Abstract
A filter disk assay is described, which measures the penetration of antifungal agents through Candida biofilms. The technique involves forming a colony biofilm on a polycarbonate membrane filter, and capping it with a second, smaller membrane filter followed by a wetted paper disk of the type used in zone-of-inhibition assays. The entire assembly is transferred to agar medium containing the antifungal agent of interest. During subsequent incubation, the drug diffuses out of the agar and through the biofilm ‘sandwich’ to the moistened paper disk. The drug concentration in the disk can be determined by measuring the zone of growth inhibition that it produces on medium seeded with an indicator strain of Candida albicans in standard bioassays. Additional procedures are outlined for determining the viabilities of drug-treated biofilms and for examining biofilm morphology by scanning electron microscopy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Calderone, R. A. (ed.) (2002) Candida and Candidiasis. ASM Press, Washington, D.C.
Kojic, E. M., and Darouiche, R. O. (2004) Candida infections of medical devices. Clin. Microbiol. Rev. 17, 255–267.
Douglas, L. J. (2003) Candida biofilms and their role in infection. Trends Microbiol. 11, 30–36.
Donlan, R. M. (2001) Biofilms and device-associated infections. Emerg. Infect. Dis. 7, 277–281.
Hawser, S. P., and Douglas, L. J. (1995) Resistance of Candida albicans biofilms to antifungal agents in vitro. Antimicrob. Agents Chemother. 39, 2128–2131.
Baillie, G. S., and Douglas, L. J. (1998) Effect of growth rate on resistance of Candida albicans biofilms to antifungal agents. Antimicrob. Agents Chemother. 42, 1900–1905.
Baillie, G. S., and Douglas, L. J. (1998) Iron-limited biofilms of Candida albicans and their susceptibility to amphotericin B. Antimicrob. Agents Chemother. 42, 2146–2149.
Baillie, G. S., and Douglas, L .J. (1999) Candida biofilms and their susceptibility to antifungal agents. Meth. Enzymol. 310, 644–656.
Baillie, G. S., and Douglas, L. J. (1999) Role of dimorphism in the development of Candida albicans biofilms. J. Med. Microbiol. 48, 671–679.
Chandra, J., Mukherjee, P. K., Leidich, S. D., Faddoul, F. F., Hoyer, L. L., Douglas, L. J., and Ghannoum, M. A. (2001) Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J. Dent. Res. 80, 903–908.
Chandra J., Kuhn, D. M., Mukherjee, P. K., Hoyer, L. L., McCormick, T., and Ghannoum, M. A. ( 2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J. Bacteriol. 183, 5385–5394.
Lewis, R. E., Kontoyiannis, D. P., Darouiche, R. O., Raad, I. I., and Prince, R. A. (2002) Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model of Candida catheter-related bloodstream infection. Antimicrob. Agents Chemother. 46, 3499–3505.
Ramage, G., VandeWalle, K., Wickes, B. L., and Lopez-Ribot, J. L. (2001) Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob. Agents Chemother. 45, 2475–2479.
Ramage, G., VandeWalle, K., Wickes, B. L., and Lopez-Ribot, J. L. (2001) Biofilm formation by Candida dubliniensis. J. Clin. Microbiol. 39, 3234–3240.
Andes, D., Nett, J., Oschel, P., Albrecht, R., Marchillo, K., and Pitula, A. (2004) Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect. Immun. 72, 6023–6031.
Gilbert, P., Maira-Litran, T., McBain, A. J., Rickard, A. H., and Whyte, F. W. (2002) The physiology and collective recalcitrance of microbial biofilm communities. Adv. Microb. Physiol. 46, 203–256.
Al-Fattani, M. A., and Douglas, L. J. (2004) Penetration of Candida biofilms by antifungal agents. Antimicrob. Agents Chemother. 48, 3291–3297.
Anderl, J. N., Franklin, M. J., and Stewart, P. S. (2000) Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob. Agents Chemother. 44, 1818–1824.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Douglas, L.J. (2009). Penetration of Antifungal Agents Through Candida Biofilms. In: Cihlar, R.L., Calderone, R.A. (eds) Candida albicans. Methods in Molecular Biology, vol 499. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-151-6_5
Download citation
DOI: https://doi.org/10.1007/978-1-60327-151-6_5
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-760-0
Online ISBN: 978-1-60327-151-6
eBook Packages: Springer Protocols