1) The study found that treating Candida albicans biofilms first with the azole antifungal fluconazole, followed by the echinocandin caspofungin, led to a significant decrease in the efficacy of caspofungin against the biofilms. 2) This phenomenon occurred due to stress responses in the biofilm cells induced by the fluconazole exposure. A key player in these stress responses is the heat shock protein Hsp90. 3) Depleting Hsp90 in the cells eliminated the decreased efficacy of caspofungin against biofilms pretreated with fluconazole. The stress response regulator calcineurin was also found to be involved but the cell wall

1. In Vitro Study of Sequential Fluconazole and Caspofungin Treatment
against Candida albicans Biofilms
Semanti Sarkar, Priya Uppuluri, Christopher G. Pierce, Jose L. Lopez-Ribot
Department of Biology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, Texas, USA
Candida albicans biofilms are generally considered to be resistant to azole antifungal agents but susceptible to echinocandins.
We demonstrate that in a sequential therapy regimen, treatment with fluconazole first followed by caspofungin leads to a signifi-
cant decrease of the efficacy of this echinocandin. Cellular stress responses induced by high fluconazole concentrations and me-
diated by Hsp90 and calcineurin play an important role in this phenomenon.
Candida spp. are among the most frequent causes of nosoco-
mial infections in hospitals in the United States and world-
wide (1–3). This rise in incidence is at least in part related to the
organism’s ability to produce biofilm infections on medical de-
vices (4, 5). Candida albicans biofilms are known to display high
levels of resistance to most major classes of antifungal agents, in-
cluding azoles and polyenes (6). In contrast, therapeutic concen-
trations of caspofungin and other echinocandins display excellent
activity against C. albicans biofilms (6–9).
We have previously reported a trend toward antagonism when
fluconazole and caspofungin are used in combination against C.
albicans biofilms (10). In the present study, we examined whether
this antagonistic interaction is also manifested in biofilms sequen-
tially treated with fluconazole first followed by caspofungin. C.
albicans strain SC5314 biofilms grown overnight under static con-
ditions in the wells of 96-well microtiter plates as previously de-
scribed by our group (11, 12) were first treated with various con-
centrations of fluconazole for 24 h. As expected, these biofilms
were completely resistant to the drug (sessile MIC80 [SMIC80] Ͼ
512 ␮g/ml) as revealed by measuring metabolic activity using a
XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazoli-
um-5-carboxanilide] reduction assay (11, 12). On the other hand,
duplicate biofilms treated with a range of concentrations of caspo-
fungin were found to be completely sensitive to caspofungin (Fig.
1A). In a sequential antifungal drug therapy regimen, i.e., treat-
ment of mature C. albicans biofilms by fluconazole first, for 24 h,
followed by another 24 h of caspofungin treatment (using a check-
erboard pattern of concentrations as described in reference 10),
we observed a significant decrease in the efficacy of this echino-
candin, thereby considerably diminishing its otherwise excellent
in vitro antibiofilm activity (Fig. 1A). We found that this dimin-
ished activity was directly dependent on the concentration of flu-
conazole used: biofilms pretreated with higher concentrations of
fluconazole (Ͼ16 ␮g/ml) demonstrated higher resistance to
caspofungin. On the other hand, caspofungin was highly effective
against biofilms pretreated with fluconazole concentrations Ͻ 4
␮g/ml (Fig. 1A).
We expanded these observations to other potential combina-
tions of clinically used azole and echinocandin agents. As seen in
Table 1, the phenomenon of increased echinocandin resistance of
biofilms after exposure to an azole derivative is not unique to
fluconazole and caspofungin but was also manifested in the case of
sequential treatment with fluconazole followed by either micafun-
gin or anidulanfungin, as well as in the case of preexposure to a
different azole, voriconazole, followed by caspofungin treatment.
As in the case of fluconazole-caspofungin sequential treatment, in
all instances this phenomenon of increased echinocandin resis-
tance was observed after the biofilms had been exposed to rela-
tively high concentrations of the azole derivatives. We also note
that multiple clinical isolates of C. albicans (13) displayed in-
creased resistance to caspofungin after fluconazole pretreatment,
while this effect was not manifested by other non-albicans Can-
dida spp., including C. glabrata, C. dubliniensis, and C. parapsilosis
(not shown). Together, these results would seem to corroborate
the increased ability of C. albicans to adapt and respond to envi-
ronmental stresses which has made this fungus such a formidable
opportunistic pathogen (14).
A similar sequential treatment regimen was performed with
biofilms formed under conditions of shear stress. In this set of
experiments, biofilms of C. albicans strain SC5314 were developed
on silicone strips using a flow biofilm model (15) for 12 h before
being subjected to media containing fluconazole (500 ␮g/ml) for
another 12 h. At this point, the metabolic activity of portions of
the biofilm was measured using the XTT assay. As expected, the
biofilms were found to be completely resistant to fluconazole.
However, on subsequent treatment of these biofilms with media
containing caspofungin (0.125 ␮g/ml, a concentration that was
fully active against control biofilms in the absence of fluconazole),
the biofilms that had been preexposed to fluconazole were found
to be resistant to this concentration of caspofungin. However, the
induced resistance to caspofungin did not manifest once cells dis-
persed from the biofilms: when tested following CLSI procedures,
dispersed cells (obtained as described before by our group [16])
remained fully susceptible to caspofungin, with MICs in the range
of 1 ␮g/ml that were virtually identical to those seen with dis-
persed cells from biofilms unexposed to fluconazole and with
planktonic cells, thereby indicating that this phenomenon is re-
Received 12 August 2013 Returned for modification 10 September 2013
Accepted 5 November 2013
Published ahead of print 11 November 2013
Address correspondence to Jose L. Lopez-Ribot, jose.lopezribot@utsa.edu.
S.S. and P.U. contributed equally to this article.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01745-13
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2. stricted to sessile cells within the biofilms and may not be retained
by the cells once they disperse out of the biofilm community.
Through evolution, C. albicans has developed mechanisms
that allow this opportunistic pathogen to cope with a variety of
environmental stresses and adapt to diverse niches within the host
(14, 17). Thus, we hypothesize that the induced resistance to
caspofungin seen subsequent to fluconazole treatment might have
been due to a cellular stress response subsequent to the exposure
of biofilm cells to fluconazole. In fact, these cellular stress re-
sponses have been involved in the development of resistance to
both azoles and echinocandins, mostly controlled by the Hsp90
molecular chaperone (14, 17–19) and, more recently, we have
reported on the important role of Hsp90 in mediating fungal bio-
film resistance (20). Therefore, our original investigations into the
mechanism(s) of induced caspofungin resistance subsequent to
fluconazole treatment focused first on a potential role of Hsp90.
Briefly, we used a tetracycline-regulatable HSP90 strain where
Hsp90 levels can be regulated by doxycycline (DOX) in growth
medium (21). In the absence of DOX (when HSP90 is being ex-
pressed), the tet-HSP90 strain behaved similarly to the wild-type
strains of C. albicans in that the biofilms were completely resistant
to fluconazole and sensitive to caspofungin (Fig. 1B). Also, simi-
larly to control biofilms, under these conditions the tet-HSP90
biofilms displayed elevated caspofungin resistance after prior ex-
posure to fluconazole (Fig. 1B). However, when DOX was added
into the biofilm growth medium, leading to a depletion of Hsp90
levels, we found that the induced resistance to the echinocandin
was virtually abolished (Fig. 1C). Overall, these results highlight
that C. albicans Hsp90 plays an important role in the induced
resistance to caspofungin displayed by cells within the biofilms
subsequent to fluconazole pretreatment.
Hsp90 helps C. albicans cells to survive the lethal effect of the
antifungal drugs by stabilizing key regulators of cellular signaling
(14, 17, 21). Thus, to further dissect the mechanism(s) by which
Hsp90 regulates the induced resistance in the sequential flucona-
zole-caspofungin treatment, we utilized strains lacking specific
downstream effectors of Hsp90. In particular, both calcineurin, a
Ca2ϩ
-calmodulin-activated protein phosphatase, and Mkc1, the
terminal mitogen-activated protein kinase (MAPK) in the protein
kinase C (PKC) signaling cascade, represent major Hsp90 client
proteins involved in antifungal drug resistance in C. albicans, in-
cluding during the biofilm mode of growth (14, 20, 22). To this
end, we examined the C. albicans mutant strain lacking the cata-
lytic subunit of calcineurin (⌬cna1 cna1) (19) in our sequential
checkerboard antifungal drug assay. As expected, the biofilms
formed by the calcineurin mutant were more sensitive to flucona-
zole with an SMIC50 of 32 ␮g/ml (Fig. 2A). These biofilms were
also hypersensitive to caspofungin. We found that pretreatment of
the calcineurin mutants with fluconazole did not translate into
subsequent caspofungin resistance (Fig. 2A). In fact, despite the
fluconazole exposure, these biofilms were still hypersensitive to
caspofungin.
As mentioned before, Mkc1 is an important determinant of the
PKC-cell wall integrity pathway (23). Since the primary target of
caspofungin is ␤-1,3-glucan in the cell wall (24), we questioned if any
compensatorymechanismsinthecellwallmayalsoinfluencebiofilm
resistance to caspofungin after fluconazole treatment. However, as
shown in Fig. 2B and in stark contrast to results obtained using the
calcineurin mutant, fluconazole pretreatment still resulted in in-
ducedresistancetocaspofunginofbiofilmsformedbythemkc1mkc1
mutant strain (25). These results clearly indicate that Mkc1, the ter-
minal MAPK in the PKC signaling cascade, is dispensable for the
induction of caspofungin resistance after exposure to fluconazole.
FIG 1 (A) Biofilms of C. albicans SC5314 were grown in 96-well microtiter
plates in RPMI medium at 37°C. After 24 h, cells were washed with phosphate-
buffered saline (PBS) to remove nonadherent cells and fresh medium was
added with various concentrations of antifungal drugs in a checkerboard for-
mat. First, fluconazole (FLC) was added in all but the first column of the wells,
which was replenished with RPMI medium. After 24 h of fluconazole pretreat-
ment, biofilms were washed once with PBS and then treated with different
concentrations of caspofungin (CAS) for an additional 24 h. Metabolic activity
of the biofilm cells was measured using the XTT assay. (B and C) Genetic
compromise of HSP90 results in sensitivity to caspofungin despite fluconazole
pretreatment. HSP90 levels were regulated in the C. albicans tetracycline-reg-
ulatable (⌬hsp90 tet HSP90) strain by the presence and absence of DOX. In the
absence of DOX (when HSP90 was being expressed), the strain showed a re-
sponse similar to that of the SC5314 wild-type strain with induction of caspo-
fungin resistance. However, in the presence of DOX (lacking HSP90 expres-
sion), the biofilms were sensitive to caspofungin subsequent to fluconazole
exposure. Color gradients are representative of biofilm viability such that light
colors depict maximum metabolic activity (low inhibition) and the darkest
color represents maximum inhibition of biofilm viability.
TABLE 1 Preexposure to azole antifungal agents induces subsequent
resistance to echinocandin derivatives in C. albicans biofilms, as
measured by SMIC80 using the XTT reduction assaya
Sequential treatment
SMIC80 (␮g/ml)a
No azole
(echinocandin
only)
Low
azole
concn
Medium
azole
concn
High
azole
concn
Fluconazole-caspofungin 0.25 0.25 Ͼ2 Ͼ2
Fluconazole-micafungin 0125 0.125 Ͼ2 Ͼ2
Fluconazole-anidulafungin 0.125 0.125 Ͼ2 Ͼ2
Voriconazole-caspofungin 0.25 0.5 Ͼ2 Ͼ2
a
The low, medium, and high azole concentrations were 1, 64 and 512 ␮g/ml for
fluconazole and 0.25, 16, and 128 ␮g/ml for voriconazole, respectively.
Sarkar et al.
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3. Overall, this report reveals that, in a sequential antifungal drug
therapy regimen in vitro, prior treatment of C. albicans biofilms
with an azole leads to a significant decrease in the efficacy of echi-
nocandin agents, almost completely abolishing their otherwise ex-
cellent in vitro antibiofilm activity. This activity is dependent on
the concentration of azole used, in that biofilms pretreated with
higher concentrations of azole derivatives demonstrated higher
resistance to echinocandins under both static and flow conditions.
This phenomenon is related to the induction of cell stress re-
sponses mediated by Hsp90 and its client protein calcineurin but
not by Mkc1. If confirmed in vivo, these observations may have
profound implications for the clinical management of patients
with candidiasis, further advocating for the use of echinocandins
as first-line therapy when a biofilm etiology is suspected.
ACKNOWLEDGMENTS
This work was supported by a grant from Merck & Co., Inc.
We thank Leah E. Cowen for C. albicans mutant strains and Rick
Kirkpatrick and the Fungus Testing Lab at UTHSCSA for isolates of non-
albicans Candida spp.
The content of this work is solely our responsibility and does not
necessarily represent the official views of the founder.
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