Role for Cell Density in Antifungal Drug Resistance in Candida albicans Biofilms

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 ؋ 10 3 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 ؋ 10 8 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 ؋ 10 3 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.

Characteristics of Candida albicans Biofilms Grown in a Synthetic Urine Medium

“…C. albicans biofilms in vitro as well as in vivo are known to be resistant to all major classes of antifungal drugs except echinocandins and liposomal formulations of amphotericin B (9,21). Drug resistance has been partially attributed to increased cell density in biofilms (17). We found that despite being architecturally simpler and thinner than RPMI medium biofilms, the SU medium biofilms were still completely resistant to fluconazole and displayed increased resistance to amphotericin B.…”

“…The pH was adjusted to 5.8, and the mixture was sterilized by filtration. RPMI medium (RPMI 1640 medium supplemented with L-glutamine and buffered with morpholinepropanesulfonic acid; Angus Buffers and Chemicals, Niagara Falls, NY), also containing 2% glucose (wt/vol) and adjusted to a final pH of 7.2, was used as a control for comparisons since C. albicans biofilms grown in this medium have been described by many different groups and have been extensively characterized (10,17,18).Two different in vitro models were used to assess biofilm growth and drug susceptibility. The first was a microtiter plate-based model that utilizes polystyrene, flat bottomed, 96-microtiter plates (Corning Inc.) as a substrate, as previously described (18).…”

Transcription factor Efg1 contributes to the tolerance of Candida albicans biofilms against antifungal agents in vitro and in vivo

“…A second set of C. albicans mutants consisted of Defg1 (HLC52), the reintegrant Defg1(EFG1) (HLC74), and the corresponding wild-type (WT) CAF2 (Lo et al, 1997). Strain DSY1050, in which all major efflux pumps encoding CDR1, CDR2 and MDR1 are inactivated (Perumal et al, 2007), and corresponding WT strain SC5314 were from Gillum et al (1984). The growth medium used was yeast extract-peptone-glucose (dextrose) (YPD; 1 % yeast extract, 2 % peptone, 2 % glucose).…”

“…2). These data indicate that the miconazole sensitivity of Defg1 sessile cells is probably due to increased To determine whether efflux pumps are involved in governing miconazole tolerance of C. albicans biofilms, we investigated the miconazole sensitivity of sessile C. albicans DSY1050 cells, lacking all the major efflux pumps (CDR1, CDR2, MDR1) (Perumal et al, 2007), by using the CV quantification method as described above. We found that the miconazole sensitivity of C. albicans DSY1050 sessile cells was significantly increased compared to sessile cells of the corresponding WT SC5314 cells upon treatment with miconazole ranging from 0.15 to 2.4 mM.…”

Optimizing a Candida Biofilm Microtiter Plate Model for Measurement of Antifungal Susceptibility by Tetrazolium Salt Assay

“…It is the method most commonly utilized for quantitative measurement of Candida biofilm mass, growth, and response to drug therapy (1,9,12,18,20,26,28,37). Other techniques used to assay the biofilm cell burden include [ 3 H]leucine incorporation, fluorescein diacetate, crystal violet staining, viable counts, dry weight measurements, and imaging using confocal or electron microscopy (5,8,10,35,36).…”