Molecular Studies on Voriconazole-Resistance in a Clinical Isolate of aspergillus fumigatus.

E. MANAVATHU, A. ESPINEL-INGROFF, G. ALANGADEN, P. CHANDRASEKAR

Author address: 

Wayne State University, Detroit, MI, 2 Medical College of Virginia, Virginia Commonwealth University, Richmond, VA

Abstract: 

aspergillus fumigatus MCV/VCU-350 (AF-350) is a clinical isolate obtained from a patient suffering from invasive aspergillosis. In vitro susceptibility studies showed that AF-350 is highly resistant to voriconazole (VCZ) and ravuconazole (MIC-0 = 16 µg/ml), moderately resistant to itraconazole (ITZ) (MIC-0 = 2 µg/ml) and posaconazole (MIC-0 = 1 µg/ml), but susceptible to amphotericin B (MIC-0 = 0.5 µg/ml) and the echinocandins caspofungin and micafungin (MIC-2 = 0.03-0.06 µg/ml). Growth of AF-350 was not inhibited at low concentrations (0.25-2 µg/ml) of VCZ and complete inhibition of growth was obtained only at 16 µg/ml. Using 3H-ITZ and a highly sensitive bioassay for VCZ we determined the accumulation of ITZ and VCZ in AF-350 mycelia and the results were compared with that of the susceptible isolate W73355. Both the susceptible isolate and AF-350 accumulated similar amounts of ITZ and VCZ suggesting that efflux may not be responsible for VCZ resistance in AF-350. To further investigate the possible mechanism(s) of VCZ resistance in AF-350, we characterized the CYP51A and CYP51B genes encoding P450 14a-sterol demethylases A (CYP51Ap) and B (CYP51Bp) in A. fumigatus. The amino acid sequences were deduced. The primary structures of CYP51Ap and CYP51Bp were compared with those of ten VCZ susceptible clinical isolates obtained from the United States (n = 4), South Africa (n = 3) and India (n = 3). A single nucleotide mutation at codon 448 (GGT to AGT) resulting in G448S was found in CYP51Ap of AF-350, but not in any of the susceptible clinical isolates. No amino acid change was found in AF-350 CYP51Bp. G448 of A. fumigatus CYP51Ap is homologous to G464 of Candida albicans CYP51p which is presumed to be in the heme-binding region associated with the active site of demethylase. These data show that G448S variation is a critical amino acid change responsible for VCZ resistance in the clinical isolate of A. fumigatus.
2003

abstract No: 

M-392

Full conference title: 

43rd Interscience Conference on Antimicrobial Agents
    • ICAAC 43rd