Arch Dermatol Res
DOI 10.1007/s00403-016-1701-4
ORIGINAL PAPER
Ketoconazole inhibits Malassezia furfur morphogenesis in vitro
under filamentation optimized conditions
Sirida Youngchim1 • Joshua D. Nosanchuk2 • Siriporn Chongkae1
Nongnuch Vanittanokom1
Received: 19 May 2016 / Revised: 22 October 2016 / Accepted: 16 November 2016
 Springer-Verlag Berlin Heidelberg 2016
Abstract Malassezia furfur, a constituent of the normal
human skin flora, is an etiological agent of pityriasis ver-
sicolor, which represents one of the most common human
skin diseases. Under certain conditions, both exogenous
and endogenous, the fungus can transition from a yeast
form to a pathogenic mycelial form. To develop a stan-
dardized medium for reproducible production of the
mycelial form of M. furfur to develop and optimize sus-
ceptibility testing for this pathogen, we examined and
characterized variables, including kojic acid and glycine
concentration, agar percentage, and pH, to generate a
chemically defined minimal medium on which specific
inoculums of M. furfur generated the most robust fila-
mentation. Next, we examined the capacity of ketocona-
zole to inhibit the formation of M. furfur mycelial form.
Both low and high, 0.01, 0.05 and 0.1 lg/ml concentrations
of ketoconazole significantly inhibited filamentation at
11.9, 54.5 and 86.7%, respectively. Although ketoconazole
can have a direct antifungal effect on both M. furfur yeast
and mycelial cells, ketoconazole also has a dramatic impact
on suppressing morphogenesis. Since mycelia typified the
pathogenic form of Malassezia infection, the capacity of
ketoconazole to block morphogenesis may represent an
additional important effect of the antifungal.
Keywords Malassezia furfur  Morphogenesis 
Ketoconazole  Filament
& Sirida Youngchim
syoungchim@gmail.com
1
Department Microbiology, Faculty of Medicine, Chiang Mai
University, Chiang Mai 50200, Thailand
2
Department of Medicine (Infectious Diseases), Albert
Einstein College of Medicine, Bronx, NY 10461, USA
•
Introduction
The lipophilic yeast Malassezia furfur is a member of the
normal microbiota of human skin. Although M. furfur is
typically a harmless commensal, it has also been associated
with an array of diseases, ranging from the pigmentation
disorder pityriasis versicolor to atopic dermatitis and
catheter-associated sepsis [4, 12, 16]. Several environ-
mental conditions, such as increased temperature and/or
humidity, are associated with an increased incidence of
disease and these environmental alterations are linked to
changes in sweat production that promotes M. furfur
growth [2, 17, 33].
M. furfur is categorized as a dimorphic fungus as it can
change morphology from a yeast form to a mycelial phase
[10]. During human commensal conditions and in standard
cultures, M. furfur primarily exists in the yeast form, which
is considered the saprophytic phase. The simultaneous
presence of hyphal elements and budding yeast cells is
characteristic of the parasitic or infectious stage, such as
found in tinea versicolor. The hyphal stage is believed to
play a predominant role in the pathogenesis of pityriasis
versicolor as hyphae are abundantly present in diseased
tissues from patients with this disorder [10, 24].
As part of our efforts to develop a new reproducible
methodology for defining susceptibilities for M. furfur, we
have focused on defining a medium on which the fungus
has the highest frequency of filamentation. We recently
demonstrated that mycelial growth can be reproducibly
achieved on minimal medium (MM) with L-3,4-dihydrox-
yphenylalanine (L-DOPA) and kojic acid, a tyrosinase
inhibitor [38]. In the present work, we additionally defined
the optimal concentration of glycine, agar percentage and
pH for the assay medium as well as determined the most
effective inoculum to maximize mycelia growth. Although
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several azoles are effective in routine clinical treatment of
M. furfur, ketoconazole remains a frequent standard choice
for the topical treatment of Malassezia-related conditions
[8]. In addition, ketoconazole is currently used in various
common topical over-the-counter preparations, including
foams, gels, and creams [8, 32]. Several studies have
reported that ketoconazole’s in vitro activity against
Malassezia spp. is extremely potent compared to the
majority of azole derivatives [3, 11, 28, 37]. Hence, we
examined the impact of ketoconazole on filamentation on
our optimized medium and demonstrated that this anti-
fungal significantly inhibits morphogenesis. In sum, we
show that our optimized defined medium reproducibly
permits a high degree of filamentation of M. furfur and that
this medium can be used for characterizing growth effects,
such as the inhibition of morphogenesis with ketoconazole,
which suggests that this medium may be a platform for a
reproducible and clinically valuable susceptibility
methodology.
Materials and methods
Fungal strain and media
Under an approved protocol for collecting microbial iso-
lates, M. furfur MM 2557 was isolated from the skin
scrapings of a patient with pityriasis versicolor in the
Dermatological Clinic, Maharaj Nakorn Chiang Mai
Hospital, Chiang Mai University, Thailand. M. furfur was
identified by biochemical characteristics, molecular bio-
logical technique [13] and matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF
MS) [20]. Malassezia yeast cells were then maintained by
monthly subculture on modified Dixon medium (mDixon:
1 l of distilled water; 36 g malt extract; 6 g peptone and
20 g ox bile (all obtained from Difco); 10 ml Tween 40,
2 ml glycerol, 2 ml olive oil (all obtained from Sigma);
0.05 gm chloramphenicol (Amresco); and 15.0 gm agar
(Difco) with a pH 6.0 [14]. To prepare inoculum, M. furfur
was grown on modified Dixon agar for 3 days at 32 C,
harvested and suspended in 3 ml of sterile distilled water
followed by dilution to the desired concentrations.
A defined cultural medium for mycelial production
in M. furfur
To induce mycelial production, M. furfur was cultured on
variations of our previously described chemically defined
minimal medium (MM) agar (containing 50 mM glycine,
10.0 mM MgSO4 and 29.4 mM KH2PO4, pH 4.6) supple-
mented with lipids, 1.0 mM L-DOPA with 1000 lg/ml
kojic acid [38]. In these studies, we determined the optimal
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Arch Dermatol Res
condition to efficiently and reproducibly trigger mycelia
production in M. furfur by adjusting the inoculum and
concentrations of glycine as well as varying the pH of the
assay medium and the percentage of agar. The cultures
were incubated at 30 C for 5 days under microaerophilic
conditions (6–12% of O2) in an anaerobic plastic jar with a
microaerobic atmosphere generating system (Pack Micro-
Aero, Mitsubishi Gas Chemical Co. Inc).
Yeast suspensions of M. furfur were prepared at 105, 106
and 107 cells/ml and a volume of 0.3 ml of each concen-
tration was applied on the mycelial induction medium and
then incubated under microaerophilic conditions. Addi-
tionally, medium was prepared with glycine at 10, 50, 100,
200 mM and kojic acid was adjusted to 500, 1000 and
1500 lg/ml. The pH values of 4.6 and 5.5 were compared.
Finally, agar concentrations between 0.5 and 2.0% were
tested.
After the incubation at 30 C for 5 days, the plates were
observed for M. furfur mycelial synthesis under a light
microscopy (magnification 10009). For each condition, a
total of 1000 cells, yeast and hyphae, were counted to
determine the percentage of each as well as the length of
hyphal filamentation using the NIS-D imaging software.
All of the experiments were done twice with triplicate
repeated measurements.
In vitro effect of ketoconazole on filament inhibition
of M. furfur
To study the inhibitory effect of ketoconazole on mycelial
production, different concentrations of ketoconazole, 0.01,
0.05 and 0.1 lg/ml, were added to the optimized mycelial
induction medium, and then 0.3 ml of the optimized con-
centration of M. furfur were inoculated onto this medium.
The plates were incubated at 30 C for 5 days under
microaerophilic conditions. Controls plates were made
with DMSO at the concentration equivalent to that required
to dissolve ketoconazole were included in our assays.
These experiments were done twice with triplicate repeated
measurements. To confirm the effect of ketoconazole on
mycelial inhibition, two more isolates of M. furfur NBRC
0656 and M. furfur 133 isolated from healthy volunteer
were included in the study.
For each concentration of ketoconazole, the plates were
observed by light microscopy and the percentages of fila-
ment inhibition in M. furfur with or without ketoconazole
were calculated. The percentage of filament inhibition was
analyzed as follow:
% of filament inhibition
 
1  % of filament production of test
¼ 100 
% of filament production of control
Arch Dermatol Res
In vitro susceptibility testing
The susceptibility of M. furfur to ketoconazole was deter-
mined by following the standard method of CLSI M27-A3
with modifications by Rojas et al. [29] to support the
growth of lipophilic yeast. The assay medium was RPMI
1640 (Sigma-Aldrich, USA) containing 1.8% glucose
(Difco), 1% peptone (Difco), 0.5% ox bile (Difco), 0.5%
malt extract (Difco), 1% glycerol (Sigma-Aldrich, Ger-
many), 0.5% Tween 40 (Sigma), 0.05% Tween 80 (Sigma),
and 250 mg/l chloramphenicol (Amresco). Briefly, the
inoculum of M. furfur was prepared in sterile saline solu-
tion and adjusted by spectrophotometry at 530 nm to get
107 CFU/ml and then diluted in 1:100 with the assay
medium to get a final concentration of 0.5–2.5 9 105 CFU/
ml, as confirmed by viable colony counts on modified
Dixon agar. A volume of 100 ll of M. furfur suspension
was added into a 96-well microtiter plate containing 100 ll
aliquots of ketoconazole at two times of the final drug
concentrations ranged from 0.003 to 16 lg/ml. The test
microtiter plate was incubated for 4 days at 32 C. The
MIC endpoint for ketoconazole was defined as the lowest
concentration at which there was a C50% inhibition of
growth as compared with the growth control (drug-free).
Candida albicans ATCC 900028, quality control strain,
was included to confirm the reproducibility of the result.
Statistical analysis
The results presented are the means with standard errors.
Statistical analyses were carried out using one-way analy-
sis of variance (ANOVA). A p value\0.05 was considered
statistically significant.
Results
Optimal condition to induce mycelial production
in M. furfur
In our study, certain key factors impacting mycelial pro-
duction of M. furfur were investigated, including the
inoculum of M. furfur, the concentrations of glycine and
kojic acid, the pH of the assay medium and the percentage
of agar. For the inoculum of M. furfur, a concentration of
107 cells/ml produced the highest percentage of filament
production compared to concentrations of 105 and 106 -
cells/ml in all of concentrations of kojic acid, 500, 1000,
and 1500 lg/ml (Fig. 1a), which similarly increased fila-
mentation in a dose dependent manner for each inoculum.
However, no significant differences were found at
107 cells/mL of inoculum when compared between 1000
and 1500 lg/ml of kojic acid. Therefore, a 107 cells/ml of
inoculum and 1000 lg/ml kojic acid were then used in the
following experiments. Moreover, the addition of higher
concentrations of glycine demonstrated significantly
increasing percentages of filament synthesis in M. furfur,
except at 50 mM glycine which showed no significant
difference compared to 100 mM glycine (Fig. 1b). Based
on this result, the concentration of glycine was used at
50 mM for our next study. For kojic acid, filamentation
was significantly increased at concentrations of 100 and
1000 lg/ml at pH 4.6 compared to pH 5.5 (Fig. 1c). In
contrast, at 250 lg/ml of kojic acid, there was a signifi-
cantly higher percentage of filamentation at pH 5.5. We
also examined the effect of varying the agar concentration
from 0.5 to 2.0%. The percent of filamentation was sig-
nificantly higher in 0.5% agar compared to the other con-
centrations (Fig. 1d). Moreover, the length of filaments
was increased in 0.5% agar compared to the higher agar
concentrations (Fig. 1e). This determination was made by
dividing the filaments into three categories, \10, 10–20
and 20–40 lm. Concomitant with the increase in longer
filaments in the 0.5% agar, this condition also reduced the
number of filaments \10 lm in length compared to the
other conditions. There were no significant differences in
the percentages of filaments 10–20 lm in length at the
concentrations examined.
In vitro effects of ketoconazole on filament inhibition
of M. furfur
We used the optimized MM (containing 50 mM glycine,
10.0 mM MgSO4 and 29.4 mM KH2PO4, pH 4.6) con-
sisting of lipid supplements and 1.0 mM L-DOPA with of
1000 lg/ml kojic acid in 0.5% agar and used an inoculum
of 0.3 ml of a yeast suspension consisting of 107 M. furfur
yeast cells for assessing the effects of ketoconazole on
filamentation of M. furfur. Significantly, the results of our
study clearly establish that ketoconazole dramatically
impedes formation of mycelia by M. furfur. Inhibition was
dose dependent with 0.1 lg/ml ketoconazole having the
greatest effect, resulting in 87.4% reduction in hyphal
formation (Figs. 2, 3). In addition, ketoconazole at 0.1 lg/
ml inhibited the mycelial synthesis in M. furfur NBRC
0656 and M. furfur 133 at 80.74 and 78.17%, respectively.
DMSO, the diluent for ketoconazole, had no effect on the
mycelial transformation of M. furfur (data not shown).
In vitro susceptibility testing
Both MIC50 and MIC90 of values obtained for ketoconazole
against M. furfur were 0.0625 lg/ml.
123

Fig. 1 The effect of different concentrations of inocula and kojic acid
(a), glycine (b), pH (c), and agar (d, e) on filament production of M.
furfur MM 2557 in microaerobic conditions. Each bar represents the
mean ± SEM of three independent experiments, each performed in
duplicate. In a *p \ 0.05 comparing an inoculum of 0.3 ml of 107 M.
furfur to conditions using 105 and 106 cultured on the same
concentration of kojic acid. b *p \ 0.05 comparing M. furfur
filamentation at 50 mM glycine compared to 10 and 200 mM glycine
but no significant difference (p [ 0.05) was found at the
Discussion
PV is a chronic superficial fungal infection caused by
Malassezia spp., it is still unclear which species are asso-
ciated. Although most studies carried out in recent years
confirmed M. globosa as the predominating species found
in PV lesions, at least in temperate climates [4, 26].
According to geographical region with variation in the
distribution of Malassezia species, M. furfur appears to be
the most common one of PV in the tropics [23], such as
Indonesia [21], India [34] and Thailand [18].
M. furfur is an anthropophilic fungus of the normal
physiological human skin, but under certain circum-
stances it manifests as a pathogenic agent of several skin
disorders. Environmental factors, such as high tempera-
ture and relative humidity, and endogenous factors, such
as greasy skin, sweating, heredity and
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Arch Dermatol Res
concentrations of 50 mM glycine and 100 mM glycine. c An asterisks
indicates a p value of \0.05 for a comparison of the percentage of
filaments between pH 4.6 and pH 5.5 at the same concentration of
kojic acid. d *p \ 0.05 comparing the percentage of filaments on
0.5% agar with that on 1.0, 1.5 and 2.0% agar. e p \ 0.05 comparing
the percentages of 20–40 lm filaments on 0.5% agar to those on 1.0,
1.5 and 2.0% agar, while §p \ 0.05 comparing the percentages of
\10 lm filaments between 0.5% agar and 2.0% agar
immunosuppressive conditions, have been suggested as
playing a role in facilitating the transformation of M.
furfur from the saprophytic phase to the parasitic phase
[12, 25]. Although our results reveal a correlation
between inoculum size and mycelial production in M.
furfur in vitro, a prior report has shown that Malassezia
density in pityriasis versicolor patients was similar to
that measured in healthy control subjects [15], suggest-
ing that this disease may be related to impaired host
responses to the organism rather than its overgrowth.
However, based on the sampling method this report
used, the contact plates collected only Malassezia on the
superficial skin, which likely underestimated the myce-
lial forms of Malassezia that are typically present in the
deeper layers of the skin in this disease. Hence, future
studies are required to accurately define the impact of M.
furfur burden on disease induction and progression in the
Arch Dermatol Res

previously reported that humidity enhances the volume

Fig. 2 The effect of ketoconazole on different concentrations, 0.01,
0.05 and 0.1 lg/ml on inhibition of M. furfur MM2557 filamentation.
Each bar represents the mean ± SD of three independent experi-
ments, each performed in duplicate. *p \ 0.05 was compared with
control, without ketoconazole
various conditions associated with M. furfur. Humidity
can increase the volume of skin secretions, which in
turn, may stimulate M. furfur growth and morphogene-
sis. Humidity is presented as a major factor for the
increased prevalence of M. furfur cutaneous diseases in
the tropics and in the summer in more temperate cli-
mates [19, 27, 35], with an incidence rate as high as
40% in some areas [31]. Pérez Blanco et al. [25].
of skin secretions, which, in turn, may stimulate M
furfur growth and facilitate infection on the skin. Cor-
responding to the concept of higher humidity and M.
furfur filamentation, our studies clearly show that the
highest mycelial production in M. furfur occurs at 0.5%
agar in comparison to the higher percentages of agar
tested. Furthermore, supporting the impact of moisture
on this process, humidity was suggested to play an
important role in the mycelial induction of in Pity-
rosporum orbiculare and P. ovale [6].
Most studies have reported that the average pH of nat-
ural skin surface is 4.7, with a wide range from pH 4.0 to
7.0; skin with pH values below 5.0 provides a better milieu
for resident skin microflora to adhere [22]. Our present
study demonstrates the effect of pH on inducing filamen-
tation in M. furfur. Our finding that the defined chemical
medium at pH 4.6 induced the greatest degree of fila-
mentation is in line with the favorable value of acidic skin
pH that promotes the attachment of human skin microbiota.
The role of glycine in hyphal induction of M. furfur was
first reported by Dorn and Roehnert [5], but the detailed
condition was unclear. This is similar with a later finding
by Saadatzadeh et al. [30] who found that glycine was
more effective than other amino acids, such as methionine,
histidine or tryptophan, for the induction of mycelium in
M. furfur and M. obtusa. Additionally, glycine also

Fig. 3 The effect of

ketoconazole on M. furfur MM
2557. M. furfur was cultured in
MM with L-DOPA and
1000 lg/ml of kojic acid with
different concentrations of
ketoconazole. a Control
(without ketoconazole), b–
d ketoconazole at
concentrations 0.01, 0.05 and
0.1 lg/ml, respectively. Bar
represents 5 lm

123

effectively induced hyphal formation in M. globosa [36].
Currently, we confirmed that glycine can be involved in
mycelial production of M. furfur in a dose dependent
manner. Glycine, a common nitrogen source in the skin,
was formerly shown to stimulate rapid growth leading to an
exponential increase in biomass of M. furfur [1]. Hypo-
thetically, glycine appears to have an indirect effect on
mycelial production by increasing the biomass and then
enhancing filamentation in M. furfur.
Malassezia yeast cells can produce melanin both in vitro
and during infection, as demonstrated by Masson-Fontana
silver staining [9] and labeling with a melanin-specific
monoclonal antibody (MAb) [38]. In addition, L-DOPA, a
phenolic substance, was earlier confirmed to be an
important compound involved in both melanization and
yeast-mycelial transformation in M. furfur. Based on
mycelial induction, kojic acid also affected filamentation in
M. furfur in a dose dependent manner, which confirmed in
our previous study [38].
In the present study, ketoconazole was significantly
effective in interfering with the production of hyphae in M.
furfur. Similarly, the study of Faergemann et al. [7] pre-
viously demonstrated that 1.0 lg/ml ketoconazole reduced
the production of hyphae in M. sympodialis from 15 to 2%
using human stratum corneum in vitro. In addition to
ketoconazole, an investigational triazole R126638, at
concentration 1.0 lg/ml, also decreased mycelial synthesis
of Malassezia [7]. However, our newly established
methodology is significantly easier to reproduce and per-
mits greater throughput, which makes it an exciting plat-
form for future studies of this phenomenon. Nevertheless,
these findings establish that a focus on inhibition of fila-
mentation may be particularly beneficial in the context of
Malassezia diseases. Indeed, the aim of antifungal treat-
ments of pityriasis versicolor may be more effective if we
do not aim to eliminate all of the Malassezia from the skin,
but to instead restore the population dynamics to the
commensal yeast-predominant microbiota. In conclusion,
our model for the study of in vitro inhibition of hyphae in
M. furfur has significant utility for screening antimycotic
agents against the pathogenic mycelial form of the yeast.
Additionally, our data suggests that ketoconazole’s efficacy
in Malassezia dermatophytoses may be due to its capacity
to impede morphogenesis combined with its cidal activity.
Acknowledgements This study was financially supported by the
Research Fund of the Faculty of Medicine, Chiang Mai University,
Chiang Mai, 50200, Thailand. JDN is partly supported by NIH
AI52733.
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts of
interest.
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Arch Dermatol Res
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