Fungal Genetics and Biology 132 (2019) 103254

Contents lists available at ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier.com/locate/yfgbi

Review

Facilitators of adaptation and antifungal resistance mechanisms in clinically T

relevant fungi
Margriet W.J. Hokkena,b, , B.J. Zwaanc, W.J.G. Melchersa,b, P.E. Verweija,b
⁎

a
Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Geert Grooteplein Zuid 10, 6525GA Nijmegen,
the Netherlands
b
Center of Expertise in Mycology Radboudumc/CWZ, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Geert Grooteplein Zuid 10,
6525GA Nijmegen, the Netherlands
c
Department of Plant Sciences, Laboratory of Genetics, Wageningen University, Droevendaalsesteeg 1, 6708PB Wageningen, the Netherlands

ARTICLE INFO ABSTRACT

Keywords: Opportunistic fungal pathogens can cause a diverse range of diseases in humans. The increasing rate of fungal
Aspergillus spp. infections caused by strains that are resistant to commonly used antifungals results in difficulty to treat diseases,
Candida spp. with accompanying high mortality rates. Existing and newly emerging molecular resistance mechanisms rapidly
Cryptococcus spp. spread in fungal populations and need to be monitored. Fungi exhibit a diversity of mechanisms to maintain
Antifungal resistance mechanisms
physiological resilience and create genetic variation; processes which eventually lead to the selection and spread
Antifungal compounds
Adaptation
of resistant fungal pathogens. To prevent and anticipate this dispersion, the role of evolutionary factors that
Mutation rate drive fungal adaptation should be investigated.
Reproduction In this review, we provide an overview of resistance mechanisms against commonly used antifungal com-
pounds in the clinic and for which fungal resistance has been reported. Furthermore, we aim to summarize and
elucidate potent generators of genetic variability across the fungal kingdom that aid adaptation to stressful
environments. This knowledge can lead to recognizing potential niches that facilitate fast resistance develop-
ment and can provide leads for new management strategies to battle the emerging resistant populations in the
clinic and the environment.

1. Introduction Additionally, organ/tissue transplant patients are at high-risk for

1.1. The rise of fungal diseases and resistance as a clinical challenge
Fungi are widespread and may cause a wide spectrum of diseases in
humans (Chotirmall et al., 2014; Latgé, 2001; Schmiedel and Zimmerli,
2016), with most infections occurring by the opportunistic pathogens
Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans.
Typically, in healthy humans, fungal infections are efficiently eradi-
cated by the immune system. Therefore, fungal infections are more
common in patients with immunodeficiencies, or patients who have
underlying chronic illnesses such as asthma, leukemia, or cystic fibrosis
(Bhatt et al., 2011; Denning et al., 2014). Frequent exposure to airborne
fungi can additionally lead to inflammatory responses rather than in-
vasive infections, such as asthma caused by Cladosporium spp. and Al-
ternaria spp., or inflamed alveoli caused by the pathogen Pneumocystis
jiroveci (Cordonnier et al., 2016; Denning et al., 2014; Hoving and Kolls,
2017).
fungal infections (Khan et al., 2015; Shoham and Marr, 2014). Fur-
thermore, the causative agents of endemic mycoses cause infections in
both healthy and immunocompromised humans and occur in specific
ecological niches (Malcolm and Chin-Hong, 2013; Salzer et al., 2018).
Factors such as prolonged lung colonization and frequent exposure
have proved to greatly influence the severity of these fungal infections.
Since the introduction of the first antifungal compounds in the late
1960s, resistant fungal isolates have been reported in many countries
worldwide (Buil et al., 2019; Hagiwara, 2018; Snelders et al., 2008;
Sorrell and Chen, 2007; Webb et al., 2018). As a result, the incidence of
invasive infections caused by resistant fungi has increased over the past
decades (Lestrade et al., 2019; Wiederhold, 2017) and antifungal re-
sistance increasingly becomes a concern for clinicians.
Although treating invasive fungal infections with antifungals is es-
sential to increase survival, treatment options remain limited. This is
because most compounds that are effective against fungi are toxic to
mammalian cells due to their shared cellular eukaryotic structures. The

⁎
Corresponding author.
E-mail address: margriet.hokken@radboudumc.nl (M.W.J. Hokken).

https://doi.org/10.1016/j.fgb.2019.103254
Received 12 June 2019; Received in revised form 16 July 2019; Accepted 16 July 2019
Available online 19 July 2019
1087-1845/ © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M.W.J. Hokken, et al.
Table 1
The major antifungal classes and their cellular targets.
Chemical class Compounds
Azoles Fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole,
ketoconazole
Echinocandins Anidulafungin, caspofungin, micafungin
Polyenes Amphotericin B, nyastatin, natamycin
Allylamines Terbinafine, naftifine
Pyrimidine analogs Flucytosine
introduction of the triazoles and the echinocandins presented more
treatment options in the 1990s, but these advances were readily fol-
lowed by the emergence of triazole and echinocandin resistant fungal
pathogens (Denning et al., 1997; Ksiezopolska and Gabaldón, 2018;
Moudgal et al., 2004; Perfect and Cox, 1999; Verweij et al., 1998).
Present-day antifungal treatment constitutes five main classes of anti-
fungals, which comprise the polyenes, imidazoles and trizoles, allyla-
mines, echinocandins and the compound 5-flucytosine (Chen and
Sorrell, 2007; Ghannoum and Rice, 1999). For an overview of common
antifungal compounds used in the clinic and their cellular targets, see
Table 1.
1.2. The adaptive potential of fungi
Fungi can quickly adapt to new and challenging environments in
nature, equipping them with intrinsic defenses to overcome cellular
stress induced by antifungal compounds (; Berkow and Lockhart, 2018;
Ksiezopolska and Gabaldón, 2018; Pré et al., 2018). Adaptation in this
sense is described as overcoming environmental challenges by gaining
beneficial mutations or adjusting cellular physiology. How quickly
these adaptations arise and spread after natural selection, depends on
forces that drive evolution itself such as (a)sexual reproduction, ploidy
changes, and genetic stability. These processes greatly influence the
ability to develop and pass on resistance mechanisms, allowing re-
sistance to spread through the population, regionally and globally.
The variety of molecular mechanisms that were discovered over the
last decades have not only given us insight in the many ways in which
fungal pathogens can exhibit intrinsic or acquired resistance (Sanglard,
2019), but has given us insights in the origin of drug-resistant fungal
isolates as well.
In this review, we aim to give an overview of the resistance me-
chanisms currently discovered in fungal pathogens. In addition, the
factors that drive evolution and spread of resistant fungal isolates are
discussed. We hope this contributes to a greater understanding towards
resistant fungal communities, and ultimately to the prevention and
improved treatment of severe fungal infections.
2. Antifungal compounds
2.1. Chemical classes and their mode of action
The antifungal compounds that are currently used in clinical
therapy inhibit cell wall biosynthesis, interfere with sterol metabolism,
or interfere with pyrimidine metabolism (Perfect, 2018). They inhibit
mechanisms uniquely present in fungi or target proteins that share little
homology with their mammalian counterparts.
2.1.1. Azoles
The azole class compounds disrupt ergosterol biosynthesis by in-
hibiting the sterol 14α-demethylase, which is a cytochrome p450 en-
zyme (Bossche et al., 1986; Liu et al., 2016). The first-generation tria-
zoles fluconazole and itraconazole are commonly used to treat Candida
spp. infections and nail infections respectively (Martin, 1999; Nelson
et al., 1994; Richardson, 1997). Fluconazole is often used as a
2
Fungal Genetics and Biology 132 (2019) 103254
Target pathway Molecular target
Ergosterol biosynthesis 14α-sterol demethylase
1,3 β-glucan biosynthesis 1,3 β-glucan synthase complex catalytic
subunit
Ergosterol biosynthesis Ergosterol in fungal membranes
Ergosterol biosynthesis Squalene epoxidase
Pyrimidine salvage pathway DNA/RNA molecules
maintenance therapy for cryptococcal meningitis in HIV patients and
non-HIV patients (Zhao et al., 2018). Second-generation triazoles in-
clude voriconazole and posaconazole, which are both used for the
treatment and prophylaxis of invasive aspergillosis (IA) respectively
(Lat and Thompson, 2011; Sandherr and Maschmeyer, 2011). Both
drugs are currently used for prophylaxis, although posaconazole treat-
ment resulted in less side effects than voriconazole according to a re-
cent study (Tang et al., 2018). In 2015, isavuconazole was approved
and showed efficacy similar to voriconazole against IA and has been
licensed for treatment of mucormycosis (Jenks et al., 2018).
Azole resistance was first reported in the clinic in 1986 in C. albicans
(Smith et al., 1986), and has now been found in many pathogenic
species including A. fumigatus, Histoplasma capsulatum and C. neofor-
mans (Denning et al., 1997, Lamb et al., 1999).
2.1.2. Echinocandins
The echinocandins micafungin, caspofungin, and anidulafungin
target fungal cell wall synthesis by inhibiting FKS1, a glucan synthase
which is essential in the biosynthesis of 1,3 ß-glucans in the fungal cell
wall (Walker et al., 2010). They prove effective against invasive can-
didiasis, exert moderate activity against Aspergillus spp., but are in-
effective in infections caused by Fusarium spp., Cryptococcus spp. or
mucorales (Eschenauer et al., 2007; Maligie and Selitrennikoff, 2005).
Echinocandin resistance has been reported extensively in many Candida
spp., (Jensen et al., 2013; Park et al., 2005; Perlin, 2007), and has also
been found in A. fumigatus (Jiménez-Ortigosa et al., 2017). Prevalence
of echinocandin resistant isolates was observed to be < 3% in 2010 in
various Candida isolates (Castanheira et al., 2010), but a more recent
study observed a prevalence of > 12% in C. glabrata clinical isolates
(Alexander et al., 2013).
2.1.3. Polyenes
The polyene compounds, which include amphotericin B and nyas-
tatin, bind to ergosterol specifically, which causes disruption of the
membrane integrity with subsequent cellular leakage as a consequence.
Furthermore, amphotericin B induces oxidative damage (Mesa-Arango
et al., 2012). Amphotericin B is a fungicidal against many different
fungi, but due to renal failure as a side-effect it is a second-line treat-
ment (Sawaya et al., 1995). Resistance to amphotericin B is not com-
monly reported, although a recent study observed increasing resistance
rates in a hospital in Brazil, where 27% of the A. fumigatus isolates had
elevated MICs (> 2 mg/L) for amphotericin B (Luzia et al., 2018).
Amphotericin B resistance may possibly arise when genes in the er-
gosterol biosynthesis pathway carry mutations. This eventually results
in strains with little to no ergosterol in their membranes, and sub-
sequent amphotericin B resistance as the target molecule is absent from
the cells. This mechanism has been described for C. tropicalis, C. lusi-
taniae, C. albicans, A. terreus and C. neoformans (Forastiero et al., 2013;
Kelly et al., 1997, 1995; Walsh et al., 2003; Young et al., 2003).
2.1.4. Allylamines
The allylamines inhibit the squalene epoxidase enzyme, which is
essential in the ergosterol biosynthesis pathway. They are commonly
used to treat skin and nail infections (Mukherjee et al., 2003). A
M.W.J. Hokken, et al. Fungal Genetics and Biology 132 (2019) 103254

1. Biofilm formation 2. Loss of mitochondrial function 3. Stress signaling Increased

Mdm31
caspofungin/fluconazole tolerance
Azole resistance Tom70
Calcineurin
drug resistance increased activation
IMM OMM

Complex I Hsp90 Regulates HOG signaling

Hyphal development

IMM
Cell wall integrity

hsp90
transcription

4. Mutations in cell wall biosynthesis genes 5. Inhibited pyrimidine salvage pathway

1

2 3 Fcy1 Fur1
5FC

4
7+8 5FU
Fks1 Fks2 5 5FC resistance
Rho1 fcyB
6 Inhibition of protein synthesis
regulates disturbs
Inhibition of DNA synthesis
β-(1,3)-GS complex

6. Increased drug efflux 7. Upregulation and mutation of target genes 8. Lipid metabolism bypass
in ergosterol biosynthesis Azole resistance
Bind to Polyene resistance
Cdr1b

inactivation

incorporation
Cyp51A Erg1

Membrane
Cyp51A Erg3
AtrR
cdr1b drug resistance TR TR cyp51A Azole resistance 14α-methyl sterols Prevention of
Transcription Transcription Allylamine resistance toxic sterol production

Mutation 5FC Allylamines Azoles Echinocandins Polyenes Ergosterol

Fig. 1. Antifungal resistance mechanisms found in fungi. 1: Biofilm formation reduces penetration of antifungal compounds into the population. 2: Loss of mi-
tochondrial complex I was shown to result in azole resistant isolates. 3: Enhanced production of Hsp90 induces stress-signaling pathways and leads to enhanced
tolerance for azoles and echinocandins. 4: Mutations in 1,3 β-glucan synthase complex catalytic subunits prevent interference in cell wall biosynthesis by echino-
candins. 5: Mutations in the cytosine permease FcyB prevent flucytosine from entering the cell. Mutations in the cytosine deaminase Fcy1 or the uracil phos-
phoribosyltransferase Fur1 prevent conversion of flucytosine to the toxic 5-fluorouracil. 6: Increased activity of drug-efflux transporters result in less intracellular
drug accumulation. 7: Mutations in the squalene expoxidase erg1 or the 14α-sterol demethylase Cyp51A prevent activity of allylamines and azoles, respectively.
Upregulation of the 14α-sterol demethylase has evolved together with target site alterations and ensures enzyme availability. 8: Absence of the sterol desaturase Erg3
prevents the production of toxic sterols if Cyp51A is inhibited by azole compounds. Non-toxic sterol intermediates are incorporated in the membrane instead.

relatively high resistance rate of 32% was observed in 63 Trichophyton
interdigitale isolates in India (Singh et al., 2018), but is as yet unclear
whether resistance is rising elsewhere. Resistant isolates were shown to
have single amino acid changes in the target gene, erg1. Another study
suggested that terbinafine resistance in A. nidulans resulted from
overexpression of salA, a salicylate 1-monooxygenase which facilitates
napthalene degradation, which may use terbinafine as a substrate as it
contains a napthalene ring (Graminha et al., 2004).
2.1.5. Pyrimidine analogs
5-Flucytosine (5FC) is a pyrimidine analog used in combination
therapy, but studies have shown that many fungal pathogens are able to
rapidly develop 5FC resistance (Charlier et al., 2016). In the cell, 5FC is
converted to toxic 5-fluorouracil, which interferes with DNA and RNA
structures (Perfect, 2018). Resistant isolates that were found include C.
glabrata, A. fumigatus, and C. gatti (Edlind and Katiyar, 2010; Gsaller
et al., 2018; Vu et al., 2018).
2.2. In vitro resistance development to low dosages of antifungal compounds
Resistance development is shown to result more likely from selec-
tion at sublethal concentrations of the drug, then from exposure to
above-MIC concentrations in A. fumigatus (Escribano et al., 2012; Zhang
et al., 2017b). Sublethal concentrations supposedly permit the fungus to
still grow and reproduce, among others through (adaptive) phenotypic
plasticity, to allow natural selection to act. Various studies have de-
scribed antifungal resistance induction in a laboratory setting at various
drug concentrations, to investigate how rapid resistance can be induced
and which mechanisms will arise in the exposed isolates (Cowen et al.,
2000; Huang et al., 2011).
De novo induction of voriconazole resistance has been shown to be
possible already after five weeks culturing which started with sublethal
concentrations at 0.125 mg L−1, although no cyp51A/B mutations were
found in the resistant A. fumigatus isolates (Händel et al., 2015; Verweij
et al., 2009). In another study, cross-resistant A. fumigatus isolates were
obtained already after three weeks of culturing in the presence of the
fungicide difenoconazole at a concentration of 1 mg L−1 (Zhang et al.,
2017b).

3
M.W.J. Hokken, et al.
In T. rubrum, azole resistance was induced in vitro after one year of
passaging isolates at sub MIC concentrations (Hryncewicz-Gwóźdź
et al., 2013). In a different study, no itraconazole or efinaconazole re-
sistance was observed in T. rubrum after 24 weeks of culturing (Iwata
et al., 2014).
Another study included four pairs of C. glabrata isolates which
consisted of one susceptible and one resistant isolate, retrieved from
four patients which received cumulative doses of 6, 10, 12, and 12.2 g
of fluconazole. Interestingly, sampling times between the susceptible
and the resistant isolates were 15, 35, 40 and 60 days, respectively,
implying that lower treatment doses lead to a faster resistance devel-
opment (Sanguinetti et al., 2005). In vitro micafungin resistance was
observed after exposure to increasing concentrations of the drug
starting at 0.015 μg/ml (Bordallo-Cardona et al., 2017). Resistance
mutations developed when drug concentrations were close to the MIC,
on average after 3 days.
These findings support the statement that initial exposure to lower
drug concentrations lead to a faster resistance development, although
resistant isolates have been recovered from patients that have received
prophylaxis with itraconazole for a longer period of time, even if serum
levels were higher than the MIC according to the EUCAST guidelines
(Pilmis et al., 2018). Multi-resistant A. fumigatus is often recovered from
the environment, for instance compost heaps, which contain a very low
concentration of agricultural azoles used for crop protection. Due to
cross-resistance, these isolates are also resistant for medical azoles
(Snelders et al., 2012; Zhang et al., 2017b). The TR34/L98H and TR46/
Y121F/T289A cyp51A resistance conferring genotypes that likely have
originated in the environment, have spread and are the cause of
treatment failure in patients with invasive aspergillosis (Fisher et al.,
2018; Garcia-Rubio et al., 2017; Lestrade et al., 2019; Zhang et al.,
2017a).
3. Mechanisms of antifungal resistance
Fungal pathogens have developed a variety of molecular mechan-
isms that enable them to exhibit intrinsic or acquired resistance to all
antifungal compounds currently used. For an overview of the me-
chanisms highlighted in this review, see Fig. 1.
3.1. Biofilm formation
Many pathogenic yeasts and molds have the ability to form a bio-
film, which works as a shield against host defense and antifungal
compounds and increases adherence to the host surface (Fanning and
Mitchell, 2012). A biofilm consists of a network of cells that are asso-
ciated to each other or a surface, which produce extracellular matrix
(ECM) that provides protection against a stressful environment
(Ramage et al., 2012). The ECM formed is thought to prevent the dif-
fusion of antifungals to the cells. Furthermore, a high amount of ex-
tracellular DNA was found in the EMC in C. albicans biofilms, which is
thought to contribute to the structure and formation of the biofilm
(Martins et al., 2010). Biofilms formed by C. albicans exhibit very high
MICs against multiple antifungals after only 72 h of growth (Chandra
et al., 2001). Biofilms also conferred high levels of resistance against
antifungals of various classes in Trichosporon asahii, A. fumigatus and C.
neoformans (Bonaventura et al., 2006; Kaur and Singh, 2015; Martinez
and Casadevall, 2015; Müller et al., 2011). A recent study showed that
in high- biofilm forming isolates of C. albicans, various amino acid
metabolism pathways were significantly up-regulated compared to low-
biofilm forming isolates. The AAT1 gene encoding an aspartate ami-
notransferase was found to play a central role in these metabolic
pathways, and its significant up-regulation in high- biofilm forming
isolates suggests it could be a potential target for antifungal compound
development and as a biomarker for these high- biofilm forming isolates
(Rajendran et al., 2016).
4
Fungal Genetics and Biology 132 (2019) 103254
3.2. Structural target site alterations
The acquisition of mutations in genes encoding antifungal targets
has resulted in resistance in many pathogenic fungi. Non-synonymous
SNPs lead to amino-acid alterations, an altered structure and reduced
binding affinity of the antifungal to the target (Balkis et al., 2002), thus
the enzyme can remain functional. This mechanism has evolved in
isolates that were exposed to echinocandins, allylamines, and is the
resistance mechanism found in pan-azole resistant A. fumigatus isolates
that have spread across all continents (Hagiwara et al., 2016b; Snelders
et al., 2008). Frequently reported amino acid substitutions that confer
azole resistance in A. fumigatus include positions G54, G138, F216,
M220, and G448 (Camps et al., 2012a,b; Chowdhary et al., 2017;
Vermeulen et al., 2013). Furthermore, specific point mutations are as-
sociated with a tandem repeat in the promoter region (Snelders et al.,
2008; Van Der Linden et al., 2013). This repeat confers upregulation of
cyp51A expression, as a duplication of this region provides an addi-
tional binding site for the transcriptional regulator SrbA, which acti-
vates expression of cyp51A (Hortschansky et al., 2016). Additionally,
azole resistance through increased transcription of cyp51A is also ob-
served in isolates harboring a P88L mutation in the HapE subunit of the
CBC complex (Camps et al., 2012a,b). The TR34/L98H and the TR46/
Y121F/T289A containing isolates were shown to be highly resistant to
itraconazole and voriconazole, respectively (Snelders et al., 2008; Van
Der Linden et al., 2013). Various studies have highlighted that specific
point mutations cause the Cyp51A enzyme to be more unstable (Paul
et al., 2017; Song et al., 2018), which could explain why a tandem
repeat is essential for a fully resistant phenotype, as unstable enzymes
can cause a decrease in overall fitness.
In Candida spp., there is much more variation in ERG11 point mu-
tations, but never accompanied by tandem repeat region variation (Fan
et al., 2018; Flowers et al., 2015). The most occurring SNPs that were
proved to confer azole resistance include Y132, K143, E266, and G464
(Flowers et al., 2015). In C. neoformans, the single amino acid sub-
stitution G484S was shown to confer resistance against fluconazole
(Bosco-Borgeat et al., 2016; Rodero et al., 2003).
In isolates resistant for the allylamine terbinafine, amino acid sub-
stitutions are often found in the squalene epoxidase, the target of ter-
binafine. In A. fumigatus, a F389L substitution in the ergA gene was
found to confer resistance to terbinafine (Rocha et al., 2006). Terbi-
nafine is commonly used to treat dermatophyte infections, but a recent
study observed a high prevalence of terbinafine resistant T. interdigitale
in India (Singh et al., 2018). They described the known F397L sub-
stitution, and the L393F substitution in the squalene epoxidase gene
erg1. This last substitution has already been confirmed to result in
terbinafine resistance, as this substitution was introduced in the squa-
lene epoxidase of C. albicans, and heterologously expressed in S. cere-
visiae, which resulted in a resistant phenotype (Osborne et al., 2005).
Echinocandin resistance through non-synonymous SNPs has been
found in the FKS1 gene, which encodes the 1,3 β-glucan synthase, one
of the main components of the fungal cell wall. In resistant isolates of C.
albicans, the most common amino acid substitutions found is a serine
substitution in codon 645 or an arginine substitution in codon 1361
(Balashov et al., 2006; Laverdière et al., 2006; Park et al., 2005). These
mutations are also found in other Candida spp. (Perlin et al., 2015).
Although echinocandin resistance in A. fumigatus is rare, in a clinical
isolate that was retrieved after micafungin treatment failure, a F675S
substitution was found in the FKS1 gene (Jiménez-Ortigosa et al.,
2017). It is apparent that point mutations that confer echinocandin
resistance in the FKS1 gene are present in regions 641–649 and
1345–1365 (Walker et al., 2010). Echinocandin resistance in C. glabrata
is mediated by point mutations in FKS1 or in its paralog FKS2, although
only FKS1 point mutations were reported to result in resistant isolates
in C. albicans and C. dubliniensis (Katiyar et al., 2012; Prigent et al.,
2017). The latter study showed that in three patients, corresponding to
8% of all patients treated with echinocandins, resistance arose within
M.W.J. Hokken, et al.
one month of therapy.
As described above, the antifungal 5-flucytosine (5FC) is currently
only used in combination therapy, as it quickly induces resistance
(Vermes et al., 2000). This resistance often results when the Fcy2 per-
mease is mutated which prohibits 5FC to enter the cell, or when the
Fur1 or Fcy enzymes are mutated, so that the conversion of 5FC to the
toxic 5-fluorouracil is prevented. Although low-level resistance may
occur due to minor amino acid substitution in the Fcy2 permease, full
resistance is only achieved with mutations that result in a null-pheno-
type for Fcy2 or Fur1 in C. albicans (Edlind and Katiyar, 2010).
3.3. Metabolic bypass
A lesser observed mechanism to gain antifungal resistance, en-
compasses the prevention of accumulating toxic metabolites by the
inactivation of another enzyme in the pathway. In C. albicans, this
mechanism has proven to confer resistance against azole compounds
(Kelly et al., 1997). When the sterol Δ5,6-desaturase ERG3 is nonfunc-
tional, 14α-methylated sterol intermediates are not converted to toxic
sterol intermediates, preventing disturbance of the cellular membranes.
Furthermore, this inactivation does not always lead to a decrease in
virulence (Vale-Silva et al., 2012). Similar mechanisms were found in
the ERG2 and ERG6 genes of C. albicans (Vincent et al., 2013). Many
non-cyp51A azole resistant isolates are not analyzed for mutations in
their ERG3 gene, so this resistance mechanism could be more wide-
spread than currently believed. However, deletion mutants of erg3A and
erg3B were created in A. fumigatus and did not lead to any differences in
antifungal drug susceptibility, although significant differences in er-
gosterol content were found in all strains (Alcazar-fuoli et al., 2006).
This could be explained by the differences in ergosterol biosynthesis
found between fungal taxa (Alcazar-Fuoli and Mellado, 2012).
3.4. Overexpression of efflux-pumps
The ability of fungal pathogens to excrete antifungal compounds
works through overexpression of specific efflux pumps and is a re-
sistance mechanism found in many fungal species. Although this me-
chanism is widespread, it is not found in echinocandin or polyene re-
sistant fungal isolates, as these are not substrates of efflux pumps and
exercise fungicidal activity on the outside of the cell (Cannon et al.,
2009). Increased gene expression feeds the synthesis of more efflux
pump protein complexes results in increased antifungals transportation
to outside the cell. These transporters usually belong to the ABC or MFS
transporter family (Perlin et al., 2014) and blocking these transporters
reduces the MICs significantly (Holmes et al., 2016).
Azole resistance through up-regulated efflux pump expression was
reported for the MDR1 (Multi-Drug Resistant), CDR1, and CDR2
(Candida-Drug Resistant) genes in many Candida spp. (Choi et al., 2016;
Moran et al., 1998; Sanglard et al., 2001, 1997). Homologs of these
transporters are also present in A. fumigatus and have shown to confer
azole resistance (White, 1997). Furthermore, many unknown putative
drug-efflux transporters were shown to be up-regulated in A. fumigatus
after exposure to itraconazole (Hokken et al., 2019). These transporters
should be investigated to elucidate their potential role in developing
azole resistance. The C. neoformans and C. gatti genome also contain
MDR1 homologs which were shown to facilitate azole efflux (Basso
et al., 2015), however, the role of other known efflux pumps was never
fully elucidated in these species.
Gain-of-function mutations in the Mrr1, Upc2, and Cap1 transcrip-
tion factors that regulate MDR1 expression, induced fluconazole re-
sistance in C. albicans (Schubert et al., 2011). Furthermore, the Tac1
transcription factor (TF) was shown to regulate the expression of the
CDR1 and CDR2 efflux-pumps, and the development of homozygosity
for this TF results in strong up-regulation of these pumps (Coste et al.,
2006, 2004). In A. fumigatus, transcription factor AtrR was found to
regulate cyp51A and cdr1B expression, emphasizing the important role
5
Fungal Genetics and Biology 132 (2019) 103254
regulatory proteins can play in for instance azole resistance (Hagiwara
et al., 2017).
An overview of the regulatory networks determining efflux-pump
expression can be found in a review by Paul and Moye-Rowley (2014).
Resistance to terbinafine via increased drug-efflux has not been re-
ported extensively. A transcriptome study in C. albicans highlighted
several up-regulated transporter genes among which MFS1 and CDR1,
after exposure to 4 mg/L terbinafine (Yue-bin et al., 2007). Ad-
ditionally, terbinafine resistance through efflux-pump upregulation has
not been reported in Trichophyton spp. (Yamada et al., 2017).
In C. glabrata, the expression of membrane transporters CgFLR1
CgFLR2 resulted in a 5FC resistant phenotype. Deletion of these
transporters led to accumulation of intracellular 5FC, indicating that
these transporters play a direct role in 5FC efflux (Pais et al., 2016).
Aside from drug-efflux machineries, membrane transporters have
been identified that confer antifungal resistance via their decreased
drug-influx capacity. Recently, it has been found in A. fumigatus that the
main importer of 5FC, the FcyB transporter, is upregulated at pH5
which explains the limited activity of this drug at pH7 (Gsaller et al.,
2018).
3.5. Mitochondrial alterations possibly facilitate resistance
Various studies have elucidated potential roles for the mitochondria
to be involved in antifungal drug resistance (Brun et al., 2004; CHAB-
ASSE et al., 2009; Sanglard et al., 2001). A C. glabrata isolate with loss
of mitochondrial function showed increased fluconazole resistance up
to 50 μg/ml, an effect that was appointed to the differential expression
of azole-resistance related genes due to loss of mitochondrial function
(Sanglard et al., 2001).
A study on A. fumigatus strains that harbor an E180D mutation in
the 29.9KD subunit of the mitochondrial complex I showed that these
strains were azole resistant, although also less virulent. The strain had
an MIC of 2–4 mg/l itraconazole, whereas the parental strain had an
MIC of 0.25 mg/l. This finding is supported by the fact that inactivation
of complex I through chemical inhibitors also confers azole resistance.
The authors suggest that this comes from a rebalancing of the hypoxic
response. Upon addition of azoles, the hypoxic response is ‘unin-
tentionally’ activated by dysfunctional oxygen sensing because of sterol
biosynthesis inhibition, although oxygen levels are normal (Bromley
et al., 2016). As the parental strain displays a reduced activity of
complex I when hypoxia is induced, the strain with the mutated com-
plex I is thought to restore its growth through this mechanism when
azoles are present.
A recent study on difenaconazole resistant A. fumigatus isolates
conducted by Zhang et al., described mitochondrial DNA mutations in a
superoxide dismutase, the inner membrane protein Mdm31 and the
outer membrane protein Tom70 (Zhang et al., 2017b). These studies
suggest that there might be a direct relation between mitochondrial
dysfunction and azole resistance.
3.6. Activation of stress pathways
Cellular stress signaling is essential to survive stressful conditions in
the environment, such as high salt concentrations or high temperatures
(Hagiwara et al., 2016a). The mechanisms that help cells cope with
stress, can also provide protection against drug-induced stress condi-
tions (Cowen and Steinbach, 2008). One of the conserved key reg-
ulators in stress signaling is the Hsp90 chaperone, which is involved in
protein folding and stabilizing many proteins involved in signal trans-
duction (Cowen, 2015). In A. fumigatus and A. terreus, Hsp90 inhibitors
lead to increased sensitivity to caspofungin. Furthermore, reduced
functioning of Hsp90 was shown to increase azole susceptibility in C.
albicans (Cowen, 2009).
M.W.J. Hokken, et al.
3.7. Adjustment of membrane homeostasis
Eukaryotic membranes are dynamic structures that contain many
lipid species that contribute to membrane integrity and maintenance.
Azoles and amphotericin B have an impact on membrane home-
ostasis by disturbing cellular ergosterol content, which leads to mem-
brane destabilization and subsequent lysis of the cell
(Georgopapadakou et al., 1987). Therefore, it is suggested that the cell
adjust its membrane homeostasis to make it more rigid, or more fluid.
This can for instance be achieved by changing the ratio of phospholipid
species (PLs), as all these PLs have different chemico-physical proper-
ties which contribute to the nature of the membrane (Renne and de
Kroon, 2018). Additionally, sterols and sphingolipids also contribute to
membrane integrity and are thought to function together in complexes
(Guan et al., 2009). In C. albicans, depletion of either ergosterol or
sphingolipids was shown to increase drug-susceptibilities, an effect that
was appointed to the increased permeability of the membrane. Fur-
thermore, a decreased surface localization of the drug-efflux transporter
Cdr1p was found in S. cerevisiae cells with disturbed sterol-sphingolipid
interactions.
In another study, it was demonstrated that null-mutations in the
FEN1 or FEN12 genes confer azole resistance in C. albicans (Gao et al.,
2018). These enzymes synthesize sphingolipid precursors, and in-
activation resulted in a significant increase in various ceramide pre-
cursors, and an overall increase of sphingolipid species.
4. Origins of antifungal resistance development
The emergence of antifungal resistance mechanisms is the result of
natural selection after the origination of beneficial genetic of physio-
logic adaptations to antifungal compounds. The arise of these adapta-
tions is dependent on processes that influence genetic and physiological
changes, such as genetic stability and sexual reproduction, and will be
discussed below. For an overview of the processes that contribute to
adaptations to antifungal compounds, see Fig. 2.
4.1. Increased antifungal drug resilience through phenotypic plasticity
Cells have the ability to change their metabolism as a response to
changing conditions, as they are not able to move away from the un-
favorable environment, containing for instance antifungal compounds.
When these responses allow cells to grow and divide when the
Mutator strains
Normal strain
AGTCGGCATTCGAA
Wildtype
Time
AGTCCGCATTCGAA
Aneuploidy
Mutator strain
AGGCCTGATCCGAA
Msh2
Generators of genomic variance
Population dynamics
G1 G2 G3
Phenotypic Maintainance of
plasticity multiple genotypes
- FCZ CDR1B
No regulation
EFH1
Up-regulation Increased
Up-regulation colonization
+ FCZ CDR1B
Fig. 2. Generation of genetic and physiologic variability in the population facilitates selection to antifungal agents.
Fungal Genetics and Biology 132 (2019) 103254
environment is not ideal, this process is referred to as adaptive phe-
notypic plasticity (APP) (Stern et al., 2007). Before any mutations arise
in the genome, the cells need to maintain growth and reproduction to
give themselves a chance to develop hard-wired (i.e. genetic) resistance
mutations. One way by which insight into these processes can be
gained, is through measuring the differences in gene expression, as
these are controlled by regulatory genes that are often mediated
through environmental impulses (Aubin-Horth and Renn, 2009;
Schlichting and Piglucci, 1993; Schlichting and Smith, 2002). Differ-
ential gene expression after drug exposure can lead to increased sur-
vival chances through the activation of compensatory mechanisms. This
gives the fungus a chance to develop resistance mutations by main-
taining growth and reproduction. Various studies highlight differences
in gene expression after exposure to antifungal compounds.
A recent study using A. fumigatus clinical isolates exposed to itra-
conazole demonstrated the up-regulation of specific efflux pumps, dif-
ferential expression patterns in ergosterol and phospholipid biosynth-
esis routes, and in MAPK cascade proteins (Hokken et al., 2019).
Another study investigated the gene expression of C. albicans after
caspofungin exposure, and found that chitin production was increased
(Walker et al., 2015). Compensatory mechanisms further include acti-
vation of the calcineurin pathway, which activates several chitin syn-
thases upon caspofungin exposure (Munro et al., 2007; Walker et al.,
2015).
In a recent transcriptomic analysis in C. glabrata, isogenic azole
susceptible and resistant isolates were assessed. It was found that ex-
pression of the adhesin-encoding EPA3 gene was increased by 4-fold in
the resistant isolate, and that overexpression of this gene promoted
biofilm formation and decreased accumulation of radiolabeled clo-
trimazole (Cavalheiro et al., 2018). Finally, a particular example
highlighted that these transcriptomic alterations are not only tem-
porary; after culturing C. glabrata isolates in rich medium containing
50 μg/ml fluconazole, both the efflux transporter encoding genes CDR1
and CDR2 were up-regulated, and remained up-regulated for up to 200
generations after switching to drug-free medium (Sanglard et al., 2001).
These changes in gene expression help facilitate fungal resilience
against antifungal agents.
4.2. Mutation frequencies and mutation rates
The rate in which spontaneous mutations arise in an organism
greatly determines the genetic variability, and thus the chance that
Increased
mutation frequency Selection of resistant fungi
HGT UV
In patients and environment
TTGACAGAACCG ROS
?
ATGACCGAAGCG
Heterokaryon
Parasexual
Sexual reproduction reproduction
a
α Ascospore
Hyphal + nuclear
formation
fusion
Genetic
reshuffling Haploidization
2n 1n
6
M.W.J. Hokken, et al.
resistance conferring mutations are present in the population. Various
studies have suggested a possible influence of the environment on
mutational rates (Galhardo et al., 2012; Hoffman and Hercus, 2000).
Stressful conditions could increase the mutational rate because main-
tenance and repair pathways function at a lower level, which results in
an increased generation of mutations over time. Most of these muta-
tions will be detrimental, but there is also a higher chance to develop
mutations which confer resistance to antifungal compounds.
As exposure to antifungal compounds in patients or in the en-
vironment creates a stressful environment for pathogenic fungi, these
mutational processes should be looked into. Processes that can be in-
vestigated to gain insight into the capabilities of a species to generate
mutations are the mutation frequency and the mutational rate of which
it is derived.
The mutation frequency is determined by calculating the ratio of
viable colonies growing on drug-containing plates over the starting
inoculum (Shields et al., 2019), whereas the mutation rate is the fre-
quency of new mutations in general, in a single gene or cell over a
certain time. For instance, the mutation rate of S. cerevisiae was de-
termined to be 0.0027 per generation; the chance of mutation per base
pair was set at 2.2 × 10−10, multiplied by the total genome size which
counts 1.2 × 107 basepairs (Drake et al., 1998; Gou et al., 2019). Al-
though mutation frequency is much easier to determine and does not
require whole genome sequencing, the latter method is able to distin-
guish between the mutational rates of different genes. This led to the
observation that the mutational rate can vary across the genome (Lang
and Murray, 2008).
The mutation frequency is thus dependent on the intrinsic muta-
tional rate of a species, but external factors which confer stressful
growth conditions were also shown to influence this number.
In C. albicans, the spontaneous mutation frequency appeared to
differ between exposure to the various echinocandins, as treatment
with caspofungin seemed to result into resistant isolates more often
than the other echinocandins (Shields et al., 2019), although they share
their cellular target.
Another particular example comes from a study conducted by Lamb
et al., where various wild strains of Penicillium lanosum and A. niger
were isolated from two different sides of the same mountain (Lamb
et al., 2008). The south-facing side of the mountain received up to
800% more damaging solar radiation than the north-side of the
mountain, which resulted in two very different environments. The
isolates that were taken from the south-side appeared to have a
2.44–8.86. fold higher mutation frequency in controlled laboratory
conditions, than the isolates that originated from the much milder,
north-side. These results possibly indicate that stressful environments
generate more genomic diversity in filamentous fungi.
Mutation rates are currently only determined for S. cerevisae and
Schizosaccharomyces pombe (Drake et al., 1998; Farlow et al., 2015). For
filamentous fungi, mutation rates are more difficult to determine be-
cause of their typical hyphal growth (Baracho and Baracho, 2003), al-
though knowledge about mutational rates for each species could give
valuable insights into the chances of developing antifungal resistance.
4.3. The development of mutator strains
The occurrence of mutator strains is a phenomenon that is observed
in various yeasts (Healey et al., 2016; Nicolas et al., 2014). Most often
by defects in DNA repair mechanisms, these strains gain mutations
quickly which increases their chances to develop resistance mutations.
In a study conducted by Lang et al, the mutation rate of a mutator strain
of S. cerevisiae was observed to be a 225-fold higher than the wildtype.
This was determined by genomic analysis of a strain harboring a
knockout of the msh2 gene, a core component of the DNA mismatch
repair machinery (Lang et al., 2013). The same gene was found to have
a single-base deletion at position 1689 in C. neoformans, which led to an
increased mutation rate. Although a decrease of genome stability could
7
Fungal Genetics and Biology 132 (2019) 103254
also lead to a decreased fitness, no changes in virulence were found
(Boyce et al., 2017). Furthermore, more than half of the isolates of
clinical C. glabrata isolates contained a mutation in the MSH2 gene, and
msh2Δ strains were shown to develop resistance to caspofungin sig-
nificantly faster than parental strain containing a wildtype MSH2 gene
(Healey et al., 2016). Generation of a msh2Δ/msh2Δ C. albicans strain in
the laboratory also resulted in elevated mutational frequencies on flu-
conazole-containing medium. Furthermore, rad50Δ/rad50Δ and
pms1Δ/pms1Δ strains were also shown to exhibit a higher appearance of
resistant colonies, emphasizing the important role of double-strand
break repair and the DNA damage repair machineries (Legrand et al.,
2007).
However, in another study, clinical S. cerevisiae isolates retrieved
from patients harboring a genotype which was previously indicated to
exhibit a mutator phenotype in vitro, were only observed to have slight
increase in mutational rate (Demogines et al., 2008; Skelly et al., 2017).
Up to date, no mutator phenotypes of Aspergillus spp. have been found
nor emerged in the clinic, although laboratory strains with defective
DNA repair machinery by deletion of atrA or atmA were shown to ac-
quire voriconazole resistance more frequently (dos Reis et al., 2017).
Mutator phenotypes are more likely to be found in fungi with a
haploid genome, as the phenotype will be determined by only one set of
chromosomes. If haploid mutator cells with acquired resistance muta-
tions would undergo (para)sexual reproduction, rearrangement of ge-
netic material could lead to offspring harboring only the beneficial
resistance mutation, without suffering possible fitness costs of an in-
stable genome.
4.4. Chromosomal aneuploidy
Antifungal resistance development is greatly driven by the ability to
generate genomic variance in the gene pool. Polyploid cells often ra-
pidly generate genetic diversity within a population due to their in-
stability (Gerstein et al., 2015). Many yeasts have the ability to lose or
gain certain segments of chromosomes, or even complete chromosomes
(Gordon et al., 2011). Chromosomal aneuploidy facilitated antifungal
resistance in various pathogenic fungi, and in some cases even their
virulence (Kwon-chung and Chang, 2012).
Pathogenic species such as C. albicans display this genome flexibility
which can lead to antifungal resistance. The trisomy of chromosome 5
was seven times as prevalent in fluconazole-resistant C. albicans isolates
as in fluconazole-sensitive isolates, and these chromosomes also dis-
played a high segmental aneuploidy (Selmecki et al., 2006). The an-
euploid chromosomes found were the trisomy of chromosome 7, and
the isochromosome 5 which consists of two copies of the left arm of the
original chromosome (Selmecki et al., 2009). Caspofungin tolerance
was also found in aneuploid C. albicans isolates, which either lost one
copy of chromosome 5, or showed combined monosomy of the left arm
and trisomy of the right arm of chromosome 5 (Yang et al., 2017). In-
terestingly, stress due to chemicals was shown to increase the rate at
which spontaneous aneuploids occurred by 100-fold in S. cerevisiae,
although inhibition of HSP90 resulted in the highest chromosome in-
stability and subsequent aneuploidy (Chen et al., 2012). Resistance of
the haploid C. neoformans to fluconazole was appointed to the doubling
of chromosome 1, 4, 10 or 12 (Sionov et al., 2010). Recently, this an-
euploidy was discovered to be the result of the inability to degrade the
septum after cell division, resulting in cells with increased DNA content
(Altamirano et al., 2017). These large cells have been described as
‘Titan cells’ and have been found to have higher chance of surviving
various stressors such as fluconazole, oxidative stress induced by H2O2
and osmotic stress induced by high NaNO3 concentrations. Further-
more, their haploid progeny were shown to display higher fluconazole
MIC values than the progeny of regular cells (Gerstein et al., 2015). In
addition, it was demonstrated that Titan cells can be induced in vitro by
exposure to FCS, a compound that induces capsule growth. Interest-
ingly, bronchial alveolar lavage (BAL) was also able to induce titan cell
M.W.J. Hokken, et al.
formation, due to the presence of biomarkers for bacterial pepti-
doglycan, which could facilitate adaptation to antifungal compounds
(Dambuza et al., 2018).
Although aneuploidy can also result in deleterious defects and fit-
ness loss in fungi (Oromendia et al., 2008), these results also indicate
that aneuploidy can lead to an increase in genetic variability and an-
tifungal resistance.
4.5. (Para)sexual reproduction
Pathogenic fungi can have various methods of reproduction, which
potentially aid the development and spread of resistance mechanisms.
The formation of asexual conidia provides an easy method for the
fungus to spread new mutations that originated during hyphal growth,
whereas the formation of sexual spores leads to increased genetic
variability due to reshuffling of chromosomes, which facilitates adap-
tation to a changing environment (Heitman, 2015; Hoekstra, 2005;
Verweij et al., 2016a,b). For sexual reproduction to occur, fusion of
hypha of two different mating types is usually required, although C.
neoformans and C. albicans evolved a form of sexual reproduction in
which only one mating type is required (Alby et al., 2009; Bui et al.,
2008). Alternatively, parasexual reproduction can occur when fungal
cells or hypha undergo cellular fusion and form heterokaryons, where
potential recombination of genetic material occurs (Pontecorvo et al.,
1953). Parasexual reproduction is hypothesized to occur in long lasting
mycelia in for instance CF patients, as there must be various genotypes
present which are still clonally related (Verweij et al., 2016a,b). A re-
cent study showed recovery of A. fumigatus heterokaryons from patients
with chronic diseases. The heterokaryons were exposed to antifungals
and had a higher growth rate than heterozygous diploids. These find-
ings implicate that the potency to develop heterokaryons results in a
higher phenotypic plasticity, and a higher chance to adapt to antifungal
compounds (Zhang et al., 2019).
Sexual ascospores of A. fumigatus were shown to be more resistant to
draught, oxidative stress, and heat (Wyatt et al., 2013), making them
more resilient to several stresses. However, not all species show this
fitness advantage in sexual spores, as A. nidulans does not produce
stress-resistant ascospores (Schoustra et al., 2010). Nevertheless, a re-
cent study showed that sexual reproduction in A. fumigatus might be
accountable for the increased length of the tandem repeat in isolates
collected from compost samples. Through unequal meiotic crossing
over, mispairing of the tandem repeat region can lead to even longer
repeats which result in a higher copy number of cyp51A transcripts and
increased resistance to azoles (Zhang et al., 2017a). In C. neoformans,
clonal cells of the α mating type were shown to mate with themselves in
α-α unisexual reproduction cycle. This form of reproduction frequently
lead to offspring with aneuploid genomes, leading to fitness advantages
against azole compounds when an extra copy of chromosome 9 or 10
was obtained (Ni et al., 2013). In C. albicans, the parasexual cycle was
shown to create fusants of homozygous cells, which displayed a higher
growth rate then their parent cells (Zhang et al., 2015).
Thus, sexual and parasexual reproduction are processes which
generate genetic variability and have shown to lead to fitness increases
in various examples. They promote potential adaptation, although to
date a direct link with increased antifungal resistance was only found in
A. fumigatus and C. neoformans.
4.6. Population dynamics
The evolution of a drug-resistant strain from a drug-susceptible
population depends on multiple processes described earlier, which lead
to genetic variation in a population. Additionally, the size of a popu-
lation affects the evolution of antifungal resistance. In a large popula-
tion, the chances are higher that beneficial mutations arise per gen-
eration, which can spread in the population through sporulation or
sexual recombination (Huang and Kao, 2012). Furthermore, the
8
Fungal Genetics and Biology 132 (2019) 103254
generation of genetic variability through parasexual reproduction is
hypothesized to occur more likely in older, long-lasting fungal colonies,
if different genotypes are present in the population (Verweij et al.,
2016a,b). Observations on the properties of fungal populations revealed
that there was a high growth rate variability in clinical isolates from
Candida spp. on fluconazole (15 μg/ml) and itraconazole (0.71 μg/ml)
(Schoofs et al., 1997), implying high genetic variation in the popula-
tion, which could explain the differences in antifungal drug tolerance. A
genetic analysis of 2026 A. fumigatus isolates where 6% of all isolates
was azole resistant, showed that the azole susceptible populations ex-
hibited little genetic differences, but the azole resistant isolates dis-
played a much higher level of genetic differences (Ashu et al., 2017).
Another study on A. fumigatus lineages derived from culturing in the
presence of the fungicide difenoconazole, observed the rise of different
morphotypes in one lineage. Variation between morphotypes was found
in colony size, sporulation speed and in mycelial density. The mixed
population of morphotypes was shown to confer faster cross-resistance
to medical azoles than isolated morphotypes from the population
(Zhang et al., 2017b). As fungal isolates often encompass a population
with mixed morphotypes, this example shows that there could be fre-
quency-dependent selection involved in a population resulting in
maintenance of various genotypes. This process leads to a more stable
community which increases the chances of survival in stressful en-
vironments, making these fungal populations difficult to combat.
A study conducted by Huang et al. also demonstrated maintenance
of several genoypes in a population (Huang and Kao, 2012). Evolu-
tionary dynamics that were studied in an isogenic C. albicans population
exposed to variable doses of fluconazole, showed that various adaptive
events occurred in the proximity of fluconazole. Most adaptive events
resulted in a higher MIC for fluconazole, but prior adaptations some-
times persisted in the population.
Notably, wildtype clinical C. albicans isolates can influence their
population size by regulation of the transcription factor EFH1, which
determined the level of gastrointestinal (GI) tract colonization.
Overexpression of EFH1 resulted in a reduced colonization, which led
to a more commensal growth behavior rather than an invasive one,
which would result in invasive candidiasis (White et al., 2007). Cells
that overexpress EFH1 showed a reduced colonization of the GI tract,
whereas null mutants were shown to lead to a higher level of coloni-
zation. The factors that influence fungal population sizes should be
monitored, as it could impose a survival mechanism that cells utilize
when encountering stressful environments.
4.7. Horizontal gene transfer in fungi
Finally, evolution of resistance mechanisms through exchange of
genetic material within a population is shown to be highly mediated by
horizontally transferred genes (HTG) in prokaryotes, although this
process also takes place in eukaryotes to a lesser extent (Collins and
Saville, 1990; Keeling and Palmer, 2008; Lerminiaux and Cameron,
2018). A study conducted by Nguyen et al., identified 273 HTG from
prokaryotic origin in the A. fumigatus genome, and 542 HTG in the A.
flavus genome (Nguyen et al., 2015). Although these genes are mostly
involved in secondary metabolism, a study was done on an A. fumigatus
strain which was identified to harbor AfuMGTC, a potential virulence
gene of bacterial origin, although this gene did not increase virulence in
A. fumigatus (Gastebois et al., 2011). In another study, an investigation
of the pan-genomes of fungal species elucidated that the majority of
HTG found in A. fumigatus (391) and C. neoformans (420) came from
other fungal species. These genes were identified to originate from
closely related species or species that share the same ecological niche
(McCarthy and Fitzpatrick, 2019).
Although no HGT of antifungal resistance has been reported yet,
these studies highlight the potential role HGT could play in the devel-
opment of antifungal resistance.
M.W.J. Hokken, et al.
5. Concluding remarks
The emergence of antifungal drug resistance mechanisms results
from adaptation, and the ability to create genetic variation within a
population.
It is crucial that these mechanisms are detected quickly, to be able
to anticipate on resistant populations and adjust treatment strategies
accordingly. New resistance mechanisms are likely to continue to arise
against all antifungal compounds, and understanding the underlying
evolutionary processes remains crucial to recognize situations that can
facilitate the development of resistance mechanisms. The rising re-
sistance rates around the world emphasize this and worldwide sur-
veillance should be increased to monitor resistance rates (Chowdhary
and Meis, 2018; Lestrade et al., 2019; Verweij et al., 2016a,b).
Awareness for environmental ‘hotspots’ that facilitate resistance de-
velopment is important, as for instance, there is a strong link between
the use of agricultural fungicides and the emergence of resistant fungal
populations (Brilhante et al., 2019; Fisher et al., 2018; Snelders et al.,
2009; Verweij et al., 2009; Zhang et al., 2017b). It is critical to monitor
the use of agricultural fungicides, to limit resistance development in the
environment (Verweij et al., 2009). The use of the same or similar
molecules for different applications, i.e. crop protection and medical
treatments, increases the risk of resistance selection that may affect the
effectiveness of the compounds for other applications.
The chances of resistance mechanisms originating in a population
that is exposed to antifungal compounds, would supposedly remain
limited if a second antifungal from a different chemical class is present,
as the mutational chances are much smaller that two resistance me-
chanisms in different pathways arise at the same instant. Although the
combined use of amphotericin B and 5-flucytosine was shown to reduce
mortality in cryptococcal menigitis, solid evidence is lacking for the
effectiveness of the combined use of other compounds in fungal infec-
tions, as these studies require time and are costly. Combinations of
antifungals should less frequently lead to the emergence of resistant
populations. Synergistic effects between antifungal compounds should
be looked into, as this could even lead to a drop in MICs, possibly
permitting the use of lower dosages (Campitelli et al., 2017).
Finally, several studies suggest the influence of environmental stress
on mutational rates (Galhardo et al., 2012; Lamb et al., 2008). If in-
creased cellular stress does indeed lead to the faster adaptation through
increased mutational rates, and mutation frequencies appear to differ
between compounds, it is essential to look into these mechanisms to
facilitate safe compound development.
Antifungal resistance develops when fungal species adjust their
physiology or develop beneficial mutations during therapy, or via en-
vironmental exposure. This inevitable process of adaptation should be
restrained as much as possible, by unraveling the mechanistic and
evolutionary factors that contribute to the spread of resistant fungal
pathogens. Understanding these factors will ultimately contribute to the
development of new therapies and may improve treatment options for
patients suffering from fungal diseases worldwide.
Formatting of funding sources
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors
References
Alby, K., Schaefer, D., Bennett, R.J., 2009. Homothallic and heterothallic mating in the
opportunistic pathogen Candida albicans. Nature 460, 890–893. https://doi.org/10.
1038/nature08252.
Alcazar-Fuoli, L., Mellado, E., 2012. Ergosterol biosynthesis in Aspergillus fumigatus: Its
relevance as an antifungal target and role in antifungal drug resistance. Front.
Microbiol. 3, 1–6. https://doi.org/10.3389/fmicb.2012.00439.
Alcazar-fuoli, L., Mellado, E., Buitrago, M.J., Lopez, J.F., Joan, O., Cuenca-estrella, J.M.,
Juan, L., Garcia-effron, G., Grimalt, J.O., Rodriguez-tudela, J.L., 2006. Aspergillus
9
Fungal Genetics and Biology 132 (2019) 103254
fumigatus C-5 Sterol Desaturases Erg3A and Erg3B : role in sterol biosynthesis and
antifungal drug susceptibility aspergillus fumigatus C-5 sterol desaturases Erg3A and
Erg3B: role in sterol biosynthesis and antifungal drug susceptibility. Antimicrob.
Agents Chemother. 5, 453–460. https://doi.org/10.1128/AAC.50.2.453.
Alexander, B.D., Johnson, M.D., Pfeiffer, C.D., Jiménez-Ortigosa, C., Catania, J., Booker,
R., Castanheira, M., Messer, S.A., Perlin, D.S., Pfaller, M.A., 2013. Increasing echi-
nocandin resistance in candida glabrata: Clinical failure correlates with presence of
FKS mutations and elevated minimum inhibitory concentrations. Clin. Infect. Dis. 56,
1724–1732. https://doi.org/10.1093/cid/cit136.
Altamirano, S., Fang, D., Simmons, C., Sridhar, S., Wu, P., Sanyal, K., Kozubowski, L.,
2017. Fluconazole-induced ploidy change in cryptococcus neoformans results from
the uncoupling of cell growth and nuclear division. mSphere 2, 1–18. https://doi.
org/10.1128/msphere.00205-17.
Ashu, E.E., Hagen, F., Chowdhary, A., Xu, J., Meis, J.F., 2017. Global population genetic
analysis of Aspergillus fumigatus. MSphere 2, 1–13.
Aubin-Horth, N., Renn, S.C.P., 2009. Genomic reaction norms: Using integrative biology
to understand molecular mechanisms of phenotypic plasticity. Mol. Ecol. 18,
3763–3780. https://doi.org/10.1111/j.1365-294X.2009.04313.x.
Balashov, S.V., Park, S., Perlin, D.S., 2006. Assessing resistance to the echinocandin an-
tifungal drug caspofungin in Candida albicans by profiling mutations in FKS1.
Antimicrob. Agents Chemother. 50, 2058–2063. https://doi.org/10.1128/AAC.
01653-05.
Balkis, M.M., Leidich, S.D., Mukherjee, P.K., Ghannoum, M.A., 2002. Mechanisms of
fungal resistance: an overview. [Review] [102 refs]. Drugs 62, 1025–1040.
Baracho, M.S., Baracho, I.R., 2003. An analysis of the spontaneous mutation rate mea-
surement in filamentous fungi. Genet. Mol. Biol. 26, 83–87.
Basso Jr, L.R., Gast, C.E., Bruzual, I., Wong, B., 2015. Identification and properties of
plasma membrane azole efflux pumps from the pathogenic fungi Cryptococcus gattii
and Cryptococcus neoformans. J. Antimicrob. Chemother. 70, 1396. https://doi.org/
10.1093/JAC/DKU554.
Berkow, E.L., Lockhart, S.R., 2018. Activity of novel antifungal compound APX001A
against a large collection of Candida auris. The Journal of Antimicrobial che-
motherapy 73 (11), 3060–3062. https://doi.org/10.1093/jac/dky302.
Bhatt, V.R., Viola, G.M., Ferrajoli, A., 2011. Invasive fungal infections in acute leukemia.
Ther. Adv. Hematol. 2, 231–247. https://doi.org/10.1177/2040620711410098.
Bonaventura, G. Di, Pompilio, A., Picciani, C., Iezzi, M., Antonio, D.D., Piccolomini, R.,
2006. Biofilm formation by the emerging fungal pathogen trichosporon asahii: de-
velopment architecture, and antifungal resistance. Antimicrobial Agents and
Chemotherapy 50, 3269–3276. https://doi.org/10.1128/AAC.00556-06.
Bordallo-Cardona, M., Escribano, P., de la Pedrosa, E., Marcos-Zambrano, L., Cantón, R.,
Bouza, E., Guinea, J., 2017. Vitro exposure to increasing micafungin concentrations
easily promotes echinocandin resistance in Candida glabrata isolates. Antimicrob.
Agents Chemother. 61, 1–5.
Bosco-Borgeat, M.E., Taverna, C.G., Vivot, W., Murisengo, O.A., Mazza, M., Córdoba, S.,
Davel, G., 2016. Amino acid substitution in Cryptococcus neoformans lanosterol 14-
α-demethylase involved in fluconazole resistance in clinical isolates. Rev. Argent.
Microbiol. 48, 137–142. https://doi.org/10.1016/j.ram.2016.03.003.
Bossche, H. Vanden, Bellens, D., Cools, W., Gorrens, J., Marichal, P., Verhoeven, H.,
Willemsens, G., Coster, R. De, Beerens, D., Haelterman, C., Coene, M., Lauwers, W.,
Jeune, L. Le, 1986. Cytochrome P- 450: Target for ltraconazole. Drug Dev. Res. 298,
287–298.
Boyce, K.J., Wang, Y., Verma, S., Shakya, V.P.S., Xue, C., Idnurm, A., 2017. Mismatch
repair of DNA replication errors contributes to microevolution in the pathogenic
fungus cryptococcus neoformans. MBio 8. https://doi.org/10.1128/mbio.00595-17.
Brilhante, R.S., de Alencar, L.P., Bandeira, S.P., Sales, J.A., de Jesus Evangelista, A.J.,
Serpa, R., de Aguiar, Cordeiro R., de Aquino, Pereira-Neto W., Sidrim, J.J., Castelo,
D.D., Rocha, M.F., 2019. Exposure of Candida parapsilosis complex to agricultural
azoles: An overview of the role of environmental determinants for the development of
resistance. Sci. Total Environ. 650, 1231–1238. https://doi.org/10.1016/j.scitotenv.
2018.09.096.
Bromley, M., Johns, A., Davies, E., Fraczek, M., Gilsenan, J.M., Kurbatova, N., Keays, M.,
Kapushesky, M., Gut, M., Gut, I., Denning, D.W., Bowyer, P., 2016. Mitochondrial
complex I is a global regulator of secondary metabolism, virulence and azole sensi-
tivity in fungi. PLoS One 11, 1–22. https://doi.org/10.1371/journal.pone.0158724.
Brun, S., Berges, T., Poupard, P., Vauzelle-Moreau, C., Renier, G., Chabasse, D., Bouchara,
J.-P., 2004. Mechanisms of azole resistance in petite mutants of Candida glabrata.
Antimicrob. Agents Chemother. 48, 1788–1796. https://doi.org/10.1128/aac.48.5.
1788-1796.2004.
Bui, T., Lin, X., Malik, R., Heitman, J., Carter, D., 2008. Isolates of Cryptococcus neo-
formans from infected animals reveal genetic exchange in unisexual, α mating type
populations. Eukaryot. Cell 7, 1771–1780. https://doi.org/10.1128/EC.00097-08.
Buil, J.B., Snelders, E., Denardi, L.B., Melchers, W.J.G., Verweij, P.E., 2019. Trends in
azole resistance in aspergillus fumigatus, the Netherlands, 1994–2016. Emerg. Infect.
Dis. 25, 176–178. https://doi.org/10.3201/eid2501.171925.
Campitelli, M., Zeineddine, N., Samaha, G., Maslak, S., 2017. Combination antifungal
therapy: a review of current data. J. Clin. Med. Res. 9, 451–456. https://doi.org/10.
14740/jocmr2992w.
Camps, S.M.T., Dutilh, B.E., Arendrup, M.C., Rijs, A.J.M.M., Snelders, E., Huynen, M.A.,
Verweij, P.E., Melchers, W.J.G., 2012a. Discovery of a hapE mutation that causes
azole resistance in aspergillus fumigatus through whole genome sequencing and
sexual crossing. PLoS One 7, e50034. https://doi.org/10.1371/journal.pone.
0050034.
Camps, S.M.T., Van Der Linden, J.W.M., Li, Y., Kuijper, E.J., Van Dissel, J.T., Verweij,
P.E., Melchers, W.J.G., 2012b. Rapid induction of multiple resistance mechanisms in
Aspergillus fumigatus during azole therapy: a case study and review of the literature.
Antimicrob. Agents Chemother. 56, 10–16. https://doi.org/10.1128/AAC.05088-11.
M.W.J. Hokken, et al.
Cannon, R.D., Lamping, E., Holmes, A.R., Niimi, K., Baret, P.V., Keniya, M.V., Tanabe, K.,
Niimi, M., Goffeau, A., Monk, B.C., 2009. Efflux-mediated antifungal drug resistance.
Clin. Microbiol. Rev. 22, 291–321. https://doi.org/10.1128/cmr.00051-08.
Castanheira, M., Woosley, L.N., Diekema, D.J., Messer, S.A., Jones, R.N., Pfaller, M.A.,
2010. Low prevalence of fks1 hot spot 1 mutations in a worldwide collection of
candida strains. Antimicrob. Agents Chemother. 54, 2655–2659. https://doi.org/10.
1128/AAC.01711-09.
Cavalheiro, M., Costa, C., Silva-Dias, A., Miranda, I.M., Wang, C., Pais, P., Pinto, S.N.,
Mol-Homens, D., Sato-Okamoto, M., Takahashi-Nakaguchi, A., Silva, R.M., Mira,
N.P., Fialho, A.M., Chibana, H., Rodrigues, A.G., Butler, G., Teixeira, M.C., 2018. A
transcriptomics approach to unveiling the mechanisms of in vitro evolution towards
fluconazole resistance of a Candida glabrata clinical isolate. Antimicrob. Agents
Chemother. 63, 1–17.
Chabasse, D., Planchenault, C., Defontaine, A., Hallet, J.-N., Declerk, P., Bouchara, J.-P.,
2009. In-vitro resistance to azoles associated with mitochondrial DNA deficiency in
Candida glabrata. J. Med. Microbiol. 48, 663–670. https://doi.org/10.1099/
00222615-48-7-663.
Chandra, J., Kuhn, D.M., Mukherjee, P.K., Hoyer, L.L., Cormick, T.M.C., Ghannoum, M.A.,
2001. Biofilm formation by the fungal pathogen Candida albicans: development,
architecture, and drug resistance. J. Bacteriol. 183, 5385–5394. https://doi.org/10.
1128/JB.183.18.5385.
Charlier, C., Sissy, E., Bachelier-bassi, S., Scemla, A., Quesne, G., Sitterlé, E., Legendre, C.,
Lortholary, O., Bougnoux, M., 2016. Acquired flucytosine resistance during combi-
nation therapy with caspofungin and flucytosine for Candida glabrata cystitis.
Antimicrob. Agents Chemother. 60, 662–665. https://doi.org/10.1128/AAC.02265-
15.Address.
Chen, C.A., Sorrell, T.C., 2007. Antifungal agents. Med. J. Aust. 187.
Chen, G., Bradford, W.D., Seidel, C.W., Li, R., City, K., Physiology, I., Boulevard, R., City,
K., 2012. Induction of aneuploidy. Nature 482, 246–250. https://doi.org/10.1038/
nature10795.Hsp90.
Choi, M.J., Won, E.J., Shin, J.H., Kim, S.H., Lee, K., Kim, M.-N., Lee, K., Shin, M.G., Suh,
S.P., Ryang, D.W., Im, Y.J., 2016. Resistance mechanisms and clinical features of
fluconazole-nonsusceptible Candida tropicalis isolates compared with fluconazole-
less-susceptible isolates. Antimicrob. Agents Chemother. 60, 3653–3661. https://doi.
org/10.1128/aac.02652-15.
Chotirmall, S.H., Mirkovic, B., Lavelle, G.M., McElvaney, N.G., 2014. Immunoevasive
aspergillus virulence factors. Mycopathologia 178, 363–370. https://doi.org/10.
1007/s11046-014-9768-y.
Chowdhary, A., Meis, J.F., 2018. Emergence of azole resistant Aspergillus fumigatus and
One Health: time to implement environmental stewardship. Environ. Microbiol. 20,
1299–1301. https://doi.org/10.1111/1462-2920.14055.
Chowdhary, A., Sharma, C., Meis, J.F., 2017. Azole-resistant aspergillosis: epidemiology,
molecular mechanisms, and treatment. J. Infect. Dis. 216, S436–S444. https://doi.
org/10.1093/infdis/jix210.
Collins, R.A., Saville, B.J., 1990. Independent transfer of mitochondrial chromosomes and
plasmids during unstable vegetative fusion in Neurospora. Nature 345, 177–179.
https://doi.org/10.1038/345177a0.
Cordonnier, C., Cesaro, S., Maschmeyer, G., Einsele, H., Peter Donnelly, J., Alanio, A.,
Hauser, P., Lagrou, K., Melchers, W.J.G., Helweg-Larsen, J., Matos, O., Bretagne, S.,
Maertens, J., Agrawal, S., Kibbler, C., Pagliuca, A., Ward, K., Akova, M., Herbrecht,
R., Mallet, V., Ribaud, P., Aljurf, M., Averbuch, D., Engelhard, D., Berg, T., Cornely,
O., Penack, O., van Boemmel, F., von Lilienfeld-Toal, M., Blennow, O., Ljungman, P.,
Bruggemann, R., Donnelly, P., Kullberg, B.J., Melchers, W., Calandra, T., Hirsch, H.,
Marchetti, O., Orasch, C., Tissot, F., Castagnola, E., Girmenia, C., Mikulska, M.,
Pagano, L., Viscoli, C., De La Camara, R., Duarte, R., Munoz, P., Drgona, L.,
Hargreaves, R., Hubacek, P., Kouba, M., Racil, Z., Klyasova, G., Pettrikos, G., Roilides,
E., Skiada, A., Rizzi-Puechal, V., Sinko, J., Slavin, M., Styczynski, J., Tweddle, L.,
Wood, C., 2016. Pneumocystis jiroveci pneumonia: Still a concern in patients with
haematological malignancies and stem cell transplant recipients. J. Antimicrob.
Chemother. 71, 2379–2385. https://doi.org/10.1093/jac/dkw155.
Coste, A., Turner, V., Ischer, F., Morschhäuser, J., Forche, A., Selmecki, A., Berman, J.,
Bille, J., Sanglard, D., 2006. A mutation in Tac1p, a transcription factor regulating
CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate
antifungal resistance in Candida albicans. Genetics 172, 2139–2156. https://doi.org/
10.1534/genetics.105.054767.
Coste, A.T., Karababa, M., Ischer, F., Bille, J., Sanglard, D., 2004. TAC1, transcriptional
activator of CDR genes, is a new transcription factor involved in the regulation of
Candida albicans ABC transporters CDR1 and CDR2. Eukaryot. Cell 3, 1639–1652.
https://doi.org/10.1128/EC.3.6.1639-1652.2004.
Cowen, L.E., 2015. Hsp90 Potentiates the rapidevolution of new traits: drugresistance in
diverse fungi. Science (80-.) 309, 1–6. https://doi.org/10.1126/science.1118370.
Cowen, L.E., 2009. Hsp90 orchestrates stress response signaling governing fungal drug
resistance. PLoS Pathog. 5, e1000471. https://doi.org/10.1371/journal.ppat.
1000471.
Cowen, L.E., Sanglard, D., Calabrese, D., Surjusingh, C., Anderson, J.B., Kohn, L.M., 2000.
Population genomics of drug resistance in experimental populations of Candida al-
bicans. J. Bacteriol. 99, 9284–9289.
Cowen, L.E., Steinbach, W.J., 2008. Stress, drugs, and evolution: the role of cellular
signaling in fungal drug resistance. Eukaryot. Cell 7, 747–764. https://doi.org/10.
1128/ec.00041-08.
Dambuza, I.M., Drake, T., Chapuis, A., Zhou, X., Correia, J., Taylor-Smith, L., LeGrave, N.,
Rasmussen, T., Fisher, M.C., Bicanic, T., Harrison, T.S., Jaspars, M., May, R., Brown,
G., Yuecel, R., MacCallum, D.M., Ballou, E.R., 2018. The Cryptococcus neoformans
Titan cell is an inducible and regulated morphotype underlying pathogenesis. PLOS
Pathog. 47, 377–384. https://doi.org/10.1097/00010694-193905000-00005.
Demogines, A., Smith, E., Kruglyak, L., Alani, E., 2008. Identification and dissection of a
10
Fungal Genetics and Biology 132 (2019) 103254
complex DNA repair sensitivity phenotype in baker’s yeast. PLoS Genet. 4. https://
doi.org/10.1371/journal.pgen.1000123.
Denning, D.W., Pashley, C., Hartl, D., Wardlaw, A., Godet, C., Del Giacco, S., Delhaes, L.,
Sergejeva, S., 2014. Fungal allergy in asthma–state of the art and research needs.
Clin. Transl. Allergy 4, 1–23. https://doi.org/10.1186/2045-7022-4-14.
Denning, D.W., Venkateswarlu, K., Oakley, K.L., Anderson, M.J., Manning, N.J., Stevens,
D.A., Warnock, D.W., Kelly, S.L., 1997. Itraconazole resistance in Aspergillus fumi-
gatus. Antimicrob. Agents Chemother. 41, 1364–1368.
dos Reis, T.F., Silva, L.P., de Castro, P.A., de Lima do Carmo, P.B.R.A., Marini, M.M., da
Silveira, J.F., Ferreira, B.H., Rodrigues, F., Malavazi, I., Goldman, G.H., 2017. The
influence of genetic stability on aspergillus fumigatus virulence and azole resistance.
G3: Genes Genom. Genet. 8, 265–278. https://doi.org/10.1534/g3.117.300265.
Drake, J.W., Charlesworth, B., Charlesworth, D., Crow, J.F., 1998. Rates of spontaneous
mutation. Genetics 78, 1209–1221. online ISSN: 1943-2631. Source: https://www.
genetics.org/content/148/4/1667.
Edlind, T.D., Katiyar, S.K., 2010. Mutational analysis of flucytosine resistance in Candida
glabrata. Antimicrob. Agents Chemother. 54, 4733–4738. https://doi.org/10.1128/
AAC.00605-10.
Eschenauer, G., DePestel, D.D., Carver, P.L., 2007. Comparison of echninocandin anti-
fungals. Ther. Clin. Risk Manag. 3, 71–97. https://doi.org/10.2147/tcrm.2007.3.
1.71.
Escribano, P., Recio, S., Peláez, T., González-Rivera, M., Bouza, E., Guinea, J., 2012. In
vitro acquisition of secondary azole resistance in Aspergillus fumigatus isolates after
prolonged exposure to itraconazole: Presence of heteroresistant populations.
Antimicrob. Agents Chemother. 56, 174–178. https://doi.org/10.1128/AAC.
00301-11.
Fan, X., Xiao, M., Zhang, D., Huang, J.-J., Wang, H., Hou, X., Zhang, L., Kong, F., Chen,
S.C.-A., Tong, Z.-H., Xu, Y.-C., 2018. Molecular mechanisms of azole resistance in
Candida tropicalis isolates causing invasive candidiasis in China. Clin. Microbiol.
Infect. https://doi.org/10.1016/j.cmi.2018.11.007.
Fanning, S., Mitchell, A.P., 2012. Fungal biofilms. PLoS Pathog. 8, 1–4. https://doi.org/
10.1371/journal.ppat.1002585.
Farlow, A., Long, H., Arnoux, S., Sung, W., Doak, T.G., Nordborg, M., Lynch, M., 2015.
The spontaneous mutation rate in the fission yeast Schizosaccharomyces pombe.
Genetics 201, 737–744. https://doi.org/10.1534/genetics.115.177329.
Fisher, M.C., Hawkins, N.J., Sanglard, D., Gurr, S.J., 2018. Health and food security.
Science (80-.) 742, 739–742.
Flowers, S.A., Colón, B., Whaley, S.G., Schuler, M.A., David Rogers, P., 2015.
Contribution of clinically derived mutations in ERG11 to azole resistance in Candida
albicans. Antimicrob. Agents Chemother. 59, 450–460. https://doi.org/10.1128/
AAC.03470-14.
Forastiero, A., Pelaez, T., Lopez, J.F., Grimalt, J.O., Cuesta, I., Zaragoza, O., Mellado, E.,
2013. Candida tropicalis antifungal cross-resistance is related to different.
Antimicrob. Agents Chemother. 57, 4769–4781. https://doi.org/10.1128/AAC.
00477-13.
Galhardo, R.S., Hastings, P.J., Rosenberg, S.M., 2012. Mutation as a stress response and
the regulation of evolvability Rodrigo. Crit. Rev. Biochem. Mol. Biol. https://doi.org/
10.1080/10409230701648502.Mutation.
Gao, J., Wang, H., Li, Z., Wong, A.H.-H., Wang, Y.-Z., Guo, Y., Lin, X., Zeng, G., Wang, Y.,
Wang, J., 2018. Candida albicans gains azole resistance by altering sphingolipid
composition. Nat. Commun. 9, 4495. https://doi.org/10.1038/s41467-018-06944-1.
Garcia-Rubio, R., Cuenca-Estrella, M., Mellado, E., 2017. Triazole resistance in aspergillus
species: an emerging problem. Drugs 77, 599–613. https://doi.org/10.1007/s40265-
017-0714-4.
Gastebois, A., Blanc Potard, A.-B., Gribaldo, S., Beau, R., Latgé, J.P., Mouyna, I., 2011.
Phylogenetic and functional analysis of aspergillus fumigatus MGTC, a fungal protein
homologous to a bacterial virulence factor. Appl. Environ. Microbiol. 77, 4700–4703.
https://doi.org/10.1128/aem.00243-11.
Georgopapadakou, N.H., Dix, B.A., Smith, S.A., Freudenberger, J., Funke, P.T., 1987.
Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candida
albicans. Antimicrob. Agents Chemother. 31, 46–51. https://doi.org/10.1128/AAC.
31.1.46.
Gerstein, A.C., Fu, M.S., Mukaremera, L., Li, Z., Ormerod, K.L., Fraser, J.A., Berman, J.,
Nielsen, K., 2015. Polyploid titan cells produce haploid and aneuploid progeny to
promote stress adaptation. MBio 6, 1–14. https://doi.org/10.1128/mBio. 01340-15.
Editor.
Ghannoum, M.A., Rice, L.B., 1999. Antifungal agents: mode of action, mechanisms of
resistance, and correlation of these mechanisms with bacterial resistance. Clin.
Microbiol. Rev. 12, 501–517. Source: https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC88922/.
Gordon, J.L., Byrne, K.P., Wolfe, K.H., 2011. Mechanisms of chromosome number evo-
lution in yeast. PLoS Genet. 7. https://doi.org/10.1371/journal.pgen.1002190.
Gou, L., Bloom, J.S., Kruglyak, L., 2019. The genetic basis of mutation rate variation in
yeast. Genetics 211, 731–740. https://doi.org/10.1534/genetics.118.301609.
Graminha, M.A.S., Rocha, E.M.F., Prade, R.A., Martinez-Rossi, N.M., 2004. Terbinafine
resistance mediated by salicylate 1-monooxygenase in Aspergillus nidulans.
Antimicrob. Agents Chemother. 48, 3530–3535. https://doi.org/10.1128/AAC.48.9.
3530-3535.2004.
Gsaller, F., Furukawa, T., Carr, P.D., Rash, B., Bertuzzi, M., Bignell, E.M., Bromley, M.J.,
2018. Mechanistic basis of ph-dependent 5-flucytosine resistance in aspergillus fu-
migatus. Antimicrob. Agents Chemother. 1–9.
Guan, X.L., Souza, C.M., Pichler, H., Schaad, O., Kajiwara, K., Wakabayashi, H., Ivanova,
T., Castillon, G.A., Piccolis, M., Abe, F., Loewith, R., Funato, K., Wenk, M.R.,
Riezman, H., 2009. Functional interactions between sphingolipids and sterols in
biological membranes regulating cell physiology. Mol. Biol. Cell 20, 2083–2095.
https://doi.org/10.1091/mbc.E08.
M.W.J. Hokken, et al.
Hagiwara, D., 2018. Current status of azole-resistant Aspergillus fumigatus isolates in East
Asia: China, Japan, Korea and Taiwan. Med. Mycol. 59, 71–76.
Hagiwara, D., Miura, D., Shimizu, K., Paul, S., Ohba, A., Gonoi, T., Watanabe, A., Kamei,
K., Shintani, T., Moye-Rowley, W.S., Kawamoto, S., Gomi, K., 2017. A novel Zn2-Cys6
transcription factor AtrR plays a key role in an azole resistance mechanism of as-
pergillus fumigatus by co-regulating cyp51A and cdr1B expressions. PLoS Pathogens.
https://doi.org/10.1371/journal.ppat.1006096.
Hagiwara, D., Sakamoto, K., Abe, K., Gomi, K., 2016a. Signaling pathways for stress re-
sponses and adaptation in Aspergillus species: stress biology in the post-genomic era.
Biosci. Biotechnol. Biochem. 80, 1667–1680. https://doi.org/10.1080/09168451.
2016.1162085.
Hagiwara, D., Watanabe, A., Kamei, K., Goldman, G.H., 2016b. Epidemiological and
genomic landscape of azole resistance mechanisms in aspergillus fungi. Front.
Microbiol. 7, 1382. https://doi.org/10.3389/fmicb.2016.01382.
Händel, N., De La Sayette, S., Verweij, P.E., Brul, S., ter Kuile, B.H., 2015. De novo in-
duction of resistance against voriconazole in Aspergillus fumigatus. J. Glob.
Antimicrob. Resist. 3, 52–53. https://doi.org/10.1016/j.jgar.2015.01.001.
Healey, K.R., Zhao, Y., Perez, W.B., Lockhart, S.R., Sobel, J.D., Farmakiotis, D.,
Kontoyiannis, D.P., Sanglard, D., Taj-Aldeen, S.J., Alexander, B.D., Jimenez-Ortigosa,
C., Shor, E., Perlin, D.S., 2016. Prevalent mutator genotype identified in fungal pa-
thogen Candida glabrata promotes multi-drug resistance. Nat. Commun. 7, 1–10.
https://doi.org/10.1038/ncomms11128.
Heitman, J., 2015. Evolution of sexual reproduction: a view from the fungal kingdom
supports an evolutionary epoch with sex before sexes. Fungal Biol. Rev. 29, 108–117.
https://doi.org/10.1016/j.fbr.2015.08.002.
Hoekstra, R.F., 2005. Why sex is good. Nature 434, 571. https://doi.org/10.1021/
jp0449511.
Hoffman, A., Hercus, M.J., 2000. Environmental stress as an evolutionary force.
Bioscience 50, 217–226.
Hokken, M.W.J., Zoll, J., Coolen, J.P.M., Zwaan, B.J., Verweij, P.E., Melchers, W.J.G.,
2019. Phenotypic plasticity and the evolution of azole resistance in Aspergillus fu-
migatus; an expression profile of clinical isolates upon exposure to itraconazole. BMC
Genom. 1–17.
Holmes, A.R., Cardno, T.S., Strouse, J.J., Ivnitski-Steele, I., Keniya, M.V., Lackovic, K.,
Monk, B.C., Sklar, L.A., Cannon, R.D., 2016. Targeting efflux pumps to overcome
antifungal drug resistance. Future Med. Chem. 8, 1485–1501. https://doi.org/10.
4155/fmc-2016-0050.
Hortschansky, P., Haas, H., Huber, E.M., Groll, M., Brakhage, A.A., 2016. The CCAAT-
binding complex (CBC) in Aspergillus species. Biochim. Biophys. Acta – Gene Regul.
Mech. https://doi.org/10.1016/j.bbagrm.2016.11.008.
Hoving, J.C., Kolls, J.K., 2017. New advances in understanding the host immune response
to Pneumocystis. Curr. Opin. Microbiol. 40, 65–71. https://doi.org/10.1016/j.mib.
2017.10.019.
Hryncewicz-Gwóźdź, A., Kalinowska, K., Plomer-Niezgoda, E., Bielecki, J., Jagielski, T.,
2013. Increase in resistance to fluconazole and itraconazole in trichophyton rubrum
clinical isolates by sequential passages in vitro under drug pressure. Mycopathologia
176, 49–55. https://doi.org/10.1007/s11046-013-9655-y.
Huang, M., Kao, K.C., 2012. Population dynamics and the evolution of antifungal drug
resistance in Candida albicans. FEMS Microbiol. Lett. https://doi.org/10.1111/j.
1574-6968.2012.02587.x.
Huang, M., McClellan, M., Berman, J., Kao, K.C., 2011. Evolutionary dynamics of candida
albicans during in vitro evolution. Eukaryot. Cell 10, 1413–1421. https://doi.org/10.
1128/ec.05168-11.
Iwata, A., Watanabe, Y., Kumagai, N., Katafuchi-Nagashima, M., Sugiura, K., Pillai, R.,
Tatsumi, Y., 2014. In vitro and in vivo assessment of dermatophyte acquired re-
sistance to efinaconazole, a novel triazole antifungal. Antimicrob. Agents Chemother.
58, 4920–4922. https://doi.org/10.1128/AAC.02703-13.
Jenks, J.D., Salzer, H.J.F., Prattes, J., Krause, R., Buchheidt, D., Hoenigl, M., 2018.
Spotlight on isavuconazole in the treatment of invasive aspergillosis and mucormy-
cosis: design, development, and place in therapy. Drug Des. Devel. Ther. 12,
1033–1044. https://doi.org/10.2147/DDDT.S145545.
Jensen, R.H., Johansen, H.K., Arendrup, M.C., 2013. Stepwise development of a homo-
zygous S80P substitution in Fks1p, conferring echinocandin resistance in Candida
tropicalis. Antimicrob. Agents Chemother. 57, 614–617. https://doi.org/10.1128/
AAC.01193-12.
Jiménez-Ortigosa, C., Moore, C., Denning, D.W., Perlin, D.S., 2017. Emergence of echi-
nocandin resistance due to a point mutation in the fks1 gene of aspergillus fumigatus
in a patient with chronic. Pulmonary Aspergillosis 61, 1–6.
Katiyar, S.K., Alastruey-Izquierdo, A., Healey, K.R., Johnson, M.E., Perlin, D.S., Edlind,
T.D., 2012. Fks1 and Fks2 are functionally redundant but differentially regulated in
Candida glabrata: implications for echinocandin resistance. Antimicrob. Agents
Chemother. 56, 6304–6309. https://doi.org/10.1128/aac.00813-12.
Kaur, S., Singh, S., 2015. Biofilm formation by Aspergillus fumigatus. J. Music Ther. 52,
2–9. https://doi.org/10.3109/13693786.2013.819592.
Keeling, P.J., Palmer, J.D., 2008. Horizontal gene transfer in eukaryotic evolution. Nat.
Rev. Genet. 9, 605–618. https://doi.org/10.1038/nrg2386.
Kelly, S., Lamb, D., Kelly, D., Manning, N., Loeffler, J., Hebart, H., Schumacher, U.,
Einsele, H., 1997. Resistance to fluconazole and cross-resistance to amphotericin B in
Candida albicans from AIDS patients caused by defective sterol Δ5,6 -desaturation.
FEBS Lett. 400, 80–82. https://doi.org/10.1016/s0014-5793(96)01360-9.
Kelly, S.L., Lamb, D.C., Corran, A.J., Baldwin, B.C., Kelly, D.E., 1995. Mode of action and
resistance to azole antifungals associated with the formation of 14 alpha-methy-
lergosta-8,24(28)-dien-3 beta,6 alpha-diol. Biochem. Biophys. Res. Commun. https://
doi.org/10.1006/bbrc.1995.1272.
Khan, A., El-Charabaty, E., El-Sayegh, S., 2015. Fungal infection in renal transplant pa-
tients. Med. Mycol. Curr. Trends Futur. Prospect. 7, 110–146. https://doi.org/10.
11
Fungal Genetics and Biology 132 (2019) 103254
1201/b18707.
Ksiezopolska, E., Gabaldón, T., 2018. Evolutionary emergence of drug resistance in
Candida opportunistic pathogens. Genes (Basel) 9, 461. https://doi.org/10.3390/
genes9090461.
Kwon-chung, K.J., Chang, Y.C., 2012. Aneuploidy and drug resistance in pathogenic
fungi. PLoS Pathogens 8, 8–11. https://doi.org/10.1371/journal.ppat.1003022.
Lamb, B.C., Mandaokar, S., Bahsoun, B., Grishkan, I., Nevo, E., 2008. Differences in
spontaneous mutation frequencies as a function of environmental stress in soil fungi
at “Evolution Canyon” Israel. Proc. Natl. Acad. Sci. U. S. A. 105, 5792–5796. https://
doi.org/10.1073/pnas.0801995105.
Lamb, D., Kelly, D., Kelly, S., 1999. Molecular aspects of azole antifungal action and
resistance. Drug Resist. Updat. 2, 390–402. https://doi.org/10.1054/drup.1999.
0112.
Lang, G.I., Murray, A.W., 2008. Estimating the per-base-pair mutation rate in the yeast
saccharomyces cerevisiae. Genetics 82, 67–82. https://doi.org/10.1534/genetics.
107.071506.
Lang, G.I., Parsons, L., Gammie, A.E., 2013. Mutation rates, spectra, and genome-wide
distribution of spontaneous mutations in mismatch repair deficient yeast. G3: Genes
Genom Genet. 3, 1453–1465. https://doi.org/10.1534/g3.113.006429.
Lat, A., Thompson, G., 2011. Update on the optimal use of voriconazole for invasive
fungal infections. Infect. Drug Resist. 43. https://doi.org/10.2147/idr.s12714.
Latgé, J.P., 2001. The pathobiology of Aspergillus fumigatus. Trends Microbiol. 9,
382–389. https://doi.org/10.1016/S0966-842X(01)02104-7.
Laverdière, M., Lalonde, R.G., Baril, J.G., Sheppard, D.C., Park, S., Perlin, D.S., 2006.
Progressive loss of echinocandin activity following prolonged use for treatment of
Candida albicans oesophagitis. J. Antimicrob. Chemother. 57, 705–708. https://doi.
org/10.1093/jac/dkl022.
Legrand, M., Chan, C.L., Jauert, P.A., Kirkpatrick, D.T., 2007. Role of DNA mismatch
repair and double-strand break repair in genome stability and antifungal drug re-
sistance in Candida albicans. Eukaryot. Cell 6, 2194–2205. https://doi.org/10.1128/
EC.00299-07.
Lerminiaux, N.A., Cameron, A.D.S., 2018. Horizontal transfer of antibiotic resistance
genes in clinical environments. Can. J. Microbiol. 65, 34–44. https://doi.org/10.
1139/cjm-2018-0275.
Lestrade, P.P., Bentvelsen, R.G., Schauwvlieghe, A.F.A.D., Schalekamp, S., van der
Velden, W.J.F.M., Kuiper, E.J., van Paassen, J., van der Hoven, B., van der Lee, H.A.,
Melchers, W.J.G., de Haan, A.F., van der Hoeven, H.L., Rijnders, B.J.A., van der Beek,
M.T., Verweij, P.E., 2019a. Voriconazole resistance and mortality in invasive
Aspergillosis: A multicenter retrospective cohort study. Clin. Infect. Dis. 68,
1463–1471. https://doi.org/10.1093/cid/ciy859.
Lestrade, P.P.A., Meis, J.F., Melchers, W.J.G., Verweij, P.E., 2019b. Triazole resistance in
Aspergillus fumigatus: recent insights and challenges for patient management. Clin.
Microbiol. Infect. https://doi.org/10.1016/j.cmi.2018.11.027.
Liu, M., Zheng, N., Li, D., Zheng, H., Zhang, L., Ge, H., Liu, W., 2016. Cyp51A-based
mechanism of azole resistance in Aspergillus fumigatus: Illustration by a new 3D
structural model of Aspergillus fumigatus CYP51A protein. Med. Mycol. 54, 400–408.
https://doi.org/10.1093/mmy/myv102.
Luzia, F.R., Lais, L., Maria, P., Moretti, L., Pham, C.D., Lockhart, S.R., Zaninelli, A., 2018.
Surveillance for azoles resistance in Aspergillus spp. highlights a high number of
amphotericin B-resistant isolates. Mycoses 360–365. https://doi.org/10.1111/myc.
12759.
Malcolm, T.R., Chin-Hong, P.V., 2013. Endemic mycoses in immunocompromised hosts.
Curr. Infect. Dis. Rep. 15, 536–543. https://doi.org/10.1007/s11908-013-0387-4.
Maligie, M.A., Selitrennikoff, C.P., 2005. Cryptococcus neoformans resistance to echi-
nocandins: (1,3)β-glucan synthase activity is sensitive to echinocandins. Antimicrob.
Agents Chemother. 49, 2851–2856. https://doi.org/10.1128/AAC.49.7.2851-2856.
2005.
Martin, M.V., 1999. The use of fluconazole and itraconazole in the treatment of Candida
abicans infections: a review. J. Antimicrob. Chemother. 44, 429–437. https://doi.
org/10.1093/jac/44.4.429.
Martinez, L.R., Casadevall, A., 2015. Biofilm Formation by Cryptococcus neoformans.
Microbiol. Spectr. 6, 513–521. https://doi.org/10.1128/microbiolspec.MB-0006-
2014.Correspondence.
Martins, M., Uppuluri, P., Thomas, D.P., Cleary, I.A., Henriques, M., Lopez-Ribot, J.L.,
Oliveira, R., 2010. Presence of extracellular DNA in the Candida albicans biofilm
matrix and its contribution to biofilms. Mycopathologia 169, 323–331. https://doi.
org/10.1007/s11046-009-9264-y.Presence.
McCarthy, C.G.P., Fitzpatrick, D.A., 2019. Pan-genome analyses of model fungal species.
Microb. Genom. 1–23. https://doi.org/10.1099/mgen.0.000243.
Mesa-Arango, A.C., Scorzoni, L., Zaragoza, O., 2012. It only takes one to do many jobs:
Amphotericin B as antifungal and immunomodulatory drug. Front. Microbiol. 3,
1–10. https://doi.org/10.3389/fmicb.2012.00286.
Moran, G.P., Sanglard, D., Donnelly, S.M., Shanley, D.B., Sullivan, D.J., Coleman, D.C.,
1998. Identification and expression of multidrug transporters responsible for fluco-
nazole resistance in Candida dubliniensis. Antimicrob. Agents Chemother. 42,
1819–1830.
Moudgal, V., Little, T., Boikov, D., Vazquez, J.A., 2004. Multiechinocandin- and multi-
azole-resistant Candida parapsilosis isolates serially obtained during therapy for
prosthetic valve endocarditis. Antimicrob. Agents Chemother. 49, 767–769. https://
doi.org/10.1128/AAC.49.2.767.
Mukherjee, P.K., Leidich, S.D., Isham, N., Leitner, I., Ryder, N.S., Ghannoum, M.A., 2003.
Clinical Trichophyton rubrum strain exhibiting primary resistance to terbinafine.
Antimicrob. Agents Chemother. 47, 82–86. https://doi.org/10.1128/AAC.47.1.82.
Müller, F.M.C., Seidler, M., Beauvais, A., 2011. Aspergillus fumigatus biofilms in the
clinical setting. Med. Mycol. 49, 96–100. https://doi.org/10.3109/13693786.2010.
502190.
M.W.J. Hokken, et al.
Munro, C.A., Selvaggini, S., Bruijn, I De, Walker, L., Lenardon, M.D., Gerssen, B., Milne,
S., Brown, A.J.P., Gow, N.A.R., 2007. The PKC, HOG and Ca2+ signalling pathways
co-ordinately regulate chitin synthesis in Candida albicans. Mol. Microbiol. 63,
1399–1413. https://doi.org/10.1111/j.1365-2958.2007.05588.x.
Nelson, M.R., Fisher, M., Cartledge, J., Rogers, T., Gazzard, B.G., 1994. The role of azoles
in the treatment and prophylaxis of cryptococcal disease in HIV infection. AIDS 8,
651–654. https://doi.org/10.1097/00002030-199405000-00011.
Nguyen, M., Ekstrom, A., Li, X., Yin, Y., 2015. HGT-finder: a new tool for horizontal gene
transfer finding and application to Aspergillus genomes. Toxins (Basel) 7,
4035–4053. https://doi.org/10.3390/toxins7104035.
Ni, M., Feretzaki, M., Li, W., Floyd-Averette, A., Mieczkowski, P., Dietrich, F.S., Heitman,
J., 2013. Unisexual and heterosexual meiotic reproduction generate aneuploidy and
phenotypic diversity de novo in the yeast cryptococcus neoformans. PLoS Biol. 11.
https://doi.org/10.1371/journal.pbio.1001653.
Nicolas, A.G., Serero, A., Legoix-Né, P., Jubin, C., Loeillet, S., 2014. Mutational landscape
of yeast mutator strains. Proc. Natl. Acad. Sci. 111, 1897–1902. https://doi.org/10.
1073/pnas.1314423111.
Oromendia, A.B., Dodgson, S., Amon, A., 2008. Aneuploidy causes proteotoxic stress in
yeast. Genetics 26, 2696–2708. https://doi.org/10.1101/gad.207407.112.Compton.
Osborne, C.S., Leitner, I., Favre, B., Neil, S., Osborne, C.S., Leitner, I., Favre, B., Ryder,
N.S., 2005. Amino acid substitution in trichophyton rubrum squalene epoxidase as-
sociated with resistance to terbinafine amino acid substitution in trichophyton ru-
brum squalene epoxidase associated with resistance to terbinafine. Antimicrob.
Agents Chemother. 49, 2840–2844. https://doi.org/10.1128/AAC.49.7.2840.
Pais, P., Pires, C., Costa, C., Okamoto, M., Chibana, H., Teixeira, M.C., 2016. Membrane
proteomics analysis of the Candida glabrata response to 5-flucytosine: Unveiling the
role and regulation of the drug efflux transporters CgFlr1 and CgFlr2. Front.
Microbiol. 7, 1–14. https://doi.org/10.3389/fmicb.2016.02045.
Park, S., Kelly, R., Kahn, J.N., Robles, J., Hsu, M.J., Register, E., Li, W., Vyas, V., Fan, H.,
Abruzzo, G., Flattery, A., Gill, C., Chrebet, G., Parent, S.A., Kurtz, M., Teppler, H.,
Douglas, C.M., Perlin, D.S., 2005. Specific substitutions in the echinocandin target
Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp.
isolates. Antimicrob. Agents Chemother. 49, 3264–3273. https://doi.org/10.1128/
AAC.49.8.3264-3273.2005.
Paul, S., Diekema, D., Moye-Rowley, W.S., 2017. Contributions of both ATP-binding
cassette transporter and Cyp51A proteins are essential for azole resistance in
Aspergillus fumigatus. Antimicrob. Agents Chemother. 61, 1–10.
Paul, S., Moye-Rowley, W.S., 2014. Multidrug resistance in fungi: Regulation of trans-
porter-encoding gene expression. Front. Physiol. 5 (APR), 1–14. https://doi.org/10.
3389/fphys.2014.00143.
Perfect, J.R., 2018. The antifungal pipeline: a reality check. Nat. Rev. Drug Discovery 16,
603–616. https://doi.org/10.1038/nrd.2017.46.The.
Perfect, J.R., Cox, G.M., 1999. Drug resistance in Cryptococcus neoformans. Drug Resist.
Updat. 2, 259–269. https://doi.org/10.1054/drup.1999.0090.
Perlin, D.S., 2007. Resistance to echinocandin-class antifungal drugs. Drug Resist. Updat.
https://doi.org/10.1016/j.drup.2007.04.002.Resistance.
Perlin, D.S., Shor, E., Zhao, Y., 2015. Update on antifungal drug resistance. Curr. Clin.
Microbiol. Rep. 2, 84–95. https://doi.org/10.1007/s40588-015-0015-1.
Perlin, M.H., Andrews, J., Toh, S.S., 2014. Essential letters in the fungal alphabet: ABC
and MFS transporters and their roles in survival and pathogenicity. Adv. Genet.
https://doi.org/10.1016/B978-0-12-800271-1.00004-4.
Pilmis, B., Garcia-Hermoso, D., Alanio, A., Catherinot, E., Scemla, A., Jullien, V.,
Bretagne, S., Lortholary, O., 2018. Failure of voriconazole therapy due to acquired
azole resistance in Aspergillus fumigatus in a kidney transplant recipient with chronic
necrotizing aspergillosis. Am. J. Transplant. 18, 2352–2355. https://doi.org/10.
1111/ajt.14940.
Pontecorvo, G., Roper, J.A., Chemmons, L.M., Macdonald, K.D., Bufton, A.W.J., 1953.
The genetics of Aspergillus nidulans. Adv. Genet. 5, 141–238. https://doi.org/10.
1016/S0065-2660(08)60408-3.
du Pré, S., et al., 2018. Effect of the Novel Antifungal Drug F901318 (Olorofim) on
Growth and Viability of Aspergillus fumigatus. Antimicrobial agents and
Chemotherapy 62 (8). https://doi.org/10.1128/AAC.00231-18.
Prigent, G., Nawel, A., Levesque, E., Fekkar, A., Costa, J., Anbassi, S. El, Duvoux, C.,
Merle, J., Dannaoui, E., Botterel, F., 2017. Echinocandin Resistance in Candida
Species isolates from Liver Transplant recipients. Antimicrob. Agents Chemother. 61,
1–10.
Rajendran, R., May, A., Sherry, L., Kean, R., Williams, C., Jones, B.L., Burgess, K.V.,
Heringa, J., Abeln, S., Brandt, B.W., Munro, C.A., Ramage, G., 2016. Integrating
Candida albicans metabolism with biofilm heterogeneity by transcriptome mapping.
Sci. Rep. 6, 1–11. https://doi.org/10.1038/srep35436.
Ramage, G., Rajendran, R., Sherry, L., Williams, C., 2012. Fungal biofilm resistance. Int.
J. Microbiol. https://doi.org/10.1155/2012/528521.
Renne, M.F., de Kroon, A.I.P.M., 2018. The role of phospholipid molecular species in
determining the physical properties of yeast membranes. FEBS Lett. 592, 1330–1345.
https://doi.org/10.1002/1873-3468.12944.
Richardson, M.D., 1997. Effect of lamisil and azole antifungals in experimental nail in-
fection. Dermatology 302.
Rocha, E.M.F., Gardiner, R.E., Park, S., Martinez-Rossi, N.M., Perlin, D.S., 2006. A
Phe389Leu substitution in ErgA confers terbinafine resistance in Aspergillus fumi-
gatus. Antimicrob. Agents Chemother. 50, 2533–2536. https://doi.org/10.1128/
AAC.00187-06.
Rodero, L., Mellado, E., Rodriguez, A.C., Salve, A., Guelfand, L., Cahn, P., Cuenca-Estrella,
M., Davel, G., Rodriguez-Tudela, J.L., 2003. G484S amino acid substitution in la-
nosterol 14-α demethylase (ERG11) is related to fluconazole resistance in a recurrent
cryptococcus neoformans clinical isolate. Antimicrob. Agents Chemother. 47,
3653–3656. https://doi.org/10.1128/AAC.47.11.3653-3656.2003.
12
Fungal Genetics and Biology 132 (2019) 103254
Salzer, H.J.F., Burchard, G., Cornely, O.A., Lange, C., Rolling, T., Schmiedel, S., Libman,
M., Capone, D., Le, T., Dalcolmo, M.P., Heyckendorf, J., 2018. Diagnosis and man-
agement of systemic endemic mycoses causing pulmonary disease. Respiration 96,
283–301. https://doi.org/10.1159/000489501.
Sandherr, M., Maschmeyer, G., 2011. Pharmacology and metabolism of voriconazole and
posaconazole in the treatment of invasive aspergillosis – review of the literature. Eur.
J. Med. Res. 139–144.
Sanglard, D., 2019. Finding the needle in a haystack: Mapping antifungal drug resistance
in fungal pathogen by genomic approaches. PLoS Pathogens 1–9.
Sanglard, D., Ischer, F., Bille, J., 2001. Role of ATP-binding-cassette transporter genes in
high-frequency acquisition of resistance to azole antifungals in Candida glabrata.
Antimicrob. Agents Chemother. 45, 1174–1183. https://doi.org/10.1128/AAC.45.4.
1174-1183.2001.
Sanglard, D., Ischer, F., Monod, M., Bille, J., 1997. Cloning of Candida albicans genes
conferring resistance to azole antifungal agents: Characterization of CDR2, a new
multidrug ABC transporter gene. Microbiology 143, 405–416. https://doi.org/10.
1099/00221287-143-2-405.
Sanguinetti, M., Posteraro, B., Fiori, B., Ranno, S., Torelli, R., Fadda, G., 2005. Unknown –
Unknown – Zał_3B_TABELA PARAMETROW.pdf.pdf. Society 49, 668–679. https://
doi.org/10.1128/AAC.49.2.668.
Sawaya, B.P., Briggs, J.P., Schnermann, J., 1995. Amphotericin B nephrotoxicity: the
adverse consequences of altered membrane properties. J. Am. Soc. Nephrol. 6,
154–164.
Schlichting, C.D., Piglucci, M., 1993. Control of phenotypic plasticity via regulatory
genes. Am. Nat. 142, 366–370.
Schlichting, C.D., Smith, H., 2002. Phenotypic plasticity: linking molecular mechanisms
with evolutionary outcomes. Evol. Ecol. 16, 189–211.
Schmiedel, Y., Zimmerli, S., 2016. Common invasive fungal diseases: an overview of
invasive candidiasis, aspergillosis, cryptococcosis, and Pneumocystis pneumonia.
Swiss Med. Weekly 1–12. https://doi.org/10.4414/smw.2016.14281.
Schoofs, A., Odds, F.C., Colebunders, R., Ieven, M., Wouters, L., Goossens, H., 1997.
Isolation of Candida species on media with and without added fluconazole reveals
high variability in relative growth susceptibility phenotypes. Antimicrob. Agents
Chemother. 41, 1625–1635. https://doi.org/10.1128/aac.41.8.1625.
Schoustra, S., Rundle, H.D., Dali, R., Kassen, R., 2010. Fitness-associated sexual re-
production in a filamentous fungus. Curr. Biol. 20, 1350–1355. https://doi.org/10.
1016/j.cub.2010.05.060.
Schubert, S., Barker, K.S., Znaidi, S., Schneider, S., Dierolf, F., Dunkel, N., Aïd, M.,
Boucher, G., Rogers, P.D., Raymond, M., Morschhäuser, J., 2011. Regulation of efflux
pump expression and drug resistance by the transcription factors Mrr1, Upc2, and
Cap1 in Candida albicans. Antimicrob. Agents Chemother. 55, 2212–2223. https://
doi.org/10.1128/aac.01343-10.
Selmecki, A., Forche, A., Berman, J., 2006. Aneuploidy and isochromosome formation in
drug-resistant Candida albicans. Strain 313, 367–370.
Selmecki, A.M., Dulmage, K., Cowen, L.E., Anderson, J.B., Berman, J., 2009. Acquisition
of aneuploidy provides increased fitness during the evolution of antifungal drug re-
sistance. PLoS Genet. 5, 1–16. https://doi.org/10.1371/journal.pgen.1000705.
Shields, R.K., Kline, E.G., Healey, K.R., Kordalewska, M., Perlin, D.S., Nguyen, M.H.,
Clancy, C.J., 2019. Spontaneous mutational frequency and fks mutation rates vary by
echinocandin agent against Candida glabrata. Antimicrob. Agents Chemother. 1–9.
Shoham, S., Marr, K.A., 2014. Invasive fungal infections in solid organ transplant re-
cipients. Future Microbiol. 71, 3831–3840. https://doi.org/10.1158/0008-5472.
CAN-10-4002.BONE.
Singh, A., Masih, A., Khurana, A., Kumar, P., Gupta, M., Hagen, F., Meis, J.F., Chowdhary,
A., 2018. High terbinafine resistance in Trichophyton interdigitale isolates in Delhi
India harbouring mutations in the squalene epoxidase gene. Mycoses 477–484.
https://doi.org/10.1111/myc.12772.
Sionov, E., Lee, H., Chang, Y.C., Kwon-Chung, K.J., 2010. Cryptococcus neoformans
overcomes stress of azole drugs by formation of disomy in specific multiple chro-
mosomes. PLoS Pathog. 6, e1000848. https://doi.org/10.1371/journal.ppat.
1000848.
Skelly, D.A., Magwene, P.M., Meeks, B., Murphy, H.A., Murphy, H.A., 2017. Known
mutator alleles do not markedly increase mutation rate in clinical Saccharomyces
cerevisiae strains. Proc. R. Soc. B: Biol. Sci. 284 (1852).
Smith, K.J., Warnock, D.W., Kennedy, C.T.C., Johnson, E.M., Hopwood, V., van Cutsem,
J., Vanden Bossche, H., 1986. Azole resistance in candida albicans. Med. Mycol. 24,
133–144. https://doi.org/10.1080/02681218680000201.
Snelders, E., Camps, S.M.T., Karawajczyk, A., Schaftenaar, G., Kema, G.H.J., van der Lee,
H.A., Klaassen, C.H., Melchers, W.J.G., Verweij, P.E., 2012. Triazole fungicides can
induce cross-resistance to medical triazoles in Aspergillus fumigatus. PLoS One 7.
https://doi.org/10.1371/journal.pone.0031801.
Snelders, E., Rijs, A.J., Kema, G.H., Melchers, W.J., Verweij, P.E., 2009. Possible en-
vironmental origin of resistance of Aspergillus fumigatus to medical triazoles. Appl.
Environ. Microbiol. 75, 4053–4057. https://doi.org/10.1128/AEM.00231-09.
Snelders, E., Van Der Lee, H.A.L., Kuijpers, J., Rijs, A.J.M.M., Varga, J., Samson, R.A.,
Mellado, E., Donders, A.R.T., Melchers, W.J.G., Verweij, P.E., 2008. Emergence of
azole resistance in Aspergillus fumigatus and spread of a single resistance me-
chanism. PLoS Med. 5, 1629–1637. https://doi.org/10.1371/journal.pmed.0050219.
Song, J., Zhang, S., Lu, L., 2018. Fungal cytochrome P450 protein Cyp51: What we can
learn from its evolution, regulons and Cyp51-based azole resistance. Fungal Biol. Rev.
32, 131–142. https://doi.org/10.1016/j.fbr.2018.05.001.
Sorrell, T., Chen, S., 2007. Impact of antifungal resistance in Australia. Microbiol. Aust.
171–175.
Stern, S., Dror, T., Stolovicki, E., Brenner, N., Braun, E., 2007. Genome-wide transcrip-
tional plasticity underlies cellular adaptation to novel challenge. Mol. Syst. Biol. 3,
1–9. https://doi.org/10.1038/msb4100147.
M.W.J. Hokken, et al.
Tang, L., Yang, X.F., Qiao, M., Zhang, L., Tang, X.W., Qiu, H.Y., Wu, D.P., Sun, A.N., 2018.
Posaconazole vs. voriconazole in the prevention of invasive fungal diseases in pa-
tients with haematological malignancies: A retrospective study. J. Mycol. Med. 28,
379–383. https://doi.org/10.1016/j.mycmed.2017.11.003.
Vale-Silva, L.A., Coste, A.T., Ischer, F., Parker, J.E., Kelly, S.L., Pinto, E., Sanglard, D.,
2012. Azole resistance by loss of function of the sterol Δ5,6- desaturase gene (ERG3)
in Candida albicans does not necessarily decrease virulence. Antimicrob. Agents
Chemother. 56, 1960–1968. https://doi.org/10.1128/AAC.05720-11.
Van Der Linden, J.W.M., Camps, S.M.T., Kampinga, G.A., Arends, J.P.A., Debets-
Ossenkopp, Y.J., Haas, P.J.A., Rijnders, B.J.A., Kuijper, E.J., Van Tiel, F.H., Varga, J.,
Karawajczyk, A., Zoll, J., Melchers, W.J.G., Verweij, P.E., 2013. Aspergillosis due to
voriconazole highly resistant Aspergillus fumigatus and recovery of genetically re-
lated resistant isolates from domiciles. Clin. Infect. Dis. 57, 513–520. https://doi.org/
10.1093/cid/cit320.
Vermes, A., Guchelaar, H.-J., Dankert, J., 2000. Flucytosine: a review of its pharma-
cology, clinical indications, pharmacokinetics, toxicity and drug interactions. J.
Antimicrob. Chemother. 171–179.
Vermeulen, E., Lagrou, K., Verweij, P.E., 2013. Azole resistance in Aspergillus fumigatus:
a growing public health concern. Curr. Opin. Infect. Dis. 26, 493–500. https://doi.
org/10.1097/QCO.0000000000000005.
Verweij, P.E., Lestrade, P.P.A., Melchers, W.J.G., Meis, J.F., 2016a. Azole resistance
surveillance in Aspergillus fumigatus: Beneficial or biased? J. Antimicrob.
Chemother. 71, 2079–2082. https://doi.org/10.1093/jac/dkw259.
Verweij, P.E., Snelders, E., Kema, G.H., Mellado, E., Melchers, W.J., 2009. Azole re-
sistance in Aspergillus fumigatus: a side-effect of environmental fungicide use?
Lancet Infect. Dis. 9, 789–795. https://doi.org/10.1016/S1473-3099(09)70265-8.
Verweij, P.E., Voss, A., Meis, J.F., 1998. Resistance of aspergillus fumigatus to itraco-
nazole. Scand. J. Infect. Dis. 30, 642–643.
Verweij, P.E., Zhang, J., Debets, A.J.M., Meis, J.F., van de Veerdonk, F.L., Schoustra, S.E.,
Zwaan, B.J., Melchers, W.J.G., 2016b. In-host adaptation and acquired triazole re-
sistance in Aspergillus fumigatus: a dilemma for clinical management. Lancet. Infect.
Dis. 16, e251–e260. https://doi.org/10.1016/S1473-3099(16)30138-4.
Vincent, B.M., Lancaster, A.K., Scherz-Shouval, R., Whitesell, L., Lindquist, S., 2013.
Fitness Trade-offs Restrict the Evolution of Resistance to Amphotericin B. PLoS Biol.
11, e1001692. https://doi.org/10.1371/journal.pbio.1001692.
Vu, K., Thompson, G.R., Roe, C.C., Sykes, J.E., Dreibe, E.M., Lockhart, S.R., Meyer, W.,
Engelthaler, D.M., Gelli, A., 2018. Flucytosine resistance in Cryptococcus gattii is
indirectly mediated by the FCY2-FCY1-FUR1 pathway. Med. Mycol. 56, 857–867.
https://doi.org/10.1093/mmy/myx135.
Walker, L.A., Gow, N.A.R., Munro, C.A., 2010. Fungal echinocandin resistance. Fungal
Genet. Biol. 47, 117–126. https://doi.org/10.1016/j.fgb.2009.09.003.
Walker, L.A., Lee, K.K., Munro, C.A., Gow, N.A.R., 2015. Caspofungin treatment of
Aspergillus fumigatus results in ChsG-dependent upregulation of chitin synthesis and
the formation of chitin-rich microcolonies. Antimicrob. Agents Chemother. 59,
5932–5941. https://doi.org/10.1128/AAC.00862-15.
Walsh, T.J., Petraitis, V., Petraitiene, R., Field-Ridley, A., Sutton, D., Ghannoum, M., Sein,
T., Schaufele, R., Peter, J., Bacher, J., Casler, H., Armstrong, D., Espinel-Ingroff, A.,
Rinaldi, M.G., Lyman, C.A., 2003. Experimental pulmonary aspergillosis due to as-
pergillus terreus: pathogenesis and treatment of an emerging fungal pathogen re-
sistant to amphotericin B. J. Infect. Dis. 188, 305–319. https://doi.org/10.1086/
377210.
13
Fungal Genetics and Biology 132 (2019) 103254
Webb, B.J.K., Ferraro, J.P., Rea, S., Kaufusi, S., Goodman, B.E., Spalding, J., 2018. epi-
demiology and clinical features of invasive fungal infection in a US Health Care
Network. Open Forum Infect. Dis. 5, 2–9. https://doi.org/10.1093/ofid/ofy187.
White, S.J., Rosenbach, A., Lephart, P., Nguyen, D., Benjamin, A., Tzipori, S., Whiteway,
M., Mecsas, J., Kumamoto, C.A., 2007. Self-regulation of Candida albicans population
size during GI colonization. PLoS Pathog. 3, 1866–1878. https://doi.org/10.1371/
journal.ppat.0030184.
White, T.C., 1997. Increased mRNA levels of ERG16, CDR, and MDR1 correlate, with
increases in azole resistance in Candida albicans isolates from a patient infected with
human immunodeficiency virus. Antimicrob. Agents Chemother. 41, 1482–1487.
Wiederhold, N.P., 2017. Antifungal resistance: current trends and future strategies to
combat. Infect. Drug Resist. 10, 249–259. https://doi.org/10.2147/IDR.S124918.
Wyatt, T.T., Wösten, H.A.B., Dijksterhuis, J., 2013. Fungal spores for dispersion in space
and time. Adv. Appl. Microbiol. https://doi.org/10.1016/B978-0-12-407672-3.
00002-2. 1st ed., Elsevier Inc.
Yamada, T., Maeda, M., Alshahni, M.M., Tanaka, R., Yaguchi, T., Bontems, O., Salamin,
K., Fratti, M., Monod, M., 2017. Terbinafine resistance of trichophyton clinical iso-
lates caused by specific point mutations in the squalene epoxidase gene. Antimicrob.
Agents Chemother. 61, 1–13. https://doi.org/10.1128/aac.00115-17.
Yang, F., Zhang, L., Wakabayashi, H., Myers, J., Jiang, Y., Cao, Y., Jimenez-Ortigosa, C.,
Perlin, D.S., Rustchenko, E., 2017. Tolerance to caspofungin in Candida albicans is
associated with at least three distinctive mechanisms that govern expression of FKS
genes and cell wall remodelling. Antimicrob. Agents Chemother. 61, 1–13.
Young, L.Y., Hull, C.M., Heitman, J., 2003. Disruption of ergosterol biosynthesis confers
resistance to amphotericin B in Candida lusitaniae. Antimicrob. Agents Chemother.
47, 2717–2724. https://doi.org/10.1128/AAC.47.9.2717-2724.2003.
Yue-bin, Z., Yuan-shu, Q., Hong-ni, G.U., Epidemiological, S., 2007. Genome-wide ex-
pression profiling of the response to terbinafine in Candida albicans using a cDNA
microarray analysis. Chin. Med. J. (Engl.) 120, 807–813.
Zhang, J., Snelders, E., Zwaan, B.J., Schoustra, S.E., Meis, J.F., Van Dijk, K., Hagen, F.,
Van Der Beek, M.T., Kampinga, G.A., Zoll, J., Melchers, W.J.G., Verweij, P.E., Debets,
A.J.M., 2017a. A novel environmental azole resistance mutation in Aspergillus fu-
migatus and a possible role of sexual reproduction in its emergence. MBio 8, 1–13.
https://doi.org/10.1128/mBio. 00791-17.
Zhang, J., Snelders, E.E., Zwaan, B.J., Schoustra, S.E., Kuijper, E.J., Arendrup, M.C.,
Melchers, W.J.G., Verweij, P.E., Debets, A.J.M., 2019. Relevance of heterokaryosis
for adaptation and azole-resistance development in Aspergillus fumigatus. Proc. R.
Soc. B Biol. Sci. 286. https://doi.org/10.1098/rspb.2018.2886.
Zhang, J., Van Den Heuvel, J., Debets, A.J.M., Verweij, P.E., Melchers, W.J.G., Zwaan,
B.J., Schoustra, S.E., 2017b. Evolution of cross-resistance to medical triazoles in
Aspergillus fumigatus through selection pressure of environmental fungicides. Proc.
R. Soc. B Biol. Sci. 284. https://doi.org/10.1098/rspb.2017.0635.
Zhang, N., Magee, B.B., Magee, P.T., Holland, B.R., Rodrigues, E., Holmes, A.R., Cannon,
R.D., Schmid, J., 2015. Selective advantages of a parasexual cycle for the yeast
Candida albicans. Genetics 200, 1117–1132. https://doi.org/10.1534/genetics.115.
177170.
Zhao, H.Z., Wang, R.Y., Wang, X., Jiang, Y.K., Zhou, L.H., Cheng, J.H., Huang, L.P.,
Harrison, T.S., Zhu, L.P., 2018. High dose fluconazole in salvage therapy for HIV-
uninfected cryptococcal meningitis. BMC Infect. Dis. 18, 1–8. https://doi.org/10.
1186/s12879-018-3460-7.