Accepted Article

DR. RAFAEL PEREIRA (Orcid ID : 0000-0002-6495-2671)

Article type : - Review Article

BIOFILM OF Candida albicans: FORMATION, REGULATION AND RESISTANCE Rafael

Pereira1, Raquel Oliveira dos Santos Fontenelle2, Erika Helena Salles de Brito3 e Selene Maia de
Morais4

1- Graduate Program in Biotechnology, Microbiology Laboratory (LABMIC), Vale do Acaraú

State University, Sobral, Ceará, Brazil. https://orcid.org/0000-0002-6495-2671.
2-Microbiology Laboratory (LABMIC), Vale do Acaraú State University, Sobral, Ceará, Brazil.
http://orcid.org/0000-0002-8865-5954.
3- Institute of Health Sciences of University for International Integration of Afro-Brazilian
Lusophony, Redenção, Ceará, Brazil.
4- Graduate Program in Biotechnology, Laboratory of Chemistry of Natural Products (LQPN),
Ceará State University, Fortaleza, Ceará, Brazil. https://orcid.org/0000-0002-2766-3790.

Correspondence
Raquel Oliveira dos Santos Fontenelle
E-mail: raquelbios@yahoo.com.br. Microbiology Laboratory (LABMIC), Vale do Acaraú State
University, Sobral, Ceará, Brazil.

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/JAM.14949
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Accepted Article
Abstract
Candida albicans is the most common human fungal pathogen, causing infections that range from
mucous membranes to systemic infections. The present article provides an overview of C.
albicans, with the production of biofilms produced by this fungus, as well as reporting the classes
of antifungals used to fight such infections, together with the resistance mechanisms to these
drugs. C. albicans is highly adaptable, enabling the transition from commensal to pathogen due to
a repertoire of virulence factors. Specifically, the ability to change morphology and form biofilms
is central to the pathogenesis of C. albicans. Indeed, most infections by this pathogen are
associated with the formation of biofilms on surfaces of hosts or medical devices, causing high
morbidity and mortality. Significantly, biofilms formed by C. albicans are inherently tolerant to
antimicrobial therapy, so the susceptibility of C. albicans biofilms to current therapeutic agents
remains low. Therefore, it is difficult to predict which molecules will emerge as new clinical
antifungals. The biofilm formation of C. albicans has been causing impacts on susceptibility to
antifungals, leading to resistance, which demonstrates the importance of research aimed at the
prevention and control of these clinical microbial communities.

Keywords: Resistance; Biofilms; Infection; Pathogenesis; Virulence.

Introduction
Fungal infections caused by Candida species are emerging as a major health problem,
causing high mortality rates and medical costs for governments and hospitalized patients.
Mortality can be attributed to the increasing occurrence of invasive systemic candidiasis and cases
of septicemia, especially in immunocompromised patients (Medici and Poeta 2015; Nami et al.
2018; Sakagami et al. 2019).

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Accepted Article Systemic diseases caused by Candida are the fourth leading cause of nosocomial
infections of the bloodstream. Candida species that reside in healthy hosts include C. albicans, C.
glabrata, C. tropicalis, C. parapsilosis and C. krusei (Spampinato and Leonardi 2013).
C. albicans is the main cause of candidiasis in most clinical situations (Armstrong et al.
2017). It is the third most commonly isolated microorganism from bloodstream infections in
hospitalized patients (Shields et al. 2018). It is an opportunistic pathogen that resides in oral and
conjunctival flora, as well as in the gastrointestinal and genitourinary tracts (Nami et al. 2018).
When the host becomes immunocompromised, C. albicans can cause superficial infection, as well
as septicemia. C. albicans is a polymorphic organism that undergoes morphological transition
between forms of yeast (blastoconidium), pseudohyphae and hyphae, depending on the
environment (Wisplinghoff et al. 2004; Alexander et al. 2013).
The ability of the C. albicans species to infect several host niches is supported by a wide
range of virulence factors and fitness attributes. Virulence factors are the ability of morphological
transition between forms of yeast and hyphae, the expression of adhesins and invasins on the cell
surface, the formation of biofilms, phenotypic exchange and the secretion of hydrolytic enzymes
(Nicholls et al. 2011).
Many factors contribute to the increase in invasive fungal infections (IFI). The most
important is the constantly expanding immunocompromised population. Infection can be due to
the rupture of the host barrier (for example, severe burns), neutropenia, cancer and acquired
immunodeficiency syndrome (AIDS). Other factors can contribute to IFI, such as invasive medical
procedures, catheters, total parenteral nutrition, mechanical ventilation, prolonged hospitalization,
treatment with steroids, hyperglycemia, use of broad-spectrum antibiotics for long periods and
intake of subinhibitory concentrations of antifungals (Pfaller and Diekema 2007; Ha et al. 2011).
The present review provides an overview of the species C. albicans associated with the
production of biofilms produced by this fungus, as well as reporting the classes of antifungals used
to fight such infections, together with the resistance mechanisms against these drugs.

Biofilm formation by C. albicans

Biofilms are complex three-dimensional structures composed of a community of central
microbial cells (a single species or mixed species) linked to host tissue or abiotic surfaces and
imbedded in an extracellular polysaccharide substance (EPS), which provides protection to the
microorganisms (Al-Fattaniand Douglas 2006; Ghannoum et al. 2015).

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Accepted Article In recent decades, research in the field of microbial biofilms has gained increasing
momentum and emerging scientific evidence has changed understanding of way microbial life
occurs. Although microorganisms have traditionally been studied in free-floating (planktonic)
cultures or as colonies grown on the surfaces of nutrient agar culture media, it is now accepted that
biofilms are the preferred growth state and probably the “natural” one for most microorganisms
(Davey and O'Toole 2000; Kolter and Greenberg 2006; Nobile and Johnson 2015).
The differences between biofilms and planktonic cells extend beyond their metabolic
activities. For example, several secreted proteases are upregulated in biofilm cells, but not in
planktonic cells, which results in distinct proteolytic cleavage profiles for each growth state
(Winter et al. 2016). When compared to "standard" planktonic cells, yeast-like cells that disperse
from mature biofilms appear to be more virulent and have a greater ability to adhere to surfaces to
form new biofilms (Uppuluri et al. 2010).
Most diseases caused by C. albicans are associated with the formation of biofilms on
abiotic or host surfaces (Nett and Andes 2006; Mathe and Van Dijck 2013). More significantly, C.
albicans is adept at adhering to catheters and several medical implants, and is currently classified
by the Centers for Disease Control and Prevention, United States, as the third most commonly
isolated bloodstream pathogen in hospitalized patients with a mortality rate of up to 50%
(Wisplinghoff et al. 2004; Tournu and Van Dijck 2012; Mathe and Van Dijck 2013).
The formation of biofilms makes treatment difficult and contributes to high rates of
morbidity and mortality, thus representing one of the main virulence factors that contribute to the
pathogenesis of candidiasis (Tumbarello et al. 2012; Rajendran et al. 2016). In vitro experiments
showed that the development of biofilm occurs in a series of sequential steps over a period of 24 to
48 h (Ricicova et al. 2010; Mathe and Van Dijck 2013). In general, the biofilm development
process of C. albicans can be divided into four main stages: adherence, proliferation, maturation
and dispersion (Blankenship and Mitchell 2006; Lohse et al. 2018).
In the adhesion stage, yeast cells isolated adhere to the substrate to form a basal layer of
cells. The cell proliferation stage involves formation of filaments, where yeast cells begin to
stretch and develop into filamentous hyphae. This is the initiation step by which cells change their
morphology and invade the host mucosa or plastic or other polymeric surfaces in inert medical
devices. An arsenal of hydrolytic enzymes, such as proteinases, hemolysins and phospholipase, is
secreted by C. albicans, which allows the fungus to invade host tissue or other solid substrates.
Secreted aspartyl proteases (SAPs), comprising a family of ten genes (SAP 1-10), are the most

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 studied among the many secreted enzymes (Mathé and Van Dijck 2013; Tsui, Kong and Jabra-
Accepted Article
Rizk 2016).
In the maturation stage, the production of hyphae is accompanied by the secretion of
extracellular polymeric substances (EPS). The EPS of C. albicans biofilm is complex, where the
main polysaccharides include α-mannan, β-1,6-glucan and β-1,3-glucan (Mitchell, Zarnowski and
Andes 2016). Although β-1,3 glucan is a minor constituent, it is the main polysaccharide of the
matrix linked to biofilm resistance to antifungals, as it can block the drug’s diffusion (Taff et al.
2013).
Although the extracellular matrix is partially self-produced and secreted by C. albicans
cells in the biofilm, it can also contain environmental aggregates such as structural components of
C. albicans and lysed host cells, specific host cells associated with or recruited to the area and
incorporated into the biofilm, such as erythrocytes, epithelial cells, urothelial cells and neutrophils,
so the biofilm can vary widely, depending on the location in the host (Nobile and Mitchell 2007;
Nett et al. 2015).
The biofilm matrix acts as a physical barrier to protect against the environment and
provides structural integrity to the biofilm. It is critical to the resistance of mature biofilms against
mechanical breakage. The presence of the matrix is more developed during and after the
maturation stage (Nobile and Johnson 2015; Gulati and Nobile 2016).
Hyphal invasion into tissues is driven by physical hydrostatic forces, which trigger
cytoplasmic forces. Cells can communicate with other cells through quorum sensing mechanisms,
and one of the most studied detection molecules that regulate biofilm formation is farnesol (Polke
et al. 2015). The phenomenon of quorum sensing, which depends on the interaction between small
diffusible signaling molecules with transcription activator proteins, associating gene expression
with cell density (Mallick and Bennett 2013; Avbelj et al. 2016; Wongsuk et al. 2016).
Farnesol inhibits the formation of hyphae, so by promoting the formation of yeast cells, it
can help the dispersion of biofilms (Ramage et al. 2002; Finkel and Mitchell 2011; Krom et al.
2015). In the physiology of C. albicans, farnesol plays multiple roles as a signaling molecule, in
addition to having harmful effects on host cells and other microorganisms (Albuquerque and
Casadevall 2012).
The discovery of the involvement of farnesol in the quorum sensing of C. albicans was a
notable advance in the study of eukaryotes. The existence of quorum sensing systems revealed that
lipids (oxylipines), peptides (pheromones), alcohols (tyrosol, farnesol, tryptophan and 1-

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 phenylethanol), acetaldehydes, in addition to some volatile compounds, are actively involved in
Accepted Article
the quality control of fungi, regulating various key functions such as pathogenesis, morphogenesis
and filament formation (Cottier and Mühlschlegel 2011; Albuquerque and Casadevall 2012; Polke
et al. 2015; Hirota et al. 2016).
Several studies have shown that in high concentrations, farnesol triggers a classic process
of apoptosis in mammalian cells with C. albicans (Scheper et al. 2008; Shirtliff et al. 2009).
Subsequent studies characterized the mechanism involving oxidative stress and the accumulation
of intracellular reactive oxygen species (ROS) mediated by farnesol, resulting in cell death (Zhu et
al. 2011). Together, these findings are intriguing, since they indicate the existence of a
sophisticated role of farnesol in the formation of fungal biofilms, possibly involving programmed
cell death (Tsui et al. 2016).
Finally, in the dispersion stage, yeast cells are released from the mature biofilm and can
spread to distant locations to start a new biofilm formation cycle. The dispersion stage of the
biofilm is of immense clinical relevance, as the newly released cells of the mature biofilm are able
not only to initiate new cycles of biofilm formation, but also to enter the bloodstream to establish a
focus of infection. This is the reason why biofilm formation is closely associated with candidemia
and clinically disseminated invasive candidiasis (Tournu and Van Dijck 2012).
During these stages of biofilm development in the laboratory, the growth medium is kept
constantly agitated to prevent floating cells from settling on the surface, or the cells are kept
continuously flowing over the biofilm to mimic the flow conditions normally present in the host
(Gulati and Nobile 2016).

Genetic regulation and biofilm formation by C. albicans

A transcriptional regulatory network, consisting of six main regulatory genes (Efg1, Bcr1,
Brg1, Ndt80, Tec1 and Rob1), is involved in controlling the biofilm formation process. These
regulators were discovered via screening a library of mutants and in vivo studies in animal models.
Efg1 and Tec1 are involved in the regulation of cell morphology, while Ndt80 is involved in
biofilm formation and the functions of Bcr1 lack of cells with hyphae in the biofilm structure.
Meanwhile, Brg1 and Rob1 are present only in a species closely linked to C. albicans. On the
other hand, evolutionary analysis suggests that the transcriptional circuit for the biofilm network in
C. albicans has recently evolved, where extensive changes in the cis-regulatory sequence and

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 regulators such as Brg1 and Rob1 are necessary for this modernized biofilm circuit. (Fox and
Accepted Article
Nobile 2012; Nobile et al. 2012).
Other regulatory genes are needed for specific processes in biofilm formation in C.
albicans, such as hyphal growth (e.g., Hwp1), extracellular matrix production (Gsc1 and Mnn1)
and drug resistance (Cdr1 and Mdr1). The functions of most genes have yet to be determined,
since most of them do not have clear homology with genes characterized from other organisms
(Nobile et al. 2012).
The cells in which the mentioned genes have been deleted exhibit a variety of phenotypes
in relation to biofilms, including reduced thickness of mature biofilms (for example, nd80
deletion), extremely thin biofilms (Efg1 deletion), decrease in biofilm thickness (deletion of Rfx2)
and reduced adhesion to the solid substrate (Rfg1) (Nobile et al. 2012; Fox et al. 2015).
Transcriptional profile studies of clinical isolates of C. albicans, with varying abilities to form
biofilms in vitro, revealed that strains that formed thicker biofilms had increased expression of
specific hyphal genes, such as Hwp1, compared to strains that formed thinner biofilms (Rajendran
et al. 2016).
The specific genes involved in the adherence phase, both in the initial stages of biofilm
formation and in the later stages of maturation, play an important role in the formation and
maintenance of the biofilm. For example, a group of ten genes (including Als1, Als2, Als4 and
Pga6) is over-regulated at the beginning of biofilm formation, while a different set of ten genes,
including Iff4, Iff6, Pga32 and Pga55, is regulated at later times (Fox et al. 2015).
The dispersion of C. albicans in the environment (mainly as yeast-shaped cells) occurs
throughout the formation of the biofilm, with a greater number of cells being dispersed when the
biofilm reaches maturity. The transcriptional regulators Ume6, Nrg1 and Pes1 (also known as
Nop7) are all linked to this dispersion. Overexpression of Ume6 reduces dispersion, while
overexpression of Nrg1 or Pes1 increases the number of cells released from the biofilm (Uppuluri
et al. 2010; Nobile et al. 2014).
Currently, there are two known regulators of biofilm matrix production in C. albicans:
Rlm1 and Zap1 (Gulati and Nobile 2016). The exclusion of Rlm1 causes a reduction in matrix
thickness, while the exclusion of Zap1 leads to an increase in the accumulation of extracellular
matrix material, probably due to the positive regulation of two glucoamylase enzymes, Gca1 and
Gca2 (Nobile et al. 2009; Nett et al. 2011).

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 Resistance mechanisms of C. albicans to antifungals
Accepted Article There is a problem related with antimycotic treatment, which may be due to several
factors, leading to clinical resistance. Clinical resistance occurs when the fungus is not sensitive to
an antifungal in vivo due to the lack of therapeutic success of the drug or because it reaches
immunodepressed or neutropenic patients that have infected tissue or closed abscesses, which can
be affected by nosocomial contamination leading to the formation of biofilms (El-Azizi, Farag and
Khardori 2015).
C. albicans causes infection with high mortality in immunocompromised people, leading
to an increased need to develop broad-spectrum fungicidal drugs. However, the prolonged use of
antifungal drugs by patients results in the selection of resistant C. albicans, strains, making them
less susceptible to drugs (Dawson, Intapa and Jabra-Rizk 2011).
Antifungal resistance is based on different mechanisms, namely: (i) reduction of the
drug's intracellular accumulation; (ii) decreased affinity/processivity of the target for the drug; and
(iii) reduction of the drug's effect. In particular, the resistance mechanism will be different
depending on the mode of action of antifungal compounds. The cellular and molecular
mechanisms that support resistance against antifungal classes have been discussed in the literature
(Kanafani and Perfect 2008; Perlin 2009; Peman, Canton and Ingroff-Espinel 2009; Vandeputte,
Ferrari and Coste 2012). Other resistance mechanisms are efflux pumps, mutations in coding
genes, modification of enzymatic activities.
Unlike the wide diversity of antibiotics, with numerous known classes and several modes
of action against different bacterial targets, current antifungals are extremely limited (Gulati and
Nobile 2016). Only four main classes of antifungal drugs are used to treat most fungal infections,
namely: azole derivatives, polyenes, echinocandins and nucleoside analogs (Tab. 1) (Fox et al.
2015). When biofilm is present, resistance to classic antifungals is multifactorial and mechanically
complex, further limiting the therapeutic arsenal (Gulati and Nobile 2016).

Mechanisms of Resistance to Azoles

Azoles comprise the largest family of antifungals used against Candida species. The first
azoles used in clinical practice were clotrimazole (Fig. 1A) and miconazole (Fig. 1B), which were
approved for use in 1969, followed by ketoconazole (Fig. 1C) in 1981 (Allen et al. 2015). These
three drugs are all imidazoles, since they have an imidazole ring in their structure. The usefulness

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 of clotrimazole and miconazole as antifungals has been limited by their inhibitory effect on human
Accepted Article
liver Cytochrome P450 enzymes (Hoekstra et al. 2014). In response to this, in the early 1990s two
triazoles, fluconazole (Fig. 1D) and itraconazole (Fig. 1E), were introduced in the market, showing
improved pharmacokinetic profiles, a broader spectrum of antifungal activity and a lesser
inhibitory effect against the human CYP450 system (Allen et al. 2015).
In the early 2000s, voriconazole emerged, showing greater activity against the non-
albicans Candida species more resistant to azoles compared to fluconazole and itraconazole.
Today, imidazoles are used mainly for the treatment of superficial candidiasis, while triazoles are
preferred for the treatment of invasive candidiasis (Arendrup et al. 2013; Allen et al. 2015).
These drugs bind through the nitrogen group in their azole ring to the heme group of the
target protein and block the demethylation of lanosterol C-14, leading to the replacement of
methylated sterols in the membrane. The inhibition of this enzyme results in a decrease in the
ergosterol content in the membrane and in the accumulation of toxic methylated intermediates,
with the consequent interruption of the fungal cell membrane function, inhibition of growth, and in
some cases cell death (Odds et al. 2003; Xiao et al. 2004; Akins 2005).
The main mechanism responsible for the high level of resistance to azoles is the
overexpression of cell membrane efflux pumps (Rogers and Barker 2003; Holmes et al. 2008),
which transport membrane-associated transport proteins that act to prevent the intracellular
accumulation of drugs, thus avoiding toxic levels that would otherwise kill the cell (Kanafani and
Perfect 2008; Spampinato and Leonardi 2013; Sanguinetti, Posteraro and Lass-Florl 2015; Nami et
al. 2018).
ABC type pumps and the main facilitating transporters pumps are responsible for
decreasing the accumulation of azoles inside yeast cells, actively translocating the compounds
across the cell membrane. (Rodriguez et al. 2008). The mechanism of resistance to triazole most
frequently found among clinical isolates of C. albicans is the positive regulation or overexpression
of the CDR1 and CDR2 genes (Prasad et al. 2015).
Due to this overexpression of efflux pumps, cross-resistance between azole derivatives is
often observed in C. albicans, both in vitro and clinically. Resistance occurs when there are
changes in the morphology of the microorganism, so that the antifungal drugs commonly used are
unable to perform fungal lysis. This process is due to the transcription of mutant genes, which in
Candida species include an alteration that has as a mold the clonal descent resulting from the
absence of sexual recombination, leading to the attainment of fungal resistance through the

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 plasticity of the genome and increased rates of mitotic mutation and recombination (Ford et al.
Accepted Article
2015).
In planktonic cells, these efflux pumps are typically regulated in response to antifungal
drugs. However, in biofilms they are regulated positively in the first hours of adherence and
remain regulated during the biofilm’s development, even in the absence of an antifungal drug
(Nobile et al. 2012; Nobile and Johnson 2015).

Mechanisms of Resistance to Polyenes

Polyenes are fungicide belonging to a class of natural compounds with a heterocyclic
amphipathic molecule (one side with a hydrophilic charge and the other side with a hydrophobic
charge). They target ergosterol in the fungal membrane, where they enter the lipid bilayers and
create pores that impair the integrity of the membrane, allowing small molecules to diffuse
through the membrane, resulting in cell death (Peman, Canton and Espinel-Ingro 2009). Nystatin
(Fig. 1F) and amphotericin B (Fig. 1G) belong to this group. Nystatin has a slightly narrower
spectrum of activity than amphotericin B, but it is active against various yeasts and fungi of the
species (Chandrasekar 2011).
Resistance to amphotericin B is quite uncommon and usually results from mutations in
the ERG3 gene (which encodes C-5 sterol desaturase, an enzyme involved in ergosterol
biosynthesis), which decreases the concentration of ergosterol in the fungal membrane (Kelly et
al. 1997). Amphotericin B resistance can also be mediated by increased catalase activity, with
decreased susceptibility to oxidative damage (Sokol-Anderson, Brajtburg and Medo 1986).
Among C. albicans isolates, amphotericin B resistance is still very rare (Arendrup 2014; Faria-
Ramos et al. 2014; Castanheira et al. 2017).
Despite its potent effect against C. albicans, the use of amphotericin B is limited by its
nephrotoxicity. Although safer formulations have been developed to transport this medication
(mainly based on the use of liposomes), its high cost remains an impediment, so it is used mainly
in second-line therapy (Pappas et al. 2016).

Mechanisms of Resistance to Echinocandins

Echinocandins are considered the first-line therapy for invasive Candida infections
(Pappas et al. 2016). These compounds are fungicides in vitro against yeasts. Currently, three

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 agents are available for clinical use: caspofungin (Fig. 1H), micafungin (Fig. 1I) and anidulafungin
Accepted Article
(Fig. 1J). They inhibit β- (1,3) glucan synthase, an enzyme complex located in the plasma
membrane of fungal cells (Agarwal et al. 2003; Denning 2003; Perlin 2007; Peman, Canton and
Espinel-Ingro 2009).
This enzyme has at least two subunits, Fks1, the catalytic subunit, and Rho, a GTP-
binding protein that regulates glucan synthase activity. They are responsible for the production of
β- (1,3) glucan synthase, essential for fungi, since it is one of the main components of the cell wall
(Perlin 2007). The safety profile of echinocandins is excellent, with few reported adverse events
and drug interactions. Despite their much higher cost, echinocandins are replacing fluconazole as
the antifungal agents of choice in intensive care units (ICUs) (Meyer et al. 2007). Due to their
safety profile and fungicidal activity, echinocandins are often used as the primary treatment for
invasive candidiasis (Pappas et al. 2016).
The occurrence of resistance to echinocandins in Candida spp. has been attributed to
mutations in the Fks1 gene, the catalytic subunit of β- (1,3) -glucan synthase, and to a lesser extent
Fks2, resulting in amino acid substitutions in Hs1 and Hs2 (Hoekstra et al. 2014). In C. albicans,
mutations encoding alterations in Fks1, Hs1 S645P, S629P, S654P, F641S, F641I and Fks1 are
common (Kritikos et al. 2018; Pfaller et al. 2019).

Mechanisms of Resistance to Pyrimidine Analogs

The fluoropyrimidine most commonly used in the treatment of candidiasis is 5-

flucytosine (5-FC) (Fig. 1L), the only representative of this class, which penetrates fungal cells
through cytosine transporters, and is subsequently metabolized by the pyrimidine rescue pathway
in 5-fluorouracil (5-FU), considered the active form of 5-FC. 5-FU incorporates RNA, causing
premature chain termination, and inhibits the activity of thymidylate synthase, an enzyme essential
for DNA synthesis (Vermes et al. 2000; Kritikos et al. 2018). Due to its toxic effects, 5-FC is
administered to patients in low concentration and in combination with other antifungals (Pappas et
al. 2016). The resistance among Candida correlates with mutations in the enzyme uracil
phosphoribosyltransferase (Fur1p), which becomes unable to convert 5-fluorouracil to 5-
fluorouridine monophosphate (Akins 2005).
Regarding the relationship between antifungals and C. albicans biofilms, resistance is
multifactorial and mechanically complex, but largely due to three main factors: the positive

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 regulation of efflux pumps, the presence of the extracellular matrix, and the existence of
Accepted Article
recalcitrant cells, metabolically inactive, also referred to as "persistent" cells. These mechanisms,
for example, render azole derivatives and the classic formulation of polyenes, ineffective against
C. albicans biofilms, further limiting the drugs that can be used to treat these infections (Gulati
and Nobile 2016).
The biofilm extracellular matrix is a major contributor to antifungal drug resistance in C.
albicans biofilms. The matrix acts as a physical barrier to drug penetration and as a stabilizer of
the overall biofilm architecture (Nett et al. 2007; Nobile and Johnson 2015). There is a clear need
for the development of new antifungal therapies that are effective against biofilm formation. The
increase in drug resistance is providing strong impetus to research to understand the mechanisms
of improved tolerance, infections associated with biofilms, and antimicrobial therapy (Tournu and
Van Dijck 2012). Table 2 describes the main mechanisms of antifungal resistance in C. albicans.

Conclusion
Although numerous in vitro biofilm model systems have been crucial development of this
type of microbial organization, as well as cellular phenotypes and drug resistance, it is precise to
accurately explain the multiplicity of host and infection site variables involved in infection
establishment.
Recent advances in the profiling of gene expression and manipulation have led to an
understanding of the regulatory pathways and mechanisms involved in the formation of biofilms
and drug resistance in C. albicans. These studies have revealed new mechanisms and signs in the
formation of C. albicans biofilms and the associated drug resistance, favoring therapeutic
prediction.
The increase in resistance to existing antifungals has also prompted research to better
understand the molecular mechanisms that lead to this panorama, including therapeutic targets.
The success of treating C. albicans infections is generally more difficult when there is an
established biofilm, and infections by biomaterials continue to be an increasingly alarming
problem of conventional therapy.
Therefore, it is crucial to explore alternative strategies to overcome the limitations of
current therapies against fungal infections associated with biofilms. In this respect, recent analyses
of gene-drug interactions suggest that the focus on the development of antifungals targeting

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 specific pathogens can lead to more potent and effective therapies, especially to treat invasive
Accepted Article
fungal infections.

Disclosure statement
The authors report no conflict of interest

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Conflict of Interest
The authors have no conflict of interests.

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Figure 1. Chemical structure of antifungals currently available. A- Clotrimazole; B- Miconazole; C- Ketoconazole; D- Fluconazole; E- Itraconazole;

F- Nystatin; G- Amphotericin B; H- Caspofungin; I- Micafungin; J- Anidulafungin and L- Flucytosine (continue)

AZOLE DERIVATIVES

A B C D

POLYENES

F G
Figure 1. Chemical structure of antifungals currently available. A- Clotrimazole; B- Miconazole; C- Ketoconazole; D- Fluconazole; E- Itraconazole;

F- Nystatin; G- Amphotericin B; H- Caspofungin; I- Micafungin; J- Anidulafungin and L- Flucytosine (continue)

ECHINOCANDIN
H I J

PYRIMIDINE ANALOGS

L
Table 1. Classes of antifungals currently available.

This table is a modified version from Tsui, Kong and Jabra-Rizk. (2016)
CLASSES OF DRUGS ANTIFUNGALS MECHANISM OF ACTION RESISTANCE OF C. REFERENCES

albicans

Inhibition of lanosterol 14 α- Expression regulated by Nailis et al. 2010

Azoles Fluconazole demethylase (ERG11; ergosterol ERG genes Mathe and Van Dijck, 2013

Clotrimazole biosynthesis). Gulati and Nobile 2016

Miconazole

Ketoconazole

Itraconazole

Binds to ergosterol in the fungal cell Replacement of cell Ghannoum and Rice 1999

Nystatin membranes; formation of membrane sterols Mathe and Van Dijck 2013

Polyene Amphotericin B transmembrane pores, resulting in loss Gulati and Nobile 2016

of membrane integrity and

interruption of the ion gradient.

 Inhibition of β-1,3-glucan synthase Regulated expression of Walsh 2002

Echinocandin Caspofungin glucan biosynthesis genes Williams and Lewia 2011

Micafungin Gulati and Nobile 2016

Anidulafungin

Inhibition of DNA and RNA synthesis Mutations in enzymes that Mathe and Van Dijck 2013

in fungal cells. catalyze pyrimidine Gulati and Nobile 2016

analogs, such as Fur1.

Flucytosine
Nucleoside analogues
Increased synthesis of

pyrimidine nucleotides that

competitively inhibit

analogs.
Table 2. Mechanisms of antifungal resistance in C. albicans

This table is a modified version from Tsui, Kong and Jabra-Rizk. (2016)
FORM MECHANISM PHYSIOLOGICAL EFFECT REFERENCES

Reduced efficacy of antifungal

Planktonic Low growth rate drugs against inactive/slow growing Baillie and Douglas 1998

cells.

Persistent cells Metabolic numbness, cells become LaFleur et al. 2006

highly tolerant to antifungals. Bink et al. 2011

Prevents the spread of antifungals Al-Fattani and Douglas 2006

Biofilm Polysaccharide extracellular matrix through biofilm; binds with Nett et al. 2010

fluconazole in the matrix. Mitchell et al. 2015

Nett et al. 2007

Taff et al. 2012

Drug target modification The target substrate is mutated, Kelly et al. 1997

preventing the inhibitory effect of Martel et al. 2010

antifungal drugs. Nolte et al. 1997

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the cell. Ramage et al. 2002

Both ways Taff et al. 2013

Mukherjee et al. 2003

Mateus et al. 2004

Lepak et al. 2006

Sanglard et al. 1995

Cell density A larger number of cells requires a

higher dose of antifungals for Perumal et al. (2007)

effectiveness.