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BIOFILMS – THE NEW MICROBIAL ORDER

 

 

Biofilms are the most common mode of bacterial growth in nature and are highly resistant to antibiotics (relevant in clinical infections). This form of microbial growth has been studied in a wide range of scientific disciplines including biomedicine, water engineering and evolutionary biology (2, 3, 14, 18, 20, 29, 31, 43, 52).A large number of studies have been performed targeted at the bacterial biofilms (41, 42, 49). However, little attention has been paid to medically relevant fungal biofilms. Transplantation procedures, immunosuppression, the use of chronic indwelling devices and prolonged intensive care unit stays have increased the prevalence of fungal disease. Fungi most commonly associated with such disease episodes are in the genus Candida, most notably C. albicans, which causes both superficial and systemic disease. Even with current antifungal therapy, mortality of patients with invasive candidiasis can be as high as 40 percent (50). Candidiasis is usually associated with indwelling medical devices (e.g. dental implants, catheters, heart valves, vascular bypass grafts, ocular lenses, artificial joints and central nervous system shunts), which can act as substrates for biofilm growth. The tenacity with which Candida infects indwelling biomedical devices makes it necessary to remove the catheter to treat the associated infections. Biofilm formation is also critical in the development of denture stomatitis, a superficial form of candidiasis that affects 65% of edentulous individuals (11, 12). Despite the use of antifungal drugs to treat denture stomatitis, infection is often re-established soon after treatment (35). These clinical observations emphasize the importance of biofilm formation to both superficial and systemic candidiasis and the inability of current antifungal therapy to cure such diseases.

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Biofilms and Catheter-Related Bloodstream Infections

Bloodstream infections (BSI) are a significant cause of morbidity and mortality in hospitalized patients (8, 36, 45)The use of central venous catheters (CVCs) in current therapeutic practice has been found to be responsible for more than 90% of these BSI (51), resulting in 20-30% mortality among hospitalized patients with CVC-related BSI (CR-BSI) (25, 26, 40)The micro-organisms most commonly associated with CR-BSI are Staphylococcus aureus, Candida albicans, coagulase-negative staphylococci, and aerobic gram-negative bacilli (8, 25, 32, 38). A nearly five-fold increase in the rate of nosocomial fungal BSI was reported for the period 1980 - 1990, in which Candida species were the most commonly isolated fungi (9). C. albicans, followed by C. parapsilosis, are the two predominant Candida species linked to intravascular CR-BSI (27, 40). The increased incidence of serious fungal infections is reflected in the prevalence of Candida cardiovascular infections such as native- and prosthetic-valve endocarditis, aortic graft infection, and arterial embolization (32).

The pathogenesis of most CR-BSI is complex and multifactorial, and in various studies has been linked to (a) duration of catheter retention (short-term, <14 days; or long-term, >14 days), (b) type of catheter material (Teflon, silicone elastomer, polyurethane, etc.), and (c) adherence/colonization properties of the organism (13, 21, 40) Most cases of CR-BSI involve colonization of micro-organisms on catheter surface where they eventually become embedded in a biofilm – an extensive community of organisms enclosed within a “slimy” extracellular matrix (ECM)(44). Supporting evidence in this regard is found in studies where biofilms have been demonstrated on right heart flow-directed catheters, endocardial pacemaker leads, and CVCs (22, 30, 34, 37, 38). In one study, direct microscopic examination by scanning electron microscopy showed that extensive biofilms were formed on 88% of CVCs that had been placed in intensive care unit patients (44). Thus, CR-BSI can often be the result of micro-organisms colonizing intravascular catheter surfaces to form biofilms, which serve as a nidus for infection. Biofilms are phenotypically different from planktonic (suspended) cells (41), and the most important manifestation of these differences is a significantly decreased susceptibility of biofilms to antimicrobials, a feature found to be true for both bacterial and fungal (5, 15, 16) biofilms.

Bacterial Biofilms

Bacterial biofilms formed by Pseudomonas aeruginosa, P. fluorescens, Escherichia coli, and Vibrio cholerae have been studied in some detail (41). A number of mechanisms including (a) drug efflux pumps (23), (b) drug diffusion (24), and (c) penetration through the ECM (1) have been proposed as mechanisms for the resistance of bacteria growing  as biofilms (10). However, none of these mechanisms alone can explain the phenomenon of increased resistance associated with biofilms. Other studies have identified genetic components required to form single-species bacterial biofilms and resulted in the identification of quorum sensing signals in P. aeruginosa biofilms (17, 19, 41, 42, 46).

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Candida albicans Biofilms

A plethora of information is available on bacterial biofilms which have led to their characterization in some detail (17, 19, 41, 42). In contrast, the field of fungal biofilms is still in its infancy. Candida biofilms have been shown to be resistant to the action of clinically important antifungal agents, including amphotericin B and fluconazole (Flu) (4, 5, 15, 16). Recently, our group established a reproducible and clinically relevant model of C. albicans biofilms, using dentures as well as catheter disks (15).  We and others, using scanning electron microscopy and fluorescence microscopy, showed that C. albicans biofilms grown on catheter material consist of yeasts, hyphae, and pseudohyphae arranged in a bilayer structure (6, 15) In the catheter model, there appeared to be a dense, basal yeast layer that anchor the biofilm to the catheter surface, and an overlying but more open, hyphal layer. A matrix of ECM surrounded the cells within a biofilm (15), and the synthesis of this material increased markedly when developing biofilms were subjected to a liquid flow (7). Recently, our group showed that  C. albicans produces quantitatively more biofilm than other Candida species (33, 39).

Our initial work on fungal biofilms involved development and characterization of C. albicans biofilms formed on two common bioprosthetic materials: polymethylmethacrylate, which is used in construction of dentures and silicone elastomer, a model material used for indwelling devices including catheters. The availability of well-characterized, reproducible biofilm models is essential to understand the nature of Candida biofilms and perform studies of biofilm formation and antifungal drug resistance. Recently, using the polymethylmethacrylate biofilm model, we showed that biofilm-grown C. albicans cells are highly resistant to antifungal agents such as fluconazole, nystatin, amphotericin B and chlorhexidine (16, published in the Journal of Dental Research), similar to previous observations reported for catheter-associated C. albicans biofilms (5, 28). We identified biofilm growth phases, determined the architectural organization and correlated antifungal resistance with biofilm development (15). The use of physiologic parameters and comparison to biofilms from patient specimens demonstrated the clinical relevance of our observations. We also compared C. albicans biofilm formation with that of Saccharomyces cerevisiae and performed an initial assessment of differential gene expression between planktonic and biofilm-grown Candida cells. Results from these studies were published in Journal of Bacteriology (15).

In a separate study on comparison of biofilm formation ability of C. albicans and C. parapsilosis, we showed that C. albicans produces quantitatively larger and more complex biofilms than other species, including C. parapsilosis. This work was recently published in Infection and Immunity (33).

These results supports the notion that true biofilms involve both the production of specific extracellular materials and special cellular functions. The information derived from these studies will further our understanding of Candida biofilm biology as well as antifungal resistance and may lead to novel therapies for biofilm-based diseases.

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LITERATURE CITED

   1.   Anderl, J. N., M. J. Franklin and P. S. Stewart. 2000. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob.Agents Chemother. 44:1818-1824.

   2.   Austin, J. W. and G. Bergeron. 1995. Development of bacterial biofilms in dairy processing lines. J.Dairy Res. 62:509-519.

   3.   Bagge, N., O. Ciofu, L. T. Skovgaard and N. Hoiby. 2000. Rapid development in vitro and in vivo of resistance to ceftazidime in biofilm-growing Pseudomonas aeruginosa due to chromosomal b-lactamase. APMIS 108:589-600.

   4.   Baillie, G. S. and L. J. Douglas. 1998. Effect of growth rate on resistance of Candida albicans biofilms to antifungal agents. Antimicrob.Agents Chemother. 42:1900-1905.

   5.   Baillie, G. S. and L. J. Douglas. 1999. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol. 310:644-656.

   6.   Baillie, G. S. and L. J. Douglas. 1999. Role of dimorphism in the development of Candida albicans biofilms. J.Med.Microbiol. 48:671-679.

   7.   Baillie, G. S. and L. J. Douglas. 2000. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J.Antimicrob.Chemother. 46:397-403.

   8.   Banerjee, S. N., T. G. Emori, D. H. Culver, R. P. Gaynes, W. R. Jarvis, T. Horan, J. R. Edwards, J. Tolson, T. Henderson and W. J. Martone. 1991. Secular trends in nosocomial primary bloodstream infections in the United States, 1980-1989. National Nosocomial Infections Surveillance System. Am.J.Med. 91:86S-89S.

   9.   Beck-Sagué, C. M. and W. R. Jarvis. 1993. Secular trends in the epidemiology of nosocomial fungal infetions in the United States, 1980-1990. National Nosocomial Infections Surveillance System. J.Inf.Dis. 167:1247-1251.

  10.   Brooun, A., S. Liu and K. Lewis. 2000. A dose-response study of antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob.Agents Chemother. 44:640-646.

  11.   Budtz-Jorgensen, E. 1990. Etiology, pathogenesis, therapy, and prophylaxis of oral yeast infections. Acta Odontol.Scand. 48:61-69.

  12.   Budtz-Jorgensen, E. 1990. Histopathology, immunology, and serology of oral yeast infections. Diagnosis of oral candidosis. Acta Odontol.Scand. 48:37-43.

  13.   Calderone, R. A. and P. C. Braun. 1991. Adherence and receptor relationships of Candida albicans. Microbiol.Rev. 55:1-20.

  14.   Carr, J. H., R. L. Anderson and M. S. Favero. 1996. Comparison of chemical dehydration and critical point drying for the stabilization and visualization of aging biofilm present on interior surfaces of PVC distribution pipe. J.Appl.Bacteriol. 80:225-232.

  15.   Chandra, J., D. M. Kuhn, P. K. Mukherjee, L. L. Hoyer, T. McCormick and M. A. Ghannoum. 2001. Biofilm formation by the fungal pathogen Candida albicans - development, architecture and drug resistance. J.Bacteriol. 180: 5385-5394.

  16.   Chandra, J., P. K. Mukherjee, S. D. Leidich, F. F. Faddoul, L. L. Hoyer, L. J. Douglas and M. A. Ghannoum. 2001. Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J.Dent.Res. 80:903-908.

  17.   Costerton, J. W. 1995. Overview of microbial biofilms. J.Ind.Microbiol. 15:137-140.

  18.   Costerton, J. W. 2001. Cystic fibrosis pathogenesis and the role of biofilms in persistent infection. Trends Microbiol. 9:50-52.

  19.   Costerton, J. W., Z. Lewandowski, D. E. Caldwell, D. R. Korber and H. M. Lappin-Scott. 1995. Microbial biofilms. Annu.Rev.Microbiol. 49:711-745.

  20.   Costerton, J. W., P. S. Stewart and E. P. Greenberg. 1999. Bacterial biofilms: a common cause of persistent infections. Science 284:1318-1322.

  21.   Crump, J. A. and P. J. Collignon. 2000. Intravascular catheter-associated infections. Eur.J.Clin.Microbiol.Infect.Dis. 19:1-8.

  22.   Dato, V. M. and A. S. Dajani. 1990. Candidemia in children with central venous catheters: role of catheter removal and amphotericin B therapy. Pediatr.Infect.Dis.J. 9:309-314.

  23.   De Kievit, T. R., M. D. Parkins, R. J. Gillis, R. Srikumar, H. Ceri, K. Poole, B. H. Iglewski and D. G. Storey . 2001. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob.Agents Chemother. 45:1761-1770.

  24.   Dunne, W. M., E. O. Mason and S. L. Kaplan. 1993. Diffusion of rifampin and vancomycin through a Staphylococcus epidermidis biofilm. Antimicrob.Agents Chemother. 37:2522-2526.

  25.   Emori, T. G., S. N. Banerjee, D. H. Culver, R. P. Gaynes, T. C. Horan, J. R. Edwards, W. R. Jarvis, J. S. Tolson, T. S. Henderson, W. J. Martone and . 1991. Nosocomial infections in elderly patients in the United States, 1986-1990. National Nosocomial Infections Surveillance System. Am.J.Med. 91:289S-293S.

  26.   Gaynes, R. P., W. J. Martone, D. H. Culver, T. G. Emori, T. C. Horan, S. N. Banerjee, J. R. Edwards, W. R. Jarvis, J. S. Tolson, T. S. Henderson and . 1991. Comparison of rates of nosocomial infections in neonatal intensive care units in the United States. National Nosocomial Infections Surveillance System. Am.J.Med. 91:192S-196S.

  27.   Groeger, J. S., A. B. Lucas, H. T. Thaler, H. Friedlander-Klar, A. E. Brown, T. E. Kiehn and D. Armstrong. 1993. Infectious morbidity associated with long-term use of venous access devices in patients with cancer. Ann.Intern.Med. 119:1168-1174.

  28.   Hawser, S. P. and L. J. Douglas. 1995. Resistance of Candida albicans biofilms to antifungal agents in vitro. Antimicrob.Agents Chemother. 39:2128-2131.

  29.   Hellio, C., G. Bremer, A. M. Pons, Y. Le Gal and N. Bourgougnon. 2000. Inhibition of the development of microorganisms (bacteria and fungi) by extracts of marine algae from Brittany, France. Appl.Microbiol.Biotechnol. 54:543-549.

  30.   Hollingsed, M. J., J. M. Morales, P. T. Roughneen and K. D. Burch. 1997. Surgical management of catheter tip thrombus: surgical therapy for right atrial thrombus and fungal endocarditis (Candida tropicalis) complicating paediatric sickle-cell disease. Perfusion 12:197-201.

  31.   Hood, S. K. and E. A. Zottola. 1997. Adherence to stainless steel by food borne microorganisms during growth in model food systems. Int.J.Food Microbiol. 37:145-153.

  32.   Kuehnert, M. J., E. Clark, S. R. Lockhart, D. R. Soll, J. Chia and W. R. Jarvis. 1998. Candida albicans endocarditis associated with a contaminated aortic valve allograft: implications for regulation of allograft processing. Clin.Infect.Dis. 27:688-691.

  33.   Kuhn, D. M., J. Chandra, P. K. Mukherjee and M. A. Ghannoum. 2002. Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect.Immun. 70: 878-888.

  34.   Leung, W. H., C. P. Lau, Y. T. Tai, C. K. Wong and C. H. Cheng. 1990. Candida right ventricular mural endocarditis complicating indwelling right atrial catheter. Chest 97:1492-1493.

  35.   MacEntee, M. I. 1985. The prevalence of edentulism and diseases related to dentures - a literature review. J.Oral Rehabil. 12:195-207.

  36.   Maki, D. G. and L. A. Mermel. 1998. Infections due to infusion therapy, In Hospital Infections. Bennett, J. V. and Brachman, P. S., (eds.) > pp. 689-724.  Lippincott-Raven, Philadelphia, PA.

  37.   Martino, P., A. Micozzi, M. Venditti, G. Gentile, C. Girmenia, R. Raccah, S. Santilli, N. Alessandri and F. Mandelli. 1990. Catheter-related right-sided endocarditis in bone marrow transplant recipients. Rev.Infect.Dis. 12:250-257.

  38.   Mermel, L. A., B. M. Farr, R. J. Sherertz, I. I. Raad, N. O'Grady, J. S. Harris and D. E. Craven. 2001. Guidelines for the management of intravascular catheter-related infections. Clin.Infect.Dis. 32:1249-1272.

  39.   Mukherjee PK, Chandra J, Kuhn DM, Walker CA, Ghannoum MA: Analysis of differential expression of proteins between the biofilm and planktonic forms of Candida albicans. 2002

  40.   Nicastri, E., N. Petrosiillo, P. Viale and G. Ippolito. 2001. Catheter-related bloodstream infections in HIV-infected patients. Ann.N.Y.Acad.Sci. 946:274-90.:274-290.

  41.   O'Toole, G., H. B. Kaplan and R. Kolter. 2000. Biofilm formation as microbial development. Annu.Rev.Microbiol. 54:49-79.

  42.   O'Toole, G. A., L. A. Pratt, P. I. Watnick, D. K. Newman, V. B. Weaver and R. Kolter. 1999. Genetic approaches to study of biofilms. Methods Enzymol. 310:91-109.

  43.   Ortega-Morales, O., J. Guezennec, G. Hernandez-Duque, C. C. Gaylarde and P. M. Gaylarde. 2000. Phototrophic biofilms on ancient Mayan buildings in Yucatan, Mexico. Curr.Microbiol. 40:81-85.

  44.   Passerini, L., K. Lam, J. W. Costerton and E. G. King. 1992. Biofilms on indwelling vascular catheters. Crit Care Med. 20:665-673.

  45.   Pittet, D., D. Tarara and R. P. Wenzel. 1994. Nosocomial bloodstream infection in critically ill patients. Excess length of stay, extra costs, and attributable mortality. JAMA 271:1598-1601.

  46.   Pratt, L. A. and R. Kolter. 1999. Genetic analyses of bacterial biofilm formation. Curr.Opin.Microbiol. 2:598-603.

  47.   Stewart, P. S. and J. W. Costerton. 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358:135-138.

  48.   Vorachit, M., K. Lam, P. Jayanetra and J. W. Costerton. 1993. Resistance of Pseudomonas pseudomallei growing as a biofilm on silastic discs to ceftazidime and co-trimoxazole. Antimicrob.Agents Chemother. 37:2000-2002.

  49.   Watnick, P. and R. Kolter. 2000. Biofilm, city of microbes. J.Bacteriol. 182:2675-2679.

  50.   Wey, S. B., M. Mori, M. A. Pfaller, R. F. Woolson and R. P. Wenzel. 1988. Hospital-acquired candidemia. The attributable mortality and excess length of stay. Arch.Intern.Med. 148:2642-2645.

  51.   Widmer, A. 1993. IV-related infections, In Prevention and Control of Nosocomial Infections. Wenzel, R. P., (ed.) > pp. 556-579.  Williams & Wilkins, Baltimore.

  52.   Xu, K. D., G. A. McFeters and P. S. Stewart. 2000. Biofilm resistance to antimicrobial agents. Microbiology 146:547-549.

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