Introduction

The majority of studies on biofilms are concerned with the formation and structure of biofilms (see Korber et al., Chapter 1), the direct effects of microbial activities on surfaces, such as in metal corrosion (see Hamilton, Chapter 9), dental caries (see Marsh, Chapter 18) tissue invasion, or indirect effects of biofilms on fluid frictional resistance, heat exchange across metal surfaces, substrate transformations and biocide resistance of the biofilm organisms on biomaterials implanted in human patients (see McLean et al., Chapter 16) and on water distribution pipeline surfaces (Characklis & Marshall 1990; Anwar et al. 1992). Extensive reviews have dealt with the activities of microorganisms at surfaces (Marshall 1971; Stotzky 1986; van Loosdrecht et al. 1990; Fletcher 1991), but little on the actual genetic responses of microorganisms at surfaces. The aim of this chapter is to consider the environmental conditions existing at gel–air and solid–water interfaces, as well as in biofilms, and to relate these conditions to possible genetic responses in the immobilized bacteria.

Properties of surfaces

Bacteria at surfaces or, more broadly, at interfaces are exposed to environmental conditions not found in an aqueous phase. An interface is defined, in physicochemical terms, as the boundary between two phases in a heterogeneous system. The most common interfaces to which bacteria are exposed in nature and under laboratory conditions are solid–water interfaces (stones, soil particles, ship hulls, pipeline surfaces, glass and plastic culture vessels), although air–water (surface of bodies of water, bubbles), oil–water (oil spills) and solid–air (intertidal zones, agar surfaces) interfaces also are encountered. The concept of bacterial behaviour at various interfaces has been treated in detail by Marshall (1976).

In any scientific examination that addresses a subject as basic as the mode of growth of bacteria it is prudent to begin by considering the successful prokaryotic communities that clearly predated the development of the eukaryotic cell. During the millions of years in which bacteria constituted the only life form on Earth, we visualize an extremely oligotrophic aquatic environment in which specific ecosystems were impacted by many factors (e.g. heat, acid) hostile to their survival. It is the nature of aquatic systems to flow from one ecosystem to another and we can imagine a primitive stream connecting permissive and non-permissive bacterial habitats in the nascent Earth. Once bacterial cells had evolved, the planktonic (floating) mode of growth would deliver them from one habitat to another until they perished in the first non-permissive locus. The sessile mode of growth as attached bacteria would allow these primitive organisms to colonize a permissive habitat and persist therein. Biofilm formation would allow these sessile organisms to trap and retain scarce organic compounds and to develop a focused attack on complex or refractory nutrients whose processing required time and/or the cooperation of one or more bacterial species. Biofilm formation would also change the microenvironment at the colonized surface in a colonized habitat and render its inhabitants less susceptible to hostile chemical, physical, or even biological (e.g. bacteriophage) factors. Each colonized habitat would become a stable crucible of genetic adaption and physiological cooperativity that would flourish in its own location but would also shed its component organisms as planktonic cells so that, if they survived, they could establish a similar integrated biofilm community in any permissive habitat downstream.

Introduction

Cholangitis consists of bacterial infection of bile in the biliary system. The syndrome of acute cholangitis has been well recognized since Charcot (1877) described the classical triad of pain, fever and jaundice in these patients. It is an important cause of abdominal emergency cases and septicaemia with a high rate of morbidity and mortality (Li et al. 1985; French et al. 1990). Biliary obstruction, due to gallstones obstructing the bile ducts, or to benign or malignant stricture of the biliary tract, is an essential element in the development of cholangitis. In the past decade, endoscopic drainage by biliary stenting has become a standard procedure in palliation for inoperable biliary malignancies and some cases of large biliary stones causing obstructive jaundice. Unfortunately, there is an increased incidence of cholangitis related to the blockage of the biliary stents with the use of this technique (Huibregtse et al. 1986; Cotton 1990). Some studies have revealed that the pathogenesis of pigment gallstones (Stewart et al. 1987; Leung et al. 1988), and the blockage of the biliary stents (Leung et al. 1988; Speer et al. 1988) are closely related to the formation of bacterial biofilms in which the glycocalyx enclosed microcolonies coalesce to form an adherent structure (Jacques et al. 1987). This chapter reviews the present knowledge of the microbial ecology of the biliary system, formation of bacterial biofilm from bacterial infection within this system, the pathogenesis of brown biliary pigment stone and blockage of the biliary stent.

Biofilms and the physiology of microorganisms within them

Microbiologists have been accustomed until recently to think of microbes as homogeneous cultures grown in well mixed containers ranging in size from shake flasks to large industrial continuously stirred tank reactors. The investigative tool of choice has been the chemostat which is homogeneous not only in spatial terms but, when operating at a steady state, in time as well.

The only other traditional badge of the microbiologist is the colony. This is much more representative of the natural ecosystem since it is a microbiological aggregate dominated by diffusion gradients. For example oxygen only penetrates about 25–35 μm into a rapidly growing young colony (Wimpenny & Coombs 1983).

Microbial ecosystems are generally spatially heterogeneous implying that solutes move down concentration gradients between sources and sinks. Such gradients are found over a huge range of dimensions, from nanometres for pH gradients around clay lattices to hundreds of metres in the case of oxygen gradients in the Black Sea. These scale factors are illustrated, generally for oxygen, in Table 5.1.

Biofilms have been defined in many different ways; however, perhaps the simplest view is that it is a microbial aggregate that forms at phase interfaces. The most common biofilms appear at solid–water interfaces epitomized by the epilithon that forms on submerged rocks in streams and other water bodies. Generally such biofilm development follows a fairly standard life history. Clean surfaces become coated with a conditioning film consisting of organic molecules, for example proteins or polysaccharides. A little later individual cells attach to the surface, first loosely and reversibly and then firmly and irreversibly.

Introduction

Traditional microbiological investigations have focused on the culture and analysis of pure cell lines of bacteria, in either batch or chemostat culture. However, it has been clearly established that in nature, disease and industry, the majority of bacteria exist attached to surfaces within biofilms (Costerton et al. 1978, 1987; Lappin-Scott & Costerton 1989; Characklis et al. 1990a). Furthermore, it has also been established that the bacteria which exist in biofilms, termed sessile bacteria, are inherently different from bacteria existing in the planktonic state. In the sessile state, bacteria may express different genes, alter their morphologies, grow at different rates, or produce extracellular polymers in large amounts (Costerton et al. 1978; Wright et al. 1988; Gilbert et al. 1990; Dagostino et al. 1991; McCarter et al. 1992). One significant consequence of sessile growth is that biofilm bacteria are more resistant to medical and industrial control strategies than their planktonic counterparts (Brown et al. 1988; Nichols 1989; Eng et al. 1991; Blenkinsopp et al. 1992).

The development of complex attached and aggregated communities is also important for the survival and reproductive success of microorganisms. These communities have been considered to act as reservoirs for diverse species, sites of specific limited niches, and protective refuges from competition, predation or harsh environmental conditions, allowing otherwise poor competitors to survive. Integration into a biofilm or bioaggregate may be regarded as a survival strategy beyond that of maximizing or increasing the growth rate.

Introduction

The rhizosphere

The rhizosphere encloses the zone of soil around a plant root in which the plant root exerts an influence on the growth and distribution of microorganisms. An important source of microbial growth limiting nutrients are the products of rhizodeposition, which include exudates, secretions, lysates and gases (Whipps & Lynch 1985). The increase in the specific growth rate of microorganisms in response to increased organic carbon input has been shown to result in a 5 to 10-fold increase in the number of bacteria when compared with that of the population in the bulk soil (Rouatt et al. 1960; Rovira & Davey 1974). Rhizosphere microorganisms largely depend on root products for their carbon and energy supply (Merckx et al. 1986), and maximum microbial population density occurs at the root surface or rhizoplane (Clarke 1949). This is a consequence of the presence of the highest concentration of growth limiting nutrient at the rhizoplane. Rhizosphere microorganisms do not form a continuous layer on the root surface, but occur in microcolonies (Newman & Bowen 1974; Rovira & Campbell 1975). This microbial cover has been estimated to be below 10% (Rovira et al. 1974; Bowen & Rovira 1976; Bowen & Theodorou 1979). The concentrations of growth limiting nutrient and microbial numbers decrease as a function of radial distance from the root, and under optimal conditions, with adequate growth limiting nutrient supply, the total bacterial numbers would be limited by space. Chemotaxis towards plant root exudates and extracts is a well established phenomenon (Morris et al. 1992), enabling motile bacteria to migrate towards the root through chemotaxis. Non-motile bacteria accumulate in response to an elevated specific growth rate.

The primary concept of this series of books is to produce volumes covering the integration of plant and microbial biology in modern biotechnological science. Illustrations abound: for example, the development of plant molecular biology has been heavily dependent on the use of microbial vectors, and the growth of plant cells in culture has largely dawn on microbial fermentation technology. In both of these cases the understanding of microbial processes is now benefitting from the enormous investments made in plant biotechnology. It is interesting to note that many educational institutions are also beginning to see things in this way and are integrating departments previously separated by artificial boundaries.

Many definitions have been proposed for biotechnology but the only one which has specifically defined environmental biotechnology is that of the European Federation of Biotechnology as The specific application of biotechnology to the management of environmental problems, including waste treatment, pollution control and integration with nonbiological technologies. The study of microbial biofilms is clearly an excellent illustration of environmental biotechnology. The manipulation and control of biofilms is of great interest to industries, including agriculture, chemicals and healthcare.

One of the leaders in the study of biofilms has been Bill Costerton, especially in his early studies when he produced superb electron micrographs to demonstrate the fascinating microbial assemblages which developed in biofilms. However, he rapidly proceeded to demonstrate important physiological functions which occured in these interesting layers. In 1986, Hilary Lappin-Scott joined him to work partly in Cambridge and partly in Calgary on the biofilms associated with oil wells, so starting a long and productive association.

Introduction

Urinary tract infections (UTI) of the lower urinary tract are a common problem causing significant morbidity in females and males (Nickel 1990). Most of the causal organisms are Gram negative bacilli such as Escherichia coli and Proteus mirabilis, or Gram positive Enterococcus and Streptococcus spp., which have their reservoir in the gastrointestinal tract. The other major source of UTI pathogens is direct transmission through sexual activity. These organisms first colonize the introitus and the periurethral area before entering the bladder or prostate. There is an indigenous population of Gram positive, acid producing lactobacilli in this environment which under normal circumstances appears to enhance the urinary defence mechanisms and inhibit the successful progression of the enterics into the urethra and bladder (Chan et al. 1985; Reid et al. 1987, 1990a). When the balance between enteric uropathogens and host defences is upset, uropathogens are able to ascend through the urethra into the bladder, the prostate (in males) and less often into the kidneys where they colonize and cause infection. Usually only the bladder is infected giving rise to simple, uncomplicated acute cystitis. This condition is readily treated by several standard antibiotic regimes (Nickel 1990). Problems arise when this infection spreads from the bladder into other organs such as the kidneys (pyelonephritis) or prostate (prostatitis), or induces the formation of calculi (struvite urolithiasis: McLean et al. 1988, 1992). In these cases, significant tissue damage may occur, possibly causing permanent damage to renal function, which can be life threatening.

Introduction

Wastewater treatment is an essential component of our social order, for without it we soon find our communities suffering from waterborne disease, and our ecosystems suffering unwanted change. For over a century wastewater treatment systems have been designed to increase microbial growth in order to remove organic carbon and other nutrients, while limiting the release of suspended solids into receiving waters. Optimizing design parameters for these goals has been a successful strategy for many conventional wastewaters. However, in the past two decades we have confronted an increasing variety of non-conventional wastewaters in the form of chemical and industrial process effluents and landfill leachates. Legislation has been enacted in many countries that specifies the levels of toxic organic and inorganic chemicals that may be released into the environment. For example, in Canada the provinces of British Columbia, Ontario and Quebec have enacted strict regulations dealing with the discharge of chlorinated organics in pulp mill wastewaters. The long term cost to Canadian industry of this legislation plus proposed new regulations further limiting the chronic toxicity of wastewater discharges will be in the billions of dollars. These costs are already exerting pressure on the industries concerned to solve toxicity problems in the most efficient way possible.

There are three components to a successful toxic wastewater treatment process: knowledge of the nature of the toxic chemicals, an understanding of the concentrations of these chemicals that affect target organisms in receiving waters, and knowledge of the conditions required for growth of toxin degrading microorganisms in the treatment system. The first two of these have been placed on a sound scientific footing using the techniques of analytical chemistry and toxicology.

Introduction

Biofilms possess a number of distinctive characteristics that both define their inherent interest as biological systems worthy of study, and determine their very considerable practical importance in a wide range of environmental, industrial and medical processes. For example, in most natural ecosystems they are comprised not of individual organisms growing in axenic culture, but rather of mixed communities of species with differing but complementary metabolic capabilities. Such consortia demonstrate structural as well as functional organization giving rise to localized microenvironments, each with a particular combination of organisms and physicochemical conditions. The ubiquity of bacterial biofilms extends to their normal association with metal substrata in such situations as ships' hulls and marine structures, water and petroleum transmission lines, and a wide range of process equipment.

In biocorrosion, biofilms are of central importance to the processes involved, with the following individual features of their structure and function assuming particular significance. (i) The substratum on which the biofilm is built may also become a substrate in respect of acting as a source of metabolic energy (H2 oxidation). (ii) Heterogeneities in the horizontal dimension (colonial growth or patchiness) can establish localized electrochemical corrosion cells through the creation of oxygen concentration or differential aeration cells. (iii) The most significant vertical heterogeneity arises from the development of anaerobic regions at the base of the biofilm which can support the growth of sulphate reducing bacteria (SRB). (iv) The extracellular-polymeric substances, which constitute the main mass of the biofilm and underpin the maintenance of these heterogeneities and microenvironments, can also influence corrosion more directly by metal binding and/or retention of corrosion products.

Introduction

In order to examine whether or not the flora of the healthy adult female urogenital tract has any role in protecting a host from infection, and thereby performing a probiotic function, we must first outline the formation, composition and fluctuations of the flora. This is not a simple task as factors such as age and hormonal status influence the type and quantity of organisms present. In simple terms, the establishment of the flora can be seen to follow the path outlined in Fig. 17.1. This figure is based upon epidemiological studies of the urogenital flora (Reid et al. 1990b, c; Sadhu et al. 1989) and a theory for maintenance and causation of infection.

The primary colonizers comprise organisms such as lactobacilli, Gram positive cocci and diphtheroids which have dominated the flora from puberty. In general, the secondary colonizers can comprise a number of species, including potential pathogenic coliforms, Escherichia coli, coagulase negative staphylococci, Klebsiella, Proteus sp. and other Gram positive and Gram negative bacteria. Depending upon the virulence of these secondary colonizers, the host may be able to maintain an infection free state or succumb to the pathogens which then infect the bladder or vagina.

Morphological and structural analyses of the urogenital flora adherent to the epithelia have shown the presence of many distinct organisms, often interacting and coaggregating, in micro colonies or diffuse patterns on the cells (Sadhu et al. 1989; Reid et al. 1990c). Figure 17.2 illustrates this adherence, primarily dominated by Lactobacillus species. The presence of glycocalyx material intertwined between the cells is evident.

In vitro studies have to some extent mimicked this coaggregation or cooperativity between lactobacilli and other urogenital organisms, including potential pathogens.

Introduction

Growth of microorganisms in close association with soft animal and plant tissue surfaces or upon non-living surfaces such as pipework, submerged materials or particulates in soil (Costerton et al. 1987), leads to the generation of microbial biofilms. In vivo biofilms often represent mono cultures (for example, Staphylococcus epidermidis infections of indwelling medical devices), whilst in other situations they are more likely to be comprised of diverse species and represent not only bacteria but also algae, protozoa and fungi. Organization of microbial populations into biofilms is thought to confer many advantages upon the component organisms (Costerton et al. 1985). These include in vivo protection from host immune defences, and conferment of some degree of homeostasis with respect to the physicochemical environment for growth. The extent to which the glycocalyx may modify the growth environment of the cells depends greatly upon the thickness of the biofilm and the density of the cells within it. In this latter respect, whilst the glycocalyx can function as an ion exchange column and exclude large, highly charged molecules, most solutes will equilibrate across it to be accessed by the resident cell population. Diffusion limitation by the glycocalyx, together with localized high densities of cells, will create gradients across the biofilm; thus, for biofilms established upon impervious surfaces, biological demand for oxygen creates within them microaerophilic/anaerobic conditions within the depth of the film yet aerobic conditions at the surface. Similar secondary metabolite, pH and nutrient gradients will also be established across the thickness of the biofilm.

Cells at different parts of the biofilm will experience different nutrient and physicochemical environments.

Waterborne microorganisms

The supply of good quality potable water has been of concern for many years and is of paramount importance for public health. Between 1911 and 1937 there were 20 outbreaks of waterborne disease in the United Kingdom, with 80% of these being due to enteric fever, and the remainder to dysentery and gastroenteritis. The implication that the water supply was the source of a large outbreak of typhoid fever in Croydon, Surrey in 1937, led to the chlorination of public water supplies. This treatment dramatically improved the quality of water supplied to the public and produced improvements in health. Despite chlorination there have been 21 incidents of disease associated with public water supplies since 1937, one of which was due to paratyphoid (George et al. 1972).

The importance of bacterial quality as a measure of water quality has been recognized for many years. Of 495 colonies identified from the water phase, 448 were Gram negative bacteria such as coliforms, Alcaligenes spp. and Pseudomonas spp. (Shannon & Wallace 1944). A diverse range of microorganisms, including Pseudomonas, Flavobacterium, Achromobacter, Klebsiella, Bacillus, Corynebacterium, Mycobacterium, Spirillium, Clostridium, Arthrobacter, Gallionella and Leptothrix spp., is present in potable drinking water (Geldreich et al. 1972), but it is generally considered that these bacteria are not harmful. However, Flavobacterium spp. and Pseudomonas spp. are known opportunistic pathogens, and P. aeruginosa is the major cause of hospital acquired infections (Favero et al. 1971). A total of 15 outbreaks of waterborne disease were identified in the UK between 1977 and 1986 (Galbraith et al. 1987), with Campylobacter enteritis and viral gastroenteritis accounting for 66% of outbreaks.