Chapter 5

Penetration of Antifungal Agents Through Candida Biofilms

L. Julia Douglas

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 asecond, 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. Duringsubsequent incubation, the drug diffuses out of the agar and through the biofilm ‘sandwich’ to the moistenedpaper disk. The drug concentration in the disk can be determined by measuring the zone of growth inhibitionthat 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 examiningbiofilm morphology by scanning electron microscopy.

Key words: Biofilm, Candida, antifungal agent, drug penetration, scanning electron microscopy.

1. Introduction

During recent years, Candida albicans, together with related Candidaspecies, has become one of the commonest agents of hospital-acquiredinfections (1, 2). Many of these are implant-associated infections, inwhich adherent fungal populations, or biofilms, are formed on thesurfaces of medical devices including catheters, prosthetic heart valvesand endotracheal tubes (3, 4). Biofilm cells are organized into struc-tured communities enclosed within a matrix of extracellular material.They are phenotypically distinct from planktonic (suspended) cells;most notably, they are significantly less susceptible to antifungal agents(5–15). This property makes implant infections difficult to treat andoften the implant must be removed.

The mechanisms of biofilm resistance to antimicrobialagents are not fully understood. One long-standing hypothesis is

Ronald L. Cihlar, Richard A. Calderone (eds.), Candida albicans: Methods and Protocols, vol. 499� Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009DOI 10.1007/978-1-60327-151-6_5 Springerprotocols.com

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that the biofilm matrix resists drug penetration by forming areaction–diffusion barrier (16), and that only the surface layersare exposed to a lethal dose of the agent. The extent to which thematrix acts as a barrier to drug diffusion would depend on thechemical nature of both the antimicrobial agent and the matrixmaterial. This protocol allows the penetration of antifungal agentsthrough Candida biofilms to be investigated (17) using a filterdisk assay modified from one previously reported for bacterialbiofilms (18). The procedure is also suitable for measuring drugpenetration through mixed-species biofilms (17). In addition,methods for determining the viabilities of drug-treated biofilmcells, and for examining the biofilms by scanning electron micro-scopy, are described.

2. Materials

2.1. Preparation of

Biofilm Inoculum

1. Slope cultures of Candida species on Sabouraud dextrose agar(Difco).

2. Yeast nitrogen base (YNB) medium (Difco) containing 50 mMglucose: batches of 50 mL of medium in 250-mL Erlenmeyerflasks.

3. Wash buffer: 0.15 M phosphate-buffered saline (PBS; pH 7.2).

2.2. Biofilm Formation 1. Polycarbonate membrane filters (Whatman): diameter 25 mmand pore size 0.2 mm. Sterilize by exposure to UV radiation for15 min on both sides.

2. YNB agar containing 50 mM glucose in Petri dishes.

2.3. Antifungal Agents 1. Flucytosine (Sigma), fluconazole (Pfizer) and caspofungin(Merck) solutions in sterile distilled water, prepared immediatelybefore use.

2. Voriconazole (Pfizer) and amphotericin B (Sigma) solutions indimethyl sulfoxide, prepared immediately before use. Otherantifungal agents may be used, dissolved fresh in the appropriatesolvent.

2.4. Penetration of

Biofilms by Antifungal

Agents

1. Antifungal-agent-supplemented agar: using a sterile filtrationunit (Sartorius), filter the drug solution into culture medium(YNB agar containing 50 mM glucose) buffered with 0.165 Mmorpholinepropanesulfonic acid (Sigma) to pH 7, and keptmolten at 50�C. High drug concentrations are used (seeNote 1).

2. Polycarbonate membrane filters (Whatman): diameter 13 mmand pore size 0.2 mm. Sterilize by exposure to UV radiation for15 min on both sides.

38 Douglas

3. Paper concentration disks (Becton Dickinson): diameter6 mm. Sterilize by exposure to UV radiation for 15 min onboth sides.

4. Plates of YNB agar containing 200 mM glucose and seededwith 150 mL of a standardized suspension of planktonic C.albicans (used here as an indicator organism and adjusted toan optical density at 520 nm of 1.0).

2.5. Viable Counts

of Biofilm Cells

Exposed to Antifungal

Agents

1. Plates of YNB agar containing 200 mM glucose.2. Dilution buffer: 0.15 M PBS (pH 7.2).

2.6. Scanning

Electron Microscopy

1. 2.5% (v/v) Glutaraldehyde in PBS.2. 1% (w/v) Osmium tetroxide.3. 1% (w/v) Uranyl acetate.4. Series of ethanol solutions ranging, in 10% increments, from

30% (v/v) ethanol in distilled water to dried absolute ethanol.

3. Methods

3.1. Preparation

of Biofilm Inoculum

1. Batches of YNB medium containing 50 mM glucose (50 mL in250-mL Erlenmeyer flasks) are inoculated from fresh Candidaslopes and incubated at 37�C for 24 h in an orbital shakeroperating at 60 rpm.

2. Cells are harvested and washed twice in PBS.3. Before use in biofilm experiments, all washed cell suspensions

are adjusted to an optical density at 600 nm of 0.2.4. If mixed-species biofilms are to be tested (see Note 2), equal

volumes of the standardized suspension of each organism aremixed immediately before use.

3.2. Biofilm Formation 1. Biofilms are grown on membrane filters resting an agar culturemedium in Petri dishes. Sterile polycarbonate membrane filters(diameter 25 mm) are placed on the surface of YNB agarcontaining 50 mM glucose (see Note 3).

2. A standardized cell suspension (50 mL) is applied to the surfaceof each membrane and the plates are incubated at 37�C.

3. After 24 h, the membrane-supported biofilms are transferredto fresh agar for a further 24 h, giving a total incubation time of48 h for biofilm formation.

3.3. Penetration

of Biofilms by

Antifungal Agents

1. After biofilm formation on membrane filters, smaller sterilepolycarbonate membrane filters (diameter 13 mm) are carefullyplaced on top of the 48-h-old biofilms.

Drug Penetration of Candida Biofilms 39

2. Sterile paper concentration disks (diameter 6 mm) are moistenedwith growth medium (normally 29mL; see Note 4) and positionedon top of the 13-mm-diameter membranes.

3. Biofilms sandwiched between the membranes and moisteneddisks are transferred to plates of antifungal-agent-containingmedium using sterile forceps and incubated at 37�C for timeperiods ranging from 60 min to 6 h (see Fig. 5.1 and Note 5).

4. The amount of antifungal agent, which penetrates each biofilmand reaches the concentration disk, is determined by usingthe disk in a standard drug diffusion assay. After the appropriateexposure time, concentration disks are removed from the biofilm‘sandwiches’ and placed on plates of YNB agar containing200 mM glucose (see Note 6), which have been seeded withthe indicator strain of C. albicans. The plates are incubated at37�C for 24 h.

5. Zones of growth inhibition are measured and used todetermine the concentration of active antifungal agent in thedisks by the reference to a standard curve prepared by usingdrug solutions of different concentrations but fixed volumes(see Note 7).

6. Control assays are included where concentration disks areplaced on the two-membrane system to which no cells havebeen added, i.e., the unit without the biofilm.

7. The drug concentration that penetrates the biofilm (C) isdivided by the drug concentration determined for the control(C0) to provide a normalized penetration curve (see Figs. 5.2and 5.3, and Note 8).

A

B

C

D

E

A

B

C

D

E

A

B

C

D

E

Fig. 5.1. Experimental system used to determine the penetration of antifungal agentsthrough biofilms. The biofilm (B ) is formed on a 25-mm-diameter membrane filter (A )resting on YNB agar. A second, smaller filter (C ) is placed on top of the biofilm, and amoistened concentration disk (D ) is positioned on top of the second filter. The entireassembly is then transferred to antifungal-containing agar (E ).

40 Douglas

3.4. Viable Counts of

Biofilm Cells Exposed

to Antifungal Agents

1. After incubation on antifungal-agent-containing agar, biofilmcells are gently scraped from the membranes with a sterilescalpel and resuspended in 10 mL of PBS.

2. Serial dilutions (10—1–10–6) of each biofilm cell suspensionare prepared in PBS.

3. Triplicate samples (0.1 mL) of the 10–4, 10–5 and 10–6 dilutionsare spread on YNB agar containing 200 mM glucose and theseplates are incubated at 37�C for 24 h.

4. In control assays, membranes are transferred to growthmedium containing no antifungal agent and viable countsdetermined as above.

0.00.10.20.30.40.50.60.70.80.91.0

a) b)

0 60 120 180 240 300 360

Time (min)

C/C

o

0.00.10.20.30.40.5

0.60.70.80.91.0

0 60 120 180 240 300 360

Time (min)

C/C

o

Fig. 5.2. Penetration of flucytosine through biofilms of (a) C. albicans GDH 2346 (closed triangles ), C. albicans GDH 2023(open squares ), and C. albicans GRI 682 (open circles ); (b) C. krusei (open squares ), C. glabrata (open diamonds ), C.parapsilosis (closed triangles) and C. tropicalis (closed circles). Error bars indicate the standard errors of the means. C,drug concentration on distal edge of biofilm; Co, drug concentration measured in control. Reproduced from Ref. (17 ) withpermission from the American Society of Microbiology.

0.00.10.20.30.40.5

0.60.70.80.91.0

a) b)

0 60 120 180 240 300 360

Time (min)

C /C

o

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 60 120 180 240 300 360

Time (min)

C/C

o

Fig. 5.3. Penetration of fluconazole through biofilms of (a) C. albicans strain GDH 2346 (closed triangles ), C. albicans GDH2023 (open squares), and C. albicans GRI 682 (open circles ); (b) C. krusei (open squares ), C. glabrata (open diamonds ),C. parapsilosis (closed triangles) and C. tropicalis (closed circles ). Error bars indicate the standard errors of the means. C,drug concentration on distal edge of biofilm; Co, drug concentration measured in control. Reproduced from Ref. (17 ) withpermission from the American Society of Microbiology.

Drug Penetration of Candida Biofilms 41

3.5. Scanning

Electron Microscopy

1. Biofilms formed on polycarbonate membranes are fixed in2.5% (v/v) glutaraldehyde in PBS for 1 h at room temperatureand immersed in 1% (w/v) osmium tetroxide for 1 h. They arethen washed three times with distilled water (3 mL), treatedwith 1% (w/v) uranyl acetate for 1 h and washed again withdistilled water (3 mL).

2. Dehydration of the samples is achieved by immersion in a seriesof ethanol solutions ranging, in 10% increments, from 30% (v/v)ethanol in distilled water to dried absolute ethanol.

3. The final step before gold coating is drying. Simple air-drying ina desiccator will suffice for many purposes, although it can leadto a significant loss of the biofilm extracellular matrix. A typicalelectron micrograph of a membrane-supported biofilm is shownin Fig. 5.4.

4. Notes

1. Drug concentrations are selected on the basis of their ability togive large zones of growth inhibition in control assays for drugpenetration. Examples of recommended concentrations would

Fig. 5.4. Scanning electron micrograph of a 48-h membrane-supported biofilm of C.tropicalis. Arrows indicate extracellular matrix material. Bar, 10 mm. Reproduced fromRef. (17 ) with permission from the American Society of Microbiology.

42 Douglas

be flucytosine, 30 times the MIC for planktonic cells of thesame strain; fluconazole, 60 times the MIC; voriconazole,220 times the MIC; and amphotericin B, 60 times the MIC.

2. The protocol can also be used to test drug penetration throughmixed-species biofilms. In such cases, a medium should beselected that is best able to support the growth of both the species.For example, tryptic soy broth (Difco) supports the growth ofboth Candida species and Staphylococcus species, and is a suitablechoice for propagation of biofilms containing both the organisms.

3. If mixed-species biofilms are to be tested, a suitable solid mediumshould be selected. Tryptic soy agar is an excellent medium forCandida/Staphylococcus biofilms.

4. Because of an occasional variation in disk thickness, a slightlylower or higher volume of medium is sometimes required tosaturate the disks. Wetting of the disks helps prevent thecapillary action of the antifungal medium through the biofilms.

5. Exposure times of 60, 90, 120, 180, 240 and 360 min areconvenient for most purposes.

6. YNB agar containing 200 mM glucose, rather than 50 mM,gives optimal lawn production by the indicator strain.

7. A standard curve is obtained by plotting the log of the drugconcentration used against the diameter of the zone of growthinhibition.

8. Colony biofilms formed on membrane filters appear to lackthe water channels that typically surround matrix-enclosedmicrocolonies in many other biofilms. The possibility thatdrugs simply move through water channels without reachingcells deep within the microcolonies is, therefore, largely elimi-nated by using this model system.

References

1. Calderone, R. A. (ed.) (2002) Candida andCandidiasis. ASM Press, Washington, D.C.

2. Kojic, E. M., and Darouiche, R. O. (2004)Candida infections of medical devices. Clin.Microbiol. Rev. 17, 255–267.

3. Douglas, L. J. (2003) Candida biofilmsand their role in infection. Trends Microbiol.11, 30–36.

4. Donlan, R. M. (2001) Biofilms and device-associated infections. Emerg. Infect. Dis. 7,277–281.

5. Hawser, S. P., and Douglas, L. J. (1995)Resistance of Candida albicans biofilms toantifungal agents in vitro. Antimicrob. AgentsChemother. 39, 2128–2131.

6. Baillie, G. S., and Douglas, L. J. (1998)Effect of growth rate on resistance of

Candida albicans biofilms to antifungalagents. Antimicrob. Agents Chemother.42, 1900–1905.

7. Baillie, G. S., and Douglas, L. J. (1998) Iron-limited biofilms of Candida albicans and theirsusceptibility to amphotericin B. Antimicrob.Agents Chemother. 42, 2146–2149.

8. Baillie, G. S., and Douglas, L .J. (1999)Candida biofilms and their susceptibility toantifungal agents. Meth. Enzymol. 310,644–656.

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Drug Penetration of Candida Biofilms 43

and Ghannoum, M. A. (2001) Antifungalresistance of candidal biofilms formed on den-ture acrylic in vitro. J. Dent. Res. 80,903–908.

11. Chandra J., Kuhn, D. M., Mukherjee, P. K.,Hoyer, L. L., McCormick, T., and Ghannoum,M. A. ( 2001) Biofilm formation by the fungalpathogen Candida albicans: development,architecture, and drug resistance. J. Bacteriol.183, 5385–5394.

12. Lewis, R. E., Kontoyiannis, D. P., Darouiche,R. O., Raad, I. I., and Prince, R. A. (2002)Antifungal activity of amphotericin B, fluco-nazole, and voriconazole in an in vitro modelof Candida catheter-related bloodstreaminfection. Antimicrob. Agents Chemother.46, 3499–3505.

13. Ramage, G., VandeWalle, K., Wickes, B.L., and Lopez-Ribot, J. L. (2001) Stan-dardized method for in vitro antifungalsusceptibility testing of Candida albicansbiofilms. Antimicrob. Agents Chemother.45, 2475–2479.

14. Ramage, G., VandeWalle, K., Wickes, B. L., andLopez-Ribot, J. L. (2001) Biofilm formation byCandida dubliniensis. J. Clin. Microbiol. 39,3234–3240.

15. Andes, D., Nett, J., Oschel, P., Albrecht, R.,Marchillo, K., and Pitula, A. (2004) Develop-ment and characterization of an in vivo centralvenous catheter Candida albicans biofilmmodel. Infect. Immun. 72, 6023–6031.

16. Gilbert, P., Maira-Litran, T., McBain, A. J.,Rickard, A. H., and Whyte, F. W. (2002) Thephysiology and collective recalcitrance ofmicrobial biofilm communities. Adv. Microb.Physiol. 46, 203–256.

17. Al-Fattani, M. A., and Douglas, L. J. (2004)Penetration of Candida biofilms by antifungalagents. Antimicrob. Agents Chemother. 48,3291–3297.

18. Anderl, J. N., Franklin, M. J., and Stewart, P. S.(2000) Role of antibiotic penetration limitationin Klebsiella pneumoniae biofilm resistance toampicillin and ciprofloxacin. Antimicrob.Agents Chemother. 44, 1818–1824.

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