Candidal infections are common opportunistic infections in compromised patients and manifest both as superficial and systemic diseases. Superficial infections are by far the most prevalent. For instance, 60% of denture wearers over the age of 60 years in countries such as Denmark suffer from Candida-associated denture stomatitis, and more than 90% of HIV-infected individuals develop oral candidosis at some time during their disease.

The adhesion of micro-organisms to host mucosal surfaces is a prerequisite for successful colonization and infection, and its importance in the pathogenesis of many fungal infections is widely recognized. Attachment enables the organisms to avoid dislodgement by the cleansing action of mucosal secretions, and facilitates infection. Various in vitro studies and animal studies have provided evidence for the propensity of Candida spp. to adhere to mucosal surfaces and their presence in infections. The process of candidal adhesion to host surfaces is rather complex, and involves both biological and non-biological factors such as adhesins, London–van der Waals attraction forces and hydrophobic interactions. Cell-surface hydrophobicity is considered to be a physical force involved in non-specific candidal adhesion that is of a lower affinity than specific interactions. It is believed that hydrophobic interactions are inextricably involved in the adherence of both bacteria and fungi to host surfaces. Thus a number of workers have studied this phenomenon in Candida spp.

Candidal stomatitis is most commonly treated with the polyene antifungal agent nystatin, which is used topically. Imidazoles such as ketoconazole and the newer triazole fluconazole are used to treat the condition if recalcitrant. But with the increasing number of patients compromised by HIV infection, haematological malignancy, and aggressive cytotoxic therapy the use of other antimycotics such as the DNA analogue, 5-fluorocytosine, has also become popular.

Despite the availability of a spectrum of antifungal agents for the treatment of oropharyngeal candidosis, failure of therapy is not uncommon as the efficacy of treatment is dependent upon many factors. In the mouth the diluent effect of saliva and the cleansing action of the oral musculature often tend to reduce the availability of the agents to below that of the effective therapeutic concentration. Thus the organisms are only suboptimally exposed to the antifungal agent and the drug concentration is likely to vary in different niches of the mouth. Moreover, the formation of Candida biofilms on oral surfaces may also contribute to the failure of drug therapy. Another reason for failure is the inaccessibility of topical antimycotics to fungal elements within the epithelium.

There are a few studies on the effect of antibiotics on the cell-surface hydrophobicity of bacteria. Though it is recognizsed that this hydrophobicity is an important attribute in the process of candidal adhesion to biological and non-biological surfaces, no information is available on the effects of antimycotics on the surface hydrophobicity of oral C. albicans isolates after limited exposure to subcidal concentrations. As the yeasts undergo only a limited exposure to the antifungals during topical treatment of oral candidosis, and as agents that modulate the surface hydrophobicity of the organisms might alter their pathogenic potential, our main aim now was to compare the cell-surface hydrophobicity of 10 oral C. albicans isolates after their limited exposure to subcidal concentrations of nystatin, 5-fluorocytosine, ketoconazole and fluconazole.

While previous researchers have demonstrated a positive correlation between the surface hydrophobicity of Candida spp. and their adherence to buccal epithelial cells, it is not clear how this relation is affected by limited exposure of yeasts to antimycotics. Thus, our secondary aim was to correlate the findings from the hydrophobicity studies with those for candidal adhesion from one of our previous investigations that employed identical experimental procedures and C. albicans isolates.

Ten isolates of oral C. albicans were studied: BM 20617, BS 742, BU 1010, BU 47204 and CA 0202 were from HIV-positive patients, and isolates designated 128981 LA, 106083, 118416, 108315 and 98917 were from HIV-negative patients. The organisms were identified by the germ-tube test and the commercially available API 20 (API System, Vercieu, France) Candida identification kits. Stock cultures were maintained at ?20°C. After recovery these were maintained on Sabouraud dextrose agar, stored at 4–6°C, during the experimental period.

Nystatin (Sigma) was dissolved in dimethylsulphoxide and absolute ethanol (3:2 ratio). Ketoconazole (Janssen, Beerse, Belgium) and fluconazole (Pfizer, Groton, CT, USA) were dissolved in dimethylsulphoxide, while 5-fluorocytosine (Sigma) was dissolved in sterile distilled water. All agents were prepared initially as 10,000?g/ml solutions and stored at ?20°C before use. During the 1-h exposure, the antifungals were suspended/diluted in RPMI 1640 medium buffered with 0.165 M MOPS [3-(N-morpholino) propanesulphonic acid] containing -glutamine and lacking sodium bicarbonate (Sigma), dissolved in 1 liter of sterile distilled water and adjusted to a pH of 7.2 and filter-sterilized. This liquid RPMI 1640 was stored at 2–8°C for 2–3 months.

The MIC obtained with the 10 isolates of C. albicans in a previous study were used here. In brief, the MIC for the 10 isolates with the four drugs were determined by the broth dilution technique according to McGinnis and Rinaldi (1996), by performing 2-fold serial dilutions of the drug in microtitre plates using an inoculum of 1–5×10⁵ c.f.u./ml. The MIC was determined visually and spectrophotometrically at 595 nm after 24 h of incubation at 37°C. The MIC was defined as the lowest concentration of the drug that inhibited the growth of yeast cells, as indicated by the absence of turbidity (optically clear). In determining the MIC for ketoconazole and fluconazole, a slight modification of the technique was used as these drugs exhibited a phenomenon known as trailing. Trailing occurs when the turbidity continually decreases as the concentration of the drug increases but the suspension fails to become optically clear. Thus, for ketoconazole and fluconazole the MIC was considered as the lowest concentration of the drug with a slight haziness in the well. Subculturing from optically clear/turbid wells was used to confirm the turbidity results.

All experiments were repeated on two separate occasions with duplicate determinations on each occasion.

Yeast cells, maintained on Sabouraud dextrose agar, were inoculated on to fresh plates and incubated overnight for 24 h before use. The organisms were harvested and a suspension prepared in sterile PBS, pH 7.4, at 520 nm to an optical density of 1.5. From this suspension, 1 ml was added to tubes containing 4 ml of RPMI broth (control) and 4 ml of RPMI/drug solution (test) in which the drug concentrations varied from four to eight times the MIC. The subcidal concentrations used were: nystatin ×6 MIC, 5-fluorocytosine ×8 MIC, ketoconazole ×4 MIC, and fluconazole ×4 MIC. This gave a cell suspension of 10⁶–10⁷ cells/ml in each assay tube.

The tubes were then incubated at 37°C for 1 h in a rotary incubator. After this limited exposure the drugs were removed by two cycles of dilution with sterile PBS and centrifugation for 10 min at 3000×g. The supernatant was then completely decanted and the pellets resuspended in 5 ml of sterile PBS.

Viable counts of the control and the test were done by spiral plating after drug removal and control suspensions were reconstituted as needed to obtain a cell concentration comparable to the test.