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Biomaterials and Tissue Engineering in Urology

Biomaterials and Tissue Engineering in Urology

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Biomaterials and Tissue Engineering in Urology

1,123 pages
12 hours
Apr 29, 2009


Urology is the branch of medicine dealing with disorders or diseases of the male genitor-urinary tract and the female urinary tract. This important book summarises the wealth of recent research on the use of biomaterials and tissue engineering to treat urological disorders.

Part one reviews the fundamentals with chapters on such topics as biofilms and encrustation formation. Part two then discusses recent advances in biomaterials and design of urological devices such as metal ureteral stents, self-lubricating catheter materials and penile implants. Chapters in Part three address urological tissue engineering with coverage of themes such as artificial and natural biomaterials, nano-technology and placental stem cells for tissue engineering the regeneration of urological tissue and organs.

With its eminent editors and international team of contributors, Biomaterials and tissue engineering in urology is an invaluable resource to researchers of urological biomaterials, devices and regenerative medicine in both industry and academia, as well as an important reference for medical practitioners.
  • Provides a comprehensive review of biomaterials and tissue engineering in urology
  • Explores the fundamentals of urology, focusing on biofilms and encrustation and formation
  • Discusses recent advances in biomaterials and the design of urological devices, catheters and stents
Apr 29, 2009

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Biomaterials and Tissue Engineering in Urology - Elsevier Science


Part I



Chapter 1: Introduction to biofilms in urology

Chapter 2: In vivo models for ureteral stents

Chapter 3: Models for the assessment of biofilm and encrustation formation on urological materials


Introduction to biofilms in urology

P. CADIEUX and G. WIGNALL,     University of Western Ontario, Canada

R. CARRIVEAU,     University of Windsor, Canada


Despite significant resources and several decades of research aimed at their prevention and treatment, biofilm-associated infections continue to be the major cause of urological device failure. Numerous strategies have been targeted towards improving device design, biomaterial composition, surface characteristics and drug elution, but have been largely thwarted by microorganisms and their arsenal of attachment, host evasion, antimicrobial resistance and dissemination strategies. This is not entirely surprising considering that natural biofilm formation has been occurring for billions of years and remains a significant element of microbe survival and evolution. Thus, the fact that biofilms develop on and in the biomaterials and tissues of humans is largely an extension of this natural tendency and underscores why they are so difficult to combat. Biofilm structure and composition intrinsically offer a protective environment for microorganisms, shielding them from the shear stress of urine flow, attack by the host immune system and antimicrobials. Furthermore, many biofilm organisms go into a metabolically quiescent state that renders them more tolerant to antibiotics and host immune factors able to penetrate the biofilm matrix. Finally, most organisms causing biofilm-associated urinary tract infections originate from the host’s own oral cavity, skin, gastrointestinal and urogenital tracts and therefore have already adapted to many host defense mechanisms. Ultimately, while biofilms continue to hold the upper hand with respect to recurrent infections and urinary tract biomaterial use, significant progress has been made in understanding these dynamic microbial communities and novel approaches offer promise for biofilm prevention and removal. These include novel device designs, antimicrobials, anti-adhesive coatings, biodegradable polymers and biofilm-disrupting compounds and therapies.

Key words



urinary tract infection

ureteral stent

urethral catheter


conditioning film

antibiotic resistance

1.1 Introduction

Biofilm-related infections remain the major cause of urological device failure despite millions of dollars and several decades of research targeted at their prevention and eradication. The plethora of strategies aimed at improving device design, biomaterial composition, surface characteristics and drug delivery have been generally thwarted by microbes and their abundance of attachment, host evasion, antimicrobial resistance, survival and dissemination strategies. It is important to acknowledge that the formation of biofilms by microorganisms in nature has been going on for billions of years and remains a major part of their current survival and evolution on the planet. Thus, the fact that biofilms develop on and in the tissues and biomaterials of humans is simply an extension of this natural tendency and largely explains why they are so difficult for us to combat. Furthermore, the majority of organisms that cause biofilm-related urinary tract infections originate from our own skin, oral cavity and gastrointestinal tract, and thus have already adapted to many of our defenses. In this chapter, we will first discuss biofilms at their most basic level including why and how they form. It is essential to understand the purpose, formation and structure of both natural and medical biofilms if we are to tackle the clinical problems associated with them and attempt to prevent and eradicate them from patients. Biofilms in medicine in general will follow and then transition into our focus of biofilms in urology and their clinical relevance. We will discuss the basic hydrodynamics of the upper and lower urinary tract along with how biofilms affect, and are affected by, urinary flow. Prosthetic versus non-prosthetic infections will be described as well as the ability of biofilms to resist antimicrobials, host urinary tract defenses and the immune system. The development and implications of chronic infections will be commented on and the effects that biofilms and biofilm-related encrustation have on them. This will lead into current treatment and prevention strategies including novel biomaterial surfaces and design, coatings, antimicrobial strategies and the promising field of biofilm-disrupting agents. We argue that although microbial biofilms continue to hold the upper hand in terms of recurrent infections and the use of biomaterials within the urinary tract, significant progress has been made in understanding these microbial communities and new strategies offer promise in the field.

1.2 What are biofilms and why do they form?

A biofilm can be defined as a community of microorganisms (bacterial, fungal, algal) attached to the interface of a liquid and a surface, and enveloped within a matrix of exopolysaccharides and other biological constituents (Costerton, 2007a; Jass et al., 2003). In the simplest terms, a biofilm is merely a mechanism used by microorganisms to remain in a favorable environment and promote their survival and reproduction within that environment. Thus, on a very basic level, a urinary biofilm on a ureteral stent is no different than those of the thermophilic bacterium Thermus aquaticus at a deep sea hydrothermal vent or Serratia marcescens on your shower wall (Langsrud et al., 2003; Stramer and Starzyk, 1978). In general, if a micro-organism comes in contact with a surface and successfully attaches to it, and the right growth conditions exist (temperature, nutrients, pH, osmolarity, etc.), it will adhere more securely and begin multiplying and forming a biofilm. In addition, biofilms provide a degree of protection for a microbial population. Although microorganisms can survive and multiply very efficiently as single cells within a liquid milieu (such as uropathogens in urine), they are completely exposed to any detrimental environmental conditions that might arise (i.e. increased temperature, host factors, antibiotics) and may be swept away from a favorable environment via shear forces within the surrounding fluid (such as during micturition). Thus, if a single-celled (planktonic) population is exposed to a sudden lethal change in temperature or high concentration of a toxic compound, all cells in the population may be killed. The structure and makeup of a biofilm protects many cells within the population by physically shielding them from the surrounding environment and inducing changes in gene expression that result in metabolic dormancy and/or increased resistance to many inhibitory substances (Donlan, 2003; Fujiwara et al., 1998). Although biofilms are far more complex in reality than described here, it is obvious that their formation improves microorganism population survival, growth and reproduction through multiple mechanisms. It is therefore not surprising that biofilms are relatively ubiquitous throughout every natural environment on the planet, ’from tropical leaves to desert bolders’ (Costerton, 2007a), or more relevant here, from the vaginal epithelia to urinary tract devices. Clearly, biofilms are the greatest challenge faced by medicine and industry in terms of treating wounds, using biomaterials and designing compounds for the prevention and treatment of infection.

1.3 Biofilm formation and structure

At a macroscopic level, most biofilms appear fairly simple in structure, as uncomplicated clusters, chains and/or layers of organisms haphazardly attached to a surface and intermixed with extracellular slime. Based upon this view, one might consider biofilm development simply as the repeated process of single organisms from the surrounding fluid adhering themselves to an object with little or no purpose. However, upon closer examination, it becomes clear that in the majority of cases, this is far from the truth. Biofilms, especially those formed in nutrient-limiting environments, are complex, highly structured communities designed to maximize survival, reproduction and spread. They can be homogeneous, consisting of only one specific organism, or heterogeneous, comprising two or more different organisms. In nature, as in urology, both types of biofilms are commonly found and the type that will occur in any specific situation depends upon multiple factors – chiefly the properties of the surface and microorganisms present, the ability of the surrounding milieu to support and inhibit their growth and the relationship the organisms have with each other. As one examines these communities in more detail, it becomes evident that biofilm complexity is largely a planned behavior and not simply a matter of architecture. It would appear that every organism within a biofilm has a purpose, each contributing to the overall good of the entire population. In fact, some organisms within biofilms will undergo a type of ’apoptosis’ or programmed cell death under certain conditions, likely to supply nutrients to the population or contribute DNA and other compounds to the matrix (Allesen-Holm et al., 2006; Whitchurch et al., 2002). Furthermore, other cells will either alter their phenotype or enter a state of dormancy (termed ’persisters’) that renders them tolerant to lethal factors such as antibiotics (Keren et al., 2004; Lewis, 2001; Lewis, 2007; Shah et al., 2006). In this sense, a biofilm can almost be thought of as a separate entity, a multicellular organism comprising diversely differentiated cells throughout, all with a common goal (Lewis, 2001; Sauer et al., 2002). This complexity and adaptability are likely driven by the rapid environmental changes that microorganisms face on a continual basis, whether in nature or in association with human tissues and biomaterials, and offers some explanation as to why medical biofilms are virtually impossible to eradicate completely once formed.

All biofilms start with an organism’s attachment to a surface (Fig. 1.1). This can be a single cell, a group of cells that grow in multiples or commonly autoaggregate, or a matrix-enclosed cluster of cells sloughed off a previous biofilm. At the time of this initial attachment, success will depend upon multiple factors, some of bacterial origin and others environmental. The major bacterial determinants are surface factor expression and cell viability. Bacteria express numerous appendages, receptor ligands and polysaccharides that can become involved in adherence to a surface such as fimbriae (pili), lipopolysaccharides, exopolysaccharides, specific protein ligands and flagella (Christiansen et al., 1989; Costerton, 2007a; Jass et al., 2003; Reid et al., 1992). The possession and expression of these factors can vary widely between different types of bacteria, even down to the strain level. As the bacterium approaches the surface, one or several of these factors will contact the surface and a reversible attachment will take place, dependent upon not only those factors but also the environmental properties of the surface and surrounding fluid as well (i.e. surface hydrophobicity, energy and charge, fluid shear stress, temperature, osmolarity, etc.). This attachment allows time for additional bacterial factors to adhere and stimulates changes in gene expression within the organism that drive polysaccharide production and other events that lead to irreversible adherence (De Kievit and Iglewski, 1999; Sauer et al., 2002; Stoodley et al., 2002b; Ziebuhr et al., 1999). In the case of a biomaterial, once this irreversible attachment has taken place and a biofilm begins to form, at present it is virtually impossible to eradicate.

1.1 Schematic of bacterial biofilm formation and development outlining the processes and stages involved.

The initial organism-to-surface attachment is further complicated during biomaterial use by the presence of biological constituents in the surrounding fluid (i.e. blood, urine), which not only provide nutrients for the microbes but also adhere to the biomaterial themselves and form what is called a ’conditioning film’ (Fig. 1.2) (Chew et al., 2006; Choong et al., 2001; Jass et al., 2003; Reid et al., 1992). This film negatively impacts the host in three critical ways. Firstly, it masks the intended surface properties of the device and may block the elution of an impregnated antimicrobial or anti-biofilm compound. Secondly, it provides a plethora of different molecules for the organism to attach to, which increases its ability to make and maintain initial contact with the surface. Finally, it provides a direct source of nutrients for the organism immediately where attachment and biofilm formation occur. It is mainly two aspects of the bacterium–biomaterial interaction, namely multiple bacterial attachment strategies and the constant deposition of a urinary conditioning film that have made it so difficult to produce biofilm-resistant prostheses for the urinary tract.

1.2 Scanning electron micrograph of a sterile urinary conditioning film formed after 24 hours on a titanium oxide surface.

As described previously, biofilm configuration and development are highly dependent on the local growth conditions (Costerton, 2007a; Jass et al., 2003). They can form fairly uncomplicated arrangements such as mats to highly complex spatial structures with fluid filled pores and channels (Fig. 1.3). Spatial variations can range from the bacterial scale (>10 μm microscale) to the meso-scale (50 μm–1 mm), and in the right environment they can even approach macro-scales (>1 mm). Thus, there is no one standard structure of a biofilm and a multitude of arrangements exist including mushrooms, towers, honeycombs and streamers. Furthermore, it is not uncommon to find that the same organism produces biofilms of very dissimilar structure and density under different environmental conditions (Jackson et al., 2001). In general, relatively flat and unstructured biofilms tend to develop in nutrient-rich habitats while more highly structured, complex films form when nutrients are restricted (Costerton, 2007a). This is likely due, at least in part, to the fact that when nutrients are scarce, elaborate structures maximizing surface area and fluid flow will be required; while when ample nutrients are available in the surrounding fluid and buried within the matrix, these elaborate structures are not required.

1.3 Klebsiella pneumoniae biofilm on a ureteral stent removed from an infected patient showing water channels that permit the rapid movement of nutrients and wastes in and out of the film.

1.3.1 Hydrodynamic effects on biofilm formation and structure

The hydrodynamics of the local aqueous environment also plays a key role in determining biofilm structure. This is fundamentally due to the supply of dissolved substrates delivered by the flow, as well as the nature and magnitude of the mechanical forces imparted on the biomass by it (Nguyen et al., 2005; Stoodley et al., 1999b; Stoodley et al., 2002a). Beyenal and Lewandowski (2002) proposed that biofilms arrange their internal structure according to the velocity of the flow they are grown in. They discovered that biofilms grown in high velocity gradient flows would adapt their internal structure to bolster their resistance to elevated shear stress. The strength increase comes about with an increase in biofilm density which slows down the internal mass transfer rate, thus reducing the nutrient transfer to deeper layers. Similarly, biofilms grown at low flow velocities evolve higher effective diffusivity but are not as dense and subsequently exhibit a weaker resistance to shear stress. In addition to biofilm formation, hydrodynamic forces can influence biofilm migration. In a study of Pseudomonas aeruginosa biofilms, Purevdorj et al. (2002) depicted a unique mechanism of biofilm locomotion developed under hydrodynamic stress. They demonstrated that biofilms could move along solid surfaces while remaining attached to these surfaces. This was unlike the more commonly known propagation process via fluid-borne detached planktonic (free floating) cells; this surface-associated mechanism allowed the spread of the whole biofilm structure. With diffusion of the entire structure comes the preserved resistance to various antibiotics and disinfectants, since the intermediate step of planktonic release is not required (Stewart and Costerton, 2001). Such a creeping biofilm flow may be an important consideration for infection in catheters and stents.

Formation of a biofilm often represents a flow impediment. The interaction of flow and biofilm has most commonly been modeled with the biofilm simulated as a solid object around which the flow must navigate. Fluids indeed flow around biofilm structures but flow also exists through the extracellular polymer substance (EPS) matrix and microchannels that can form within the film (Fig. 1.3) (Nguyen et al., 2005; Stoodley et al., 1994). This multi-scale environment can make for subtle but potentially significant complications in models that attempt to simulate the flow around and through biofilms. Microscopic flow effects through biofilms can affect the local mesoscopic flow field (Nguyen et al., 2005). The challenge in fully quantifying the effects of flow in, at, and around biofilms is largely a result of the complex mechanical material properties that a biofilm exhibits.

The hydrodynamic impact of biofilm accumulation is well documented, particularly in the infrastructure engineering realm. In general, hydrodynamic resistance is a function of the frictional resistance of the fluid boundary (surface drag), and the losses due to a difference between upstream and downstream pressures around an obstacle – caused by boundary layer separation (Debler, 1990). Thus, in the context of biofilms we may encounter increased skin friction and form friction from biofilm structural geometry (ripples, dunes, streamers) (Stoodley et al., 1999a; Towler et al., 2007). Even the simplest flat biofilm geometry can lead to urological implications, specifically with regard to flow through implants. The resistance to idealized flow through a circular conduit is proportional to the fifth power of the conduit diameter. That is to say, hydraulic losses are very sensitive to reductions in diameter. Reductions in device diameter owing to biofilm longitudinal and circumferential propagation may mean reduced urinary flow rates should the necessary compensatory increase in pressure gradient to combat losses across the device not be available.

In considering the local environmental conditions of the urinary tract, the above findings would suggest that relatively flat films possessing high effective diffusion rates would tend to develop on urinary prostheses, as ample nutrient levels and low flow velocities generally exist immediately around them. Studies investigating ureteral stents inserted for up to 128 days support this, finding that biofilms formed in the absence of encrustation did not induce blockage of any of the devices, even in patients whose biofilms developed over several months (Reid et al., 1992). Furthermore, those devices examined using scanning electron microscopy revealed relatively flat biofilms with depths in the micrometer range. Thus, in the majority of cases, it would appear that the major clinical difficulties associated with urinary biofilms are not due to device failure but more to biofilm-associated infections and their resilience. However, this would not be the case for biofilms associated with urease-producing organisms, since the rapid alka-linization of the urine they induce would lead to rapid encrustation and subsequent blockage of the device due to the formation of more crystalline biofilms with higher densities (Morris et al., 1999; Stickler et al., 1998). These organisms, particularly Proteus mirabilis, are covered in more detail in Chapter 7, Proteus mirabilis biofilm formation and catheter design, by David Stickler, an expert in the field.

1.3.2 Communication within biofilms

The success of microorganism survival in general is largely owed to their ability to sense even minute environmental changes and make rapid adjustments. In order to achieve this, microorganisms have developed a multitude of ligand–receptor and sensor–response regulator systems for assessing their local conditions and eliciting a response (Khorchid and Ikura, 2006; Mascher et al., 2006). These triggers can work alone or in combination and may result in the organism altering a number of biological processes including growth rate, movement, membrane permeability, efflux rates, exopolysaccharide production and cell lysis. With this in mind it is understandable that microorganisms, especially those within biofilms, would constantly assess their own population dynamics and adjust their phenotypes accordingly. The best characterized example of cell–cell communication is that of quorum sensing in the marine bacterium Vibrio fischeri, which resides in a symbiotic relationship within the light organs of several marine creatures (Dunlap, 1999). When a critical number of organisms (quorum) is achieved within the organ, a factor secreted by the bacterium (inducer) builds up to a level where it binds to a sufficient number of surface receptors to trigger the expression of a bioluminescent protein important to the fish for avoiding predators at night. These inducer–receptor systems and/or the like have since been found in virtually all microorganisms tested and have been shown to regulate a multitude of biological processes including virulence and biofilm formation, with Pseudomonas aeruginosa the most studied (Christensen et al., 2007; Erickson et al., 2002; Murray et al., 2007; Rumbaugh et al., 2000). Typical quorum-sensing compounds for Gram-negative and -positive bacteria are acylated homoserine lactones (Erickson et al., 2002; Hentzer et al., 2002) and processed oligo-peptides (Abraham, 2006), respectively. Multiple studies have further argued that quorum-sensing systems may be used specifically by biofilm organisms to mobilize to more amenable locations within the film (Hansen et al., 2000), modify the local environment (Allison and Gilbert, 1995), and attract and detach organisms to and from the biofilm, respectively (Stoodley et al., 2001a; Stoodley et al., 2001b).

Another potential means by which cell–cell signaling occurs within biofilms is through electrical pulses sent along microbially produced ’nanowires’. The production and utility of such structures was first observed in biofilms of Schewanella oneidensis (Gorby et al., 2006), a versatile, environmental organism infrequently isolated from human opportunistic infections. These nanowires have since been demonstrated in Geobacter sulfurreducens (Reguera et al., 2006) as well, a metal-reducing bacterium with multiple bioremediation applications. From this it can be hypothesized that these structures are not limited to these organisms and may instead be a fairly common characteristic of biofilms. This could allow for the rapid transfer of both energy and information throughout the biofilm, potentially resulting in global or partitioned phenotypic changes.

1.4 Biofilms in general medicine

The human body is covered with bacteria, inside and out, such that our own cells are roughly outnumbered 10 to 1. It is largely due to this fact that the human body has evolved multiple strategies to deal with these potential invaders, from bodily mechanics to the development of a complex immune system, as well as the procurement of commensal microorganisms to inhabit our skin and oral, respiratory, gastro-intestinal and urogenital tracts. Despite these tactics, biofilms remain an extremely difficult mechanism for humans to cope with in terms of both pathogenic and opportunistically pathogenic microorganisms. The CDC (Centers for Disease Control and Prevention) reports that approximately 65% of all infections in developed countries are caused by biofilms, making apparent the fact that biofilm formation plays a key role in many clinical and sub-clinical infections (Hall-Stoodley et al., 2004). Biofilms have been identified in virtually every system in the human body, and prosthetic devices – including artificial joints, urinary catheters and heart valves – are prone to the formation of biofilms once exposed to pathogens and present a particular dilemma for clinicians (Gristina, 1987). In this section we briefly review some occurrences of biofilm formation in clinical infection in general.

Biofilms are primarily associated with bacterial infections, either in association with human infections or adherent to the surfaces of medical devices. That said, other pathogens have been known to form biofilms including yeasts and other fungi (Saarela et al., 2004). For instance, Candida spp. biofilms form in a similar fashion to those of bacterial species, and as with bacterial biofilms, fungal biofilms provide microbes with enhanced resistance to medical treatment with antifungal agents (Jabra-Rizk et al., 2004). The most commonly encountered biofilms in the human body reside in the oral cavity in the form of dental plaques (Marsh, 2003). In addition, chronic bacterial infections of the head and neck are commonly associated with biofilm formation. Such infections include chronic otitis media and chronic tonsillitis as well as prosthetic infections associated with endotracheal tubes and voice prostheses (Post et al., 2004). Chronic otitis media was long thought to be an inflammatory condition secondary to the fact that only one-third of infections had positive cultures. Evidence now suggests that bacterial biofilms form on the inner ear and occasionally release planktonic bacteria resulting in sporadic positive cultures (Post, 2001). This is supported by the finding that many biofilm-associated organisms commonly resist cultivation using standard plating techniques and media (Veeh et al., 2003). Foreign materials such as endotracheal and tracheostomy tubes present surfaces that are prone to biofilm formation. Natural host defenses such as coughing are rendered ineffective by the presence of the device and biofilm formation provides antibiotic resistance leading to complications such as ventilator acquired pneumonia.

Wound infections may be separated into acute and chronic infections. Acute wound infections are encountered in situations such as surgical wounds where pathogens are introduced into the tissues at the time of surgery. Despite preventative measures such as preoperative skin preparation with antimicrobial solutions, vigorous hand washing and sterile surgical technique it is estimated that roughly 3% of surgical wounds become infected (Horan et al., 1993). It is likely that virtually all surgical incisions are contaminated with bacteria, primarily with Staphylococcus spp. from the skin; however relatively few of these wounds become clinically infected. Factors such as the site of surgery (i.e. skin, bowel), skill of the surgical team, host factors and the virulence of the bacteria determine whether wound colonization results in infection (Fry and Fry, 2007). Chronic wound infections such as those associated with diabetic foot ulcers and venous leg ulcers are a significant cause of morbidity and mortality around the world. Infection of these wounds is well known to impede the process of wound healing and adequate antimicrobial therapy is essential to prevent complications such as sepsis or limb loss. It has been shown that many chronic infections result from bacterial biofilm formation, a process that likely contributes to the difficulty in their treatment (Costerton et al., 1999; Donlan and Costerton, 2002; James et al., 2008; Percival and Bowler, 2004). In general, evidence suggests that while biofilms are prevalent in chronic wounds they are relatively rare in acute wounds (James et al., 2008).

Burn injuries present a different sort of acute wound that may result in life-threatening infection. Death from systemic sepsis is the most common cause of mortality among burn patients (Mayhall, 2003). Thermal injuries are easily colonized by organisms such as Staphylococcus spp. and Pseudomonas aeruginosa. Strategies to prevent and/or treat infected burn wounds include systemic and topical antimicrobial treatments and dressings. There is evidence to support the role of biofilm formation in burn wounds enhancing bacterial resistance to treatment. Efforts have been made to develop topical therapies to both reduce biofilm formation and provide bactericidal action for the treatment of burn infections (Martineau and Dosch, 2007).

Patients with valvular heart disease, both native and prosthetic, have also been the focus of significant biofilm research. Native valve endocarditis results from interaction between the endothelial lining and circulating bacteria, primarily streptococci, although fungi have been identified as well. Additionally, infection of prosthetic heart valves (prosthetic valve endocarditis (PVE)) may affect up to 4% of patients with these devices (Douglas and Cobbs, 1992). Colonization of bacteria is most likely to occur at the sights of implantation of these valves where the tissue has been injured. PVE may be divided into early and late, with early infections being primarily caused by coagulase-negative staphylococci thought to result from contamination. Late PVE (>12 months) may also include S. aureus, enterococci, and fungi among pathogens (Donlan and Costerton, 2002).

As with endotracheal tubes and prosthetic heart valves, virtually every type of prosthetic used in the human body is prone to biofilm formation and infection. Strategies to prevent or eliminate biofilms will be discussed in greater detail later in this chapter. As discussed, the most commonly employed strategy to date, once prosthetic infection has been identified, is the complete removal and replacement of the prosthetic device. The disadvantages of this approach include the greater risk to the patient with increasing surgery, the risk of persistent infection and the greater technical difficulty of replacing the prosthetic in a previous surgical site. As we turn the discussion to the importance of biofilms in genitourinary infections, it will become increasingly important to distinguish between biofilms associated with prosthetic versus non-prosthetic infections.

1.5 Biofilms in urology

1.5.1 Hydrodynamics of the upper and lower urinary tract

In the upper urinary tract, urine flows largely peristaltically from the kidney to the bladder. Although modern research on this process has been active since Boyarski (Boyarsky et al., 1971), many details of urine transport remain unresolved (Vahidi and Fatouraee, 2007). This may be a result of the complexity of the transport process, which is not completely peristaltic. A second component of the flow is driven purely by a pressure gradient developed between the renal pelves and the bladder. This second flow component can be problematic should pressures in the bladder exceed those at the renal pelves. Furthermore, computational analysis has demonstrated that the normal initiation of peristalsis can itself momentarily reverse the flow in segments of the ureter nearest its upstream entrance (Vahidi and Fatouraee, 2007). These potential reflux conditions can lead to a reverse flow of urine, along with potential bacteria and toxins, from the bladder to renal pelves and on to the kidneys (Bykova and Regirer, 2005). Since the placement of a ureteral stent relaxes the annular muscles at the stent–ureter interface, subsequently disabling the peristaltic urine transport, it can further encourage this backward flow. Under these conditions urine flow, through and around the stent, becomes completely driven by the pressure differential that exists between the pelves and the bladder. This reduced and at times retrograde flow enhances the ability of biofilms to migrate along the device toward the kidneys, potentially leading to pyelonephritis (Choisy et al., 1998; Choong and Whitfield, 2000; Cummings et al., 2004). Importantly, it has been shown in mathematical modeling that perforating holes common to most stents act to reduce this potential for reflux (Cummings et al., 2004). Recent numerical modeling of a stented ureter revealed that these perforations or pass-through holes are generally inactive during flow, only to become stimulated once there is a fouling or blockage (Tong et al., 2007).

In general, urine flow from the bladder via the urethra has received little theoretical attention (Bykova and Regirer, 2005). Only relatively recently (Lecamwasam et al., 1999), experiments on surgically separate canine urethras revealed that urethral flow was most likely laminar. It has been speculated that the flow may briefly or sporadically transition to a non-laminar state in humans. Common flow rates in the urethra vary between 9 and 21 ml/s in adult males and between 10 and 18 ml/s in females. With the implementation of a catheter the characteristics of lower urinary tract flow change substantially. At the downstream end, the catheter expels the urine outside of the body; at the upstream end, the catheter extends into the bladder. By way of extension into the bladder the catheter effectively holds the internal sphincter open so as to create a constant drip flow through the catheter to a collection device. This sort of low energy flow has negative consequences for the patient as the higher shear rates associated with normal urination are no longer present. High flow velocities in normal urinary flow are a result of the driving pressure built up in the bladder by a closed internal sphincter. The higher shear rates connected with these flows are often successful at flushing out many of the potentially detrimental resident bacteria. A further catheter complication stems from the balloons attached to maintain catheter position. Often, the balloon and catheter orientation can lead to the formation of a urine sump at the bottom of the bladder leading to an accumulation of bacteria and deposits. In an attempt to regain some of the advantages of the healthy urine flushing regimen, some research has focused on the development of an electronic catheter valve. The valve can be opened on a programmable time release basis or manually by the patient through a proximity activator worn as a ring on a finger. Clinical trials of this device are due to finish around 2009 (Evans-Pughe, 2005). Clearly, this is an area where substantial gains can be made for patient comfort with innovative solutions that tap microbiological, urological and engineering expertise.

1.5.2 Urinary infections associated with biofilms

Infection of the genitourinary tract has long been a source of frustration for patients and physicians. Chronic bothersome infections such as recurrent cystitis and chronic bacterial prostatitis often resist treatment despite multi-modality therapy. It is certainly reasonable to assume that the formation of biofilms in the genitourinary tract is, in part, responsible for the chronicity of these infections as well as their resistance to treatment. In this section we examine some of the basic factors involved with genitourinary infection in general, as well as several specific examples where biofilms likely play a significant role in the infectious process.

The normal human urinary tract contains multiple factors to protect against infection including the antegrade flow of sterile urine, the presence of commensal bacteria and a smooth epithelial lining (urothelium). A number of additional host strategies exist that will be discussed in greater detail further into the chapter (Section 1.7.1). The failure of any or all of these systems through aging or pathologic processes may render the host more open to pathogenic bacterial colonization and infection. For example, the distal 1 cm of the female urethra contains a biofilm composed predominantly of lactobacilli similar to those present in the vagina (Costerton, 2007b). In this setting the biofilm is actually beneficial and functions to prevent the passage of pathogenic bacteria from the environment to the bladder. The distal portion of the male urethra contains a similar biofilm barrier and is significantly longer than the female counterpart making colonization even less likely (Costerton, 2007b). Any process that compromises this initial biofilm defense may eventually lead to urinary tract infection. Such processes include the insertion of foreign bodies (i.e. urethral catheters), colonization with pathogenic bacteria and the use of broad-spectrum antimicrobials that destroy the commensal bacteria. As we turn to specific examples of genitourinary infection involving biofilms, we will distinguish between those infections involving prosthetics (i.e. penile prostheses, urinary catheters, ureteral stents) and those that do not (i.e. chronic bacterial prostatitis, staghorn calculi).

Chronic prostatitis (CP) and male chronic pelvic pain syndrome (CPPS) present a challenging management dilemma for clinicians. Many patients suffer with ongoing symptoms such as urinary frequency and urgency as well as pelvic and penile pain without ever conclusively discerning the cause. Patients with such symptoms are often treated with antimicrobial agents even in the absence of positive cultures in an effort to eradicate sub-clinical infection. The leading theories behind the evolution of bacterial prostatitis are the retrograde spread of bacteria from the environment through the urethra and the reflux of infected urine through prostatic ducts. Acute bacterial prostatitis may be well treated with antibiotics while pathogens have not formed colonies and biofilms. Common pathogens include Escherichia coli, Klebsiella spp., enterobacteria, Pseudomonas aeruginosa, Staphylococcus spp. and Proteus spp., among others (Donlan and Costerton, 2002). Studies in animal models of prostatitis suggest the formation of glycocalyx-encased microcolonies of bacteria adherent to prostatic mucosa layers (Nickel et al., 1990). Nickel and Costerton (1993) examined prostatic biopsies in men with a history of chronic bacterial prostatitis. Bacteria were found attached to prostatic ductal walls, particularly P. aeruginosa. Others studies have clearly demonstrated the presence of biofilms in men who failed treatment for chronic bacterial prostatitis (Domingue and Hellstrom, 1998). It is a logical conclusion that biofilm formation is, at least in part, responsible for the failure of treatment in many men with chronic bacterial prostatitis. In the future, strategies for the prevention or disruption of these biofilms may significantly enhance our ability to manage this challenging disease.

Staghorn renal calculi are stones that fill much of the collecting system of the kidney and may result in serious complications such as urinary obstruction, renal failure or sepsis. These stones are typically associated with urea-splitting bacteria (primarily Proteus spp.) and, if left untreated, may result in renal loss or even death. Proteus vulgaris produces biofilms and struvite crystals that may lead to petrification of the urinary collecting system (Costerton, 2007b). Patients with staghorn calculi frequently have recurrent urinary tract infections that respond initially to antibiotics but almost certainly recur so long as the stone remains untreated. Successful treatment of this condition requires complete removal of all visible stone, usually through percutaneous renal surgery followed by antibiotic therapy to sterilize the urine and prevent recurrent infection and biofilm formation.

In North America alone more than 100 million urinary tract devices (urethral catheters, ureteral stents, penile prostheses) are inserted each year, translating into millions of device-associated infections and billions of dollars in additional health care expenditure annually (Jacobsen et al., 2008). One of the major problems associated with these foreign bodies is that they present novel, non-host surfaces on which bacteria can colonize and form biofilms. Urethral catheters are commonly used during surgery to monitor urinary output and do not typically result in clinical infection as there is a sufficiently short indwelling time and bacteria are unlikely to form biofilms. Conversely, many patients require long-term urethral catheterization and may suffer from chronic recurrent bladder infections. It has been estimated that the risk of bacteriuria increases by roughly 10% for each day that a urethral catheter is in place and that virtually all catheterized patients will develop urinary tract infection when the catheter remains in place beyond 28 days (Stickler, 1996). Not only does the colonization of urinary catheters lead to urinary tract infection but it may also affect the functioning of the catheter through obstruction of the lumen with encrustation (Liedl, 2001; Stickler et al., 1993; Stickler, 1996). Bacteria spread in a retrograde fashion and tend to progress faster through the lumen of the catheter than on the external surface (Nickel et al., 1985). Ureteral catheters (i.e. ureteral stents) are likewise subject to biofilm development, infection and encrustation. These devices may be colonized despite sterile urine cultures, again drawing attention to the lack of success in cultivating biofilm organisms (Lifshitz et al., 1999). The major bacterial species involved in catheter and stent infections are Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecalis and Staphylococcus aureus. Ureteral stent biofilms involving several of these pathogens are shown in Fig. 1.4. In addition, there are countless opportunistic pathogens that can form biofilms on these devices and cause infection depending upon the patient’s health and immune status, as well as microbial exposure. Management of patients with long-term catheterization and stenting remains a challenge. In most situations, only patients at risk for sepsis or those with symptomatic urinary tract infections should be actively treated while those with isolated bacteriuria may be observed. Routine device changes may help to reduce bacterial load and allow for more effective antibiotic therapy when required, since mature biofilms may be more difficult to treat than those less established (Anwar et al., 1989).

1.4 Panels show biofilms formed as a result of infection with (a) Candida albicans, (b) Enterococcus faecalis, (c) Escherichia coli, (e) Klebsiella pneumoniae and (f) Staphylococcus aureus; panel (d) shows encrustation caused by Proteus mirabilis and panel (g) is a cross-section of a ureteral stent infected with E. coli and P. mirabilis.

Genitourinary prosthetic devices such as artificial urinary sphincters and penile prostheses are convenient targets for bacterial seeding and biofilm infection (Silverstein and Donatucci, 2003). This is typically far more devastating to the patient than catheter infection due to the possible need for device removal and replacement. Not only are these surgeries difficult, but reoperations for prosthetic devices are associated with a much higher risk of infection than a primary procedure. It is accepted that most prosthetic infections result from bacterial seeding during surgery (Silverstein and Donatucci, 2003). These bacteria may cause early clinical infections or may form biofilms on the device surface and take years before causing a clinically apparent infection. Staphylococcus epidermidis is commonly isolated from infected penile prostheses and may be present in up to 80% of these infections (Blum, 1989). Interestingly, it is likely that the majority of penile prosthetics are seeded and colonized although most will never manifest with clinical infection. Silverstein et al. (2006) examined the devices of ten patients undergoing device revision or replacement of clinically noninfected penile prostheses. Bacterial biofilm formation was identified in eight of ten prostheses with multiple associated organisms suggesting that the number of prostheses colonized with bacteria is far greater than was previously believed.

The treatment of infected prostheses has traditionally involved device removal with a course of antibiotic therapy and delayed reinsertion of a new prosthetic (Silverstein and Donatucci, 2003). Infection is associated with pronounced inflammation and corporal scarring making insertion of a new device extremely difficult (Wilson and Delk, 1995). In more recent years, it has become far more common to attempt salvage of infected penile prostheses with removal of the infected device and replacement of a new device during the same procedure with adjuvant antibiotic treatment (Brant et al., 1996). From the standpoint of biofilms this makes sense as device removal should eliminate the vast majority of established biofilms and remaining bacteria are likely to be in the planktonic state where they will be more susceptible to antimicrobials.

1.5.3 The female urogenital tract

The female urogenital tract is an interesting microbial environment, in that it consists of areas commonly inhabited by microorganisms (vagina and distal urethra) and others regarded as being sterile (upper genital and urinary tracts). Thus, while generally 10⁷–10⁸ colony forming units (CFU)/gram of fluid are present within the vagina, the bladder and uterus are, for the most part, devoid of organisms. However, since organisms residing within the vagina have access to both the upper genital and urinary tracts, the vaginal microbiota can greatly influence the development of infections throughout the entire tract. In addition, factors such as sexual relations and exposure to spermicides, pregnancy, hormone fluctuations, menstruation, hygienic procedures, antibiotic usage and constant exposure to a vast array of environmental and fecal-derived organisms make it a highly dynamic region for both the host and microbes (Reid et al., 1990). This relative instability suggests that a niche will occur at times in which potential pathogens can attach, grow, form biofilms and cause infection. Indeed, aside from vaginal infections, studies have demonstrated a significant correlation between bacterial colonization of the vagina and periurethral area and the development of urinary tract infections (Stamey et al., 1971; Stapleton et al., 2002).

In a normal, healthy vagina the pH is generally below 4.5, which largely restricts the organisms that can survive there. Understandably, acidophilic species of bacteria, such as lactobacilli, typically predominate. Indeed, members of the Lactobacillus genus are the most commonly isolated organisms from the vaginal microbiota of healthy women, and as such have been hypothesized to play a major role in maintaining this healthy status. When the numbers of vaginal lactobacilli drop, typically due to one of the events listed above, multiple types of infection can develop. These include yeast vaginitis, bacterial vaginosis (BV), aerobic vaginitis (AV) and urinary tract infections. Collectively, these infections cover the fungi as well as most non-sexually transmitted bacterial species known to inhabit the urogenital tract. While BV is predominantly associated with anaerobes – such as Gardnerella vaginalis, Mobiluncus spp., Bacteroides spp., Peptostreptococcus spp., Prevotella bivia, Mycoplasma hominis, Atopobium vaginae and Ureaplasma urelyticum (Hale et al., 2006; O’Brien, 2005) – AV is generally caused by multiple species of aerobic bacteria including Escherichia coli, Staphylococcus aureus and members of the Group B streptococci (Romanik et al., 2007). Of these, only biofilms of G. vaginalis have been extensively studied within the vagina and been shown to regularly occur (Swidsinski et al., 2005). However, the frequent recovery of bacteria-laden epithelial cells from vaginal swabs as well as the ability of many of these organisms to remain in the tract for extended periods of time strongly supports their ability to form biofilms as well (Hillier, 1993; Saidi et al., 1994; Sobel, 1990). These biofilms have the potential to be powerful nidi for infection, especially in the case of urethral catheters, as aseptic technique during device insertion has no bearing on potential colonization. Ultimately, to maintain overall urogenital tract health in women, the goal is to simultaneously support the growth of commensal Lactobacillus biofilms within the vagina while inhibiting those associated with pathogens.

1.5.4 Intracellular biofilm communities (IBCs) caused by uropathogenic escherichia coli (UPEC)

It has been known for over 25 years that certain strains of E. coli could enter urinary epithelial cells (Fukushi et al., 1979), and it is now well recognized that this mode of attack is quite common during infection with UPEC. The host’s response to these organisms is exfoliation of the bladder epithelial lining to remove these bacteria-laden cells while preparing both innate and acquired immune response modalities to deal with the remaining organisms still present. However, it was not until Scott Hultgren’s group discovered the ability of UPEC cells to migrate through the superficial umbrella cells and into underlying layers of the bladder, where they multiplied and formed bacterial communities, that the idea of intracellular UPEC biofilms was formed (Anderson et al., 2004). While all of the early work by Hultgren’s group was performed using a murine model of UPEC cystitis (Anderson et al., 2004; Justice et al., 2004), their most recent studies have demonstrated these same IBCs in the exfoliated bladder cells of women with active cystitis or a history of its recurrence (Garofalo et al., 2007; Rosen et al., 2007). This finding offers the most likely explanation for observed UPEC resistance to host immune factors and antibiotics, in addition to their propensity for inducing chronic infections in the absence of immune defects or urinary prosthetics (Mysorekar and Hultgren, 2006). Furthermore, these findings offer potential insight to other conditions and chronic infections which may use similar mechanisms. One such urinary condition is interstitial cystitis, which has been suggested to be caused by chronic infection with undetected organisms such as Enterococcus spp., despite little solid evidence (Elgavish et al., 1997). Future research is required to determine whether any connections actually

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