Abstract

In recent years there has been an increasing appreciation that microbial biofilms are ubiquitous, which has resulted in a number of studies on infectious diseases from a biofilm perspective. Biofilms are defined as structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. A wide range of biomaterials used in clinical practice have been shown to support colonization and biofilm formation by Candida spp., and the increase in Candida infections in the last decades has almost paralleled the increase and widespread use of a broad range of medical implant devices, mainly in populations with impaired host defenses. Formation of Candida biofilms has important clinical repercussions because of their increased resistance to antifungal therapy and the ability of cells within biofilms to withstand host immune defenses. Further recognition and understanding of the role of Candida biofilms in human infection should help in the clinical management of these recalcitrant infections.

Introduction

Our perception of microorganisms as unicellular life forms is primarily based on the pure-culture mode of growth. Since microorganisms can be diluted to a single cell and studied in liquid culture, this mode of growth has overwhelmingly predominated in the study of microbial physiology and pathogenesis in the research laboratory. However, the majority of microorganisms in their natural habitats are attached to surfaces within a structured biofilm ecosystem rather than existing as free-floating (planktonic) organisms (Costerton, 1987; Donlan, 2002). Biofilms are structured microbial communities attached to surfaces and encapsulated within a protective extracellular matrix (Costerton, 1995). Recently, there has been an increasing recognition of the role that microbial biofilms play in human medicine, and it has been estimated that about 65% of all human microbial infections involve biofilms (Khardori & Yassien, 1995; Costerton, 1999; Donlan, 2001a, b; Donlan & Costerton, 2002; Douglas, 2003). This has important consequences, since sessile cells in biofilms display phenotypic traits that are dramatically different from those of their planktonic counterparts, such as increased resistance to antimicrobial agents and protection from host defenses (Costerton, 1995; Gilbert, 1997; Habash & Reid, 1999).

A variety of manifestations of infections caused by Candida spp., particularly Candida albicans, are associated with the formation of biofilms on implantable medical devices (Douglas, 2002, 2003; Kumamoto, 2002). These devices provide the necessary surfaces for biofilm formation and are currently responsible for a significant percentage of clinical candidiasis. Moreover, biomaterial-related infections and bloodstream infections might be related to one another by virtue of hematogenous spread: a candidemia may stem from an indwelling device infection, where sessile (biofilm) cells are protected from the hostile environment of the host. Clearly, modern medicine, through newer and more aggressive treatment regimens, creates a high-risk environment for infection, while standard or advanced medical procedures create an opportunity for the formation of a biofilm. Once a Candida biofilm forms in vivo, removal of the substrate that is supporting the biofilm growth is almost always required to eliminate the infection. Unfortunately, in many instances removal is impossible, due to the patient's condition, the anatomic location, or underlying disease. The net effect is that Candida biofilms adversely impact on the health of these patients, with increasing frequency and severity, and with soaring economic sequelae (Beck-Sague & Jarvis, 1993,Wilson, 2002).

Formation and characteristics of Candida biofilms

Candida organisms are normal commensals of humans, which facilitates their encounter with most implanted biomaterials. In other instances, the devices become contaminated prior or during implantation due to manipulation by medical personnel. To colonize any implanted medical device, yeast cells must first adhere to biomaterial surfaces. The biomaterial properties affecting initial adhesion range from chemical properties to hydrophobicity to surface roughness. Since these biomedical devices are usually surrounded by body fluids such as urine, blood, saliva and synovial fluid, their surfaces often acquire a glycoproteinaceous conditioning film following implantation (Gristina, 1987; Francois, 1998). This conditioning film could confer chemical characteristics that are completely different from those of the original biomaterial. The initial attachment of Candida cells to biomaterials is mediated by nonspecific factors (cell surface hydrophobicity and electrostatic forces) and by specific adhesins on the fungal surface recognizing ligands in the conditioning films, such as serum proteins (fibrinogen and fibronectin) and salivary factors (for a review see Chaffin, 1998). Additionally, Candida cells can coaggregate and/or bind to bacteria already colonizing these devices (Chaffin, 1998). The initial focal attachment of individual cells to a substratum is closely followed by cell division, proliferation, and biofilm development. Mature Candida biofilms exhibit a complex three-dimensional structure and extensive spatial heterogeneity, with a typical microcolony/water channel architecture, and are encased within exopolymeric material, similar to what has been reported for bacterial biofilms (Hawser & Douglas, 1994; Hawser, 1998; Baillie & Douglas, 1999; Chandra, 2001a, b; Ramage, 2001b, c, d, 2002d; Kuhn, 2002a). This structural complexity represents an optimal spatial arrangement for influx of nutrients, disposal of waste products and establishment of microniches throughout the biofilm of microcolonies and ramifying water channels. Quorum sensing seems to play an important role in Candida biofilm formation (Ramage, 2002b).

From the clinical perspective, the most important feature of Candida biofilms is their high level of resistance to conventional antifungal therapy. Several groups have demonstrated that the Candida biofilm lifestyle leads to dramatically increased levels of resistance to the most commonly used antifungal agents, fluconazole and amphotericin B (Hawser & Douglas, 1995; Hawser, 1996; Baillie & Douglas, 1999; Chandra, 2001b; Ramage, 2001a, b, c, d, 2002c; Kuhn, 2002b). Different mechanisms may be responsible for the intrinsic resistance of Candida biofilms. These include: (1) effects of the biofilm matrix on penetration of drugs; (2) decreased growth rate and nutrient limitation; (3) expression of resistance genes, particularly those encoding efflux pumps; and (4) presence of ‘persister’ cells (Baillie & Douglas, 1998a, b, 2000; Lewis, 2001; Ramage, 2001b, c, d, 2002a, c; Kuhn, 2002b; Mukherjee, 2003; Andes, 2004; Mateus, 2004). However, the current consensus is that biofilm resistance is a complex phenomenon that cannot be explained by one mechanism alone; instead, it is multifactorial and may involve different molecular mechanisms of resistance as compared to those displayed by planktonic cells (Ramage, 2001a, 2002a, 2005; Douglas, 2002, 2003). In the case of C. albicans biofilms, it has been reported that they are up to 4000 times more resistant to fluconazole when compared to planktonic, free-floating cells (Ramage, 2001b, c, d). It is important to note that standard NCCLS broth dilution techniques for antifungal susceptibility testing use planktonic populations and will not enable prediction of antifungal efficacy against Candida biofilms (National Committee for Clinical Laboratory Standards, 1997). Thus, this may be one of the main reasons for the lack of correlation between results of antifungal susceptibility testing, as determined by NCCLS guidelines, and clinical outcome in patients suffering from these types of infection (Ghannoum, 1997; Rex, 1997). However, determining the effectiveness of different antifungal agents in this setting has important clinical implications, in that it may guide therapeutic decisions that potentially may affect the outcome of patients suffering from these difficult-to-treat infections. A recently developed microtiter plate-based biofilm model that is compatible with the 96-well platform technology has proved valuable for determination and standardization of antifungal susceptibility testing in Candida biofilms (Ramage, 2001b, c, d). Newer antifungal agents, such as the echinocandins and liposomal formulations of amphotericin B, show increased activity against Candida biofilms (Bachmann, 2002; Kuhn, 2002b; Ramage, 2002c). The echinocandins are a new class of antifungal agents that act by inhibiting synthesis of 1,3-β-d-glucan, a key step in fungal cell wall biosynthesis (Bartizal, 1997). For example, caspofungin, the first FDA-approved member of this class, displays potent in vitro activity against C. albicans biofilms, with sessile minimum inhibitory concentration values well within its therapeutic range (Bachmann, 2002; Ramage, 2002c).

Implantable medical devices in which Candida biofilms may develop

There is no question that the use of various medical devices has greatly facilitated the management of serious medical and surgical conditions. However, introduction of artificial materials into several body locations has been accompanied by the ability of microorganisms, including Candida spp., to colonize them and form biofilms that protect them from antibiotics and host defenses, leading to persistent infections. Devices such as shunts, prostheses (voice, heart valve, knee, etc.), stents, implants (lens, breast, denture, etc.), endotracheal tubes, pacemakers and various types of catheter have all been shown to support colonization and biofilm formation by Candida (Table 1). Not only does Candida colonization of biomaterials precede infection, but it can also adversely affect the function of the implanted device. A description follows of Candida biofilms on specific devices.

1

Implantable devices in which Candida biofilms develop most frequently

Device Usage per year Infection risk (%) Main Candida species 
Central and peripheral venous catheters 5 million 3–8 albicans 
  glabrata 
  parapsilosis 
Hemodialysis and peritoneal dialysis catheters 240 000 1–20 albicans 
  parapsilosis 
Urinary catheters Tens of millions 10–30 albicans 
  glabrata 
Endotracheal tubes Millions 10–25 albicans 
Intracardiac prosthetic devices 400 000 1–3 albicans 
  glabrata 
  parapsilosis 
  tropicalis 
Breast implants 130 000 1–2 albicans 
Prosthetic joints 600 000 1–3 parapsilosis 
  albicans 
  glabrata 
Neurosurgical shunts 40 000 6–15 albicans 
Voice prostheses Thousands 50–100 albicans 
  tropicalis 
Dentures >1 million 5–10 albicans 
  glabrata 
Device Usage per year Infection risk (%) Main Candida species 
Central and peripheral venous catheters 5 million 3–8 albicans 
  glabrata 
  parapsilosis 
Hemodialysis and peritoneal dialysis catheters 240 000 1–20 albicans 
  parapsilosis 
Urinary catheters Tens of millions 10–30 albicans 
  glabrata 
Endotracheal tubes Millions 10–25 albicans 
Intracardiac prosthetic devices 400 000 1–3 albicans 
  glabrata 
  parapsilosis 
  tropicalis 
Breast implants 130 000 1–2 albicans 
Prosthetic joints 600 000 1–3 parapsilosis 
  albicans 
  glabrata 
Neurosurgical shunts 40 000 6–15 albicans 
Voice prostheses Thousands 50–100 albicans 
  tropicalis 
Dentures >1 million 5–10 albicans 
  glabrata 

Central venous catheters

The use of central venous catheters has become common practice in modern medicine. Unfortunately, their increased role in patient management has been accompanied by a steady rise in catheter-related infections. Intravenous catheters are the leading cause of nosocomial bloodstream infections, and infection related to central venous catheters results in significant increases in hospital costs, duration of hospitalization, and patient morbidity. Scanning electron microscopy (SEM) and transmission electron microscopy have shown that biofilms are present in virtually all central venous catheters (on either the outside or the inner lumen) (Raad, 1998). Importantly, yeasts (mainly Candida) are the third leading cause of intravascular catheter-related infections, with the second highest colonization to infection rate and the overall highest crude mortality (Crump & Collignon, 2000). Infections may arise at any time during hospitalization. Most frequently, contamination occurs by: (1) extraluminal colonization of the catheter, which originates from the skin, either from the patient's skin microbial community or from the exogenous microbial communities of medical personnel; or (2) by hematogenous seeding from a distal site of infection, or after reaching the bloodstream from the gastrointestinal tract following cytotoxic therapy; or (3) by intraluminal colonization of the hub and lumen of the device (Raad, 1998, 2000; Crump & Collignon, 2000). Less commonly contaminated infusates may also be the origin of infection. Diagnosis is initially based on clinical findings, and treatment is usually empirical. Management of Candida catheter-related infections should include catheter removal plus treatment with antifungal therapy for at least 14 days after the last positive blood culture and when signs and symptoms of infection have resolved (Raad, 2000; Mermel, 2001; O'Grady, 2002). Failure to remove the catheter is almost inevitably associated with poor prognosis and higher mortality rates (Viudes, 2002).

Implantable venous access ports

The need for intravenous access devices for the long-term administration of anticancer therapy has grown proportionally with the increasing numbers of patients diagnosed with cancer. In these patients, infections represent the most common and feared complication of implantable venous access ports (Kurul, 2002; Chang, 2003). Candida spp. are among the most common microorganisms isolated (Chang, 2003). The differential diagnosis is often difficult, so in many cases these devices are removed on suspicion (Kurul, 2002).

Hemodialysis and peritoneal dialysis catheters

Biofilm microbial infections are common in patients undergoing treatment with hemodialysis and peritoneal dialysis (Dasgupta, 2002). The growth of Candida biofilms serves as a nidus for infection and candidemia in hemodialysis patients (Dasgupta, 2002). Infection of peritoneal dialysis catheters leading to Candida peritonitis is a common complication in patients with end-stage renal disease treated by peritoneal dialysis, with high rates of morbidity and mortality (Dasgupta, 2002; Bibashi, 2003). Clinical manifestations are nonspecific, but culture of Candida organisms in the dialysis effluent can confirm the infection. The most frequently isolated species are C. albicans and C. parapsilosis. These infections can lead to loss of ultrafiltration and the discontinuation of dialysis treatment. Treatment of these infections requires early institution of antifungal therapy and removal of the peritoneal catheter (Dasgupta, 2002; Bibashi, 2003).

Urinary catheters

Candida spp. (mainly C. albicans and C. glabrata) are frequent causative organisms of catheter-related urinary tract infection, which remains a leading cause of nosocomial infections, with significant morbidity, and additional hospital costs (Harris, 1999; Foxman, 2003). For example, Candida spp. are now the microbial pathogens that are most frequently isolated from the urine samples of patients in surgical intensive care units (ICUs), with about 10–15% of nosocomial urinary tract infections being caused by Candida (Lundstrom & Sobel, 2001). The presence of an indwelling urethral catheter represents a significant risk factor for urinary tract infection. Catheterization can cause infection by introducing organisms during the catheterization process or by allowing migration of organisms into the bladder along the surface of the catheter from the external periurethral surfaces. However, it has to be noted that the distinction between Candida colonization of the urinary tract and infection is often problematic, and asymptomatic candiduria occurs in a large proportion of catheterized ICU patients (Lundstrom & Sobel, 2001). If this is the case, it infrequently requires antifungal therapy, because morbidity is low and ascending infection and candidemia remain rare complications (Rivett, 1986). As is characteristic of biofilm-associated infections, in these asymptomatic patients candiduria tends not to be eradicated by antifungal therapy but normally resolves with catheter removal.

Intrauterine devices

Candida spp. are frequent inhabitants of the female genital tract. Fetal Candida infection is rarely described but is often associated with a retained intrauterine contraceptive device in situ, where Candida may form biofilms. Thus, the presence of foreign intrauterine bodies during pregnancy necessitates repetitive search for Candida infections and prompts adequate antifungal treatment in cases of documented infection (Roque, 1999).

Endotracheal tubes

Endotracheal tubes have been cited as potential reservoirs for infecting organisms, including Candida spp. in the respiratory tract, a major risk factor for ventilator-associated pneumonia (Koerner, 1997; Adair, 1999). Initial colonization of endotracheal tubes with Candida spp. may occur as a result of gastropulmonary reflux or from a contaminated oropharynx. Within hours of placement, microorganisms readily adhere to the surface of endotracheal tubes and eventually form mature biofilms with extensive slime production (Inglis, 1989, 1995). Infective aggregates can become dislodged from the main body of the biofilm and disseminate towards the lower respiratory tract (Inglis, 1993; Koerner, 1997).

Intracardiac prosthetic devices

Candida spp. are among the microorganisms capable of producing prosthetic valve endocarditis, a condition resulting from attachment and subsequent biofilm development on components of mechanical heart valves and surrounding heart tissue (Hauser, 2003). Contamination can occur at the time of surgery or originate from an indwelling device. Diagnosis, mostly based on blood cultures and echocardiographic findings in febrile high-risk patients, is often difficult because of the severity of patients' comorbid illnesses and the coexistence of several risk factors. Mortality rates associated with these infections are high. A combination of surgery with debridement, valve replacement and antifungal therapy normally offers the best outcome in these patients (Muehrcke, 1995). Permanent transvenous pacemakers, implantable cardioverter defibrillators and left ventricular assist devices are also susceptible to Candida infection (Giamarellou, 2002).

Neurosurgical shunts

Infections are common complications of neurosurgical shunting. Although the majority of these infections are caused by bacteria, reports of fungal infections, particularly Candida, have increased in the last few years (Montero, 2000). The biofilm etiology of these infections was demonstrated by Gower (1986) who reported SEM of an infected shunt showing a fungal biofilm adherent to the silicone elastomer tubing. Infections occurring shortly after placement are probably the result of contamination at the time of placement, but later infections may be due to transient episodes of candidemia. Signs and symptoms are often nonspecific: nausea, vomiting, and low level of activity, with declining mental status with fever being an inconsistent finding. Blood cultures are typically negative but culture of Candida organisms from the shunt liquid may be indicative of infection. Shunt obstruction may occur. Treatment consists of antifungal therapy (systemic but sometimes supplemented with intrathecal administration of antifungals) and shunt removal (Montero, 2000; Murphy, 2000).

Prosthetic joints

Prosthetic joint (mostly hip and knee arthroplasties) infections with Candida are relatively rare, with fewer than 30 cases having been reported in the English-language literature (Yang, 2001; Phelan, 2002). The main etiologic agents are C. albicans, C. parapsilosis and C. glabrata. Contamination most likely results from the inoculation of skin microorganisms at the time of implantation, but Candida organisms can also produce infection by hematogenous spread or local invasion. Management of these infections typically includes surgical procedures (thorough debridement to remove all cement involving infectious materials and necrotic tissue, which may also include removal of the implant) together with an appropriate long course of systemic antifungal therapy (Phelan, 2002).

Voice prostheses

Because the environment in which they are placed is not sterile, silicone rubber voice prostheses fitted in laryngectomized patients are subject to rapid microbial colonization and subsequent biofilm formation, most notably by Candida spp. (Neu, 1994; Ell, 1996). These prostheses often fail within months of placement because the biofilm causes malfunction (increased airflow resistance and leakage) of the valve, which needs to be replaced.

Dentures

The involvement of Candida as a potential causative agent in denture-induced stomatitis (‘rubber sore mouth’) was first described by Cahn in 1936. Candida-associated denture stomatitis is a common recurring disease, observed in c. 11–67% of otherwise healthy denture wearers (Arendorf & Walker, 1987). The etiology of the disease is multifactorial. Although current studies indicate that denture stomatitis is generally associated with the presence of Candida, other factors, such as denture cleanliness, trauma, oral bacteria and certain immune defects, may also be involved (Webb, 1998). Surface roughness and cracks within the acrylic support attachment and colonization of Candida, which represents the first step in the infectious process and ultimately results in varying grades of denture stomatitis due to injury to the adjacent mucosa (Radford, 1999). Retrieval of portions of dentures from patients suffering from denture stomatitis followed by examination by SEM allowed the visualization of Candida biofilms formed in vivo and demonstrated a role for Candida biofilms in the etiology of denture stomatitis. These biofilms consisted mainly of intricate networks of yeast cells and hyphae, deeply embedded in cracks and imperfections of the biomaterials (Ramage, 2003).

Conclusions

Candida biofilms possess unique developmental characteristics that are in stark contrast to free-floating planktonic cells. Several aspects of Candida biofilms make their formation on implanted biomaterials a highly clinically significant process. These are their increased resistance to antifungal agents, their protection from host defenses, and their role as reservoirs for persistent sources of infections. Biofilm formation may also cause device failure. Once Candida biofilms are formed on these devices, therapeutic interventions only rarely achieve clinical cure, and removal of the infected device is required, often involving surgery. Still, mortality rates associated with these types of infections remain unacceptably high. Increased awareness of the role of Candida biofilms in human medicine should positively impact on the clinical decision-making process, which directly affects patients' health and safety. New antifungal agents and preventive strategies such as antimicrobial coatings may offer new hope in the management of these recalcitrant infections.

Acknowledgements

J.L.L.-R. was the recipient of a New Investigator Award in Molecular Pathogenic Mycology from the Burroughs Wellcome Fund.

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Author notes

Editor: Rafael Sentandreu