Journal of Medical Microbiology (2015), 64, 714 – 723 DOI 10.1099/jmm.0.

000082

Antifungal activity of clotrimazole against

Candida albicans depends on carbon sources,
growth phase and morphology
Lydia Kasper,1 Pedro Miramón,13 Nadja Jablonowski,1
Stephanie Wisgott,1 Duncan Wilson,14 Sascha Brunke1,2
and Bernhard Hube1,2,3
1
Correspondence Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research
Lydia Kasper and Infection Biology – Hans Knoell Institute, Jena, Germany
lydia.kasper@leibniz-hki.de 2
Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control
and Care (CSCC), University Hospital, Jena, Germany
3
Friedrich Schiller University, Jena, Germany

Vulvovaginal candidiasis, a superficial infection caused predominantly by the pathogenic fungus

Received 1 December 2014
Accepted 6 May 2015
Candida albicans, is frequently treated with clotrimazole. Some drug formulations contain
lactate for improved solubility. Lactate may modify C. albicans physiology and drug sensitivity by
serving as a carbon source for the fungus and/or affecting local pH. Here, we explored the
effects of lactate, in combination with pH changes, on C. albicans proliferation, morphology and
clotrimazole sensitivity. Moreover, we determined the influence of growth phase and morphology
per se on drug sensitivity. We showed that utilization of lactate as a carbon source did not
promote fast fungal proliferation or filamentation. Lactate had no influence on clotrimazole-
mediated killing of C. albicans in standard fungal cultivation medium but had an additive effect
on the fungicidal clotrimazole action under in vitro vagina-simulative conditions. Moreover,
clotrimazole-mediated killing was growth-phase and morphology dependent. Post-exponential
cells were resistant to the fungicidal action of clotrimazole, whilst logarithmic cells were
sensitive, and hyphae showed the highest susceptibility. Finally, we showed that treatment of
pre-formed C. albicans hyphae with sublethal concentrations of clotrimazole induced a
reversion to yeast-phase growth. As C. albicans hyphae are considered the pathogenic
morphology during mucosal infections, these data suggest that elevated fungicidal activity of
clotrimazole against hyphae plus clotrimazole-induced hyphae-to-yeast reversion may help to
dampen acute vaginal infections by reducing the relative proportion of hyphae and thus shifting
to a non-invasive commensal-like population. In addition, lactate as an ingredient of clotrimazole
formulations may potentiate clotrimazole killing of C. albicans in the vaginal microenvironment.

INTRODUCTION Vulvovaginal candidiasis (VVC) is arguably the most

common superficial infection in otherwise healthy indi-
The fungus Candida albicans is both a harmless commensal
viduals, and C. albicans is the Candida sp. that is most
on human mucosal surfaces of the oral, gastrointestinal and frequently associated with VVC (Achkar & Fries, 2010;
vaginal tracts, and an important human pathogen. Under Mendling & Brasch, 2012). Overall, 75% of women experi-
certain predisposing conditions, C. albicans can cause ence at least one episode of VVC in their lifetime, and
superficial but also life-threatening systemic infections. 5–10% suffer from recurrent VVC (Sobel, 2007).
Acute VVC can be treated topically with polyenes, ciclo-
3Present address: Department of Microbiology and Molecular Genetics,
pirox olamine or azoles (Mendling & Brasch, 2012; Sobel,
University of Texas – Houston Medical School, Houston, TX, USA.
2013). Azoles, which inhibit fungal ergosterol biosynthesis,
4Present address: Aberdeen Fungal Group, School of Medical Scien- have proven to be effective in short-term treatment of
ces, University of Aberdeen, Aberdeen AB25 2ZD, UK. vaginal candidiasis (Edelman & Grant, 1999), and clotri-
Abbreviations: VVC, vulvovaginal candidiasis; VSM, vagina-simulative mazole is one of the azole antifungals used most commonly
medium. in this context (Plempel, 1982; Sobel, 2013). We have
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shown recently that low levels of clotrimazole reduce but
do not block fungal growth, and thus can effectively inhibit
C. albicans-induced damage of vaginal epithelial cells in vitro
(Wächtler et al., 2011). Furthermore, we found that these
low clotrimazole levels are sufficient to dampen the vaginal
inflammation response to C. albicans infection (Wilson
et al., 2013).
A 500 mg vaginal ovule of clotrimazole for 1-day treatment
(Canesten GYN Once Kombi; Bayer) is supplied in an acid
formulation containing lactate, the ion salt of lactic acid,
to improve drug solubility and bioavailability (Mendling &
Plempel, 1982). Lactic acid is a natural component of vagi-
nal secretions (Owen & Katz, 1999) and is believed to be
generated mainly by vaginal lactobacilli (Boskey et al.,
2001). Clotrimazole ovule application may lead to a rise in
vaginal lactate levels and may therefore affect C. albicans
growth and clotrimazole sensitivity: altered levels of lactic
acid/lactate may change the local pH in the vaginal cavity,
which may have an impact on growth and membrane integ-
rity of the infecting C. albicans cells. For example, the
environmental pH affects the morphogenetic transition of
C. albicans between the yeast and hyphal growth form
(Biswas et al., 2007), which is known to be critical for patho-
genesis of candidal vaginitis (Sobel et al., 1984). Recent
studies have shown that growth on lactate as a carbon
source affects C. albicans cell-wall properties, interaction
with immune cells and virulence (Ene et al., 2012a, b, 2013).
In this study, we analysed the possible effects of lactate in
clotrimazole formulations on growth and drug sensitivity
of C. albicans. We investigated the effect of pH changes as
well as the impact of fungal growth phase and morphology
on the clotrimazole antifungal activity. Finally, we deter-
mined the clotrimazole action under conditions mimicking
the physiological environment in the vaginal cavity, by using
a vagina-simulative medium (VSM) (Owen & Katz, 1999;
Sosinska et al., 2008) at CO2 and O2 levels typical for the
vagina.
METHODS
Strains and growth conditions. Experiments were performed
with the C. albicans clinical isolate SC5314 (Gillum et al., 1984).
Yeasts were cultured overnight at 30 uC and 180 r.p.m. in liquid
YPD medium (1% yeast extract, 1% peptone, 2% glucose,) to post-
exponential growth phase. To obtain yeasts in exponential growth
phase, overnight pre-cultures were diluted to an OD600 of 0.2 in fresh
YPD and incubated for 4 h at 30 uC at 180 r.p.m. Liquid media with
different carbon sources were prepared based on defined minimal
medium [1| yeast nitrogen base (YNB) with ammonium sulfate; BD
Biosciences], and 2% glucose (111 mM; Roth) or 2% D/L -lactate
(178 mM; Sigma) was added as carbon source. The media were
buffered with 75 mM sodium tartrate (pH 3.0, 3.5, 4.0, 4.5 or 5.0),
75 mM potassium phosphate (pH 6.0) or 75 mM HEPES (pH 7.0
or 7.5). None of these buffers can be utilized as a carbon source by
C. albicans (data not shown).
Microbial growth under vagina-simulative conditions was monitored
in modified VSM (Owen & Katz, 1999; Sosinska et al., 2008) con-
sisting of 58 mM NaCl, 18 mM KOH, 2 mM Ca(OH)2, 1.75 mM
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Determinants of C. albicans clotrimazole sensitivity
glycerol, 6.7 mM urea, 33 mM glucose and 6.7 g YNB l21, as well as
lactic acid (22 mM) and acetic acid (17 mM), at a pH of 4.2. Cells in
VSM were cultured in vagina-typical gas conditions of 6% CO2 and
7% O2 (Sosinska et al., 2008). Clotrimazole (Bayer Vital GmbH) was
added from a 25 mM stock in DMSO.
Yeast growth analyses. Growth curve experiments were performed
in 1|YNB medium (see above) with yeasts inoculated from a log-
arithmic YPD pre-culture at a starting OD600 of 0.05. Growth was
measured by monitoring OD600 per well every 30 min in a 200 ml
volume in a 96-well plate (TPP) using a Tecan Infinite M200
microplate reader at 30 uC with intermittent shaking. Generation
times were calculated after subtracting the OD600 of medium controls.
C. albicans survival assays. Survival in the presence of clo-
trimazole was assayed for yeasts inoculated from logarithmic or post-
exponential YPD pre-cultures (see above) to a cell number of
5|105 ml21 in 1|YNB medium or VSM (see above). Cells were
incubated in glass vials with rotary shaking for 5 h. For evaluation of
survival, cells were plated directly after inoculation (0 h) as well as
after 5 h incubation on YPD plates, and c.f.u. were counted to cal-
culate viable cell numbers ml21. The survival rate was then deter-
mined as the percentage of the 0 h value: %5(viable cell count at
5 h/viable cell count at 0 h)|100.
Hyphal growth analysis. Hyphal growth was analysed by inoculat-
ing an overnight YPD pre-culture to a cell density of 4|104 cells
ml21 into 1|YNB medium buffered to pH 7.5 (see above) or RPMI
with fetal bovine serum (RPMI+FBS). Filamentation was induced by
a temperature change to 37 uC at 5% CO2 with contact induction on
plastic (24-well plates; TPP). After fixation with 4% paraformaldehyde
(Histofix; Roth), the morphology was evaluated by inverse microscopy
(Axio Vert.A1; Zeiss).
Statistical analysis. The graphs show means+ SD of at least three
experimental replicates. A statistical analysis was performed with a
two-way ANOVA followed by an unpaired Student’s t-test with
Bonferroni multiple comparison correction. Significant differences
were determined at Pv0.05, Pv0.01 or Pv0.001.
RESULTS
Influence of lactate and pH on yeast and hyphal
growth
Lactate in clotrimazole drug formulations may serve as a
carbon source for C. albicans at the site of treatment or
lead to pH changes, which in turn could influence fungal
growth behaviour. To determine the impact of lactate/
lactic acid and pH changes on C. albicans in vitro, we mon-
itored proliferation and filamentation in YNB-based mini-
mal medium over a pH range of 3.0–7.5 with lactate as the
sole carbon source. At pH values above 4.0, lactate is pre-
sent as an ion salt, whilst at lower pH values, lactic acid
(pKA53.86) is present in the undissociated form. Glucose
as a preferential carbon source for C. albicans was used
for comparison. The results are summarized in Table 1.
As expected, yeast growth in medium containing lactate as a
carbon source was generally reduced compared with growth
in glucose-containing medium, as shown by prolonged gen-
eration times. In addition, in contrast to glucose-containing
cultures, growth in lactate-containing cultures was more pH
715
L. Kasper and others

Table 1. Summarized phenotypes of C. albicans yeast and filamentous growth depending on pH and carbon source

Results are shown as means¡SD of three experimental replicates. Significant differences: *P,0.05; ** P,0.01; *** P,0.001 between glucose and
lactate samples at the same pH; #P,0.05; ##P,0.01; ###P,0.001 compared with pH 4.5 as a vagina-typical pH.

pH Yeast generation time (h)D Filamentation (%)Dd

Glucose Lactate Glucose Lactate

3.0 1.49¡0.15*** 11.28¡3.41### 0.67¡1.15 0.33¡0.58

3.5 1.29¡0.08** 6.27¡1.85 4.33¡3.51 1.00¡1.00
4.0 1.38¡0.30 4.83¡0.43 4.00¡6.08 3.00¡5.20
4.5 1.42¡0.13 4.13¡0.52 4.67¡7.23 1.00¡1.00
5.0 1.52¡0.06 5.27¡1.19 3.67¡3.79 1.33¡2.31
6.0 1.42¡0.05 4.09¡0.76 55.31¡26.25*** ### 5.25¡3.82
7.0 1.46¡0.06** 6.80¡1.07 54.20¡31.63* ### 24.12¡16.44
7.5 1.59¡0.15*** 8.32¡4.46## 79.63¡17.19*** ### 30.06¡25.58#

DCells were grown in 16YNB minimal medium at 30 8C (yeast growth) or 37 8C and 5 % CO2 (filamentous growth) with 2 % glucose or 2 % lactate
as the carbon source.
dFilamentation rate (percentage of hyphal and pseudohyphal cells on total cell count) was determined after 6 h.

dependent and was thus significantly reduced at strongly
acidic pH (pH 3) and neutral/alkaline pH (pH 7.5) (Table
1). Of note, a combination of lactate and glucose led to a sig-
nificant reduction in yeast growth compared with glucose
alone (Fig. 1).
Induction of filamentous growth was analysed under
hyphae-inducing conditions (37 uC and 5% CO2 on plastic).
As observed previously (Biswas et al., 2007), pH values
below 6.0 did not support filamentation of C. albicans,
whilst filaments (hyphae and pseudohyphae) were present
at pH 6.0, 7.0 and 7.5 after 6 h (Table 1). This was also true
after 3 and 8 h (data not shown). The filamentation rate
(percentage of cells forming filaments) in lactate-containing
0.3
Growth (OD600)
media was much lower than in glucose-containing media
(Table 1), and filaments were generally much shorter
(16.0+ 6.9 mm for lactate; 46.5 + 9.9 mm for glucose after
6 h, pH 7.0). A combination of glucose and lactate, how-
ever, allowed similar filamentation as with glucose alone
(data not shown).
Taken together, lactate as the sole carbon source did not
promote fast yeast growth or induce significant filamenta-
tion. In combination with glucose, lactate had a slight
inhibitory effect on yeast growth. Changes in pH were
well tolerated by glucose-grown yeasts but led to decreased
growth with lactate at very low or high pH values.
Influence of lactate and pH on growth inhibition
and killing of C. albicans by clotrimazole
As shown in our previous studies, clotrimazole levels in the
low micromolar range reduce yeast as well as filament
growth in a fungistatic fashion, by inhibition of ergosterol

***
2 % Glucose biosynthesis (Wächtler et al., 2011). Clotrimazole concen-
0.2
2 % Glucose + trations above 100 mM are known to have fungicidal effects
2 % lactate on yeasts, probably by a distinct mechanism independent
0.1
2 % Lactate
of ergosterol biosynthesis via direct physicochemical mem-
brane damage (Iwata et al., 1973; Mendling et al., 1982; Sud
0.0 & Feingold, 1981). By affecting C. albicans growth and
0 2 4 6 8 10 12 14 16 18 20 22
Time (h) morphology, lactate may interfere with these fungistatic
and fungicidal drug actions of clotrimazole.

Fig. 1. Growth with lactate as the sole or combinatory carbon

To determine the effect of pH and carbon source on the
source. Logarithmic C. albicans pre-cultures were inoculated in 16 fungistatic activity of clotrimazole, we recorded growth
YNB medium with 2 % glucose, 2 % lactate or 2 % glucose+2 % curves in the presence of 1, 10 or 100 mM clotrimazole
lactate to an OD600 of 0.05. Growth was monitored at 30 8C. The (Fig. 2a). Cell densities reached in the absence of the
OD600 is shown after subtraction of initial OD600 at time point 0 h. drug were lower in lactate-grown cultures than in glu-
Significant differences in OD600 (***P,0.001) between 2 % cose-grown cultures, consistent with the slow growth rate
glucose+2 % lactate and 2 % glucose were observed at time with lactate as a carbon source (Table 1, Fig. 1). In the pre-
points 5 to 22 h. sence of glucose, a significant growth reduction compared
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 Determinants of C. albicans clotrimazole sensitivity

(a)
Glucose Lactate
0.4 0.4
pH 3.0
*** pH 3.5
0.3 0.3

Growth (OD600)
Growth (OD600)

pH 4.0
***

**
*** pH 4.5
0.2 *** 0.2 pH 5.0

**
*** **
***
pH 6.0
0.1 0.1 pH 7.0
pH 7.5
0.0 0.0

µM

µM
µM

µM
SO

SO
µM

µM
d

ed
te

at
DM

DM
0

0
10

10
a

1
10

10
tre

tre
Un

Un
(b)

pH 4.5 pH 7.5
1000 1000 DMSO
### ###
*** *** 100 µM
** # 400 µM
*** #
Survival (%)

Survival (%)

100 *** 100 ** **

***

10 10

1 1
se

se

e
e

ce

ce
at

at
at

at
co

co
ur

ur
ct

ct
ct
ct

so

so
lu

lu
La

La
la
la
G

+
+

n
o

o
se

e
rb

rb
s
ca

ca
co

co
lu

lu
o

o
G

G
N

Fig. 2. Effect of carbon source and pH on growth inhibition of C. albicans by clotrimazole. (a) Logarithmic C. albicans pre-
cultures were inoculated in 16 YNB medium with 2 % glucose or 2 % lactate to an OD600 of 0.05. Growth was monitored in
the presence of 1, 10 or 100 mM clotrimazole or DMSO (vehicle control) at 30 8C to promote growth in the yeast form.
Shown is the difference in the maximum and minimum OD600 reached during 24 h incubation. (b) Logarithmic C. albicans
pre-cultures were inoculated in 16 YNB medium with 2 % glucose, 2 % lactate, 2 % glucose+2 % lactate or without a car-
bon source, buffered to pH 4.5 or 7.5, to a starting cell density of 56105 ml21. Cells were incubated for 5 h at 30 8C with
100 or 400 mM clotrimazole or DMSO. Shown are survival rates on a logarithmic scale, calculated from c.f.u. counts of yeasts
plated before and after clotrimazole treatment. Significant differences: **P,0.01, ***P,0.001, compared with DMSO controls
at the respective pH; #P,0.05, ###P,0.001, compared with pH 4.5 as a typical pH of the vagina.

with untreated control samples was evident at all three clotrimazole, expected to be in the lower limit of fungicidal
tested clotrimazole concentrations. Lactate-containing cul- drug action, and 400 mM clotrimazole, expected to have
tures showed a growth reduction for clotrimazole concen- clear fungicidal effects (Sud & Feingold, 1981). C.f.u. num-
trations of 10 and 100 mM but not for 1 mM. Similar trends bers after treatment were compared with the initial inocu-
were seen for the different pH conditions. lum to detect possible fungicidal drug effects (c.f.u. counts
To determine whether the fungicidal action by clotrimazole lower than that of the inoculum; % survival v100%).
on yeast cells was influenced by lactate or pH, we Yeast proliferation (% survival w100%) was observed in
monitored C. albicans survival by recording the number control samples containing no drug in the presence of glu-
of c.f.u. by plating cells recovered after 5 h incubation cose as carbon source (glucose or glucose+lactate) but not
with clotrimazole. As relevant concentrations with clearly in the presence of lactate alone or without any carbon
discernible effects on fungal growth, we chose 100 mM source (DMSO-treated samples; Fig. 2b). Treatment with
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L. Kasper and others

100 mM clotrimazole was not sufficient to cause fungal kill- with a physiological pH of 4.0–4.5 (Linhares et al., 2011).
ing at pH 4.5 and even allowed yeast proliferation in glu- To determine the influence of physiological and altered
cose-containing samples. However, a low fungicidal effect lactate concentrations in this system, we modified the
was observed in samples buffered to pH 7.5. In contrast, VSM-typical lactate concentration of 22 mM to 0, 22,
400 mM clotrimazole caused clear fungicidal effects inde- 50 or 100 mM. Experiments were performed at 37 uC
pendent of pH and carbon source. under vagina-typical gas conditions (6% CO2 and 7% O2)
These data showed that growth with either glucose or lac- (Sosinska et al., 2008) and were compared with atmospheric
tate as a carbon source can be inhibited by clotrimazole. gas conditions (*0.04% CO2 and *21% O2). As described
Furthermore, the sensitivity of C. albicans to the fungicidal previously, under these conditions, the cells grew pre-
activity of clotrimazole was not overly influenced by lactate dominantly in non-hyphal forms (Sosinska et al., 2008).
in standard cultivation medium. We monitored fungal survival (i.e. fungicidal drug activity)
by recording the number of c.f.u. from cells recovered
Lactate affects clotrimazole sensitivity of after 5 h treatment with clotrimazole and comparing
C. albicans under vagina-simulative conditions with the number of c.f.u. of the initial inoculum. Similar
to our experiments in 1|YNB medium with comparable
In vitro experiments often do not reflect key physiological pH (Fig. 2b, pH 4.5), we observed a low inhibitory effect
aspects of the in vivo setting. In the vaginal cavity, for of 100 mM clotrimazole when incubated under atmospheric
example, other nutrients and carbon sources besides gas conditions. Importantly, under vagina-simulative gas
glucose and lactate are likely to affect C. albicans growth, conditions, the sensitivity of C. albicans to the drug was
morphology and drug sensitivity. To improve analysis of
increased (Fig. 3). Survival was lowest with 22 mM lactate.
clotrimazole sensitivity in an in vitro system, we made
In addition, vagina-typical lactate levels promoted growth
use of a VSM, mimicking the physiological conditions
in DMSO-treated control samples, as cells in medium
during growth in the vaginal cavity. The medium compo-
with 22 mM lactate reached the highest viable cell count
sition is based on known concentrations and constituents
under both gas conditions. Treatment with 400 mM clotri-
of the vaginal fluid of healthy pre-menopausal women
mazole led to complete killing of the yeast independent of
(Owen & Katz, 1999; Sosinska et al., 2008). Besides lactate
lactate or gas conditions.
and glucose, the medium contains glycerol and acetic acid
as potential carbon sources for C. albicans, and is buffered In conclusion, under in vitro vagina-simulative conditions,
to a pH of 4.2. This pH reflects the expected situation lactate had a positive effect on the fungicidal action of
during vaginal infection, as Candida vaginitis is associated clotrimazole.

VSM atmospheric VSM 6 % CO2 7 % O2

1000 1000
### DMSO
### 100 µM
100 *** 400 µM
Survival (%)

Survival (%)

# 100
*
* *
10 10
***

*** *** *** *** *** ** **

0 0 0 0 0 0 0 0
1 1
te

te

te

e
at

at

at

at

at
ta

ta
ct

ct

ct

ct

ct

ct
ac

ac
la

la

la

la

la

la
tl

tl
M

M
ou

ou
m

m
ith

ith
22

50

22

50

0
W

W
10

10

Fig. 3. Clotrimazole sensitivity of C. albicans under vagina-simulative conditions. Logarithmic C. albicans pre-cultures were
inoculated in VSM with 0, 22, 50 or 100 mM lactate at a starting cell number of 56105 ml21. Clotrimazole was added at a
concentration of 100 or 400 mM. Control samples contained DMSO (vehicle control). Cells were incubated for 5 h at 37 8C
under atmospheric (,0.04 % CO2 and ,21 % O2) or vagina-typical (6 % CO2, 7 % O2) gas conditions. Shown are survival
rates on a logarithmic scale, calculated from c.f.u. counts of yeasts plated before and after clotrimazole treatment. Significant
differences: *P,0.05, **P,0.01, ***P,0.001 compared with untreated controls at the respective lactate concentration;
#
P,0.05, ###P,0.001 compared with corresponding sample without lactate.
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 Determinants of C. albicans clotrimazole sensitivity

(a)
pH 4.5 pH 7.5
1000 1000 DMSO
# 100 µM
*** ##
* * 400 µM
Survival (%)

Survival (%)
100 100

10 10

1 1
e

e
te

te
e

ce

ce
os

os
at

at
ta

ta
ur

ur
ct

ct
c

c
c

c
so

so
lu

lu
la

la
La

La
G

G
+

+
on

on
e

e
rb

rb
os

os
ca

ca
c

c
lu

lu
o

o
G

G
N

N
(b)

pH 4.5 pH 7.5
1000 1000 ### DMSO
*** *** 100 µM
### 400 µM
Survival (%)

Survival (%)

100 100
*

10 *** 10 ### ###

*** ***
***
0 0 0 0 0
1 1
se

ce

se

ce
at

at

at

at
co

co
ur

ur
ct

ct

ct

ct
so

so
lu

lu
la

La

la

La
G

G
+

+
on

on
se

se
rb

rb
co

co
ca

ca
lu

lu
o

o
G

G
N

Fig. 4. C. albicans growth phase affects clotrimazole sensitivity. Post-exponential (a) or logarithmic (b) C. albicans pre-
cultures were inoculated in 16 YNB medium with 2 % glucose, 2 % lactate, 2 % glucose+2 % lactate or without a carbon
source, buffered to pH 4.5 or 7.5, at a starting cell density of 56105 ml21. Cells were incubated for 5 h with 100 or
400 mM clotrimazole or DMSO at 30 8C (a) or 37 8C (b). Shown are survival rates on a logarithmic scale, calculated from
c.f.u. counts of yeasts plated before and after clotrimazole treatment. Significant differences: *P,0.05, ***P,0.001, com-
pared with DMSO controls at the respective pH; #P,0.05, ##P,0.01, ###P,0.001, comparing samples at pH 4.5 and 7.5.

Clotrimazole sensitivity depends on growth phase presented in Fig. 2(b) were with logarithmic pre-cultures.
and morphology In contrast, with yeasts pre-grown to post-exponential phase,
At the onset of treatment of VVC, the vaginal C. albicans clotrimazole was not fungicidal under any of the tested
population is likely to consist of a mixture of cells in carbon sources and pH conditions at 30 uC (Fig. 4a).
different growth phases (actively growing or stationary To test the influence of fungal morphology, cells were
phase cells) and morphologies (yeast or hyphal growth). cultured under conditions that promote hypha forma-
We therefore investigated the effect of lactate on clotrima- tion (pH 7.5, 37 uC) in the absence or presence of clotri-
zole antifungal activity against different growth phases and
mazole. Strikingly, under these conditions, clotrimazole
morphologies of C. albicans. As C. albicans grows predomi-
exhibited exceptionally high fungicidal activity (Fig. 4b,
nantly in non-hyphal forms in VSM medium (Sosinska
et al., 2008), we used standard YNB-based minimal right panel). Parallel samples at pH 4.5 (Fig. 4b, left
medium for these experiments. C. albicans cells were incu- panel) showed no such fungicidal effect at 100 mM clotri-
bated in the respective media in the presence or absence of mazole but, compared with incubation at 30 uC (Fig. 2b,
clotrimazole, and survival was monitored by recording left panel), a higher sensitivity to 400 mM clotrimazole
c.f.u. from cells recovered after 5 h incubation. The fungi- was observed. The differences in drug sensitivity were inde-
cidal effects of 400 mM clotrimazole on yeasts at 30 uC pendent of the presence of lactate.
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L. Kasper and others
Taken together, drug sensitivity was strongly affected by
growth phase and temperature, with increased sensitivity
of logarithmic-phase cells compared with post-exponential-
phase cells and stronger fungicidal effects at 37 uC. Maximum
sensitivity to clotrimazole was achieved under conditions
promoting hyphal growth.
Inhibition of hyphal elongation and maintenance
by clotrimazole
As hyphal morphology plays a critical role in the pathogen-
esis of candidal vaginitis (Sobel et al., 1984), we aimed
to directly study the effect of clotrimazole on hyphal
growth. Previous experiments focused on the induction of
filamentation in the presence of the drug (Odds et al.,
1985; Wächtler et al., 2011). However, during treatment of
VVC, a significant portion of the C. albicans population
has probably already formed hyphae. We were thus inter-
ested in the impact of clotrimazole on pre-formed hyphae.
We incubated C. albicans under hyphae-inducing conditions
(on plastic, 37 uC, neutral pH) in 1|YNB medium with 2%
glucose or in strong hyphae-inducing RPMI with 10% fetal
bovine serum (RPMI+FBS). After an initial 3 h incubation
in the absence of drug, the formed hyphal cells were exposed
to clotrimazole or DMSO (control) for 3 h. Fresh medium
without clotrimazole was then added to follow the resump-
tion of hyphal growth after treatment for a further 3 h.
Experiments in 1|YNB medium showed that even low clo-
trimazole concentrations (1 mM) were sufficient to inhibit
hyphal elongation. Hyphal length was significantly reduced
in clotrimazole-treated samples as compared with DMSO
controls (Fig. 5a, c). Interestingly, following the removal
of clotrimazole (post-incubation 3 h), growth inhibition
was maintained. Low azole concentrations (1 and 10 mM)
permitted very low levels of hyphal elongation (24 mm at
the end of the pre-incubation to 39 mm after 3 h in the
presence of 1 mM clotrimazole), whilst 100 mM clotrimazole
completely blocked hyphal elongation. This, in addition to
the results obtained for clotrimazole-mediated killing
under similar conditions (Fig. 4b), suggested that 100 mM
clotrimazole may be fungicidal to C. albicans hyphae.
Experiments in RPMI+FBS gave similar results. Again, a
growth-inhibitory effect of low clotrimazole concentrations
was observed, whilst 100 mM clotrimazole completely blocked
hyphal growth (Fig. 5b, d). Hyphae formed in RPMI+FBS
were longer than in 1|YNB medium, and hyphal branching
was observed regularly (Fig. 5d). Interestingly, treatment with
1 or 10 mM clotrimazole reversed the hyphae growth pro-
gramme and induced growth of secondary yeast cells (yeast
cells that originate from hyphal cells; Fig. 5d, lower panels).
Taken together, these data showed that low clotrimazole
concentrations are sufficient to inhibit filament elongation
when added to hyphal cells, and can even induce reversion
to yeast morphology. Higher clotrimazole concentrations
(100 mM) completely block hyphal elongation, possibly due
to direct fungicidal action on hyphal cells.
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DISCUSSION
Influence of lactate on C. albicans growth and
clotrimazole sensitivity
The presence of lactate, either as a natural compound of
the vaginal fluid or as an ingredient in drug formulations,
can potentially alter the clotrimazole sensitivity of
C. albicans. These effects could be due to lactate-induced
pH changes or utilization of lactate as a carbon source.
The data presented here showed that, compared with glu-
cose, lactate is a poor carbon source for C. albicans, support-
ing only slow growth and not promoting a significant shift to
hyphal growth at neutral pH. Slow growth, especially at a pH
below 4.0, as well as slight inhibition of growth in glucose
cultures with added lactate can possibly be explained by
weak acid stress in presence of undissociated lactic acid
(pKA53.86) (Davis, 2009). Besides the effect of lactate on
fungal growth described here, Ene et al. (2012a) showed lac-
tate-dependent changes to C. albicans cell-surface proper-
ties, stress resistance and drug sensitivity.
We did not detect any influence of lactate on clotrimazole
sensitivity, either in the form of growth inhibition or as
fungal killing, in standard culture medium (1|YNB).
Additionally, under yeast growth-favouring conditions
(30 uC), the medium pH had little influence. This is in
agreement with previous studies that showed C. albicans
susceptibility to azoles at neutral but also low pH (Danby
et al., 2012).
In contrast, cultivation in VSM, especially under physiologi-
cal gas conditions of 6% CO2 and 7% O2, increased the sen-
sitivity of C. albicans to clotrimazole in the presence of
lactate. These data showed that clotrimazole is more effec-
tive in an environment that mimics the physiological con-
ditions during vaginal colonization than in standard
laboratory growth medium. Similar tendencies were found
for fluconazole in a previous study (Moosa et al., 2004).
One possible reason may be a synergistic action of lactate
with the acetic acid present in VSM, by causing weak acid
stress (Costa et al., 2013; Moosa et al., 2004), which may
potentiate the drug-generated stress. In addition, different
fungal replication rates in vagina-simulative compared
with standard atmospheric gas conditions, as described by
Sosinska et al. (2008), may influence drug sensitivity.
Consequently, we showed that lactate as a supplement of
clotrimazole drug formulations can be beneficial not only
for drug solubility but also by providing an additive posi-
tive effect by the inhibition of C. albicans growth. These
in vitro data can be seen as a basis for future in vivo ana-
lyses, for example in a mouse model of vaginal candidiasis
(Stevens et al., 2002), to analyse whether the additional
beneficial effect of lactate holds true in an in vivo setting
and to obtain more information on the general suitability
of lactate addition to vaginal clotrimazole preparations
for the treatment of patients.
Journal of Medical Microbiology 64
 Determinants of C. albicans clotrimazole sensitivity

(a)
YNB glucose pH 7.5 (b) RPMI + FBS
200 200
### Pre-incubation 3 h
Hyphal length (µm)

Hyphal length (µm)

150 Clotrimazole treatment 3 h
150 Post-incubation 3 h
###
100 ### 100 ###
###
*** ***
50 * ** *** *** 50
###

*** ***
*** ND
0 0
D ion
SO

10 M
1 µM
st µ M

st O
Po st-1 M
-1 µ M
µM

D on
SO

10 M
1 µM
st µ M

st O
Po st-1 M
-1 µ M
µM
µ

Po MS

Po MS

µ
i
at

at
M

M
1

Po 00

Po -1

st 0
00

Po 00

Po -1

st 0
00
ub

ub
-D

-D
c

c
in

in
e-

e-
Pr

Pr
(c)
YNB glucose pH 7.5
Pre-incubation
3h
Clotrimazole
treatment
3h
Post-incubation
3h

Untreated/DMSO 1 µm clotrimazole 10 µM clotrimazole 100 µM clotrimazole

(d) RPMI + FBS

Pre-incubation
3h
Clotrimazole
treatment
3h
Post-incubation
3h

Untreated/DMSO 1 µm clotrimazole 10 µM clotrimazole 100 µM clotrimazole

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L. Kasper and others

Fig. 5. Clotrimazole inhibits hyphal elongation and maintenance. Overnight C. albicans pre-cultures were inoculated in
16 YNB medium with 2 % glucose at pH 7.5 (a, c) or in RPMI with 10 % fetal bovine serum (RPMI+FBS) (b, d), at a start-
ing cell density of 46104 ml21 in 24-well-plates. After a pre-incubation for 3 h at 37 8C and 5 % CO2, clotrimazole (1, 10 or
100 mM) or DMSO (vehicle control) was added and cells were incubated for additional 3 h. After a washing step to remove
exogenous clotrimazole, cells were incubated for a further 3 h in fresh medium without the drug. Cells were fixed with 4 %
paraformaldehyde and analysed by inverse microscopy. (a, b) Quantification of hyphal length. Quantification was not possible
in RPMI+FBS post-incubation samples due to extensive branching (ND , not determined). Significant differences: *P,0.05,
###
**P,0.01, ***P,0.01, compared with DMSO-treated controls at the respective time points; P,0.001, compared with 3 h
sample. (c, d) Representative microscopic images. Examples of secondary yeast cells are indicated with white arrowheads.

Our observations are also relevant for understanding the
interplay of C. albicans with bacteria during vaginal coloni-
zation and disease. Lactobacilli have been shown to directly
inhibit C. albicans growth in vitro by producing lactic acid
(Boskey et al., 2001; Köhler et al., 2012). Several studies
have associated VVC with a low number of lactobacilli in
the vagina (Mendling & Brasch, 2012). In this setting,
one would expect lower levels of vaginal lactate, which
could in turn be reversed by administration of the lactate-
containing clotrimazole drug.
Influence of growth phase and morphology on
C. albicans clotrimazole sensitivity
At the onset of VVC treatment, the vaginal C. albicans
population is likely to consist of a mixture of cells in differ-
ent growth phases and in different morphological states.
These subpopulations may be differentially susceptible to
the fungistatic (inhibition of ergosterol biosynthesis) and/
or fungicidal action (direct physicochemical membrane
damage) of clotrimazole (Iwata et al., 1973; Sud & Feingold,
1981). Two earlier studies addressed this question, focusing
on distinct growth states. Whilst Niimi et al. (1985) found
an increased sensitivity of C. albicans hyphae compared
with stationary yeast cells to the fungicidal action of
clotrimazole, Beggs et al. (1987) described how stationary
cells are resistant to clotrimazole killing whilst logarith-
mic cells are killed effectively. In this study, we directly
compared the clotrimazole sensitivity of logarithmic-
and post-exponential-phase yeast cells, as well as hyphae.
Our data confirmed that fungal cells in the exponential
growth phase are much more sensitive to clotrimazole
than post-exponential-phase cells. Importantly, we demon-
strated that sensitivity of C. albicans to 100 mM clotrimazole
was strongly increased under hyphae-inducing conditions
(37 uC, pH 7.5). These data were supported by the complete
lack of elongation of hyphal cells treated with 100 mM
clotrimazole, again suggesting a direct fungicidal action
of clotrimazole against this C. albicans morphotype. Thus,
our data imply that clotrimazole-mediated killing depends
on both the growth phase and the morphological state of
C. albicans, with hyphal cells being the most sensitive.
Clotrimazole concentrations in the vaginal fluid during and
after treatment with the vaginal ovule are expected to be in
the fungicidal range (Mendling & Plempel, 1982). However,
drug levels inside vaginal epithelia encountered by invading
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fungal cells may be much lower. Interestingly, we observed
that even these low, non-lethal clotrimazole concentrations
strongly inhibited the elongation of pre-formed hyphae
and even induced a reversion to the yeast growth form.
One likely explanation for this phenomenon is an elevated
production of the quorum-sensing molecule farnesol due
to drug treatment (Hornby & Nickerson, 2004). Inhibition
of hyphal formation from yeast cells has been shown pre-
viously by addition of clotrimazole preceding the hyphae-
inducing conditions (Odds et al., 1985; Wächtler et al.,
2011). We showed here that inhibition of hyphal elongation
even occurred in C. albicans cells that had already undergone
the yeast-to-hypha transition. This is significant because, by
the time clotrimazole is administered therapeutically (i.e.
following the onset of symptoms), C. albicans cells will prob-
ably have already formed hyphae. Invasive hyphal growth
probably occurs concomitant with epithelial damage and
induction of a pro-inflammatory immune response during
acute vaginal C. albicans infection (Moyes et al., 2010).
As hyphae are more sensitive to the fungicidal action of
clotrimazole, and sublethal clotrimazole levels promote a
morphological transition from hyphae to yeast, we thus pro-
pose the following mechanisms for clotrimazole resolution
of acute vaginal infections: initial drug treatment, associated
with high levels of clotrimazole, reduces the relative pro-
portion of hyphae in the infecting C. albicans population
by selectively killing this cell type. In the vaginal micro-
environment, this strong fungicidal effect may be even
potentiated by the presence of lactate. Subsequent lower
levels of clotrimazole induce yeast over hyphal growth,
thus promoting the formation and maintenance of a non-
invasive commensal C. albicans population. This is associ-
ated with reduced vaginal cell damage and reduced inflam-
mation (Wächtler et al., 2011; Wilson et al., 2013). This is
associated with reduced vaginal cell damage and reduced
inflammation. In summary, the therapeutic activity of anti-
fungal drugs, such as clotrimazole, appears to be the net
result of a complex interplay of multiple factors, including
fungal killing, growth inhibition, morphological manipu-
lation and dampened inflammation.
ACKNOWLEDGEMENTS
This work was funded by Bayer Vital and supported by the resources
and facilities at the Hans Knoell Institute Jena, Germany. We thank
Melanie Kahl for excellent assistance with the laboratory work.
Journal of Medical Microbiology 64

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