Discrimination between susceptible and resistant strains of Candida spp. to amphotericin B

Vitale, R., Meletiadis J, Meis J, Rodriguez Tudela JL, De Hoog S,;P., Verweij


Aim: To evaluate the effect of medium and pH and the ability to distnguish between strains that are susceptible and resistance to amphotericin B. Methods: A total of 14 Candida species were evaluated, 6 resistant to AB (4 C. lusitaneae, 1 C. albicans and 1 C. tropicalis) and 8 susceptible to amphotericin B (AB) (4 C. albicans, 3 C. parapsilosis and 1 C. lusitaneae). Candida krusei ATCC 6258 and parapsilosis ATCC 22019 were used as control strains. The following media were used: RPMI and AM3 buffered with MOPS at pH 5 and 7; AM3 buffered with acetate at pH 5 and buffered with phosphate at pH 7. Glucose was added to each media to achieve a final concentration of 2%. AB concentration ranged between to 16-0,016 µg/ml. Inocula were preparated spectrophotometrically by adjusting the transmitance to 75-77% in order to obtain 1-5 x 106 CFU/ml. The microtitration plates were inoculated with a final inoculum of 1-5 x 103 CFU/ml and inculated at 35ºC for 24 and 48 h. The MIC was defined as the lowest concentration of drug that inhibited all visible growth. The pH of the drug-free well was measured after 24 and 48h. Minimal fungicidal concentration (MFC) was also determined by subculturing 0.1 ml from each well with no visible growth onto drug-free Sabouraud agar plates. All data were analyzed by Mann Whitney test. Results: The differences in geometric means between the MICs of susceptible and resistant strains were always higher when AM3 used than RPMI 1640. For AM3 after 24h the differences were larger at pH 5 than pH 7 with the largest difference axhibited in AM3 buffered with MOPS (3 times, p=0.004). After 48 h the discrimination between susceptible and resistant strains was better at pH 7 with the largest difference exhibited in AM3 buffered with phosphate (3.19 times, p=0.0013) although the AM3 buffered with MOPS showed similar results (2.96 times, p=0.0007). When RPMI 1640 beffered at pH 7 with MOPS used, the difference were larger after 24h (1.33 times, p=0.0027) than after 48h but always lower compared with AM3. The discrimination in MFC better in AM3 compared with RPMI especially when it was buffered with phosphate at pH 7 (2.38 times, p=0.02). Concerning of the measurement of pH, pH 7 was more stable than pH 5. At pH 5 the MIC’s were higher than those at pH 7.

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6th Congress of the European Confederation of Medical Mycology Societies
    • ECMM 6th (2000)