ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2010, p. 3505–3508
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 8
Role of Fks1p and Matrix Glucan in Candida albicans Biofilm
Resistance to an Echinocandin, Pyrimidine, and Polyene?
Jeniel E. Nett,1,3Kyler Crawford,1Karen Marchillo,1and David R. Andes1,2*
Departments of Medicine,1Medical Microbiology and Immunology,2and Cellular and Molecular Biology,3
University of Wisconsin, Madison, Wisconsin
Received 16 February 2010/Returned for modification 22 April 2010/Accepted 24 May 2010
Candida infections frequently involve drug-resistant biofilm growth on device surfaces. Glucan synthase
gene FKS1 has been linked to triazole resistance in Candida biofilms. We tested the impact of FKS1
modulation on susceptibility to additional antifungal classes. Reduction of FKS1 expression rendered
biofilms more susceptible to amphotericin B, anidulafungin, and flucytosine. Increased resistance to
anidulafungin and amphotericin B was observed for biofilms overexpressing FKS1. These findings suggest
that Candida biofilm glucan sequestration is a multidrug resistance mechanism.
In hospital settings, Candida spp. often cause disease by
adhering to the surface of a medical device and adapting to
a biofilm lifestyle (7, 10). Biofilms consist of cells attached to
a surface and embedded in a protective matrix produced by
the organisms (5). C. albicans biofilm cells are phenotypi-
cally distinct, and their ability to survive exposure to high
antifungal concentrations presents a serious therapeutic di-
lemma (1, 2, 11, 14, 19–21). Biofilm cells exhibit up to
1,000-fold-increased resistance relative to free-floating, or
planktonic, cells (3, 9, 12, 18).
Glucan synthesis by Fks1p has been implicated in C. albicans
biofilm resistance to the azole drug fluconazole (17). FKS1
disruption was found to reduce manufacture and deposition of
?-1,3-glucan in the biofilm matrix, resulting in susceptibility to
fluconazole. The matrix glucan was shown to sequester the
triazole, preventing it from reaching its target. The mechanism
is biofilm specific and has been studied only for the triazoles.
The purpose of this study was to determine the role of
FKS1 in C. albicans biofilm resistance to other available
antifungal drug classes. We chose to study three strains with
differing expressions of FKS1 and concomitant variations in
matrix glucan. The strains included a heterozygous deletion
mutant (FKS1/fks1?), an FKS1 overexpression mutant
(TDH3-FKS1) with one FKS1 allele under the control of
TDH3 promoter and one allele intact, and a reference strain
(4, 17). Finally, because FKS1 is essential in C. albicans, a
conditional TET-FKS1 mutant was also included (22). The
TET-FKS1 strain has one allele deleted and one allele under
the control of a tetracycline- or doxycycline-repressible pro-
moter. An echinocandin (anidulafungin), flucytosine, and
amphotericin B deoxycholate were selected for their differ-
ent mechanisms of action.
For biofilm antifungal susceptibility testing, C. albicans
biofilms were grown in 96-well polystyrene plates as previ-
ously described (16, 20). Wells were inoculated with 106
cells/ml in RPMI medium-MOPS (morpholinepropanesul-
fonic acid). After an adherence period (6 or 24 h), biofilms
were washed with phosphate-buffered saline (PBS). Fresh
media and antifungals were applied, and plates were incu-
bated for an additional 24 h at 37°C. The concentration
ranges included those above and below the planktonic MIC
values and included 0.001 to 0.125 ?g/ml anidulafungin, 0.03
to 8 ?g/ml flucytosine, and 0.008 to 2 ?g/ml amphotericin B
deoxycholate (13). After 24 h of incubation at 30°C, an XTT
5-carboxanilide] reduction assay was performed and end-
points were determined spectrophotometrically at 492 nm as
a measure of cell metabolic activity (16, 20).
For the FKS1/fks1? strain, the TDH3-FKS1 strain, and the
reference strain, we measured the impact of antifungal wells
compared to the no-drug control wells. The impact of doxycy-
cline repression of FKS1 on antifungal susceptibility during
biofilm formation was similarly examined using the TET-FKS1
strain with a doxycycline concentration range of 1 to 240 ng/ml
in a 96-well checkerboard format. After adherence, biofilms
were incubated in the presence of the doxycycline and antifun-
gal in combination for 24 h prior to the XTT assay. For plank-
tonic studies, MICs were determined two times in duplicate
and measured visually using CLSI endpoints (15).
Anidulafungin was the most effective against parent C.
albicans biofilms, while flucytosine had minimal or no activ-
ity at the highest concentration tested (Fig. 1). The biofilm
formed by the FKS1/fks1? heterozygote was more suscepti-
ble to flucytosine and anidulafungin, with drug impact at 2-
to 8-fold-lower concentrations. Heterozygous FKS1 disrup-
tion did not impact amphotericin B activity in this assay
design. To determine if a difference for amphotericin B
might be due to the phase of growth, a later phase of biofilm
growth (24 h) was tested. By this method, FKS1/fks1? bio-
films were more susceptible to amphotericin B than refer-
ence strain biofilms were, but the difference was less than
that observed for the other antifungal drug classes (not
shown). For example, treatment with amphotericin B at 0.25
?g/ml decreased FKS1/fks1? biofilms by 80%, compared to
60% for the reference strain (P ? 0.05; Student’s t test).
The TET-FKS1 strain recapitulated the phenotypes for
* Corresponding author. Mailing address: 600 Highland Ave., H4/
572 Clinical Sciences Center, Department of Medicine, University of
Wisconsin, Madison, WI 53792. Phone: (608) 263-1545. Fax: (608)
263-4464. E-mail: firstname.lastname@example.org.
?Published ahead of print on 1 June 2010.
FIG. 1. Impact of FKS1 modulation on antifungal susceptibility in C. albicans biofilms. FKS1-modulated biofilms were grown in 96-well plates
for 6 h and treated with serial dilutions of anidulafungin (A), flucytosine (B), or amphotericin B deoxycholate (C) for an additional 24 h. Endpoints
were assessed using an XTT assay, and data are shown as percentages of biofilm growth relative to growth of untreated controls. Assays were
performed in triplicate, and each error bar represents one standard error. Statistical significance was determined by analysis of variance with
pairwise comparisons using the Holm-Sidak method.*, P ? 0.05.
3506NETT ET AL.ANTIMICROB. AGENTS CHEMOTHER.
susceptibility to echinocandin and flucytosine, with 4- to
8-fold-lower drug concentrations effective for the condition
with doxycycline repression of FKS1 (Fig. 2). Interestingly,
modulation of FKS1 by doxycycline or heterozygous disrup-
tion did not render biofilms more susceptible to amphoter-
icin B. The explanation for this difference is not clear. Doxy-
cycline did not impact C. albicans reference strain growth or
drug susceptibility at the concentrations used in these ex-
periments (data not shown).
FKS1 overexpression had a similar but lesser impact on
biofilm susceptibility to anidulafungin (Fig. 1). Increased
resistance to flucytosine was not detectable by these over-
expression assays, due to the profound resistance of the
reference biofilm at the highest concentrations. The TDH3-
FKS1 overexpression biofilm exhibited a marked increased
resistance to amphotericin B, supporting a role for glucan in
polyene biofilm resistance.
Importantly, modulation of FKS1 did not impact plank-
tonic susceptibility to the various antifungals based on stan-
dard CLSI testing and interpretation (Table 1) (15). Be-
cause the drug target of anidulafungin is Fks1p, we
considered the possibility that genetically modifying expres-
sion and regulation of this gene may directly impact suscep-
tibility to the compound (6). For example, echinocandin
resistance in planktonic cells has been linked to altered
Fks1p kinetics due to point mutations in several hot spots
(8). However, the FKS1 heterozygote was similarly suscep-
tible to echinocandin in this planktonic assay, while the
strain was more susceptible to echinocandin in the biofilm
assay relative to the parent strain, again suggesting a bio-
film-specific mode of action (Table 1).
FKS1 has been linked to C. albicans resistance through a
mechanism specific to biofilms. Investigations using flucon-
azole and amphotericin B suggest that this process involves
antifungal sequestration by the matrix glucan (16, 17, 23).
Modulation of FKS1, through either inhibition or overex-
pression, impacted biofilm susceptibility to all the antifungal
agents tested. As observed for FKS1 and fluconazole resis-
tance, this mechanism appears to be biofilm specific, since
disruption of FKS1 has no impact on planktonic resistance.
Our findings indicate that FKS1 similarly impacts biofilm
resistance to other antifungal drug classes, possibly through
the same mechanism.
We thank C. Douglas, A. Mitchell, and C. Nobile for strains and
This work was supported by the National Institutes of Health (grant
1. Al-Fattani, M. A., and L. J. Douglas. 2006. Biofilm matrix of Candida albi-
cans and Candida tropicalis: chemical composition and role in drug resis-
tance. J. Med. Microbiol. 55:999–1008.
2. Baillie, G. S., and L. J. Douglas. 1998. Effect of growth rate on resistance of
Candida albicans biofilms to antifungal agents. Antimicrob. Agents Che-
TABLE 1. Impact of FKS1 modulation on drug susceptibility
of planktonic cellsa
MIC (?g/ml) for strain type
Amphotericin B deoxycholate
aMICs were determined using the CLSI method and endpoints.
FIG. 2. Impact of doxycycline repression of FKS1 on antifungal
susceptibility in C. albicans biofilms. TET-FKS1-modulated biofilms
were grown in 96-well plates for 6 h and treated for 24 h with serial
dilutions of anidulafungin (A), flucytosine (B), or amphotericin B
deoxycholate (C) in combination with doxycycline by using a checker-
board format. Endpoints were assessed using an XTT assay, and data
are shown as percentages of biofilm growth relative to growth of
untreated controls. Checkerboard assays were performed in duplicate,
and results from one assay replicate are shown.
VOL. 54, 2010 Fks1p IN C. ALBICANS BIOFILM MULTIDRUG RESISTANCE 3507
3. Chandra, J., D. M. Kuhn, P. K. Mukherjee, L. L. Hoyer, T. McCormick, and Download full-text
M. A. Ghannoum. 2001. Biofilm formation by the fungal pathogen Candida
albicans: development, architecture, and drug resistance. J. Bacteriol. 183:
4. Davis, D., R. B. Wilson, and A. P. Mitchell. 2000. RIM101-dependent and-
independent pathways govern pH responses in Candida albicans. Mol. Cell.
5. Donlan, R. M. 2001. Biofilm formation: a clinically relevant microbiological
process. Clin. Infect. Dis. 33:1387–1392.
6. Douglas, C. M., J. A. D’Ippolito, G. J. Shei, M. Meinz, J. Onishi, J. A.
Marrinan, W. Li, G. K. Abruzzo, A. Flattery, K. Bartizal, A. Mitchell, and
M. B. Kurtz. 1997. Identification of the FKS1 gene of Candida albicans as the
essential target of 1,3-beta-D-glucan synthase inhibitors. Antimicrob. Agents
7. Douglas, L. J. 2003. Candida biofilms and their role in infection. Trends
8. Garcia-Effron, G., S. Park, and D. S. Perlin. 2009. Correlating echinocandin
MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida
albicans: implications for interpretive breakpoints. Antimicrob. Agents Che-
9. Hawser, S. P., and L. J. Douglas. 1994. Biofilm formation by Candida species
on the surface of catheter materials in vitro. Infect. Immun. 62:915–921.
10. Kojic, E. M., and R. O. Darouiche. 2004. Candida infections of medical
devices. Clin. Microbiol. Rev. 17:255–267.
11. Kuhn, D. M., and M. A. Ghannoum. 2004. Candida biofilms: antifungal
resistance and emerging therapeutic options. Curr. Opin. Invest. Drugs
12. Mah, T. F., B. Pitts, B. Pellock, G. C. Walker, P. S. Stewart, and G. A.
O’Toole. 2003. A genetic basis for Pseudomonas aeruginosa biofilm antibiotic
resistance. Nature 426:306–310.
13. McDonnell, G. E. 2007. Antisepsis, disinfection, and sterilization: types,
action, and resistance. ASM Press, Washington, DC.
14. Mukherjee, P. K., J. Chandra, D. M. Kuhn, and M. A. Ghannoum. 2003.
Mechanism of fluconazole resistance in Candida albicans biofilms: phase-
specific role of efflux pumps and membrane sterols. Infect. Immun. 71:4333–
15. NCCLS/CLSI. 2002. Reference method for broth dilution antifungal suscep-
tibility testing. Document M27-A2, 2nd ed. National Committee for Clinical
Laboratory Standards, Wayne, PA.
16. Nett, J., L. Lincoln, K. Marchillo, R. Massey, K. Holoyda, B. Hoff, M.
VanHandel, and D. Andes. 2007. Putative role of beta-1,3 glucans in Candida
albicans biofilm resistance. Antimicrob. Agents Chemother. 51:510–520.
17. Nett, J. E., H. Sanchez, M. T. Cain, and D. Andes. 2010. Genetic basis of
Candida biofilm resistance due to drug sequestering matrix glucan. J. Infect.
18. O’Toole, G. A. 2003. To build a biofilm. J. Bacteriol. 185:2687–2689.
19. Ramage, G., S. Bachmann, T. F. Patterson, B. L. Wickes, and J. L. Lopez-
Ribot. 2002. Investigation of multidrug efflux pumps in relation to flucon-
azole resistance in Candida albicans biofilms. J. Antimicrob. Chemother.
20. Ramage, G., K. Vande Walle, B. L. Wickes, and J. L. Lopez-Ribot. 2001.
Standardized method for in vitro antifungal susceptibility testing of Candida
albicans biofilms. Antimicrob. Agents Chemother. 45:2475–2479.
21. Ramage, G., K. VandeWalle, S. P. Bachmann, B. L. Wickes, and J. L.
Lopez-Ribot. 2002. In vitro pharmacodynamic properties of three antifungal
agents against preformed Candida albicans biofilms determined by time-kill
studies. Antimicrob. Agents Chemother. 46:3634–3636.
22. Roemer, T., B. Jiang, J. Davison, T. Ketela, K. Veillette, A. Breton, F.
Tandia, A. Linteau, S. Sillaots, C. Marta, N. Martel, S. Veronneau, S.
Lemieux, S. Kauffman, J. Becker, R. Storms, C. Boone, and H. Bussey. 2003.
Large-scale essential gene identification in Candida albicans and applications
to antifungal drug discovery. Mol. Microbiol. 50:167–181.
23. Vediyappan, G., T. Rossignol, and C. d’Enfert. 2010. Interaction of Candida
albicans biofilms with antifungals: transcriptional response and binding of
antifungals to beta-glucans. Antimicrob. Agents Chemother. 54:2096–2111.
3508 NETT ET AL.ANTIMICROB. AGENTS CHEMOTHER.