Biofilms (2005) 2, 63–71 C© 2005 Cambridge University Press
doi:10.1017/S1479050505001687 Printed in the United Kingdom
Adhesion and biofilm formation of
Candida albicans on native and
Pluronic-treated polystyrene
K. E. Wesenberg-Ward1∗, B. J. Tyler2 and J. T. Sears1
ABSTRACT
Candida albicans forms part of the normal human flora whose growth
is usually restricted by the normal flora bacteria and the host’s immune
system. It is an opportunistic fungal pathogen that causes infections in
immunocompromised individuals, mechanical trauma victims and iatrogenic
patients. Candida albicans can ingress the human host by adhering to a
plastic surface (i.e. prosthetic devices, catheters, artificial organs, etc.) that
is subsequently implanted, and forms a protective biofilm that provides a
continuous reservoir of yeast to be hematogenously dispersed. In order for
the medical profession to battle device-related infections, initial adhesion and
biofilm formation of C. albicans needs to be better understood. There has been
some skepticism as to whether the initial adhesion events bear any relationship
to subsequent biofilm formation. Thus, to better comprehend the relationship
between the initial adhesion rates and growth rate and biofilm formation, these
events were studied on two different, well-defined culture surfaces, native
polystyrene and Pluronic F127-conditioned polystyrene. The adhesion studies
determined that Pluronic F127 adsorption dramatically reduced the adhesion
of C. albicans to polystyrene. The biofilm growth studies, analyzed by confocal
scanning laser microscopy, revealed that Pluronic F127 decreased the biofilm
surface coverage, cluster group size, thickness and the presence of hyphal
elements over the untreated polystyrene. These findings indicate that the effect
of a material’s surface chemistry on the initial adhesion process has a direct
influence on subsequent biofilm formation.
* Corresponding author:
Dr K. E. Wesenberg-Ward
Department of Chemical Engineering
Montana State University–Bozeman
Bozeman, MT 59717
USA
T 1 406 570 7201
F 1 406 994 5308
E Karemw@aol.com
1 Department of Chemical Engineering,
Montana State University–Bozeman,
Bozeman, MT 59717, USA
2 Department of Chemical and Fuels
Engineering, University of Utah,
Salt Lake City, UT 84112, USA
INTRODUCTION
Approximately one in five U.S. citizens has a long-term
implanted medical device. One of the most common
complications associated with implanted medical devices
is infectious biofilms. Most studies of infectious biofilms
have been concerned with bacterial biofilms. Device-
centered infections are quite resistant to the body’s
immune system, antibiotics and antifungal agents
(Costerton et al., 1999). Since one-quarter of the implant
infections are caused by the yeast Candida albicans, the
necessity for gaining insight into the initial adhesion
process and ensuing biofilm development becomes
apparent. Candidiasis, the infection caused by species of
Candida, with C. albicans being the primary etiologic
agent, represents an opportunistic disease. Candida
albicans exists as a normal commensal of the human
gastrointestinal and genitourinary tracts and mucosa.
The infection stems from an overgrowth of this normal
human flora. Groups susceptible to the disease include
immunocompromised individuals, mechanical trauma
victims and those undergoing iatrogenic procedures
(Odds, 1979; Kuby, 1994).
Several possible factors are thought to contribute
to the virulence of C. albicans. Those relevant to this
study include hyphal formation, adherence properties
and variable characteristics such as the dynamic cell
surface. Hyphal formation is one of three different
morphogenetic processes that C. albicans can undergo.
The other processes include blastospore formation
and pseudohyphal formation. The morphological state
depends on the pH, incubation temperature, inoculum
size, and the composition of the growth medium (Odds,
1979; Kuby, 1994).
Candida albicans can invade the human host by
adhering to a plastic surface (i.e. prosthesis, catheter,
prosthetic valve, etc.) with subsequent formation of a
protective biofilm and is then dispersed by means of the
vascular system. By themselves, antifungal agents are not
capable of preventing and controlling yeast bloodstream
infections (Hawser & Douglas, 1994; Zhang et al., 1997).
Combating candidal infections requires a strategy that
combines the use of antifungal agents along with blocking
of adhesion and biofilm growth. A well-defined culture
surface provides a way to study adhesion and biofilm
structure. Control over the chemical features of the surface
Adhesion and biofilm formation of C. albicans 63
64 K. E. Wesenberg-Ward et al.
allows the influence of chemistry on cell behavior to be
studied.
Other investigators have shown that Pluronic condi-
tioning on a surface inhibits the adhesion of bacteria
(Bridgett et al., 1992; Portoles et al., 1994) and proteins
(Harper et al., 1991; Alexandridis & Hatton, 1995;
Freij-Larsson et al., 2000) to polymer surfaces. Pluronic
F127 is an amphiphilic A-B-A triblock copolymer. A is
poly(ethylene oxide) (PEO), the hydrophilic segment,
and B is poly(propylene oxide) (PPO), the hydrophobic
segment. Pluronic F127 consists of 98 PEO segments and
67 PPO segments. Pluronic is thought to adhere to the
plastic surface via the hydrophobic portion in such a
manner that the hydrophilic portion extends into the
bulk fluid, imparting a hydrophilic character to the plastic
surface. Pluronic F127 is a trade name given to industrial
and pharmaceutical grades of Poloxamer 407. This study
shows that Pluronic F127 adsorption dramatically reduces
the adhesion of C. albicans to polystyrene (PS). The
relationship between initial adhesion rates and the long-
term growth rates and biofilm formation of C. albicans is
still highly controversial. Therefore, the objectives of this
work were to compare initial adhesion and subsequent
biofilm formation on native polystyrene and Pluronic-
conditioned PS by C. albicans. The biofilm development
was investigated using confocal scanning lasermicroscopy
(CSLM).
MATERIALS AND METHODS
Culture surface preparation
The well-defined culture surfaces included native PS and
Pluronic F127-conditioned PS (PLPS). The PS surface
employed was either a cleaned PS coupon or a PS surface
formed on a silanized glass coverslip by spin coating. The
PS couponswere 0.5 in. (1.27 cm) diameter disks punched
from a 1/16 in. (0.16 cm) thick sheet. The coupons were
cleaned prior to experimentation or manipulation. The
PS coupons were cleaned by being placed in high-
performance liquid chromatography grade hexane and
swirled for approximately 5 s. The coupons were then
allowed to dry. After drying, the PS coupons were set
in a beaker containing methanol and sonicated at 0 ◦C for
approximately 5min. The PS couponswere removed from
the methanol and placed in a clean Pyrex glass container.
Glass coverslips (43mm× 61mm) were prepared for
spin coating by soaking in 5% (v/v) dimethyldichlorosi-
lane in toluene for aminimum of 3 h and then rinsed with
methanol to remove the excess silanizing agent, rinsed
with nanopure water, and allowed to dry at ambient
conditions. The silanized coverslips were then spin coated
with 5wt% polystyrene (average Mr 230 000) in toluene.
The PS spin-coated coverslips were prepared 24 h prior to
experimentation or manipulation.
The PS coupons were utilized in the adhesion
experiments, while the spin-coated PS coverslips were
utilized in the biofilm growth experiments. The spin-
coated PS surface was needed to accommodate a flow cell
design for the biofilm growth experiments thatminimized
plugging and expedited analysis with CSLM.
The Pluronic F127-treated PS surfaces were generated
by soaking cleanPS coupons or coverslips in a 4%Pluronic
F127 solution in phosphate-buffered saline (PBS) for a
minimum of 3 h. The PBS solution was 0.01 M and had
a pH between 7.15 and 7.25. The conditioned PS samples
were either used directly or stored for no longer than
1week.
Surface composition
X-ray photoelectron spectroscopy (XPS) was used to
confirm the surface chemical composition. XPS analysis
wasperformedwith aPHI5600XPS system.Theoperating
pressure in the analysis chamber was in the 10−9 torr
range. A monochromatized X-ray source (Al Kα anode,
1486.6 eV)was operated at 15 kV and 300W. The aperture
was set to 4, corresponding to analysis of a spot 800 µm in
diameter. Charge neutralizationwas achieved using a low-
energy electron gun. All binding energies were referenced
to the hydrocarbon component (C–C/C–H) of the C1s
peak, which was set to 285.0 eV. A pass energy of 58.7 eV
was used.
Cells and culture media
Candida albicans CA1 isolates were maintained in
a subzero temperature freezer. Every month a new
subculture was generated using the streak plate method
of isolation. Following a 48 h incubation period at 35 ◦C,
a single colony was removed from the isolation plate and
transferred to a slant tube containing Sabouraud dextrose
agar, generated following the manufacture’s instructions.
The inoculated slant tubes were incubated for 48 h at
35 ◦C and then kept at approximately 4 ◦C. This culture
of C. albicans was used to perform adhesion and biofilm
growth experiments. Each inoculum of C. albicans for
experimentation was grown in (normal) GYEP (5%
glucose, 0.3% yeast extract, 1% bacto-peptone) broth at
35 ◦Cand160 revolutions/min for 24 h.Thefirst inoculum
was removed from a slant tube and placed in 100ml of
sterile GYEP broth. The second inoculum was obtained
from the initial broth culture of which 0.25ml was placed
in 100ml of sterile GYEP broth. The GYEP broth was
sterilized by autoclaving for 15 to 20min.
The C. albicans cells used in the growth and adhesion
experiments were obtained from the second inoculum
following a24 h incubationperiod (stationaryphase).Two
4ml portions of cells were removed from the broth culture
and spun for 90 s at ambient conditions. The cells were
rinsed three times with 2ml of sterile, refrigerated 0.01M
PBS. After the final rinse, 2ml of sterile PBS were added to
each portion of cells. The cells were combined to generate
a little more than 4ml of cells in PBS, which were pelleted
and kept on ice prior to use. The concentration of cells
in this solution was determined using a hemacytometer.
This solution of cells was then used to generate a new
solution of cells, with a concentration of approximately
107 cells/ml, to be utilized in the flow cell experiments.
Adhesion and biofilm formation of C. albicans 65
Adhesion and biofilm growth experiments
Theadhesionexperimentswereperformed inaTeflonflow
cell device containing the material of interest. A Pluronic
F127-treated PS coupon or native PS coupon was set in
the well of the Teflon flow cell. Initially, PBS was pumped
through the flow cell using a peristaltic pump to establish
the desired flow rate, focus the Olympus microscope, and
remove air from the flow cell assembly. The yeast cells, at
a concentration of approximately 1× 107 cells/ml, were
then introduced into the flow cell. A shear rate of 16 s−1
was selected, based on arterial wall shear rates that ranged
from10 to1000 s−1.Aflowrateof 1.5ml/minwasnecessary
to achieve the specified wall shear rate as determined by
the design of the flow cell. The adhesion process was
conducted at room temperature and observed for 1 h.
The progress of the experiment was monitored using an
Olympus microscope (phase contrast microscopy in the
reflected light mode) fitted with a 20× objective under
10× or 15×magnification and recorded through Imaging
Program for Windows r©.
The biofilm growth experiments on the culture surface
were conducted in a Kynar flow cell device that was
maintained at approximately 37 ◦C. The sample to be
studied was placed in the flow cell and then disinfected
with a 5% bleach solution for 15min followed by rinsing
with PBS for 15–30min, which also allowed for the
creation of the required flow rate. Following a 30min
seeding (adhesion) period, the cells within the flow cell
were fed a sterile, cell-free reduced GYEP broth (0.05%
glucose, 0.03% yeast extract, 0.1% bacto-peptone), for
approximately 48 h to allow for the development of a
biofilm. Acridine orange (AO), a cationic dye, binds to
DNA and RNA and can be used in CSLM. The biofilm
cultivated after the 48 h growth period was stained with
a 0.05% AO solution in PBS for 15–30min, depending
on the length of time needed for AO penetration of the
biofilm. The excess AO was removed by pumping PBS
through the flow cell until the effluent ran predominantly
clear and the biofilm could now be imaged.
Pluronic F127 toxicity analysis
Toxicity tests were conducted by generating growth curves
for CA1. The growth curves were produced for suspension
cultures at 37 ◦C in reduced GYEP broth either with or
without 0.04% (v/v) Pluronic F127. Growth curves were
also generated for suspension cultures at 37 ◦C in normal
GYEP broth with 0.0004%, 0.04% (v/v), or no Pluronic
F127.
Owing to the difference in the effect of chemicals
on planktonic cells versus their sessile counterparts, the
impact of Pluronic F127 adsorbed onto the surface on
cell viability was examined using propidium iodide (PI).
Cells on or near the surface following the initial adhesion
event (60min) and extended biofilm growth (48 h) were
stained with 25 µg PI/ml. Staining “live” cells and “dead”
cells tested the reliability of PI in identifying non-viable
cells of C. albicans. The “live” cells were obtained from
a 35 ◦C stationary phase suspension culture. The “dead”
cells were obtained by autoclaving the “live” cells for 15
to 20 min. Both groups of cells were stained with 25 µg
PI/ml and loaded into a hemacytometer. The cells in one
of the four corner blocks were counted and the percentage
of compromised cells was established. Compromised cells
fluoresced red under ultraviolet (UV) light as observed
using an Olympus microscope fitted with a 20× objective
(Olympus DplanApo 20 UV) under 15×magnification.
In staining heat-killed cells with PI, all of the cells counted
fluoresced red, whereas only 2% of CA1 cells fluoresced
red. On the basis of these results, PI appeared to be a
reliable measure of whether cells within the biofilm were
compromised.
Biofilm imaging
CSLM was used to obtain a three-dimensional image of
the C. albicans biofilm. The biofilm was examined using
a Leica DMRXE microscope with a 63× dry objective
0.70 PL Fluotar or with a 20× dry objective Nplan having
working distances of 2.0mm and 2.52mm, respectively,
dual Mitsubishi Diamond Pro 91 TXMmonitors, and the
Leica TCS NT imaging program. The CSLM was fitted
with an argon ion laser, 488 nmwavelength, that was used
to excite the AO. When bound to DNA, AO has excitation
and emissionwavelengths of 500 nm and 526 nm, respect-
ively. The excitation and emission wavelengths for AO
bound to RNA are 460 nm and 650 nm, respectively.
Images were acquired at 4–5µm z-intervals, with the pin-
hole setting at 1.00. On average, 7–10 regions were
examined during each of three experiments for the two
materials. An average of 4–12 optical sections, 158.7 µm×
158.7 µm, were collected, depending on the thickness of
the biofilm. Each optical section was averaged twice in
an effort to eliminate visual noise. The last visible layer
next to the surface was the last image that was obtained.
That is to say, an image of the surface itself was not
procured. This becomes important when we discuss the
locationof the thickest portionof thebiofilm.Quantitative
analysis of the biofilmwas performedusing theUTHSCSA
ImageTool program (developed at the University of Texas
Health Science Center at San Antonio, Texas, USA, and
available over the the Internet by anonymous FTP from
<ddsdx.uthscsa.edu/dig/itdesc.html>) (Silyn-Roberts &
Lewis, 1997).
RESULTS AND DISCUSSION
To confirm the presence of the desired chemical species
on the surfaces, the native PS and PLPS were subjected
to XPS analysis. The surfaces were prepared 24 h prior
to examination to ensure that the samples were dry,
preventing out-gassing of the sample, and to minimize
the possibility for contamination. Table 1 presents the
carbon and oxygen surface concentrations. The surface
oxygen on PS was the result of oxidation of the surface
upon exposure to air. The increase in the surface oxygen
content of PS upon treatment with Pluronic F127 was
attributed to the presence of ether-type carbon (C-O-C)
evidenced by a C1s component near 286.5 eV.
66 K. E. Wesenberg-Ward et al.
Table 1: XPS atomic concentrations
Concentration (%)
Pluronic F127-conditioned
Element Pristine polystyrene polystyrene
C1s 98.40 89.60
O1s 1.60 10.40
The results of the adhesion experiments of C. albicans
to PS and PLPS are summarized in Fig. 1, which shows
the number of cells adhering to an area of 1mm2 after
exposure for 1 h to a cell concentration of approximately
1× 107 cells/ml at a rate of 1.5ml/min. The number of
cells that adhered to PLPS (116 +− 31 cells/mm2) was
Fig. 1: Adhesion of C. albicans (Ca) to treated (PLPS) and untreated
(PS) polystyrene after 60 min of exposure to a cell concentration of
approximately 1 × 107 cells/ml at a rate of 1.5 ml/min. Values are expressed
as mean number of cells per unit area. The error bar represents the
standard deviation.
significantly less than that which adhered to PS (16 241+−
540 cells/mm2).
The results from the 1 h adhesion experiments
prompted the question of whether the influence of
Fig. 2: (a) Biofilm formation on pristine polystyrene at 48 h: the first cell layer was at the surface and the consecutive layers given in approximately 5 µm
increments. (b) Biofilm formation on Pluronic F127-conditioned polystyrene at 48 h: the first cell layer was at the surface and consecutive layers given in
approximately 4 µm increments.
Adhesion and biofilm formation of C. albicans 67
Fig. 2: Continued.
Pluronic F127 on initial cell adhesion persists during the
formation of the biofilm. The biofilms that formed on
the pristine PS and on the Pluronic F127-treated PS were
strikingly different. Fig. 2 displays CSLM images for a
C. albicans biofilm formed on PS and PLPS following 48 h
of growth at approximately 37 ◦C. TheCSLM images show
four layers of a biofilm, beginning at thefirst layer of visible
cells and extending towards the bulk fluid. The first layer
of visible cells was determined by scanning through the
biofilm until no cells were observed and then scanning
back up to the first layer of visible cells.
If the attachedcells are termedabiofilm,Fig. 3 shows the
percentage surface coverage for biofilm (48 h) formation
onPS andPLPS as a function of distance above the first cell
layer.Height zero represents thefirst visible layer of cells on
the surface.As yeast cells have an average diameter of 5 µm,
this seems to indicate that the greatest surface coverage
occurs at the second cell layer beyond the substratum;
however, it may also be that the biofilm actually begins
on average near the 5 µm point and the reason some cells
are observed below this point may be due to an artifact
such as the surface not being completely level. Also, as
the biofilm was scanned, some cells appeared clearly at
one level and faintly at levels above and below, and the
distorted appearance of the cells may be a consequence
of passing the laser through air–solid–liquid interfaces.
These factorsmay then influence the initial layer imageand
the biofilm thickness obtained from the confocal imaging.
The heterogeneous nature of the biofilm on PS resulted
in a surface coverage ranging from approximately 10%
to approximately 60% at its apex, and the total thickness
ranged from 20 to 60 µm. This thickness is similar to that
expected from a mature bacterial biofilm (50–100µm).
The percentage surface coverage reached a peak at a height
of approximately 5 µm from the surface layer of cells and
tapered off into the bulk fluid. The average maximum
surface coverage for the three experiments was 21%. On
PLPS, a very spotty “biofilm” was formed and less than
1% of the surface was covered. At the maximum surface
coverage of 0.5% on PLPS and 21% on PS, there is a very
observable difference.
Does this indicate that PLPS will stop C. albicans
biofilm formation in the long term? The number of
cells on PLPS changed very little over the 48 h growth
period whereas the biofilms grown on native polystyrene
increased dramatically over what was observed during
the initial attachment stage. It is known that bacterial
biofilms can slowly colonize a surface even though initial
colonization is reduced by some treatments. However, this
is due to the degradation of the surface and interactions
between cell clusters. Pluronic F127-treated surfaces are
inherently resistant, since it is the oxygen/hydrophilic
68 K. E. Wesenberg-Ward et al.
Fig. 3: Mean percentage surface coverage versus distance from the first
cell layer for PS and PLPS. (a) Three experiments with an average of
10 regions of the biofilm formed on pristine polystyrene per experiment.
(b) Four experiments with an average of seven regions of the biofilm
formed on Pluronic F127-conditioned polystyrene per experiment. The
bars on the graph represent the standard deviation of the coverage due
to heterogeneity of the biofilm.
surface that seems to be the resistant phase, and the
attached yeast clusters are far apart and unlikely to interact
and foul. Preliminary results on the adhesionofC. albicans
to oxygen-plasma-treated PS revealed limited attachment
(768–448 cells/mm2). As long as the oxygen/hydrophilic
surface remains and is not removed from the surface, it is
very likely that the surfacewill not foul. ThePEO segments
should ideally be covalently grafted to the surface.
Candida albicans tends to form cell clusters. The
various cluster groups within each layer of the biofilm
were therefore classified into different cluster sizes.
Representative images and the cluster sizes that were
considered are shown in Fig. 4. The smallest cluster group
consisted of single cells and budding cells. The bar graphs
displayed in Fig. 5a,b indicate the cluster group fraction
of the total cluster area in each layer of the biofilm. Both
graphs reveal in general that the largest of the total cluster
groups (gray) represent the greatest fraction of the total
cluster area in regions close to the surface, and the smallest
cluster groups (diagonal hatching) occupy the greatest
fraction of the total cluster area in regions distant from
the surface. The appearance of large cluster groups on
PLPS has decreased dramatically in comparison to that
associated with the biofilms formed on untreated PS.
The recalcitrance of bacterial biofilms is thought to be a
multicellular endeavor.Degradation of hydrogenperoxide
by catalase produced by bacterial cells requires a concerted
effort by a group of cells. Similarly, the activity of some
Cluster size 3–20 µm2
Cluster size 20–40 µm2
Cluster size 40–60 µm2
Cluster size 60–80 µm2
Cluster size 80–100 µm2
Cluster size 100+ µm2
Fig. 4: Examples of each cluster size used in quantifying the biofilm that
formed on treated and untreated polystyrene.
antibiotics requires oxygen. The cells on the perimeter
of the biofilm consume the oxygen and thereby protect
their deeper neighbors (Netting, 2001; Stewart, 2001).
Biofilmbacteria generate signal transduction proteins that
gather information from the environment and relay it
to chromosomal elements. This form of communication
allows for a group virulence response. This type of gene
regulation, termed “quorum sensing and response”, re-
quires a sufficientpopulation size (Davies et al., 1998;Reid,
1999). Biofilms also act as a diffusion barrier, with the
maximum diffusion distance increasing as the cluster size
increases (El-Azizi&Khardori, 1999; Jenkinson&Lappin-
Scott, 2001). Thus smaller clusters and/or the absence of
biofilm cell clusters would seem to necessarily restrict the
multicellular response thought to support biofilms.
The formation of hyphal elements within the biofilm
was frequently observed on native PS. However, upon
treatment with Pluronic F127, the expression of hyphal
elements was scarce. The CSLM images displayed in Fig. 6
reveal the typical presence of hyphal elements within
a biofilm formed on native PS (Fig. 6a) and the rare
appearance of hyphal elements within a biofilm formed
on PLPS (Fig. 6b).
Hyphal formation, along with being a major virulence
factor, has been shown to be necessary for formation
of normal biofilms by C. albicans. Ramage et al. (2002)
demonstrated the importance of the EFG1 gene in the
development ofC. albicans filamentous form and biofilms
that display the typical three-dimensional architecture.
Similarly, Baillie & Douglas (1999) showed that a
C. albicans mutant strain (1001–92′) unable to produce
hyphae also formed atypical biofilms. The EFG1 mutant
strains formed a thinly distributed layer of elongated
cells. The hyphal-mutant strain utilized by Baillie &
Adhesion and biofilm formation of C. albicans 69
1
0.8
0.6
0.4
0.2
0
Fr
ac
tio
n 
of
to
ta
l c
hi
st
er
 a
re
a
3–
20
20
–4
0
40
–6
0
60
–8
0
80
–1
00
10
0+
Chister size (µm2)
3–
20
20
–4
0
40
–6
0
60
–8
0
80
–1
00
10
0+
Chister size (µm2)
36.0
31.5
27.0
22.5
18.0
13.5
9.5
4.5
0
Distance from surface (µm2)
24.0
20.0
16.0
12.0
8.0
4.0
0
Distance from surface (µm2)
(a)
1
0.8
0.6
0.4
0.2
0
Fr
ac
tio
n 
of
to
ta
l c
hi
st
er
 a
re
a
(b)
Fig. 5: Cluster group fraction of the total cluster area in each layer of the biofilm formed on (a) pristine polystyrene averaged for eight regions of the
biofilm on each of three experiments and (b) Pluronic F127-conditioned polystyrene averaged for seven regions of the biofilm.
Douglas developed a monolayer of compact yeast cells.
The biofilms generated by the mutant strains in both
studies were found to be resistant to the antifungal
agent amphotericin B. The EFG1 mutant biofilms
were also resistant to the antifungal agent fluconazole.
However, the EFG1 mutant strain had sessile minimum
inhibitory concentrations at 80% inhibition of 1 µg/ml by
comparison with 8 µg/ml for strain CAF-2, which served
as the control. The response of the cells that attached to
the Pluronic F127-treated surface to antifungal agents was
not measured.
In an attempt to determine whether Pluronic F127
is toxic to C. albicans, the surfactant (0.04% (v/v)) was
added to the growth medium of a suspension culture as
described in Materials and Methods, above. The growth
profile for C. albicans grown in medium containing
Pluronic F127was comparedwith those generated for cells
grown in the absence of the surfactant. The results from
analysis of variance indicated that Pluronic F127 (0.04%
(v/v)) significantly decreased the log concentration of
cells (P< 0.0001) and decreased the 24 h plateau level
by approximately one-third. However, because of the
differences in chemical sensitivity observed between
sessile cells and their planktonic counterparts (Hawser &
Douglas, 1995; Chandra et al., 2001), we investigated the
potential effect of Pluronic F127 on cells within a biofilm,
formed on a surface modified by adsorption of Pluronic
F127.
70 K. E. Wesenberg-Ward et al.
Fig. 6: Hyphal formation on pristine polystyrene (a) and on Pluronic F127-conditioned polystyrene (b) after 48 h of biofilm growth.
Observation of PI-stained biofilms using CSLM
revealed that CA1 biofilms formed on PS had regions
that fluoresced red (i.e. were stained with PI and therefore
presumably compromised) in most of the areas of the
biofilmexamined.However, the regions stained byPIwere
small in comparison with the total area of the biofilm. In
contrast, regions of CA1 biofilms generated on PLPS that
were stained by PI constituted a greater portion of the
total cellular area. However, less than half of the regions
examined were stained. Those cellular components that
fluoresced red were hyphal elements, but not all hyphal
elements observed were stained. One should also consider
the findings of others that Pluronic alters the permeability
of cells (Laouar et al., 1996). Therefore the cells that
are stained may be stained as a result of increased cell
permeability and not as a consequence of cell death. On
the basis of these results, it is not evident that Pluronic
F127 is toxic to the yeast cells on the surface. The anti-
adhesive quality of Pluronic F127 towards C. albicans is
probably due to steric stabilization of the surface, as has
been indicated for bacteria and proteins, rather than a
product of toxicity (Bridgett et al., 1992; Alexandridis &
Hatton, 1995; Freij-Larsson et al., 2000).
To summarize, images were taken during development
of a 48 h biofilm, and the thickness of the biofilm on
an untreated substratum was 40–50 µm, a reasonable
thickness for a mature biofilm. This work established that
Pluronic F127 addition to PS reduced the adhesion of
C. albicans to PS by more than two orders of magnitude.
Pluronic F127 treatment similarly diminished the mean
surface coverage from 21% to 0.5% at approximately 5 µm
(the maximum visible cell layer on the surface). Pluronic
F127 treatment reduced hyphal formation, an important
virulence factor and also curtailed the formation of large
clusters of cells, perhaps by minimizing the adhesion. It
was also shown that the smallest cluster groups represent
the greatest fractionof the total area of clusters throughout
the biofilm. These results can be used, with some caution,
to indicate that Pluronic F127 treatment of a surface can
inhibit the formation of a fungal biofilm of C. albicans.
Adhesion and biofilm formation of C. albicans 71
Long-term studies are needed to establish whether the
effect is important on implant devices. On the basis
of these studies, it appears that the effect of surface
chemistry on the initial adhesion events persists during
the development of the biofilm – limited adhesion results
in limited biofilm growth.
ACKNOWLEDGEMENTS
This work was supported by: the Center for Biofilm
Engineering, Montana State University–Bozeman; the
ICAL, Montana State University–Bozeman; NIH Grant
R01 DE 013231-01; and UTHSCSA ImageTool and NIH
Image J.
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