Penetration of Candida Biofilms by Antifungal Agents

A filter disk assay was used to investigate the penetrationof antifungal agents through biofilms containing single andmixed-species biofilms containing Candida. Fluconazole permeatedall single-species Candida biofilms more rapidly than flucytosine.The rates of diffusion of either drug through biofilms of threestrains of Candida albicans were similar. However, the ratesof drug diffusion through biofilms of C. glabrata or C. kruseiwere faster than those through biofilms of C. parapsilosis orC. tropicalis. In all cases, after 3 to 6 h the drug concentrationat the distal edge of the biofilm was very high (many timesthe MIC). Nevertheless, drug penetration failed to produce completekilling of biofilm cells. These results indicate that poor antifungalpenetration is not a major drug resistance mechanism for Candidabiofilms. The abilities of flucytosine, fluconazole, amphotericinB, and voriconazole to penetrate mixed-species biofilms containingC. albicans and Staphylococcus epidermidis (a slime-producingwild-type strain, RP62A, and a slime-negative mutant, M7) werealso investigated. All four antifungal agents diffused veryslowly through these mixed-species biofilms. In most cases,diffusion was slower with biofilms containing S. epidermidisRP62A, but amphotericin B penetrated biofilms containing theM7 mutant more slowly. However, the drug concentrations reachingthe distal edges of the biofilms always substantially exceededthe MIC. Thus, although the presence of bacteria and bacterialmatrix material undoubtedly retarded the diffusion of the antifungalagents, poor penetration does not account for the drug resistanceof Candida biofilm cells, even in these mixed-species biofilms.

INTRODUCTION

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Introduction

Candida albicans is the major fungal pathogen of humans (10).During recent years this organism, together with related Candidaspecies, has become one of the commonest agents of hospital-acquiredinfections (18). Many of these are implant-associated infections,in which adherent microbial populations, or biofilms, are foundon the surfaces of devices, including catheters, prostheticheart valves, endotracheal tubes, and joint replacements (15,17). Such infections can be caused by a single microbial speciesor by a mixture of fungal or bacterial species (13, 28). Individualorganisms in biofilms are embedded within a matrix of frequentlyslimy, extracellular polymers and typically display a phenotypethat is very different from that of planktonic (free-floating)cells. In particular, biofilm cells are significantly less susceptibleto antimicrobial agents (16, 17, 19, 34). As a result, drugtherapy for an implant infection may be futile, and often, theonly solution is mechanical removal of the implant (13).

Various model systems have been used to investigate the propertiesof Candida biofilms in vitro (17). These range from simple assayswith catheter disks to more complex flow systems, such as theperfused biofilm fermentor (7). Biofilms of C. albicans usuallyconsist of a mixture of yeasts, hyphae, and pseudohyphae andmay have a basal yeast layer that anchors the biofilm to thesurface (8). The cells are surrounded by a matrix of extracellularpolymeric material, the synthesis of which markedly increaseswhen developing biofilms are exposed to a liquid flow (24).Results from several studies have shown that Candida biofilmsare resistant to clinically important antifungal agents, includingamphotericin B, fluconazole, flucytosine, itraconazole, andketoconazole (6, 11, 12, 23, 31, 37, 38). Newer azoles (voriconazoleand ravuconazole) are also ineffective against biofilms (29),although some antibiofilm activity has been demonstrated withthe echinocandin caspofungin in vitro (4, 29, 39). Mixed Candida-Staphylococcusbiofilms are similarly resistant to fluconazole, and there isevidence that the bacteria can enhance Candida resistance (1).

The mechanisms of biofilm resistance to antimicrobial agentsare not fully understood. One long-standing hypothesis for theresistance of bacterial biofilms is that the matrix materialrestricts drug penetration by forming a reaction-diffusion barrier(19) and that only the surface layers of a biofilm are exposedto a lethal dose of antibiotic. The extent to which the matrixacts as a barrier to drug diffusion would depend on the chemicalnature of both the antimicrobial agent and the matrix material.Several research groups have investigated antibiotic penetrationin Pseudomonas aeruginosa biofilms (26, 30, 42, 44). The overallconclusion from that work was that fluoroquinolones penetrateP. aeruginosa biofilms readily, whereas the penetration of aminoglycosidesis retarded. Further studies suggested that aminoglycosidesdiffuse more slowly because they bind to matrix polymers suchas alginate (20, 21, 36). Analogous investigations with Candidaspecies have not been reported. However, the drug susceptibilityprofiles of C. albicans biofilms incubated statically (whichhave relatively little extracellular matrix material) were comparedwith those of biofilms incubated with gentle shaking (whichproduce much more matrix material). Biofilms grown with or withoutshaking did not exhibit significant differences in susceptibilitiesto amphotericin B, flucytosine, or fluconazole, suggesting thatdrug resistance is unrelated to the extent of matrix formation(9).

In the study described here, we have investigated the penetrationof antifungal agents through Candida biofilms using a filterdisk assay adapted from the technique reported by Anderl etal. (3) for bacterial biofilms. The abilities of flucytosineand fluconazole to permeate biofilms of C. albicans and relatedCandida species were evaluated in this way. In parallel, theviabilities of drug-treated biofilm cells were determined. Thedrug penetration of mixed-species biofilms containing C. albicansand Staphylococcus epidermidis (a slime-producing wild-typestrain and a slime-negative mutant) was also assessed. S. epidermidisis the organism most frequently isolated from bacterial implant-associatedinfections and has also been found in polymicrobial infectionswith C. albicans (28). Voriconazole and amphotericin B, as wellas flucytosine and fluconazole, were used in these mixed-speciesexperiments.

MATERIALS AND METHODS

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Materials and Methods

Organisms. C. albicans GDH 2346 (NCYC 1467) and GDH 2023 were originallyobtained from patients with denture stomatitis at Glasgow DentalHospital; strain GRI 682 was from a vaginal smear at GlasgowRoyal Infirmary. C. glabrata AAHB 12, C. tropicalis AAHB 73,and C. parapsilosis AAHB 4479 were isolated from patients withline infections at Crosshouse Hospital, Kilmarnock, Scotland.C. krusei was obtained from a clinical specimen and came fromthe Regional Mycology Reference Laboratory, Glasgow, Scotland.All strains were maintained on slopes of Sabouraud dextroseagar (Difco) and were subcultured monthly. Every 2 months, cultureswere replaced by new ones freshly grown from freeze-dried stocks.

Two strains of S. epidermidis (RP62A and M7) were maintainedon Colombia blood agar (Oxoid). Strain RP62A (ATCC 35984) isa known slime producer; strain M7 is a slime-negative mutantobtained after chemical mutagenesis of S. epidermidis RP62Awith mitomycin C (41). The growth rate, initial adherence, cellwall composition, surface characteristics, and antimicrobialsusceptibility profile of strain M7 are indistinguishable fromthose of wild-type strain RP62A (41).

Medium and culture conditions. All Candida species were grown in yeast nitrogen base (YNB)medium (Difco) containing 50 mM glucose. Batches of medium (50ml in 250-ml Erlenmeyer flasks) were inoculated from fresh slopesand incubated at 37°C for 24 h in an orbital shaker at 60rpm. Cells were harvested and washed twice in 0.15 M phosphate-bufferedsaline (PBS; pH 7.2). Before use in biofilm experiments, allwashed cell suspensions were adjusted to an optical densityat 600 nm of 0.2.

Tryptic soy broth (Difco) was selected as the liquid mediumbest able to support the growth of both fungi and bacteria.C. albicans GDH 2346 and the two strains of S. epidermidis (strainsRP62A and M7) grew at similar rates in this medium (1). Cultureswere inoculated from fresh slopes and incubated with shakingat 37°C for 24 h. Cells were harvested, washed twice inPBS, and suspended to an optical density at 600 nm of 0.2 priorto use in biofilm experiments. For mixed-species biofilms, equalvolumes of the standardized suspension of each organism weremixed immediately before use.

Biofilm formation. Biofilms were grown on membrane filters resting on agar culturemedium in petri dishes. For experiments with Candida species,polycarbonate membrane filters (diameter, 25 mm; pore size 0.2µm; Whatman) were sterilized by exposure to UV radiationfor 15 min on both sides prior to inoculation and were thenplaced on the surface of YNB agar containing 50 mM glucose.Tryptic soy agar was used for S. epidermidis and mixed-speciesbiofilms. A standardized cell suspension (50 µl) was appliedto the surface of each sterile membrane. All plates were incubatedat 37°C for 24 h. The membrane-supported biofilms were thentransferred to fresh agar for a further 24 h, giving a totalincubation time of 48 h for biofilm formation.

Penetration of biofilms by antifungal agents. Four clinically important antifungal agents were used in thisstudy. Flucytosine and amphotericin B were obtained from Sigma.Fluconazole and voriconazole were kindly donated by Pfizer Limited.All drug solutions were prepared immediately before use. Flucytosineand fluconazole were dissolved in sterile distilled water andthen added to molten culture medium at 50°C by use of asterile filtration unit (Sartorius) to create antifungal agent-supplementedagar for the biofilm experiments. Voriconazole and amphotericinB were dissolved in dimethyl sulfoxide and filtered into thegrowth medium. The medium was buffered to pH 7 with 0.165 Mmorpholinepropanesulfonic acid buffer (Sigma). High drug concentrationswere used in the antifungal agent-supplemented agar. They wereselected on the basis of their ability to give large zones ofgrowth inhibition in control assays for drug penetration outlinedbelow. The concentrations used were as follows: flucytosine,6 µg/ml (30 times the MIC for planktonic C. albicans GDH2346); fluconazole, 24 µg/ml (60 times the MIC); voriconazole10 µg/ml (220 times the MIC); and amphotericin B, 78 µg/ml(60 times the MIC).

Penetration of antifungal agents through biofilms was assessedby a modification of the filter disk technique described previouslyfor bacterial biofilms (3). After biofilm formation on membranefilters, smaller polycarbonate membrane filters (diameter, 13mm; pore size, 0.2 µm; Whatman) were sterilized by exposureto UV radiation for 15 min on both sides and were then carefullyplaced on top of the 48-h-old biofilms. Paper concentrationdisks (diameter, 6 mm; Becton Dickinson) were also sterilizedby exposure to UV radiation for 15 min per side and then moistenedwith growth medium (normally 29 µl) prior to placementon top of the 13-mm-diameter membranes. Because of an occasionalvariation in disk thickness, a slightly higher or lower volumeof medium was sometimes required to saturate the disks. Wettingof the disks helped prevent the capillary action of the antifungalmedium through the biofilms. Biofilms sandwiched between themembranes and moistened disks were transferred to antifungalagent-containing agar medium. All plates were incubated forspecified exposure times, namely, 60, 90, 120, 180, 240, or360 min.

The amount of antifungal agent which had penetrated each biofilmand which had reached the concentration disk was determinedby using the disk in a standard drug diffusion assay. Platesof YNB agar containing 200 mM glucose were seeded with 150 µlof a standardized suspension of planktonic C. albicans GDH 2346(used here as an indicator organism and adjusted to an opticaldensity at 520 nm of 1.0). After the appropriate exposure time,concentration disks were removed from the biofilm "sandwiches"and placed on the seeded plates, which were then incubated at37°C for 24 h. The zones of growth inhibition were measuredand used to determine the concentration of active antifungalagent in the disks by reference to a standard curve preparedby using drug solutions of different concentrations but fixedvolumes. All drug penetration assays were carried out in duplicateon at least two separate occasions. In control assays, concentrationdisks were placed on the two-membrane system to which no cellshad been added, i.e., the unit without the biofilm. The drugconcentration that penetrated the biofilms (C) was divided bythe drug concentration determined for the controls (C₀) to providea normalized penetration curve (3).

Viable counts of biofilm cells exposed to antifungal agents. After biofilm formation on 25-mm-diameter membrane filters,biofilms were capped with sterile, 13-mm-diameter filters, transferredto antifungal agent-containing agar, and incubated at 37°Cfor 6 h (the maximum exposure period in drug penetration assays)or 24 h. After incubation, biofilm cells were gently scrapedfrom the membranes with a sterile scalpel and resuspended in10 ml of PBS. Serial dilutions (10^–1 to 10^–6) ofeach biofilm cell suspension were then prepared. Triplicatesamples (0.1 ml) of the 10^–4, 10^–5, and 10^–6dilutions were spread on YNB agar containing 200 mM glucose,and the plates were incubated at 37°C for 24 h. In controlassays, the membranes were transferred to growth medium containingno antifungal agent.

Scanning electron microscopy (SEM). Biofilms of C. tropicalis and mixed-species biofilms of C. albicansGDH 2346 and S. epidermidis M7 formed on polycarbonate membraneswere fixed with glutaraldehyde and then treated with osmiumtetroxide and uranyl acetate as described previously (22). Afterdehydration in a series of ethanol solutions, samples were airdried in a desiccator for 48 h, coated with gold with a Polaroncoater, and viewed under a Philips 500 scanning electron microscope.

RESULTS

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Results

In this study, we adapted a novel, filter disk assay devisedby Anderl et al. (3) to investigate the penetration of antifungalagents through single- and mixed-species biofilms containingCandida. The technique involves the formation of a 48-h-oldcolony biofilm on a polycarbonate membrane filter and the cappingof this biofilm with a second, smaller membrane filter and thena wetted paper disk of the type used in zone-of-inhibition bioassays.The assembly, which represents a primitive diffusion cell, istransferred to agar medium containing the antifungal agent.During subsequent incubation, the drug diffuses out of the agarand through the biofilm sandwich to the moistened paper disk.The drug concentration in the disk can finally be determinedby measuring the zone of growth inhibition that it produceson medium seeded with an indicator strain of C. albicans instandard bioassays. The great advantage of this system is thatbecause there is physical access to both sides of the biofilm,the penetration of solutes can be measured directly. Moreover,colony biofilms appear to lack the water channels that typicallysurround matrix-enclosed microcolonies in many other biofilms(46). The possibility that drugs simply move through water channelswithout reaching cells deep within the microcolonies is thereforelargely eliminated by using this model system.

Flucytosine penetration through Candida biofilms. The levels of flucytosine penetration of biofilms of three C.albicans strains were similar, but they were initially lowerwith strain GDH 2346 (Fig. 1a). The drug concentration at thedistal edge of the biofilm (i.e., distal with respect to theagar) was approximately 50% of that of the control value after180 min. After 240 min it had reached 63 to 78% of that of thecontrol value (Fig. 1a).

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FIG. 1. Penetration of flucytosine through biofilms of C. albicans GDH 2346 (closed triangles), C. albicans GDH 2023 (open squares), and C. albicans GRI 682 (open circles) (a) and through biofilms of C. krusei (open squares), C. glabrata (open diamonds), C. parapsilosis (closed triangles), and C. tropicalis (closed circles) (b). Error bars indicate the standard errors of the means. The mean C₀ after 6 h was 22.7 µg of flucytosine/ml.

When biofilms of other Candida species were used, there wasrapid penetration of the drug (approximately 50% of that forthe control after 60 min) through both C. glabrata and C. kruseibiofilms and then a decrease to a stable level of approximately30% after 120 to 360 min. By contrast, there was slow diffusionthrough C. parapsilosis biofilms (25% by 360 min; Fig. 1b).However, the slowest penetration was observed with C. tropicalisbiofilms (approximately 15% after 360 min; Fig. 1b).

Enzymatic degradation of flucytosine by C. glabrata and C. kruseidid not occur. This was demonstrated by removing membrane-supportedbiofilms from antifungal agent-containing plates and spreadinga sensitive strain (strain GDH 2346) of C. albicans onto theplates. These indicator organisms were unable to grow on anypart of the plate, including the location that had been beneaththe biofilm (results not shown).

Fluconazole penetration through Candida biofilms. Fluconazole penetration was similar and rapid for three C. albicansstrains (approximately 90% of that for the control after 60min; Fig. 2a). There was then a slight decrease and a levelingoff at 70% of the control value after 360 min. Interestingly,diffusion was much more rapid with fluconazole than with flucytosine,but the final extents of drug penetration were similar after360 min (Fig. 1a and 2a). However, this represents a higherdrug concentration with fluconazole.

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FIG. 2. Penetration of fluconazole through biofilms of C. albicans strain GDH 2346 (closed triangles), C. albicans GDH 2023 (open squares), and C. albicans GRI 682 (open circles) (a) and through biofilms of C. krusei (open squares), C. glabrata (open diamonds), C. parapsilosis (closed triangles), and C. tropicalis (closed circles) (b). Error bars indicate the standard errors of the means. The mean C₀ after 6 h was 26.6 µg of fluconazole/ml.

When non-C. albicans Candida species were used, there was rapidfluconazole penetration through biofilms of either C. glabrataor C. krusei, followed by a decrease to roughly 70% of the controlvalue (Fig. 2b). The level of drug penetration through C. parapsilosisbiofilms was zero after 60 min but then rose rapidly and leveledoff at 65% of the control value. The slowest penetration wasagain with C. tropicalis biofilms (Fig. 2b). For all non-C.albicans species, the overall level of penetration of fluconazole(55 to 85%) was higher than that of flucytosine (15 to 50%;Fig. 1b and 2b).

Penetration of antifungal agents through single- and mixed-species biofilms of C. albicans and S. epidermidis. Flucytosine diffused through biofilms of C. albicans GDH 2346fairly slowly, as noted above, with penetration of about 50%of the control value after 360 min (Fig. 3a). This value wasslightly lower than that shown in Fig. 1a (65% after 360 min).In these experiments, however, the C. albicans biofilms, likethe mixed-species biofilms, were grown on tryptic soy agar ratherthan YNB agar. By contrast, the drug penetrated S. epidermidisbiofilms very poorly (Fig. 3a); there was approximately 12%penetration after 180 min for slime-negative mutant M7 and 10%penetration after 240 min for wild-type strain RP62A. Mixedfungal-bacterial biofilms showed similarly slow and poor drugpenetration, although diffusion was more rapid with Candida-M7biofilms than with Candida-RP62A biofilms (Fig. 3a).

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FIG. 3. Penetration of flucytosine (a) and amphotericin B (b) through single- and mixed-species biofilms of C. albicans and S. epidermidis. Biofilms contained C. albicans GDH 2346 (closed triangles), S. epidermidis RP62A (closed circles), S. epidermidis M7 (closed squares), C. albicans GDH 2346 and S. epidermidis RP62A (open circles), and C. albicans GDH 2346 and S. epidermidis M7 (open squares). Error bars indicate the standard errors of the means. Mean C₀s after 6 h were 24.2 µg of flucytosine/ml and 120.6 µg of amphotericin B/ml.

Despite its low solubility in water, amphotericin B diffusedrapidly through biofilms of C. albicans GDH 2346 (65% of thecontrol value after 60 min; Fig. 3b). Penetration through S.epidermidis biofilms was slower and less extensive. With mixedfungal-bacterial biofilms, drug diffusion was also slow andpoor, but in this instance diffusion was faster through biofilmscontaining wild-type strain RP62A than through those containingslime-negative mutant M7 (Fig. 3b). Surprisingly, the C₀ ofamphotericin B (Fig. 3 legend), like that of flucytosine (Fig.1 legend), was higher than the drug concentration in the agar.The reason for this is not clear. However, it is conceivablethat during the drug penetration assay some drugs bind to thedisk cellulose (thus effectively reducing their concentrationin solution) but then are released during the zone-of-inhibitionassay.

Both fluconazole and the newer azole voriconazole diffused rapidlythrough biofilms of C. albicans GDH 2346, although penetrationby fluconazole was more extensive (Fig. 4a and b). There wasslower and poorer penetration of S. epidermidis biofilms. Thesedrugs also diffused through mixed fungal-bacterial biofilmsslowly, but fluconazole penetrated mixed biofilms to a greaterextent than any other antifungal agent tested (Fig. 4a). Thediffusion of both azoles, like that of flucytosine, was morerapid with biofilms containing S. epidermidis M7 than with thosecontaining wild-type strain RP62A (Fig. 4a and b).

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FIG. 4. Penetration of fluconazole (a) and voriconazole (b) through single- and mixed-species biofilms of C. albicans and S. epidermidis. Biofilms contained C. albicans GDH 2346 (closed triangles), S. epidermidis RP62A (closed circles), S. epidermidis M7 (closed squares), C. albicans GDH 2346 and S. epidermidis RP62A (open circles), and C. albicans GDH 2346 and S. epidermidis M7 (open squares). Error bars indicate the standard errors of the means. Mean C₀s after 6 h were 27.0 µg of fluconazole/ml and 7.9 µg of voriconazole/ml.

Effects of antifungal agents on the viability of biofilm cells. To assess the effects of the antifungal agents on biofilm cells,biofilms sandwiched between the two membranes, as in the drugpenetration assay, were exposed to antifungal agent-containingagar at 37°C for 6 h (the time period during which drugpenetration was determined) or 24 h. Antifungal agents (flucytosineor fluconazole) were present at concentrations identical tothose used in the drug penetration assay. After incubation,the numbers of viable biofilm cells were determined by a standardprocedure of serial dilution followed by plating. In no casedid drug penetration result in the complete killing of biofilmcells (Table 1). C. glabrata AAHB 12 was wholly unaffected byfluconazole after 6 h, but many strains of this species areknown to be resistant to fluconazole even when they are grownin planktonic culture (14). The results with fluconazole overallwere not unexpected, despite the high concentration used, sincethis drug is generally considered to be fungistatic only. However,a recent study (35) has demonstrated that fluconazole can befungicidal under certain conditions.

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TABLE 1. Viability of biofilm cells of Candida species after exposure to flucytosine or fluconazole for 6 or 24 h^a

SEM. Biofilms of C. tropicalis had a very slimy appearance, suggestiveof an extensive matrix, and were poorly penetrated by both flucytosineand fluconazole. C. tropicalis, whose ability to produce biofilmshas received little attention, is able to grow in the form ofyeast cells or filaments. Examination of the biofilms by SEMshowed that they consisted of a dense cell network containingboth morphological types. Many of the filaments appeared tolie parallel to each other in the form of bundles (Fig. 5).The procedure used for sample preparation allows clear visualizationof biofilm cells but normally fails to preserve the biofilmmatrix. However, fairly extensive matrix material could stillbe seen adhering to and linking some of the cells, a findingconsistent with the slimy appearance of C. tropicalis biofilms.

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FIG. 5. Scanning electron micrograph of a 48-h-old colony (membrane-supported) biofilm of C. tropicalis. Arrows indicate extracellular matrix material. Bar, 10 µm.

Previous work (1) demonstrated that both strains of S. epidermidisused here formed thick biofilms on catheter disks. However,wild-type strain RP62A, unlike mutant M7, produced abundantmatrix material, or slime. The physical interactions betweenstaphylococci and C. albicans were more easily seen with theslime-free mutant (1). A similar examination of mixed-speciesbiofilms of C. albicans GDH 2346 and S. epidermidis M7 grownon polycarbonate membranes in this study also revealed multiple,adhesive interactions between bacterial and fungal cells. Staphylococciwere clearly adherent to both yeasts and hyphae (Fig. 6).

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FIG. 6. Scanning electron micrograph of a 48-h-old colony (membrane-supported) biofilm of C. albicans GDH 2346 and S. epidermidis M7. Bar, 10 µm.

DISCUSSION

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Discussion

The mechanisms that protect microorganisms in biofilms fromantibiotics and biocides are still being elucidated. Currently,four mechanisms are under study: (i) slow penetration of theantimicrobial agent into the biofilm, (ii) an altered chemicalmicroenvironment within the biofilm leading to zones of slowor no growth, (iii) adaptive responses to environmental stress,and (iv) the existence of persister cells that are protectedfrom all types of antimicrobial insult (43). Almost all of thiswork is being done with bacterial biofilms. At present, no singlemechanism seems to account for the exceptional resistance ofbiofilm cells to a wide variety of antimicrobial agents. Instead,it is likely that two or more mechanisms operate together. Allfour mechanisms appear to depend on the multicellular natureof biofilms. For example, when an antimicrobial agent failsto penetrate a biofilm, it is because the drug is reactivelyneutralized as it diffuses into a cell cluster. This processmay involve enzymatic degradation of the drug or drug bindingto charged extracellular polymers, but it is effective onlywhen microorganisms are aggregated and exert their collectiveneutralizing activity (43).

In this study, we have investigated the penetration of antifungalagents through single- and mixed-species biofilms formed byfungal pathogens in the genus Candida. Our results demonstratedthat fluconazole permeated all single-species Candida biofilmsmore rapidly than flucytosine. The rates of diffusion of eitherdrug through biofilms of three strains of C. albicans were similar.On the other hand, the rates of drug diffusion through biofilmsof C. glabrata and C. krusei were faster than those throughbiofilms of C. parapsilosis and C. tropicalis. In all cases,the drug concentration reached at the distal edge of the biofilmwas very high. However, drug penetration failed to produce completekilling of biofilm cells even when the incubation period wasextended from 6 to 24 h. These results indicate that poor drugpenetration is not a major resistance mechanism for Candidabiofilms.

The biofilms showing the lowest levels of drug penetration,particularly with flucytosine, were those formed by C. parapsilosisand C. tropicalis. The strain of C. tropicalis used in theseexperiments, a clinical isolate, had a very slimy appearanceon solid medium, and some of this slime, or matrix material,could be seen when biofilm preparations were viewed under ascanning electron microscope. As yet, nothing is known of thechemical composition of this material, but it could play a minorrole in the drug resistance of C. tropicalis biofilms by slowingthe diffusion of antifungal agents. The matrix of C. albicansbiofilms has been isolated and shown to contain mainly carbohydrateand protein, with a relatively high proportion (16%) of glucose(9). Recent work with P. aeruginosa has led to the identificationof periplasmic glucans in biofilm cells which appear to sequesterantibiotics and slow their diffusion, perhaps preventing themfrom reaching their sites of action in the cytoplasm (33). Ithas also been postulated that the nature and amounts of extracellularglucans produced by oral streptococci from sucrose in dentalplaque are major determinants retarding acid diffusion (25).It will be interesting to determine whether the matrix materialof C. tropicalis biofilms is especially rich in glucan polysaccharides.

All four antifungal agents tested diffused very slowly throughmixed-species biofilms containing C. albicans and either thewild-type or M7 mutant strain of S. epidermidis. In most cases,diffusion was slower with biofilms containing S. epidermidisRP62A, the wild-type, slime-producing strain. Curiously, however,amphotericin B penetrated biofilms containing the M7 mutantmore slowly. In all of these experiments with mixed fungal-bacterialbiofilms, the drug concentrations reaching the distal edgesof the biofilms substantially exceeded the MIC for C. albicans.Thus, although the presence of bacteria and bacterial matrixmaterial undoubtedly retarded the diffusion of the antifungalagents, poor penetration does not account for the drug resistanceof Candida biofilm cells, even in these mixed-species biofilms.

The nature of the extracellular matrix of S. epidermidis biofilmsis not fully established. It appears to contain a polymer ofß-1,6-linked N-acetylglucosamine residues with somedeacetylated amino groups, as well as succinate and phosphatesubstituents (the intercellular polysaccharide adhesin) (32).A 140-kDa accumulation-associated protein has also been identified(27). The M7 mutant, which fails to accumulate on glass surfaces(41) but which does form biofilms on polyvinyl chloride catheterdisks (1), has been reported to lack this protein but neverthelesssynthesizes intercellular polysaccharide adhesin (27). Interactionsbetween these polymers and those produced by C. albicans inmixed-species biofilms might result in a more viscous matrix.Rheological interactions between matrix polysaccharides fromPseudomonas cepacia and P. aeruginosa have been shown to decreasethe rates of diffusion and antimicrobial activities of antibiotics(2). On the other hand, a recent study of oral biofilms containingsix microbial species, including C. albicans, suggested thatretarded diffusion of fluorescent probes through the biofilmwas due to tortuosity, i.e., the convoluted paths traversedby macromolecules during biofilm penetration (45).

Biofilm cells appear to grow slowly because of the limited availabilityof nutrients, especially at the base of the biofilm. Growthrate has therefore been considered as an important modulatorof drug activity in biofilms (17, 19). A perfused fermentorwas used to generate C. albicans biofilms at different growthrates, and the susceptibility of the biofilm cells to amphotericinB was compared with that of planktonic organisms grown at thesame rates in a chemostat. The results indicated that biofilmswere resistant to the drug at all growth rates tested, whereasplanktonic cells were resistant only at low growth rates (5).An alternative mechanism of drug resistance might be upregulationof genes coding for multidrug efflux pumps in biofilm cells.C. albicans possesses two different types of efflux pump: ATP-bindingcassette transporters and major facilitators, which are encodedby CDR and MDR genes, respectively. Recent work has shown thatgenes encoding both types of pump are indeed upregulated duringbiofilm formation and development. However, mutants carryingsingle or double deletion mutations in some of these genes werehighly susceptible to fluconazole when they were growing planktonicallybut retained the resistant phenotype during biofilm growth (40).Overall, it seems probable that drug resistance in Candida biofilms,like that in bacterial biofilms, is a complex process involvingmore than one mechanism.

ACKNOWLEDGMENTS

Mohammed Al-Fattani is the recipient of a research studentshipfrom the Ministry of Health of Saudi Arabia.

We are indebted to Margaret Mullin for expert assistance withelectron microscopy and to Pfizer Limited for a supply of fluconazoleand voriconazole.

FOOTNOTES

* Corresponding author. Mailing address: Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom. Phone: 44 141 330 5842. Fax: 44 141 330 4600. E-mail: J.Douglas{at}bio.gla.ac.uk.

REFERENCES

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