Evaluation of Amphotericin B (AB) Activity Against Candida Spp Biofilms (BF) Using Metabolic Indicators (MTT, XTT and Resazurin).


Author address: 

Hosp. Vall d'Hebron., Barcelona, Spain.


Background:Candida spp BF are involved in refractory to treatment infections of prosthetic materials, and show reduced susceptibility to antifungals. AB activity against BF of Candida spp strains isolated from patients with catheter-related candidaemia was evaluated using tetrazolium salts (MTT, XTT) and the redox indicator resazurin (RES) Methods:2 C. tropicalis (CT),5 C. parapsilosis (CP) and 9 C. albicans (CA) strains were studied. BF were generated inoculating microtiter plates with a McFarland 2 yeast suspension in A3 broth+8% glucose. Growth and sterility (blank) controls were included. Plates were incubated (24 h, 35ºC), washed and filled with A3 broth containing two-fold dilutions of AB (128-0.25 mg/L). After incubation (35ºC, 24 h) plates were washed and A3 containing MTT 0.56 mg/L, XTT 0.2 mg/L + Menadione 0.1 μM or RES 0.0015% as appropriate was added. After incubation (3h,35ºC)absorbances of MTT (570 nm) and XTT (492 nm) and fluorescence of RES (excitation 530 nm; emission 590 nm) were read. Blank values were subtracted from those obtained for wells containing BF. % of metabolic activity (MA) of BF exposed to AB respect to MA of drug free controls was calculated. Concentrations inhibiting 50 and 90% of MA were considered BF Inhibitory Concentrations (BIC) 50 and 90 respectively. Studies were run in triplicate. Results:With MTT, geometric mean (GM) BIC50 was 0.25 mg/L for all 3 species; BIC90 was 2.33 (CA), 2.30 (CP), and 1.41 (CT). With XTT, GM BIC50/90 were 8.64/>128 (CA); 0.44/5.28 (CP) and 0.71/64 (CT). With RES MG BIC50/90 were >128/>128 (CA), 8/128 (CP) and 0.5/>128 (CT) Conclusions:Detection of AB activity against Candida BF varies depending on the metabolic indicator. 100% inhibition of MA was not achieved with AB in the range of concentrations studied; at the highest concentrations, AB was less active

abstract No: 


Full conference title: 

46th Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 46th