial localization, surfaces may have provided the protective niche in which attached cells could create a localized homeostatic environment. In a holistic sense both biofilm and planktonic phenotypes may be viewed as integrated components of prokaryote life.

Biofilms as complex differentiated communities.

According to a public announcement by the US National Institutes of Health
 , “Biofilms are medically important, accounting for over 80% of microbial infections in the body”. Yet bacterial biofilms remain poorly understood and strategies for their control remain underdeveloped. Standard antimicrobial treatments typically fail to eradicate biofilms, which can result in chronic infection and the need for surgical removal of afflicted areas. The need to create effective therapies to counter biofilm infections presents one of the most pressing challenges in anti-bacterial drug development. In this article, the mechanisms that underlie biofilm resistance to antimicrobial chemotherapy will be examined, with particular attention being given to potential avenues for the effective treatment of biofilms.

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Understanding biofilm resistance to antibacterial agents.

Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

It is well established that in nature, bacteria are found primarily as residents of surface-associated communities called biofilms. These structures form in a sequential process initiated by attachment of cells to a surface, followed by the formation of matrix-enmeshed microcolonies, and culminating in dispersion of the bacteria from the mature biofilm. In the present study, we have demonstrated that, during growth, Pseudomonas aeruginosa produces an organic compound we have identified as cis-2-decenoic acid, which is capable of inducing the dispersion of established biofilms and of inhibiting biofilm development. When added exogenously to P. aeruginosa PAO1 biofilms at a native concentration of 2.5 nM, cis-2-decenoic acid was shown to induce the dispersion of biofilm microcolonies. This molecule was also shown to induce dispersion of biofilms, formed by Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Bacillus subtilis, Staphylococcus aureus, and the yeast Candida albicans. Active at nanomolar concentrations, cis-2-decenoic acid appears to be functionally and structurally related to the class of short-chain fatty acid signaling molecules such as diffusible signal factor, which act as cell-to-cell communication molecules in bacteria and fungi.

David G. Davies

Papers

The involvement of cell-to-cell signals in the development of a bacterial biofilm

Biofilms as complex differentiated communities.

Understanding biofilm resistance to antibacterial agents.

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

A Fatty Acid Messenger Is Responsible for Inducing Dispersion in Microbial Biofilms