Ergosterol is one of the major sterol components of the yeast cell membrane and is responsible for maintaining cell integrity and function. It is the target for antifungal agents, principally AMB, whose mechanism of action is by binding to it. Therefore, cells with less content of ergosterol may lead to resistance to this compound. We studied the ergosterol content of 5 Cryptococcus neoformans (Cn) isolates from different episodes of a recurrent cryptococcal meningitis. All strains were clonally related when studied by hybridization with the CNRE 1 probe. During a 16 month period, the patient received initial AMB therapy and long term fluconazole (FCZ) treatment with doses of 400, 800, 1200 and 1600 mg/day. MICs for AMB, flucytosine, FCZ and itraconazole were determined and timed kill curves (TKC) against AMB were performed for each isolate. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane and then ergosterol was identified by its unique spectrophotometric absorbance profile (220-300 nm). Cn ATCC 90012 and ten Cn clinical isolates were used as control strains. All assays were performed by duplicate. For the first-episode isolate ergosterol content was 0.046 Â± 0.004 %w/w(%ergosterol/wet weight) similar to control ATCC strain (0.045 Â± 0.005 %w/w) and to the average of clinical isolates (0.040 Â± 0.009 %w/w). In isolates from 4th and 5th episode, after prolonged treatment with FCZ, no ergosterol could be detected. MICs of the five recurrent isolates did not show resistance for any of the antifungals tested. However, tolerance for AMB was observed in these 5 strains when using TKC. These in vivo observations seemed to correlate with reports demonstrating that in vitro preexposure of yeasts to azoles induces a decrease in ergosterol content and could ultimately lead to changes in antifungal tolerance.
Full conference title:
- ICAAC 41st