MAJOR ARTICLE

Parenteral Lipid Emulsion Induces Germination

of Candida albicans and Increases Biofil
Formation on Medical Catheter Surfaces
Kim Swindell,1 Ali Abdul Lattif,2 Jyotsna Chandra,2 Pranab K. Mukherjee,2 and Mahmoud A. Ghannoum2
1
Pediatric Infectious Diseases, Childrens Hospital, Cleveland Clinic, and 2Center for Medical Mycology, University Hospitals Case Medical Center,
and Department of Dermatology, Case Western Reserve University, Cleveland, Ohio

Candida albicans is known to form biofil on prosthetic material and has been observed on the surfaces
of indwelling medical catheters removed from patients
[1, 2]. Candida species have emerged as the fourth most
common cause of hospital-acquired bloodstream infections [3], and C. albicans is the most commonly
isolated pathogen from patients with fungemia [4].

Received 12 December 2008; accepted 3 March 2009; electronically published

23 June 2009.
Potential conflicts of interest: none reported.
Presented in part: 48th Annual Interscience Conference on Antimicrobial Agents
and Chemotherapy/46th Annual Infectious Diseases Society of America Meeting,
Washington, DC, 2528 October 2008 (abstract M-1567).
Financial support: National Institutes of Health (grants R01 DE017486-01A1 and
R01DE 13932-4); Bristol-Myers Squibb (Freedom to Discover Award to M.A.G.).
The assistance of the Confocal Scanning Laser Microscopy Core Facility (National
Cancer Institute grant P30CA43703-12) at Case Western Reserve University is
acknowledged.
Reprints or correspondence: Dr Mahmoud A. Ghannoum, Center for Medical
Mycology, Dept of Dermatology, University Hospitals of Cleveland and Case
Western Reserve University, 11100 Euclid Ave, Cleveland, OH 44106-5028
(Mahmoud.ghannoum@case.edu).
The Journal of Infectious Diseases 2009; 200:47380
 2009 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2009/20003-0020$15.00
DOI: 10.1086/600106

The biofil of C. albicans develops in 3 phases [5,

6]. In the early phase, yeast cells adhere to surfaces,
divide, and form a layer of microcolonies. In the intermediate phase, cells produce extracellular material
and differentiate into pseudohyphae and hyphae. In the
maturation phase, extracellular material increases and
the network of hyphal structures grow in parallel, adding structure to the biofilm Biofil development is
influence by several factors, including strain [79] and
growth medium [5, 10, 11].
The propensity of C. albicans to form biofil on the
surfaces of indwelling medical catheters and prosthetic
material may lead to persistent infection. Moreover,
formation of biofil by Candida bloodstream isolates
has been associated with increased virulence and mortality [9, 1214]. In the biofil state, C. albicans resists
antifungal drugs and host defenses [15, 16], making
antifungal therapy alone insufficien for cure [2, 6, 17].
Removal of the catheter is often necessary [18].
The administration of parenteral nutrition provides
hydration and nutrients to critically ill patients and to
those who cannot tolerate enteral feeding. Parenteral
Lipid Emulsion C. albicans Biof lm

JID 2009:200 (1 August) 473

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Background. The administration of parenteral nutrition, including lipid emulsion (LE), to patients via medical
catheters is an unexplained risk factor for the development of candidemia. Germination and biof lm formation
are recognized virulence determinants of Candida albicans. No studies have addressed the effect of LE on candidal
biofil production. In this study, we investigated the effect of LE on candidal germination and its ability to form
biofi m on medical catheter material.
Methods. C. albicans strain SC-5314 was grown in standard growth medium in the presence or absence a
commercially available LE. Biofilm grown on silicone-elastomer catheter discs in these media were compared for
mass by dry weight measurements. Biofil morphology was analyzed by scanning electron microscopy and confocal
laser microscopy. The effect of LE on C. albicans germination and growth was evaluated microscopically and by
determination of colony-forming units, respectively.
Results. Addition of LE to standard growth medium increased C. albicans biofil production and resulted in
observed changes in biofil morphology and architecture. Furthermore, LE induced germination and supported
the growth of C. albicans.
Conclusions. LE-inducible candidal virulence determinants, such as germination and enhanced biofi m production, may help to explain the increased risk of candidemia in patients receiving LE via medical catheters.

nutrition is infused via an indwelling medical catheter and

typically consists of both an aqueous solution (water, dextrose,
essential amino acids, electrolytes) and lipid emulsion (LE)
[19].
The administration of parenteral nutrition via indwelling
medical catheters is a well-known yet unexplained risk factor
for the development of candidemia [9, 14, 2025]. The Centers
for Disease Control and Prevention Healthcare Infection Control Practices Advisory Committee (HICPAC) guidelines state
that certain Candida species may produce slime (now commonly referred to as biofilm within catheters used to administer glucose-containing fluid and infer that slime production
may explain the increased risk of catheter-related fungemia in
patients receiving parenteral nutrition [26]. Furthermore, because of the association between fungemia and the administration of parenteral LE, HICPAC guidelines recommend completing infusion of LEs within 12 h and replacing tubing used
to administer LEs within 24 has opposed to 72 h for solutions
containing only dextrose and amino acids [26]. These recommendations are based on prior observations that Candida
species are able to grow in LEs [2736]. However, the effect of
LE on the ability of Candida to form biofilm on medical
catheters is unknown.
In an effort to gain insight into the mechanisms by which
LE may predispose a patient to candidal bloodstream infection,
in this study we investigate the effect of parenteral LE on the
growth and biofilm-fo ming ability of C. albicans by means of
an established in vitro silicone-elastomer catheter model [5,
37]. Specificall , we assessed the effect of LE on candidal growth,
474 JID 2009:200 (1 August) Swindell et al

germination, and ability to form biofilm Additionally, the effect

of LE on biofil morphology and architecture was investigated.
METHODS
Strain and growth conditions. The clinical C. albicans wildtype strain SC-5314 [38] was used in all experiments and was
maintained on Sabourad dextrose agar (SDA) (yeast extract,
peptone, and dextrose at 1:2:1]) (Difco Laboratories) or kept
at 80C for long-term storage. This C. albicans strain was
selected because it can form robust biofil on catheters and
is well characterized genetically. For all experiments, SC-5314
was grown in yeast nitrogen base (YNB) medium (Difco) supplemented with 50 mmol/L dextrose, henceforth referred to as
standard growth medium. Fifty milliliters of medium (in 250mL Erlenmeyer flasks was inoculated with SC-5314 from fresh
SDA and incubated for 24 h at 37C in an orbital water bath
shaker at 60 rpm. Cells were harvested and washed twice with
0.15 mol/L phosphate-buffered saline (PBS) (pH 7.2, Ca2+ and
Mg2+ free). Cells were resuspended in 10 mL of PBS, counted
using a hemacytometer after serial dilution, standardized, and
used immediately.
Effect of LE on germination. To determine the ability of
LE to induce germ-tube formation, C. albicans were grown
planktonically in LE-containing media. Germination rate was
compared with that of cells grown in medium containing fetal
bovine serum (FBS) (Hyclone), a known inducer of germination. Briefl , C. albicans were grown, prepared, and counted
as described above. Cells were then diluted to 1 107 cells/mL

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Figure 1. Effect of different media on the germination and growth of Candida albicans. A, C. albicans germinated in media containing 1%, 5%, or
10% lipid emulsion (LE) in Hanks balanced salt solution (HBSS). For optimal germination, cells grown in 10% fetal bovine serum (FBS) in HBSS required
120 min, whereas cells grown in 5% or 10% LE required an incubation time of 240 min. Shown are data from 3 separate experiments. Media types
were as follows: HBSS alone, 1% (vol/vol) LE in HBSS (1LEHBSS), 5% (vol/vol) LE in HBSS (5LEHBSS), 10% (vol/vol) LE in HBSS (10LEHBSS), and
10% (vol/vol) FBS in HBSS (10FBSHBSS). B, Effect on growth. C. albicans grew similarly in LE media with or without supplemental dextrose over 36
h. Shown are data from 1 representative experiment performed with 6 replicates taken from 1 culture per media type for each time point. Media
types were as follows: yeast nitrogen base (YNB) (6.7g/L in sterile water), YNB plus 50 mmol/L dextrose (YNBD), 5% (vol/vol) LE in YNBD (5LEYNBD),
10% (vol/vol) LE in YNBD (10LEYNBD), and 10% LE in YNB (10LEYNB). All values are means  standard deviations.

Table 1. Comparisons of Dry Weights of Early-Phase (6-h) and Mature-Phase (48-h)

Biofilms Formed by Candida albicans Grown in Standard Growth Medium and in Lipid
Emulsion (LE) Media
Dry weight, mg
Growth phase, media types
6h
YNB vs YNBD

Mean differencea

SE

95% CI

1.5111

0.0968

1.7906 to 1.2317

!.001

5LEYNBD vs YNBD
10LEYNBD vs YNBD
10LEYNB vs YNBD
48 h

0.5389
1.6953
0.7488

0.0968
0.0968
0.0968

0.2594 to 0.8183
1.4158 to 1.9748
1.0283 to 0.4693

!.001

YNB vs YNBD
5LEYNBD vs YNBD

1.8424
1.0214

0.2231
0.2266

2.4870 to 1.1977
0.3668 to 1.6761

!.001

10LEYNBD vs YNBD
10LEYNB vs YNBD

3.0152
1.0288

0.2266
0.2231

2.3605 to 3.6698
1.6735 to 0.3842

!.001

!.001
!.001

!.001
!.001

Differences were calculated as the value for standard growth medium (YNBD) minus that for the
indicated media type. All mean differences were significant at the P ! .05 level.

in Hanks balanced salt solution (HBSS) (Mediatech). In separate 1.5-mL tubes, 50 mL of these cells were diluted to 1 mL
in HBSS, in 10% (vol/vol) FBS in HBSS, or in 1%, 5%, or 10%
(vol/vol) LE (Intralipid 20%; Fresenius Kabi) in HBSS for a
concentration of 5 10 5 cells/mL and incubated on a rocker
at 37C for up to 4 h. At 15-min intervals, 10-mL samples from
each media type were microscopically evaluated using a hemacytometer. Total cell count and germination (define as a
germ-tube length greater than or equal to the blastospore diameter) was determined from an average of 4 observations.
One hundred to 200 cells were counted per observation. The
assays were discontinued when cell clumping due to germination disallowed counting of individual cells. Percent germinated cells (germinated cells per the total number of cells)
was calculated at each time point.
Planktonic growth in LE-containing media. To determine
the ability of LE to support growth of C. albicans, cells were
grown planktonically in LE-containing media. C. albicans was
grown, prepared, and counted as described above. Because the
range of LE concentrations within a catheter lumen includes
5% and 10% during administration of parenteral nutrition [19],
growth in medium containing either YNB (6.7 g/L in sterile
water), YNB supplemented with 50 mmol/L dextrose (YNBD),
5% (vol/vol) LE in YNBD (5LEYNBD), 10% (vol/vol) LE in
YNBD (10LEYNBD), or 10% (vol/vol) LE in YNB (10LEYNB)
was studied. C. albicans was grown in media containing LE
with or without the addition of dextrose to determine whether
LE independently supports the growth of C. albicans and to
determine any synergistic effect of addition of LE to dextrosecontaining media with respect to candidal growth. In separate
250-mL flasks cells were inoculated into 50 mL of each media

type for a fina concentration of 1 10 4 cells/mL. Cell suspensions were incubated in an orbital shaker at 60 rpm. At intervals
of 26 h, 20-mL cell suspension from each media type was
removed and serially diluted in PBS. A cell suspension (5 mL)
from each media type was inoculated onto separate SDA plates
in 6 replicates. Culture plates were grown at 37C for 24 h.
Colonies were counted, and the average number of colonyforming units was determined for each time point.
Biofil formation on silicone-elastomer discs. A model of
C. albicans biofil formation on silicone-elastomer discs has
been described elsewhere [5, 37]. Briefl , 1.5-cm-diameter silicone-elastomer disks (Cardiovascular Instrument) were placed
in 12-well tissue culture plates and incubated in FBS for 24 h
at 37C on a rocker. Disks were then washed with PBS to remove
residual FBS. To ensure uniform biofil formation across the
strongly hydrophobic disk surface, disks were immersed in 3
mL of standardized cell suspension (1 107 cells/mL) and incubated for 90 min at 37C. Next, discs were rinsed gently with
PBS to remove nonadhered cells, transferred to new 12-well
plates containing 4 mL of medium (YNB, YNBD, 5LEYNBD,
10LEYNBD, or 10LEYNB), and incubated at 37C on a rocker
for either 6 h (early-phase biofilm or 48 h (mature-phase
biofi m).
Quantitation of biofilms Quantitation of Candida biofilm was performed as described elsewhere [39] by using dry
weight measurements. Dry weight measurements represent total biofil mass, including fungal cells and extracellular material (for details on this technique, see Chandra et al [39]).
Briefl , biofilm were scraped off the surface of the disks by
means of a cell scraper (Becton Dickinson). Then, disks and
scrapers were rinsed with PBS to remove residual biof lms.
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NOTE. Media types were as follows: yeast nitrogen base (YNB) (6.7g/L in sterile water), YNB plus 50
mmol/L dextrose (YNBD), 5% (vol/vol) LE in YNBD (5LEYNBD), 10% (vol/vol) LE in YNBD (10LEYNBD),
and 10% LE in YNB (10LEYNB). CI, confidence interval; SE, standard error.

Next, the scraped material was filte ed by using a preweighed

0.45-mm-pore nitrocellulose membrane (Millipore), dried in an
incubator at 37C for 48 h, and weighed.
For dry weight measurements, control discs were prepared
for each media type and handled identically, but no Candida
cells were added.
Scanning electron microscopy. To determine the effect of
exposure to LE, the surface topography of C. albicans biofi m
was investigated using scanning electron microscopy (SEM), as
described elsewhere [5]. Briefl , biofilm grown in different
media were prepared as stated above. After growth for 6 or 48
h, biofilm were prepared for SEM. Silicone-elastomer disks
with early-phase or mature-phase biofilm were fixe with 2%
glutaraldehyde, followed by fixin with osmium tetraoxide, tannic acid, and uranyl acetate. This was followed by a series of
ethanol dehydration steps, and the prepared samples were sputter coated with Au-Pd (60:40 ratio) and viewed with a model
XL3C ESEM Philips scanning electron microscope.
Confocal scanning laser microscopy. To determine the effect of exposure to LE, architectural phenotype and morphology
of mature-phase biofilm were investigated by confocal scanning laser microscopy (CSLM). Biofil staining and CSLM
examination were performed as previously elsewhere [5, 37].
Briefl , after 48 h of growth silicone-elastomer disks on which
biofilm were developing were transferred to a 12-well plate
476 JID 2009:200 (1 August) Swindell et al

and incubated for 45 min at 37C in 2 mL of PBS containing

the fluo escent stains FUN-1 (10 mmol/L) and concanavalin A
Alexa Fluor 488 conjugate (ConA) (25 mg/mL). FUN-1 (excitation wavelength, 543 nm; emission, 560 nm [long-pass fi ter]) is converted to orange-red cylindrical intravacuolar structures by metabolically active cells, whereas ConA (excitation
wavelength, 488 nm; emission, 505 nm [long-pass fi ter]) binds
to glucose and mannose residues of cell wall polysaccharides
with green fluo escence. Control biofilm were concomitantly
grown in standard growth medium and were used to set the
laser values for red and green channels of CSLM. Settings done
for red and green channels using control biofil discs were
used for all other experimental discs.
After incubation with the dyes, silicone-elastomer disks were
flippe and placed on a 35-mm-diameter glass-bottom petri
dish (MatTek). Stained biofilm were observed with a Zeiss
LSM510 confocal scanning laser microscope equipped with argon and HeNe lasers and mounted on a Zeiss Axiovert100 M
microscope. The objective used was a water-immersion Capochromat lens (20). To determine the structure of the biofilms a series of horizontal (xy) optical sections were taken
throughout the full length of the biofilm Confocal images of
green (ConA) and red (FUN-1) fluo escence were conceived
simultaneously using a multitrack mode. Images were captured
and processed using Zeiss LSM Image Examiner (version

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Figure 2. Effect of different growth media on the surface morphology and topography of early-phase and mature-phase Candida albicans biofilms.
Analyses of scanning electron microscopy (SEM) images of early-phase (6-h) biofilms revealed differences in surface morphology and topography
between biofilm grown in standard growth medium (A), which resulted primarily in yeast cells, and biofilm grown in standard growth medium
supplemented with 10% (vol/vol) lipid emulsion (LE) (B), which resulted in densely arranged hyphal elements. Panel C shows an SEM image of maturephase (48-h) biofilm grown in standard growth medium, and panel D depicts the effect of standard growth medium supplemented with 10% (vol/vol)
LE, demonstrating differences in surface morphology. An SEM image of early-phase (6-h) biofilm grown in media containing yeast nitrogen base lacking
dextrose supplemented with 10% (vol/vol) LE (E) revealed clusters of yeast. Original magnification, 1500.

4.2.0.121) and Photoshop CS2 (version 9.0; Adobe Systems)

software.
Statistical analyses. Germination data (figu e 1A) were
combined from 3 separate experiments conducted on different
days. Planktonic growth data (figu e 1B) were obtained from
1 representative experiment consisting of 6 replicates taken
from 1 culture per media type for each time point. To compare
fungal growth in different media, colony-forming unit data
were log transformed (base 2) and analyzed by a 2-tailed linear
regression model (P p .05) that included terms for media,
time, and media-time interaction by means of SAS software
(version 9.1; SAS Institute). Dry weight measurements were
performed in 6 replicates on different days. All other statistical
analyses, including analysis of variance and post-hoc analysis
with the Bonferroni-Dunn calculation, were performed using

SPSS for Windows (version 16.0; SPSS) software. P ! .05 was

considered significant Images shown in CSLM and SEM panels
are presented without processing or retouching.
RESULTS
Induction of germ-tube formation in planktonically grown C.
albicans by LE. Because the ability of C. albicans to germinate
is recognized as a virulence determinant, we investigated the
ability of LE to induce hyphal formation in planktonically
grown C. albicans. C. albicans grown in medium containing
10% (vol/vol) FBS in HBSS achieved 94%  0.9% (mean 
standard deviation) germination at 2 h, whereas C. albicans
grown in 5% or 10% (vol/vol) LE in HBSS achieved 89% 
1.5% and 90%  1.0% germination, respectively, by 4 h (fi ure
Lipid Emulsion C. albicans Biof lm JID 2009:200 (1 August) 477

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Figure 3. Top-down (xy) reconstructed images of lipid emulsion (LE)exposed mature-phase (48-h) Candida albicans biofilms examined by confocal
scanning laser microscopy with the fluorescent stains FUN-1 and concanavalin AAlexa Fluor 488 conjugate (ConA). Extracellular material was stained
with ConA, resulting in the green color. FUN-1 is converted by metabolically active cells, resulting in the red-orange color. Biofilm grown in standard
growth medium (A) revealed both yeast and hyphal elements embedded in extracellular material. Biofilms grown in standard growth medium supplemented
with 5% (vol/vol) LE (B) and 10% (vol/vol) LE (D) revealed increasingly more compact arrangements of hyphal elements. Biofilm grown in yeast nitrogen
base lacking dextrose supplemented with 10% (vol/vol) LE revealed comparatively few hyphal elements and little extracellular material. Original
magnification, 20.

478 JID 2009:200 (1 August) Swindell et al

media lacking dextrose showed only scattered islands of yeast,

suggesting that biofi m failed to form (fi ure 2E).
Induction of morphological changes in C. albicans biofil s
exposed to LE. We used CSLM to determine the architecture
and morphological composition and organization of live, hydrated biofilm grown in the presence of LE. Confocal images
of mature-phase biofilm grown in standard growth medium
were typical of those obtained previously in our model [5, 37]
and showed both yeast and hyphal elements encased in extracellular material (figu e 3A). In contrast, biofilm grown with
the addition of 5% (figu e 3B) or 10% (figu e 3C) LE displayed
an increasing number of densely arranged hyphal elements.
CSLM images of C. albicans grown in LE media lacking dextrose
showed primarily yeast, few hyphal elements, and little extracellular material, suggesting that biofil failed to develop (fig
ure 3D).
DISCUSSION
The present study identifie virulence determinants of C. albicans, specificall germination and increased biofi m formation, which are induced by exposure to LE. Furthermore, we
observed microscopically that biofilm grown in standard
growth medium supplemented with LE exhibited an increase
in hyphal forms at both early and mature phases compared
with biofilm grown in standard growth medium or biofi ms
grown in LE-supplemented medium lacking dextrose. These
finding may help to explain the increased risk of fungemia
associated with the administration of parenteral nutrition (including LE) to patients via medical catheters.
The ability of C. albicans and other pathogens to grow in
parenteral nutrition formulations is known and is considered
to be a risk factor for the development of catheter-related
bloodstream infections in patients receiving parenteral nutrition [26]. With regard to the growth of C. albicans in parenteral
LE, our finding agree with those of several studies [2736, 40],
including those on which clinical guidelines for administration
of LE to patients via medical catheters are based [2731, 33
35]. However, our finding expand the understanding of the
behavior of C. albicans in LE by demonstrating that exposure
to LE also induces germination.
The development of hyphal structures is necessary for biofi m
development [6, 41]. Additionally, hyphal structures contribute
to virulence by facilitating adherence and tissue invasion [42
44]. Fatty acids contained in LE may play a role in C. albicans
germination. For example, Hoberg et al [45] found that exposure to cerulenin, an antibiotic that inhibits fatty acid biosynthesis in yeast, inhibited germination in C. albicans through
the inhibition of lipid biosynthesis. The addition of palmitic
acid restored germination in cerulenin-inhibited cells. Noverr
and Huffnagle [46] tested the ability of medium-chain fatty
acids, as well as a subset of long-chain fatty acids contained in

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1A). C. albicans grown in 1% (vol/vol) LE in HBSS achieved

44%  0.0% germination by 4 h. C. albicans grown in control
medium containing HBSS alone did not germinate by 4 h. Thus,
LE induced germination in C. albicans at all concentrations
studied.
Growth of planktonically grown C. albicans supported by
LE. To determine the ability of parenteral LE to support C.
albicans growth, we compared planktonic growth of C. albicans
in standard growth medium supplemented with clinically relevant concentrations of LE [19]. C. albicans grew, with or without supplemental dextrose, in each LE-containing media over
the 36-h period of study (figu e 1B). Subsequent analysis
showed no significan differences in growth rate among cells
grown in standard growth medium with or without supplemental LE and in media containing LE without supplemental
dextrose (P 1 .05 for all comparisons) (figu e 1B). As expected,
the growth rate in media containing only YNB was signif cantly
less (P ! .001) compared with all other media tested.
Increase in biofil dry weight due to exposure to LE. The
clinical virulence of some Candida species is associated with
both the ability to form biofil and the amount of biofi m
produced in vitro [9, 1214]. To determine the effect of LE on
biofil production on silicone-elastomer discs, we compared
the dry weight and metabolic activity of C. albicans biofi m
grown in standard growth medium to that of biofi m grown
in LE-containing media. In our model, supplementation of
standard growth medium with LE resulted in significantl increased early-phase and mature-phase biofil mass (table 1).
Of note, the biofil mass of C. albicans grown in LE medium
lacking dextrose was significantl less than that of C. albicans
grown in standard growth medium (table 1). Furthermore,
biofil dry weight did not differ significantl between early
and mature phase for cells grown in the absence of dextrose
(for YNB, P p .299; for 10LEYNB, P p .464).
Induction of early hyphal formation and changes to surface
topology in C. albicans biofilm exposed to LE. Because we
found that exposure to LE increases the mass of C. albicans
biofilm we hypothesized that exposure to LE may also affect
the morphological or architectural phenotype of the biofi m.
To test our hypothesis, we used SEM to visualize the surface
topology of C. albicans biofilm formed in the presence of LE.
SEM images of early-phase biofilm showed a predominance
of yeast in biofil grown in standard growth medium (fi ure
2A), whereas biofilm grown in standard growth medium supplemented with 10% LE showed extensive hyphal elements (fi ure 2B). SEM images of mature-phase biofilm showed an increased density of hyphal elements in biofilm grown in
standard growth medium supplemented with 10% LE (fi ure
2D), compared with those grown in standard growth medium
(figu e 2C). Of note, SEM images of C. albicans grown in LE

clinical studies of biofil development and the risk of candidemia wherein LE is administered separately from dextrosecontaining f uids.

Acknowledgments
We thank S. Worley and S. McIntyre for help with statistical analysis of
growth rates. We thank J. Goldfarb and C. Foster for input and guidance
and for review of the manuscript.

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Lipid Emulsion C. albicans Biof lm JID 2009:200 (1 August) 479

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parenteral LE (oleic acid and linolenic acid), to induce germination in C. albicans. When these long-chain fatty acids were
added individually to media containing FBS, they found no
increase in germination capacity over 2 h of observation compared with cells grown in FBS. In our study, C. albicans grown
in LE-containing media required 4 h to germinate to a similar
extent as cells grown in FBS; germination at 2 h was !10%.
Thus, if these fatty acids induce germination in C. albicans, the
apparent difference between these 2 studies may be attributed
to different incubation periods used.
In our study, C. albicans grown on medical catheter material
in standard growth medium supplemented with clinically relevant concentrations of LE produced significantl more biofi m
by dry weight analysis than did those grown on catheter material in standard growth medium without LE. This fi ding
may be important, given that biofil formed on the surface
of indwelling medical catheters serves as the source of fungal
cells that can seed the bloodstream, thereby promoting systemic
candidiasis [47]. Furthermore, production of biofil by C. albicans bloodstream isolates is associated with an increased risk
of mortality [14], and, in an in vitro medical catheter material
model, strain-specifi biofil production of other Candida species was associated with virulence [12]. The increased propensity to form biofil resulting from exposure to LE may be a
virulence determinant with respect to catheter-related bloodstream infections caused by C. albicans in patients receiving LE
via medical catheters.
The effect of LE on the architecture and morphology of C.
albicans biofil is reported for the firs time in this study. As
evidenced by SEM, early-phase biofilm grown in standard
growth medium supplemented with LE showed an increased
number of hyphal elements compared with early-phase biofilm
grown in standard growth medium alone. Likewise, CSLM revealed markedly more hyphal elements within the hydrated
biofil of C. albicans grown in standard growth medium supplemented with LE. As observed in prior in vitro models of
biof lm formation [5, 6], an increase in extracellular material
and the development of hyphal elements are characteristic of
biofil maturation. In our study, the observation of hyphal
elements in the early-phase biofil of LE-exposed C. albicans
may indicate an accelerated transition in biofi m morphology
resulting from exposure to LE.
Further study is needed to determine whether the lipid or
phospholipids contained in LE are involved in the mechanism
of these proposed LE-inducible virulence determinants. Dextrose may serve as the carbohydrate energy source required by
C. albicans for biofil formation. The findin that biof lm formation increased with the addition of LE to dextrose-containing growth medium but failed to form in LE medium devoid
of dextrose warrants additional study of carbon-source utilization as it relates to biofil development and may lead to

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