Investigation of the susceptibility of Candida species isolated from denture wearers to different antifungal antibiotics

The aim of this study was to determine the the prevalence of in vitro resistance amongst Candida species isolated from the oral cavity of denture wearers. The in vitro susceptibility of 156 Candida isolates to amphotericin B, fluconazole, 5fluorocytosine, caspofungin and terbinafine was determined. The Clinical Laboratory Standards Institute’ (CLSI; formally National Committee for Clinical Laboratory Standards) broth microdilution method was used and MIC50 and MIC90 determined. Candida albicans, the most frequently isolated strains, are sensitive to amphotericin (61%) and fluconazole (44%), frequently used agents in the treatment of Candida-associated denture stomatitis. A 100% susceptibility to 5-fluorocytosine was observed among the 109 isolates of C. albicans. Among non C. albicans strains only 1 Candida kefyr strain was determined as susceptible dependent upon dose for 5-fluorocytosine. Among Candida glabrata, the second most common isolate, a 100% susceptibility to caspofungin and 5-fluorocytosine were observed. Since the isolates are sensitive to Caspofungin and 5-fluorocytosine, rarely used in the treatment of oral fungal infections, it is suggested that these antifungal agents be used as alternative medicine in the treatment of oral infections especially caused by strains resistant to amphotericin B and fluconazole.

Since Candida species, which are among the normal flora microorganisms of human body, are opportunist pathogens, they can cause different clinical manifestations of candidiasis (Scardina et al., 2007).Oral candidiasis is the most common mycotic infection in the oral cavities in humans.Long-term use of prosthesis is the most important risk factor for Candida species colonization of the mucosal surfaces, this may be sufficient for the development of oral candidiasis.Oral fungal infection that causes Candida associated prosthesis stomatitis is a common disease in 50 to 60% of denture wearers (Budtz-Jorgensen, 2000;Darwazeh et al., 2001;Pıres et al., 2002).*Corresponding author.E-mail: ozlemabaci@yahoo.com.
There are several antifungal medicines which can be used topically or systemically in the treatment of oral candidiasis.(i) Polyenes (amphotericin B and nistatin) form complexes with ergosterol which open channels in the fungal membrane that cause leakage of critical intracellular constituents and subsequent cell death.(ii) Azoles (fluconazole, itraconazole, ketoconazole, etc) which inhibits cellular membrane formation by interfering with ergosterol synthesis.(iii) Another chemical employed against Candida albicans is 5-fluorocytosine whose entry to the cell is mediated by the cytosine permease.This compound is transformed to 5-fluorourasil by cytosine deaminase.Incorporation of 5-fluorourasil into RNA interrupts protein synthesis leading to cell death (Ghannoum and Rice, 1999;Farah et al., 2000;Casalınuovo et al., 2004).The purpose of this work was to determine the minimum inhibitory concentration (MIC) of amphotericin B, fluconazole, 5-fluorocytosine, caspofungin, terbinafine for Candida species isolated from individuals wearing prosthesis.

Yeast strains
156 isolates of Candida spp.were previously isolated from saliva samples, smear from palatal mucosa and dorsum of the tongue were taken from total of 110 individuals (being treated for prosthodontic treatment in Ege University, Dental Faculty) -30 individuals wearing total prosthesis, 30 removable partial prostheses, 30 fixed prostheses and 20 with natural teeth.Candida species isolated were identified by germ tube and clamydospores production, and commercially available API 20C AUX yeast identification system (BioMerieux, France) (Abaci et al., 2010).Prior to antifungal susceptibility testing, all Candida spp.were subcultured at least twice on Sabouraud dextrose agar (SDA) plates.

Quality control
Quality control was ensured by testing the CLSI recommended quality control strains Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258.

Antifungal susceptibility studies
Broth microdilution (BMD) testing was performed in accordance with the quidelines in CLSI document M27-A2 by using the spectrophotometric method of inoculum preparation, an inoculum concentration of 1.5(±1.0)x10 3 cells/ml, and RPMI 1640 medium buffered with MOPS.A 0.1-ml yeast inoculum was added to each well of the microdilution trays.
The final concentrations of the antifungal agents were 0.0625 to 32 µg/ml for amphotericin B, 0.125 to 64 µg/ml for fluconazole, 0.125 to 64 µg/ml for 5-fluorocytosine, 0.007 to 8 µg/ml for caspofungin, and 0.025 to 128 µg/ml for terbinafine.The trays were incubated at 35°C, and MIC were read after 48 h.Drug-free and yeast free controls were included.
Following incubation, the BMD wells were examined with aid of a reading mirror and the growth in each well was compared the inhibition of growth control well.The MICs were read for fluconazole Abaci and Haliki-Uztan 1399 and caspofungin as the lowest concentration of antifungal that inhibited 50% growth of the organism detected visually.The MIC of amphotericin B was defined as the lowest drug concentration causing 100% inhibition of fungal growth, 5-fluorocytosine were defined as the lowest drug concentrations at least 80% inhibition (CLSI M27-A2, 2002).Terbinafine were defined as the lowest drug concentrations at least 80% inhibition (Ryder et al., 1998;Moore et al., 2001).Antifungal activity was expressed as the MIC of each isolate to the drug.The following resistance breakpoints were used according to CLSI or based on previous investigation.
(iv) Caspofungin: The CLSI-approved MIC breakpoint for caspofungin susceptibility is 2 g/ml.There is no intermediate or dose-dependent category (Brown and Traczewski, 2008).
(v) Terbinafine: Clinically relevant breakpoints are currently not available for terbinafine.However, Ryder et al. (1998) evaluated that a breakpoint of >8 µg/ml was taken to indicate in vitro resistance.

RESULTS
109 C. albicans strains, 16 C. glabrata strains, 10 C. kefyr strains, 6 C. tropicalis strains, 3 C. sphaerica strains, 3 C. famata strains and 2 S. cerevisiae strains were identified (Abaci et al., 2010).The result for the three quality control organisms were within the quoted reference ranges (Table 1).Amphotericin B, fluconazole, 5-fluorocytosine, caspofungin and terbinafine MIC ranges of Candida species and MIC 50 and MIC 90 values, whose susceptibility are evaluated in the study, are shown in Table 2. MIC 50 : MIC at which 50% of the isolates were inhibited; MIC 90 : MIC at which 90% of isolates were inhibited.

D SCUSS ON
The most effective agents used in the treatment of Candida species are the antifungal agents belonging to polyene and azole groups.Nystatin, amphotericin B, myconazole, fluconazole, itraconazole are generally used in the management of prostheses stomatitis (Farah et al., 2000;Akpan and Morgan, 2002).Although there are several treatment alternatives, widespread administration of antifungal agents has caused fungal pathogens resistant to one or more agents to emerge.In order to avoid problems regarding resistant fungi and to develop prophylactic and therapeutic strategies, it is vital to understand the nature of antifungal drug resistance (Rogers, 2006).
It was also determined that amphotericin B prevents C. albicans adhesion to buccal epithelial cells and germ tube formation in lower concentrations.In addition, dental adhesion of denture acrylic treated with amphotericin B is also greatly prevented.The former effects may be due to the mechanism of action of amphotericin B on the candidal cell wall, while the latter may be due to the blocking of the yeast attachment sites on the denture acrylic by the drug.Subminimal inhibitory concentrations (sub-MIC) of amphotericin B reduce proteinases activity of oral C. albicans isolates (Ellepola and Samaranayake, 2000).
Fluconazole and amphotericin B are frequently used in the treatment of oral candidiasis (Rogers, 2006).However, amphotericin B have some undesirable sideeffects, such as significant renal toxicity, while prophylactic exposure to fluconazole can lead to resistance or overgrowth by naturally resistant organisms like C. krusei and C. glabrata.The limitations have led to a search for more effective antifungals.Caspofungin is a echinocandin and act by inhibiting of the synthesis of 1,3ß-D-glucan in the fungal cell wall.Caspofungin is as effective as conventional amphotericin B for treating mucosal and systemic candidiasis (Cannon et al., 1995;Nicholas et al., 2004;Chenn and Sorrell, 2007).It is known that caspofungin is as effective as fluconazole in the treatment of thrush and esophagitis.It was determined that fluconazole-resistant C. albicans isolates collected from esophagitis patients were sensitive to caspofungin (Hernandez et al., 2004).It was observed that the treatment of azole-resistant oral-esophageal candidiasis cases seen in AIDS patients with caspofungin was successful (Jokela and Kaur, 2007).In our study only 1 (0.9%) of 109 C. albicans strains was resistant to caspofungin.We also found that all of the non-albicans Candida strains were susceptible.
Terbinafine hydrochloride [(Ter-HCl), (E)-N-(6,6dimethyl-2-hepten-4-yn-yl)-N-methyl-1naphthalenemethanamide hydrochloride], is a new potent antifungal agent of the allylamine class which selectively inhibits fungal squalene epoxidase.It has a broadspectrum activity against yeast, fungi, molds (eg.Aspergillus and Penicillium species) and dermatophytes and is indicated for both oral and topical treatment of mycoses (Özcan et al., 2009).Ryder and coworkers indicates that terbinafine has good activity against at least some azole-resistant C. albicans strains.Using 80% inhibition of growth as the assay end point, clear and reproducible MICs were obtained of terbinafine for the C. albicans.In our study, all Candida isolates were determined as resistant to terbinafine, except 2 C. krusei strains were susceptible.In accordance with our study, Figueiredo et al. (2007) determined that terbinafine presented low activity for strains of C. albicans and C. tropicalis isolated from fingernail infection.It is thought that CDR1, CDR2 and BEN r which are responsible for fluconazole resistance may all impact resistance to terbinafine.CDR1 can use terbinafine as a substrate (Ghannoum and Rice, 1999).
5-fluorocytosine is rarely used in the management of oral candidiasis.The DNA analogue 5-fluorocytosine is a fungistatic agent which is highly effective against all Candida spp.In a study conducted by Blignaut et al. (2002) in order to determine the susceptibility of 589 Candida strains they collected from HIV/AIDS patients' and healthy individuals' mouths to antifungal substances, they found that only 2.3% of C. albicans strains were resistant to 5FC.In our study, all C. albicans strains were sensitive to 5-fluorocytosine.Of all the non-albicans Candida strains, only 2 C. krusei strains and 1 C. kefyr strain were found to be susceptible dependent upon dose.5 -fluorocytosine transported into the fungal cell by the action of cytosine permease, and inside the cell, is converted to form the active metabolite 5-fluorouracil by cytosine deaminase.5-fluorouracil is then incorporated into RNA in place of uracil, with resulting abnormalities of protein synthesis.In addition, it blocks thymidylate synthetase, causing inhibition of DNA synthesis.Resistance to 5-fluorocytosine may result from decreased uptake or loss of enzymatic activity responsible for conversion to 5-fluorouridylic acid (FUMP).Since the mammalian cells lack cytosine deaminase, they are not affected by the drug (Ghannoum, 1999).
It is known that different oral infections such as hyperplasic candidiasis (CHC) can develop in denture wearers.CHC development is especially of importance in terms of malignancy development in that area.Because, C. albicans induces neoplastic developments by inducting the production of carcinogenic nitrosamines in the saliva (Sitheeque and Samaranayake, 2003;Hadjieva et al., 2006).It is seen that while some of the isolates we collected were resistant to antifungal agents such as amphotericin B and fluconazole which are frequently used in the treatment of Candida-associated denture stomatitis, the isolates we collected were susceptible to caspofungin and 5 -fluorocytosine which are rarely used in the treatment of oral fungal infections.Thus, it was concluded that these antifungal agents could be used as alternative drugs in oral infections which develop due to strains especially due to strains resistant to amphotericin B and fluconazole.

Table 2 .
Minimum inhibitory concentration and MIC50 and 90 values obtained for amphotericin B, caspofungin, fluconazole, 5fluorocytosine and terbinafine for Candida spp.by the CLSI methods.

Table 2 .
Contd.It has been in use since the 1950s.It has a broad spectrum of activity.There have only been few reports on resistant C. albicans isolates.In our study, 56% (n=61) of C. albicans was found to be susceptible to amphotericin B. Recently there have been reports on resistant C. glabrata and C. krusei isolates.Resistant isolates have also been found in C. tropicalis, C. parapsilosis, and C. lusitaniae.C. glabrata is considered as intermediate or susceptible dependent upon dose