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Antimicrobial Agents and Chemotherapy, July 2007, p. 2454-2463, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.01237-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Role for Cell Density in Antifungal Drug Resistance in Candida albicans Biofilms{triangledown}

Palani Perumal,{dagger} Satish Mekala,{ddagger} and W. LaJean Chaffin*

Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

Received 3 October 2006/ Returned for modification 30 November 2006/ Accepted 25 April 2007


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ABSTRACT
 
Biofilms of Candida albicans are less susceptible to many antifungal drugs than are planktonic yeast cells. We investigated the contribution of cell density to biofilm phenotypic resistance. Planktonic yeast cells in RPMI 1640 were susceptible to azole-class drugs, amphotericin B, and caspofungin at 1 x 103 cells/ml (standard conditions) using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay. As reported by others, as the cell concentration increased to 1 x 108 cells/ml, resistance was observed with 10- to 20-fold-greater MICs. Biofilms that formed in microtiter plate wells, like high-density planktonic organisms, were resistant to drugs. When biofilms were resuspended before testing, phenotypic resistance remained, but organisms, when diluted to 1 x 103 cells/ml, were susceptible. Drug-containing medium recovered from high-cell-density tests inhibited low-cell-density organisms. A fluconazole-resistant strain showed greater resistance at high planktonic cell density, in biofilm, and in resuspended biofilm than did low-density planktonic or biofilm organisms. A strain lacking drug efflux pumps CDR1, CDR2, and MDR1, while susceptible at a low azole concentration, was resistant at high cell density and in biofilm. A strain lacking CHK1 that fails to respond to the quorum-sensing molecule farnesol had the same response as did the wild type. FK506, reported to abrogate tolerance to azole drugs at low cell density, had no effect on tolerance at high cell density and in biofilm. These observations suggested that cell density has a role in the phenotypic resistance of biofilm, that neither the drug efflux pumps tested nor quorum sensing through Chk1p contributes to resistance, and that azole drug tolerance at high cell density differs mechanistically from tolerance at low cell density.


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INTRODUCTION
 
Candida albicans is a human commensal found on mucosal and cutaneous surfaces. The organism can also cause opportunistic infections of the surfaces that it colonizes as well as of deep tissues to which it may gain access. Like the majority of microbes growing in their normal niche, C. albicans can participate in biofilm formation on mucosal surfaces as well as on device surfaces, e.g., dentures and catheters (see reviews in references 15, 16, 29, 30, 32, 37, and 56). Biofilms are a community of microorganisms attached to a surface and surrounded by extracellular matrix. One of the properties generally associated with biofilms is reduced susceptibility to many commonly used drugs (15, 16, 29, 30, 32, 37, 56). The susceptibility of biofilms to drugs has been examined in several in vitro systems. Intact biofilms formed on polyvinyl chloride catheter discs, polymethylmethacrylate (denture acrylic) pieces, silicone elastomer pieces, and polystyrene wells all showed reduced sensitivity to drugs compared to planktonic organisms (12, 14, 23, 28, 49, 52). The drugs include ketoconazole, fluconazole, itraconazole, ravuconazole, miconazole, amphotericin B, nystatin, and chlorhexidine. The effect of caspofungin is unclear, as reports differ on biofilm versus planktonic cell susceptibility (6, 14, 28). Biofilm is not resistant to lipid formulations of amphotericin B (28).

Several hypotheses have been advanced and tested without success to explain fully the reduced sensitivity to drugs. One of the first tested was the expression of drug efflux pumps. Ramage et al. (49) reported that expression of CDR1, CDR2, and MDR1 was increased during biofilm formation. They also tested the resistance of biofilms formed by homozygous single and double deletion strains, {Delta}cdr1, {Delta}cdr2, {Delta}mdr1, {Delta}cdr1 {Delta}cdr2, and {Delta}cdr1{Delta}mdr1 strains, and observed that biofilms of these strains developed the same drug resistance as did a wild-type strain. Another study was performed by Mukherjee et al. (36) with strains lacking one of the efflux pumps or both CDR-encoded pumps or the triple deletion strain lacking both CDR1 and CDR2 and MDR1. They found that at 6 h of biofilm development the double and triple mutant strains were more sensitive to drugs than was the wild-type strain but at subsequent development times the mutant strains were resistant to drugs. Transcript was lower at 48 h of biofilm development than at 12 h. Thus, this confirmed the prior report that efflux pumps did not account for resistance. Biofilm formed by a strain lacking CDR1, CDR2, MDR1, and FLU1 was also less susceptible to drugs (31). Mukherjee et al. (36) examined membrane sterol content and reported that ergosterol content was reduced at intermediate and mature phases of biofilm compared to the early phase. They suggested that drug resistance was multicomponent and phase dependent. This correlation between change in sterol content and development of resistance was not further examined.

Another hypothesis was advanced that the extracellular matrix of the biofilm inhibits penetration of the drug into the biofilm. As yet there are no mutant strains lacking matrix. However, the extent of matrix formation can be altered by shaking the biofilm during development and even more by medium flow (2, 21). When biofilms formed with (enhanced matrix) and without shaking, there was no difference in resistance, while resistance was enhanced under flow conditions (2, 9). Al-Fattani and Douglas also directly investigated the penetration of drug into biofilm (3). They reported differences in rates of penetration, but by 3 to 6 h various drugs had reached the distal surface of the biofilm at concentrations in severalfold excess of the planktonic MIC. Drug penetration through biofilm containing both bacteria (Staphylococcus epidermidis) and C. albicans was slower than that through biofilm containing C. albicans alone, although ultimately drug also reached the distal edge at levels greater than the MIC. Drug penetration of biofilm was investigated by Samaranayake et al. (59), with similar conclusions that drugs penetrated biofilms of C. albicans, Candida parapsilosis, and Candida krusei with some differences among drugs and species. The sensitivity to lipid formulation of amphotericin B also supports the ability of drugs to penetrate biofilm (28).

Baillie and Douglas (7) investigated yet another hypothesis that growth rate affected resistance. They used a perfused biofilm fermentor which allows control of growth rate. The release of daughter cells from the biofilm was determined at drug concentrations manyfold above the planktonic MIC, and amphotericin B produced a greater effect than ketoconazole, fluconazole, or flucytosine did. Response to amphotericin B was not affected by change of growth rate. At all growth rates the viability of biofilm organisms was unaffected by amphotericin B. When planktonic organisms were grown at similar rates, only the most slowly grown cells were resistant.

Most recently, two studies have reported the presence of a subpopulation of yeast cells that, after removal of the biofilm, remained adhered to the abiotic surface on which the biofilm was formed and that showed increased resistance to membrane-perturbing amphotericin B (26, 31) and chlorhexidine (31). The development of about 1% persister cells was associated with attachment to the abiotic surface and not the development of complex biofilm architecture (31).

In this study, we have investigated the hypothesis that cell density contributes to the phenotypic resistance of biofilm. The effect of inoculum size has been recognized for many years (41-43, 57, 58, 64) and with an inoculum-independent range of 2 x 102 to 5 x 105 cells/ml (4). The guidelines for cell density for susceptibility testing are within this inoculum-independent range (40). Although the cell density in a developed biofilm has not been reported, various microscopic methods show that organisms are closely clustered within the biofilm both in vitro and in vivo (5, 11, 23, 54, 59, 61). This led us to investigate the contribution of cell density to the observation of phenotypic resistance of C. albicans biofilms. We evaluated the susceptibility of planktonic yeast cells to azole-class drugs (fluconazole and ketoconazole), amphotericin B, and caspofungin over a cell density range of 1 x 103 to 1 x 108 cells/ml and of intact biofilm and dissociated biofilm over a cell density range equivalent to that of planktonic yeast cells. We also investigated the contribution of drug efflux pumps and the response to the quorum-sensing molecule farnesol and the small molecule FK506 to the susceptibilities of biofilm organisms to these same drugs.


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MATERIALS AND METHODS
 
Organisms and growth conditions. Seven strains were used in this study. SC5314, obtained from William Fonzi (Washington, DC), is a strain widely used in general and molecular studies of C. albicans and also used for biofilm studies. GDH2346 has been used in several biofilm studies, starting with reports from the Douglas laboratory (23). We also obtained three isolates, an azole-sensitive isolate, FH1, and two matched azole-resistant isolates, FH5 and FH8, from Ted White (Seattle, WA) (35) with a fluconazole MIC of 64 µg/ml. Strain DSY1050 ({Delta}cdr1::hisG/{Delta}cdr1::hisG {Delta}cdr2::hisG/{Delta}cdr2::hisG {Delta}mdr1::hisG-URA3-hisG/{Delta}mdr1::hisG) was obtained from Dominique Sanglard (Lausanne, Switzerland), and strain CHK21 ({Delta}ura3::imm434/{Delta}ura3::imm434{Delta}cachk1::hisG/{Delta}cachk1::hisG-URA3-hisG) was obtained from Richard Calderone (Washington, DC) (10). Yeast cell cultures were incubated in yeast nitrogen base with amino acids (YNB; Becton Dickinson Co., Franklin Lakes, NJ) with 50 mM glucose at room temperature overnight with shaking (180 rpm), and 100 µl of the culture was inoculated into another 50 ml fresh medium and incubated as described above until the cell density reached ~2 x 108 cells/ml. The cells were collected by centrifugation, washed, and resuspended in RPMI 1640 medium supplemented with L-glutamine and buffered with MOPS [3-(N-morpholino)-propanesulfonic acid; Sigma Chemical Co., St. Louis, MO]. The final cell density was adjusted as needed with 2 x 103 cells/ml being considered the standard suspension and various suspensions being used for testing planktonic yeast cells.

Biofilms of C. albicans strains were formed on a polystyrene surface following the protocol of Ramage et al. (52), except that biofilms were formed for 48 h. The yeast cells grown as described above were diluted in RPMI medium to a final cell concentration of 1 x 106 cells/ml, and 100 µl of this standardized cell suspension was dispensed into polystyrene microtiter wells (96-well plate). The plates were incubated at 37°C for 48 h in a moist chamber. After 48 h the biofilm was washed once with RPMI medium to remove unattached cells and 100 µl RPMI medium was added to the washed biofilm prior to the drug susceptibility testing described below. For other studies, the washed biofilm was disrupted in the 100-µl medium by scraping and aspirating it with sterile pipette tips. To determine if organisms remain attached to the well surface after dissociated cells were removed, 200 µl medium was added and the metabolic activity was determined as described below on the same plate with intact biofilm. For some experiments the resuspended biofilm organisms were used without dilution while in other cases the cell density was adjusted to about 2 x 103 cells/ml before the cells were treated with antifungal agents. As biofilms of C. albicans consist of extensively grown hyphae, pseudohyphae, and yeasts, preparing standardized cell suspensions by employing the standard cell counting procedures is extremely difficult. Therefore, we adopted a spectrophotometric method to prepare cell suspensions of the desired cell density from biofilm dissociated cells. The planktonic yeast cells of C. albicans SC5314 were grown in YNB medium at room temperature to an early stationary phase (~2 x 108 cells/ml). The cells were harvested by centrifugation, washed once, and resuspended in sterile water. The cell concentration was determined by hemacytometer counting. From this stock, suspensions with cell densities ranging from 1 x 103 to 5 x 108 cells/ml were prepared in replicate, and the absorbance was measured at 600 nm and used as a reference standard. Biofilms were also prepared on other surfaces. Discs (1-cm diameter) were cut from denture acrylic strips, a polyvinylchloride thoracic catheter, a silicone elastomer-coated latex catheter, and polystyrene. The discs were rinsed with distilled water, dried, and sterilized with ethylene oxide. The discs were placed in 24-well tissue culture plates, and 1 ml of yeast cell suspension (1 x 106 cells/ml) was inoculated onto the surfaces. The yeast cells were allowed to adhere to the surface at 37°C for 90 min. After this time the discs were gently washed with RPMI medium and transferred to a fresh plate containing 1 ml of RPMI medium per well. The plates were incubated at 37°C for 48 h, and the biofilms formed on the surface were collected with sterile cell scrapers. The cells were resuspended in RPMI, and the standardized cell suspensions were prepared as described for drug susceptibility testing.

Drug susceptibility testing and effect of FK506. A predissolved injectable form of fluconazole (200 mg/100 ml; Pfizer Inc., New York, NY) was directly diluted with RPMI 1640 medium (Mediatech, Inc., Herndon, VA) supplemented with L-glutamine and buffered with MOPS. Ketoconazole (Sigma Chemical Co., St. Louis, MO), amphotericin B (Fischer Scientific International Inc., Hampton, NH), and caspofungin (Merck & Co., Inc., Whitehouse Station, NJ) were solubilized in dimethyl sulfoxide and diluted with sterile RPMI 1640 medium to obtain stock solutions of a desired strength. The stock solutions were diluted twofold with RPMI 1640 to obtain drug concentrations from 1,024 to 0.0313 µg/ml, and 100 µl drug solution was added to the wells containing organisms. The final dimethyl sulfoxide concentration did not affect the assay. Concentrated stock solutions were frozen at –80°C until further use, and for azole-class drugs on occasion dilutions were maintained at 4°C for 24 to 48 h. Amphotericin B solution was prepared fresh for each experiment.

For testing planktonic yeast cells or undiluted or diluted dissociated biofilm organisms, 100 µl of cell suspension described above was added to wells of a microtiter plate. Intact biofilm prepared as described above was tested in the well in which the biofilm was formed. A suspension of 2 x 103 cells/ml as recommended in the CLSI standard (40) was considered the standard suspension. The organisms were incubated with 100 µl of twofold-serially diluted antifungal drugs at 37°C for 48 h. Incubation with caspofungin was for 24 h as recommended in two studies (43, 46). Organisms without added drug were used as a control. At 48 h the metabolic activity of fungal cells was determined by the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay as described by Ramage et al. (52). The XTT tetrazolium salt was dissolved at 0.5 g/liter in phosphate-buffered saline (pH 7.4), filter sterilized through an 0.2-µm filter, and stored in aliquots at –80°C. Just before use an aliquot was thawed and 10 mM menadione in acetone was added to the XTT solution to a final concentration of 1 µM. One hundred microliters of this solution was added to planktonic yeast cells, resuspended biofilm organisms, or intact biofilm in microtiter wells; mixed well; and incubated at 37°C. Cells were incubated up to 2 h, particularly with low-density cells. The incubation was reduced to 50 to 60 min for high cell density so that the color intensity was within the sensitivity of the plate reader. For a given cell density, color development was linear with time (r2 = 0.99). The reduced formazan-colored product was measured at 490 nm in a microplate reader. The lowest drug concentration which inhibited the growth by 80% was considered the MIC80. In each experiment three or more replicates were used for each determination and the average (± standard deviation) was used. MIC determinations were repeated at least twice. FK506 (Techoland Corp., Edison, NJ) was prepared as a 100x stock solution in sterile pyrogen-free water. The volumes of cell suspension, drug, and FK506 were adjusted to yield the desired final concentrations in 200 µl before incubation and the XTT assay as described above.

Development of genetic resistance. Development of genetic resistance was tested by the drop plate method. Briefly, planktonic yeast cells at high cell density (1 x 108 cells/ml) were incubated for 48 h without or with 64 µg/ml fluconazole as described above. The organisms were serially diluted 10-fold, and 5 µl of each dilution was plated on solid YNB medium described above containing 2% (wt/vol) agar without or with 0.5 µg/ml or 64 µg/ml fluconazole. Additions of YNB and fluconazole were made to the agar solution at about 45°C, and the plates were made immediately (45).


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RESULTS
 
Susceptibilities of planktonic yeast cells under standard conditions to various drugs. The susceptibilities of the strains SC5314 and GDH2346, as well as FH1 and matched fluconazole-resistant isolates FH5 and FH8, to fluconazole, ketoconazole, amphotericin B, and caspofungin were determined under standard conditions of low cell density of planktonic yeast cells (Table 1). In initial experiments the observations with the fluconazole-resistant isolates FH5 and FH8 were similar. Hence only observations for FH5 are shown and only FH5 was used in subsequent experiments. Except for the fluconazole-resistant strain, planktonic cells were sensitive to the drugs.


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  		TABLE 1. Drug susceptibilities of C. albicans strains grown and tested under various conditions

Planktonic cell density and antifungal susceptibility. The inoculum size has been reported to affect MIC determinations (41-43, 58, 64), and we began this study by determining the effect of planktonic yeast cell density on susceptibility to various drugs before examining biofilm cell density. The observations for the drug-sensitive strain SC5314 with fluconazole are shown in Fig. 1. The MIC remained at 0.25 to 0.5 µg/ml from 5 x 102 through 5 x 105 cells/ml. However, at higher cell densities, there was less inhibition and for 5 x 107 cells/ml the inhibition at 512 µg/ml was about 40%. The observations confirmed that at high cell densities planktonic cells have an increased MIC and are phenotypically resistant to fluconazole. At high cell density, planktonic yeast cells of all strains showed reduced susceptibilities to all drugs with less than 80% inhibition obtained at the highest tested concentration (Table 1). Eighty percent inhibition was not observed for amphotericin B or caspofungin. For amphotericin B, 50 to 75% inhibition was observed within the tested range with MIC50s often ≤8 µg/ml (data not shown). For caspofungin, 50% inhibition at ≤0.5 µg/ml was observed in several cases (data not shown).


Figure 1
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  		FIG. 1. Fluconazole susceptibility at different cell densities. Planktonic yeast cells of SC5314 were resuspended at different cell densities, and their susceptibility to fluconazole was determined as described in Materials and Methods. The symbol key shows the cell density in the assay.

We asked whether drug remained in the culture medium after treatment of high-density cells and, if so, whether there was enough drug to inhibit cells at low density. High-density planktonic cells of SC5314 were incubated with 64 µg/ml fluconazole in the standard assay for 48 h. The contents of several wells were collected and filter sterilized. Assuming 100% drug recovery (64 µg/ml), serial dilutions of the sterilized supernatant were added to low-density planktonic cells for the standard assay. The supernatant was inhibitory at a dilution of 1:16, which was interpreted as demonstrating the presence of drug in the recovered supernatant. However, if the 1:16 dilution was assumed to be equivalent to the 0.5 µg/ml observed for the MIC for low-density cells (Table 1), that would suggest that the concentration in the recovered supernatant was approximately 4 µg/ml.

Drug susceptibilities of biofilms. We next examined the susceptibilities of biofilms formed by the four strains SC5314, GDH2346, FH1, and FH5 to various drugs. Each of the strains formed a biofilm. The drug resistance of biofilms to fluconazole and ketoconazole for three strains is shown in Fig. 2 and given for all strains and other drugs in Table 1. For azole-class drugs, the biofilms had metabolic activity in the presence of the highest concentration of drug tested and 80% reduction was not observed. In one case a 39% reduction in activity was observed. Both amphotericin B and caspofungin failed to inhibit biofilm at 80%, although for amphotericin B 50% inhibition was observed generally at <1 µg/ml and in a few instances with caspofungin 50% inhibition was observed.


Figure 2
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  		FIG. 2. Drug susceptibility of biofilm. Biofilms of strains SC5314, GDH2346, and FH1 were formed over 48 h and then treated with fluconazole (Flu) and ketoconazole (Keto) as described in Materials and Methods.

Biofilm cell density and antifungal susceptibility. With the observation that planktonic organisms at high cell density displayed resistance to several drugs similar to that of biofilms, we predicted that dissociated biofilm organisms would have a drug susceptibility pattern similar to that of biofilms. For these experiments, after biofilm formation 100 µl medium was added to washed biofilms and some biofilms were assayed intact while others were dissociated and assayed. We determined the cell population of biofilms in the wells and tested the resuspended biofilm organisms at that density, i.e., the numbers of organisms in the well were the same. The biofilm when resuspended in 100 µl had a cell density equivalent to approximately 2 x 108 planktonic yeast cells/ml. After addition of drug solution, the cell density in the assay was equivalent to approximately 1 x 108 cells/ml. The observations with strain GDH2346 for fluconazole and ketoconazole are shown in Fig. 3 along with those for planktonic yeast cells at high cell density (1 x 108 cells/ml in assay). Observations with other drugs and strains are summarized in Table 1. As predicted, dissociated biofilm organisms, when assayed at the same cell density as was biofilm, showed a similar pattern of reduced susceptibility to antifungal drugs. These observations suggested that the activity in the biofilm assay was not associated with cells tightly adhered to the surface. This was directly examined by assessing the activity after the dissociated biofilm was removed. In some wells biofilm formed by strain SC5314 was dissociated and removed prior to assay. Wells containing intact biofilm formed in the absence of drug had 2.32 ± 0.10 A490 compared to 0.2 ± 0.5 A490 for wells from which biofilm was removed. Biofilm exposed to 64 µg/ml fluconazole had reduced activity (1.75 ± 0.06 A490) compared to untreated biofilm, while the wells from which biofilm had been removed were 0.1 ± 0.01 A490.


Figure 3
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  		FIG. 3. Drug susceptibility of high-density organisms. Biofilm of strain GDH2346 was formed for 48 h, and the intact biofilm was assayed with drug (Biofilm). Similarly formed biofilm was dissociated as described in Materials and Methods prior to assay (Dissociated). Planktonic yeast cells were resuspended at high density and tested at an equivalent density (Planktonic). The density for all tests was equivalent to approximately 1 x 108 cells/ml.

Drug susceptibility of developing biofilm. We then asked if, as the cell number increased in a developing biofilm, the developing biofilm would show the same pattern of cell-number-dependent susceptibility as did planktonic yeast cells. Development of biofilm of C. albicans SC5314 in wells of a polystyrene microtiter plate was followed for 72 h (Fig. 4A). The metabolic activity began dramatically increasing after 6 h and reached a peak at 36 h, after which it began declining at 48 h. We then tested the susceptibility of the developing biofilm to fluconazole (Fig. 4B and C) and amphotericin B (Fig. 4D and E). At each time of analysis over the 72 h, organisms were dissociated from the developing biofilm in some wells and assayed at the standard concentration of 1 x 103 cells/ml (Fig. 4C and E) while the developing biofilm in other wells was undisturbed and was assayed as intact biofilm (Fig. 4B and D). At all intervals the dissociated cells were susceptible to fluconazole while the developing biofilm was susceptible for the first 3 hours. At 6 hours the intact biofilm was somewhat less susceptible, and after 6 hours the biofilm was resistant at >8 µg/ml. Amphotericin B inhibited the growth of dissociated biofilm cells at all stages of development when incubated with dissociated cell suspensions containing 1 x 103 cells/ml. The drug also inhibited the growth of developing biofilms up to 12 h (MIC80 of 0.125 to 1 µg/ml); however, at 24 h and later the biofilms were less susceptible (MIC80, 8 to >32 µg/ml). However, 50% inhibition was observed at 0.25 to 1 µg/ml (data not shown).


Figure 4
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  		FIG. 4. Drug susceptibility of developing biofilm. Biofilm development for strain SC5314 was monitored over 72 h, and a representative plot is shown in panel A. At various intervals the susceptibility of the developing biofilm to fluconazole (B) and amphotericin B (D) was determined. At each of these intervals the developing biofilm was dissociated and dissociated biofilm organisms were tested at a density equivalent to 1 x 103 cells/ml for susceptibility to fluconazole (C) and amphotericin B (E). The symbol key for panels B to E is between panels B and C.

Susceptibilities of biofilms formed on different surfaces. To determine if the surface on which the biofilm forms contributes to the development of resistance to antifungal drugs, biofilms of C. albicans SC5314 were grown on three clinically relevant surfaces: polymethylmethacrylate (acrylic) used for dentures and silicone elastomer-coated latex and polyvinylchloride catheters. The biofilm cells harvested from these surfaces were tested with fluconazole at 1 x 103 and 1 x 108 cells/ml (Fig. 5). Fluconazole inhibited the growth of biofilm dissociated cells at 0.25 to 8 µg/ml when the drug was incubated with biofilm cell suspensions at 1 x 103 cells/ml. As observed previously, fluconazole did not inhibit the growth of biofilm dissociated cells when incubated with cell suspensions containing 1 x 108 cells/ml. The surface on which the biofilm was formed did not affect the drug susceptibility profile.


Figure 5
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  		FIG. 5. Drug susceptibility of biofilm organisms formed on different surfaces. Biofilms of strain SC5314 were formed on denture acrylic (acrylic) and silicone elastomer-coated latex (SE) and polyvinylchloride (PVC) catheters, and susceptibilities of dissociated organisms to fluconazole were tested at 1 x 103 and 1 x 108 cells/ml.

Development of a genetically resistant subpopulation. Although the development of a genetically resistant population during the treatment of high-density cells seemed unlikely, we tested this possibility by treating three cultures of high-density (1 x 108 cells/ml) planktonic yeast cells of SC5314 with 64 µg/ml fluconazole for 48 h and then testing them along with untreated cultures for growth in the absence or presence of fluconazole (0.5 µg/ml and 64 µg/ml) by the drop plate method. There was no difference between the fluconazole-treated and untreated cultures (data not shown). This supported the notion that the resistance was phenotypic rather than genotypic.

Contribution of drug efflux pumps to resistance. C. albicans drug efflux pumps CDR1, CDR2, and MDR1 have previously been reported not to contribute to the reduced susceptibility of mature biofilms (36, 49). To determine if efflux pumps contributed to the high-cell-density-dependent reduced susceptibility to various drugs, a Cdr1– Cdr2– Mdr1– strain, DSY1050, was examined (Table 1). At standard cell density both planktonic yeast cells and dissociated biofilm organisms were sensitive to all drugs tested. Biofilm formed by this strain showed the same reduced susceptibility as did strains with intact efflux pumps. Dissociated biofilm organisms tested at a high density showed reduced susceptibility similar to that of intact biofilm as did planktonic yeast cells at high cell density. This suggests that the drug efflux pumps did not contribute to the high-cell-density phenotypic resistance.

Quorum sensing. When planktonic yeast cells grow to high cell density, farnesol, a quorum-sensing molecule that can suppress hyphal formation and biofilm initiation, is formed (24, 50). A morphogenetic autoregulatory activity that similarly inhibited hyphal formation was produced by mature biofilm (50). Farnesol can induce expression of drug efflux pumps CDR1 and CDR2 (18). A Chk1– mutant strain, CHK21, defective in two-component signal transduction, does not respond to farnesol and is able to form a biofilm (27). If farnesol contributes to the reduced drug susceptibility of high-density planktonic yeast cells, then the CHK21 strain would be predicted to be susceptible to the drugs through failure to sense farnesol. However, the MIC of this strain at low cell densities of planktonic yeast cells, biofilm, and dissociated high-density biofilm organisms was similar to that of wild-type strains (Table 1). This suggested that quorum sensing through farnesol was not required for high-cell-density tolerance of fluconazole.

Inhibition of the calcineurin pathway and biofilm drug susceptibility. The calcineurin pathway has been implicated in tolerance to azoles which are fungistatic. Cyclosporine, FK506 (tacrolimus), and FK520 (an FK506 derivative), which target the calcineurin pathway, act synergistically with fluconazole, resulting in fungicidal activity (33, 34, 60). Under standard testing conditions, FK506 is synergistic with fluconazole at <0.04 µg/ml (44). We tested the possibility that FK506 could abrogate the high-cell-density resistance to fluconazole. As shown in Fig. 6, at the highest concentration of fluconazole with or without 2 µg/ml FK506 the inhibition of metabolic activity was 17% versus 15%. The addition of FK506 in the presence of fluconazole did not alter the cell response. This suggested that the tolerance mechanism of cells at high cell density differs from that at low cell density and that the calcineurin pathway is not involved.


Figure 6
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  		FIG. 6. Effect of FK506. The effect of FK506 on the fluconazole susceptibility of strain SC5314 was tested at four FK506 concentrations shown in the symbol key.


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DISCUSSION
 
In this study we have tested the hypothesis that biofilm cell density contributes to the development of phenotypic resistance and demonstrated that a correlation exists between cell density and phenotypic resistance independent of the source of the organisms.

The recommended testing for drug susceptibility of C. albicans is performed at low cell density (40). Under these conditions three wild-type strains, SC5314, GDH2346, and FH1, showed typical patterns of susceptibility to the azole-class drugs fluconazole and ketoconazole as well as amphotericin B and caspofungin (Table 1). The {Delta}cdr1 {Delta}cdr2 {Delta}mdr1 efflux pump mutant strain showed a similar pattern. When 48-h biofilm was examined, the biofilms were phenotypically resistant with higher MICs to all drugs. These observations are consistent with other reports in which the same strains have been tested (11, 23, 28, 35, 36, 51), except for caspofungin. Similar planktonic and biofilm MIC50s (0.06 to 0.5 µg/ml) for caspofungin have been observed with biofilm formed on silicone elastomer and microtiter plate wells with greater inhibition observed in kinetic and concentration-dependent studies (6, 28, 53). However, another study observed lower planktonic MICs (0.06 to 0.001 µg/ml) with 15 strains and found that these levels were ineffective with biofilm formed on silicone catheters, although at 2 µg/ml (33- to 2,000-fold greater than planktonic MIC) inhibition was observed (14). In this study, biofilm also was not susceptible to the planktonic MIC80 levels (Table 1), although the standard planktonic MIC80 was about 10-fold higher than that in the study by Cocuaud et al. (14). The basis for the difference between these studies that find similar inhibition of low-density planktonic organisms and biofilm and those that do not is unclear.

Unlike planktonic organisms, which are individual organisms, frequently in a homogeneous environment in suspension culture, a biofilm is a community of organisms in which organisms are in contact with other organisms. For mature C. albicans biofilms formed in vitro and in vivo there is a mixture of yeast cells, hyphae, and pseudohyphae in a dense network of organisms and water channels (5, 11, 22, 55, 61). Thus, unlike standard antifungal susceptibility testing of planktonic yeast cells, biofilms are tested with organisms at a much higher density. We tested planktonic yeast cells over a range of cell densities with the highest density equivalent to that of biofilm organisms (Table 1; Fig. 1). Above 2.5 x 106 cells/ml the susceptibilities to the various drugs decreased and were the same as those of biofilm at 5 x 107 cells/ml. When biofilm organisms were dissociated in 100 µl, the equivalent planktonic yeast cell density was about 2 x 108 cells/ml. When these cells were tested, the susceptibility to drugs was the same as that of biofilm (Table 1; Fig. 3). When dissociated biofilm organisms were diluted to a low cell density equivalent to that in testing planktonic yeast cells at 1 x 103 cells/ml, the biofilm organisms showed the same susceptibility as that of planktonic yeast cells at that cell density (Table 1; Fig. 4). At a high cell density for planktonic yeast cells and dissociated biofilm organisms and for biofilm the reduced susceptibilities to antifungal drugs are the same (Fig. 2; Table 1). In this study, organisms that remained adhered to the surface after biofilm dissociation had little metabolic activity and did not account for the reduced susceptibility of biofilm. Previous reports have observed that biofilms developed reduced susceptibility to drugs independently of the surface on which the biofilm formed (12, 14, 23, 28, 49, 52), with one report suggesting that the extent of reduced susceptibility may differ among supports (9). Similarly in this study, the surface on which the biofilm was formed did not affect the high- or low-cell-density susceptibility of dissociated biofilm organisms (Fig. 5). Thus, we concluded that biofilm architecture was not necessary for reduced susceptibility to azole-class drugs. At low cell densities for planktonic yeast cells and dissociated biofilm organisms the profiles for susceptibility to drugs were also the same.

Only a few studies have investigated whether when organisms are dissociated from biofilm they retain the resistance of the intact biofilm. The observations are somewhat varied. In a perfused fermentor experiment (7), approximately 2 x 104 cells/ml were incubated with amphotericin B for 1 h before being plated for viability. Under these conditions the disaggregated cells retained about 80% viability compared to intact biofilm organisms. In another study, disaggregated biofilm organisms tested at approximately 1 x 106 cells/ml showed resistance (fluconazole MIC, 256 µg/ml) compared to planktonic yeast cells (fluconazole MIC, 2 to 4 µg/ml) tested under the same conditions but less resistance than that of intact biofilm (fluconazole MIC, >1,024 µg/ml) (8). In an early study in our laboratory using a macrobroth method and measuring optical density, we observed that dissociated biofilm organisms, 106 cells/ml, were sensitive at about 2 twofold serial dilutions greater than planktonic yeast cells (63). In view of this study, we suggest that the partial resistance of the dissociated biofilm organisms reflects testing at an intermediate cell density. Two other studies have examined resuspended biofilm at low cell density. Our observations with amphotericin B are in agreement with those of Khot et al. (26), who observed a similar amphotericin B endpoint for planktonic organisms and resuspended biofilm organisms. Ramage et al. (49) found that dissociated organisms had susceptibility intermediate (256 µg/ml) between those of planktonic cells (4 µg/ml) and intact biofilm (>1,024 µg/ml). None of these studies further investigated a contribution of cell density. Four experiments were performed to investigate the basis of biofilm and high-cell-density phenotypic resistance. First our observations demonstrated that reduced susceptibility was phenotypic and not genotypic. Second, a Cdr1– Cdr2– Mdr1– strain (DSY1050) that lacks three efflux pumps developed drug resistance in biofilms that was also observed in high-density planktonic yeast cells and dissociated biofilm organisms, but sensitivity was again observed with dissociated biofilm organisms at low cell density (Table 1). The observation for biofilms is consistent with previous reports that drug efflux mutants develop drug-resistant biofilms (36, 49). Further, the planktonic yeast cells that were sensitive to azole drugs at low cell density were phenotypically resistant at high cell density (Table 1). In another experiment, the contribution of farnesol quorum sensing to high-cell-density phenotypic resistance was examined with a chk1 strain that is insensitive to farnesol. Planktonic yeast cells at low and high cell density, biofilm, and high- and low-density dissociated biofilm organisms showed the same drug sensitivity profile as did wild-type organisms (Table 1). These observations suggested that neither the tested drug efflux pumps nor farnesol sensing through Chk1p was responsible for the development of phenotypic resistance at high cell density.

Consideration of all of the observations suggested that at a high cell density, C. albicans develops tolerance for high concentrations of azole drugs and increased concentrations of amphotericin B and caspofungin. Azole drugs are fungistatic, and at low cell density C. albicans is tolerant of this class of drugs (1). At high cell density, C. albicans organisms also appeared to be tolerant of high azole drug concentrations (Table 1). When supernatant was recovered from high-cell-density organisms treated with high azole drug concentrations, the supernatant contained sufficient drug to inhibit the growth of low-cell-density organisms, although the concentration was less than that added as judged from the effective dilution that inhibited low-cell-density organisms. The apparent loss of fluconazole from the medium may be due to uptake and sequestration of drug within the cells or binding to the cell surface. We concluded that the high-cell-density organisms were exposed to a drug concentration that is effective for low-density organisms. In low-density C. albicans planktonic yeast cells, azole tolerance can be abrogated by the addition of FK506 (tacrolimus), which inhibits the calcineurin pathway through binding to FKB12 encoded by RPB1 (33, 34, 44, 60). When FK506 was added to high-density planktonic yeast cells, no synergism was observed and the organisms did not become susceptible to fluconazole (Fig. 6). This observation suggested that the high-cell-density tolerance to azole drugs differed from the low-cell-density tolerance.

One of the differences between low-density and high-density organisms in culture is the accumulation of metabolites that may have signaling or regulatory functions. In C. albicans two such molecules, farnesol and tyrosol, have been identified (13, 24), and a quorum-sensing activity with autoregulatory morphogenetic properties is produced in mature biofilm (50). During preparation of high-density cell suspensions for testing, these organisms may retain the effect from inoculum growth or secrete sufficient metabolites to affect cellular responses. Although in this study sensing of farnesol through Chk1p was not implicated in the reduced antifungal drug susceptibility, there may be other farnesol sensors affecting this response or other unidentified signaling molecules. Such molecules would be lost in dilution to low cell density from either planktonic cultures or dissociated biofilm. Upon dilution there could be a very rapid change in gene expression, inhibition of the drug tolerance mechanism, or activation of a drug susceptibility mechanism. The membrane composition and fluidity can affect susceptibility to amphotericin B and azole drugs, e.g., fluconazole (20, 25, 38, 39, 47, 48). However, whether such changes could occur with the necessary time frame is unknown. In mammalian cells signaling and protein localization in the membrane can be altered rapidly (17, 19, 62) and, if similar effects occur in the candidal membrane, would be a means to effect drug-related membrane function. If there is a critical ratio of drug to cells, the ratio at the low-cell-density MIC80 is greater than that tested at high planktonic cell density or in biofilm (28, 45, 49, 52).

The observations of this study suggested a role for cell density in the acquisition of phenotypic resistance or tolerance of biofilms to high concentrations of various antifungal drugs and suggested that this acquisition was not a unique property of biofilm formation or architecture. A similar tolerance was observed with high-density planktonic yeast cells or dissociated biofilm, while dissociated biofilm at low cell density had the same drug susceptibility profile as did low-cell-density planktonic yeast cells. The mechanism of this tolerance was not related to several drug efflux pumps, farnesol quorum sensing through Chk1p, or low-cell-density azole drug tolerance, but the tolerance is lost in a fairly short time frame in a matter of minutes.


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ACKNOWLEDGMENTS
 
We thank Ted White for discussions about biofilm phenotypic resistance to various drugs.

This study was supported by Public Health Service grant 1RO1 DE 14029 from the National Institutes of Health to W.L.C.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430. Phone: (806) 745-2545. Fax: (806) 743-2334. E-mail: LaJean.Chaffin{at}ttuhsc.edu Back

{triangledown} Published ahead of print on 14 May 2007. Back

{dagger} Present address: Center for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, India 600025. Back

{ddagger} Present address: 1209 Fox Run Dr., Plainsboro, NJ 08536. Back


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