INTRODUCTION

Uniquely in the human body, the tooth surface provides a non-shedding surface for microbial attachment. As a result, large numbers of microorganisms accumulate, together with their metabolic products, to form dental plaque, especially at occluded sites. Dental plaque was the first biofilm described by van Leeuwenhoek (1683), who noted the unusual resistance of the ‘animalcules’ he observed to treatment with vinegar (McHugh, 1999). Since this time, dental plaque has probably been the most studied of biofilms, with some justification: dental plaque is the most accessible of human-associated biofilms and is responsible for the most common diseases affecting man in the developed world – dental caries and periodontal diseases. This chapter provides a brief historical view of light and electron microscopic studies of dental plaque. The major focus of the chapter is then to describe how novel microscopic approaches are providing new insights into the structure and metabolic activities of dental plaque biofilms.

LIGHT AND ELECTRON MICROSCOPIC STUDIES OF DENTAL PLAQUE DEVELOPMENT

Since van Leewenhoek's time, vast numbers of light microscopic studies (especially of dental plaque) have examined the structure of biofilms. Furthermore, the development of electron microscopy (EM) began to unearth the diversity within, and structures of, developing dental plaque (Saxton, 1973; Listgarten, 1976, 1999).

The development of dental plaque can be divided into several arbitrary stages. Pellicle formation occurs almost instantaneously on a cleaned tooth surface (Figure 8.1a). After a cleaned tooth surface has been exposed for 4 hours to the oral environment, relatively few bacteria are found.

INTRODUCTION

Many environmental as well as host-associated microorganisms not only live as single independent cells, but are able to interact with each other and to build multicellular communities, whose architecture is determined by a self-produced extracellular matrix. Also, in the family Enterobacteriaceae, different types of multicellular behaviour have been identified, for example, in Salmonella enterica serotype Typhimurium (S. typhimurium) and Escherichia coli (Harshey and Matsuyama, 1994; Romling et al., 1998a). The rdar (red, dry and rough colony morphology) morphotype (Figure 11.1, first identified in S. enterica serotype Typhimurium (Romling et al., 1998a), represents a characteristic multicellular behaviour of S. enterica serotypes (Salmonella spp.) and E. coli in the late stationary phase of growth (Zogaj et al., 2001). Life within a community, compared with planktonic growth, is significantly different for S. enterica serotype Typhimurium and E. coli with respect to, for instance, cell density, nutrient supply, and production of architectural components (extracellular matrix formation); cell–cell communication, gene expression, and regulation are altered on various levels (Pringent-Combaret et al., 2001; Zogaj et al., 2001). An additional regulatory network, under tight control by environmental conditions, is required to coordinate the transition from the multicellular to the free-living form (Gerstel and Romling, 2001). Novel regulatory pathways involving genes with signalling domains of unknown function are part of this network (Romling et al., 2000). The impact of multicellular behaviour on the life cycle of S. enterica and E. coli has not been unambiguously proven.

INTRODUCTION

Microbial development and biofilm formation on implanted biomaterials and hospital equipment are important sources of nosocomial infections, mainly because surface-associated contaminants express biofilm-specific properties such as increased resistance to biocides, antibiotics, and immunological defences. Although it has long been recognised that the presence of a solid phase can influence many bacterial functions (ZoBell, 1943; Costerton et al., 1987; Van Loosdrecht et al., 1990), we are just beginning to understand the regulatory processes at the molecular level. There is no doubt that the identification of the structures involved in the sensing of the particular microenvironments encountered at interfaces and in developing biofilms and the description of the regulatory networks allowing the appropriate genetic responses will lead to the development of surface coatings and preventive or curative drugs able to deal with these life-threatening infections.

BIOFILM FORMATION IS A DEVELOPMENTAL PROCESS

An invidual bacterium present on, or introduced into, the human body can reach the surface of an indwelling medical device by three different mechanisms (Van Loosdrecht et al., 1990): passive transport due to air or liquid flow, diffusive transport resulting from Brownian motion, and active movement involving flagella. Although contact is, therefore, frequently a question of chance, chemotactic processes can direct motile bacteria in response to any concentration gradient that may exist in the interfacial region. Following contact, the next stage may be initial adhesion.

INTRODUCTION

For a long time bacteria were believed to exist as unicellular organisms; however, it is now realized that in nature bacteria are more often found clustered in communities. Within these communities, bacteria are capable of coordinated activity through the use of a sophisticated intercellular communication mechanism called quorum sensing (QS). The capacity to behave collectively as a group has obvious advantages, for example, migration to a more suitable environment/better nutrient supply or adopting a more favourable mode of growth such as sporulation. Recently, QS was discovered to play a role in the formation of biofilms. This latter phenomenon will be the focus of this chapter as we review our current understanding of how QS affects the complex processes of biofilm development. With respect to intercellular communication and biofilms, Pseudomonas aeruginosa is one of the most intensely studied organisms, and therefore, much of this chapter will concentrate on this bacterium.

QUORUM SENSING

QS exists in both Gram-positive and Gram-negative bacteria with obvious differences between the two systems (for reviews, see Dunny and Leonard, 1997; Fuqua, Winans, and Greenberg, 1996). Here, we will focus on Gram-negative bacteria, where the two primary components of QS systems are the autoinducer (AI) signal molecule and the transcriptional activator, or R-protein. In general, the ‘language’ used for intercellular communication is based on small, diffusible, self-generated signal molecules called AIs.

INTRODUCTION

In 1962, Nobel Laureate Frank MacFarlane Burnet wrote ‘one can think of the middle of the twentieth century as the end of one of the most important social revolutions in history, the virtual elimination of the infectious diseases as a significant factor in social life’ (Burnet, 1962). Indeed, advances in medical technology have come forth at an impressive rate. However, pathogens have adapted to and even taken advantage of the altered host environments created by modern therapies and medical devices, and new forms of microbial infections continue to challenge and confound modern medical technology. Development of microbial resistance to antibiotics and biocide disinfectants represent well-known examples of microbial adaptation. The increasing frequency with which indwelling medical devices, including intravenous catheters, replacement heart valves, prosthetic hip and knee joints, and artificial hearts, are being used represents a tremendous advancement in modern medicine, but it has also afforded certain microbes with a new opportunity to breach the primary host defences and initiate a focal point of infection. These devices penetrate protective dermal or mucosal layers and provide many pathogenic organisms with a surface on which they can attach and grow. The mode of growth that occurs, that of a community of interconnected and communicating cells known as a biofilm, allows a high density cluster of cells in various physiologic states to coexist.

INTRODUCTION

The only natural habitat for the aetiological agents of dental caries and periodontal diseases, as well as the overwhelming majority of oral bacteria, is the mouth. These organisms exist almost exclusively as constituents of the biofilms that form on the soft and hard tissues of the oral cavity and, therefore, can be thought of as ‘obligate biofilm organisms’ (Burne, 1998a). Bacteria capable of sustaining a free-living existence have sensing and differentiation systems that allow the organisms to detect acquisition of a suitable host, to alter motility and capsule expression, and to transition from oligotrophic systems to relatively nutrient-rich systems where they must confront the innate and acquired defences of a host. In contrast, oral bacteria do not have to transition between radically different environments, such as moving from the gut of a mammal to a mountain stream or decaying forest material and vice versa. Therefore, it is reasonable to predict that, through normal evolutionary processes, oral biofilm bacteria may have sloughed off many of the elaborate genetic systems for responding to the transition from the free-living state to the host-associated or biofilm state. In fact, with the completion or near completion of a number of genomes of oral bacteria, it is clear that oral bacteria have comparatively small chromosomes and lack many of the sensing and differentiation pathways that are present in organisms that can exist in a free-living state, such as Pseudomonas aeruginosa, Escherichia coli, and Yersinia pestis.

Biofilms are increasingly recognised as the preferred mode of growth of bacteria in a wide range of sites, including the many, and varied, habitats present in man. Until approximately 10 years ago, biofilms were regarded as consisting simply of an accumulation of bacteria and their products on a surface. But recent technical advances have significantly changed our understanding of biofilm organisation and function. Non-destructive methods of examining organisms in their living, hydrated state (for example, confocal laser scanning microscopy) have revealed that they have an ordered structure, often permeated by water channels which can function as a primitive circulation system. Modern molecular techniques have identified genes that are up- and down-regulated in different regions of a biofilm – this has important implications for assessing the behaviour of organisms in biofilms (for example, virulence potential, susceptibility to antimicrobiol agents, and host defence mechanisms). Furthermore, the discovery of population-dependent gene regulation (quorum sensing) in bacteria has meant revising our concept of bacteria as being independently operating cells. Quorum-regulated gene regulation is, obviously, likely to play an important role in determining the collective properties of bacteria in biofilms.

During the last few years, interest in microbial biofilms has increased dramatically and now encompasses a broad range of disciplines – microbiology, molecular biology, microscopy, medicine, engineering, ecology, and marine biology. This increased specialisation within the field has led to a need for books that focus on particular aspects of biofilms.

INTRODUCTION: THE PROBLEMS

There is increasing concern over the role played by microbial biofilms in infection. These include well-known examples of medical device-related infections such as those associated with artificial joints, prosthetic heart valves, and catheters. Indeed, recent surveys indicate that catheter-associated bacteraemia, consequent from catheter-related infection, is by far the leading cause of nosocomial bloodstream infection in intensive care units (Brub-Buisson, 2001). Many chronic infections, not related to medical devices, are now recognised to be due to bacteria either not growing and relatively dormant or growing slowly as biomasses or adherent biofilms on mucosal surfaces. Thus, the question of how to treat biofilm infections extends to many aspects of medicine. Indeed, the issue of biofilm eradication extends way beyond the infected patient, since bacteria in the environment typically exist as biofilms. These are commonly complex multispecies ecosystems associated with protozoa (Brown and Barker, 1999). The biofilm mode of growth greatly enhances the survival of the constituent microbes.

Growth as a biofilm almost always leads to a large increase in resistance to antimicrobial agents, including antibiotics, biocides, and preservatives, compared with cultures grown in suspension (planktonic) in conventional liquid media (Gilbert, Collier, and Brown, 1990; Stewart and Costerton, 2001). However, a recent paper with high density planktonic cultures indicated similar resistance to antimicrobials as did biofilm cultures (Spoering and Lewis, 2001). Currently, there is no generally agreed mechanism to account for the broad resistance to chemical agents.

THE LARGE INTESTINAL MICROBIOTA

It has been estimated that of the 1014 cells associated with the human body, approximately 90 per cent are microorganisms, and the vast majority of these organisms are bacteria growing in the large intestine (Savage, 1977). The large bowel is the main area of permanent microbial colonisation of the human gastrointestinal tract; gastric acid kills most oral and environmental microorganisms in the stomach, whereas the rapid passage of digestive materials through the upper gut does not allow time for significant bacterial growth to occur (Macfarlane and Cummings, 1991). However, the rate of movement of intestinal contents slows markedly in the large gut, which facilitates development of rich and diverse bacterial communities (Cummings, 1978; Cummings et al., 1993). The growth and metabolic activities of these microbial populations are influenced to a considerable degree by diet, as well as by the structure and physiology of the colon (Macfarlane et al., 1995).

The large intestine is an open system in the sense that food residues from the small intestine enter at one end and, together with bacterial cell mass, are excreted at the other end. Because of this, the colon is often viewed as being a continuous culture system, although only the caecum and ascending colon really exhibit characteristics of a continuous culture (Cummings et al., 1987; Macfarlane, Macfarlane, and Gibson, 1998).

INTRODUCTION

Oral Biofilms

Biofilms are surface-attached bacterial communities formed by unicellular organisms of single or multiple species. Most bacteria in their natural ecosystems colonise surfaces and are found in biofilm communities, rather than as planktonic cells. Biofilm formation is a highly structured process that occurs for numerous reasons, including protection from host immune systems, nutrient availability, and protection from harsh changes in the environment (Costerton et al., 1995; Costerton, Stewart, and Greenberg, 1999). A number of studies examining planktonic cells grown in batch culture have usually treated bacteria as unicellular species, even though they often exist in biofilms.

Although biofilm formation has been recognised and documented for approximately 100 years, we are just beginning to understand this process at the molecular level. Increasingly, bacteria have been studied as multicellular populations and, in some cases, viewed as interactive multicellular organisms (Shapiro, 1998), partly due to the fact that biofilm cells exist in a physical and physiological state that can increase their resistance to antimicrobials and mechanical forces (Costerton et al., 1995, 1999). Bacteria in a biofilm often display a dramatically different phenotype when compared with their counterparts in liquid culture. For example, biofilm cells often display higher resistance to antimicrobial agents, and they often exist in localised anoxic microenvironments and/or microenvironments that vary significantly in pH and ionic strength (Costerton et al., 1995, 1999).

INTRODUCTION

More than 200,000 primary hip and 200,000 primary knee arthroplasties are performed each year in the United States alone. Between 0.5 and 3.9 per cent of them will become infected within 10 years (Table 4.1) (An and Friedman, 1996; Stocks and Janssen, 2000). For revision total joint replacements, the infection rates can be much higher, with numbers as high as 3.2 per cent reported by Sperling et al. (2001), 12.5 per cent reported by Itasaka et al. (2001), and 17 per cent reported by Spangehl et al. (1999). Infection often causes complete failure of a total joint arthroplasty. Sepsis following total joint replacement can have catastrophic results, both physically and psychologically, for the patient, leading to failure of the arthroplasty, prolonged hospitalisation, possible amputation, and even death (Cheatle, 1991). In addition, the management of infected cases, especially those of joint replacements, is very costly (Hebert et al., 1996). Although the use of prophylactic antibiotics and greatly improved surgical techniques have decreased the infection rate of joint replacement from an average of 5.9 per cent in 1975 to 1.2 per cent in 1993 (An and Friedman, 1996), challenges still remain for better preventive and therapeutic measures. In addition to joint replacement, implant infections also occur in other orthopaedic subspecialties, such as trauma (Eijer et al., 2001) and spine (Wimmer and Gluch, 1996), with a significant impact on the patient and society.

INTRODUCTION

The past few decades have witnessed the advent of many medical advancements, which undoubtedly have aided health care professionals and ameliorated the suffering of many critically ill patients. Among these advancements are intravascular catheters, which have become indispensable for the administration of medications, especially chemotherapeutic agents, intravenous antibiotics, blood products, parenteral nutritional solutions, and fluids; for monitoring critically ill patients haemodynamically; and for providing access for haemodialysis (Flowers et al., 1989). The use of such devices is associated with an increased risk for catheter-related infections, such as local catheter site infection, thrombophlebitis, catheter-related bloodstream infections (CR-BSI), endocarditis, metastatic infections such as osteomyelitis, arthritis, endophthalmitis, and distant organ abscesses (Arnow, Quimosing, and Beach, 1993). Our understanding of the process that leads to CR-BSI has evolved over the years, and now the role of bacterial biofilms in this process is certainly undeniable.

Bacterial biofilms have always existed ubiquitously in the world around us. Costerton and Stewart describe some examples: ‘the slippery coating on a rock in a stream, dental plaque (which most of us confront daily), and the slime that inevitably materializes inside a flower vase after two or three days’ (Costerton and Stewart, 2001). In addressing the medical implications of biofilms as they relate to intravascular-catheter-related infections, we will begin by defining the magnitude of the problem of catheter-related infections, followed by addressing the role of biofilms in the pathogenesis of CR-BSI. Finally, we will review some strategies to prevent biofilm formation on central venous catheters (CVC).

INTRODUCTION

The anatomy of the urinary tract, unfortunately, allows ready access by pathogens to the urethra and beyond when normal defences are breached. The potential for urinary tract infection (UTI) is considerably enhanced by the presence of an indwelling device such as a urethral catheter that provides a conduit to the bladder (Tunney, Jones, and Gorman, 1999). This allows not only the voiding of urine, but also the ingress of microorganisms that colonise the device material and adopt a biofilm growth mode. Such ‘device-related’ infection is a frequent occurrence in the urinary tract, requiring considerable time and resource in its management. It is estimated that over 40 per cent of nosocomial infections are related to the urinary tract (Nickel, Downey, and Costerton, 1989). Despite careful aseptic management, bacteriuria arises in approximately 50 per cent of patients within 10–14 days and in all those undergoing long-term catheterisation for management of urinary retention and incontinence by 28 days. Additional complications such as blocking encrustations, stone formation, pyelonephritis, and bladder cancer may also arise in patients with asymptomatic infection (Gorman and Tunney, 1997). The elderly are particularly prone to urinary device-related infection. Residents of nursing homes undergoing long-term catheterisation are three times more likely to receive antibiotics, be hospitalised, and die within a year than matched non-catheterised residents (Kunin, Chin, and Chambers, 1987).

The obstruction of urine flow in urinary devices by crystalline encrustation is an additional clinical complication.

INTRODUCTION

Staphylococcus epidermidis is a ubiquitous commensal of human skin. In the past 20 years, this organism has emerged as a serious pathogen, causing frequent nosocomial infections. In contrast to S. aureus and except for rare cases of native valve endocarditis, S. epidermidis needs a predisposed host to cause infection. Individuals who are especially susceptible to S. epidermidis infections are immunocompromised patients, intravenous drug abusers, and, most commonly, patients with indwelling medical devices (Heilmann and Peters, 2000). Immunocompromised patients include those with malignant disease who are undergoing cytostatic and/or immunosuppressive therapy and premature newborns. In premature newborns, S. epidermidis is the most frequent cause of septicaemia with an onset later than 48 hours after birth. In drug abusers, continual injections of heroin containing crystals are believed to cause microlesions in the valvular endothelium leading to right-sided endocarditis; the damaged tissue is then infected with S. epidermidis that is introduced due to non-sterile conditions. By far, the largest number of infections caused by S. epidermidis are those associated with implanted foreign bodies such as orthopaedic devices, intravascular catheters, cerebrospinal fluid shunts, prosthetic heart valves, continuous ambulatory peritoneal dialysis (CAPD) catheters, and cardiac pacemakers (George, Leibrock, and Epstein, 1979; Karchmer, Archer, and Dismukes, 1983; Peters, Locci, and Pulverer, 1982; Peters et al., 1984; West et al., 1986). These devices are manufactured from various polymers, most commonly from polyvinylchloride, polyethylene, silicon rubber, and polyurethane.

Colonisation of the polymer surface by S. epidermidis is the most critical step in the pathogenesis of foreign-body-associated infections.