ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2007, p. 3081–3088
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 9
Paradoxical Growth Effect of Caspofungin Observed on Biofilms and
Planktonic Cells of Five Different Candida Species?
Analy S. Melo,1Arnaldo L. Colombo,1and Beth A. Arthington-Skaggs2*
Division of Infectious Diseases, Universidade Federal de Sao Paulo, Sao Paulo, Brazil,1and Mycotic Diseases Branch,
Centers for Disease Control and Prevention, Atlanta, Georgia2
Received 23 May 2007/Returned for modification 11 June 2007/Accepted 16 June 2007
The paradoxical growth (PG) of Candida sp. biofilms in the presence of high caspofungin (CAS) concentrations
was previously unknown. We sought to characterize the PG at supra-MICs of CAS among clinical Candida sp.
isolates grown as biofilms in 96-well polystyrene microtiter plates. The MICs of CAS were determined for 30 clinical
Candida sp. isolates (4 Candida albicans, 6 C. tropicalis, 7 C. parapsilosis, 8 C. orthopsilosis, and 5 C. metapsilosis
isolates) when they were grown as planktonic cells and biofilms and were defined as the lowest drug concentrations
that resulted in a prominent decrease in growth and a 50% reduction in metabolic activity, respectively. PG was
defined as a resurgence of growth (>50% of that in the drug-free growth control well) at drug concentrations above
the MIC. With the exception of C. tropicalis, all isolates displayed PG more frequently when they were grown as
biofilms than when they grown as planktonic cells. PG was undetectable among C. metapsilosis isolates in planktonic
cell MIC tests but was present in 100% of the isolates in biofilm MIC tests. The drug concentration and the number
of drug dilutions supporting PG were higher for biofilms than for planktonic cells. Microscopic changes in cell
morphology were observed among both planktonic and biofilm cells with PG. Specifically, the accumulation of
enlarged, globose cells was associated with PG, and we hypothesize that CAS-induced changes in the cell wall
composition may be the explanation.
Paradoxical growth (PG) in Candida has been described as
growth in the presence of echinocandin concentrations above
the MIC in broth microdilution susceptibility tests performed
according to Clinical Laboratory Standards Institute (CLSI;
formerly NCCLS) guidelines (25, 26, 27). The evidence to date
suggests that PG is most commonly observed with caspofungin
(CAS) and less so with other echinocandin antifungals (2). The
echinocandin antifungals exert their antifungal effect by inhib-
iting cell wall biogenesis through inhibition of ?-1,3-glucan
synthesis (13). Candida sp. isolates are generally susceptible to
CAS in vitro, with the majority of isolates inhibited at a MIC
of ?1 ?g/ml (20). Despite the growth-inhibitory activity of
CAS at low concentrations, PG at supra-MICs, followed by
growth inhibition in the presence of the highest drug concen-
trations, has been observed for some Candida sp. isolates. Cells
growing in the presence of high CAS concentrations are not
resistant on retesting but show the PG effect of the parent, i.e.,
inhibition at low CAS concentrations and a resurgence of
growth at supra-MICs (25). PG is distinct from the trailing
growth phenotype described for some Candida albicans and
Candida tropicalis isolates in broth microdilution azole anti-
fungal susceptibility tests; trailing growth refers to the reduced
but persistent growth of cells in the presence of drug concen-
trations above the MIC, whereas PG is a resurgence of growth
in the presence of supra-MICs of a drug to nearly the levels
achieved in the absence of drug. To date, studies describing PG
in Candida isolates have focused on planktonic cell cultures (2,
4, 25, 26, 27), and the in vivo significance of PG remains
We report here for the first time the ability of Candida sp.
biofilms to display PG when they are exposed to high concen-
trations of CAS in vitro. Biofilms are commonly defined as a
structured community of microbial cells attached to a surface
and encased in a self-produced organic extracellular matrix (6).
A clinically significant characteristic of microbial biofilms is
their markedly enhanced resistance to antimicrobial agents (7,
12). Many manifestations of candidiasis are associated with the
formation of biofilms on host tissue (e.g., oral thrush) and
indwelling medical devices (e.g., central venous catheter-asso-
ciated candidemia) (1). Because of the extreme difficulty in
treating catheter-associated candidemia with systemic antifun-
gal therapy, current national guidelines recommend the re-
moval of catheters infected with Candida in order to eradicate
a potential nidus of bloodstream infection (15). Catheter re-
moval, however, is not always feasible for extremely ill patients,
and therefore, treatment strategies which enable catheter sal-
vage, such as antifungal lock therapy, are desirable (19). An-
tifungal lock therapy consists of filling the catheter lumen with
an antibiotic at high concentrations and allowing it to dwell in
the device for a period of time in order to sterilize the device.
With this method, a high local concentration of an appropriate
antibiotic can be applied in the catheter lumen while avoiding
systemic toxicity and the need to monitor serum drug levels.
CAS and lipid amphotericin B are the only antifungal drugs
with demonstrable activities against Candida biofilms in vitro
(5, 11) and in vivo (21, 22) and represent promising candidates
for antifungal lock therapy for catheter-associated candidemia.
Therefore, as antifungal lock therapy with high concentrations
of CAS continues to evolve as a realistic treatment strategy for
catheter-associated candidemia, it is pertinent to understand
* Corresponding author. Mailing address: Centers for Disease Control
and Prevention, 1600 Clifton Road, NE, Mailstop G-11, Atlanta, GA
30333. Phone: (404) 639-4041. Fax: (404) 639-3546. E-mail: bskaggs@cdc
?Published ahead of print on 25 June 2007.
the phenomenon of biofilm PG in the presence of high CAS
Clinical isolates from five Candida spp. were studied, includ-
ing two newly recognized species, Candida metapsilosis and
Candida orthopsilosis. These new species are phenotypically
identical but genotypically distinct from Candida parapsilosis
and are capable of causing bloodstream infections (14, 28).
The in vitro susceptibilities of Candida sp. biofilms to CAS and
the frequency of PG in biofilms compared with the frequency
of PG in planktonic cells was studied. Microscopic examination
of the cells with PG was performed and provided clues to the
underlying mechanism(s) of PG. These studies are necessary to
understand the contribution of PG to the recalcitrant nature of
Candida biofilms in vivo and any potential risk that antifungal
lock therapy with high CAS concentrations, e.g., concentra-
tions associated with PG in vitro, poses to patients who may be
candidates for this treatment strategy.
MATERIALS AND METHODS
Isolates. Thirty clinical Candida sp. isolates, including four C. albicans, six C.
tropicalis, seven Candida parapsilosis, eight Candida orthopsilosis, and five Can-
dida metapsilosis isolates, were used. The strains were from patients from dif-
ferent Brazilian cities and were isolated from different clinical specimens, in-
cluding blood, oropharyngeal, vaginal, urine, skin, and nail specimens. They were
selected from among strains previously identified and maintained at ?70°C in
the yeast stock collection of the Special Mycology Laboratory, Universidade
Federal de Sao Paulo. For species identification, the strains were plated onto
CHROMagar Candida (CHROMagar Microbiology, Paris, France) to check the
purity and viability of all original yeast cultures. All isolates were identified on
the basis of their micromorphology on cornmeal-Tween 80 agar and biochemical
tests performed with the commercial ID32C system (bioMerieux Marcy l’Etoile,
France). Since phenotypic methods do not differentiate C. orthopsilosis and C.
metapsilosis from C. parapsilosis, the isolates identified as C. parapsilosis were
submitted to molecular testing by randomly amplified polymorphic DNA
(RAPD) analysis (with primer 1253 [5?-GTT TCC GCC C-3?]) and internal
transcribed spacer (ITS) region sequencing for species identification. Reference
strains C. parapsilosis ATCC 90018, C. orthopsilosis ATCC 96141, and C. metap-
silosis ATCC 96143 were used as controls to interpret the RAPD and ITS
Planktonic cell MICs. CAS pure powder was received as a gift from Merck &
Co., Inc. (Rahway, NJ) and was solubilized in sterile water. The susceptibility of
planktonic cells was determined by the M27-A2 method, as described previously
(18). MIC endpoints were determined after 24 h on the basis of a prominent
decrease in growth compared to that of the drug-free growth control. CLSI-
recommended quality control strains (C. krusei ATCC 6258 and C. parapsilosis
ATCC 22019) were included on each day of testing, and the MICs were within
the recommended range. MIC endpoints were also determined by using the col-
orimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)car-
bonyl]-2H-tetrazolium hydroxide (XTT) with menadione as an electron-coupling
agent to parallel the biofilm MIC endpoint determination method. The XTT-
menadione solution was prepared fresh on each day of testing by adding 1.5 ml
of XTT (1 mg/ml in sterile saline; Sigma Chemicals, St. Louis, MO) to 300 ?l
menadione solution (0.4 mM in acetone; Sigma Chemicals). After 48 h, the MICs
were determined visually; 12 ?l of XTT-menadione solution was added to each
well of the microtiter plate, including both the drug-free growth control and the
sterility control wells; and the contents of each well were mixed by pipetting them
up and down with a multichannel pipette. The plates were incubated for 1 h at
35°C in the dark. Following incubation, the microtiter plate was covered with
tape and centrifuged at 4,000 rpm for 7 min to pellet the yeast cells. One hundred
microliters of supernatant was transferred to a fresh flat-bottom microtiter plate,
and the optical density was read with a spectrophotometer at 490 nm. The MIC
obtained by the use of XTT was defined as the lowest drug concentration that
caused a 50% decrease in metabolic activity relative to that in the drug-free
growth control. PG in planktonic MIC tests was defined as metabolic activity
(20% or more of that in the drug-free control well) in the presence of drug
concentrations above the MIC.
Biofilm formation. The biofilm formation and the growth conditions used in
this study were adapted from those described elsewhere (9). Yeast strains were
cultured on Sabouraud dextrose agar at 30°C for 24 h and then subcultured into
RPMI 1640 broth medium with L-glutamine, without bicarbonate (Sigma Chem-
icals), buffered to pH 7.0 with 3-(N-morpholino)propanesulfonic acid (165 M;
Sigma Chemicals) and were grown overnight (18 h) with shaking at 120 rpm at
35°C. The cell cultures were harvested, washed twice with phosphate-buffered
saline (PBS), and adjusted to a concentration of approximately 1 ? 107cells/ml
in RPMI 1640 medium. Biofilms were produced on sterilized, polystyrene, flat-
bottomed, 96-well microtiter plates (Costar; Corning Incorporated, Corning,
NY). For the attachment phase, 100 ?l of the adjusted cell suspension was
transferred to each well. No cells were added to the final column to be used as
the negative control. The plate was incubated at 37°C for 1.5 h with shaking at 75
rpm so that the cells could attach to the surfaces of the wells. Following the
attachment phase, unattached cells were removed, the wells were washed with
150 ?l of PBS, and 100 ?l of fresh RPMI 1640 medium was added. The plate was
incubated at 37°C for 66 to 72 h with shaking at 75 rpm to allow biofilm growth.
Biofilm quantitation by XTT reduction assay. Biofilm metabolic activity was
measured by the XTT reduction assay. After biofilm formation, the wells were
washed two times with 200 ?l PBS. To each well, 200 ?l of PBS and 12 ?l of
XTT-menadione solution (prepared as described above) were added. The cul-
ture plate was incubated in the dark for 1 h at 35°C, and afterward, 100 ?l of the
reaction solution was transferred to a new flat-bottomed microtiter plate and
the absorbance was measured with a spectrophotometer plate reader at 490 nm.
The experiments were performed with five replicates for each strain. The absor-
bance values of the negative control wells (containing no cells) were subtracted
from the values of the test wells.
Biofilm MICs. Biofilms were formed in RPMI 1640 medium as described
above. After 24 h of biofilm growth, the biofilms were washed two times with PBS
and the plates were inverted onto absorbent paper to remove residual amounts
of PBS prior to challenge with CAS. CAS was diluted in RPMI 1640 medium to
yield 10 doubling serial dilutions ranging from 2 to 1,024 ?g/ml. Two hundred
microliters of each of the drug concentrations was added to the respective wells
of the microtiter plate. The biofilms were incubated with CAS for 48 h at 35°C
with shaking at 75 rpm. Wells containing biofilm but no drug served as positive
controls for each strain tested. After 48 h of drug exposure, the medium was
removed and the biofilms were washed two times with PBS. Biofilm activity was
then measured by the XTT reduction assay exactly as described above. Biofilm
MICs were calculated on the basis of a 50% reduction in metabolic activity
compared with the activity of the drug-free control. Isolates were tested in
Microscopy. Cells from the planktonic cell MIC tests were removed from the
drug-containing and the drug-free control wells and visualized with a BX40 light
microscope (Olympus, Central Valley, PA) at ?400 magnification. Evaluation of
biofilm morphology with and without CAS exposure was carried out with an
inverted light microscope (Leitz-Diavert). The flat-bottomed microtiter plates
were placed on the microscope platform and examined at ?200 magnification.
Photographs were taken with a digital camera (Nikon Corp., Melville, NY).
CAS susceptibility. (i) Planktonic cell MICs. Broth microdi-
lution susceptibility testing of planktonic cells by use of the
CLSI guidelines revealed that the CAS MICs were ?2 ?g/ml
for all isolates tested (Table 1). In addition to visual MIC
readings, the MICs of planktonic cells were also determined by
the XTT reduction assay to ensure that the differences in MICs
between planktonic and biofilm cells were not influenced by
the use of different endpoint determination methods. In all
cases, the MICs determined by the XTT reduction assay were
the same as or 1 dilution higher than those determined visually
(data not shown). The median CAS MICs were the same for
the C. parapsilosis, C. orthopsilosis, and C. metapsilosis isolates
(median MIC ? 2 ?g/ml) and were consistently 1 to 2 dilutions
higher than the MICs for the C. albicans (median MIC ? 0.5
?g/ml) and the C. tropicalis (median MIC ? 0.75 ?g/ml) iso-
lates (Table 1).
(ii) Biofilm MICs. CAS MICs for 24-h biofilms (with 48 h of
drug exposure) ranged from 2 to 512 ?g/ml (Table 1). CAS
appeared to have excellent activity against the biofilms from all
3082 MELO ET AL.ANTIMICROB. AGENTS CHEMOTHER.
five species tested; the median biofilm MIC was 2 ?g/ml for
each species, which was consistent with the planktonic cell
MICs for the same isolates. Biofilm MICs ?2 ?g/ml were
observed for 6 of 30 (20%) isolates tested, including 2 C.
tropicalis isolates, 3 C. parapsilosis isolates, and 1 C. orthopsi-
losis isolate (Table 1). Low run-to-run variability was observed
among the XTT assay results for the replicate samples tested;
standard deviations were ?20% of the mean values (data not
PG. PG, or the reemergence of growth in the presence of
drug concentrations above the MIC, was observed for plank-
tonic and/or biofilm cells of each species tested. PG was spe-
cific for CAS and was not observed in amphotericin B or
fluconazole MIC tests for planktonic or biofilm cells (data not
shown). The PG effect of Candida sp. biofilms in micafungin
and anidulafungin MIC tests was unable to be determined due
to an inability to obtain drug powders. PG was more frequently
observed in biofilm MIC tests (24 of 30 isolates; 80%) than in
planktonic cell MIC tests (12 of 30 isolates; 40%). The fre-
quency of PG among planktonic cells differed by species, as
follows: C. tropicalis (five of six isolates; 83%) ? C. parapsilosis
(three of seven isolates; 43%) ? C. orthopsilosis (three of eight
isolates; 37.5%) ?, C. albicans (one of four isolates; 25%). C.
metapsilosis isolates did not display PG in planktonic cell CAS
MIC tests. PG was more prevalent in biofilm MIC tests; and
species-specific differences in frequency were also noted: C.
albicans (four of four isolates; 100%) ? C. metapsilosis (five of
five isolates; 100%) ? C. orthopsilosis (seven of eight; 87%) ?
C. tropicalis (four of six isolates; 67%) ? C. parapsilosis (four
of seven isolates; 57%). The lower frequency of PG among C.
tropicalis and C. parapsilosis isolates in biofilm MIC tests is due
to the fact that these biofilms were less susceptible to CAS in
vitro. There was, however, evidence of a “mini-PG” effect for
the two C. tropicalis and the three C. parapsilosis isolates with
high CAS biofilm MICs. The “mini-PG” was defined as an
initial decrease in metabolic activity at a lower CAS concen-
tration (although it did not meet the criteria for the MIC),
followed by an increase in metabolic activity at a higher CAS
concentration. For these isolates, the “mini-PG” effect oc-
curred over the drug concentration range of 8 to 128 ?g/ml
(Fig. 1B and C). Interestingly, PG in planktonic cell MIC tests
did not predict PG in biofilm MIC tests. Of the 30 isolates
TABLE 1. Caspofungin MICs and PG characteristics for 30 clinical Candida sp. isolates in planktonic and biofilm growth forms
Planktonic cells Biofilms
Isolate no. Species
aMIC endpoint based on visual determination of the lowest drug concentration that produced a prominent decrease in growth relative to that for the drug-free
growth control well.
bDrug concentration(s) above the MIC with visible growth ?20% for planktonic cells and ?50% for biofilm cells relative to the growth for the drug-free growth
cMIC endpoint based on the lowest drug concentration producing a 50% reduction in metabolic activity relative to that for the drug-free growth control, as measured
by the XTT reduction assay.
dNA, no PG observed.
VOL. 51, 2007EFFECT OF CASPOFUNGIN ON CANDIDA SPECIES GROWTH3083
studied, 3 isolates (1 isolate each of C. tropicalis, C. parapsilo-
sis, and C. orthopsilosis) displayed PG in planktonic cell tests
but not in biofilm tests and 15 isolates (3 C. albicans, 2 C.
parapsilosis, 5 C. orthopsilosis, and 5 C. metapsilosis isolates)
displayed PG in biofilm tests but not in planktonic cell tests
In addition to differences in the frequency of PG between
planktonic cells and biofilms, biofilms displayed a distinct pat-
tern of PG. Specifically, biofilms displayed PG over a wider
range of CAS concentrations (3 to 4 twofold drug dilutions)
than planktonic cells (1 to 2 twofold drug dilutions) and at
higher drug concentrations (16 to 128 ?g/ml) than planktonic
cells (4 to 32 ?g/ml) (Table 1 and Fig. 1). Finally, the growth
in the PG wells relative to that in the drug-free control well for
a given isolate was higher when the isolate was grown as a
biofilm than when it was grown as planktonic cells, even though
under both growth conditions the magnitude of growth inhibi-
tion at the MIC was similar (Fig. 1). That is, PG in biofilm cells
increased to levels near that of the drug-free growth control,
whereas for planktonic cells, the metabolic activity in the PG
wells increased to 20 to 50% of that in the drug-free control
Microscopy. To investigate whether PG was associated with
changes in cellular morphology, we studied planktonic cells
(Fig. 2) and biofilms (Fig. 3) in the presence and absence of
CAS using light microscopy. CAS exposure was associated with
the inhibition of hyphae and pseudohyphae and the rounding
and clumping of yeast cells in both the planktonic and the
biofilm growth modes (Fig. 2 and 3). The appearance of en-
larged and globose “giant cells” was correlated with PG for
both biofilm and planktonic cells. The viability of the giant cells
was not investigated directly in this study, but these cells ap-
peared to be viable on the basis of microscopic evidence of
budding in some cases. Furthermore, the number of giant cells
in a given well with PG corresponded to the level of metabolic
activity in that well with PG; that is, the well with PG and the
highest metabolic activity corresponded to the well with the
greatest number of giant cells. As the metabolic activity
dropped off in the presence of higher CAS concentrations, the
number of giant cells also declined.
We have described biofilm formation, CAS susceptibility,
and prevalence of PG among isolates from five medically rel-
evant Candida species, including C. albicans, C. tropicalis, C.
parapsilosis, C. orthopsilosis, and C. metapsilosis. Previously,
Song and colleagues reported that C. orthopsilosis and C.
metapsilosis, also known as group II and group III C. parapsi-
losis, respectively, did not form biofilms, suggesting that the
FIG. 1. Metabolic activity of Candida sp. isolates as measured by XTT reduction assay of planktonic and biofilm cells following CAS challenge
for 48 h. (A) C. albicans; (B) C. tropicalis; (C) C. parapsilosis; (D) C. metapsilosis; (E) C. orthopsilosis.
3084MELO ET AL.ANTIMICROB. AGENTS CHEMOTHER.
biofilm-forming phenotype ascribed to C. parapsilosis was lim-
ited to group I isolates (23). One explanation for the discrep-
ancy in results is the difference in the biofilm growth and
quantitation methods between the two studies. In our study, we
used the chemically defined RPMI 1640 medium (pH 7.0)
containing minimal (0.2%) glucose, whereas Song et al. used a
complex medium, Sabouraud dextrose broth, supplemented
with 8% glucose (23). Studies of bacterial biofilms have shown
that nutrient density and medium osmolarity are important
environmental factors which regulate biofilm attachment,
growth, and maturation (24); and for most microbial species,
moderate nutrient limitation favors biofilm growth relative to
the growth achieved under nutrient-rich and nutrient-poor
conditions (24). Another important difference that may have
contributed to our result was our use of the XTT reduction
assay to measure biofilm metabolic activity rather than spec-
trophotometric measurement of the decreased light transmis-
sion caused by biofilm formation on microtiter plate wells.
VOL. 51, 2007EFFECT OF CASPOFUNGIN ON CANDIDA SPECIES GROWTH3085
FIG. 2. Light microscopy of representative isolates of each of the Candida spp. tested which displayed PG in planktonic MIC tests. Cells from
the drug-free control well and cells displaying PG in the presence of high CAS concentrations after 48 h incubation are shown. (A) C. albicans;
(B) C. orthopsilosis; (C) C. tropicalis; (D) C. parapsilosis.
3086MELO ET AL.ANTIMICROB. AGENTS CHEMOTHER.
XTT reduction has been widely used to measure biofilm activ-
ity and allows the detection of small differences in metabolic
activity between strains when the colorimetric reaction is mea-
sured during the linear phase of the reaction (8, 11, 16).
MIC results indicated that CAS has excellent in vitro activity
against all isolates tested in the planktonic growth mode and
against many isolates grown as biofilms. Interestingly, C. parap-
silosis biofilms were more resistant to CAS than C. orthopsilosis
and C. metapsilosis biofilms, even though they had the same
MICs in planktonic cell tests. Since CAS acts by disrupting
?-1,3-glucan synthesis, the intrinsic ability of a given strain to
compensate for the reduction of this polymer may promote
reduced caspofungin susceptibility. ?-1,3-Glucan has also been
shown to be an important constituent of the Candida biofilm
extracellular matrix, where it contributes to substrate and/or
cell-cell adherence and overall biofilm stability (3, 10). The
CAS-induced reduction of ?-1,3-glucan in the biofilm matrix
may increase the fragility of the biofilm and, likewise, the
susceptibility to antifungal killing. Furthermore, Candida
strains that have adapted to life with reduced amounts of
?-1,3-glucan in the cell wall and biofilm matrix may be less
susceptible to the antifungal effects of CAS. Quantitative com-
FIG. 3. Light microscopy of representative biofilms of each of the Candida sp. tested which displayed PG in biofilm MIC tests. Biofilms in the
drug-free control well and biofilms displaying PG in the presence of high CAS concentrations after 48 h incubation are shown. (A) C. albicans; (B) C.
orthopsilosis; (C) C. tropicalis; (D) C. metapsilosis; (E) C. parapsilosis. Arrows point to the “globose” cells frequently observed in wells displaying PG.
VOL. 51, 2007 EFFECT OF CASPOFUNGIN ON CANDIDA SPECIES GROWTH3087
parisons of the ?-1,3-glucan content in the cell wall and the Download full-text
extracellular matrix among various Candida species represent
an interesting area for further exploration.
PG following exposure to supra-MICs of CAS has previously
been described for Candida sp. isolates grown as planktonic
cells but not as biofilms (25, 27). We observed PG in 40% of the
isolates when they were tested with CAS as planktonic cells and
twice that (80%) when the same isolates were tested as biofilms,
suggesting that PG is not an uncommon phenomenon.
In this study, we used simple light microscopy to visualize
the morphological changes in planktonic and biofilm cells as-
sociated with CAS exposure and PG. Cell clumping and the
appearance of enlarged, globose cells were hallmarks of PG.
One explanation is the fungal cell wall changes due to the
reduced ?-1,3- and ?-1,6-glucan contents and increased chitin
content. A study by Stevens et al. (26) reported that cell wall
preparations from a C. albicans isolate capable of PG and
grown in the presence of supra-MICs of CAS had 81% and
73% reductions in ?-1,3- and ?-1,6-glucans contents, respec-
tively, and an 898% increase in chitin content compared to the
contents in cells grown in the absence of CAS. Furthermore,
Nakai et al. reported that the PG of Candida spp. in broth
microdilution tests with micafungin was dependent on the os-
motic conditions of the growth medium; PG was produced only
under hyperosmotic conditions (17). Understanding of the
links between PG and (i) the presence of large, rounded cells
in biofilms, (ii) the shift in the contents of the key components
of the fungal cell wall, and (iii) the dependency on the osmo-
larity of the medium will be key to understanding the basis of
echinocandin-associated PG among Candida sp. isolates and
the clinical significance of PG.
In conclusion, Candida sp. biofilms can display PG in the
presence of high concentrations of CAS and do so more readily
then planktonic cells of the same strains. The cellular morpho-
logical changes associated with PG can be observed microscop-
ically and are likely due to alterations in the fungal cell wall.
The clinical significance of PG remains unclear, yet the pro-
posed use of high concentrations of echinocandin antifungals
as catheter lock therapy for the treatment and prevention of
catheter-associated candidemia may be thwarted by the stim-
ulation of Candida biofilm growth at CAS concentrations
above the MIC. On the basis of these findings, further studies
to determine the occurrence of PG in vivo with animal models
of catheter-associated infection and antifungal lock therapy
The findings and conclusions in this report are those of the authors
and do not necessarily represent the views of the Centers for Disease
Control and Prevention/Agency for Toxic Substances and Disease
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