Abstract Fungi comprise a minor component of the oral
microbiota but give rise to oral disease in a signifi cant pro-
portion of the population. The most common form of oral
fungal disease is oral candidiasis, which has a number of
presentations. The mainstay for the treatment of oral can-
didiasis is the use of polyenes, such as nystatin and ampho-
tericin B, and azoles including miconazole, fl uconazole, and
itraconazole. Resistance of fungi to polyenes is rare, but
some Candida species, such as Candida glabrata and C.
krusei, are innately less susceptible to azoles, and C. albi-
cans can acquire azole resistance. The main mechanism of
high-level fungal azole resistance, measured in vitro, is
energy-dependent drug effl ux. Most fungi in the oral cavity,
however, are present in multispecies biofi lms that typically
demonstrate an antifungal resistance phenotype. This resis-
tance is the result of multiple factors including the expres-
sion of effl ux pumps in the fungal cell membrane, biofi lm
matrix permeability, and a stress response in the fungal cell.
Removal of dental biofi lms, or treatments to prevent biofi lm
development in combination with antifungal drugs, may
enable better treatment and prevention of oral fungal
disease.
Key words Oral candidiasis · Antifungal drug resistance ·
Biofi lms
Introduction
Oral fungal infections affect a signifi cant, and increasing,
proportion of the population.
nent of the oral microbial fl ora but certain species, mostly
belonging to the Candida genus, are routinely present at
low concentrations without causing infection.
These com-
mensal fungi are opportunistic pathogens and can cause
disease when their host becomes immunocompromised.
These infections can be superfi cial and affect the mucous
membranes, or can penetrate the epithelium and be
hematogenously disseminated with serious consequences.
Mucosal infections are seen in neonates and in the elderly,
two groups with suboptimal immune function. They also
affl ict people whose immune systems have been suppressed.
Oropharyngeal candidiasis (OPC) is common in acquired
immunodefi ciency syndrome (AIDS) patients who do
not have access to highly active antiretroviral therapy
(HAART),
whereas oral candidiasis often affects cancer
patients undergoing chemotherapy and/or radiotherapy.
When fungi penetrate the epithelial surfaces of immuno-
compromised hosts, they can cause invasive fungal infec-
tions (IFIs) that are associated with high morbidity and
mortality. The fungal genera most often associated with
IFIs are Candida, Aspergillus, and Cryptococcus.
Patients with oral fungal infections are often treated with
polyene or azole antifungal drugs.
Although azoles, such
as fl uconazole (FLC), have better solubility and less neph-
rotoxicity than polyenes, fungal azole resistance can be a
problem.
Candida glabrata and C. krusei are innately less
susceptible to azoles than other Candida species, and C.
albicans can develop azole resistance. The molecular mech-
anisms of fungal azole drug resistance have been studied
extensively,
but mostly in vitro. The majority of fungal
cells in the oral cavity, however, are associated with bio-
fi lms, and microorganisms in biofi lms are often more resis-
tant to antimicrobials than planktonic cells.
This review
investigates the contribution of molecular resistance mech-
anisms and biofi lm growth to the antifungal drug resistance
of oral fungi.
Oral fungi
Presence of fungi in the oral cavity
The human mouth supports a diverse microbiota. There are
thought to be several hundred bacterial species and several
thousand phylotypes that inhabit the oral cavity.
14,15
These
Odontology (2010) 98:15–25
© The Society of The Nippon Dental University 2010
DOI 10.1007/s10266-009-0118-3
Masakazu Niimi · Norman A. Firth · Richard D. Cannon
Antifungal drug resistance of oral fungi
M. Niimi · N.A. Firth · R.D. Cannon (*)
Department of Oral Sciences, School of Dentistry, University of
Otago, 310 Great King Street, Dunedin 9016, New Zealand
Tel.
+
64-3-479-7081; Fax
+
64-3-479-7078
e-mail: richard.cannon@otago.ac.nz
Received: November 9, 2009 / Accepted: November 28, 2009
REVIEW ARTICLE
16
estimates are based on data from culture-independent
molecular analyses of samples from healthy and diseased
mouths.
Such molecular techniques allow the identifi ca-
tion of microbial species that are diffi cult to culture and can
measure small genetic differences between related phylo-
types. One of the most commonly used methods to identify
bacteria in the oral cavity is the cloning and sequencing of
16S rRNA genes amplifi ed by polymerase chain reaction
(PCR) from DNA extracted from oral samples.
This
analysis has been taken to a greater level of sensitivity by
the advent of high-throughput pyrosequencing of PCR-
amplifi ed DNA, which does not require cloning steps. A
recent pyrosequencing analysis of bacterial 16S rDNA
extracted from human saliva and plaque identifi ed 3621 and
6888 species-level phylotypes, respectively.
Other tech-
niques used to identify and quantify oral bacteria include
real-time quantitative PCR (qPCR) and checkerboard
DNA–DNA hybridization.
There have been relatively few similar investigations of
fungi present in the oral cavity. Classically, yeast have been
identifi ed from saliva, oral swabs, plaque, oral rinses, or
concentrated oral rinses by culturing them on selective or
differential agar.
Such techniques can use chromogenic
primary isolation media such as CHROMagar for the pre-
sumptive identifi cation of the most prominent Candida
species including C. albicans, C. dubliniensis, C. glabrata, C.
krusei, C. tropicalis, and in some instances C. parapsilo-
sis.
Other culture-based growth, morphology, and bio-
chemical tests are available in kit format for the identifi cation
of fungi isolated from clinical samples. These kits include
API ID 32C, API 20C AUX, and RapID Yeast Plus.
Although these kits are relatively easy to use, the results
often show poor discrimination between possible species,
and the process involves two culturing steps that can take
up to 72 h.
Molecular identifi cation techniques allow the
rapid and sensitive detection of fungi, but these methods
are less frequently applied to the detection of fungi than to
the detection of bacteria. Techniques that have been used
include checkerboard DNA–DNA hybridization,
rDNA sequencing,
PCR,
Early culture-
based detection studies, reviewed by Odds, found yeast
present in the oral cavities of from 2.0% to 71.3% of healthy
individuals sampled, with a mean carriage rate of 25.5%.
The mean carriage rate was higher in patients (47.0%),
refl ecting underling predisposing conditions in hospitalized
individuals. The most common yeast isolated from human
mouths is C. albicans, and mean carriage rates of 17.7% and
40.6% were found in healthy individuals and patients,
respectively.
C. albicans comprised approximately 70% of
all isolates; the next most common species were C. tropica-
lis, C. glabrata, C. parapsilosis, C. krusei, C. kefyr, and C.
guilliermondii. These studies were carried out before the
discovery of C. dubliniensis.
This species is very similar,
genotypically and phenotypically, to C. albicans
and so
was probably counted as C. albicans before its discovery.
These culture-based results have been confi rmed by recent
molecular studies with C. albicans being identifi ed as the
most prevalent oral yeast, followed by C. glabrata, C. dub-
liniensis, C. tropicalis, C. krusei, C. parapsilosis, C. famata,
C. guilliermondii, and in some cases Saccharomyces cerevi-
siae.
Although other fungi that cause respiratory dis-
eases, such as Cryptococcus neoformans and Aspergillus
fumigatus, pass through the oral cavity they are rarely, if
ever, detected in oral samples, indicating that they are not
part of the normal commensal fl ora.
Thus, the oral fl ora contains relatively few fungal species.
In addition, there are very few yeast cells in mouths com-
pared to bacterial cells. The oral cavity presents many sur-
faces for colonization by oral microorganisms. Yeast can be
found on mucosal surfaces including the tongue, teeth, and
dental prostheses, in dental plaque, and in saliva.
Saliva is easily obtained and yeast in saliva can be indicative
of microbial colonization at other oral sites. An early study
found that the mean concentration of yeast in the saliva of
healthy adults was 296/ml.
More recently we have found
a similar concentration of yeast (mean, 387/ml) in the saliva
of 134 children 7–8 years old (Boyd and Cannon, unpub-
lished data); this compares with a bacterial concentration
of approximately 10
7
–10
8
/ml saliva.
Despite the low con-
centrations of yeast in the oral cavity, they can still give rise
to oral disease, usually when the host’s immune system
becomes compromised or suppressed.
are, therefore, opportunistic pathogens.
Oral fungal infections
Oral candidiasis is the most common oral mucosal fungal
infection.
There are a number of presentations of oral can-
didiasis, which can be classifi ed as acute or chronic, accord-
ing to the timeframe of the infection (Table 1).
Pseudomembranous candidiasis
Pseudomembranous candidiasis can either be acute or, in
the immunocompromised and in other groups such as long-
term users of corticosteroid inhalers (asthmatics), it can be
chronic. It most frequently affects infants, the elderly, and
the terminally ill.
It may also be an indicator of an underly-
ing serious medical condition such as diabetes, leukemia,
other malignancy, or human immunodefi ciency virus (HIV)
infection/AIDS. The clinical appearance consists of non-
adherent creamy white patches or fl ecks, which are easily
wiped off with a swab or a mouth mirror. Scraping may
produce bleeding and generally reveals an erythematous
base. The soft palate, oropharynx, tongue, buccal mucosa,
and gingiva are commonly affected.
The pseudomem-
branes consist of a mesh of Candida hyphae, entangled with
desquamated epithelial cells, fi brin, keratin, necrotic debris,
and bacteria. If pseudomembranous candidiasis extends to
the pharynx, it is termed oropharyngeal candidiasis (OPC).
This condition affl icts many AIDS patients
signifi cant infection in cancer patients being treated with
chemotherapy and/or radiotherapy.
the fi rst clinical symptom in HIV-positive patients, before
the onset of overt AIDS.
In cancer patients, the increased
incidence of OPC results from both the debilitating effects
17
of the cancer itself and from the immunosuppressive treat-
ment for the cancer.
Erythematous candidiasis
Erythematous candidiasis, similar to pseudomembranous
candidiasis, may be acute or chronic depending on its dura-
tion. The acute form frequently follows a course of broad-
spectrum antibiotics or topical antibiotics and has also been
called antibiotic sore mouth. The antibiotics probably
reduce bacterial competition in the oral cavity and allow the
overgrowth of fungi. This form of candidiasis is painful, and
patients may complain of a burning sensation in the mouth.
Erythematous candidiasis may also be associated with the
use of corticosteroid inhalers.
Chronic erythematous candidiasis occurs on the palatal
mucosa beneath full or partial maxillary dentures and is
sometimes called Candida-associated denture stomatitis or
denture sore mouth.
There is a chronic edema of the
mucosa in contact with the denture and often a sharp
demarcation between the affected and unaffected tissue.
Occasionally the edentulous mandible may be involved.
This form of candidiasis is more frequent in those who do
not remove their dentures at night and the wearers of old
dentures. The dentures can act as a reservoir for infecting
yeast, and several factors, such as surface roughness and
hydrophobicity, contribute to the ability of Candida to colo-
nize the dental acrylic.
In addition to antifungal treatment
(see below), good oral hygiene is effective in alleviating this
condition; patients should remove their dentures at night
and, following cleaning, soak them in either 2% chlorhexi-
dine gluconate or 1% sodium hypochlorite overnight.
Plaque-like/nodular candidiasis
Plaque-like/nodular candidiasis (which is also called chronic
hyperplastic candidiasis or candidal leukoplakia) is charac-
terized by irregular white plaques that cannot be removed
by scraping. It is less common than pseudomembranous or
erythematous candidiasis. Lesions are generally bilateral
and occur on the buccal mucosa near the commissures of
the lips at the level of the occlusal plane. The lesions may
also present as speckled or nodular lesions. Often biopsy is
indicated to confi rm the diagnosis because there may often
be more serious clinical signs (for example, induration or
ulceration). The frequency of epithelial dysplasia in plaque-
like/nodular candidiasis is four to fi ve times higher than that
estimated for other oral leukoplakias, and 9%–40% of
lesions develop oral cancer compared with 2%–6% in
leukoplakias in general.
Angular cheilitis
Angular cheilitis is characterized by erythema, crusting, and
cracking in the commissural regions of the lips. This
Candida-associated lesion frequently has a bacterial com-
ponent, such as Staphylococcus aureus. Predisposing factors
include defi ciency states (iron, folate, or vitamin B
12
), dia-
betes mellitus or HIV/AIDS, skin creasing resulting from
age, poor dentures with reduced vertical dimension, and
pooling of saliva in the affected areas. Angular cheilitis may
indicate that an intraoral Candida infection is present.
Median rhomboid glossitis
Another Candida-associated lesion is median rhomboid
glossitis, which often presents as a diamond-shaped lesion
on the dorsum of the tongue near the junction of the ant-
erior two-thirds and posterior one-third. An oral swab may
confi rm the presence of yeast in a mixed fl ora. A biopsy is
not necessary unless other clinical signs of major concern
are present, as the lesion often responds to antifungal
therapy.
Other Candida infections occur rarely, usually in patients
with underlying medical conditions. These infections include
chronic mucocutaneous candidiasis, cheilocandidiasis, mul-
tifocal candidiasis, and Candida endocrinopathy syndrome.
Systemic infections caused by other fungi sometimes show
oral involvement. Oral aspergillosis can involve the soft
palate, tongue, and gingiva. Lesions on the soft palate have
generally been associated with upper respiratory tract
involvement. Palatal lesions consist of oral ulceration sur-
rounded by a margin of black tissue. The gingival lesions
are painful, infl amed, and ultimately ulcerated with tissue
Table 1. Classifi cation of presentation of oral candidiasis
Candida infection
Clinical presentation
Acute
Pseudomembraneous
Multiple removable white plaques
Erythematous
Generalized redness of tissue
Chronic
Pseudomembraneous
Multiple removable white plaques
Erythematous
Generalized redness of tissue on fi tting surface of upper denture
Plaque-like/nodular
Fixed white plaques on commissures
Candida-associated
Angular cheilitis
Bilateral cracks at angles of mouth
Median-rhomboid glossitis Fixed red/white lesion, dorsum of tongue
Source: Adapted from Cannon and Firth (2006)
38
18
Cryptococcosis, caused by C. neoformans, rarely
involves the oral cavity. The oral lesion may present as
ulceration or as a nodule on the tongue, palate, gingivae, or
tooth socket following extraction. The differential diagnosis
includes squamous cell carcinoma, tuberculosis, and trau-
matic ulcer. Oral histoplasmosis may occur in either pulmo-
nary or disseminated histoplasmosis or as a primary lesion
in an otherwise healthy person. Oral histoplasmosis is
sometimes seen in patients with HIV/AIDS and may rarely
be the initial manifestation of the disease. Oral lesions can
present as single or multiple indurated ulcers or as nodular
lesions. The palate, tongue, buccal mucosa, gingiva, and lips
are the usual sites of involvement. These oral manifesta-
tions of fungal disease are rare; the most common oral
fungal diseases are forms of candidiasis.
Current treatments for oral fungal infections
There are four main antifungal drug classes with different
modes of action (Table 2). The fl uorinated pyrimidine ana-
logue 5-fl uorocytosine (5-FC) causes aberrant RNA synthe-
sis and interferes with DNA replication.
The polyenes,
such as nystatin (NYS) and amphotericin B (AMB), were
developed in the 1950s. They are heterocyclic amphipathic
molecules that insert into lipid bilayers, bind to ergosterol,
and aggregate in annuli to form pores. These pores disrupt
the fungal plasma membrane integrity and permit the effl ux
of cations such as K
+
, which results in cell death, and so the
drugs are fungicidal. Polyenes are also thought to cause
oxidative damage.
The azole antifungals interfere with
sterol biosynthesis. They inhibit the cytochrome P
450
14
α
-
lanosterol demethylase, encoded by the ERG11 gene, which
is involved in ergosterol biosynthesis. Inhibition of Erg11p
depletes the ergosterol content of membranes and results
in the accumulation of toxic sterol pathway intermediates,
which inhibit growth.
Azoles are thus usually fungistatic
for C. albicans. The fi rst azole drugs developed were the
imidazoles such as miconazole (MCZ) and ketoconazole
(KTZ).
These drugs are relatively insoluble. Imidazoles
were followed by triazoles, such as FLC, which have
increased water solubility and improved pharmacokinetic
properties. Another triazole with a wider spectrum of activ-
ity is itraconazole (ITC). The most recently developed class
of antifungals is the echinocandins. Originally obtained
from soil fungi in the 1970s, semisynthetic derivatives of the
cyclic lipopeptides have been developed such as caspofun-
gin, micafungin, and anidulafungin. These drugs non-
competitively inhibit
β
(1,3)-glucan synthase activity and
synthesis of
β
(1,3)-glucan, the major and essential compo-
nent of the fungal cell wall.
The echinocandins are only
available for parenteral application and are not currently
used for oral fungal infections.
The antifungal drugs most commonly used to treat
patients with oral fungal infections are the polyenes and
azoles.
NYS is not absorbed from the gastrointestinal tract
(GIT) and therefore it is used for topical application intra-
orally (Table 3). Unfortunately, it has an unpleasant taste,
so preparations for oral use contain fl avoring agents. NYS
comes in a number of forms including a cream, an ointment,
tablets, a suspension, a gel, a pessary, and a pastille. AMB
is also not absorbed very well from the GIT and therefore
again is generally for topical use and available in similar
formulations to NYS. AMB and NYS can be used together,
for example, NYS ointment applied to the fi tting surface of
denture and AMB lozenges in the treatment of denture-
associated chronic erythematous candidiasis, or NYS oint-
ment applied to the affected tissue and AMB lozenges in
the treatment of angular cheilitis. Not all forms of these
agents are available in all countries.
Azoles are also used for treating patients with oral can-
didiasis. MCZ is not absorbed from the GIT and is mainly
used topically. It is reported to have a bacteriostatic effect
in addition to being active against Candida and therefore is
useful in the treatment of angular cheilitis. However, it can
be absorbed topically in suffi cient amounts to interact with
warfarin drugs such as Coumadin (widely used as an anti-
coagulant). MCZ potentiates this effect, and the resultant
Table 2. Antifungal drugs, their targets, and possible resistance mechanisms
Antifungal class and examples
Primary target (mode of action)
Resistance mechanisms
Fluorinated pyrimidine analogues
e.g.,
5-Fluorocytosine
RNA and DNA synthesis (misincorporation
of 5-fl urouracil)
Mutation in Fur1p (uracil phosphoribosyl
transferase)
Polyenes
e.g.,
Nystatin
Amphotericin
B
Cell membrane ergosterol (disruption of
plasma membrane integrity, and oxidative
damage)
Induction of low membrane ergosterol content
detected in some fungi
Imidazoles
e.g.,
Miconazole
Clotrimazole
Ketoconazole
Triazoles
e.g.,
Fluconazole
Itraconazole
Ergosterol biosynthesis (inhibition of Erg11p,
involved in ergosterol biosynthesis;
conversion of Erg11p substrate into toxic
methylated sterols)
Mutations in Erg11p
Induced overexpression of Erg11p
Effl ux via ABC and MFS transporters
Tolerance to methylated sterols via mutation
in ERG3
Echinocandins
e.g.,
Caspofungin
Micafungin
Anidulafungin
Cell-wall biosynthesis (inhibition of
β
(1-3)glucan synthase)
Mutation in
β
(1-3)glucan synthase
Adapted from Cannon et al. (2009)
10
19
internal hemorrhage could potentially be fatal. Topical
preparations of clotrimazole (oral troches) and ITC (solu-
tion) can also be used for oral candidiasis.
KTZ is absorbed systemically after oral administration.
It is useful in the treatment of chronic mucocutaneous can-
didiasis and oral candidiasis in immunocompromised
patients. It can have side effects such as nausea, cutaneous
rash, pruritus, and hepatotoxicity. Because alteration to
liver function can occur, monitoring of liver enzymes is
essential during KTZ treatment. The triazole FLC has
increased water solubility and is effective in treating HIV/
AIDS-related oral candidiasis such as OPC,
which is also
a signifi cant infection in cancer patients being treated with
chemotherapy and/or radiotherapy.
The prolonged use of
azoles, however, can result in the induction, or selection, of
azole-resistant Candida strains.
Resistance of fungi to antifungal drugs
Resistance of fungi to polyenes is rare. The resistance can
be caused by a reduction in the amount of plasma membrane
ergosterol, to which polyenes bind (see Table 2). There is
primary (intrinsic) resistance in some isolates of Candida
lusitaniae, C. lipolytica, and C. guilliermondii.
terreus and A. fl avus are frequently associated with AMB
resistance in both in vitro and in vivo studies.
Although
the molecular mechanisms are not well understood, it is
clear that A. terreus has a much lower ergosterol content
than most other fungal species,
wall glucans have been shown to lead to AMB resistance in
A. fl avus.
Mutations in C. albicans ERG3, which encodes
a C-5 sterol desaturase (Erg3p), an enzyme in the ergosterol
biosynthetic pathway, lower the concentration of ergosterol
in the plasma membrane and cause AMB resistance.
These
mutations also confer cross-resistance to azoles.
There is signifi cant primary and secondary (acquired)
resistance of Candida and Aspergillus species to 5-FC, limit-
ing its utility. Resistance of clinical C. albicans isolates to
5-FC most often correlates with mutations in the enzyme
uracil phosphoribosyltransferase (Fur1p) that prevent the
conversion of 5-fl uorouracil to 5-fl uorouridine monophos-
phate (see Table 2).
Mutations in cytosine deaminase
(CaFca1p) may also contribute to resistance.
The inci-
dence of 5-FC resistance in fungi means it is primarily used
in combination with other antifungals such as AMB.
There
is a low incidence of echinocandin resistance in clinical
isolates of Candida species that are normally sensitive to
echinocandins, despite the ready in vitro selection of echi-
nocandin-resistant variants of C. albicans
or S. cerevi-
siae.
Echinocandin-resistant Candida isolates usually have
single amino acid point mutations in the
β
(1,3)-glucan syn-
thase subunit (Gsc1p) that is orthologous to S. cerevisiae
Fks1p.
There are multiple mechanisms that can give rise to
azole resistance in fungi (see Table 2). The drug target,
Erg11p, can be overexpressed or can develop point muta-
tions that reduce FLC binding.
C. albicans Erg11p that confer moderate azole resistance
are Y132H, S405F, G464S and R467K.
C. albicans growth inhibition is caused by reduction in the
ergosterol content of membranes and also by the accumula-
tion of toxic ergosterol precursors such as 14
α
-methyler-
gosta-8,24(28)-dien-3
β
,6
α
-diol. If Erg3p is inactivated by
mutation, in the presence of FLC these cells accumulate the
nontoxic sterol 14
α
-methylfecosterol.
Fungal resistance to azole drugs
High-level azole resistance in several Candida species cor-
relates with overexpression in the plasma membrane of
proteins that pump the drug out of the cell, thus reducing
intracellular azole concentrations to levels at which Erg11p
is not inhibited.
There are two main classes of effl ux
pumps: ATP-binding cassette (ABC) proteins and major
facilitator superfamily (MFS) pumps.
These membrane
proteins pump compounds across cell membranes using dif-
ferent energy sources. The ABC proteins are primary trans-
porters that use the hydrolysis of ATP. The MFS pumps are
secondary transporters that utilize the proton-motive force
across the plasma membrane.
Table 3. Antifungal treatment of oral candidiasis
Antifungal
Treatment
Candida infection
Nystatin
Cream applied to affected area 3–4 times daily
Or pastille (100 000 units) sucked after meals 4 times daily for 7 days
Or oral suspension (100 000 units) applied after meals 4 times daily for 7 days
Pseudomembraneous, erythematous,
plaque-like/nodular, angular cheilitis,
median rhomboid glossitis
Amphotericin B
Lozenge (10 mg) sucked 4 times daily for 10–28 days
Or oral suspension taken after food 4 times daily for 14 days
Pseudomembraneous, erythematous,
plaque-like/nodular, angular cheilitis,
median rhomboid glossitis
Miconazole
Oral gel applied to the affected area 3–4 times daily
Or cream applied twice daily; continue for 10 days after lesion heals
Erythematous, plaque-like/nodular,
angular cheilitis
Clotrimazole
Cream applied to the affected area 2–3 times daily for 3–4 weeks
Or solution (5 ml) 3–4 times daily for 14 days
Angular cheilitis
Ketoconazole
Tablets (200 mg) taken 1–2 times daily with food for 14 days
Chronic mucocutaneous candidiasis
Fluconazole
Capsules (100 mg) once daily for 7–14 days
Pseudomembraneous, chronic
mucocutaneous candidiasis
Itraconazole
Capsules (100 mg) once daily immediately after food for 14 days
Pseudomembraneous, chronic
mucocutaneous candidiasis
Source: Adapted from Samaranayake et al. (2009)
and Cannon and Firth (2006)
20
ABC pumps have important physiological functions and
are present in all organisms; most fungal species have mul-
tiple ABC genes.
The sequencing of the S. cerevisiae
revealed that it contains 29–30 ABC genes.
albicans has a similar number of ABC genes (27),
and C.
glabrata has approximately two-thirds that number (18).
Much larger numbers of ABC genes are found in A fumiga-
tus (49) and C. neoformans (54).
In several pathogenic
fungi the expression of ABC genes has been correlated with
increased resistance to azole antifungals. In C. albicans,
transcriptional upregulation of ABC genes CDR1 and
CDR2 has been shown, in vitro, for FLC-resistant isolates
relative to their susceptible parental strains.
pression of ABC genes in azole-resistant isolates of C. gla-
brata
and C. neoformans
has also been reported. In
C. krusei, the expression of the ABC effl ux pump ABC1 in
combination with an insensitivity of Erg11p to azoles is
thought to be responsible for its innate azole resistance
phenotype.
In general, MFS transporters are thought to contribute
less to the azole resistance of fungi than ABC pumps.
In the C. albicans genome database (CGD http://www.
candidagenome.org/),
six genes are annotated as MFS like
[MDR1 (BEN
R
), FLU1, TPO3, orf19.2350, NAG3, and
MDR97]. Of these, only Mdr1p and Flu1p have substrates
that are antifungals, and there is no evidence of FLU1
expression being associated with azole resistance in clinical
isolates. Furthermore, C. albicans Mdr1p is relatively spe-
cifi c for FLC,
whereas many azole drugs can act as sub-
strates for ABC pumps Cdr1p and Cdr2p.
Expression of
C. albicans MDR1 has been detected in both in vitro-derived
FLC-resistant mutants
and in azole-resistant clinical iso-
lates,
but it is not as frequently detected as expression
of ABC genes. In contrast, in C. dubliniensis there is a
strong association between the expression of the MFS trans-
porter Mdr1p and FLC resistance.
Most investigations of the contribution of fungal pump
expression to azole resistance have measured mRNA
expression, but this may not equate to levels of pump
protein expression, consequent to mRNA turnover. A
recent analysis of transporter protein expression in a collec-
tion of C. albicans clinical isolates with reduced FLC sus-
ceptibilities showed that ABC pump Cdr1p was expressed
in greater amounts than Cdr2p, and only one isolate showed
MFS pump Mdr1p expression.
Furthermore, it was shown
that Cdr1p rather than Cdr2p mediated most FLC effl ux
function in these clinical isolates. These studies were,
however, conducted in vitro, and the levels of pump expres-
sion required to achieve clinically signifi cant resistance, in
the complex oral environment, have not been determined.
Oral biofi lms and antifungal drug resistance
Biofi lm formation
Although saliva contains a high concentration of bacteria,
most of the microorganisms in the oral cavity are not in free
solution (planktonic) but are present in biofi lms; well-
defi ned multispecies communities embedded in a matrix
consisting largely of extracellular polymers.
Oral fungi,
which as already discussed are predominantly Candida
species, are also present in biofi lms.
can develop on medical devices such as indwelling intravas-
cular catheters, implanted devices, and prostheses and are
diffi cult to eliminate because of their antifungal drug resis-
tance.
Candida biofilms are of clinical importance in the
development of denture stomatitis,
and Candida biofi lms
can damage silicon voice prostheses and necessitate their
replacement.
The presence of Candida in the relatively
well-protected biofi lm environment may explain the ability
of the yeast to cause recalcitrant and recurrent infections.
Biofilm formation proceeds through different develop-
mental phases. The fi rst phase is microbial adhesion. C.
albicans cells can adhere to a variety of oral surfaces, includ-
ing hydroxyapatite (a model for dental enamel),
lial cells,
and can coaggregate with oral streptococci (Fig.
In many cases the adhesion of C. albicans to oral sur-
faces is promoted by saliva adsorbed either to the yeast or
to the oral surface as a pellicle.
Adhesion of C. albi-
cans to oral surfaces is mediated by cell-surface adhesins,
which are often mannoproteins. Well-characterized adhe-
sions implicated in biofi lm formation include Hwp1p
(hyphal wall protein)
and Als (agglutinin-like sequence)
family members such as Als3p.
C. albicans Bcr1p, a zinc
fi nger transcription factor, plays a signifi cant role in biofi lm
formation by controlling Als3p expression.
Other adhesins
such as Als1p, Hwp1p, and Ece1 (extent of cell elongation)
involved in biofi lm formation are also under the control
of Bcr1p.
Fungi present in saliva are often in an ovoid
yeast morphology. Once C. albicans yeast cells adhere onto
a surface, they often form germ tubes that extend into
hyphae and pseudohyphae, and the biofi lm matures and
becomes enclosed in a matrix of extracellular polysaccha-
ride (Fig. 2).
E
B
GT
C
B
M
Fig. 1. Cryo-scanning electron micrograph of Candida albicans cells
growing as a multispecies biofi lm on buccal epithelial cells. C, C. albi-
cans cell; B, bacteria; E, epithelial cell, GT, C. albicans germ tube; M,
extracellular matrix. Bar 5
μ
m. (Kindly provided by Professor M.
Tokunaga)
21
It is well known that microorganisms in biofi lms “com-
municate” and interact through the secretion and detection
of diffusible chemicals such as quorum-sensing molecules
(QSMs) and bacteriocins.
C. albicans is known to secrete
a number of quorum-sensing molecules. The best-studied
C. albicans QSM is farnesol, which represses hyphal forma-
tion at high cell density and inhibits biofi lm formation.
Farnesol is continually released from the cells during
growth, and its rate of accumulation is roughly proportional
to the cell density.
It has been proposed that QSMs are
secreted by cells in the late stages of biofi lm formation to
inhibit hyphal formation when the cell concentration is high
and therefore promote the dispersal of yeast cells to colo-
nize new environments.
Fungi, such as C. albicans, interact with bacteria within
biofi lms (see Figs. 1, 2). Synergistic and antagonistic interac-
tions occurring between Candida and bacteria contribute to
the development of mixed-species biofi lm communities. For
example, diffusible substances from Streptococcus gordonii
can overcome the hyphal-inhibitory effect of farnesol and
promote hyphal growth and biofi lm formation, leading to a
synergy in biofi lm formation.
In contrast, the interaction
between C. albicans and Pseudomonas aeruginosa is antag-
onistic; P. aeruginosa secretes homoserine lactones, QSMs
that inhibit hyphal formation and eventually kill C.
albicans.
Antifungal drug resistance of biofi lms
Candida cells in biofi lms display different phenotypes com-
pared to their planktonic counterparts in terms of their
markedly enhanced resistance to antifungal agents and pro-
tection from host defenses.
Compared to planktonic cells,
Candida biofi lms have been demonstrated to show increased
resistance to a variety of antifungal agents including AMB,
FLC, ITC, and KTC.
Minimum (growth) inhibitory con-
centrations (MICs) of such antifungals for biofi lm cells are
commonly determined using growth assays that measure
the metabolic reduction of dye 2,3-bis(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT).
The
precise mechanisms by which Candida biofilms resist the
actions of antifungal agents are not known, although several
factors have been proposed: these include altered metabolic
activity of biofi lm cells, the presence of extracellular materi-
als that reduce antifungal penetration, expression of resis-
tance genes, and the presence of a subpopulation of resistant
“persister” cells.
Comparison of the susceptibility of the
C. albicans biofi lms to AMB with that of planktonic cells
grown at the same rates in continuous culture using a
chemostat bioreactor showed that biofi lm cells were more
resistant than planktonic cells.
resistance of biofilm cells to antifungals, including AMB,
FLC, chlorhexidine, and NYS, increased with the matura-
tion of the biofi lm. The increased drug resistance was asso-
ciated with the concomitant increase in metabolic activity
of developing biofilms.
Antifungal drug resistance is acquired early in biofi lm
formation and appears to be governed by different mecha-
nisms in early and late biofi lms. The C. albicans genes
encoding ABC (Cdr1p, Cdr2p) and MFS (Mdr1p) trans-
porters are upregulated in the early stage of biofi lm forma-
tion.
However, mutants lacking one or more of the drug
effl ux pumps were FLC resistant at later stages of biofi lm
formation.
Therefore, effl ux pumps may play a role in
FLC resistance only in the early phase of biofi lm develop-
ment.
A phase-dependent expression of ABC pump
genes CDR1 and CDR2 has also been described during C.
glabrata biofi lm formation.
The sterol composition of C.
albicans biofi lms and planktonic cells has been investigated.
A signifi cantly decreased level of ergosterol in biofi lm cells
has been reported.
In contrast, an overexpression of both
ERG25 and ERG11 (also known as ERG16) was observed
for later stages of biofi lm formation.
Altered ergosterol
content of fungal cells could affect their polyene susceptibil-
ity. An in vivo biofi lm model showed that CDR1 and CDR2
mRNAs were increased in biofi lm cells, but ERG11 and
MDR1 expression did not appear to be affected in either
biofi lm or planktonic cells.
It is important to note that
fungal cells within biofi lms will experience stress because of
changes in pH and metabolic products from other microor-
ganisms. Such stress can induce physiological and genetic
responses that confer drug resistance.
Drug exclusion by the extracellular matrix is considered
to be another possible resistance mechanism for Candida
biofilms.
The extracellular matrix contains carbohy-
drate, protein, hexosamine, phosphorus, and uronic acid
and may act as a physical barrier to prevent penetration of
antifungal agents to their target sites. Indeed, it has been
shown that the matrix made a significant contribution to
drug resistance in Candida biofilms and that the composi-
tion of the matrix materials was an important resistance
determinant.
A putative role of
β
(1,3)-glucans in C. albi-
cans biofi lm resistance has been suggested because
β
(1,3)-
glucan levels increased signifi cantly in biofi lm yeast cell
walls and matrix material, compared to their planktonic
counterparts, and because exogenous biofi lm matrix and
soluble
β
(1,3)-glucan reduced the activity of FLC against
A recent study suggests that the zinc-
responsive transcription factor Zap1p, which regulates
genes encoding glucoamylases and alcohol dehydrogenases,
may control matrix formation including the amount of
β
(1,3)-glucan in C. albicans biofi lms.
“Persister” cells are
Antifungal (AF)
AF
AF
AF
Inter-microbial interactions
Biofilm extracellular
matrix
Bacteria
Yeast
Efflux
pump
Salivary
pellicle
Oral
surface
C. albicans
germ tube
Fig. 2. Interactions between fungi and other microorganisms in an oral
biofi lm that could result in antifungal (AF) resistance
22
a subpopulation of cells that can remain viable in the pres-
ence of antimicrobial drugs. Candida biofi lms have been
found to contain highly antifungal-tolerant persister cells
that are not found in planktonic cultures.
However, the
mechanism of survival of persister cells in the biofi lm is not
clearly understood. Taken together, these observations
clearly indicate that the antifungal drug resistance of bio-
fi lms is a multifactorial phenomenon.
The incorporation of fungi in oral biofi lms presents clini-
cal problems for eradication of the fungus using antifungals.
The newer classes of antifungal agents such as the echino-
candins and lipid formations of AMB, however, appear
effective against Candida biofi lms,
although there is a
report that Candida isolates from urine that were grown as
biofi lm-associated cell phenotypes were resistant to caspo-
fungin.
Other novel approaches to the control of fungal
biofi lms include the use of anti-
β
-glucan antibodies, which
have been shown to inhibit Candida adhesion and growth.
Also, the combination of calcineurin inhibitor FK506 or
cyclosporine A with FLC, which renders FLC fungicidal in
planktonic cells,
may be effective against the so-called
biofi lm persisters and could be an alternative effective anti-
fungal regimen to inhibit the development of Candida
biofi lm formation.
Conclusion
The majority of oral fungal infections are forms of candidia-
sis that respond relatively well to polyene treatment. Azoles
provide advantages in terms of their solubility and reduced
nephrotoxicity, but fungi can exhibit innate or acquired azole
resistance. The resistance of oral fungi to azoles has two com-
ponents: one is the resistance of individual cells, and the
other is resistance conferred by growth as a biofi lm. The
mechanisms of azole resistance occurring in monoculture in
vitro are well understood, and high-level resistance is often
caused by the expression of effl ux pumps. The nature of anti-
fungal resistance seen clinically in vivo is more complex: it is
multifactorial and in part the result of growth as a biofi lm.
Although effl ux pumps may play a role in azole resistance
early in biofi lm development, interactions with other micro-
organisms, the biofi lm extracellular matrix, and the response
of the fungi to stress all contribute to the drug resistance of
biofi lms. Therefore, the simple prescription of antifungal
drugs has limitations. Improved treatment of patients with
oral fungal disease may be achieved by physical removal of
biofi lms or treatments that prevent biofi lm development,
such as inhibiting fungal adhesion or cell–cell communica-
tion, in combination with fungicidal antifungal drugs.
Acknowledgments The authors gratefully acknowledge funding from
the National Institutes of Health, USA (R01DE016885), the Founda-
tion for Research Science and Technology of New Zealand (IIOF grant
UOOX0607) and a Health Science Research Grant for Research on
Emerging and Re-emerging Infections Diseases (H19-Shinko-8) from
the Ministry of Health, Labour and Welfare of Japan. The authors are
grateful to Dr. A. Holmes for her critical evaluation of the manuscript
and to Professor M. Tokunaga for providing the electron micrograph
used in Fig. 1.
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