INTRODUCTION

Most biological sciences had defined their subject organisms, grossly and at the microscopic level, long before the advent of molecular biology. For this reason, sciences like botany had characterized the structure, even at the ultrastructural level, of the cells and tissues that comprise all of the forms adopted by plants, including all vegetative and reproductive manifestations of thousands of organisms. Therefore, the detailed molecular mechanisms discovered by modern plant molecular biologists fit nicely into a framework of understanding of exactly where these mechanisms operate, in terms of the structure and function of the organism concerned. We visualize the process of transcription occurring on the cytoplasmic aspect of the endoplasmic reticulum, and we can follow the resultant peptides through their various fates, including excretion in vesicles via the Golgi apparatus. The intellectual synthesis within a field like botany is essentially very satisfying, because we could previously see the central genetic control of shape and function, and now we can understand the molecular mechanisms by which this control is exercised and we can manipulate plants using this knowledge.

The relatively new science of microbiology, which has done so much to eradicate epidemic diseases and to improve our lives, is presently reeling in confusion because it has not followed the same orderly development of concepts and techniques. At a very early stage in its development, microbiology embraced two moda operandi that served it well, but damaged it in the long term. First, following Robert Koch, we removed bacterial cells from the ecosystems in which they lived, and grew them in monospecies cultures in fluids or on agar.

INTRODUCTION

A perusal of the published literature might lead the naïve to believe that microbial biofilms are definable entities each of which, whilst possessing characteristics that are markedly dissimilar from their planktonic equivalents, has its own defined physiology and architecture. Thus it is easy to imagine, rather like for our own bodies, that the visible, physical manifestation has a degree of permanency. The reality is that the structures known as biofilms, as well as the majority of tissues that make up our bodies, are in a state of dynamic flux/turnover. Modern imaging techniques generally give us snapshots in time and only rarely give an indication of the turmoil within (Costerton et al., 1995).

It is the intention of this article to dispel any view the reader might have entertained of biofilms being static entities. Dynamics within microbial biofilm communities will be considered in terms of their spatial stability (movement/drift), and changes in biomass, genetic diversity and community function. In the latter respects, population dynamics will be considered for both short-term events, such as the formation and establishment of biofilm communities on ‘virgin’ surfaces (Geesey et al., 1992), and long-term events, with biofilms being considered as units of proliferation/evolution (Caldwell et al., 1997).

Dynamics in microbial communities can be considered on a number of distinct levels, many of which have been considered separately in this symposium volume. The first of these relates to the spatial stability of the biofilm community. Matrix polymers not only glue the biofilm to the surface but also enable spatial organization to be imposed on the community. The polymers are plastic and may be physically deformed through the imposition of shear forces from the fluid phase.

COAGGREGATION AND COADHESION

Certain molecules on the surfaces of human oral bacteria can be recognized by cognate surface components of genetically distinct cells, which bind to form networks of cell–cell interactions. When these interactions occur in suspension, they are called coaggregations (Kolenbrander, 1988). When the interaction occurs between suspended or planktonic cells and already adherent cells, it is called coadhesion (Bos et al., 1994). Coadhesion may involve the accretion of an already formed coaggregate onto a biofilm, which is an assemblage of living cells on a substratum, or onto a virgin surface.

Coaggregation among human oral bacteria was first described 30 years ago (Gibbons & Nygaard, 1970). Coaggregation is measured by several methods, including visual inspection of clumps or coaggregates after mixing dense suspensions of two cell types (Gibbons & Nygaard, 1970), turbidometric measurement of supernatant after slowspeed centrifugation to pellet the coaggregates (McIntire et al., 1978), filtration through specific pore size to separate single cells from coaggregates (Lancy et al., 1980), distribution of radiolabelled cells of one cell type in coaggregates and supernatant after slow-speed centrifugation (Kolenbrander & Andersen, 1986) and binding of a radiolabelled cell type to partner cells immobilized on a nitrocellulose membrane (Lamont & Rosan, 1990). Coaggregations may be unimodal or bimodal (Kolenbrander, 1997). Unimodal coaggregations involve protease-sensitive molecules on the cell surface of one of the partners recognizing their cognate receptors (protease-insensitive) on the other partner's cell surface. Bimodal coaggregations involve more than one of the unimodal mechanisms. For example, one partner expresses both an adhesin and a non-cognate receptor. Its partner expresses the respective cognates for the adhesin and receptor.

INTRODUCTION TO BIOFILM STRUCTURE

The general view on biofilm structure has dramatically changed during the last decade. It had been previously assumed that most biofilms are more or less homogeneous layers of micro-organisms in a slime matrix. The use of confocal scanning laser microscopy (CSLM) and computerized image analysis tools has revealed a more complex picture of biofilm morphology (Lawrence et al., 1991; Caldwell et al., 1993) and structural heterogeneities (Costerton et al., 1994; Gjaltema et al., 1994). Cell clusters may be separated by interstitial voids and channels, which create a characteristic porous structure. In some cases, biofilms grow as clusters taking a ‘mushroom’ shape, whilst in others, more compact and homogeneous layers can be observed.

Types of biofilm heterogeneity

In many biofilms, the reported non-uniformities are unidirectional and perpendicular to the substratum. Three-dimensional variation of microbial species, biofilm porosity, substrate concentration and diffusivity have been repeatedly reported in biofilm. It is becoming clear that there are many forms of heterogeneity in biofilms, and a definition of biofilm heterogeneity is needed. According to Bishop & Rittmann (1995), heterogeneity may be defined as ‘spatial differences in any parameter we think is important’. An adapted list from Bishop & Rittmann (1995) summarizes a few examples of possible biofilm heterogeneity:

1. (1) Geometrical heterogeneity: biofilm thickness, biofilm surface roughness, biofilm porosity and substratum surface coverage.

2. (2) Chemical heterogeneity: diversity of chemical solutes (nutrients, metabolic products, inhibitors), pH variations, diversity of reactions (aerobic/anaerobic, etc.).

3. (3) Biological heterogeneity: microbial diversity of species and their spatial distribution, differences in activity [growing cells, extracellular polymeric substances (EPS) producers, dead cells, etc.].

4. […]

INTRODUCTION

In nature, colonization of habitats by mixtures of bacterial populations is the rule rather than the exception. Diverse groups of micro-organisms are invariably isolated from samples from environmentally exposed habitats. Evidence is accumulating that such mixtures of organisms are not merely passive neighbours but that they are involved in a wide range of dynamic physical and metabolic interactions. Indeed, these interactions appear to be essential for the attachment, growth and survival of species at a site, and also enable organisms to persist in what often appear to be overtly hostile environments.

Such interacting mixtures of micro-organisms are termed microbial communities, and are generally found on a surface, spatially organized as a biofilm (see other chapters in this volume). Microbial communities have been described from habitats ranging from aquatic environments, anaerobic digesters, plant surfaces and soil particles, to the digestive tract of humans and animals. Although the nomenclature of the species from these diverse habitats is different, this chapter will demonstrate that the function (or niche; Alexander, 1971) of the community members is often similar.

Existence within a microbial community can have profound consequences for the component populations. These include (a) a broader habitat range for colonization, (b) an increased metabolic diversity and efficiency, so that substrates normally recalcitrant to catabolism by individual organisms can often be broken down by consortia to simpler products, (c) increased resistance to environmental stress and to host defence factors (Caldwell et al., 1997a, b; Shapiro, 1998) and, in some cases, (d) an increased ability to cause disease (pathogenic synergism; van Steenbergen et al., 1984; Brook, 1987a).

INTRODUCTION

Less than 50 years of molecular biology have documented that a few thousand genes suffice to construct prokaryotic life forms, which could be argued to be the most successful of all life forms throughout all biological evolution. These were the first to appear, and most likely the last to sustain as living organisms on earth. Molecular biology has also taught us that one explanation for the success of bacteria is the continuous evolution of not only new traits, but also of new regulatory elements, making these organisms very adaptable to a range of environments and to shifts between these. Expression of a large fraction of the genes in modern bacterial genomes is controlled at several levels – from the very specific metabolite-directed derepression scheme to global regulatory loops controlling the overall state of the cell and its interactions with the environment. Through complex webs of signal-transduction events bacteria sense their environment, and react to a large number of factors by finetuning expression of a large number of genes in apparently ‘intelligent’ ways.

It is therefore an almost logical extrapolation to assume that these simple, but highly regulated, organisms may also possess the capacity to engage in multicellular system constructions, in which features of co-ordinated organizational designs develop. The embryonic development of compartmentalized organisms with different organs and tissues from the undifferentiated fertilized egg-cell may have its parallel in prokaryotic organisms forming highly structured communities with distributed functionality based on a programmed, progressive differentiation process.

INTRODUCTION

Bacteria have been shown to exist predominantly in nature as sessile populations attached to surfaces in contact with water (Geesey et al., 1977). The numbers of bacteria attached to surfaces have been estimated to be between 1000 and 10000 times greater than the numbers of planktonic bacteria in any given environment (Watkins & Costerton, 1984).

The advantages to the micro-organisms of being attached to a surface have been largely attributed to enhanced scavenging of nutrients from both the bulk water and from the substratum with which the bacteria are associated (Marshall, 1976). Microorganisms at surfaces are considered to be exposed to higher nutrient conditions than their planktonic counterparts and, therefore, are at an advantage in the attached state. As a demonstration of this, Kjelleberg et al. (1982) exposed starved planktonic and attached marine bacteria to 2 mg l−1 of both yeast extract and tryptone. The attached population was able to grow under low nutrient conditions, while the planktonic population was not. Further investigations revealed that when adsorbed to surfaces, both low (Power & Marshall, 1988) and high (Samuelsson & Kirchman, 1990) molecular mass compounds were available for microbial growth. These and other studies strengthened the idea that differences in growth rates of surface-associated bacteria and planktonic bacteria were primarily due to differences in nutrient availability. Thus attached bacteria, while nutritionally at an advantage over their planktonic counterparts, have for many years been considered to be similar in other respects to non-attached bacteria.

Continued research began to expose differences between planktonic and attached bacteria that implied physiological alterations following attachment to a surface.

INTRODUCTION

A vast number of microbial aggregates fall into this ‘catch-all’ name – biofilm. Whether this unifying term does a great service or the opposite to a core branch of microbiology is open to some doubt – for biofilm is found in almost every environment graced with surfaces, sufficient nutrient and some water. A gentle digital examination of the waste outlet of the average kitchen sink will reveal a certain sliminess which embraces the quintessential soul of a biofilm! That ‘dirt’ which can block car windscreen washer jets is from the same stable. It does not seem necessary to list all possible examples of such structures. They range from growth on the leads of cardiac pacemakers, through biofilm attached to the inner surfaces of water distribution pipes, to the epilithon of rocks in streams and accumulated plaque on the surface of teeth.

There are almost as many definitions of biofilm as there are scientists working in the field or types of the structure itself. Any reasonable definition needs to incorporate the idea of a surface or interface on or at which microbes proliferate; it should also invoke the unifying effect of extracellular polymers which can envelop and probably protect the microbial colonies forming. It might also embrace a sense of community with the implication of emergent properties.

It is worth trying to classify a number of microbial systems that seem to be related to biofilm since they share many of its properties. In fact, members of the whole family of microbial aggregates have more in common than separates them (Table 1).

INTRODUCTION

It has been shown that microbial communities contribute extensively to the attenuation, mineralization and transport of both organic and inorganic contaminants in the environment. The development of biofilms by microbial communities is often a key factor contributing to the overall efficiency of these processes (Rothemund et al., 1996). For instance, bacterial biofilms are able to accumulate metals through various mechanisms (Marques et al., 1991; Sillitoe et al., 1994). Liehr et al. (1994) showed that biofilms formed by algae could concentrate metals at levels more than four orders of magnitude higher than those in the surrounding water.

The potential of bioremediation as an alternative to physical and chemical remediation strategies has resulted in a significant amount of research effort on degradative biofilms. Although much emphasis has been placed on the degradation of xenobiotic compounds, the knowledge gained through these studies has also contributed to an improved understanding of processes involved in the degradation of naturally occurring molecules as well as nutrient cycling in general. Tank & Webster (1998) suggested that competition for nutrients might regulate heterotrophic microbial processes in natural streams. In their study, they found that nutrient immobilization by leaves partially inhibited other heterotrophic processes, as evidenced by low microbial respiration, fungal biomass and extracellular enzyme activity among wood biofilms in the presence of leaf litter. Lawrence et al. (1998) stressed the applicability of knowledge gained through the study of naturally occurring attenuation mechanisms to remediating contaminated environments. Clearly, the study of degradative biofilms is of both fundamental and applied interest.

The study of biofilm has been embraced by the microbiological community as it recognizes the profound effect that attachment of cells and cell populations to surfaces has upon their physiology and combined metabolic potential. Particularly, growth of microbial cells as communities, associated with interfaces, has been found to more directly address the many problems and opportunities associated with micro-organisms than do planktonic mono-culture studies. Fifteen years ago, the term ‘biofilm’ was mentioned in the abstracts and titles of approximately one scientific publication per week. Today, such citations occur every few hours and the wealth of literature captured by this umbrella term has burgeoned. The term biofilm is no longer definitive; rather it is an epithet indicative of an organism's or community's relationship to its natural habitat. Biofilm research, particularly at the community level, does not lend itself to reductionist experiments. Rather, the more one approaches the perfect experiment then the less flexible and informative it sometimes becomes! Inevitably, as the complexity of the system is increased then the range of outcomes and their interpretation broaden. In selecting the contributions to this symposium volume, we have tried not only to reflect the dynamic nature of microbial communities but also to represent the wide range of diverse disciplines that have been brought to bear on this topic. We particularly hope that the book and symposium will kindle the ‘biofilm’ spirit in the young researcher.

We would like to thank all of the contributors for their input to both the meeting and to the book, and express our sincere gratitude to Melanie Scourfield of the Society for her efficient and gentle handling of the Editors in the production of this volume.

INTRODUCTION

It is increasingly evident that biofilms growing in a diverse range of medical, industrial and natural environments form a similarly diverse range of complex structures (Stoodley et al., 1999a). These structures often contain water channels which can increase the supply of nutrients to cells in the biofilm (deBeer & Stoodley, 1995) and prompted Costerton et al. (1995) to propose that the water channels may serve as a rudimentary circulatory system of benefit to the biofilm as a whole. This concept suggests that biofilm structure may be controlled, to some extent, by the organisms themselves and may be optimized for a certain set of environmental conditions. To date, most of the research on biofilm structure has been focused on the influence of external environmental factors such as surface chemistry and roughness, physical forces (that is, hydrodynamic shear) or nutrient conditions and the chemistry of the aqueous environment. However, there has been a recent increase in the number of researchers using molecular techniques to study the genetic regulation of biofilm formation and development. Davies et al. (1998) demonstrated that the structure of a Pseudomonas aeruginosa biofilm could be controlled through production of the cell signal (or pheromone) N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). In this paper, we will examine some of the research that has been conducted in our laboratories and those of others on the relative contribution of hydrodynamics, nutrients and cell signalling to the structure and behaviour of bacterial biofilms.

HYDRODYNAMICS

The hydrodynamic conditions of an aquatic environment will determine the transport rate of nutrients and planktonic cells to a surface, the shear stress acting on the biofilm and the rate of erosion of cells from the biofilm.

INTRODUCTION

Horizontal gene transfer is a ubiquitous process that allows DNA sequences to be widely disseminated in natural microbial populations. During horizontal gene transfer, micro-organisms transfer genetic material to organisms other than their descendants, distinguishing the process from vertical gene transfer in which genetic material is passed to offspring via sexual or asexual reproduction. Horizontal gene transfer is most prevalent among bacteria but is not restricted to prokaryotes; highly unrelated organisms can participate in horizontal gene transfer including single- and multi-celled eukaryotes. Presently, little is known about the rates of horizontal gene transfer in both natural and engineered biofilm environments such as water distribution systems, water and wastewater treatment plants, wetlands and biologically active soils and sediments.

Evidence for horizontal gene transfer has been gathered steadily over the last 50 years. Its most prominent manifestation is the spread of antibiotic resistance among bacteria. The genes conferring antibiotic resistance can be quickly disseminated through bacterial populations via horizontal gene transfer, confounding the development of new drugs to treat bacterial infections. Another manifestation of horizontal gene transfer is tumour formation on the surfaces of plant roots after infection by Agrobacterium tumefaciens. This bacterium has been shown to transfer its DNA to plant cells, where the DNA directs the rapid growth of plant-cell tumours and the production of chemicals that nourish the bacteria. From these studies and others, several mechanisms of gene transfer have emerged, which appear to be highly conserved.

Greater understanding of horizontal gene transfer in the environment is needed for multiple purposes. In particular, there has been rapid progress in the creation of genetically engineered micro-organisms (GEMs) to be released into the environment.

INTRODUCTION

Biofilms are notorious for their high level of resistance towards all forms of chemical treatments intended to kill or control their growth. Such resistance to antibiotics, biocides and disinfectants has been attributed to a variety of processes from which a number of dominant mechanisms have been identified. The most frequent attributions of resistance relate to the properties of the extracellular polymer matrix (glycocalyx). The diffusivity of the glycocalyx towards antimicrobial agents is only slightly less than that of water. It does not, therefore, present a significant diffusion barrier in its own right, but can restrict access of antimicrobial to the depths of the biofilm by acting as a substrate for chemically reactive agents or through non-specific binding of highly charged antimicrobial compounds. Additionally, the matrix binds extracellular enzymes, such as β-lactamases and formaldehyde lyase, which are then able to augment the small change in diffusivity through the destruction of susceptible compounds. Factors, other than the glycocalyx, responsible for the resistance properties of biofilms relate to the close proximity of cells. In a dense biofilm community, nutrients and oxygen are preferentially consumed at the periphery. In this respect, gradients of growth rate, associated with different growth-limiting nutrients, will develop across the community. During exposure to antimicrobial agents, the slower-growing cells, at the core of the biofilm and at the colonized surface, will generally out-survive the faster metabolizing, peripheral ones. Slow growth will, in addition, cause the cells to express dormant, starvation phenotypes which often overexpress non-specific defences such as shock proteins, multi-drug efflux pumps (acrAB) and also extracellular polymers.

INTRODUCTION

This chapter reviews the broad area of biofilm detachment, the mechanisms of detachment and the methods used to study this important process. Two case studies are included: the first of these focuses on the control of clinical biofilms; the second case study examines detachment in the water industry.

Biofilms are dynamic structures found in a wide variety of both natural and man-made environments. Their formation has been well studied; for example, Characklis (1990) described eight different stages of biofilm accumulation (Table 1 and Fig. 1). There has been much research into the initial attachment of micro-organisms to surfaces, including the effect of electrostatic interactions and electrochemical forces (Bos et al., 1999). The physiological changes that attaching cells undergo have also been examined; for example, the production of surface appendages such as fimbriae (Austin et al., 1998). In contrast to the work undertaken on attachment, detachment has received little attention although many researchers regard it as a crucial stage of biofilm development (Stewart, 1993; Allison et al., 1999).

Bryers (1988) classified the detachment process into four separate groups: abrasion, grazing, erosion and sloughing. Detachment from the biofilm can be directly caused by the collision or rubbing together of surfaces on which the biofilm has developed, leading to abrasive detachment. Larger organisms feeding on the biofilm can indirectly cause detachment through grazing. Erosion and sloughing refer to physical or chemical processes, which indirectly affect the biofilm structure, leading to detachment. Erosion refers to the continual removal of cells or small groups of cells from the biofilm, whereas sloughing is the loss of discrete amounts of biofilm.

THE USE OF PROSTHETIC DEVICES IN MEDICINE

Prosthetic devices are used to repair or replace a structure or function which has been damaged or is absent as a result of disease, trauma (including surgery) or congenital defect. The limits on their use are considerations of mechanical properties or function, and biocompatibility, and these are progressively being overcome as new biomaterials are developed. However, most prosthetic devices in common use are made from a narrow range of biomaterials, such as silicone elastomer, polyurethanes, fabricated polytetrafluoroethylene (Teflon) or polyethylene terephthalates (Dacron), titanium, stainless steel, ceramics and composite materials containing carbon or glass fibres.

Most prosthetic devices are used in either children or the elderly, and the proportion of the population comprising these two age groups is increasing in most developed countries. It is likely, therefore, that the absolute numbers of prosthetic devices used each year will rise rapidly. Examples of such devices are large joint (hip, knee, shoulder, etc.) replacements for arthritis, spinal fixation to stabilize the spine after cancer or trauma, prosthetic heart valves, pacemakers, vascular grafts for obstructed or weakened major arteries, voice prostheses for those whose larynx has been removed because of cancer or trauma, central venous catheters for parenteral nutrition or anticancer drug administration, catheters for peritoneal dialysis for those with renal failure, and shunts to control hydrocephalus, or pathological accumulation of fluid in the brain.

Incidence and resource implications of device-related infection

While biodeterioration or mechanical dysfunction are important complications, the most feared is infection, which occurs at widely varying rates between devices.