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Antimicrobial Agents and Chemotherapy, August 1998, p. 2146-2149, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Iron-Limited Biofilms of Candida
albicans and Their Susceptibility to Amphotericin B
George S.
Baillie and
L. Julia
Douglas*
Division of Infection and Immunity, Institute
of Biomedical and Life Sciences, University of Glasgow, Glasgow G12
8QQ, United Kingdom
Received 2 March 1998/Returned for modification 18 May
1998/Accepted 9 June 1998
 |
ABSTRACT |
Biofilms of Candida albicans were grown in vitro under
iron limitation and at a low growth rate to simulate conditions for implant-associated biofilms in vivo. Their properties were compared with those of glucose-limited biofilms grown under analogous
conditions. At steady state, the adherent cell populations of
iron-limited biofilms were double those of glucose-limited
biofilms, although the growth rates were similar (0.038 to 0.043 h
1). Both biofilm types were resistant to amphotericin B,
but daughter cells from iron-limited biofilms were significantly
more susceptible to the drug than those from glucose-limited biofilms.
| |
TEXT |
Pathogenic fungi in the genus
Candida are now widely recognized as important agents of
hospital-acquired infection. Implanted devices, particularly indwelling
intravascular catheters, are a significant risk factor and are
frequently associated with these infections (11, 13).
Catheters provide a surface on which microorganisms can form an
adherent biofilm of cells embedded in a matrix of extracellular
material (7, 8, 24). Candida biofilms have been
studied in vitro by using a simple model system in which
adherent populations are grown on the surfaces of small discs of
catheter material (14-16). Biofilms of Candida
albicans consisted of mixtures of yeasts, hyphae, and pseudohyphae
and were resistant to the action of a variety of antifungal agents, including amphotericin B and fluconazole (15). Recently, a
more complex model system, the perfused biofilm fermentor
(12), was used to investigate whether the resistance of
C. albicans biofilms to amphotericin B could be attributed
to phenotypic changes resulting from the low growth rate characteristic
of biofilm cells (3). The findings indicated that the
resistance of C. albicans biofilms to amphotericin B was not
simply due to a low growth rate under the conditions tested, where
growth was limited by the availability of the carbon source,
glucose.
In vivo, the growth of C. albicans is likely to be limited
by the availability of a different nutrient, iron (6). To
survive in vivo, pathogenic microorganisms, including C. albicans, have developed various iron-scavenging mechanisms,
notably the secretion of iron-chelating compounds termed siderophores
(18, 22, 25). Iron deprivation can affect the surface
composition of microorganisms (20, 23), which, in turn, can
alter their susceptibility to antimicrobial agents
(5). In this investigation, we have grown biofilms of
C. albicans under conditions of iron limitation and compared
their susceptibility to amphotericin B with that of glucose-limited biofilms grown at a similar rate. Biofilms were formed within cylindrical cellulose filters perfused with culture medium by using a
modification of a method previously described for bacterial biofilms
(17). This system allows the formation of Candida
biofilms at reproducible, low growth rates. Moreover, the modified
apparatus lacks stainless steel components, thus facilitating the
production of iron-limited cultures.
Growth of biofilms under conditions of iron and glucose limitation.
C. albicans GDH 2346, a denture stomatitis isolate
(23), was used in all experiments. Yeast nitrogen base
medium for biofilm growth was prepared from individual constituents and
deferrated by using Chelex 100 ion-exchange resin, as described
previously (23). Glucose (50 mM) was added as the
carbon source. Analysis by graphite furnace atomic absorption
spectrometry revealed an iron content of <0.036 µM. This
concentration limits the growth of C. albicans GDH 2346 (23) and resulted in a stationary-phase optical density of
1.3 at 540 nm in batch culture. The medium used for the growth of
biofilms under glucose-limiting conditions was yeast nitrogen
base, prepared from individual constituents without deferration and
containing 4 mM glucose; this allowed batch growth of C. albicans to a stationary-phase optical density of 1.3 at 540 nm.
Biofilms were grown on cylindrical filters consisting of compacted
cellulose fibers (Gilson safety filters, 22 by 8 mm; Anachem, Luton,
United Kingdom) by using a modification of the method described by
Hodgson et al. (17) for bacteria. Each filter was inserted into silicone tubing attached to the bottom of a disposable syringe body (2 ml) from which the plunger had been removed. Medium was pumped
directly into the vertically clamped syringe body via silicone tubing.
In this modified method there was no requirement for a stainless
steel syringe needle, thus removing a possible source of iron
contamination. Filters were prewetted with sterile saline (5 ml) and
then inoculated with an exponential-phase batch culture (10 ml)
grown under glucose-limiting or iron-limiting conditions. Inocula
were prepared by addition of samples (10 ml) of an overnight culture in the appropriate medium to fresh batches (40 ml) of prewarmed
medium and incubation at 37°C with shaking for 3 h. After
perfusion of the inoculum, the filters were perfused with medium
at a flow rate of 0.87 ml min1. The eluate passing
through the filter was collected at various time intervals, and viable
counts were made by serial dilution in 0.15 M phosphate-buffered saline
(pH 7.2) and plating in triplicate on Sabouraud dextrose agar. The
plates were incubated at 37°C for 16 h before counting. This
gave an estimate of the numbers of newly formed daughter cells. Growth
rates of biofilms (divisions hour1) were calculated by
dividing the number of daughter cells produced per hour at steady state
by the estimated adherent cell population (determined by viable counts
of resuspended biofilms). All biofilms were grown for at least 24 h under steady-state conditions before drug treatment.
Properties of iron-limited and glucose-limited biofilms.
Elution profiles of cells released from the biofilms (Fig.
1) were highly reproducible but differed
from those previously reported for glucose-limited populations
maintained in a perfused biofilm fermentor (3). The number
of cells eluted from each cylindrical filter decreased over the first
hour, but this was followed by a period of biofilm growth
during which the adherent culture attained an optimal density (Fig. 1).
Iron-limited biofilms took much longer to reach this stage (24 h) than
glucose-limited biofilms maintained at the same flow rate (6 h),
although both populations shed daughter cells at a constant rate
thereafter. By contrast, glucose-limited biofilms in a perfused biofilm
fermentor achieved a steady state after only 80 min (3).
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FIG. 1.
Elution of C. albicans from cylindrical
cellulose filters perfused with iron-limited ( ) or glucose-limited
( ) growth medium at a flow rate of 0.87 ml min1.
Results are means from two independent experiments carried out with
duplicate sampling. Standard errors were less than 10% of means.
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|
The additional time required for iron-limited biofilms to
attain an optimal density may correspond to a period of prolonged
adaptation to iron limitation of growth.
C. albicans
GDH 2346
synthesizes a siderophore of the hydroxamate type when grown
planktonically
in yeast nitrogen base medium containing low
concentrations of
iron (
22). The same growth conditions also
produce alterations
in yeast cell wall composition, as revealed by
sensitivity to
the muralytic enzyme Zymolyase and by
125I
labelling of surface proteins (
23). These physiological
changes
were all apparent after incubation of batch cultures for
48 h
at 37°C (
22,
23). A detailed study of the
kinetics of siderophore
synthesis by
C. albicans GDH 2346 has not been reported. However,
a recent investigation of this
type with
Staphylococcus aureus (
9) showed that although siderophore activity could be
detected
after 24 h, maximal levels of activity were not reached
until
cultures had been incubated for 5 days. A similarly low rate of
siderophore production by
C. albicans, with its consequences
for
yeast physiological processes, might explain the delay in
attainment
of a steady state by iron-limited biofilms.
A summary of some important features of glucose- and iron-limited
biofilms grown on cylindrical filters is presented in Table
1. Iron-limited biofilms at steady state
had an adherent cell
population which was double that of
glucose-limited biofilms.
Similarly, eluates from iron-limited biofilms
contained almost
twice as many cells as those from glucose-limited
biofilms. These
quantitative determinations were confirmed by
scanning electron
microscopy, which showed that iron-limited
biofilms were denser
and more confluent than those formed under glucose
limitation
(Fig.
2). The medium flow rate
was identical for both biofilm
types, and unsurprisingly, the
calculated growth rates were similar
(Table
1). These growth rates were
only slightly lower than that
reported for
S. aureus (0.06 h
1) with the same biofilm system (
17).
However, they were considerably
lower than that previously determined
for glucose-limited
Candida biofilms (0.2 h
1)
growing in a perfused biofilm fermentor at a comparable flow
rate
(
3). Biofilms produced in the fermentor also contained
100-fold fewer cells
(3.5 × 10
7 ± 0.30 × 10
7 CFU
[mean ± standard error]), presumably because of the
two-dimensional
nature of the filter support.
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TABLE 1.
Analysis of steady-state C. albicans biofilms
grown on cylindrical cellulose filters under conditions of
glucose or iron limitationa
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FIG. 2.
Scanning electron micrographs of C. albicans
biofilms grown for 24 h on cylindrical cellulose filters perfused
with iron-limited (A) or glucose-limited (B) growth medium. Biofilms
were prepared for microscopy as described previously (14)
except that they were air dried overnight. Bar, 10 µm.
|
|
The increased adherent cell population of iron-limited biofilms
may be related to cell surface hydrophobicity. Hydrophobic
interactions
are considered to be important in the formation and
maintenance of
stable biofilm communities, principally by promoting
cell
adhesion. Indeed, there is some evidence that dispersal of
daughter
cells may be regulated by changes in surface hydrophobicity
(
1,
2). The cell surface hydrophobicity of
C. albicans is
significantly enhanced when the yeast is grown planktonically
in
iron-restricted medium (
21). An analogous change in
iron-limited
adherent populations might produce a denser biofilm that
is better
able to resist sloughing due to medium flow.
Unexpectedly, both iron-limited and glucose-limited biofilms
consisted exclusively of yeast cells. Hyphae and pseudohyphae
appeared to be completely absent (Fig.
2). By contrast, biofilms
grown
without nutrient limitation on polyvinyl chloride catheter
discs
(
14) and glucose-limited biofilms grown on cellulose acetate
filters in the perfused biofilm fermentor (
3) contain
mixtures
of morphological forms. Iron deprivation has been shown to
inhibit
the formation of germ tubes by
C. albicans
(
23), and this may
partly account for the yeast morphology
of iron-limited biofilms.
The reason for the absence of hyphae from
glucose-limited biofilms
is less obvious; it could be related to a
possible oxygen deficiency
inside the cylindrical filter, although
such conditions usually
favor hyphal development (
19).
Alternatively, if morphogenesis
in biofilms is dependent on
contact-induced gene expression, as
suggested previously
(
14), the precise nature of the surface
may be a crucial
determinant in triggering the response.
Susceptibility of biofilm cells to amphotericin B.
The
susceptibility to amphotericin B of steady-state biofilm cells,
resuspended biofilm cells, and newly formed daughter cells was
determined by a method based on that of Evans et al. (10). Biofilm cells grown under iron-limiting conditions were compared with
those grown under glucose-limiting conditions. An amphotericin B
concentration of 0.1 µg ml1 was used, since this
concentration gave a reduction in viability of more than 80% when
planktonic cells of C. albicans GDH 2346 were
tested by the same procedure (3). Cylindrical cellulose filters with adherent biofilms were removed from the apparatus and cut
in half longitudinally. One-half of each filter was immersed in
amphotericin B solution (0.1 µg ml1; 10 ml) for 1 h at 37°C; the adherent cells were then resuspended by breaking up
the filter with a sterile aluminum rod, followed by vigorous vortexing
for 1 min. Cells on the other half of the filter were first
resuspended in sterile water (10 ml) in an identical fashion, and then
samples of the suspension (25 µl; approximately 3.5 × 108 CFU ml1 for glucose-limited biofilms and
7.5 × 108 CFU ml1 for iron-limited
biofilms) were added to amphotericin-B solution (9.975 ml; final
concentration, 0.1 µg ml1) and incubated at 37°C for
1 h. Samples of the eluate (1 ml) containing daughter cells
(approximately 3 × 106 CFU ml1 for
glucose-limited cells and 4.8 × 106 CFU
ml1 for iron-limited cells) were also added to
amphotericin-B solution (9 ml; final concentration, 0.1 µg
ml1) and incubated similarly for 1 h. Viable counts
were made on all samples by serial dilution in 0.15 M
phosphate-buffered saline (pH 7.2) and plating in triplicate on
Sabouraud dextrose agar. The plates were incubated at 37°C for
16 h before counting. Figures for percent survival were calculated
by using counts for untreated control samples processed similarly.
Colony counts of control samples before and after the 1-h incubation
period showed only very small increases in cell numbers.
Glucose-limited biofilms, like those studied previously in the perfused
biofilm fermentor (
3), were resistant to this treatment
with
amphotericin B (Fig.
3). Iron-limited
biofilms were equally
resistant. However, cells resuspended
from either biofilm type
were some 20% more susceptible than intact
biofilm populations
(Fig.
3). This partial loss of resistance with
resuspended cells
was also observed with glucose-limited biofilms
from the perfused
fermentor (
3). It may be due to dispersal
of the matrix if
the latter acts as a physical barrier to drug
penetration, as
has been suggested (
4).
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FIG. 3.
Survival of biofilm cells of C. albicans
grown in iron-limited or glucose-limited medium after treatment with
amphotericin B. Intact biofilms ( ), resuspended biofilm cells ( ),
and biofilm daughter cells ( ) were exposed to amphotericin B for
1 h, and the percent survival was estimated by viable counts.
Results are means ± standard errors of the means from three
independent experiments with viable counts done in triplicate.
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|
The major difference observed between the two types of biofilm
population lay in the susceptibility to amphotericin B of their
daughter cells. Daughter cells eluted from glucose-limited biofilms
were more susceptible than either biofilm organisms or organisms
resuspended from biofilms (Fig.
3). These results, again, are
similar
to those obtained with glucose-limited biofilms in the
perfused
biofilm fermentor (
3). However, daughter cells from
iron-limited biofilms were significantly more susceptible
(
P <
0.05) than those from glucose-limited biofilms
(Fig.
3), with
a survival of less than 20% after exposure to the drug.
Microbial biofilms on catheters and other implanted devices are thought
to constitute a reservoir of infection, resistant
both to host defense
mechanisms and to antimicrobial agents (
4,
7). Detachment of
organisms from the biofilm can give rise
to a septicemia which may
respond to conventional drug therapy.
However, biofilm cells are not
killed by such treatment and remain
as a potential source of further
infection. The results presented
here seem to support this view for
Candida implant infections.
Our model system produced
slow-growing biofilms of
C. albicans whose limiting nutrient
was iron. These conditions mimic those
found in vivo, where growth is
slow and the availability of iron
is extremely limited (
6).
Iron-limited
Candida biofilms were
resistant to amphotericin
B, whereas daughter cells formed from
them were highly susceptible to
the drug. In vivo, an acute disseminated
infection resulting from
the release of such cells would be expected
to respond rapidly to
therapeutic doses of amphotericin B, possibly
leading to the conclusion
that the problem had been resolved.
The biofilm, however, would persist
as a sessile population of
viable fungi whose eradication would require
removal of the implant.
| |
ACKNOWLEDGMENTS |
This work was supported by grant 94/22A from the Sir Jules Thorn
Charitable Trust.
We are indebted to L. Tetley and M. Mullin for assistance with scanning
electron microscopy and to D. J. Halls (Glasgow Royal Infirmary)
for atomic absorption spectrometry.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Infection and Immunity, Institute of Biomedical and Life Sciences,
Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom. Phone: 0141-330-5842. Fax: 0141-330-4600. E-mail:
J.Douglas{at}bio.gla.ac.uk.
| |
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Antimicrobial Agents and Chemotherapy, August 1998, p. 2146-2149, Vol. 42, No. 8
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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