Odontology (2010) 98:15–25 DOI 10.1007/s10266-009-0118-3

© The Society of The Nippon Dental University 2010

REVIEW ARTICLE

Masakazu Niimi · Norman A. Firth · Richard D. Cannon

Antifungal drug resistance of oral fungi

Received: November 9, 2009 / Accepted: November 28, 2009

Abstract Fungi comprise a minor component of the oral microbiota but give rise to oral disease in a significant proportion 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 candidiasis is the use of polyenes, such as nystatin and amphotericin B, and azoles including miconazole, fluconazole, 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. albicans can acquire azole resistance. The main mechanism of high-level fungal azole resistance, measured in vitro, is energy-dependent drug efflux. Most fungi in the oral cavity, however, are present in multispecies biofilms that typically demonstrate an antifungal resistance phenotype. This resistance is the result of multiple factors including the expression of efflux pumps in the fungal cell membrane, biofilm matrix permeability, and a stress response in the fungal cell. Removal of dental biofilms, or treatments to prevent biofilm development in combination with antifungal drugs, may enable better treatment and prevention of oral fungal disease. Key words Oral candidiasis · Antifungal drug resistance · Biofilms

Introduction Oral fungal infections affect a significant, and increasing, proportion of the population.1 Fungi are a minor component of the oral microbial flora but certain species, mostly belonging to the Candida genus, are routinely present at low concentrations without causing infection.2,3 These commensal fungi are opportunistic pathogens and can cause

disease when their host becomes immunocompromised. These infections can be superficial 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 afflict people whose immune systems have been suppressed. Oropharyngeal candidiasis (OPC) is common in acquired immunodeficiency syndrome (AIDS) patients who do not have access to highly active antiretroviral therapy (HAART),4 whereas oral candidiasis often affects cancer patients undergoing chemotherapy and/or radiotherapy.5 When fungi penetrate the epithelial surfaces of immunocompromised hosts, they can cause invasive fungal infections (IFIs) that are associated with high morbidity and mortality. The fungal genera most often associated with IFIs are Candida, Aspergillus, and Cryptococcus.6 Patients with oral fungal infections are often treated with polyene or azole antifungal drugs.1 Although azoles, such as fluconazole (FLC), have better solubility and less nephrotoxicity than polyenes, fungal azole resistance can be a problem.7 Candida glabrata and C. krusei are innately less susceptible to azoles than other Candida species, and C. albicans can develop azole resistance. The molecular mechanisms of fungal azole drug resistance have been studied extensively,7–10 but mostly in vitro. The majority of fungal cells in the oral cavity, however, are associated with biofilms, and microorganisms in biofilms are often more resistant to antimicrobials than planktonic cells.11–13 This review investigates the contribution of molecular resistance mechanisms and biofilm growth to the antifungal drug resistance of oral fungi.

Oral fungi Presence of fungi in the oral cavity

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: [email protected]

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

16

estimates are based on data from culture-independent molecular analyses of samples from healthy and diseased mouths.16 Such molecular techniques allow the identification of microbial species that are difficult to culture and can measure small genetic differences between related phylotypes. One of the most commonly used methods to identify bacteria in the oral cavity is the cloning and sequencing of 16S rRNA genes amplified by polymerase chain reaction (PCR) from DNA extracted from oral samples.17,18 This analysis has been taken to a greater level of sensitivity by the advent of high-throughput pyrosequencing of PCRamplified DNA, which does not require cloning steps. A recent pyrosequencing analysis of bacterial 16S rDNA extracted from human saliva and plaque identified 3621 and 6888 species-level phylotypes, respectively.14 Other techniques used to identify and quantify oral bacteria include real-time quantitative PCR (qPCR) and checkerboard DNA–DNA hybridization.18,19 There have been relatively few similar investigations of fungi present in the oral cavity. Classically, yeast have been identified from saliva, oral swabs, plaque, oral rinses, or concentrated oral rinses by culturing them on selective or differential agar.3,20 Such techniques can use chromogenic primary isolation media such as CHROMagar for the presumptive identification of the most prominent Candida species including C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. tropicalis, and in some instances C. parapsilosis.21–23 Other culture-based growth, morphology, and biochemical tests are available in kit format for the identification of fungi isolated from clinical samples. These kits include API ID 32C, API 20C AUX, and RapID Yeast Plus.23,24 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.25 Molecular identification 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,26,27 26S rDNA sequencing,28 PCR,25,29 and qPCR.30 Early culturebased 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%.31 The mean carriage rate was higher in patients (47.0%), reflecting 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.31 C. albicans comprised approximately 70% of all isolates; the next most common species were C. tropicalis, C. glabrata, C. parapsilosis, C. krusei, C. kefyr, and C. guilliermondii. These studies were carried out before the discovery of C. dubliniensis.32 This species is very similar, genotypically and phenotypically, to C. albicans33 and so was probably counted as C. albicans before its discovery. These culture-based results have been confirmed by recent molecular studies with C. albicans being identified as the most prevalent oral yeast, followed by C. glabrata, C. dubliniensis, C. tropicalis, C. krusei, C. parapsilosis, C. famata,

C. guilliermondii, and in some cases Saccharomyces cerevisiae.28–30 Although other fungi that cause respiratory diseases, 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 flora. Thus, the oral flora contains relatively few fungal species. In addition, there are very few yeast cells in mouths compared to bacterial cells. The oral cavity presents many surfaces 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.2,3,34,35 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.34 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, unpublished data); this compares with a bacterial concentration of approximately 107–108/ml saliva.36,37 Despite the low concentrations 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.2,3 These oral fungi are, therefore, opportunistic pathogens.

Oral fungal infections Oral candidiasis is the most common oral mucosal fungal infection.1 There are a number of presentations of oral candidiasis, which can be classified as acute or chronic, according 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 longterm users of corticosteroid inhalers (asthmatics), it can be chronic. It most frequently affects infants, the elderly, and the terminally ill.1 It may also be an indicator of an underlying serious medical condition such as diabetes, leukemia, other malignancy, or human immunodeficiency virus (HIV) infection/AIDS. The clinical appearance consists of nonadherent creamy white patches or flecks, 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.38 The pseudomembranes consist of a mesh of Candida hyphae, entangled with desquamated epithelial cells, fibrin, keratin, necrotic debris, and bacteria. If pseudomembranous candidiasis extends to the pharynx, it is termed oropharyngeal candidiasis (OPC). This condition afflicts many AIDS patients39 and is also a significant infection in cancer patients being treated with chemotherapy and/or radiotherapy.5,40 OPC is frequently the first clinical symptom in HIV-positive patients, before the onset of overt AIDS.40 In cancer patients, the increased incidence of OPC results from both the debilitating effects

17 Table 1. Classification of presentation of oral candidiasis Candida infection Acute Pseudomembraneous Erythematous Chronic Pseudomembraneous Erythematous Plaque-like/nodular Candida-associated Angular cheilitis Median-rhomboid glossitis

Clinical presentation Multiple removable white plaques Generalized redness of tissue Multiple removable white plaques Generalized redness of tissue on fitting surface of upper denture Fixed white plaques on commissures Bilateral cracks at angles of mouth Fixed red/white lesion, dorsum of tongue

Source: Adapted from Cannon and Firth (2006)38

of the cancer itself and from the immunosuppressive treatment for the cancer.

Erythematous candidiasis Erythematous candidiasis, similar to pseudomembranous candidiasis, may be acute or chronic depending on its duration. The acute form frequently follows a course of broadspectrum 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.1 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.1 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 colonize the dental acrylic.41 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% chlorhexidine gluconate or 1% sodium hypochlorite overnight.38

Plaque-like/nodular candidiasis Plaque-like/nodular candidiasis (which is also called chronic hyperplastic candidiasis or candidal leukoplakia) is characterized 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 confirm the diagnosis because there may often be more serious clinical signs (for example, induration or ulceration). The frequency of epithelial dysplasia in plaquelike/nodular candidiasis is four to five 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.38

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 component, such as Staphylococcus aureus. Predisposing factors include deficiency states (iron, folate, or vitamin B12), diabetes 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 anterior two-thirds and posterior one-third. An oral swab may confirm the presence of yeast in a mixed flora. 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, multifocal 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 surrounded by a margin of black tissue. The gingival lesions are painful, inflamed, and ultimately ulcerated with tissue

18 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 Polyenes e.g., Nystatin Amphotericin B Imidazoles e.g., Miconazole Clotrimazole Ketoconazole Triazoles e.g., Fluconazole Itraconazole Echinocandins e.g., Caspofungin Micafungin Anidulafungin

RNA and DNA synthesis (misincorporation of 5-flurouracil) Cell membrane ergosterol (disruption of plasma membrane integrity, and oxidative damage) Ergosterol biosynthesis (inhibition of Erg11p, involved in ergosterol biosynthesis; conversion of Erg11p substrate into toxic methylated sterols)

Mutation in Fur1p (uracil phosphoribosyl transferase) Induction of low membrane ergosterol content detected in some fungi

Cell-wall biosynthesis (inhibition of β(1-3)glucan synthase)

Mutation in β(1-3)glucan synthase

Mutations in Erg11p Induced overexpression of Erg11p Efflux via ABC and MFS transporters Tolerance to methylated sterols via mutation in ERG3

Adapted from Cannon et al. (2009)10

necrosis.38 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 traumatic ulcer. Oral histoplasmosis may occur in either pulmonary 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 manifestations 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 fluorinated pyrimidine analogue 5-fluorocytosine (5-FC) causes aberrant RNA synthesis and interferes with DNA replication.7,8 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 efflux of cations such as K+, which results in cell death, and so the drugs are fungicidal. Polyenes are also thought to cause oxidative damage.7,9,42 The azole antifungals interfere with sterol biosynthesis. They inhibit the cytochrome P450 14αlanosterol 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.7,8 Azoles are thus usually fungistatic for C. albicans. The first azole drugs developed were the imidazoles such as miconazole (MCZ) and ketoconazole

(KTZ).43 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 activity 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 caspofungin, micafungin, and anidulafungin. These drugs noncompetitively inhibit β(1,3)-glucan synthase activity and synthesis of β(1,3)-glucan, the major and essential component of the fungal cell wall.44 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.1 NYS is not absorbed from the gastrointestinal tract (GIT) and therefore it is used for topical application intraorally (Table 3). Unfortunately, it has an unpleasant taste, so preparations for oral use contain flavoring 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 fitting surface of denture and AMB lozenges in the treatment of dentureassociated chronic erythematous candidiasis, or NYS ointment 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 candidiasis. 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 sufficient amounts to interact with warfarin drugs such as Coumadin (widely used as an anticoagulant). MCZ potentiates this effect, and the resultant

19 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 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 Pseudomembraneous, erythematous, plaque-like/nodular, angular cheilitis, median rhomboid glossitis Erythematous, plaque-like/nodular, angular cheilitis Angular cheilitis

Amphotericin B

Miconazole

Ketoconazole Fluconazole

Oral gel applied to the affected area 3–4 times daily Or cream applied twice daily; continue for 10 days after lesion heals 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 Tablets (200 mg) taken 1–2 times daily with food for 14 days Capsules (100 mg) once daily for 7–14 days

Itraconazole

Capsules (100 mg) once daily immediately after food for 14 days

Clotrimazole

Chronic mucocutaneous candidiasis Pseudomembraneous, chronic mucocutaneous candidiasis Pseudomembraneous, chronic mucocutaneous candidiasis

Source: Adapted from Samaranayake et al. (2009)1 and Cannon and Firth (2006)38

internal hemorrhage could potentially be fatal. Topical preparations of clotrimazole (oral troches) and ITC (solution) can also be used for oral candidiasis. KTZ is absorbed systemically after oral administration. It is useful in the treatment of chronic mucocutaneous candidiasis 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,39 which is also a significant infection in cancer patients being treated with chemotherapy and/or radiotherapy.5,40 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.42 Aspergillus terreus and A. flavus are frequently associated with AMB resistance in both in vitro and in vivo studies.45–49 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,48,49 and alterations in cellwall glucans have been shown to lead to AMB resistance in A. flavus.42 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.50 These mutations also confer cross-resistance to azoles.7,50 There is significant primary and secondary (acquired) resistance of Candida and Aspergillus species to 5-FC, limiting 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-fluorouracil to 5-fluorouridine monophosphate (see Table 2).8 Mutations in cytosine deaminase (CaFca1p) may also contribute to resistance.51 The incidence of 5-FC resistance in fungi means it is primarily used in combination with other antifungals such as AMB.52 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 echinocandin-resistant variants of C. albicans53,54 or S. cerevisiae.55 Echinocandin-resistant Candida isolates usually have single amino acid point mutations in the β(1,3)-glucan synthase subunit (Gsc1p) that is orthologous to S. cerevisiae Fks1p.56,57 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 mutations that reduce FLC binding.7,8,58,59 Common mutations in C. albicans Erg11p that confer moderate azole resistance are Y132H, S405F, G464S and R467K.60–62 Azole-induced C. albicans growth inhibition is caused by reduction in the ergosterol content of membranes and also by the accumulation of toxic ergosterol precursors such as 14α-methylergosta-8,24(28)-dien-3β,6α-diol. If Erg3p is inactivated by mutation, in the presence of FLC these cells accumulate the nontoxic sterol 14α-methylfecosterol.

Fungal resistance to azole drugs High-level azole resistance in several Candida species correlates 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.59,61,63 There are two main classes of efflux pumps: ATP-binding cassette (ABC) proteins and major facilitator superfamily (MFS) pumps.10 These membrane proteins pump compounds across cell membranes using different energy sources. The ABC proteins are primary transporters that use the hydrolysis of ATP. The MFS pumps are secondary transporters that utilize the proton-motive force across the plasma membrane.

20

ABC pumps have important physiological functions and are present in all organisms; most fungal species have multiple ABC genes.10 The sequencing of the S. cerevisiae genome64 revealed that it contains 29–30 ABC genes.65,66 C. albicans has a similar number of ABC genes (27),67 and C. glabrata has approximately two-thirds that number (18).68 Much larger numbers of ABC genes are found in A fumigatus (49) and C. neoformans (54).69,70 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.71–73 Overexpression of ABC genes in azole-resistant isolates of C. glabrata74,75 and C. neoformans76,77 has also been reported. In C. krusei, the expression of the ABC efflux pump ABC1 in combination with an insensitivity of Erg11p to azoles is thought to be responsible for its innate azole resistance phenotype.78,79 In general, MFS transporters are thought to contribute less to the azole resistance of fungi than ABC pumps.10 In the C. albicans genome database (CGD http://www. candidagenome.org/),80 six genes are annotated as MFS like [MDR1 (BENR), 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 specific for FLC,7,81 whereas many azole drugs can act as substrates for ABC pumps Cdr1p and Cdr2p.82 Expression of C. albicans MDR1 has been detected in both in vitro-derived FLC-resistant mutants63 and in azole-resistant clinical isolates,61,73,83 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 transporter Mdr1p and FLC resistance.84 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 collection of C. albicans clinical isolates with reduced FLC susceptibilities showed that ABC pump Cdr1p was expressed in greater amounts than Cdr2p, and only one isolate showed MFS pump Mdr1p expression.85 Furthermore, it was shown that Cdr1p rather than Cdr2p mediated most FLC efflux function in these clinical isolates. These studies were, however, conducted in vitro, and the levels of pump expression required to achieve clinically significant resistance, in the complex oral environment, have not been determined.

defined multispecies communities embedded in a matrix consisting largely of extracellular polymers.11 Oral fungi, which as already discussed are predominantly Candida species, are also present in biofilms.13,86 Candida biofilms can develop on medical devices such as indwelling intravascular catheters, implanted devices, and prostheses and are difficult to eliminate because of their antifungal drug resistance.87 Candida biofilms are of clinical importance in the development of denture stomatitis,41 and Candida biofilms can damage silicon voice prostheses and necessitate their replacement.86,88 The presence of Candida in the relatively well-protected biofilm environment may explain the ability of the yeast to cause recalcitrant and recurrent infections. Biofilm formation proceeds through different developmental phases. The first phase is microbial adhesion. C. albicans cells can adhere to a variety of oral surfaces, including hydroxyapatite (a model for dental enamel),89 to epithelial cells,90 and can coaggregate with oral streptococci (Fig. 1).91 In many cases the adhesion of C. albicans to oral surfaces is promoted by saliva adsorbed either to the yeast or to the oral surface as a pellicle.89,90,92,93 Adhesion of C. albicans to oral surfaces is mediated by cell-surface adhesins, which are often mannoproteins. Well-characterized adhesions implicated in biofilm formation include Hwp1p (hyphal wall protein)94 and Als (agglutinin-like sequence) family members such as Als3p.95 C. albicans Bcr1p, a zinc finger transcription factor, plays a significant role in biofilm formation by controlling Als3p expression.96 Other adhesins such as Als1p, Hwp1p, and Ece1 (extent of cell elongation) involved in biofilm formation are also under the control of Bcr1p.96 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 biofilm matures and becomes enclosed in a matrix of extracellular polysaccharide (Fig. 2).13

B

M C GT

E

B

Oral biofilms and antifungal drug resistance Biofilm 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 biofilms; well-

Fig. 1. Cryo-scanning electron micrograph of Candida albicans cells growing as a multispecies biofilm on buccal epithelial cells. C, C. albicans cell; B, bacteria; E, epithelial cell, GT, C. albicans germ tube; M, extracellular matrix. Bar 5 μm. (Kindly provided by Professor M. Tokunaga)

21 Inter-microbial interactions Antifungal (AF)

Biofilm extracellular matrix

AF

Bacteria

AF Efflux pump

Yeast

AF

Oral surface

C. albicans germ tube Salivary pellicle

Fig. 2. Interactions between fungi and other microorganisms in an oral biofilm that could result in antifungal (AF) resistance

It is well known that microorganisms in biofilms “communicate” and interact through the secretion and detection of diffusible chemicals such as quorum-sensing molecules (QSMs) and bacteriocins.11 C. albicans is known to secrete a number of quorum-sensing molecules. The best-studied C. albicans QSM is farnesol, which represses hyphal formation at high cell density and inhibits biofilm formation.97,98 Farnesol is continually released from the cells during growth, and its rate of accumulation is roughly proportional to the cell density.97 It has been proposed that QSMs are secreted by cells in the late stages of biofilm formation to inhibit hyphal formation when the cell concentration is high and therefore promote the dispersal of yeast cells to colonize new environments.99 Fungi, such as C. albicans, interact with bacteria within biofilms (see Figs. 1, 2). Synergistic and antagonistic interactions occurring between Candida and bacteria contribute to the development of mixed-species biofilm communities. For example, diffusible substances from Streptococcus gordonii can overcome the hyphal-inhibitory effect of farnesol and promote hyphal growth and biofilm formation, leading to a synergy in biofilm formation.100 In contrast, the interaction between C. albicans and Pseudomonas aeruginosa is antagonistic; P. aeruginosa secretes homoserine lactones, QSMs that inhibit hyphal formation and eventually kill C. albicans.101

Antifungal drug resistance of biofilms Candida cells in biofilms display different phenotypes compared to their planktonic counterparts in terms of their markedly enhanced resistance to antifungal agents and protection from host defenses.12 Compared to planktonic cells, Candida biofilms have been demonstrated to show increased resistance to a variety of antifungal agents including AMB, FLC, ITC, and KTC.102 Minimum (growth) inhibitory concentrations (MICs) of such antifungals for biofilm cells are commonly determined using growth assays that measure the metabolic reduction of dye 2,3-bis(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT).103 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 biofilm cells, the presence of extracellular materials that reduce antifungal penetration, expression of resistance genes, and the presence of a subpopulation of resistant “persister” cells.12,102 Comparison of the susceptibility of the C. albicans biofilms to AMB with that of planktonic cells grown at the same rates in continuous culture using a chemostat bioreactor showed that biofilm cells were more resistant than planktonic cells.104 The emergence of drug resistance of biofilm cells to antifungals, including AMB, FLC, chlorhexidine, and NYS, increased with the maturation of the biofilm. The increased drug resistance was associated with the concomitant increase in metabolic activity of developing biofilms.105 Antifungal drug resistance is acquired early in biofilm formation and appears to be governed by different mechanisms in early and late biofilms. The C. albicans genes encoding ABC (Cdr1p, Cdr2p) and MFS (Mdr1p) transporters are upregulated in the early stage of biofilm formation.106,107 However, mutants lacking one or more of the drug efflux pumps were FLC resistant at later stages of biofilm formation.98,108 Therefore, efflux pumps may play a role in FLC resistance only in the early phase of biofilm development.98,107 A phase-dependent expression of ABC pump genes CDR1 and CDR2 has also been described during C. glabrata biofilm formation.109 The sterol composition of C. albicans biofilms and planktonic cells has been investigated. A significantly decreased level of ergosterol in biofilm cells has been reported.107 In contrast, an overexpression of both ERG25 and ERG11 (also known as ERG16) was observed for later stages of biofilm formation.110 Altered ergosterol content of fungal cells could affect their polyene susceptibility. An in vivo biofilm model showed that CDR1 and CDR2 mRNAs were increased in biofilm cells, but ERG11 and MDR1 expression did not appear to be affected in either biofilm or planktonic cells.111 It is important to note that fungal cells within biofilms will experience stress because of changes in pH and metabolic products from other microorganisms. Such stress can induce physiological and genetic responses that confer drug resistance.112 Drug exclusion by the extracellular matrix is considered to be another possible resistance mechanism for Candida biofilms.12,102 The extracellular matrix contains carbohydrate, 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 composition of the matrix materials was an important resistance determinant.113 A putative role of β(1,3)-glucans in C. albicans biofilm resistance has been suggested because β(1,3)glucan levels increased significantly in biofilm yeast cell walls and matrix material, compared to their planktonic counterparts, and because exogenous biofilm matrix and soluble β(1,3)-glucan reduced the activity of FLC against planktonic cells.114 A recent study suggests that the zincresponsive 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 biofilms.115 “Persister” cells are

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a subpopulation of cells that can remain viable in the presence of antimicrobial drugs. Candida biofilms have been found to contain highly antifungal-tolerant persister cells that are not found in planktonic cultures.116 However, the mechanism of survival of persister cells in the biofilm is not clearly understood. Taken together, these observations clearly indicate that the antifungal drug resistance of biofilms is a multifactorial phenomenon. The incorporation of fungi in oral biofilms presents clinical problems for eradication of the fungus using antifungals. The newer classes of antifungal agents such as the echinocandins and lipid formations of AMB, however, appear effective against Candida biofilms,117–119 although there is a report that Candida isolates from urine that were grown as biofilm-associated cell phenotypes were resistant to caspofungin.120 Other novel approaches to the control of fungal biofilms include the use of anti-β-glucan antibodies, which have been shown to inhibit Candida adhesion and growth.121 Also, the combination of calcineurin inhibitor FK506 or cyclosporine A with FLC, which renders FLC fungicidal in planktonic cells,122 may be effective against the so-called biofilm persisters and could be an alternative effective antifungal regimen to inhibit the development of Candida biofilm formation.123

Conclusion The majority of oral fungal infections are forms of candidiasis 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 components: one is the resistance of individual cells, and the other is resistance conferred by growth as a biofilm. The mechanisms of azole resistance occurring in monoculture in vitro are well understood, and high-level resistance is often caused by the expression of efflux pumps. The nature of antifungal resistance seen clinically in vivo is more complex: it is multifactorial and in part the result of growth as a biofilm. Although efflux pumps may play a role in azole resistance early in biofilm development, interactions with other microorganisms, the biofilm extracellular matrix, and the response of the fungi to stress all contribute to the drug resistance of biofilms. Therefore, the simple prescription of antifungal drugs has limitations. Improved treatment of patients with oral fungal disease may be achieved by physical removal of biofilms or treatments that prevent biofilm development, such as inhibiting fungal adhesion or cell–cell communication, in combination with fungicidal antifungal drugs. Acknowledgments The authors gratefully acknowledge funding from the National Institutes of Health, USA (R01DE016885), the Foundation 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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