Journal of Investigative and Clinical Dentistry (2016), 7, 149–157

ORIGINAL ARTICLE
Oral Microbiology

Impact of brief and sequential exposure to nystatin,

amphotericin B, ketoconazole, and fluconazole in
modulating adhesion traits of oral Candida dubliniensis
isolates
Arjuna N.B. Ellepola1, Bobby K. Joseph1, Lakshman P. Samaranayake2, H.M.H.N. Bandara3 & Zia U.
Khan4
1 Departments of Bioclinical and Diagnostic Sciences, Faculty of Dentistry, Kuwait University, Jabriya, Kuwait
2 School of Dentistry, University of Queensland, Brisbane, Qld, Australia
3 College of Pharmacy, University of Texas at Austin, Austin, TX, USA
4 Department of Microbiology, Faculty of Medicine, Kuwait University, Jabriya, Kuwait

Keywords Abstract
adhesion, antifungal agent, Candida Aim: Candida adherence is implicated in the pathogenesis of oral candidosis.
dubliniensis, oral candidosis, polyene. Adhesion to buccal epithelial cells (BEC), germ tube (GT) formation, and rela-
tive cell surface hydrophobicity (CSH) are colonization attributes of candidal
Correspondence
Dr A. Ellepola, Department of Bioclinical
pathogenicity. Candida dubliniensis (C. dubliniensis) is allied with recurrent oral
Sciences, Faculty of Dentistry, Kuwait candidosis, which can be treated with nystatin, amphotericin B, ketoconazole,
University, Jabriya, Safat 13110, Kuwait. and fluconazole. Due to the diluent effect of saliva and the cleansing effect of
Tel: +965-2463-6714 the oral musculature in the oral cavity C. dubliniensis isolates undergo brief
Fax: +965-2532-6049 and sequential exposure to antifungal agents during therapy. Thus, in the pres-
Email: arjuna@hsc.edu.kw ent study, we evaluated the adhesion to BEC, GT formation, and the CSH of
oral isolates of C. dubliniensis following brief and sequential exposure to nysta-
Received 8 April 2014; accepted 17 August
2014.
tin, amphotericin B, ketoconazole, and fluconazole.
Methods: After determining the minimum inhibitory concentration (MIC) of
doi: 10.1111/jicd.12132 the aforementioned drugs, 20 oral isolates of C. dubliniensis were briefly (1 h),
and sequentially (10 days) exposed to subcidal concentrations of these drugs.
Following drug removal, adhesion to BEC, GT formation, and CSH of these
isolates were determined.
Results: The percentage reduction of adhesion to BEC, GT formation, and
CSH of the isolates following exposure to antifungal agents were as follows:
nystatin: 53.55%, 33.98%, and 29.83% (P < 0.001); amphotericin B: 53.84%,
36.23%, and 28.97% (P < 0.001); ketoconazole: 37.43%, 20.51%, and 16.49%
(P < 0.001); and fluconazole: 8.93% (P < 0.001), 1.6%, and 0.63% (P > 0.05).
Conclusions: Brief and sequential exposure of C. dubliniensis to antifungal
agents would continue to wield an antifungal effect by altering its adhesion
attributes, and elucidate possible pharmacodynamics by which antifungal agents
might operate in modulating candidal adherence.

isolated from the oral cavity of diabetic patients and from

Introduction
Candida dubliniensis (C. dubliniensis) is now well recog-
nized as an opportunistic pathogen associated with recur-
rent oral candidiasis in AIDS patients. It has also been
the sputum of cystic fibrosis patients. The fact that C. dub-
liniensis has been isolated from the upper respiratory tract
specimens, blood, as well as from a case of endocarditis
involving a prosthetic aortic valve, suggests that it can

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Antimycotics and adhesion of Candida
disseminate to other sites as well.1–5 In addition, resistance
to fluconazole has been discerned in C. dubliniensis iso-
lates obtained from AIDS patients, and stable fluconazole
resistance can be readily persuaded in C. dubliniensis fol-
lowing exposure to the drug in vitro.6 Furthermore, a
breakthrough in C. dubliniensis fungemia has found to
have transpired in a patient during prolonged exposure to
voriconazole.7 More recently, it was revealed that longitu-
dinal genotyping of C. dubliniensis isolates from HIV-
infected patients might acquire itraconazole resistance,
even in the absence of prior azole therapy.8
Adherence of Candida to host mucosal surfaces is a
major determinant of successful microbial colonization
and subsequent infection, and its critical role in the patho-
genesis of oral candidiasis is well recognized. Such attach-
ment enables the organisms to avoid dislodgement due to
the cleansing action of mucosal secretions, and facilitates
infection. Various in vitro and animal studies have pro-
vided evidence for a relationship between the proclivity of
Candida species to adhere to mucosal surfaces and their
presence in infections.9 Therefore, candidal adherence to
human buccal epithelial cells (BEC) is considered the criti-
cal initial step in the pathogenesis of oral candidiasis. In
addition, germ tubes (GT), which mark the onset of hyphal
growth, have been implicated in the pathogenesis of candi-
diasis, as these cylindrical extrusions, unlike the blasto-
spore form, are known to facilitate yeast adherence to
epithelial cells and impart resistance to phagocytic killing.10
Furthermore GT tend to promote the aggregation of
Candida cells and the bridging of adjacent hyphal elements,
thereby bringing a large battery of organisms into intimate
contact with the oral epithelium. Candida hyphae have also
been shown to penetrate dentinal tubules along cracks of
tooth surfaces, enabling the organism to invade dental hard
tissues.11 Apart from the aforementioned biological factors,
microbial cell surface hydrophobicity (CSH), which
contributes to hydrophobic interactions between cells and
surfaces, is thought to be an important non-biological
factor associated in the adherence of Candida to inert sur-
faces.12 Studies have also shown that hydrophobic yeasts
are more virulent than their hydrophilic counterparts.13
Statistically-significant correlations between CSH and can-
didal adhesion to BEC and denture acrylic surfaces have
also been reported.14,15
Many antifungal drugs are available for the treatment
of oral candidiasis. These include polyene antifungals,
such as nystatin and amphotericin B, and azoles, such as
ketoconazole (an imidazole) and fluconazole (a triazole).
However, the luent effect of saliva and the cleansing effect
of the oral musculature in the oral cavity tend to reduce
the availability of these antimycotics below that of the
effective therapeutic concentrations, thereby compromis-
ing therapeutic efficacy.16–18 Thus, the opportunistic
150
A.N.B. Ellepola et al.
yeasts might undergo brief and sequential exposure to
antifungal drugs during the drug-dosage regimen, a sce-
nario all too familiar in the niches of the oral cavity.16–18
Such transient exposure to antifungals might affect the
aforementioned adhesion traits of C. dubliniensis isolates.
To our knowledge, there is no information on the impact
of brief and sequential exposure to antimycotic agents on
the colonization traits of oral C. dubliniensis isolates. Tak-
ing together the aforementioned information, as well as
the findings of a recent prevalence study where oral
C. dublinienis isolates had the highest prevalence rate
among non-albicans oral Candida species in Kuwait,19 the
aim of the present study was to determine the effect of
such brief and sequential exposure to four antifungal
drugs — nystatin, amphotericin B, ketoconazole, and
fluconazole — on three candidal adhesion traits, such as
adhesion to BEC, GT formation, and CSH of oral isolates
of C. dubliniensis.
Materials and methods
Organisms
Twenty oral isolates of C. dubliniensis recovered from the
oral rinse samples from patients attending the Kuwait
University Dental Clinic (KUDC) for dental treatment in
a previous study were included in the current study.19
None of the patients from whom the isolates were recov-
ered had oral candidosis. These patients were not on any
antimicrobial therapy and had not taken any antimicrobi-
als for at least 6 months prior to obtaining the oral rinse
samples. Initially, all the yeast isolates were tested for GT
formation. Thereafter, the colony characteristics were
observed using CHROMagar Candida medium (Becton
Dickinson, Sparks, MD, USA) and carbohydrate assimila-
tion profiles obtained using the VITEK 2 yeast identifica-
tion system (BioMerieux, Crappone, France). The identity
of C. dubliniensis was confirmed by the production of
rough colonies with hyphal fringes and chlamydospores
on simplified sunflower seed agar, and by using seminest-
ed polymerase chain reaction amplification of the inter-
nally-transcribed spacer (ITS)-2 region of rDNA, followed
by direct DNA sequencing of the ITS region of rDNA.
Antifungal agents and media
Antifungal agents were prepared as reported previ-
ously.16–18 Briefly, nystatin, amphotericin B, ketoconazole,
and fluconazole (Sigma, St Louis, MO, USA) were dis-
solved in DMSO. These antimycotic agents were prepared
initially as 10 000 lg/mL and stored at 20°C before use.
They were suspended/diluted in the following medium
during the exposure period (1 h) of yeasts. RPMI-1640
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A.N.B. Ellepola et al.
medium buffered with 0.165 M MOPS (3-[N-morpholi-
no] propanesulfonic acid) containing L-glutamine and
lacking sodium bicarbonate (Sigma) was dissolved in 1 L
sterile, distilled water, adjusted to a pH of 7.2, and filter
sterilized. This liquid RPMI-1640 was stored at 2–8°C for
2–3 months. The stock solution was used to obtain drug
concentrations (i.e. 29 minimum inhibitory concentra-
tion [MIC]) used in the experiments.
Determination of minimum inhibitory concentration
Antifungal susceptibility values for nystatin were deter-
mined by the broth dilution technique, as described pre-
viously, with the same isolates used in this study by
performing twofold serial dilutions of the drug in microt-
itre plates using an inoculum of 1–5 9 105 c.f.u./mL.16
The MIC was determined visually and spectrophotometri-
cally at 595 nm following 24 h incubation at 37°C. The
MIC was defined as the lowest concentration of the drug
that inhibited the growth of yeast cells, as indicated by
the absence of turbidity (optically clear). The MIC was
read independently by two independent observers. Can-
dida albicans (C. albicans) American Type Culture Collec-
tion (ATCC) 90028 was used as a reference strain. All
experiments were repeated on two separate occasions with
duplicate determinations on each occasion.
Antifungal susceptibility values for amphotericin B, ke-
toconazole, and fluconazole were determined by E tests, as
described previously, with the same isolates used in this
study by performing these tests according to the manufac-
turer’s recommendations (AB BIODISK, Solna, Swe-
den).17–19 Each test isolate was freshly subcultured. Five
isolated colonies were uniformly suspended in sterile
saline, and turbidity was adjusted to 0.5 McFarland stan-
dard. The McFarland standard 0.5 is approximately equal
to 1–5 9 106 cells/mL. This inoculum was swabbed onto
the agar plates (150 mm diameter) and allowed to dry for
10–15 min before the E-test strips were applied. RPMI-
1640 agar supplemented with 2% glucose and buffered
with MOPS (0.165 M, pH 7.0) was used for susceptibility
testing, according to the method recommended by the
Clinical and Laboratory Standards Institute (formerly
National Committee for Clinical Laboratory Standards).
The plates were incubated at 35°C, and the MIC was read
after 24–48 h of incubation. The point where inhibition
ellipses intercepted the scale on the antifungal strip was
taken as the MIC for each test isolate: complete inhibition
(100%) of growth for amphotericin B, and marked
decrease in growth intensity (80%) for fluconazole and
ketoconazole. Reference strains of C. albicans ATCC 90028
and Candida parapsilosis (C. parapsilosis) ATCC 22019
were used for quality control of the susceptibility testing.
Interpretive susceptibility breakpoints for fluconazole were
ª 2014 Wiley Publishing Asia Pty Ltd
Antimycotics and adhesion of Candida
those recommended by Clinical and Laboratory Standards
Institute document M27-A2. Due to the lack of defined
susceptibility breakpoints for amphotericin B and ketoco-
nazole, an isolate was considered susceptible if it had an
MIC breakpoint of ≤1.0 lg/mL for amphotericin B and
0.125 lg/mL for ketoconazole, as determined in previous
studies.17–19
Preparation of cell suspension for the adhesion to buccal
epithelial cells, germ tube formation, and relative cell
surface hydrophobicity
A previously-described method was used for this pur-
pose.20,21 Briefly, yeast cells were maintained in Sabouraud
dextrose agar (SDA), inoculated onto fresh plates, and
incubated overnight at 37°C for 24 h prior to use. The
organisms were harvested, and a cell suspension prepared
in sterile phosphate-buffered saline (PBS) at pH 7.4 to an
optical density of 1.5 at 520 nm. From this cell suspension,
1 mL was added to tubes containing 4 mL RPMI broth
(control) and 4 mL RPMI/drug solution (test), in which
the drug concentrations were twice the MIC values. This
yielded a cell suspension of 106 cells/mL in each assay tube.
The tubes were then incubated at 37°C for 1 h in a rotary
incubator. Following this limited exposure, the drugs were
removed by two cycles of dilution with sterile PBS and cen-
trifuged for 10 min at 3000 9 g. Afterwards, the superna-
tant was completely decanted, and the pellets were
resuspended in 5 mL sterile PBS; 100 lL of this cell sus-
pension was then inoculated onto SDA plates and incu-
bated at 37°C for 24 h. Thereafter, the organisms were
harvested and once again exposed to the drug, as men-
tioned previously. This procedure was performed for 10
consecutive days, after which cultures of the drug-treated
Candida (test), as well as Candida unexposed to antifungal
agents (control), were inoculated onto SDA plates and
incubated at 37°C for 24 h. These cultures were then main-
tained in SDA, stored at 4–6°C, prior to being used for the
adhesion to BEC, GT formation, and CSH assays. This
procedure allowed Candida exposed to the drug to recover
and grow after the final treatment of antifungal drugs.
The aforementioned procedure for drug removal has
been shown to reduce the concentration of the drug as
much as 10 000-fold, thereby minimizing any carry-over
effect of the drug following its removal.16–18,20,21 Viable
counts of the control and the test were performed after
each procedure of drug removal. As the procedure of
drug removal effectively eliminated any carry-over effect,
there was virtually no difference in the viable counts of
the control and the tests following exposure to the already
diluted subtherapeutic concentrations of the drug, as
observed in previous studies.16–18,20,21 In addition, under
Gram stain, Gram-positive yeast cells were seen when
151
Antimycotics and adhesion of Candida
Candida were observed under light microscopy before
brief exposure to these antifungals, and did not reveal any
observable changes after brief and sequential exposure to
these drugs (i.e. before the adhesion to BEC, GT forma-
tion, and CSH assays were performed) compared to the
cells observed before the exposure.
Adhesion to buccal epithelial cell assay
A previously-used adhesion assay was performed with
slight amendments.16,18 Briefly, human BEC from four
adults were collected by gently rubbing the inner aspect of
the right and left buccal mucosa with sterile cotton swabs.
These were then dispersed in sterile PBS. The pooled BEC
suspension was washed in PBS to remove any attached
organisms, by centrifugation at 3500 9 g for 10 min. The
BEC were thereafter resuspended in PBS to obtain a con-
centration of 1 9 105 cells/mL by hemocytometer count-
ing. For the adhesion assay, 0.5 mL BEC and 0.5 mL
Candida suspension following brief exposure to the drugs
were mixed gently in tubes and incubated at 37°C for 1 h.
The Candida/BEC suspension was diluted in 4 mL sterile
PBS. The BEC were harvested onto 12-lm-pore-size poly-
carbonate filters and washed gently with sterile PBS to
remove any unattached Candida cells. Each filter was
placed on a glass slide and removed gently after 10 sec.
The preparation on the glass side was air dried and stained
with Gram’s stain. The number of adherent yeast cells was
quantified by light microscopy at 4009 magnification.
Fifty sequential BEC were observed for adherent Candida
cells. Clumped, folded, or overlapping BEC were excluded,
as done in previous experiments.16,18
Microscopic quantification of germ-tube forming cells
(germ-tube assay)
A previously-used method for GT induction was per-
formed.16,17 RPMI-1640 medium with L-glutamine
(Sigma) was chosen for the assay because it effectively
induces GT formation. For GT induction, 250 lL of yeast
suspensions of the control (prior to drug exposure) and
test (following brief and sequential exposure) was added
to 1 mL RPMI-1640 medium with L-glutamine and incu-
bated at 37°C for 90 min. Afterwards, the tube was vor-
tex mixed for 10 sec, and a drop of each cell suspension
was placed on a Neubauer hemocytometer chamber and
covered with a cover slip for the quantification of GT.
Thereafter, 300 yeast cells in contiguous fields were
counted (under 409 magnification), and the percentage
of GT-forming cells was calculated. A previously-used cri-
terion was used for counting.16,17 The following criteria
were used: (a) only yeast cells with a GT, without con-
striction at the junction between the cell and the elonga-
152
A.N.B. Ellepola et al.
tion, were counted; (b) clumped cells with GT were
excluded; and (c) pseudo-hyphae-forming yeast cells were
excluded.
Relative cell surface hydrophobicity assay
A biphasic aqueous-hydrocarbon assay, previously used
for the assessment CSH on oral Candida species, was
used in the current study.15–17 In brief, 5 mL of the con-
trol (prior to drug exposure) and test (following brief
and sequential exposure) yeast suspensions were adjusted
spectrophotometrically to an optical density (OD) of
0.442–0.448 at 520 nm to obtain a cell concentration of
McFarland standard 7.0. For each organism tested (with
and without exposure to nystatin), 2.5 mL volumes of
suspension were added to two sterile glass test tubes
(16 9 150 mm; 20 mL), representing one test and one
control. A total of 0.5 mL xylene was added to each test
suspension. The test and the controls were placed in a
water bath at 37°C for 10 min to equilibrate, then in
turn vortex mixed for 30 sec and returned to the water
bath for a further 30 min to allow the immiscible xylene
and aqueous phases to separate. The lower, aqueous
phase of the sample was carefully removed using a pip-
ette and transferred to a clean test tube. Any traces of
contaminating xylene that might have been carried over
in the pipette or bound to the yeast were removed by
bubbling air through the suspension at a rate of
180 mL/min for 2 min. The absorbance was measured
spectrophotometrically as before at 520 nm following
vortex mixing for 5 sec. The hydrophobicity was
expressed as described previously, as the percentage
reduction in OD of the test suspension compared with
the control.15–17 Thus, the greater the change in absor-
bance, the greater the shift in yeasts from the bulk med-
ium to the interface (i.e. the more hydrophobic the yeast
strain). Suspensions without xylene were used as the
negative controls.
All experiments were repeated on three separate occa-
sions with duplicate determinations on each occasion.
C. dubliniensis reference strain CD36 was also used in the
above assays.
Statistical analysis
The effect of antimycotic agents on Candida isolates was
statistically analyzed. The data obtained from all three
adhesion to BEC, GT formation, and relative CSH assays
were analyzed using ANOVA Dunnett’s t-tests, which treat
one group as a control (unexposed to antimycotics), and
compare all other groups (exposed to antimycotics)
against it. A P value of <0.05 was considered statistically
significant.
ª 2014 Wiley Publishing Asia Pty Ltd
A.N.B. Ellepola et al. Antimycotics and adhesion of Candida

pared to the controls that were not exposed, this resulted

Results
in a 33.98%, 36.23%, and 20.51% significant (P < 0.001)
The MIC (lg/mL) values of 20 isolates of C. dubliniensis percentage reduction with nystatin, amphotericin B, and
to nystatin, amphotericin B, ketoconazole, and fluconaz- ketoconazole, respectively. However, the 1.6% reduction
ole prior to exposure and following brief and sequential observed with fluconazole was not statistically significant
exposure to the respective drugs were as follows: for nys- (Table 2).
tatin, the MIC ranged from 0.09 to 0.78; for amphotericin The relative CSH of the of the of the 20 C. dubliniensis
B, the MIC range was between 0.002 and 0.125; for isolates that were not exposed to the drugs and following
ketoconazole, it ranged from 0.002 to 0.012; and for brief and sequential exposure to nystatin, amphotericin B,
fluconazole, the MIC range was between 0.016 and 0.38. ketoconazole, and fluconazole was 17.4, 12.21, 12.36,
Thus, all isolates were susceptible to the drugs tested 14.53, and 17.12, respectively, thereby resulting in a
before and after exposure to the respective drugs. 29.83%, 28.97%, and 16.49% significant (P < 0.001) per-
The adhesion to BEC (yeasts/50 BEC) of the 20 C. dub- centage reduction, with nystatin, amphotericin B, and
liniensis isolates not exposed to the drugs and following ketoconazole, respectively. However, the 0.63% reduction
brief and sequential exposure to nystatin, amphotericin B, observed with fluconazole was not significant (Table 3).
ketoconazole, and fluconazole was 215.72, 100.20, 99.57,
134.98, and 196.46, respectively. Compared to the controls
Discussion
that were not exposed, this resulted in a 53.55%, 53.84%,
37.43%, and 8.93% significant (P < 0.001) percentage Candida dubliniensis is now well-known as an emerging
reduction, respectively, for all drugs tested (Table 1). pathogen associated with oral candidiasis. Particular atten-
The percentage of GT-positive cells of the of the 20 tion has been given to experimenting candidal adhesion to
C. dubliniensis isolates not exposed to the drugs and fol- BEC of the oral mucosa, as it is intimately allied with all
lowing brief and sequential exposure to nystatin, ampho- forms of oral candidosis.9 Another phenotypic characteris-
tericin B, ketoconazole, and fluconazole was 26.28%, tic associated with candidal adhesion is the formation of
17.35%, 16.76%, 20.89%, and 25.86%, respectively. Com- GT, which marks the onset of hyphal growth and the
Table 1. Adhesion to BEC of oral Candida
Isolate Control Nystatin Amphotericin B Ketoconazole Fluconazole
dubliniensis isolates (yeast cells/50 BEC) fol-
lowing brief (1 h) and sequential (10 days) CD1 223.7 94.6 96.3 138.2 198.7
exposure to antifungal drugs CD2 235.3 99.3 97.8 141.8 199.3
CD3 200.4 92.7 97.4 148.1 187.4
CD4 210.8 93.6 91.1 129.7 183.2
CD5 203.6 90.6 92.4 123.4 181.1
CD6 211.5 98.5 100.4 141.4 201.6
CD7 228.3 107.8 109.2 145.8 208.7
CD8 200.4 92.3 93.5 133.7 192.6
CD9 233.4 112.8 114.5 139.3 208.5
CD10 209.2 96.7 93.2 137.4 195.6
CD11 201.7 90.4 97.4 129.5 193.4
CD12 234.3 115.3 111.9 143.2 207.6
CD13 202.2 92.3 91.7 120.7 189.8
CD14 221.8 108.6 104.8 131.9 201.4
CD15 201.9 94.2 93.8 130.7 190.3
CD16 206.7 98.8 92.3 128.5 193.7
CD17 212.3 105.2 101.8 122.7 181.2
CD18 229.5 116.1 118.5 142.5 198.7
CD19 216.2 97.5 94.6 130.3 201.8
CD20 231.1 106.7 98.7 140.8 214.6
Mean 215.72 100.20 99.57 134.98 196.46
SEM 2.85 1.88 1.82 1.78 2.11
SD 12.75 8.40 8.13 7.98 9.42
Percentage reduction 53.55 53.84 37.43 8.93
Significance (P) <0.001 <0.001 <0.001 <0.001

All values indicate the average of experiments repeated on three separate occasions with dupli-
cate determinations on each occasion (i.e. average of six values).
BEC, buccal epithelial cells; SD, standard deviation; SEM, standard error of mean.

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Antimycotics and adhesion of Candida A.N.B. Ellepola et al.

Table 2. Percentage of germ tube-positive

Isolate Control Nystatin Amphotericin B Ketoconazole Fluconazole
cells in 20 oral Candida dubliniensis isolates
CD1 24.56 16.82 17.33 21.04 24.33 following brief (1 h) and sequential (10 days)
CD2 29.33 18.65 17.92 24.79 28.92 exposure to antifungal drugs
CD3 21.52 14.78 13.76 16.03 20.87
CD4 28.24 19.43 17.18 23.32 26.19
CD5 31.41 23.45 21.89 24.84 32.33
CD6 22.87 14.44 15.22 18.22 23.54
CD7 21.32 12.29 11.91 16.09 20.89
CD8 27.43 19.73 18.22 22.76 28.01
CD9 29.14 22.72 23.08 24.21 28.53
CD10 32.67 22.87 20.73 25.63 31.22
CD11 21.92 14.02 12.96 16.71 22.08
CD12 25.63 16.69 15.92 21.11 24.62
CD13 24.22 15.44 16.06 20.06 24.97
CD14 27.68 18.31 17.48 19.73 26.31
CD15 23.83 12.77 14.02 18.22 24.55
CD16 29.28 18.83 19.63 24.66 28.08
CD17 22.65 12.77 11.92 16.66 22.03
CD18 24.77 14.51 14.22 18.83 22.91
CD19 30.64 22.66 20.77 24.87 31.93
CD20 26.43 15.78 14.93 20.04 24.76
Mean 26.28 17.35 16.76 20.89 25.86
SEM 0.77 0.81 0.73 0.74 0.78
SD 3.45 3.61 3.26 3.30 3.51
Percentage reduction 33.98 36.23 20.51 1.60
Significance (P) <0.001 <0.001 <0.001 >0.05

All values indicate the average of experiments repeated on three separate occasions with dupli-
cate determinations on each occasion (i.e. average of six values).
SD, standard deviation; SEM, standard error of mean.

ability to transform from the blastospore phase to the
hyphal phase.22 This could be another reason for the path-
ogenic nature of C. dubliniensis. For instance, candidal
hyphae are thigmotropic in nature and traverse along sur-
face irregularities both in vivo and in vitro, thus facilitating
in the custody of the organism in hostile habitats, such as
the oral cavity.11 Apart from the aforesaid biological phe-
notypic traits, the relative CSH of Candida is considered a
non-biological physical force of critical importance con-
cerning candidal adhesion. For instance, Hazen and Hazen
verified that hydrophobic Candida are more virulent than
their hydrophilic counterparts.23 Shibl et al.24 and Rama-
dan et al.25 showed that the reduction in CSH following
limited exposure to antimicrobials promoted increased
ingestion of microbes by polymorphonuclear leukocyte
(PMNL), thus increasing the susceptibility of the organ-
isms to the killing effect of PMNL. Hydrophobic cells also
exhibited greater adherence to epithelial cells and extracel-
lular matrix proteins, and decreased susceptibility to phag-
ocytic killing.26 Furthermore, enhanced virulence of
hydrophobic cells over hydrophilic cells could be due to
the potential of hydrophobic cells to bind to various
organs following clearance from the bloodstream.26
In the present study, the MIC values of all tested anti-
fungal agents before and following brief and sequential
exposure to the respective drugs were within the range of
the reference strain, which implies that the isolates tested
were susceptible to the drugs, despite multiple episodes of
drug exposure. Furthermore, these values were within the
MIC range obtained in previous investigations of C. dub-
liniensis isolates.16–19 Therefore, it could be speculated
that such brief (1 h), sequential (10 days) exposure to
subtherapeutic concentrations of antifungal agents does
not have a significant impact on the susceptibility of the
isolates, and is unlikely to induce development of drug
resistance, despite such exposure.
The suppression of adhesion to BEC and GT production
by limited exposure to the polyene antifungal agents (nys-
tatin and amphotericin B) could be related to the mecha-
nism of action of these polyenes on the Candida cell wall.
Polyenes bind to the sterol components in the cell wall of
Candida and make it more permeable, as mentioned in
previous studies.16,21 It has also been suggested that the
formation of sterols or their precursors could be inhibited
by polyenes.16,21 Studies have demonstrated dynamic
changes in the ultrastructural features of the cell wall dur-
ing morphogenic transformation to GT, and have shown
that the cell wall of GT possesses stratification comparable
to that of the blastospore wall.27 Other studies have shown
internally-collapsed cells with an intact cell wall leaving

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A.N.B. Ellepola et al. Antimycotics and adhesion of Candida

Table 3. Relative cell surface hydrophobicity

Isolate Control Nystatin Amphotericin B Ketoconazole Fluconazole
of 20 oral Candida dubliniensis isolates fol-
lowing brief (1 h) and sequential (10 days) CD1 16.78 11.38 11.92 14.53 16.02
exposure and subsequent removal of antifun- CD2 16.97 11.22 12.06 14.02 16.54
gal drugs CD3 17.81 12.73 12.01 13.26 16.77
CD4 18.38 12.81 11.63 13.45 18.78
CD5 21.23 14.87 15.66 18.44 20.95
CD6 16.45 11.92 11.04 13.94 16.81
CD7 16.96 11.06 11.88 13.61 16.22
CD8 17.43 12.03 11.91 14.03 16.98
CD9 21.62 15.02 14.91 17.34 22.04
CD10 17.32 12.98 12.61 14.14 17.55
CD11 17.81 12.02 11.34 14.08 16.76
CD12 16.43 11.94 12.07 13.96 17.02
CD13 16.22 11.32 12.11 14.22 16.06
CD14 16.81 10.96 11.95 14.37 17.05
CD15 21.84 15.93 15.77 18.91 21.26
CD16 15.02 11.21 11.97 13.68 15.96
CD17 15.61 11.08 11.06 14.03 15.23
CD18 16.07 11.67 12.05 13.48 15.91
CD19 16.16 11.24 12.19 13.22 16.33
CD20 15.07 10.78 11.07 13.93 15.63
Mean 17.40 12.21 12.36 14.53 17.29
SEM 0.45 0.33 0.31 0.37 0.43
SD 1.99 1.47 1.40 1.65 1.94
Percentage reduction 29.83 28.97 16.49 0.63
Significance (P) <0.001 <0.001 <0.001 >0.05

All values indicate the average of experiments repeated on three separate occasions with dupli-
cate determinations on each occasion (i.e. average of six values).
SD, standard deviation; SEM, standard error of mean.

“ghost-like” cells and deflated Candida cells following
exposure to subcidal concentrations of nystatin.28 In addi-
tion, microbial structures that contribute to CSH include
outer membrane protein, lipoprotein, phospholipid, lipo-
polysaccharide, and fimbriae. Drugs, such as polyenes, that
modify these structural features could reduce CSH of
microbes.24,25 In the case of C. albicans, it has been shown
that CSH correlates with the concentration of fibrils in the
exterior layer of the cell wall. As C. dubliniensis isolates are
phenotypically similar to C. albicans isolates, the observed
suppression of CSH elicited by polyenes on C. dubliniensis
could also be related to the aforementioned pharmacody-
namics of the polyenes on the cell wall of C. dubliniensis.
In previous studies, we demonstrated that a single, brief
exposure to polyenes induced an approximately 75%
reduction of adhesion to BEC, almost complete abrogation
of GT formation by approximately 95%, and approxi-
mately 34% reduction in CSH in the same oral isolates of
C. dubliniensis used in the present study.16–18 This higher
percentage of suppressive effect by the polyenes on all
tested adhesion traits of C. dubliniensis following brief, sin-
gle exposure compared to brief and sequential exposure
could be due the fact that previous studies were conducted
soon after drug removal when isolates were in a state of
polyene-induced metabolic shock, whereas the current
study was conducted after a recovery period of these iso-
lates, resulting in a lesser impact on the adhesion attributes
tested. Irrespective of these differences, it is reasonable to
ascertain that by affecting the cell wall structures polyene,
antifungals continue to subdue both biological (adhesion
to BEC and GT formation) and non-biological (CSH)
adhesion traits of C. dubliniensis isolates.
For ketoconazole, brief and sequential exposure of
Candida to this azole antifungal drug resulted in a statisti-
cally-significant reduction of their adhesion to BEC, GT
formation, as well as suppression of its CSH, although to a
lesser extent than exposure to the polyene antifungals. In
previous studies, we demonstrated that single, brief expo-
sure to ketoconazole induced an approximately 50%
reduction of adhesion to BEC, abrogation of GT formation
by approximately 18%, and approximately 21% reduction
in candidal CSH in the same oral isolates of C. dubliniensis
used in the present study.16–18 This slightly higher percent-
age of the suppressive effect by ketoconazole on adhesion
to BEC and candidal CSH of C. dubliniensis following
brief, single exposure could be once again due the fact that
these studies were conducted soon after drug removal
when isolates were in a state of drug-induced metabolic
shock, compared to the current study, which was con-
ducted after a recovery period of these isolates. However,

ª 2014 Wiley Publishing Asia Pty Ltd 155

Antimycotics and adhesion of Candida
although the exact reason cannot be speculated, there was
hardly any difference in the ability of ketoconazole to sup-
press candidal GT formation irrespective of the exposure
procedure (18% following brief single exposure vs 20% fol-
lowing brief and sequential exposure). Irrespective of these
differences, the suppressive effects observed might also be
related to the mode of action of ketoconazole. These drugs
alter the fungal cell membranes by blocking the 14 a-deme-
thylation step in the biosynthesis of ergosterol. The conse-
quent depletion of ergosterol and the accumulation of 14
a-methyl-sterols lead to alterations in a number of mem-
brane-associated functions, which could be the reason for
the observed suppressive effect on adhesion to BEC, GT
formation, and relative CSH of C. dublinienis isolates.17,18
In contrast to ketoconazole, the other azole drug (fluco-
nazole), induced a significant suppression on adhesion to
BEC, but virtually had no impact on the suppression of GT
formation and candidal CSH. Although fluconazole is
remarkably more effective than ketoconazole in the man-
agement of candidal infection, its growth inhibitory activ-
ity against Candida isolates in vitro is less than that of
ketoconazole,29 which could be due to the subtle changes
between the two azoles; ketoconazole being a imidazole,
and fluconazole being a triazole. Although the tested
C. dubliniensis isolates remained susceptible to fluconazole
even after brief and sequential exposure to the drug, it has
been shown that fluconazole resistance can be readily
induced in C. dubliniensis following exposure to the drug
in vitro.6 Thus, it can be speculated that the aforemen-
tioned differences between the two azoles could be the rea-
son behind the reduced ability of fluconazole to suppress
the adhesion attributes, as seen in the current study. In
addition, similar to polyenes and ketoconazole, in previous
studies, we demonstrated that single, brief exposure to
fluconazole induced an approximately 29% reduction of
adhesion to BEC, suppression of GT formation by approxi-
mately 12%, and approximately 9% reduction in CSH in
the same oral isolates of C. dubliniensis used in the present
study.16–18 This higher percentage of suppressive effect on
all adhesion traits by fluconazole on C. dubliniensis isolates
following brief, single exposure, similar to that of the poly-
enes, might yet again be due the fact that these studies were
conducted soon after drug removal, when Candida were in
a state of fluconazole-induced metabolic shock, whereas
the current study was conducted after a recovery period of
these isolates, resulting in a lesser fluconazole impact on
the adhesion attributes tested.
The isolation, identification and
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(research grant no. DB 01/11 and DB 02/11). The techni-
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